Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Animal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4-15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.     

Survey of Vietnamese Peanuts,Corn and Soil for the Presence of <Emphasis Type="Italic">Aspergillus flavus</Emphasis> and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis>

Aspergillus flavus and Aspergillus parasiticus cause perennial infection of agriculturally important crops in tropical and subtropical areas. Invasion of crops by these fungi may result in contamination of food and feed by potent carcinogenic aflatoxins. Consumption of aflatoxin contaminated foods is a recognised risk factor for human hepatocellular carcinoma (HCC) and may contribute to the high incidence of HCC in Southeast Asia. This study conducted a survey of Vietnamese crops (peanuts and corn) and soil for the presence of aflatoxigenic fungi and used microsatellite markers to investigate the genetic diversity of Vietnamese Aspergillus strains. From a total of 85 samples comprising peanut (25), corn (45) and soil (15), 106 strains were isolated. Identification of strains by colony morphology and aflatoxin production found all Vietnamese strains to be A. flavus with no A. parasiticus isolated. A. flavus was present in 36.0% of peanut samples, 31.1% of corn samples, 27.3% of farmed soil samples and was not found in virgin soil samples. Twenty-five per cent of the strains produced aflatoxins. Microsatellite analysis revealed a high level of genetic diversity in the Vietnamese A. flavus population. Clustering, based on microsatellite genotype, was unrelated to aflatoxin production, geographic origin or substrate origin.     

Rapid analysis of Jatropha curcas gene functions by virus-induced gene silencing

Jatropha curcas L. is a small, woody tree of the Euphorbiaceae family. This plant can grow on marginal land in the tropical and subtropical regions and produces seeds containing up to 30% oil. Several Asian countries have selected Jatropha for large scale planting as a biodiesel feedstock. Nevertheless, Jatropha also possesses several undesirable traits that may limit its wide adoption. An improved understanding of plant development and the regulation of fatty acid (FA) and triacylglyceride biosynthesis in Jatropha is particularly facilitative for the development of elite crops. Here, we show that a tobacco rattle virus (TRV) vector can trigger virus-induced gene silencing (VIGS) in Jatropha. Our optimized method produced robust and reliable gene silencing in plants agroinoculated with recombinant TRV harbouring Jatropha gene sequences. We used VIGS to investigate possible functions of 13 Jatropha genes of several functional categories, including FA biosynthesis, developmental regulation and toxin biosynthesis, etc. Based on the effects of VIGS on the FA composition of newly emerged leaves, we determined the function of several genes implicated in FA biosynthesis. Moreover, VIGS was able to discriminate independent functions of related gene family members. Our results show that VIGS can be used for high-throughput screening of Jatropha genes whose functions can be assayed in leaves.     

Extracting biologically significant patterns from short time series gene expression data

Background  

Time series gene expression data analysis is used widely to study the dynamics of various cell processes. Most of the time series data available today consist of few time points only, thus making the application of standard clustering techniques difficult.     

Distribution and abundance of marine turtles in the Socialist Republic of Viet Nam

To establish baseline data on the distribution, abundance and threats to marine turtles in Viet Nam we conducted surveys with local fishers, community members and provincial Ministry of Fisheries staff from 17 of Viet Nam’s 29 coastal provinces. These data indicate that five species of marine turtle reside in Viet Nam’s waters (loggerhead, olive ridley, leatherback, green and hawksbill turtles), and four species nest on Viet Nam’s beaches (all of the above except the loggerhead turtle). It is evident from these data that significant declines have occurred in both foraging and nesting populations of all five marine turtle species found in Viet Nam. The greatest current threats to marine turtle populations in Viet Nam are habitat degradation, the accidental and opportunistic of turtles capture by fishers and the direct take of nesting females and their eggs. Successful conservation efforts have been made in recent years through collaboration between international Non Government Organisations and several Vietnamese Government Ministries. Continued success of these projects and the development and implementation of marine conservation policy will depend upon building awareness among Government employees, fishers and the general public about marine turtle biology, ecology, and the need to protect them.     

Genetic association of the antiviral restriction factor TRIM5alpha with human immunodeficiency virus type 1 infection

The innate antiviral factor TRIM5alpha restricts the replication of some retroviruses through its interaction with the viral capsid protein, leading to abortive infection. While overexpression of human TRIM5alpha results in modest restriction of human immunodeficiency virus type 1 (HIV-1), this inhibition is insufficient to block productive infection of human cells. We hypothesized that polymorphisms within TRIM5 may result in increased restriction of HIV-1 infection. We sequenced the TRIM5 gene (excluding exon 5) and the 4.8-kb 5' putative regulatory region in genomic DNA from 110 HIV-1-infected subjects and 96 exposed seronegative persons, along with targeted gene sequencing in a further 30 HIV-1-infected individuals. Forty-eight single nucleotide polymorphisms (SNPs), including 20 with allele frequencies of >1.0%, were identified. Among these were two synonymous and eight nonsynonymous coding polymorphisms. We observed no association between TRIM5 polymorphism in HIV-1-infected subjects and their set-point viral load after acute infection, although one TRIM5 haplotype was weakly associated with more rapid CD4(+) T-cell loss. Importantly, a TRIM5 haplotype containing the nonsynonymous SNP R136Q showed increased frequency among HIV-1-infected subjects relative to exposed seronegative persons, with an odds ratio of 5.49 (95% confidence interval = 1.83 to 16.45; P = 0.002). Nonetheless, we observed no effect of individual TRIM5alpha nonsynonymous mutations on the in vitro HIV-1 susceptibility of CD4(+) T cells. Therefore, any effect of TRIM5alpha polymorphism on HIV-1 infection in primary lymphocytes may depend on combinations of SNPs or on DNA sequences in linkage disequilibrium with the TRIM5alpha coding sequence.     

The enzymes of a marine bacterial isolate from the brown alga <Emphasis Type="Italic">Sargassum polycystum</Emphasis> agardh, 1821, that catalyzes the transformation of polyanionic oligo-and polysaccharides

A search for enzymes involved in the degradation of polyanionic polysaccharides (fucoidans and alginic acid) was conducted among bacterial epiphytes of the brown alga Sargassum polycystum that grows in the territorial waters of the Socialist Republic of Vietnam. Two resistant bacterial strains, F10 and F14, have been isolated from the algal microflora that degrade the thallus of the alga under laboratory conditions. These bacterial strains differed in the morphological, physiological, and biochemical characteristics and in the composition of enzymes. The strains were studied for the ability to synthesize intracellular oligo-and polysaccharide hydrolases and alginate lyases. The optimal conditions for the growth of bacterial strain F14 and the biosynthesis of fucoidanase and polymannuronate-specific alginate lyase were determined. The partially purified alginate lyase was stable at a temperature up to 40°C and had an optimal pH 6.0 and an optimal temperature 35°C.     

A century of landscape disturbance and urbanization of the San Francisco Bay region affects the present-day genetic diversity of the California Ridgway’s rail (<Emphasis Type="Italic">Rallus obsoletus obsoletus</Emphasis>)

Fragmentation and loss of natural habitat have important consequences for wild populations and can negatively affect long-term viability and resilience to environmental change. Salt marsh obligate species, such as those that occupy the San Francisco Bay Estuary in western North America, occupy already impaired habitats as result of human development and modifications and are highly susceptible to increased habitat loss and fragmentation due to global climate change. We examined the genetic variation of the California Ridgway’s rail (Rallus obsoletus obsoletus), a state and federally endangered species that occurs within the fragmented salt marsh of the San Francisco Bay Estuary. We genotyped 107 rails across 11 microsatellite loci and a single mitochondrial gene to estimate genetic diversity and population structure among seven salt marsh fragments and assessed demographic connectivity by inferring patterns of gene flow and migration rates. We found pronounced genetic structuring among four geographically separate genetic clusters across the San Francisco Bay. Gene flow analyses supported a stepping stone model of gene flow from south-to-north. However, contemporary gene flow among the regional embayments was low. Genetic diversity among occupied salt marshes and genetic clusters were not significantly different. We detected low effective population sizes and significantly high relatedness among individuals within salt marshes. Preserving genetic diversity and connectivity throughout the San Francisco Bay may require attention to salt marsh restoration in the Central Bay where habitat is both most limited and most fragmented. Incorporating periodic genetic sampling into the management regime may help evaluate population trends and guide long-term management priorities.     

Enzymatic and molecular characterization of an endo-1,3-β-d-glucanase from the crystalline styles of the mussel Perna viridis

The retaining endo-1,3-β-d-glucanase (EC 3.2.1.39) was isolated from the crystalline styles of the commercially available Vietnamese edible mussel Perna viridis. It catalyzes hydrolysis of β-1,3-bonds in glucans and enables to catalyze a transglycosylation reaction. Resources of mass-spectrometry for analysis of enzymatic products were studied. cDNA sequence of endo-1,3-β-d-glucanase was determined by RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) methods. The cDNA of 1380bp contains an open reading frame of 1332bp encoding a mature protein of 328 amino acids. On basis of amino acid sequence analysis endo-1,3-β-d-glucanase was classified as a glycoside hydrolase of family 16.