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.

[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.

[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.

Template synthesized molecularly imprinted polymer nanotube membranes for chemical separations.

In this report, we describe the synthesis of a molecularly imprinted polymer (MIP) nanotube membrane, using a porous anodic alumina oxide (AAO) membrane by surface-initiated atom transfer radical polymerization (ATRP). The use of a MIP nanotube membrane in chemical separations gives the advantage of high affinity and selectivity. Furthermore, because the molecular imprinting technique can be applied to different kinds of target molecules, ranging from small organic molecules to peptides and proteins, such MIP nanotube membranes will considerably broaden the application of nanotube membranes in chemical separations and sensors. This report also shows that the ATRP route is an efficient procedure for the preparation of molecularly imprinted polymers. Furthermore, the ATRP route works well in its formation of MIP nanotubes within a porous AAO membrane. The controllable nature of ATRP allows the growth of a MIP nanotube with uniform pores and adjustable thickness. Thus, using the same route, it is possible to tailor the synthesis of MIP nanotube membranes with either thicker MIP nanotubes for capacity improvement or thinner nanotubes for efficiency improvement.

[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.

Protein recognition via surface molecularly imprinted polymer nanowires.

In this paper, we present a technique for the preparation of polymer nanowires with the protein molecule imprinted and binding sites at surface. These surface imprinting nanowires exhibit highly selective recognition for a variety of template proteins, including albumin, hemoglobin, and cytochrome c. This recognition may be through a multistep adsorption, with the specificity conferred by hydrogen bonding and shape selectivity. Due to the protein imprinted sites are located at, or close to, the surface; these imprinted nanowires have a good site accessibility toward the target protein molecules. Furthermore, the large surface area of the nanowires results in large protein molecule binding capacity of the imprinted nanowires.

Direct analysis of mercury in Traditional Chinese Medicines using thermolysis coupled with on-line atomic absorption spectrometry.

The purpose of this study is to develop and apply a mercury analyzer system capable of quantitative analysis of mercury in Traditional Chinese Medicines (TCM) drugs in the concentrations range from ng g(-1) to mg g(-1). No sample pre-treatment was needed and this greatly simplifies the analytical procedure and minimizes potential sources of contamination. The precisions of analyzing solid mercury standard sample and real TCM materials were 2.1% and 2.5-8.2%, respectively; and the recovery based on the analysis of standard reference materials ranged from 95.2% to 105%. The performance of the method has been compared with inductively coupled plasma-mass spectrometry (ICP-MS) technique and excellent agreements were observed between the two methods. The method has been applied to the investigation of Hg content in several TCM drugs containing or not containing cinnabar. Mercury concentration in the same TCM products differs widely with different manufacturers, suggesting that external contamination and the Hg presence in raw herbal materials are the main sources of Hg. In addition, comparison of mercury thermal releasing profiles between TCM drug and cinnabar suggests that mercury conversion from cinnabar to biological matrices-bound Hg could occur because of the aid of other ingredients in the formulated drug.

Investigation of zinc binding metallothioneins' polymerization in tris(hydroxymethyl)-aminomethane buffer by coupling of size exclusion chromatography with electrospray ionization mass spectrometry.

Polymerization of metallothioneins (MTs) is one of the commonly encountered puzzles in researching the structure and function of metallothioneins. In this work, a method involving SEC coupled with negative ion electrospray ionization mass spectrometry (ESI-MS) detection has been developed for the study of zinc binding MTs' polymerization in tris(hydroxymethyl)-aminomethane (TRIS) acetate buffer at physiological pH. This hyphenated technique allows separating the different polymeric states of MTs by SEC, followed by on-line identification of the individual MT subisoforms in each polymeric peak by ESI-MS detection. Purified MT subisoforms (MT-2d and MT-2a), MT-2d and MT-2a mixture and rabbit liver MT complexes were investigated in the experiments to confirm the results obtained. From the results, both oxidative polymerization and non-oxidative oligomerization were found. The cystein-dependent oxidation results in the tetrameric peak as shown in the chromatograms of oxidized MT-2d, and stable dimeric and monomeric of MT were detected in this peak by MS. For the dimeric and trimeric peaks, different MT subisoforms were detected. In the five major subisoforms detected in rabbit liver MT complexes, MT-2a and MT-2c exist primarily as trimer, while MT-2e, MT-2d and MT-1a exist mainly as dimer. Our results suggest that in the three kinds of polymers, dimer, trimer and tetramer that were found in samples, the tetramer comes from the oxidation of MT molecular; for the dimer and trimer resulting from cystein independent oligomerization, they are closely associated with the charge of subisoform.

[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.

Surface molecularly imprinted nanowires for biorecognition.

Herein we present a novel method for preparation of surface molecularly imprinted size-monodisperse nanowires. The imprint molecule is immobilized on the pore walls of a silane-treated nanoporous alumina membrane. The nanopores are then filled with the monomer mixture, and the polymerization is initiated. The alumina membrane is subsequently removed by chemical dissolution, leaving behind polypyrrole nanowires with glutamic acid binding sites situated at the surface. These nanowires can be dissolved in aqueous media, and their applications therefore should be compatible with procedures in which biological antibodies might otherwise be used. For example, the analyte molecule can be tagged with various markers, such as fluorescence probes and enzymes, whereby the problem of steric hindrance is avoided. Furthermore, these surface-imprinted nanowires are likely suited for imprinting and recognition of large-molecular-weight peptides and proteins. Related work is currently being undertaken in our laboratory.

[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.

[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.

Molecularly imprinted sol-gel nanotubes membrane for biochemical separations.

In this study, we report a simple procedure for applying molecular imprinting functional groups to the inner surfaces of the template-synthesized sol-gel nanotubes for chemical separation of estrone. The silica nanotubes were synthesized within the pores of nanopore alumina template membranes using a sol-gel method by simultaneous hydrolysis of a silica monomer-imprinted molecule complex and tetraethoxysilane (TEOS). A covalent imprinting strategy was employed by generating a sacrificial spacer through the reaction of the isocyanate group of 3-(triethoxysilyl)propyl isocyanate and a phenol moiety of estrone to form a thermally cleavable urethane bond. This allowed us to remove the imprinted estrone by simple thermal reaction and to simultaneously introduce functional groups into the cavity formed by the silica nanotubes. Experiments indicated that estrone could be bound selectively by such an approach and have a binding affinity of 864 +/- 137 (n = 3).

Investigation of metal-binding metallothioneins in the tissues of rats after oral intake of cinnabar.

The multi-metal-binding MT fractions in rat tissues after oral intake of cinnabar were characterized by hyphenated size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). With the increase of both the feeding time and dosage of cinnabar the amounts of Hg-binding MT fractions in rat kidney of groups fed cinnabar increased significantly compared with the control group. Meanwhile, more Cu-binding MT were synthesized in the rat kidney, which confirmed the manipulating effect of MT in the homeostasis of Cu for detoxification of nephritic mercury. Although the Hg-binding MT fractions in rat liver of all cinnabar groups were almost independent of cinnabar dosage (2.5-5.0 g kg(-1) bw) and feeding time (2-4 weeks), the amount was much higher than that of the control group. The amounts of Hg-MT in other rat tissues of the cinnabar groups and the control group were compared and their significance is discussed.

Magnetite-containing spherical silica nanoparticles for biocatalysis and bioseparations.

The simultaneous entrapment of biological macromolecules and nanostructured silica-coated magnetite in sol-gel materials using a reverse-micelle technique leads to a bioactive, mechanically stable, nanometer-sized, and magnetically separable particles. These spherical particles have a typical diameter of 53 +/- 4 nm, a large surface area of 330 m(2)/g, an average pore diameter of 1.5 nm, a total pore volume of 1.427 cm(3)/g and a saturated magnetization (M(S)) of 3.2 emu/g. Peroxidase entrapped in these particles shows Michaelis-Mentan kinetics and high activity. The catalytic reaction will take place immediately after adding these particles to the reaction solution. These enzyme entrapping particles catalysts can be easily separated from the reaction mixture by simply using an external magnetic field. Experiments have proved that these catalysts have a long-term stability toward temperature and pH change, as compared to free enzyme molecules. To further prove the application of this novel magnetic biomaterial in analytical chemistry, a magnetic-separation immunoassay system was also developed for the quantitative determination of gentamicin. The calibration for gentamicin has a working range of 200-4000 ng/mL, with a detection limit of 160 ng/mL, which is close to that of the fluorescent polarization immunoassay (FPIA) using the same reactants.

[Preparation and characterization of Radix Salvia reference material for heavy metals under GAP control].

OBJECTIVE: To prepare and characterize the heavy metal reference material of radix salvia planted in Zhongjiang, Sichuan province under the good agricultural procedure (GAP). METHOD: After being prepared, the reference material was digested with 65% HNO3 and H2O2 by microwave-assisted oven systems, and the heavy metals in Radix salvia reference material were accurately determined by inductively coupled plasma mass spectrometry (ICP-MS). RESULT: The measuring method was validated by running certificated reference materials, including bush twigs and leaves (GBW07602) and tea twigs and leaves (GBW07605) under the same conditions. The recoveries of the elements mostly ranged from 90% to 110%, and the RSD was within 5%. The measurement of radix salvia reference material was carried out by different laboratories with ICP-MS and by several time intervals in one year for the stability. The results showed that the concentration of the heavy metals provided were accurate and the reference material was stable. CONCLUSION: The reference material is suitable to be the criterions of heavy metals for radix salvia in the qualities controlling, and is also suitable to be the criterion of poisonous heavy metals of other herbs in the administration of GAP.

[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.

[pH sensors based on rubbery ormosils preparation and their spectrum studies].

A new type of methyl substituted ormosils as a matrix for bromophenol blue (BPhB) and bromocresol green (BCG) is described. The new ormosils combine features of classical TEOS sol-gel material such as solvability in organic solvent and those of sol-gel glasses such as transparent and a porous structure, the ormosils also make a good mechanical stability. The influence of the conditions during the polymerisation process on the photochemical properties of BPhB and BCG has been studied. This sol-gel material was wed to immobilize pH-sensitive absorption dyes, bromothymol blue and bromocresol green, to prepare pH sensing films. The several aspects of the sensing films, including the leaching of the dye from gel, response time to different pH buffer solution, absorption spectra and the improvement of the immobilization of the dyes to filmo, were also discussed.