Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus

Middle East respiratory syndrome coronavirus (MERS‐CoV) is a single‐stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS‐CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS‐CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS‐CoV. Using a recombinant full‐length S protein, an indirect ELISA was developed and found to detect MERS‐CoV‐specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS‐CoV S protein. Neither ELISA system exhibited significant intra‐assay or inter‐assay variation, indicating good reproducibility. Moreover, the inter‐day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS‐CoV.

     

Prevalence of Human Melioidosis on Hainan Island in China

Eight human cases of melioidosis were diagnosed at Hainan People's Hospital over a period of one year. Four of the cases were of septicemia form, while the rest were of chronic form with abscesses in different organs. All the isolates were intrinsically resistant to cefazolin, cefuroxime, and gentamicin, while being rather sensitive to ampicillin/sulbactam, ticarcillin/clavulanic acid, and imipenem. A serological survey of exotoxin antibodies at different farms showed that Xinglong was a farm seriously devastated by Burkholderia pseudomallei, while the mountainous farm of Licai had the lowest prevalence (P < 0.01). From the results of serological survey and melioidosis case distribution, it could be clearly seen that melioidosis predominantly exists in coastal plain regions around this island, where the altitude above sea-level is below 100 m, the annual rainfall is up to 2,300 mm and a rather warm climate in coldest months of December and January.     

An approach to bioassess pelagic ciliate biodiversity at different taxonomic resolutions in response to various habitats in the Amundsen Sea (Antarctica)

The rapid melting of glaciers and loss of sea ice will result in changes in habitat conditions that may drive substantial changes in biodiversity. In order to bioassess the changing polar ecosystem and evaluate biological conservation, pelagic ciliate communities at different taxonomic resolutions were studied at five habitats in the Amundsen Sea during the austral summer from December 2010 to January 2011. Distinctive spatial patterns were observed in the communities among the five habitats (oceanic areas, transitional areas, polynyas, edges of glaciers, and edges of sea ice) in response to environmental variability (e.g., temperature, salinity, chlorophyll a, and nutrients). The distributions in the numbers of different taxonomic levels and of three biodiversity indices (Shannon-Wiener H′, Pielou’s J′, and Margalef D) also revealed clear spatial variability with the maximum mean species number and indices in the polynya and maximum genus and family numbers in the transitional area. The presence/absence of data at taxonomic resolutions up to the family level provided sufficient information to evaluate the ecological patterns of pelagic ciliate communities and could accurately reflect habitat variations. The k-dominance curves illustrated clearly that maximum diversity was presented in the polynya at the species level and in the transitional area at the genus and family level. We suggest that the diversity at higher taxonomic resolutions should be considered more in future monitoring. Our findings provide basic data and an approach toward answering important questions about biological conservation, especially the biodiversity at various taxonomic resolutions in response to the increasing climate changes in polar ecosystems.     

Application of denaturing high-performance liquid chromatography for rice variety identification and seed purity assessment

Recent DNA sequencing projects and the establishment of high-throughput assays have provided an abundance of sequence information and data on nucleotide polymorphisms in rice. Based on previously identified single-nucleotide polymorphisms (SNPs) and insertions/deletions, we employed denaturing high-performance liquid chromatography (DHPLC) to genotype rice varieties using a chromatographic pattern-based strategy. In this study, 12 amplicons harboring multiple and informative SNPs were screened. PCR products of the 12 amplicons from 47 rice varieties were analyzed by DHPLC and DNA sequencing. Each homozygous sample with a single peak pattern in the initial DHPLC analysis was mixed with zhenshan97 for a second DHPLC analysis. The 12 amplicons were found to be polymorphic across the hybrids, and mixed homozygous samples with 43 distinct DHPLC elution profiles detected. Sequence analysis confirmed that the distinct DHPLC patterns corresponded to different DNA sequences. A set of distinct characteristic profiles in six amplicons differentiated between all of the hybrids, inbred lines, and restorer lines and produced unique a fingerprint for these lines. In addition, we found that the DHPLC pattern of the hybrid was in accordance with the results obtained by DHPLC analysis of a mixed sample of the two parents. These results demonstrate that DHPLC can be efficiently applied for the rapid and automated identification of diverse rice varieties and could possibly be utilized for seed genetic purity testing on a high-throughput scale.     

S1P stimulates chemotactic migration and invasion in OVCAR3 ovarian cancer cells

OVCAR3 ovarian cancer cells express three sphingosine 1-phosphate (S1P) receptors, S1P(1), S1P(2), and S1P(3), but not S1P(4). Stimulation of OVCAR3 cells with S1P induced intracellular calcium increases, which were partly inhibited by VPC 23019 (an S1P(1/3) antagonist). S1P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S1P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S1P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S1P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 23019 (an S1P(1/3) antagonist) completely inhibited S1P-induced chemotaxis. S1P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 23019. Taken together, this study suggests that S1P stimulate chemotactic migration and cellular invasion, and VPC 23019-sensitive S1P receptor(s) might be involved in the processes.     

Arginine 177 is involved in Mn(II) binding by manganese peroxidase.

Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric state were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K(m) values for Mn(II) were approximately 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K(m) values for the substrates, H(2)O(2) and ferrocyanide, and the k(cat) values for Mn(II) and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn(II) were approximately 10-fold lower than for wild-type MnP. In addition, the K(D) values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn(II) for the mutant enzymes were approximately 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn(II) were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn(II) to the oxidized heme apparently is not affected.