A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

High susceptibility of human dendritic cells to avian influenza H5N1 virus infection and protection by IFN-alpha and TLR ligands

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-alpha. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.     

Novel Chloroplast Microsatellite (cpSSR) Markers for Genetic Diversity Assessment of Cultivated and Wild Hevea Rubber

Understanding the genetic diversity and population structure of Hevea is of great importance for managing its conservation and for utilization of rubber genetic resources. In this study, we investigated the genetic diversity and population structure of eight populations of Hevea rubber genotypes from Malaysia (MY), India (IN), Sri Lanka (LK), Indonesia (ID), France (FR), Thailand (TH), Brazil (BR), and China (CN), in addition to individual primary clones, using 10 nuclear and 11 polymorphic novel chloroplast microsatellite markers (nSSRs and cpSSRs, respectively). The BR population exhibited the greatest genetic diversity (H _e ) for both nSSR (0.841) and haploid genetic diversity (cpSSR; 0.207) markers. Bayesian analysis was applied to infer genetic structure based on nSSR data using STRUCTURE software, and a neighbor-joining tree was used to construct an unrooted phylogram based on Nei’s genetic distance that clustered these Hevea genotypes into three groups. Six polymorphic cpSSR markers produced 13 alleles and eight chlorotypes. Seven chlorotypes, A, B, C, D, E, F, and H were detected among Brazilian populations from Acre (AC), Rondônia (RO), and Mato Grosso (MT) locations. The G and H chlorotypes were found in Asiatic genotypes or Wickham clones and only one genotype, 15 AC-F-7 38-62 from AC location. These results provide valuable data for in situ or ex situ conservation and utilization of germplasm collections for breeding programs.

     

Studies of H5N1 influenza virus infection of pigs by using viruses isolated in Vietnam and Thailand in 2004

To determine whether avian H5N1 influenza viruses associated with human infections in Vietnam had transmitted to pigs, we investigated serologic evidence of exposure to H5N1 influenza virus in Vietnamese pigs in 2004. Of the 3,175 pig sera tested, 8 (0.25%) were positive for avian H5N1 influenza viruses isolated in 2004 by virus neutralization assay and Western blot analysis. Experimental studies of replication and transmissibility of the 2004 Asian H5N1 viruses in pigs revealed that all viruses tested replicated in the swine respiratory tract but none were transmitted to contact pigs. Virus titers from nasal swabs peaked on day 2, and low titers were detected in the liver of two of the four pigs tested. Our findings indicate that pigs can be infected with highly lethal Asian H5N1 viruses but that these viruses are not readily transmitted between pigs under experimental conditions.     

Dengue infection in children in Ratchaburi, Thailand: a cohort study. II. Clinical manifestations

Background

Dengue infection is one of the most important mosquito-borne diseases. More data regarding the disease burden and the prevalence of each clinical spectrum among symptomatic infections and the clinical manifestations are needed. This study aims to describe the incidence and clinical manifestations of symptomatic dengue infection in Thai children during 2006 through 2008.

Study Design

This study is a school-based prospective open cohort study with a 9,448 person-year follow-up in children aged 3–14 years. Active surveillance for febrile illnesses was done in the studied subjects. Subjects who had febrile illness were asked to visit the study hospital for clinical and laboratory evaluation, treatment, and serological tests for dengue infection. The clinical data from medical records, diary cards, and data collection forms were collected and analyzed.

Results

Dengue infections were the causes of 12.1% of febrile illnesses attending the hospital, including undifferentiated fever (UF) (49.8%), dengue fever (DF) (39.3%) and dengue hemorrhagic fever (DHF) (10.9%). Headache, anorexia, nausea/vomiting and myalgia were common symptoms occurring in more than half of the patients. The more severe dengue spectrum (i.e., DHF) had higher temperature, higher prevalence of nausea/vomiting, abdominal pain, rash, diarrhea, petechiae, hepatomegaly and lower platelet count. DHF cases also had significantly higher prevalence of anorexia, nausea/vomiting and abdominal pain during day 3–6 and diarrhea during day 4–6 of illness. The absence of nausea/vomiting, abdominal pain, diarrhea, petechiae, hepatomegaly and positive tourniquet test may predict non-DHF.

Conclusion

Among symptomatic dengue infection, UF is most common followed by DF and DHF. Some clinical manifestations may be useful to predict the more severe disease (i.e., DHF). This study presents additional information in the clinical spectra of symptomatic dengue infection.     

Tropism of Avian Influenza A (H5N1) Virus to Mesenchymal Stem Cells and CD34+ Hematopoietic Stem Cells

The presence of abnormal hematologic findings such as lymphopenia, thrombocytopenia, and pancytopenia were diagnosed in severe cases of avian influenza A H5N1. Whether direct viral dissemination to bone marrow (BM) cells causes this phenomenon remains elusive. We explore the susceptibility of the two stem cell types; hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) isolated from human BM cells or cord blood, to infection with avian H5N1 viruses. For the first time, we demonstrated that the H5N1 virus could productively infect and induce cell death in both human stem cell types. In contrast, these activities were not observed upon human influenza virus infection. We also determined whether infection affects the immunomodulatory function of MSCs. We noted a consequent dysregulation of MSC-mediated immune modulation as observed by high cytokine and chemokine production in H5N1 infected MSCs and monocytes cocultures. These findings provide a better understanding of H5N1 pathogenesis in terms of broad tissue tropism and systemic spread.     

Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction

Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program.     

Generating and characterizing monoclonal and polyclonal antibodies against avian H5N1 hemagglutinin protein

Accurate and timely diagnoses are central to H5N1 infection control. Here we describe the cloning and expression of the HA1 protein of the A/Vietnam/1203/04 strain in a bacterial system to generate mono-/polyclonal antibodies. All of the eight generated monoclonal antibodies recognized the same linear epitope on the top globular region of the HA structure—a highly conserved epitope among all circulating H5N1 clades identified by amino acid alignment. Results from immunofluorescence staining and Western blotting indicate that all monoclonal antibodies interacted with a denatured form of HA proteins, while the resultant polyclonal antibodies recognized both denatured and native HA proteins on H5N1 reverse-genetics (RG) viruses. Results from flow cytometry and microneutralization assays indicate that the polyclonal antibodies blocked viral binding and neutralized H5N1-RG viruses. Our results may prove useful to establishing future H5N1 mono-and polyclonal antibodies, and perhaps contribute to the development of an alternative H5N1 vaccine.