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Polarization-contrast microscopy coupled with an atomic force microscope is utilized to attain far-field optical images of the multipolar surface plasmon resonance (SPR) modes of single gold nano-rod. Modulated standing modes resulted from the interference of longitudinal SPR modes and incident light are observed and studied. By counting the average distance of adjacent beats on this single gold nano-rod, the wave vector of longitudinal SPR modes can be obtained. We found a linear relationship between the wave vectors of the incident light and the induced SPR modes. Experimental results demonstrate a feasible way on acquiring plasmonic optical properties from an individual single gold nano-rod.

The present investigation aims at the synthesis of (Y2-xEuxBiy)O3 red phosphor materials using the combinatorial chemistry method. We developed square-type arrays consisting of 90 compositions to obtain the optimum composition of co-dopants in a Y2O3 host. The optimized composition was found to be (Y2-xEuxBiy)O3 (x = 0.16-0.18, y = 0.08-0.10). The screening of the compositions was investigated by analysis of the emission spectrum under 365 nm UV excitation arising from the energy transfer between Bi3+ and Eu3+ ions. The results of the photoluminescence excited by ultraviolet agree well with the conventional-scale synthesis results, indicating that the combinatorial screening method is fast, reliable, reproducible, and applicable to the study of powder materials with relatively quick calcinations at high synthesis temperatures.

In this paper, an endophytic strain B-001 against tobacco bacterial wilt (Ralstonia solanacarum) was isolated from the stem of healthy tobacco in R. solanacarum-infected fields, which had a stronger inhibitory effect on some kinds of gram-positive bacteria, gram-negative bacteria, and pathogenic fungi. This strain belonged to Bacillus, and its 16S rDNA after PCR and sequencing had an accession of GenBank being DQ444283. The 16S rDNA phylogenetic tree was constructed with MEGA3, and compared with the published 16S rDNA sequences of relative bacteria species. B-001 had a 99.2% sequence similarity with Bacillus subtilis (DQ415893). According to the morphological, physiological and biochemical characteristics, and based on phylogenetic analysis, B-001 was identified as a strain of B. subtilis. Field experiments in Guiyang and Ningxiang counties of Hunan Province showed that in 2005 and 2006, the control efficacy of B-001 on R. solanacarum ranged from 40.03% to 78. 14%, better than that of Streptomycini.

This study investigates the validity of questionnaire-based self-reports of illicit drug use by comparing with a two-tiered urinalysis among patients at emergency departments. Questions on the use of alcohol and drugs were administered to patients recruited on a continual basis for 2weeks at the emergency department of two hospitals in northern Taiwan. Positive tests of initial urinalysis using fluorescence polarization immunoassay were further confirmed by gas chromatography/mass spectrometry. In a total of 1502 patients interviewed, 632 (42%) also provided a urine sample. Among those with urine samples, the positive rate of urinalysis was 1.4% for amphetamine-type drugs and 1.6% for opiates. Among those with positive urinalysis, a false-negative rate ranged from 66.6% for amphetamines to 70.0% for opiates. Meanwhile, all the self-reported current uses of either amphetamines or opiates were confirmed by urinalysis. The results indicate that the false-negative rates of questionnaire-based, self-reported current use of illicit drug are around two thirds and the false-positive rates are negligible, which might be useful for the calibration of estimates from epidemiological surveys.

This investigation describes the formation of a metal nanorattle with a pure metal shell by varying experimental parameters. The galvanic replacement reaction between silver and chloroauric acid was adopted to prepare hollow metal nanoparticles. This approach is extended to produce nanorattles of Au cores and Au shells by starting with Au(core)Ag(shell) nanoparticles as templates. The effect of temperature on the nanostructure of the final product is also considered. The composition of the shell in nanorattles can be controlled by varying the reaction temperature (to form pure gold or gold-silver alloy, for example). X-ray absorption fine structure spectroscopy is conducted to elucidate the fine structure of these nanoparticles. Partial alloying between the Au core and the Ag shell is observed by extended X-ray absorption fine structure (EXAFS).

In this study, we examined the amount-dependent change in morphology in a series of Au-Pt bimetallic nanoparticles synthesized using chemical reduction. The amount of Au precursor was kept constant throughout the experiment. The Au/Pt molar ratio was varied from 1/1 to 1/4 to synthesize Pt shell layers with different thicknesses. We observed a remarkable shift of the surface plasmon band at around 410 nm. By high-resolution transmission electron microscopy (HRTEM) and energy-dispersive spectrometry (EDS), the composition of the shell layer was found to be Pt-enriched Au-Pt alloy. As the concentration of Pt increases, Pt clusters (ca. 1.8 nm in diameter) form a string-like shape on the surface of nanoparticles.

Potassium antimonite precipitation was used to located calcium in the egg cells (before and after anthesis), zygotes and proembryos of lettuce (Lactuca sativa L.). A few calcium precipitates (ppts) were located in the small vacuoles of cytoplasm of egg cell at 3 d before anthesis, when egg cells just formed. Then the small vacuoles fused to form some bigger vacuoles in egg cell at 2d before anthesis. Calcium ppts increased evidently in the cytoplasm and nucleus of egg cells at this time. At 1d before anthesis, a biggest vacuole located at the micropyle end of the cell and its nucleus was pushed toward the chalazal end of the cell, which made an evident cellular polarity. The number of calcium ppts in the egg cell markedly decreased, suggesting that change of calcium distribution may be related to the development of egg cell. After anthesis and before fertilization, calcium ppts were still few in the egg cells, and most of them were accumulated in the nucleus, especially in the vacuoles of nucleolus. At 4h after anthesis, egg cell was fertilized and the wall at the chalazal end of egg cell was formed completely. Calcium ppts evidently increased again in egg cell, and some big ppts appeared in the karyoplasm of nucleus and abundant small ppts in the large vacuole. At 9h after anthesis, zygote completed its first division. Calcium ppts in the nucleus and cytoplasm of two-celled proembryo began to decrease, and only some ones accumulated in the vacuoles of nucleolus. At 18h after anthesis, zygote divided several times and became a multi-celled proembryo. Calcium ppts in the cells of proembryo ulteriorly diminished but there were many ppts on the surface of proembryo. The result indicates that calcium in egg cell, zygote and the cells of proembryo orderly changes its temporal and spatial position, which suggests that calcium may play a role during the development of egg cell and zygote.

Potassium antimonite was used to locate calcium in the anther of lettuce (Lactuca sativa L) during its development. At the early stage of anther development there were few calcium granules in microspore mother cells and the cells of anther wall. After meiosis of microspore mother cells, calcium granules first appeared in the tapetal cells in which some small secretive vacuoles containing many calcium granules were formed and secreted into locule. Then, the tapetal cells began to degenerate. At the late stage of microspore, tapetal cells completely degenerated and its protoplast masses moved into anther locule with many calcium granules. Few calcium granules were precipitated in the microspores just being released from tetrad, but some on the surface of exine. Then calcium granules appeared in the nucleus and cytoplasm of early microspores, as wall as in the exine. When microspores formed some small vacuoles containing some calcium granules, and then the small vacuoles fused to form a large vacuole, the calcium granules in the nucleus and cytoplasm evidently decreased, microspore developed to the late stage. The result suggested that calcium is related to the formation of large vacuole in microspores. The wall of microspore also is a main location of calcium granules during its developing. At early microspore some calcium granules began to accumulate in exine, which suggested calcium related with exine formation. At late stage of microspore, most of calcium granules were mainly deposited on the surface of exine. After the first mitosis of microspores, the large vacuole of bicellular pollen disappeared and calcium granules in the large vacuole went back to cytoplasm again. When bicellular pollen synthesized starches some calcium granules appeared on the surface of starches, which suggested calcium may regulate starch synthesis. With amount of starches increasing, calcium granules disappeared from pollen cytoplasm and only some of them located on the surface of pollen.

Potassium antimonite was used to deposit calcium in the young ovule of lettuce (Lactuca sativa L.) at megasporogenesis stage to study the relationship between calcium and megaspore degeneration. At the megaspore mother cell stage, few calcium granules were formed in the cell (Plate I-1, 2). After meiosis of megaspore mother cell and forming an arrayed tetrad in a line (Plate I-3), three megaspores degenerated one by one from the micropyle end. In the process of degeneration, the numbers of calcium granules decreased in the three megaspores. After the first megaspore degenerated, the number of calcium granules decreased in the second megaspore, which began to degenerate (Plate II-7, 8). The third megaspore also had its number of calcium granules diminishing before it degenerated (Plate III-13, 14). The fourth megaspore always accumulated many calcium granules in the cytoplasm during its development (Plate IV-17, 18) and finally becomes functional one that will develop into an embryo sac (Plate IV-20). Megaspore degeneration is a process of programmed cell death which may be closely related with change in calcium content: when a megaspore of tetrad decreases calcium content the cell begins to degenerate, and when calcium increases in the cell, it will continue to develop into a functional megaspore. This is the first report about calcium distribution in megaspores of a tetrad during megasporogenesis in higher plants and will open a door to study the physiological function of calcium in megasporogenesis.

Potassium antimonite was used to deposit calcium in the stigma and style of lettuce (Lactuca sativa L.) before and after pollination. The stigma of lettuce is two splits. Abundant calcium granules are displayed in the wall of papillae on the receptive surface of stigma before and after pollination, which may facilitate pollen germination. However, a few calcium granules in the wall of epidermis cell on no-receptive surface. Calcium distribution in style presents a gradient in transmitting tissue and parenchyma cells from the top to the base of the style before pollination. After pollination, calcium in transmitting tissue distinctly increased and its gradient distribution became more evident. Pollen tubes grow in the intercellular gaps of transmitting tissue. When pollen tubes grew into transmitting tissue, calcium granules in parenchyma around transmitting tissue decreased, suggesting a calcium movement was controlled by pollen tubes. The calcium gradient distribution also appeared in the trachea of vascular bundle of style. In general, calcium in style displays a feature of time-special distribution: transmitting tissue doesn't need much more calcium that is only stored in the parenchyma before pollination. However, calcium in parenchyma cells may be transported to transmitting tissue and make the latter contain more calcium to form an evident calcium gradient and meet the requirement of pollen tubes directionally growing after pollination. This is the second sample of calcium gradient existing in style, which was found by using potassium antimonite method.

The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.

The hepatitis E virus (HEV) capsid consists of a single structural protein, a portion of which is engaged in isosahedral contact to form a basal shell, and another portion in dimeric contact to form the homodimers protruding from the shell. Previous studies revealed that homodimers of the truncated HEV capsid proteins, E2 (amino acids 394-606) and p239 (amino acids 368-606), model dominant antigenic determinants of HEV. Immunization with these proteins protected rhesus monkeys against the virus, and three monoclonal antibodies against the homodimers could neutralize HEV infectivity and/or immune-capture of the virus. Furthermore, homodimers of p239 further interact to form particles of 23 nm diameter, rendering it an efficacious candidate vaccine. In light of this we postulate that the interactions involved in the formation of the homodimers and particles might be similar to those involved in assembly of the virus capsid. Presently, mutational analysis was carried out to identify these sites of interactions. The site of dimeric interactions was located to a cluster of six hydrophobic amino acids residues, Ala597, Val598, Ala599, Leu601, and Ala602; furthermore, the site involved in particle formation was located at amino acids 368-394. The possibility that these sites are also involved in assembly of the virus capsid is supported by the fact that they are located at two major and highly conserved hydrophobic regions of the HEV structural protein.

Aim: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein. This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS., Methods: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His-Mut+) by electroporation. His(+)Mut(+) recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively., Results: All of the recombinants were His(+)Mut(+) after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains. The final optimal concentration of methanol was 20 mL/L, the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved, and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum. The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in E.coli when the same rSCoVN protein quantity was used., Conclusion: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GS115. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum. This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.

Two-dimensional nanoparticle self-assemblies provide a means of tuning the optical properties by manipulation of the fabrication parameters, thereby controlling the light-matter interaction. We report fabrication of self-assembled monolayers of colloidal gold nanoparticles on organosilane-coated substrates and a study of the linear and nonlinear optical properties. With increase in particle coverage of the gold monolayer self assemblies, an additional band in the absorption spectrum due to the collective particle surface plasmon resonance could be seen. The intensity and lambda(max) of this feature depends on the interparticle spacing and particle aggregation. Z-scan measurements show a saturable absorption behavior and a negative nonlinear refractive index of the monolayer films. A strong correlation between particle density and the linear and nonlinear optical properties is established.

Production of Hepatitis E Virus capsid protein by high cell density culture in recombinant E. coli has been studied in 10L and 30L fermentors. The effects of different factors on growth and producing recombinant protein of E. coli have been studied by batch culture, such as different media, the ratio of phosphate and Magnesium sulfate. Comparison of fermentation performance for recombinant E. coli in different fed-methods culture has been investigated by fed-batch culture. The effects of inducing at different stages of growth and time of inducing on growth and producing recombinant protein, also obtained by fed-batch culture. At last, the solubility of inclusion body in different urea concentrations also has been obtained by fed-batch culture. The results show that the concentration of phosphate and Magnesium sulfate in the optimal media is 80mmol/L and 20mmol/L in batch culture respectively, that induction with 1.0mmol/L IPTG at mid log phase (about 45 OD at 600nm) is suitable for growth and recombinant protein expression, the cells were approaching stationary growth phase and the maximum cell OD at 600nm of 80 was achieved in 5h of fed-batch culture, and the expression level is 29.74%. The results also indicate that the solubility of inclusion body in 4mol/L urea solution induced at 37 degrees C reaches 14mg/mL, over 80% inclusion body was resolved. The culture process achieved in 10L fermentor could be successfully scaled up to 30L fenmentor with good reproducibility.

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.

Hepatitis E is a main cause of acute viral hepatitis in developing countries where it occurs as sporadic cases and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. The approximately 7.5 kb positive-sense single-strand RNA genome includes three open reading frames (ORFs), one of which (ORF2) is postulated to encode the major viral capsid protein (pORF2) of 660 amino acid residues. We earlier showed that a bacterially expressed peptide, designated as NE2, located from amino acid residues 394 to 606 of ORF2, was found to aggregate into homodimer to at least hexamer. To understand the interface domains within this peptide vital for dimerization and formation of major neutralizing epitopes, NE2 protein underwent terminal-truncated and site-directed mutation. The hydrophobic region, ORF2 aa597-aa602 (AVAVLA), played a key role in oligomerization. Any amino acid residue of this region replaced with glutamic acid residue, the peptide can not refold as homodimer and/or oligomer. The immunoreactivities of these mutant peptides, blotted with anti-HEV neutralizing monoclonal antibody (8C11) and convalescent human sera, show associated to the formation of homodimer. The intermolecular contact region on homodimer was investigated by chemical cross-linking of two site-directed cysteines. When the alanine on aa597 site mutated with cysteine, two different homodimers were found in SDS-PAGE analysis. One (42kD) can be disassociated with 8mol/L urea, which is postulated to form by virtue of hydrophobic interaction, and the other (60kD) falls apart with the reductant DTT present. The exact conformation, generating the cross-linking reaction of cysteines, was further investigated by induced-oxidation on monomer and hydrophobic homodimer of A597C protein with GSH/GSSG. And the results revealed, it is the conformation of hydrophobic homodimer that induces the disulfide bond come into being, instead of the one of monomer. So the aa597 site was verified to be located on interface domain of hydrophobically interacting homodimeric complex. To evaluate the biological significance of hydrophobicity of interface domain, we searched natural variations as to the region on all available databases with NCBI blast program. All variations on these amino acid residues kept higher hydrophobicity, which suggests that the hydrophobic domain is critical for the assemblage and propagation of HEV. NE2 N-terminal deletions up to aa458 had no effect on dimerization and took no exact part in formation of major neutralizing epitopes, but the fragment may act as helper for the formation of major neutralizing epitopes on NE2. Interestingly, the C-terminus aa605-aa660 of ORF2 can also act as helper instead of the N-terminus of NE2. This study suggests an interface domain of NE2 might be vital for HEV capsomer assembly and formation of major neutralizing epitopes. These results may offer clues to the rational design of recombinant anti-HEV vaccine.

A fragment of hepatitis E virus open reading frame-2(ORF2), located from amino acid residues 394 to 604, was expressed in E. coli. The recombinant protein NE2 was found to form homodimer mostly in SDS-PAGE, which can be dissociated to monomers when treated with urea, and it was recognized more strongly in its dimeric form than the monomer by HEV reactive human serum in Western blotting. Besides, many aggregated form of NE2 from dimer to at least hexamer can be seen in MALDI-TOF-MS. And when the hydrated dynamic semidiameter of NE2 moleculars in PBS was measured as about 4 nm by Dynamic Light Scattering (DLS), being equal to tetramer, but with high polydispersity, which suggested that the NE2 moleculars were existed in PBS in many different sizes. These results suggested that the recombinant NE2 can aggregate into several oligomer forms, the association in the dimer is most strong, and dimers can assemble further to form some super-structure.