Pseudomonas plecoglossicida fliP gene affects the immune response of Epinephelus fuscoguttatus ♀×Epinephelus lanceolatus ♂ to infection.

Pseudomonas plecoglossicida is a pathogen that causes visceral white spot disease in a variety of teleosts. The protein encoded by fliP gene is involved in the assembly of bacterial flagella, which plays a vital role in bacterial pathogenicity. However, the roles of the fliP gene on the host immune response remain unclear. Here, we compared the pathogenicity of fliP gene-deleted (ΔfliP) strain, fliP gene-complemented (C-ΔfliP) strain and wild-type (NZBD9) strain of P. plecoglossicida to hybrid grouper (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), and explored the impacts of fliP gene on the immune response of hybrid grouper to P. plecoglossicida infection by using RNA-seq. In this study, the grouper in the ΔfliP strain-infected group had a 30% higher survival rate than those in the NZBD9 strain-infected group. In addition, the deletion of fliP gene decreased bacterial load in the spleen, intestine, liver as well as head kidney of hybrid grouper and the tissues damage were weakened. Moreover, the infection of hybrid grouper spleen by the ΔfliP strain induced 1,189 differential expression genes compared with the counterpart infected by NZBD9 strain. KEGG enrichment analysis showed that 9 immune-related pathways, 5 signal transduction pathways, and 3 signaling molecules and interaction pathways were significantly enriched. qRT-PCR analysis revealed that the ΔfliP strain mainly up-regulated the expression of inflammation related genes (IL-6, IL-12, IL-1ß, IL-10, CXCL8, CXCL10) and immune regulation related genes (TLR2, P65, MyD88, P85, AKT), but down-regulated the expression of cell death related genes (FoxO1, Bim, PLK2 and LDHA) during infection. Based on the above results, fliP gene contributed to the pathogenicity of P. plecoglossicida to hybrid grouper (E. fuscoguttatus ♀ × E. lanceolatus ♂), deletion of fliP gene promoted the inflammation and immune response of hybrid grouper to P. plecoglossicida infection, which accelerating host clearance of pathogen and reducing tissue damages.

Function of the rpoD gene in Pseudomonas plecoglossicida pathogenicity and Epinephelus coioides immune response.

Pseudomonas plecoglossicida is a Gram-negative pathogenic bacterium that causes visceral white spot disease in several marine fish species, resulting in high mortality and financial loss. Based on previous RNA sequencing (RNA-seq) results, rpoD gene expression is significantly up-regulated in P. plecoglossicida during infection, indicating that rpoD may contribute to bacterial pathogenicity. To investigate the role of this gene, five specific short hairpin RNAs (shRNAs) were designed and synthesized based on the rpoD gene sequence, with all five mutants exhibiting a significant decrease in rpoD gene expression in P. plecoglossicida. The mutant with the highest silencing efficiency (89.2%) was chosen for further study. Compared with the wild-type (WT) P. plecoglossicida strain NZBD9, silencing rpoD in the rpoD-RNA interference (RNAi) strain resulted in a significant decrease in growth, motility, chemotaxis, adhesion, and biofilm formation in P. plecoglossicida. Silencing of rpoD also resulted in a 25% increase in the survival rate, a one-day delay in the onset of death, and a significant decrease in the number of white spots on the spleen surface of infected orange-spotted groupers (Epinephelus coioides). In addition, rpoD expression and pathogen load were significantly lower in the spleens of E. coioides infected with the rpoD-RNAi strain than with the WT strain of P. plecoglossicida. We performed RNA-seq of E. coioides spleens infected with different P. plecoglossicida strains. Results showed that rpoD silencing in P. plecoglossicida led to a significant change in the infected spleen transcriptomes. In addition, comparative transcriptome analysis showed that silencing rpoD caused significant changes in complement and coagulation cascades and the IL-17 signaling pathway. Thus, this study revealed the effects of the rpoD gene on P. plecoglossicida pathogenicity and identified the main pathway involved in the immune response of E. coioides.

The contribution of exbB gene to pathogenicity of Pseudomonas plecoglossicida and its interactions with Epinephelus coioides.

To study the roles of the exbB gene in Pseudomonas plecoglossicida during interactions with Epinephelus coioides, five short hairpin RNAs (shRNAs) were designed and synthesized to silence the exbB gene in P. plecoglossicida which resulted in significant reductions in exbB mRNA expression. The mutant with the best silencing efficiency (89.3%) was selected for further study. Silencing exbB in the exbB-RNA interference (RNAi) strain resulted in a 70% increase in the survival rate and a 3-day delay in the onset of infection in E. coioides. Silencing of the exbB gene also resulted in a significant decrease in the number of white spots on the spleen surface and in the spleen pathogen load. The results of dual RNA-seq showed that exbB silencing in P. plecoglossicida also resulted in a significant change in both the pathogen and host transcriptomes in the spleens of infected E. coioides. Comparative transcriptome analysis showed that silencing exbB caused significant changes in multiple signaling molecules and interaction- and immune system-related genes in E. coioides. Gene silencing also resulted in the differential expression of flagellar assembly and the bacterial secretion system in P. plecoglossicida during the infection period, and most of the DEGs were down-regulation. These host-pathogen interactions may make it easier for E. coioides to eliminate the exbB-RNAi strain of P. plecoglossicida, suggesting a significant decrease in the pathogenicity of this strain. These results indicated that exbB was a virulence gene of P. plecoglossicida which contributed a lot in the pathogen-host interactions with E. coioides.

Integration of RNA-seq and RNAi reveals the contribution of znuA gene to the pathogenicity of Pseudomonas plecoglossicida and to the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important pathogen in aquaculture and causes serious economic losses. Our previous study indicated that znuA gene might play an important role in the pathogenicity of P. plecoglossicida. Five shRNAs were designed and synthesized to silence the znuA gene of P. plecoglossicida. Two of the five mutants of P. plecoglossicida exhibited significant reduction in the expression level of znuA mRNA with different efficiencies. The mutant with the highest silencing efficiency of 89.2% was chosen for further studies. Intrapleural injection of the znuA-RNAi strain at a dose of 105  cfu/fish did not cause the death of Epinephelus coioides, and no significant signs were observed at the spleen surface of infected E. coioides, while the counterpart E. coioides infected by the same dose of wild-type strain of P. plecoglossicida all died in 5 days post-infection (dpi). The expression of znuA gene of znuA-RNAi strain in E. coioides was always lower than that in wild-type strain of P. plecoglossicida. The pathogen load in the early stage of infection was higher than that in the later stage of infection. Although the infection of the znuA-RNAi strain of P. plecoglossicida could induce the production of antibodies in E. coioides, it failed to produce a good immune protection against the infection of wild-type strain of P. plecoglossicida. Compared with the transcriptome data of E. coioides infected by the wild-type strain of P. plecoglossicida, the transcriptome data of E. coioides infected by the znuA-RNAi strain of P. plecoglossicida have altered significantly. Among them, KEGG enrichment analysis showed that the focal adhesion pathway was significantly enriched and exhibited the largest number of 302 DEMs (differentially expressed mRNAs). These results showed that the immune response of E. coioides to P. plecoglossicida infection was significantly affected by the RNAi of znuA gene.

Dual RNA-seq reveals the effect of the flgM gene of Pseudomonas plecoglossicida on the immune response of Epinephelus coioides.

Pseudomonas plecoglossicida is an important and highly pathogenic bacterium for aquaculture and causes serious losses. The expression level of flgM was found to be significantly upregulated post-infection compared with in vitro results, which was confirmed by quantitative real-time PCR. RNAi significantly reduced the expression level of flgM mRNA of P. plecoglossicida. Compared with infection with the wild-type strain, infection with the flgM-RNAi strain resulted in a delay in death and a 75% reduction in the mortality of Epinephelus coioides, followed by alleviation of the symptoms in E. coioides spleen. Moreover, compared with infection with the wild-type strain, infection with the flgM-RNAi strain of P. plecoglossicida resulted in a significant change in the transcriptome of the spleens of infected E. coioides and P. plecoglossicida. KEGG analysis for E. coioides showed that genes of 17 immune pathways were most affected by flgM-RNAi of P. plecoglossicida. Among them, the expression of mhc2, zap70, rhoh, tlr2, ca79a, hcst and cd32 in E. coioides spleen was predicted to be negatively related to flgM in P. plecoglossicida but positively related to genes involved in communication, metabolism and motility.

Dual RNA-Seq reveals the role of a transcriptional regulator gene in pathogen-host interactions between Pseudomonas plecoglossicida and Epinephelus coioides.

Pseudomonas plecoglossicida is a highly pathogenic bacterium for maricultured fish and causes serious losses. A transcriptional regulator gene RK21_RS10315 was found up-regulated during the whole infection process, which was confirmed by qRT-PCR. Five shRNA were designed to silence RK21_RS10315 gene, and the gene expression was reduced up to 96.1%. Compared with the counterpart infected with wild type strain, the infection of RK21_RS10315-RNAi strain resulted in the death time delay, and 90% reduction in mortality of Epinephelus coioides, as well as the alleviation in the symptoms of E. coioides spleen. Moreover, compared with the fish infected with wild type strain, the infection of RK21_RS10315-RNAi strain of P. plecoglossicida resulted in a significant change both in transcriptome of spleen of infected E. coioides and P. plecoglossicida. The KEGG analysis showed that genes of 16 immune pathways in E. coioides were affected by the silence of RK21_RS10315 of P. plecoglossicida. Among them, intestinal immune network for IgA production pathway and leukocyte transendothelial migration pathway were more prominent than other pathways. 19 euk-DEMs in these immune pathways had varying degrees of correlation with 19 pro-DEMs, and the expression of ipxA, grpE, yhbJ, truD and suhB from 19 pro-DEMs were predicted more related to RK21_RS10315 in P. plecoglossicida.

Transcriptomic and metabolomic insights into the role of the flgK gene in the pathogenicity of Pseudomonas plecoglossicida to orange-spotted grouper (Epinephelus coioides).

Pseudomonas plecoglossicida is the pathogen responsible for visceral white spot disease in large yellow croaker (Larimichthys crocea) and orange-spotted grouper (Epinephelus coioides). Previously, RNA sequencing showed that P. plecoglossicida flgK gene expression was significantly up-regulated in orange-spotted grouper spleens during infection. To explore the role of flgK in P. plecoglossicida pathogenicity, RNA interference (RNAi) was performed to silence the P. plecoglossicida flgK gene, and the mutant (flgK-RNAi strain) with the best silencing efficiency (89.40%) was chosen for further study. Results showed that flgK gene silencing significantly attenuated P. plecoglossicida motility, adhesion, and biofilm formation. Compared to those fish infected with the wild-type strain of P. plecoglossicida, orange-spotted grouper infected with the flgK-RNAi strain showed a 55% increase in the survival rate and a one-day delay in time of first death, with fewer pathogens in the spleen and fewer white spots on the spleen surface. RNAi of flgK significantly affected the transcriptome and metabolome of the spleen in infected orange-spotted grouper. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the C-type lectin receptor signaling pathway was the most significantly changed immune-related pathway and the mitogen-activated protein kinase (MAPK) signaling pathway was related to multiple immune-related pathways. Furthermore, arginine biosynthesis and glycerophospholipid metabolism were the most significantly changed metabolism-related pathways. These findings suggest that flgK is a virulence gene of P. plecoglossicida. Furthermore, flgK appears to be involved in the regulation of motility, adhesion, and biofilm formation in P. plecoglossicida, as well as in the regulation of inflammatory and immune responses of orange-spotted grouper to P. plecoglossicida infection.

The Effect of tonB Gene on the Virulence of Pseudomonas plecoglossicida and the Immune Response of Epinephelus coioides.

Pseudomonas plecoglossicida is the causative agent of "visceral white spot disease" in cultured fish and has resulted in serious economic losses. tonB gene plays a crucial role in the uptake of nutrients from the outer membranes in Gram-negative bacteria. The previous results of our lab showed that the expression of tonB gene of P. plecoglossicida was significantly upregulated in the spleens of infected Epinephelus coioides. To explore the effect of tonB gene on the virulence of P. plecoglossicida and the immune response of E. coioides, tonB gene of P. plecoglossicida was knocked down by RNAi; and the differences between the wild-type strain and the tonB-RNAi strain of P. plecoglossicida were investigated. The results showed that all of the four mutants of P. plecoglossicida exhibited significant decreases in mRNA of tonB gene, and the best knockdown efficiency was 94.0%; the survival rate of E. coioides infected with the tonB-RNAi strain was 20% higher than of the counterpart infected with the wild strain of P. plecoglossicida. Meanwhile, the E. coioides infected with the tonB-RNAi strain of P. plecoglossicida carried less pathogens in the spleen and less white spots on the surface of the spleen; compared with the wild-type strain, the motility, chemotaxis, adhesion, and biofilm formation of the tonB-RNAi strain were significantly attenuated; the transcriptome data of E. coioides infected with the tonB-RNAi strain were different from the counterpart infected with the wild strain of P. plecoglossicida; the antigen processing and presentation pathway and the complement and coagulation cascade pathway were the most enriched immune pathways. The results indicated that tonB was a virulence gene of P. plecoglossicida; tonB gene was involved in the regulation of motility, chemotaxis, adhesion, and biofilm formation; tonB gene affected the immune response of E. coioides to P. plecoglossicida infection.

[Nonradioactive iodine-labeled antibodies-inductively coupled plasma mass spectrometry for immunoassay].

In the present study, the system of nonradioactive iodine-labeled-antibodies linking inductively coupled plasma mass spectrometry for immunoassay was reported. The goat-anti-Escherichia coli and goat anti rabbit were considered as simulant antigen and antibody respectively in order to establish a new method of immunoassay by inductively coupled plasma mass spectrometry which has the advantage of high sensitivity, low detection limit and preferable linearity range. During the experiment, the N-bromosuccinimide, a mild oxidant, was used to oxidize the non-radioactive iodine (127 I) that labeled the protein. The method of nonradioactive iodine labeled protein was established and the best labeling condition was explored. The compound of I was purified by Sephadex G50 column chromatography, then the stability and activity were examined. The results showed that the labeling program was simple, reaction time was within two minutes, the labeling yield achieved 63.12% and none of I shed from the compound after 96 hours. The simulant antigen and antibody reacted on polystyrene microtiter plate and the I was detected by ICP-MS, the detection limit of the method was 0.12 mg x L(-1), relative standard deviation (n = 9) was less than 3% and the linearly dependent coefficient was 0.998 7. This system can also be used in analysis of other protein, nucleic acid and so on.

PMMA/PDMS valves and pumps for disposable microfluidics.

Poly(methyl methacrylate) (PMMA) is gaining in popularity in microfluidic devices because of its low cost, excellent optical transparency, attractive mechanical/chemical properties, and simple fabrication procedures. It has been used to fabricate micromixers, PCR reactors, CE and many other microdevices. Here we present the design, fabrication, characterization and application of pneumatic microvalves and micropumps based on PMMA. Valves and pumps are fabricated by sandwiching a PDMS membrane between PMMA fluidic channel and manifold wafers. Valve closing or opening can be controlled by adjusting the pressure in a displacement chamber on the pneumatic layer via a computer regulated solenoid. The valve provides up to 15.4 microL s(-1) at 60 kPa fluid pressure and seals reliably against forward fluid pressure as high as 60 kPa. A PMMA diaphragm pump can be assembled by simply connecting three valves in series. By varying valve volume or opening time, pumping rates ranging from nL to microL per second can be accurately achieved. The PMMA based valves and pumps were further tested in a disposable automatic nucleic acid extraction microchip to extract DNA from human whole blood. The DNA extraction efficiency was about 25% and the 260 nm/280 nm UV absorption ratio for extracted DNA was 1.72. Because of its advantages of inexpensive, facile fabrication, robust and easy integration, the PMMA valve and pump will find their wide application for fluidic manipulation in portable and disposable microfluidic devices.

Portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea, and characteristics of bacterial adhesion to mucus.

The portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea is via the intestinal tract rather than gill or skin according to the kinetics of the bacterial adhesion to different mucus. The different effects on adhesion caused by proteolytic enzymes and heat treatment might be due to the different chemical compositions of mucus. Adhesion of V. alginolyticus to mucus depends on concerted action of bacterial surface structures such as cell-surface proteins, somatic antigens, flagella, etc. In addition, starvation and monosaccharides, especially fructose, inhibit the bacterial adhesion to the mucus. Knowledge of these adhesive characteristics should be very useful for designing more efficacious prophylactic strategies.

[Total selenium and se species in vegetables measured with hydride generation-atomic fluorescence spectrometry].

The amounts of total selenium and Se-species including organic and inorganic selenium in vegetables were measured with the method of hydride generation-atomic fluorescence spectrometry. The instrumental parameters and analytical conditions were optimized. In order to obtain the maximum fluorescence signal, the effects of the concentrations of HCI and KBH4 in carrier solution, the concentrations of HCl and HNO3 in sample medium, and the interference from foreign ion (Cu) on the signal were mainly investigated. Under the optimum conditions, the detection limit estimated with 3-fold standard deviation by 11 replicates of procedure blank was 0.35 ng x g(-1). The recovery tested by adding standards ranged from 97.6% to 101%. After being digested with HNO3 by microwave, selenium in several kinds of vegetables was measured. The results indicated that the total amounts of Se in the vegetable samples were low except straw mushroom, in which the content of Se was 0.151 microg x g(-1) (dry weight). In addition, the species of organic selenium were the main existing forms in vegetables.

[Determination of trace mercury species in water and soil samples with atomic fluorescence spectrometry].

With hydride generation-cold atomic fluorescence spectrometry (HG-AFS), the method of determining trace mercury species in water and soil samples in Jimei, Xiamen city, China was established. The content of inorganic mercury in water was measured by sample direct injection, while the total mercury was measured after digestion with the reagents of KBrO3-KBr. The soil samples were digested with microwave for total mercury measurement. Sequential extraction procedure was carried out for determining different mercuric species in soil samples. The results indicated that the mercury concentration of wastewater from chemical laboratory exceeded the limit of the integrated wastewater discharge standard of China (GB 8978-1996). It is one of the serious pollution sources of mercury in environment. The mercury contents from soil samples including the sideward soil of highway, the sea sediment and the garden soil were under the limits of relative national standards of China. However, attention should be paid to the accumulation of mercury in garden soil due to the artificial pollution. Meanwhile, the average recoveries for water and soil samples tested with adding standards were 93.7% and 93.8%, respectively. Meanwhile, the detection limits estimated with 3-fold standard deviation were 0.000 8 microg x L(-1) for water and 0.072 3 microg x kg(-1) for soil, respectively. The results indicated that the established method, with the merits of high sensitivity and precision, was suitable for the measurement of trace mercury species in environmental samples.

Base-Promoted Cascade C-C Coupling/N-α-sp3C-H Hydroxylation for the Regiospecific Synthesis of 3-Hydroxyisoindolinones.

A base-promoted cascade reaction for the regiospecific synthesis of substituted 3-hydroxyisoindolinones under transition-metal-free conditions is developed. The base-mediated C-C bond coupling and N-α-sp3C-H bond hydroxylation are involved in this procedure, which features high regioselectivity, efficiency, and environmental friendliness. Various substituted 3-hydroxyisoindolinones, including some bioactive molecules, were provided in up to 93% yield for 28 examples.

Determination of trace metals and analysis of arsenic species in tropical marine fishes from Spratly islands.

Trace metal contents in 38 species of tropical marine fishes harvested from the Spratly islands of China were determined by microwave digestion and inductively coupled plasma mass spectrometry analysis. Arsenic species were determined by high-performance liquid chromatography and inductively coupled plasma mass spectrometry analysis. The average levels of Al, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, Cd, Pb, and U in the fish samples were 1.683, 0.350, 0.367, 2.954, 36.615, 0.087, 0.319, 1.566, 21.946, 20.845, 2.526, 3.583, 0.225, 0.140, and 0.061mg·kg-1, respectively; Fe, Zn, and As were found at high concentrations. The trace metals exhibited significant positive correlation between each other, with r value of 0.610-0.852. Further analysis indicated that AsB (8.560-31.020mg·kg-1) was the dominant arsenic species in the fish samples and accounted for 31.48% to 47.24% of the total arsenic. As(III) and As(V) were detected at low concentrations, indicating minimal arsenic toxicity.

[Preparation and characterization of chitosan membrane for solid phase microextraction technique].

Chitosan is a kind of natural polymers containing plenty of amido and hydoxy. The chitosan membrane is tough, and since the partition coefficient of chlorophyll between chitosan membrance and water is as high as 9,090, the chitosan membrane may beeasily manufactured as solid-phase microextraction membrane for the analysis of chlorophyll. The system may reach a complete equilibrium in 80 min. Then the membrane can be desorbed completely in 30 min in 5% NaOH with ultrasonic. Both IR and XRD indicated that hydrogen bonds between the molecules of chitosan were weakened and amido and hydoxy were considered as the keys during the process of extracting chlorophyll.

[Study on Hg2+ distribution and speciation in different tissues of rats].

Mercury is one of the important pollutants that threaten people health greatly in environment. The purpose of this paper was to determine mercury distribution and mercury binding proteins in different tissues of rat fed orally with mercuric chloride by ICP-MS and SEC-UV-ICP-MS. The result showed that liver and kidney induced large amount of metallothioneins that was found to bind to mercury, copper and zinc after mercury intake in stomach and intestines. The metallothioneins induced may be prior to combine the mercury so that it would decrease mercury binding with the other proteins that would disable their normal function.

[Fabrication of relative humidity optical fiber sensor based on nafion-crystal violet sensing film].

With two kinds of sensing methods for relative humidity based on fluorescence and visible absorption respectively, crystal violet was opted as the molecule probe for humidity. The optical chemical sensing film for humidity was prepared when crystal violet was embedded in the Nafion gel. The relative humidity sensor was fabricated after the sensing film had been coupled with other components, such as optical fiber and detector, which possessed short response time ( < 2 min), high sensitivity (< or = 5% RH), wide dynamic range (30%-100%) and good reversibility (RSD < or = 2.6%) for relative humidity at 640 nm wavelength.

[Determination of mercury in Chinese medicinal material and biological samples using pyrolysis atomic absorption spectrometry].

In this paper a rapid and simple method using pyrolysis coupled with atomic absorption spectrometry for the analysis of total mercury in Chinese medicinal material and biological samples is presented. No sample digestion was needed and this greatly simplifies the analytical procedure and minimizes potential sources of contamination. Under optimum conditions, the reproducibility of the method was 2.1% for peak area and 9.1% for peak height. The detection limit (3sigma) was 6.3 ng x g(-1), and the recovery was within the range of 95%-105%. Several standard reference materials were analyzed and the results were obtained with satisfaction. The performance of the method was compared with inductively coupled plasma-mass spectrometry (ICP-MS), and excellent agreements were observed between these two methods.

[Speciation of 9 trace elements in a traditional Chinese medicine long dan cao by flow injection-inductively coupled plasma mass spectrometry with a focused microwave assisted extraction system].

A method based on focused microwave extraction for leaching 9 trace elements in traditional Chinese medicine Long Dan Cao was introduced. An online C18 enrichment-separation system and an HP4500 ICP mass spectrometer with a flow injection system was established for the separation and determination of inorganic speciation and organic speciation of the selected elements. Orthogonal design was applied for the optimization of microwave leaching conditions. The results showed that the temperature was the major factor for the leaching recoveries.