Comparison of the levels of bacterial diversity in freshwater, intertidal wetland, and marine sediments by using millions of illumina tags.

Sediment, a special realm in aquatic environments, has high microbial diversity. While there are numerous reports about the microbial community in marine sediment, freshwater and intertidal sediment communities have been overlooked. The present study determined millions of Illumina reads for a comparison of bacterial communities in freshwater, intertidal wetland, and marine sediments along Pearl River, China, using a technically consistent approach. Our results show that both taxon richness and evenness were the highest in freshwater sediment, medium in intertidal sediment, and lowest in marine sediment. The high number of sequences allowed the determination of a wide variety of bacterial lineages in all sediments for reliable statistical analyses. Principal component analysis showed that the three types of communities could be well separated from phylum to operational taxonomy unit (OTU) levels, and the OTUs from abundant to rare showed satisfactory resolutions. Statistical analysis (LEfSe) demonstrated that the freshwater sediment was enriched with Acidobacteria, Nitrospira, Verrucomicrobia, Alphaproteobacteria, and Betaproteobacteria. The intertidal sediment had a unique community with diverse primary producers (such as Chloroflexi, Bacillariophyta, Gammaproteobacteria, and Epsilonproteobacteria) as well as saprophytic microbes (such as Actinomycetales, Bacteroidetes, and Firmicutes). The marine sediment had a higher abundance of Gammaproteobacteria and Deltaproteobacteria, which were mainly involved in sulfate reduction in anaerobic conditions. These results are helpful for a systematic understanding of bacterial communities in natural sediment environments.

Effects of polymerase, template dilution and cycle number on PCR based 16 S rRNA diversity analysis using the deep sequencing method.

BACKGROUND: The primer and amplicon length have been found to affect PCR based estimates of microbial diversity by pyrosequencing, while other PCR conditions have not been addressed using any deep sequencing method. The present study determined the effects of polymerase, template dilution and PCR cycle number using the Solexa platform. RESULTS: The PfuUltra II Fusion HS DNA Polymerase (Stratagene) with higher fidelity showed lower amount of PCR artifacts and determined lower taxa richness than the Ex Taq (Takara). More importantly, the two polymerases showed different efficiencies for amplifying some of very abundant sequences, and determined significantly different community structures. As expected, the dilution of the DNA template resulted in a reduced estimation of taxa richness, particularly at the 200 fold dilution level, but the community structures were similar for all dilution levels. The 30 cycle group increased the PCR artifacts while comparing to the 25 cycle group, but the determined taxa richness was lower than that of the 25 cycle group. The PCR cycle number did not changed the microbial community structure significantly. CONCLUSIONS: These results highlight the PCR conditions, particularly the polymerase, have significant effect on the analysis of microbial diversity with next generation sequencing methods.

Dengue Fever in mainland China.

Dengue is an acute emerging infectious disease transmitted by Aedes mosquitoes and has become a serious global public health problem. In mainland China, a number of large dengue outbreaks with serious consequences have been reported as early as 1978. In the three decades from 1978 to 2008, a total of 655,324 cases were reported, resulting in 610 deaths. Since the 1990s, dengue epidemics have spread gradually from Guangdong, Hainan, and Guangxi provinces in the southern coastal regions to the relatively northern and western regions including Fujian, Zhejiang, and Yunnan provinces. As the major transmission vectors of dengue viruses, the biological behavior and vectorial capacity of Aedes mosquitoes have undergone significant changes in the last two decades in mainland China, most likely the result of urbanization and global climate changes. In this review, we summarize the geographic and temporal distributions, the serotype and genotype distributions of dengue viruses in mainland China, and analyze the current status of surveillance and control of vectors for dengue transmission.

Identification and characterization of Schistosoma japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis.

The pathogenesis of schistosomiasis is mainly caused by egg-induced granuloma formation and subsequent fibrosis. If Schistosoma japonicum infections could be detected in the early stage, especially before the egg deposition in the host tissues, the development of severe pathologic lesions might be prevented efficiently. The present study identified and characterized S. japonicum Sjp40, a potential antigen candidate for the early diagnosis of schistosomiasis. From the S. japonicum cercariae cDNA library, a clone encoding Sjp40 was identified by screening with the pooled rabbit sera collected on day 21 postinfection. Then, the recombinant Sjp40 protein (rSjp40) and monoclonal antibodies (McAbs) anti-rSjp40 were developed. The expression profiles of Sjp40 at 3 stages of S. japonicum, including egg, cercariae, and adult, were also determined at both mRNA and protein level, which displayed that the expression pattern of Sjp40 varied at different stages. Quantification of circulatory anti-Sjp40 IgG in the infected mice sera by time-resolved fluoroimmunoassay showed a statistically significant increase on days 21, 28, 35, and 42 postinfection compared with the mice sera prior to infection and the control mice. It was further confirmed by Western blot that all 8 clones of anti-rSjp40 McAbs could react specifically with the native antigen in S. japonicum cercariae, and rSjp40 could be recognized by the pooled infected mouse sera on days 21, 28, 35, and 42 postinfection as well as the pooled patient sera with acute schistosomiasis japonica. These findings indicated that Sjp40 and its antibodies are detectable from the host at a relatively early phase (day 21 postinfection with S. japonicum) and suggested that Sjp40 is a potential antigen candidate for the early diagnosis of schistosomiasis.

[Isolation, identification and analysis of the expression profile of miRNAs in Aedes albopictus].

OBJECTIVE: To verify the miRNA in Aedes albopictus and characterize the expression profile of several miRNAs across all the life stages of Aedes albopictus. METHODS: Based on the published miRNA sequences of Anopheles gambiae and Aedes aegypti, 6 DIG-labeled antisense probes were synthesized. The total RNAs from Aedes albopictus in 6 developmental stages (embryo, early larvae, late larvae, pupa, male and female adults) were extracted with a mirVana miRNA isolation kit, loaded onto 15% denaturing polyacrylamide gel and hybridized with the appropriate DIG-labeled probes. RESULTS: Northern blotting detected 5 miRNAs in Aedes albopictus, of which mir-9a was mainly expressed in embryo and larva stages, let-7 in pupa and adult stages, miR-184 in all life stages, mir-M1 only in the embryos and miR-1175 in all the life stages except for embryos. The expression profiles of these miRNAs in Aedes albopictus were similar to those in D. melanogaster and An.stepheni. miR-1174 was not detected in any of the developmental stages of Aedes albopictus. CONCLUSION: These results present the first direct experimental evidence of miRNA in Aedes albopictus. The expression profiles of the analyzed miRNAs in Aedes albopictus showed stage specificity and conservation with other mosquitoes. Further studies on the functions of these miRNAs may offer new insights in mosquito biology and may lead to novel approaches to the development of insecticides.

[Development and identification of monoclonal antibodies against the recombinant P38 antigen of Schistosoma japonicum].

OBJECTIVE: To express P38 of Schistosoma japonicum in Escherichia coli BL21 and develop the monoclonal antibodies (McAb) against rSjP38. METHODS: The recombinant plasmid pET32(a)-P38 was transformed into E. coli BL21 (DE3). 1 mmol/L IPTG (isopropyl-beta D-thiogalactopyranoside) was used to induce the expression of the recombinant rSjP38. The rSjP38 was soluble in supernatant after sonication and further purified by His-Ni chromatography. BALB/c mice were immunized with the purified rSjP38 and hybridomas were generated with traditional technique. McAbs were screened by ELISA with limited dilution. The subtype and specificity of McAb were identified by kit and Western blot respectively. RESULTS: Eight hybridoma cell lines secreting monoclonal antibodies to rSjP38 were obtained. The subtype of all the 8 McAbs are IgG1. Western blotting showed that the 8 McAbs reacted strongly and specifically with native antigen (P38) of Schistosoma japonicum. CONCLUSIONS: Eight hybridoma cell lines secreting highly specific McAbs against P38 have been established.

[A colloidal gold immunochromatographic assay for detecting p38 antigen of Schistosome japonicum].

OBJECTIVE: To establish a colloidal gold immunochromatographic assay (GICA) for detecting Schistosome japonicum (Sj). METHODS: Eight monoclonal antibodies (mAbs) against Sj p38 antigen (1A6, 3C4, 3D12, 6F10, 6G12, 9H6, 9G7 and A5H) prepared previously were purified by protein-G affinity chromatography. The affinity constant (K(aff)) was determined by indirect enzyme-linked immunosorbent assay. All the mAbs were labeled with horseradish peroxidase by sodium oxidation method and the mAb pairs with high affinity and stability were identified according to their optical density at 450 nm (OD450). Four mAbs (1A6, 6G12, 9H6 and 9G7) were chosen for colloidal gold labeling. GICA was then performed by further optimization of the labeling and the conditions were determined. The sera of mice at different infection stages were examined with GICA dipstick, with the sera collected before infection and those of Toxoplasma gondii-infected mice as negative controls. RESULTS: The purity of the 8 mAbs was higher than 95% with K(aff) ranging from 2.8x10(-10) to 1x10(-8) mol/L. 9G7 coating (2.5 mg/ml) as the capture antibody and detection with 1A6 (diluted at 1:4) as the labeling antibody was determined as the best reaction model. With this combination, the positivity rates of the detection were 40%, 50%, 60% and 80% for mouse sera collected at 3, 4, 5 and 6 weeks after Schistosome japonicum infection, respectively, without positive results for the negative control samples. CONCLUSION: GICA established in this study is characterized by simplicity, rapidity and good sensitivity, and the prepared rSjP38 dipstick can test the circulating antigen SjP38 in early stage of infection.

Preparation and identification of monoclonal antibodies against human brain-derived neurotrophic factor.

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against human brain-derived neurotrophic factor (BDNF). METHODS: BALB/C mice were immunized with purified BDNF protein in conjunction with acid-treated Salmonella (antigen:thallus=1:5), and mAbs were subsequently derived by hybridoma technique. RESULTS: Three hybridoma cell lines secreting anti-BDNF mAbs were obtained, designated as B1, B2 and 4D1 respectively and all categorized into IgG1 subtype. The titers of the mAbs in the ascitic fluid of the rats ranged from 1x10(6) to 1x10(5), and their relative affinities were B2>B1>4D1. CONCLUSION: mAbs against BDNF are successfully prepared, which can be instrumental for further study of the expression and distribution of BDNF in vivo, and the detection of BDNF produced by genetic engineering.

[Detection of apoptosis with gel eletrophoresis combined with computerized gel analysis system].

OBJECTIVE: To explore a new method for detecting cellular apoptosis. METHODS: Using routine electrophoresis assisted by computerized gel documentation analysis system(GDAS), apoptosis of the macrophages treated with dexamethasone was qualitatively and then quantitatively detected. RESULTS: Apoptosis occurred in the macrophages treated with dexamethasone, and DNA fragmentation of the apoptotic macrophages was visualized on 1% agarose gel electrophoresis. The apoptosis rates as determined by computerized GDAS were consistent with the results obtained from flow cytometry that showed typical apoptosis peak. CONCLUSION: Agarose gel electrophoresis assisted by computerized GDAS was relatively simple and less costly in qualitative and quantitative detection of apoptosis with precision.