Global Analysis Reveals the Crucial Roles of DNA Methylation during Rice Seed Development.

Seed development is an important process of reproductive development and consists of embryo and endosperm development; both comprise several key processes. To determine and investigate the functions of the dynamic DNA methylome during seed development, we profiled the DNA methylation genome wide in a series of developmental stages of rice (Oryza sativa) embryo and endosperm by methylcytosine immunoprecipitation followed by Illumina sequencing. The results showed that embryo is hypermethylated predominantly around non-transposable element (TE) genes, short DNA-TEs, and short interspersed TEs compared with endosperm, and non-TE genes have the most diverse methylation status across seed development. In addition, lowly expressed genes are significantly enriched in hypermethylated genes, but not vice versa, confirming the crucial role of DNA methylation in suppressing gene transcription. Further analysis revealed the significantly decreased methylation at early developing stages (from 2 to 3 d after pollination), indicating a predominant role of demethylation during early endosperm development and that genes with a consistent negative correlation between DNA methylation change and expression change may be potentially directly regulated by DNA methylation. Interestingly, comparative analysis of the DNA methylation profiles revealed that both rice indica and japonica subspecies showed robust fluctuant profiles of DNA methylation levels in embryo and endosperm across seed development, with the highest methylation level at 6 d after pollination (2 d after pollination of endosperm in japonica as well), indicating that a complex and finely controlled methylation pattern is closely associated with seed development regulation. The systemic characterization of the dynamic DNA methylome in developing rice seeds will help us understand the effects and mechanism of epigenetic regulation in seed development.

Molecular and immune response characterizations of a novel AIF and cytochrome c in Litopenaeus vannamei defending against WSSV infection.

Apoptosis inducing factor (AIF) and cytochrome c (CYC) are two mitochondrial apoptogenic factors. In the present study, the cDNA sequences of AIF (LvAIF) and CYC (LvCYC) were cloned from Pacific white shrimp, Litopenaeus vannamei. The LvAIF was 1664 bp, including a 5'-terminal untranslated region (UTR) of 154 bp, an open reading frame (ORF) of 1323 bp encoding a polypeptide of 440 amino acids (aa) and a 3' UTR of 187 bp. The LvCYC was 582 bp, including a 50 bp 5' UTR, a 315 bp ORF encoding for 104 aa, and a 217 bp 3' UTR. The deduced protein of LvAIF contained a conserved Pyr_redox and AIF_C domain at the N-terminal and the predicted LvCYC included a conservative cytochrome_C domain, respectively. Phylogenetic analysis revealed that LvAIF belonged to AIF1 subfamily and showed a close relationship with AIF1 from vertebrates and LvCYC showed the closest relationship with its counterparts from shrimp Marsupenaeus japonicus. Tissue expression profiles showed that both LvAIF and LvCYC existed in most tissues, with the most predominant expression of LvAIF in intestine, then followed muscle and the weakest expression in gill. The highest expression of LvCYC was detected in muscle, and the weakest expression was in hemocytes. Additionally, after white spot syndrome virus (WSSV) infection, the significant up-regulation of LvAIF, LvCYC and caspase 3 transcripts and the increase of pro-caspase 3 and active-caspase 3 protein were detected at most time points (P < 0.05). However, all of the three genes down-regulated in hemocytes in the early stage after WSSV infection. WSSV proliferation and shrimp mortality showed a time-dependent manner and the production of ROS in hemocytes were significantly increased at 6 and 24 h after infection. Our results showed that the apoptotic genes AIF, CYC and caspase 3 might play crucial roles in hepatopancreas, however, the production of ROS in hemocytes might be important in shrimp defense against WSSV infection.

[Determination of Wine Varieties with NIR and Fusion of Multiple Classifiers].

The conventional qualitative analysis of near infrared spectroscopy (NIR) commonly uses one single classification model. This paper focused on the fusion of multiple classifiers based on different single classifiers by using the fused classifier to determine different varieties of red-wines. NIR spectra of 170 red-wine samples were collected by using Fourier transform near-infrared spectrometer. Red-wine classification models were established respectively, based on PLS-DA, SVM, Fisher and Ada-Boost. Then these models were selected to obtain some different base classifiers according to Diversity Measure Feature Selective (DMFS). The highest accuracy rate of determining different varieties of red-wine test samples of four single base classifiers was up to 88.24%, and at the same time the lowest discriminant accuracy rate was 81.18%. At last, we got the fused classifier, which combined four base classifiers with weighted voting principle, and determined its test set again by using the fused classifier. The final classification accuracy rate for red-wine varieties increased to 92.94%, In contrast with one single classifier, the lowest misjudged number of fused classifiers decreased from 9 to 6.These results suggested that the performance of fused classifier is superior to one single classifier. It is feasible to use fused classifier combined with near infrared spectroscopy to determine different varieties of red-wines.

[Long-fragment RNA inhibits hepatitis B virus gene replication and expression in HepG2.2.15 cells].

To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernate were detected by ELISA. The HBV DNA in the supernate was quantified by real-time PCR. After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621+/-0.027, 3.399+/-0.018 and 2.232+/-0.187 respectively; the levels of HBeAg were 0.749+/-0.019, 1.548+/-0.025 and 1.570+/-0.044 respectively and the levels of HBV DNA were 1.597+/-0.082, 3.381+/-0.297 and 3.610+/-0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P value is less than 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z = -0.866) and HBV DNA (Z = -1.155) levels in the culture supernate but slightly increased the HBsAg level (Z = -2.309). Antisense RNA might be a useful tool to repress HBV replication.

[Up-regulation of major histocompatibility complex class I-related molecules A (MICA) induced by 5-aza-2'-deoxycytidine].

OBJECTIVE: Major histocompatibility complex class I C-related molecules A and B (MICA and MICB) are innate immune system ligands for the NKG2D receptor expressed by natural killer cells and activated CD8(+)T cells. Our previous study showed that 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, can induce the expression of MICB and sensitized cells to NKL-cell-mediated cytolysis. The aim of this study was to determine the expression level of MICA in HepG2 cells (an HCC cell line) and L02 cells ( a normal liver cell), and to investigate the effect of 5-aza-dC on MICA expression in HepG2 cells. METHODS: Cells were treated with 5-aza-dC, caffeine and ATM-specific siRNA. The cell surface MICA protein on HepG2 cells and L02 cells was determined using flow cytometry. The mRNA level was detected using real time RT-PCR. RESULTS: MICA was undetectable on the surface of L02 cells, but was highly expressed on HepG2 cells. MICA expression was upregulated in response to 5-aza-dC treatment (P less than 0.05), and the upregulation of MICA was partially prevented by pharmacological or genetic inhibition of ataxia telangiectasia mutated (ATM) kinase (P less than 0.05). CONCLUSION: Our data suggest that 5-aza-dC induces the expression of MICA by a DNA damage-dependent mechanism.

[Studies on chemical constituents in fresh fleshy scaleleaf of Lilium lancifolium].

OBJECTIVE: To study the chemical constituents in fresh fleshyscaleaf of Lilium lancifolium. METHOD: The constituents were separated. by various kinds of chromatography and their structures were identified on the basis of spectral analysis. RESULT: Ten compounds were identified regaloside A (1), regaloside C (2), methyl-a-D-mannopyranosid (3), methyl-ca-D-glucopyranoside (4), (25R, 26R) -26-methoxyspirost-5-ene-3p-yl-O-ca-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranoside (5), (25R)-spirost-5-ene-3beta-yl-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->6)]-beta-D-glucopyranoside (6), (25R, 26R)-17alpha-hydroxy-26-methoxyspirost-5-ene-3beta-yl-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyra nosyl-(1-->6)]-beta-D-glucopyranoside (7), daucosterol (8), adenoside (9), berberine (10). CONCLUSION: All compounds except 1 and 3 were isolated from this species for the first time, and berberine was first reported in genus Lilium.

Packed-fiber solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry for determination of diethylstilbestrol, hexestrol, and dienestrol residues in milk products.

A sensitive analytical method based on packed-fiber solid-phase extraction and high performance liquid chromatography-tandem mass spectrometry (PF SPE-HPLC-MS/MS) has been developed for determination of three synthetic stilbenes in milk. The stilbenes are extracted with acetonitrile, using sodium chloride, and purified with PF SPE using a cartridge containing electrospun polystyrene nanofibers. Parameters affecting the efficiency of PF SPE, such as pH and amount of salt, were optimized. Under optimal conditions, the limits of detection and quantification were 5-13pg/g and 15-37pg/g, respectively. Absolute recoveries varied between 60% and 85% at three different levels. The method was successfully applied for the determination of estrogenic stilbenes in a total of 69 milk samples. The method is sensitive and cost-effective in stilbene detection, and has potential in quality control of dairy products.

[Seasonal variation of soil respiration and its components in tropical rain forest and rubber plantation in Xishuangbanna, Yunnan].

By using trenching method and infrared gas analyzer, this paper studied the seasonal variation of soil respiration (SR), including root respiration (RR) and heterotrophic respiration (HR), in tropical seasonal rain forest (RF) and rubber (Hevea brasiliensis) plantation (RP) in Xishuangbanna of Yunnan, China. The results showed that the SR and HR rates were significantly higher in RF than in RP (P < 0.01), while the RR rate had less difference between the two forests. Soil temperature and moisture were the key factors affecting the SR, RR and HR. The SR and HR rates in the two forests were rainy season > dry-hot season > foggy season, but the RR rate was rainy season > foggy season > dry-hot season in RF, and foggy season > rainy season > dry-hot season in RP. The contribution of RR to SR in RF (29%) was much lower than that in RP (42%, P < 0.01), while the contribution of HR to SR was 71% in RF and 58% in RP. When the soil temperature at 5 cm depth varied from 12 degrees C to 32 degrees C, the Q10 values for SR, HR, and RR rates were higher in RF than in RP. HR had the highest Q10 value, while RR had the lowest one.

Phenolic glucosides from Alangium plantanifolium.

A novel phenolic glucoside alangitanifoliside A (1) together with two known phenolic glucosides 4',6'-O-(S)-hexahydroxydiphenylsalicin (2) and salicin (3), and gallic acid were isolated from stem barks of Alangium plantanifolium. Their structures were determined by spectroscopic and chemical methods. The structure of 1 was elucidated to be 1-O-[2-(1-hydroxy-6-oxocyclohex-2-ene-1-carboxymethyl)-phenyl]-4,6-O-[(S)-,4,4',5,5' 6,6'-hexahydroxydiphenoyl]-beta-D-glucopyranose.

Furostanol oligoglycosides from Asparagus cochinchinensis.

Three new furostanol oligoglycosides, named aspacochioside A (1), B (2) and C (3), together with the known compound 3-O-[(alpha-L-rhamnopyranosyl-(1 --> 4))(beta-D-glucopyranosyl)]-26-O[beta-D-glucopyranosyl]-(25S)-5beta-spirostane-3beta-ol were isolated from the roots of Asparagus cochinchinensis. Their structures were elucidated by spectroscopic techniques (IR, HR-ESIMS, ESIMS/MS, ID and 2D NMR) and chemical methods as 3-O-[(alpha-L-rhamnopyranosyl-(1 --> 4))(beta-D-glucopyranosyl)]-26-O-[beta-D-glucopyranosyl]-(25S)-5beta-furostane-3beta,22alpha,26-triol (1), 3-O-[(alpha-L-rhamnopyranosyl-(1 --> 4))(beta-D-glucopyranosyl)]-26-O-[beta-D-glucopyranosyl]-22alpha-methoxy-(25S)-5beta-furostane-3beta,26-diol (2), and 3-O-[(alpha-L-rhamnopyranosyl-(1 --> 4))(beta-D-glucopyranosyl)]-26-O-[beta-D-glucopyranosyl]-(25S)-5beta-furost-20(22)-en-3beta,26-diol (3).