Etching of Ag nanoparticles triggered bidirectional regulation for electrochemiluminescence ratiometric immunoassay.

A highly efficient ratiometric electrochemiluminescence (ECL) immunoassay was explored by bidirectionally regulating the ECL intensity of two luminophors. The immunoassay was conducted in a split-type mode consisting of an ECL detection procedure and a sandwich immunoreaction. The ECL detection was executed using a dual-disk glassy carbon electrode modified with two potential-resolved luminophors (g-C3N4-Ag and Ru-MOF-Ag nanocomposites), and the sandwich immunoreaction using glucose oxidase (GOx)-modified SiO2 nanospheres as labels was carried out in a 96-well plate. The Ag nanoparticles (NPs) acted as bifunctional units both for triggering the resonance energy transfer (RET) with g-C3N4 and for accelerating the electron transfer rate of the Ru-MOF-Ag ECL reaction. When the H2O2 catalyzed by GOx in the 96-well plate was transferred to the dual-disk glass carbon electrode, the doped Ag NPs in the two luminophors could be etched, thus destroying the RET between C3N4 and the accelerated reaction to Ru-MOF, resulting in an opposite trend in the ECL signal outputted from the dual disks. Using the ratio of the two signals for quantification, the constructed immunosensor for a model target, i.e. myoglobin, exhibited a low detection limit of 4.7 × 10-14 g/mL. The ingenious combination of ECL ratiometry, bifunctional Ag NPs, and a split-type strategy effectively reduces environmental and human errors, offering a more precise and sensitive analysis for complex samples.

H2O2-activated independently bidirectional regulation for a sensitive and accurate electrochemiluminescence ratiometric analysis.

Potential-resolved electrochemiluminescence (ECL) ratiometric analysis has become a research hotspot in bioassays by virtue of its good accuracy, versatility and specificity. Current ECL ratiometry mainly focuses on the competition for the co-reactant or quantitative analysis using a variable signal and a changeless signal; the disorganized change or small difference between the two signals may affect the accuracy and sensitivity of detection. In this study, we have developed a novel ECL ratiometric sensor based on the bidirectional regulation of two independent co-reaction systems by H2O2. H2O2 as a bidirectional moderator permits the ECL signals of the cathode and anode to independently change in opposite trends, which greatly enhances the organization and difference between the two signals. The ratio of the two signals is used to realize the quantitative analysis of myoglobin (MyO) with a good linear relationship between log(ECLcathode/ECLanode) and log CMyO in the range of 1.0 × 10-13 to 1.0 × 10-7 g mL-1. The detection limit is 4.0 × 10-14 g mL-1. Furthermore, it showed excellent performance in the determination of MyO in human serum samples. The proposed biosensor provides some developments for the sensitive and accurate detection of disease markers.

The substrate-dependent regulatory effects of the AfeI/R system in Acidithiobacillus ferrooxidans reveals the novel regulation strategy of quorum sensing in acidophiles.

A LuxI/R-like quorum sensing (QS) system (AfeI/R) has been reported in the acidophilic and chemoautotrophic Acidithiobacillus spp. However, the function of AfeI/R remains unclear because of the difficulties in the genetic manipulation of these bacteria. Here, we constructed different afeI mutants of the sulfur- and iron-oxidizer A. ferrooxidans, identified the N-acyl homoserine lactones (acyl-HSLs) synthesized by AfeI, and determined the regulatory effects of AfeI/R on genes expression, extracellular polymeric substance synthesis, energy metabolism, cell growth and population density of A. ferrooxidans in different energy substrates. Acyl-HSLs-mediated distinct regulation strategies were employed to influence bacterial metabolism and cell growth of A. ferrooxidans cultivated in either sulfur or ferrous iron. Based on these findings, an energy-substrate-dependent regulation mode of AfeI/R in A. ferrooxidans was illuminated that AfeI/R could produce different types of acyl-HSLs and employ specific acyl-HSLs to regulate specific genes in response to different energy substrates. The discovery of the AfeI/R-mediated substrate-dependent regulatory mode expands our knowledge on the function of QS system in the chemoautotrophic sulfur- and ferrous iron-oxidizing bacteria, and provides new insights in understanding energy metabolism modulation, population control, bacteria-driven bioleaching process, and the coevolution between the acidophiles and their acidic habitats.

Liposome-assisted enzyme catalysis: toward signal amplification for sensitive split-type electrochemiluminescence immunoassay.

Developing an efficient signal amplification strategy is very important to improve the sensitivity of bioanalysis. In this paper, a liposome-assisted enzyme catalysis signal amplification strategy was developed for electrochemiluminescence (ECL) immunoassay of prostate specific antigen (PSA) in a split-type mode. The sandwich immunoreaction occurred in a 96-well plate, and glucose oxidase (GOx) encapsulated and antibody-modified liposomes were used as labels. The ECL detection was carried out using a rGO-Au NP modified glassy carbon electrode (GCE). The large amount of generated H2O2, i.e. the coreactant of the luminol system, and the excellent catalytic behavior of rGO-Au NPs greatly boosted the ECL signal, resulting in the signal amplification. The developed ECL immunosensor for detecting PSA achieved a wider linear range from 1.0 × 10-13 to 1.0 × 10-8 g mL-1 and a detection limit of 1.7 × 10-14 g mL-1. The application of the proposed strategy was demonstrated by analyzing PSA in human serum samples with recoveries from 89.0% to 113.0%, and relative standard deviations (RSDs) were less than 6.6%. This work provides a new horizon to expand the application of liposomes for ECL bioanalysis.

Ferric Uptake Regulator Provides a New Strategy for Acidophile Adaptation to Acidic Ecosystems.

Acidophiles play a dominant role in driving elemental cycling in natural acid mine drainage (AMD) habitats and exhibit important application value in bioleaching and bioremediation. Acidity is an inevitable environmental stress and a key factor that affects the survival of acidophiles in their acidified natural habitats; however, the regulatory strategies applied by acidophilic bacteria to withstand low pH are unclear. We identified the significance of the ferric uptake regulator (Fur) in acidophiles adapting to acidic environments and discovered that Fur is ubiquitous as well as highly conserved in acidophilic bacteria. Mutagenesis of the fur gene of Acidithiobacillus caldus, a prototypical acidophilic sulfur-oxidizing bacterium found in AMD, revealed that Fur is required for the acid resistance of this acidophilic bacterium. Phenotypic characterization, transcriptome sequencing (RNA-seq), mutagenesis, and biochemical assays indicated that the Acidithiobacillus caldus ferric uptake regulator (AcFur) is involved in extreme acid resistance by regulating the expression of several key genes of certain cellular activities, such as iron transport, biofilm formation, sulfur metabolism, chemotaxis, and flagellar biosynthesis. Finally, a Fur-dependent acid resistance regulatory strategy in A. caldus was proposed to illustrate the ecological behavior of acidophilic bacteria under low pH. This study provides new insights into the adaptation strategies of acidophiles to AMD ecosystems and will promote the design and development of engineered biological systems for the environmental adaptation of acidophiles.IMPORTANCE This study advances our understanding of the acid tolerance mechanism of A. caldus, identifies the key fur gene responsible for acid resistance, and elucidates the correlation between fur and acid resistance, thus contributing to an understanding of the ecological behavior of acidophilic bacteria. These findings provide new insights into the acid resistance process in Acidithiobacillus species, thereby promoting the study of the environmental adaptation of acidophilic bacteria and the design of engineered biological systems.

Enhancing peptaibols production in the biocontrol fungus Trichoderma longibrachiatum SMF2 by elimination of a putative glucose sensor.

Trichoderma spp. are main producers of peptide antibiotics known as peptaibols. While peptaibols have been shown to possess a range of biological activities, molecular understanding of the regulation of their production is largely unclear, which hampers the production improvement through genetic engineering. Here, we demonstrated that the orthologue of glucose sensors in the outstanding biocontrol fungus Trichoderma longibrachiatum SMF2, TlSTP1, participates in the regulation of peptaibols production. Deletion of Tlstp1 markedly impaired hyphal growth and conidiation, but significantly increased peptaibols yield by 5-fold for Trichokonins A and 2.6-fold for Trichokonins B. Quantitative real-time polymerase chain reaction analyses showed that the increased peptaibols production occurs at the transcriptional levels of the two nonribosomal peptide synthetase encoding genes, tlx1 and tlx2. Transcriptome analyses of the wild type and the Tlstp1 mutant strains indicated that TlSTP1 exerts a regulatory effect on a set of genes that are involved in a number of metabolic and cellular processes, including synthesis of several other secondary metabolites. These results suggest an important role of TlSTP1 in the regulation of vegetative growth and peptaibols production in T. longibrachiatum SMF2 and provide insights into construction of peptaibol-hyperproducing strains through genetic engineering.

The σ54-dependent two-component system regulating sulfur oxidization (Sox) system in Acidithiobacillus caldus and some chemolithotrophic bacteria.

The sulfur oxidization (Sox) system is the central sulfur oxidization pathway of phototrophic and chemotrophic sulfur-oxidizing bacteria. Regulation and function of the Sox system in the chemotrophic Paracoccus pantotrophus has been elucidated; however, to date, no information is available on the regulation of this system in the chemolithotrophic Acidithiobacillus caldus, which is widely utilized in bioleaching. We described the novel tspSR-sox-like clusters in A. caldus and other chemolithotrophic sulfur-oxidizing bacteria containing Sox systems. The highly homologous σ54-dependent two-component signaling system (TspS/R), upstream of the sox operons in these novel clusters, was identified by phylogenetic analyses. A typical σ54-dependent promoter, P1, was identified upstream of soxX-I in the sox-I cluster of A. caldus MTH-04. The transcriptional start site (G) and the -12/-24 regions (GC/GG) of P1 were determined by rapid amplification of cDNA ends (5'RACE), and the upstream activator sequences (UASs; TGTCCCAAATGGGACA) were confirmed by electrophoretic mobility shift assays (EMSAs) in vitro and by UAS-probe-plasmids assays in vivo. Sequence analysis of promoter regions in tspSR-sox-like clusters revealed that there were similar σ54-dependent promoters upstream of the soxX genes. Based on our results, we proposed a TspSR-mediated signal transduction and transcriptional regulation pathway for the Sox system in A. caldus. The regulation of σ54-dependent two-component systems (TCSs) for Sox pathways were explained for the first time in A. caldus, A. thiooxidans, T. tepidarius, and T. denitrificans, indicating the significance of modulating the sulfur oxidization in these chemolithotrophic sulfur oxidizers.

Rational design of an "OFF-ON" phosphorescent chemodosimeter based on an iridium(III) complex and its application for time-resolved luminescent detection and bioimaging of cysteine and homocysteine.

Biothiols, such as cysteine (Cys) and homocysteine (Hcy), play very crucial roles in biological systems. Abnormal levels of these biothiols are often associated with many types of diseases. Therefore, the detection of Cys (or Hcy) is of great importance. In this work, we have synthesized an excellent "OFF-ON" phosphorescent chemodosimeter 1 for sensing Cys and Hcy with high selectivity and naked-eye detection based on an Ir(III) complex containing a 2,4-dinitrobenzenesulfonyl (DNBS) group within its ligand. The "OFF-ON" phosphorescent response can be assigned to the electron-transfer process from Ir(III) center and C^N ligands to the DNBS group as the strong electron-acceptor, which can quench the phosphorescence of probe 1 completely. The DNBS group can be cleaved by thiols of Cys or Hcy, and both the (3)MLCT and (3)LC states are responsible for the excited-state properties of the reaction product of probe 1 and Cys (or Hcy). Thus, the phosphorescence is switched on. Based on these results, a general principle for designing "OFF-ON" phosphorescent chemodosimeters based on heavy-metal complexes has been provided. Importantly, utilizing the long emission-lifetime of phosphorescence signal, the time-resolved luminescent assay of 1 in sensing Cys was realized successfully, which can eliminate the interference from the short-lived background fluorescence and improve the signal-to-noise ratio. As far as we know, this is the first report about the time-resolved luminescent detection of biothiols. Finally, probe 1 has been used successfully for bioimaging the changes of Cys/Hcy concentration in living cells.

Liposome-assisted chemical redox cycling strategy for advanced signal amplification: A proof-of-concept toward sensitive electrochemiluminescence immunoassay.

This work presents a novel signal amplification strategy for electrochemiluminescence (ECL) biosensor based on liposome-assisted chemical redox cycling for in situ formation of Au nanoparticles (Au NPs) on TiO2 nanotubes (TiO2 NTs) electrode. The system was exemplified by ascorbic acid (AA)-loaded liposome, the redox cycling of AA utilizing tris (2-carboxyethyl) phosphine (TCEP) as reductant, and the use of Au nanoclusters (Au NCs)/TiO2 NTs as working electrode to implement the ECL detection of prostate specific antigen (PSA). Specifically, the AA-loaded liposomes were used as tags to label the captured PSA through a sandwich immunoreaction. After the lysate of the liposome was transferred onto the interface of Au NCs/TiO2 NTs in the presence of Au3+ and TECP, the chemical redox cycling was triggered. In the cycling, Au3+ was directly reduced in situ by AA to form Au NPs on Au NCs/TiO2 NTs electrode, whereas the oxidation product of AA was reduced by TCEP to regenerate AA. The large loading capacity of the liposome and chemical redox cycling resulted in the incomplete reduction of the Au NCs to Au NPs on the TiO2 NTs electrode, enhancing the ECL intensity greatly. The multiple signal amplification strategy achieved an ultrasensitive detection for PSA with a detection limit down to 6.7 × 10-15 g mL-1 and a wide linear concentration range from 1.0 × 10-14 to 1.0 × 10-8 g mL-1. It is believed that this work is anticipated to extend the employment of advanced chemical redox cycling reaction in the field of ECL bioassays.

A large perturbation on geometry structures, excited state properties, charge-injection and -transporting abilities of Ir(III) complexes by different substituents on ligands: a DFT/TDDFT study.

The molecular geometries, electronic structures, photophysical properties, charge-injection and -transporting abilities of a series of Ir(III) complexes with different carrier-transporting substituents, such as carbazole, oxadiazole and dimesitylboryl groups, are investigated theoretically using density functional theory (DFT) and time-dependent DFT (TDDFT) calculations to understand the influence of these substituents on the optical and electronic properties of Ir(III) complexes and to explore how to improve the optoelectronic properties of the complexes. It is found that the introduction of substituents can stabilize both HOMOs and LUMOs and induce variations in the energy gap between HOMO and LUMO. The introduction of hole-transporting carbazole substituent induces the blue-shift of absorption spectrum and improves the hole-injection and -transporting performances of complex. The introduction of electron-transporting oxadiazole substituent and electron-accepting dimesitylboryl substituent induces the red-shift in absorption spectra of complexes, improves their charge transfer abilities and leads to the better balance between the hole- and electron-transporting abilities. Through Lewis acid/base interactions between B atom and F(-), the electronic properties of 4 show dramatic changes in the presence of F(-) and thus 4 can also be used as selective phosphorescent F(-) probe.

Isolation and Identification of Chromium Reducing Bacillus Cereus Species from Chromium-Contaminated Soil for the Biological Detoxification of Chromium.

Chromium contamination has been an increasing threat to the environment and to human health. Cr(VI) and Cr(III) are the most common states of chromium. However, compared with Cr(III), Cr(VI) is more toxic and more easily absorbed, therefore, it is more harmful to human beings. Thus, the conversion of toxic Cr(VI) into Cr(III) is an accepted strategy for chromium detoxification. Here, we isolated two Bacillus cereus strains with a high chromium tolerance and reduction ability, named B. cereus D and 332, respectively. Both strains demonstrated a strong pH and temperature adaptability and survival under 8 mM Cr(VI). B. cereus D achieved 87.8% Cr(VI) removal in 24 h with an initial 2 mM Cr(VI). Cu(II) was found to increase the removal rate of Cr(VI) significantly. With the addition of 0.4 mM Cu(II), 99.9% of Cr(VI) in the culture was removed by B. cereus 332 in 24 h. This is the highest removal efficiency in the literature that we have seen to date. The immobilization experiments found that sodium alginate with diatomite was the better method for immobilization and B. cereus 332 was more efficient in immobilized cells. Our research provided valuable information and new, highly effective strains for the bioremediation of chromium pollution.

Essential Role of σ Factor RpoF in Flagellar Biosynthesis and Flagella-Mediated Motility of Acidithiobacillus caldus.

Acidithiobacillaceae, an important family of acidophilic and chemoautotrophic sulfur or iron oxidizers, participate in geobiochemical circulation of the elements and drive the release of heavy metals in mining associated habitats. Because of their environmental adaptability and energy metabolic systems, Acidithiobacillus spp. have become the dominant bacteria used in bioleaching for heavy metal recovery. Flagella-driven motility is associated with bacterial chemotaxis and bacterial responses to environmental stimuli. However, little is known about how the flagellum of Acidithiobacillus spp. is regulated and how the flagellum affects the growth of these chemoautotrophic bacteria. In this study, we analyzed the flagellar gene clusters in Acidithiobacillus strains and uncovered the close relationship between flagella and the sulfur-oxidizing systems (Sox system). The σ28 gene (rpoF) knockout and overexpression strains of Acidithiobacillus caldus were constructed. Scanning electron microscopy shows that A. caldus ΔrpoF cells lacked flagella, indicating the essential role of RpoF in regulating flagella synthesis in these chemoautotrophic bacteria. Motility analysis suggests that the deletion of rpoF resulted in the reduction of swarming capability, while this capability was enhanced in the rpoF overexpression strain. Both static cultivation and low concentration of energy substrates (elemental sulfur or tetrathionate) led to weak growth of A. caldus ΔrpoF cells. The deletion of rpoF promoted bacterial attachment to the surface of elemental sulfur in static cultivation. The absence of RpoF caused an obvious change in transcription profile, including genes in flagellar cluster and those involved in biofilm formation. These results provide an understanding on the regulation of flagellar hierarchy and the flagellar function in these sulfur or iron oxidizers.

Sulfur Oxidation in the Acidophilic Autotrophic Acidithiobacillus spp.

Sulfur oxidation is an essential component of the earth's sulfur cycle. Acidithiobacillus spp. can oxidize various reduced inorganic sulfur compounds (RISCs) with high efficiency to obtain electrons for their autotrophic growth. Strains in this genus have been widely applied in bioleaching and biological desulfurization. Diverse sulfur-metabolic pathways and corresponding regulatory systems have been discovered in these acidophilic sulfur-oxidizing bacteria. The sulfur-metabolic enzymes in Acidithiobacillus spp. can be categorized as elemental sulfur oxidation enzymes (sulfur dioxygenase, sulfur oxygenase reductase, and Hdr-like complex), enzymes in thiosulfate oxidation pathways (tetrathionate intermediate thiosulfate oxidation (S4I) pathway, the sulfur oxidizing enzyme (Sox) system and thiosulfate dehydrogenase), sulfide oxidation enzymes (sulfide:quinone oxidoreductase) and sulfite oxidation pathways/enzymes. The two-component systems (TCSs) are the typical regulation elements for periplasmic thiosulfate metabolism in these autotrophic sulfur-oxidizing bacteria. Examples are RsrS/RsrR responsible for S4I pathway regulation and TspS/TspR for Sox system regulation. The proposal of sulfur metabolic and regulatory models provide new insights and overall understanding of the sulfur-metabolic processes in Acidithiobacillus spp. The future research directions and existing barriers in the bacterial sulfur metabolism are also emphasized here and the breakthroughs in these areas will accelerate the research on the sulfur oxidation in Acidithiobacillus spp. and other sulfur oxidizers.

Model-based evaluation of 5 Chinese medicine recipes in the treatment of H5N1 influenza virus infection in BALB/c mice.

OBJECTIVE: To investigate whether Chinese medicine (CM) recipes could ameliorate H5N1 influenza virus infection in BALB/c mice model. METHODS: BALB/c mice were orally administrated with 5 CM recipes (removing toxin, tonifying qi, cooling blood, laxation, and compound recipes), oseltamivir, or saline solution respectively for 5 consecutive days after the infection of H5N1 influenza virus. Series of indices were employed to evaluate the amelioration of the 5 CM recipes on infection, including clinical assessment, gross observation, histopathologic findings, cytokine levels and viral burden in the lungs. RESULTS: Two CM recipes (cooling blood and compound recipes) could postpone the death period of the mice infected with high-dose H5N1 influenza virus (P< 0.05). And for the mice infected with low-dose H5N1 influenza virus, CM recipes could significantly reduce the mortality and inhibit viral proliferation in the lungs as compared with the control group (P<0.05). There was no significant difference in lung coefficients between the treatment and the control groups, but histopathological findings in the lungs were improved in CM recipes groups compared to control group findings. A transient increase was observed in pro-inflammatory and anti-inflammatory cytokines during the first 6 days of infection. The levels of interleukin (IL)-12p40 and interferon-gamma of the treatment groups were significantly lower than that of the control group at day 3 post-infection (P<0.05), while only compound recipe were significantly lower in level of tumor necrosis factor alpha than the control (P<0.05). The level of IL-10 of the control was higher than others, and the differences between the control and cooling blood, removing toxin recipes were significant (P<0.05). CONCLUSIONS: Results of this study suggested the potentials of the CM recipes in ameliorating influenza virus infection by suppressing viral proliferations, improving histopathological lesions, and inhibition of over expression of inflammatory cytokines.

The Two-Component System RsrS-RsrR Regulates the Tetrathionate Intermediate Pathway for Thiosulfate Oxidation in Acidithiobacillus caldus.

Acidithiobacillus caldus (A. caldus) is a common bioleaching bacterium that possesses a sophisticated and highly efficient inorganic sulfur compound metabolism network. Thiosulfate, a central intermediate in the sulfur metabolism network of A. caldus and other sulfur-oxidizing microorganisms, can be metabolized via the tetrathionate intermediate (S4I) pathway catalyzed by thiosulfate:quinol oxidoreductase (Tqo or DoxDA) and tetrathionate hydrolase (TetH). In A. caldus, there is an additional two-component system called RsrS-RsrR. Since rsrS and rsrR are arranged as an operon with doxDA and tetH in the genome, we suggest that the regulation of the S4I pathway may occur via the RsrS-RsrR system. To examine the regulatory role of the two-component system RsrS-RsrR on the S4I pathway, ΔrsrR and ΔrsrS strains were constructed in A. caldus using a newly developed markerless gene knockout method. Transcriptional analysis of the tetH cluster in the wild type and mutant strains revealed positive regulation of the S4I pathway by the RsrS-RsrR system. A 19 bp inverted repeat sequence (IRS, AACACCTGTTACACCTGTT) located upstream of the tetH promoter was identified as the binding site for RsrR by using electrophoretic mobility shift assays (EMSAs) in vitro and promoter-probe vectors in vivo. In addition, ΔrsrR, and ΔrsrS strains cultivated in K2S4O6-medium exhibited significant growth differences when compared with the wild type. Transcriptional analysis indicated that the absence of rsrS or rsrR had different effects on the expression of genes involved in sulfur metabolism and signaling systems. Finally, a model of tetrathionate sensing by RsrS, signal transduction via RsrR, and transcriptional activation of tetH-doxDA was proposed to provide insights toward the understanding of sulfur metabolism in A. caldus. This study also provided a powerful genetic tool for studies in A. caldus.

[Construction of an engineered Acidithiobacillus caldus with high-efficiency arsenic resistance].

Using the recombinant technique in vitro, a new arsenic resistance plasmid pSDRA4 was constructed by subcloning the arsenic resistance genes from plasmid pUM3 into the wide-host-range IncQ plasmid pMMB24 with the hybrid trp-lac ( tac ) promoter, and followed by deleting the regulative gene of the promoter, the lacIQ gene. Then plasmid pSDRA4 was introduced from E. coli into extremely acidophilic obligately chemolithotrophic Acidithiobacillus caldus by conjugative transfer with a frequency of( 1.444 +/- 0.797) x 10(-4), and the engineered strain of Acidithiobacillus caldus (pSDRA4) for biomining was constructed. The successful transfer demonstrates the development of a conjugational system between strains of E. coli and A. caldus. The recombinant plasmid pSDRA4 is stable in A. caldus. Compared with wild type A. caldus, the level of the arsenic resistance of A. caldus (pSDRA4) is greatly raised from 10mmol/L to 45mmol/L.

[Isolation, identification and genetic analysis of a murine norovirus strain].

Murine norovirus (MNV) was first discovered in mice in 2003. MNV is a member of the genus Norovirus in the family Caliciviridae. It is one of the most important and prevalent pathogens of laboratory mice, and almost all mouse strains are susceptible to MNV infection. In this study, a MNV strain was isolated from the cecal contents of infected mice and identified by the cytopathic effect (CPE) assay, virus plaque assay, 50% tissue culture infectious dose (TCID50) assay, electron microscopy, indirect immunofluorescence assay (IFA) and nucleotide sequencing. On infection, the RAW264.7 cell line showed obvious cytopathic effects within 24 to 48 hours post-inoculation, as infected cells became rounded, bright and shrunken, with ultimate disintegration of the cell sheet. After the isolation of the MNV virus, the virus was plaque-purified in RAW264.7 cells. The TCID50 of the virus was 10(5.25/0.1 mL. Electron microscopic observations of the purified virus showed the presence of spherical and non-enveloped viral particles that were 30 to 35 nm in diameter. According to the identification results, the isolate was named as MNV Guangzhou/K162/09/CHN. Thereafter, five overlapping gene fragments that covered the entire open reading frame (ORF) were amplified by RT-PCR, and the 3'-untranslated region (UTR) and 5'-UTR were amplified using the 3'-rapid amplification of cDNA ends (RACE) and the 5'-RACE method, respectively. Each of the gene fragments were cloned and sequenced, and whole genome sequences of the strain were obtained by assembling the cDNA fragment sequences. The results showed that the length of the complete genome was 7 380 nucleotides (GenBank accession number: HQ317203). The comparison of nucleotide and deduced amino acid sequences of the isolate was performed against other MNV strains in the GenBank database. A phylogenetic tree based on VP1 nucleotide sequences was constructed using MEGA5.0 software. The homology of nucleotides between the MNV Guangzhou/K162/09/CHN strain and other MNV isolates ranged from 87.4% to 89.7%. Phylogenetic analysis showed that there was a close genetic relationship between the Guangzhou/K162/09/CHN strain and MNV strains isolated from Japan (S7-P2 and S7-PP3 isolates), Korea (K4 isolate), and Germany (Berlin/04/06/DE and Berlin/05/06/DE isolates). This is the first report of the isolation and identification of MNV in China, and the first report of the genetic analysis of its complete genome.

[Model index observations in SIVmac251-infected rhesus macaques].

In this study, five rhesus macaques were inoculated intravenously with SIVmac251 to establish a model of simian autoimmune deficiency syndrome (SAIDS). Peripheral blood samples were collected at different time points to monitor changes in the total T cell number and T lymphocyte subset. Plasma viral loads, cytokine expression levels and anti-SIV antibody levels were also assayed to acquire certain basic indexes to evaluate disease progression in the rhesus macaque SAIDS model. During the acute stage of infection, plasma viral loads reached a peak at week 1 post-inoculation and lasted for approximately 3 to 44 weeks. The CD3+ CD4+ T lymphocyte count in peripheral blood also transitorily decreased. During the same period, the level of interferon-gamma show an increasing trend, whereas IL-12 levels decreased; IL-2, IL-4, IL-10 and TNF-alpha were maintained at normal levels or could not be detected. During the asymptomatic and ARC phases, plasma viral loads persisted above 10(4) RNA copies/mL and either increased or declined during the later stages of disease; CD3+ CD4+ counts showed a steadily declining trend and the ratio of CD4 to CD8 decreased during late-stage disease. Moreover, antibodies against viral proteins were detected in the plasma and showed a significant increasing trend, while there were no apparently changes in the levels of IFN-gamma, IL-12, IL-2, IL-4, IL-10 and TNF-alpha. In conclusion, the characteristics of the SIV animal models in our study are similar to those of patients with AIDS. Therefore, the rhesus macaque SIVmac251 infection models can be applied for further studies into AIDS.

[Expression of quorum-sensing related genes during Enterococcus faecalis biofilm formation].

OBJECTIVE: To investigate the relationship between the expression of the quorum-sensing related genes during Enterococcus faecalis(Ef) biofilm formation. METHODS: Ef biofilms model was established in vitro and film formation process was observed by confocal laser scanning microscope at 6, 12, 24 and 48 hours respectively.Quantification of biofilms was achieved by staining with crystal violet.Real-time fluorescence quantitative PCR method was used to detect the expression of fsrB, gelE and sprE genes in the process of Ef biofilm formation. RESULTS: A lot of live and dead bacteria unevenly distributed in Ef biofilm. The quantity of biofilms increased with time within 24 hours and was 0 h:0.00 ± 0.00, 6 h:1.09 ± 0.13, 12 h:2.10 ± 0.79, 24 h:3.30 ± 0.13, which was significantly different among the 4 time period(P < 0.05). The quantity of biofilm at 48 h(3.51 ± 0.01) increased slightly compared with 24 h(3.30 ± 0.13) , but did not show significant difference.Quantitative real-time PCR showed that the expression of quorum-sensing related fsrB increased with time within 24 hours and was 0 h:9.98 ± 0.46, 6 h:23.45 ± 1.13, 12 h:47.30 ± 2.49, 24 h: 331.30 ± 2.18, which was significantly different among the 4 time period(P < 0.05). The expression of gelE was 0 h: 6.54 ± 0.73, 6 h: 14.26 ± 1.24, 12 h: 37.47 ± 2.35, 24 h:264.80 ± 5.10(P < 0.05). The expression of sprE was 0 h: 7.72 ± 0.74, 6 h: 21.15 ± 0.96, 12 h:49.87 ± 3.18, 24 h:441.89 ± 7.74, which was significantly different among the 4 time period(P < 0.05). CONCLUSIONS: The fsrB, gelE and sprE genes are closely related to the biofilm formation in Ef.

Simulation of dust particles in dual-frequency capacitively coupled silane discharges.

The behavior of nanoparticles in dual-frequency capacitively coupled silane discharges is investigated by employing a one-dimensional self-consistent fluid model. The numerical simulation tries to trace the formation, charging, growth, and transport of dust particles during the discharge, under the influences of the high- and low-frequency electric sources, as well as the gas pressure. The effects of the presence of the nanoparticles and larger anions on the plasma properties are also discussed, especially, for the bulk potential, electron temperature, and densities of various particles. The calculation results show that the nanoparticle density and charge distribution are mainly influenced by the voltage and frequency of the high-frequency source, while the voltage of the low-frequency source can also exert an effect on the nanoparticle formation, compared with the frequency. As the discharge lasts, the electric potential and electron density keep decreasing, while the electron temperature gets increasing after a sudden drop.