Expression of rice gall dwarf virus outer coat protein gene (<Emphasis Type="Italic">S8</Emphasis>) in insect cells

To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.     

In vitro anti-influenza virus activities of sulfated polysaccharide fractions from <Emphasis Type="Italic">Gracilaria lemaneiformis</Emphasis>

In this paper, in vitro anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were investigated. Cytotoxicities and antiviral activities of Gracilaria lemaneiformis polysaccharides (PGL), Gracilaria lemaneiformis polysaccharide fraction-1 (GL-1), Gracilaria lemaneiformis polysaccharide fraction-2 (GL-2) and Gracilaria lemaneiformis polysaccharide fraction-3 (GL-3) were studied by the Methyl thiazolyl tetrazolium (MTT) method, and the inhibitory effect against Human influenza virus H1-364 induced cytopathic effect (CPE) on MDCK cells were observed by the CPE method. In addition, the antiviral mechanism of PGL was explored by Plaque forming unit (PFU), MTT and CPE methods. The results showed: i) Cytotoxicities were not significantly revealed, and H1-364 induced CPE was also reduced treated with sulfated polysaccharide fractions from Gracilaria lemaneiformis; ii) Antiviral activities were associated with the mass percentage content of sulfate groups in polysaccharide fractions, which was about 13%, in polysaccharides (PGL and GL-2) both of which exhibited higher antiviral activity; iii) A potential antiviral mechanism to explain these observations is that viral adsorption and replication on host cells were inhibited by sulfated polysaccharides from Gracilaria lemaneiformis. In conclusion, Anti-influenza virus activities of sulfated polysaccharide fractions from Gracilaria lemaneiformis were revealed, and the antiviral activities were associated with content of sulfate groups in polysaccharide fractions.     

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.     

Characterization of variants in the promoter of BZLF1 gene of EBV in nonmalignant EBV-associated diseases in Chinese children

Background

SEN virus is a blood-borne, circular ssDNA virus and possessing nine genotypes (A to I). Among nine genotypes, SENV-D and SENV-H genotypes have the strong link with patients with unknown (none-A to E) hepatitis infections. Infection with blood-borne viruses is the second important cause of death in thalassemic patients. The aim of this study was to determine the frequency of SENV-D and SENV-H genotypes viremia by performing nested-PCR in 120 and 100 sera from healthy blood donors and thalassemic patients in Guilan Province, North of Iran respectively. Also, to explicate a possible role of SEN virus in liver disease and established changes in blood factors, the serum aminotransferases (ALT and AST) and some of the blood factors were measured.

Results

Frequency of SENV-D, SENV (SENV-H or SENV-D) and co-infection (both SENV-D and SENV-H) viremia was significantly higher among thalassemic patients than healthy individuals. Frequency of SENV-H viremia was significantly higher than SENV-D among healthy individuals. In comparison to SENV-D negative patients, the mean of mean corpuscular hemoglobin was significantly higher in SENV-D positive and co-infection cases (P < 0.05). The means of AST and ALT were significantly higher in thalassemic patients than healthy blood donors, but there were not any significant differences in the means of the liver levels between SENV-positive and -negative individuals in healthy blood donors and thalassemic patients. High nucleotide homology observed among PCR amplicon's sequences in healthy blood donors and thalassemic patients.

Conclusions

The high rate of co-infection shows that different genotypes of SENV have no negative effects on each other. The high frequency of SENV infection among thalassemic patients suggests blood transfusion as main route of transmission. High frequency of SENV infection in healthy individuals indicates that other routes rather than blood transfusion also are important. Frequency of 90.8% of SENV infection among healthy blood donors as well as high nucleotide homology of sequenced amplicons between two groups can probably suggest that healthy blood donors infected by SENV act partly as a source of SENV transmission to the thalassemic patients. In conclusion, SENV-D isolate in Guilan Province may be having a pathogenic agent for thalassemic patients.     

Parameter inference for discretely observed stochastic kinetic models using stochastic gradient descent

Background  

Stochastic effects can be important for the behavior of processes involving small population numbers, so the study of stochastic models has become an important topic in the burgeoning field of computational systems biology. However analysis techniques for stochastic models have tended to lag behind their deterministic cousins due to the heavier computational demands of the statistical approaches for fitting the models to experimental data. There is a continuing need for more effective and efficient algorithms. In this article we focus on the parameter inference problem for stochastic kinetic models of biochemical reactions given discrete time-course observations of either some or all of the molecular species.     

Identification and mapping of PmG16, a powdery mildew resistance gene derived from wild emmer wheat

The gene-pool of wild emmer wheat, Triticum turgidum ssp. dicoccoides, harbors a rich allelic repertoire for disease resistance. In the current study, we made use of tetraploid wheat mapping populations derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16) to identify and map a new powdery mildew resistance gene derived from wild emmer wheat. Initially, the two parental lines were screened with a collection of 42 isolates of Blumeria graminis f. sp. tritici (Bgt) from Israel and 5 isolates from Switzerland. While G18-16 was resistant to 34 isolates, Langdon was resistant only to 5 isolates and susceptible to 42 isolates. Isolate Bgt#15 was selected to differentiate between the disease reactions of the two genotypes. Segregation ratio of F_2-3 and recombinant inbreed line (F₇) populations to inoculation with isolate Bgt#15 indicated the role of a single dominant gene in conferring resistance to Bgt#15. This gene, temporarily designated PmG16, was located on the distal region of chromosome arm 7AL. Genetic map of PmG16 region was assembled with 32 simple sequence repeat (SSR), sequence tag site (STS), Diversity array technology (DArT) and cleaved amplified polymorphic sequence (CAPS) markers and assigned to the 7AL physical bin map (7AL-16). Using four DNA markers we established colinearity between the genomic region spanning the PmG16 locus within the distal region of chromosome arm 7AL and the genomic regions on rice chromosome 6 and Brachypodium Bd1. A comparative analysis was carried out between PmG16 and other known Pm genes located on chromosome arm 7AL. The identified PmG16 may facilitate the use of wild alleles for improvement of powdery mildew resistance in elite wheat cultivars via marker-assisted selection.     

Genetic relationships between resistances to Fusarium head blight and crown rot in bread wheat (Triticum aestivum L.)

Fusarium head blight (FHB) and crown rot (CR) are two wheat diseases caused by the same Fusarium pathogens. Progress towards CR resistance could benefit from FHB-resistant germplasm if the same genes are involved in resistance to these two different diseases. Two independent studies were conducted to investigate the relationship between host resistances to these two diseases. In the first study 32 genotypes were assessed and no significant correlation between their reactions to FHB and CR was detected. The second study was based on a QTL analysis of a doubled haploid population derived from a variety with resistance to both diseases. Results from this study showed that loci conferring resistance to FHB and CR are located on different chromosomes. Together, these results suggest that, despite a common aetiology, different host genes are involved in the resistance against FHB and CR in wheat. Thus, although it is possible that genes affecting both diseases may exist in other germplasm or under different conditions, separate screening seems to be needed in identifying sources of CR resistance.     

Effects of organic matter incorporation on nitrous oxide emissions from rice-wheat rotation ecosystems in China

Organic matter addition is thought to be an important regulator of nitrous oxide (N₂O) emissions from croplands. Contradictory effects, however, were reported in previous studies. To investigate the effects of crop residue management on N₂O emissions from rice-wheat rotation ecosystems, we conducted field experiments at three sites (Suzhou, Wuxi and Jiangdu) in the Yangtze River Delta, using static chamber and gas chromatography methods. Our data show that N₂O emissions throughout the rice season from plots treated with wheat straw application at a high rate (WS) prior to rice transplanting (1.1–2.0 kg N ha^?1) were significantly lower (P?<?0.05) than those from the control plots without organic matter addition or added with wheat straw at a moderate rate (1.6–2.9 kg N ha^?1). Furthermore, the WS treatments had a residual inhibitory effect on N₂O emissions in the following non-rice season, which consistently resulted in significantly lower emissions (P?<?0.05) compared to the control treatments (2.2–3.1 vs. 3.9–5.6 kg N ha^?1). In comparison to the control treatments, the WS treatments reduced both the seasonal and annual direct emission factors of the applied nitrogen (EF_d) by 50–68% (mean: 57%). The addition of compost (aerobically composted rice or wheat straw harvested in the last rotation) reduced the seasonal and annual EF_ds by 29–32%. Over the entire rice-wheat rotation cycle, annual N₂O emissions from the fertilized fields at the three sites ranged from 3.3?±?0.3 to 16.8?±?0.6 kg N ha^?1, with a coefficient of variation (CV) of 61%. Similarly, the EF_ds during the rice-wheat rotation cycle ranged from 0.4% to 2.5%, with a CV of 67%. These high spatial variations might have been related to: variations in soil properties, such as texture and soil organic carbon; management practices, such as straw treatments (i.e., compost versus fresh straw) and weather conditions, such as precipitation and rainfall distribution. Our results indicate that the incorporation of fresh wheat straw at a high rate during the rice season is an effective management practice for the mitigation of N₂O emissions in rice-wheat rotation systems. Whether this practice is also effective in reducing the overall global warming potential of net N₂O, CH₄ and CO₂ emissions needs to be seen through further studies.