Comparative genome-wide DNA methylation analysis reveals epigenomic differences in response to heat-humidity stress in Bombyx mori.

DNA methylation is an important epigenetic modification and has been shown to be involved in the response to abiotic stress. However, there are few studies on DNA methylation in insect response to environmental signals. In this study, we conducted a comprehensive comparative analysis of DNA methylation profiles between two silkworm strains with significantly different resistance to heat and humidity by whole-genome bisulfite sequencing (WGBS). We identified, in total, 2934 differentially methylated regions (DMRs) between RT_48h (resistant strain with high-temperature/humidity treatment for 48 h) and ST_48h (sensitive strain with high-temperature/humidity treatment for 48 h) under cytosine context (CG), which corresponded to 1230 DMR-related genes (DMGs), and the DMRs were primarily located in the gene body (exon and intron) region. Gene ontology (GO) and KEGG analysis showed that these DMGs were most significantly enriched in binding, cellular metabolic process, and RNA transport pathways. Moreover, 10 DMGs have been revealed to be involved in the heat-humidity stress response in the silkworm. The results of this study indicated that DNA methylation plays crucial roles in silkworm response to environmental stressors and provides important clues to identify key resistance genes in silkworm under high-temperature/humidity stress response.

[Inhibitory effects of an antisense PCDGF vector on proliferation and invasion of highly malignant ovarian cancer cells and the related mechanism].

OBJECTIVE: To investigate the inhibitory effects of an antisense PC cell derived growth factor (PCDGF) vector on proliferation and invasion of highly malignant ovarian cancer cell lines Sw626 and A2780 cells, and preliminarily explore the related mechanisms. METHODS: MTT assay and Boyden chamber in vitro invasion assay were employed to detect the changes of proliferation and invasion ability in the Sw626 and A2780 cells transfected with anti-sense PCDGF. The expression levels of cyclin D1 and CDK4 proteins before and after transfection were detected by Western blotting. The effects on the expression and activity of MMP-2 were evaluated by quantitative RT-PCR and zymography, respectively. RESULTS: Comparing with the blank group, the proliferation inhibition rate of the Sw626 and A2780 cells transfected with anti-sense PCDGF was 72.9% and 70.9%, respectively, and the invasion ability was inhibited by 62.9% and 59.0%, respectively. The levels of cyclin D1 and CDK4 protein expression in antisense PCDGF transfected cells were 0.38 +/- 0.08 and 0.37 +/- 0.13, respectively, all significantly lower than 0.84 +/- 0.11 and 0.64 +/- 0.11, respectively, in the blank group (P < 0.01). The MMP-2 mRNA expression level in antisense PCDGF transfected cell group was 0.66 +/- 0.11, not significantly decreased in comparison with 0.89 +/- 0.09 in the blank group (P > 0.05), but the activity of MMP-2 was inhibited significantly. CONCLUSION: The antisense PCDGF vector may inhibit markedly the proliferation and invasion of highly malignant ovarian cancer cells, and partially reverses their malignant phenotype. It seems to be related with down-regulating the expression of cyclin D1 and CDK4 and inhibiting the activity of MMP-2. Our findings indicate that PCDGF may become a new target for antisense gene therapy of ovarian cancer.

Ethyl 4-(4-cyano-phen-yl)-6-methyl-2-thioxo-1,2,3,4-tetra-hydro-pyrimidine-5-carboxyl-ate.

The asymmetric unit of the title compound, C(15)H(15)N(3)O(2)S, contains two independent mol-ecules corresponding to the R and S enanti-omers. The dihydro-pyrimidinone rings adopt a flattened boat conformation. One of the ethyl groups is disordered over two orientations with occupancy factors of 0.700 (7) and 0.300 (7). In the crystal structure, mol-ecules are linked by inter-molecular N-H⋯O hydrogen-bonding inter-actions into one-dimensional chains along the c-axis direction. The chains are further connected by N-H⋯S hydrogen bonds, forming a three-dimensional network.

4-Cyano-benzaldehyde thio-semi-carbazone.

The mol-ecule of the title compound, C(9)H(8)N(4)S, adopts an E configuration about both the C=N and C-NH bonds. In the crystal structure, adjacent mol-ecules are linked by inter-molecular N-H⋯S hydrogen-bonding inter-actions, forming chains running parallel to the b axis.

3-Nitro-benzaldehyde thio-semicarbazone.

The mol-ecule of the title compound, C(8)H(8)N(4)O(2)S, adopts an E configuration about both the C-N bonds. In the crystal structure, adjacent mol-ecules are linked by inter-molecular N-H⋯S hydrogen-bonding inter-actions, forming chains running parallel to the b axis.

[Screen of media for insect cells (HzAm1) growth and its adaptation to low serum culture].

Insect cell-baculovirus system is a useful tool for both insecticidal virus production and the expression of medically useful foreign genes. Serum-free culture or low serum culture for insect cells is essential and significant. Media for the growth of insect cells HzAm1 from three kinds of commercial media TC-100,GRACE and IPL-41 with 10% serum were investigated and screened. The result shows that medium TC-100 is the most suitable medium for the growth of cells HzAm1. Adaptation of insect cells HzAm1 to cultures in medium TC-100 with serum reduction from 10% to 1% was carried out as cultures were supplemented with lactalbumin hydrolysate and yeastolate etc. Growth of insect cells HzAml in medium TC-100 with serum 1%, lactalbumin hydrolysate and yeastolate was well.

Multi-peak phenomenon of insect cell infection with baculovirus at low multiplicity of infection.

In this communication we report the infection of armyworm Spodoptera frugiperda IPLB-Sf-21 cells with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus at low multiplicity of infection (MOI). The temporal variation of the extra-cellular virus and of the unstained cell was followed. The series of peaks in the virus concentration and the unstained cells count were used in order to infer the dynamic mechanism of the infection at low MOI. This mechanism can be used as the basis for the future formulation of a mathematical model of the process.

A mathematical model of baculovirus infection on insect cells at low multiplicity of infection.

The expression efficiency of the insect cells-baculovirus system used for insecticidal virus production and the expression of medically useful foreign genes is closely related with the dynamics of infection. The present studies develop a model of the dynamic process of insect cell infection with baculovirus at low multiplicity of infection (MOI), which is based on the multi-infection cycles of insect cell infection at low MOI described. A mathematical model for the amount of viruses released from primary infected cells and the amount of free viruses before secondary infected cells release viruses has been developed. Comparison of the simulation results with the experimental data confirms qualitatively that this model is highly reasonable before secondary infected cells release viruses. This model is considered as a base for further modeling the entire complicated infection process.

[The transcriptional regulation study on human delta globin gene with CAAT box C-->T point mutation in its promoter].

OBJECTIVE: To study the transcriptional regulation of human delta globin gene with C-->T point mutation at -64 in its promoter. METHODS: Human delta globin genes including wild CAAT box and mutant CAAT box (-64C-->T) were separately cloned into eukaryotic expression vector pcDNA3.1 (-)/Myc-His A which was cut out the strong promoter CMV, transfected MEL cells, and induced by DMSO to express. The transcriptional regulation of human delta globin gene was analysed using semi-quantitative RT-PCR. RESULTS: The expression level of human delta globin gene with mutant CAAT box was 2.2-fold as high as that with wild CAAT box. CONCLUSION: The defective CAAT box of human delta globin gene promoter region may be one of the major reasons for its low expression level.