RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells

Glycoproteins have various biological functions including enzymatic activity, protein stability and others. Due to the presence of paucimannosidic N-linked glycans, recombinant proteins from an insect cell expression system may not be suitable for therapeutic use. Because baculovirus expression systems (BESs) are used to produce recombinant proteins, it is of interest to modify the endogenous N-glycosylation pathway in insects to mimic that of mammals. Using a soaking RNAi sensitive cell line, BmN4-SID1, has enabled us to suppress Bombyx mori FDL (BmFDL), an N-linked glycan-specific β-N-acetylglucosaminidase. Western blotting and MALDI-TOF MS demonstrated that the BmFDL depletion almost completely converted the paucimannosidic structures of the recombinant proteins produced by BES into a complex-type structure. This highly efficient, simple and low-cost method can be used for mass production of secretion proteins with complex-type N-linked glycans.     

The HEX1 Gene of Fusarium graminearum Is Required for Fungal Asexual Reproduction and Pathogenesis and for Efficient Viral RNA Accumulation of Fusarium graminearum Virus 1

The accumulation of viral RNA depends on many host cellular factors. The hexagonal peroxisome (Hex1) protein is a fungal protein that is highly expressed when the DK21 strain of Fusarium graminearum virus 1 (FgV1) infects its host, and Hex1 affects the accumulation of FgV1 RNA. The Hex1 protein is the major constituent of the Woronin body (WB), which is a peroxisome-derived electron-dense core organelle that seals the septal pore in response to hyphal wounding. To clarify the role of Hex1 and the WB in the relationship between FgV1 and Fusarium graminearum, we generated targeted gene deletion and overexpression mutants. Although neither HEX1 gene deletion nor overexpression substantially affected vegetative growth, both changes reduced the production of asexual spores and reduced virulence on wheat spikelets in the absence of FgV1 infection. However, the vegetative growth of deletion and overexpression mutants was increased and decreased, respectively, upon FgV1 infection compared to that of an FgV1-infected wild-type isolate. Viral RNA accumulation was significantly decreased in deletion mutants but was significantly increased in overexpression mutants compared to the viral RNA accumulation in the virus-infected wild-type control. Overall, these data indicate that the HEX1 gene plays a direct role in the asexual reproduction and virulence of F. graminearum and facilitates viral RNA accumulation in the FgV1-infected host fungus.     

Impact of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> inoculation on indigenous bacterial communities during agricultural waste composting

This research was conducted to distinguish between the separate effects of the Phanerochaete chrysosporium inoculation and sample property heterogeneity induced by different inoculation regimes on the indigenous bacterial communities during agricultural waste composting. P. chrysosporium was inoculated during different phases. The bacterial community abundance and structure were determined by quantitative PCR and denaturing gradient gel electrophoresis analysis, respectively. Results indicated a significant stimulatory effect of P. chrysosporium inoculation on the bacterial community abundance. The bacterial community abundance significantly coincided with pile temperature, ammonium, and nitrate (P?<?0.006). Variance partition analysis showed that the P. chrysosporium inoculation directly explained 20.5 % (P?=?0.048) of the variation in the bacterial communities, whereas the sample property changes induced by different inoculation regimes indirectly explained up to 35.1 % (P?=?0.002). The bacterial community structure was significantly related to pile temperature, water-soluble carbon (WSC), and C/N ratio when P. chrysosporium were inoculated. The C/N ratio solely explained 7.9 % (P?=?0.03) of the variation in community structure, whereas pile temperature and WSC explained 7.7 % (P?=?0.026) and 7.5 % (P?=?0.034) of the variation, respectively. P. chrysosporium inoculation affected the indigenous bacterial communities most probably indirectly through increasing pile temperature, enhancing the substrate utilizability, and changing other physico-chemical factors.     

A modified metabolic model for mixed culture fermentation with energy conserving electron bifurcation reaction and metabolite transport energy

A modified metabolic model for mixed culture fermentation (MCF) is proposed with the consideration of an energy conserving electron bifurcation reaction and the transport energy of metabolites. The production of H₂ related to NADH/NAD⁺ and Fdred/Fdox is proposed to be divided in three processes in view of energy conserving electron bifurcation reaction. This assumption could fine‐tune the intracellular redox balance and regulate the distribution of metabolites. With respect to metabolite transport energy, the proton motive force is considered to be constant, while the transport rate coefficient is proposed to be proportional to the octanol–water partition coefficient. The modeling results for a glucose fermentation in a continuous stirred tank reactor show that the metabolite distribution is consistent with the literature: (1) acetate, butyrate, and ethanol are main products at acidic pH, while the production shifts to acetate and propionate at neutral and alkali pH; (2) the main products acetate, ethanol, and butyrate shift to ethanol at higher glucose concentration; (3) the changes for acetate and butyrate are following an increasing hydrogen partial pressure. The findings demonstrate that our modified model is more realistic than previous proposed model concepts. It also indicates that inclusion of an energy conserving electron bifurcation reaction and metabolite transport energy for MCF is sound in the viewpoint of biochemistry and physiology. Biotechnol. Bioeng. 2013; 110: 1884–1894. © 2013 Wiley Periodicals, Inc.     

Tenuibacillus halotolerans sp. nov., a novel bacterium isolated from a soil sample from a salt lake in Xinjiang, China and emended description of the genus Tenuibacillus

A Gram-positive, moderately halotolerant, rod-shaped bacterium, designated YIM 94025^T, was isolated from a soil sample from a salt lake in Xinjiang province, north-west China. Strain YIM 94025^T was observed to grow at 25–45 °C (optimum 37 °C), 0–22 % NaCl (optimum 2–10 %) and pH 6.0–9.0 (optimum pH 8.0). Phylogenetic analyses based on 16S rRNA gene sequences revealed that the organism belongs to the genus Tenuibacillus and exhibited sequence similarity of 98.0 % to the closest type strain, Tenuibacillus multivorans AS 1.3442^T. The predominant menaquinone was found to be MK-7; the cell-wall peptidoglycan diamino acid was meso-diaminopimelic acid; the polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid and an unknown lipid; and the major fatty acids were found to contain iso-C_15:0, anteiso-C_15:0 and iso-C_16:0. The chemotaxonomic characteristics of strain YIM 94025^T are consistent with those of the genus Tenuibacillus. The level of DNA–DNA relatedness value between YIM 94025^T and T. multivorans AS 1.3442^T was 36.6 ± 4.5 %. The G+C content of the strain YIM 94025^T was determined to be 38.5 %. Based on the comparative analysis of physiological, biochemical and chemotaxonomic data, as well as DNA–DNA hybridization results, the isolate is considered to represent a novel species of the genus Tenuibacillus, for which the name Tenuibacillus halotolerans sp. nov., is proposed, with the type strain of YIM 94025^T (=CCTCC AB 2012860^T = KCTC 33046^T).     

Discovery of a small molecular compound simultaneously targeting RXR and HADC: Design,synthesis, molecular docking and bioassay

Retinoid X receptor (RXR) and Histone deacetylase (HDAC) are considered important targets for anti-cancer therapy due to their crucial roles in genetic or epigenetic regulations of cancer development and progression. Here, we have designed and synthesized a novel compound which targets both RXR and HADC. This dual-targeting agent is derived from bexarotene and suberoylanilide hydroxamic acid (SAHA), prototypical RXR agonist and HDAC inhibitor, respectively. Molecular docking studies demonstrate that this agent has a relatively strong affinity to RXR and HADC. Importantly, it presents the potentials of activation of RXR and inhibition of HDAC in both cell-free and whole-cell assays, and displays anti-proliferative effect on representative cancer cell lines and drug-resistant cancer cell lines.     

Characterization of Tudor-sn-containing granules in the silkworm,Bombyx mori

The Tudor-sn protein, which contains four staphylococcal nuclease domains and a Tudor domain, is a ubiquitous protein found in almost all organisms. It has been reported that Tudor-sn in mammals participates in various cellular pathways involved in gene regulation, cell growth, and development. In insects, we have previously identified a Tudor-sn ortholog in the silkworm, Bombyx mori, and detected its interactions between with Argonaute proteins. The role of Tudor-sn in silkworm, however, still remains largely unknown. In this study, we demonstrated that silkworm Tudor-sn is a stress granule (SG) protein, and determined its interactions with other SG proteins using Bimolecular Fluorescence Complementation assay and Insect Two-Hybrid method. Depletions of Argonaute proteins and SG-marker protein Tia1 by RNAi impaired the involvement of Tudor-sn in the SG formation. Protein domain deletion analysis of Tudor-sn demonstrated that SN2 is the key domain required for the aggregation of Tudor-sn in SGs.     

响应面分析法优化当归粗多糖提取工艺

选取岷县当归药材为原料,采用水提醇沉法进行多糖提取,以当归粗多糖得率为指标,探讨加水量、回流提取时间、水提液的浓缩比、醇沉后所达含醇比例对当归多糖得率的影响。在单因素分析的基础上,采用响应面分析法(RSM)确定了当归粗多糖最佳提取条件为:加水量837.6 mL/100 g,浓缩后溶液体积为228.12 mL,最终含乙醇浓度为65.80%,回流提取时间2 h,在此条件下预测当归粗多糖得率理论的最佳值为10.44%,实际验证值为10.40%,两者相符,说明RSM法分析的可靠性。     

Bio-Plex悬浮芯片系统检测两种实验兔白介素基因的差异表达

目的采用液相悬浮芯片系统同时测定实验兔圆小囊中IL-1β、IL-1R1、IL=8、IL-8RA和IL=15各基因的表达情况,并对该方法进行评价。方法利用Affymetrix的Panomics QuantiGene Plex2．0Assay中bDNA信号放大和多磁珠分析技术,来同时检测两种实验兔圆小囊中多重mRNA并定量。建立实验兔免疫相关白介素基因的液相悬浮芯片检测方法。结果可同时检测IL-1β、IL=1R1、IL-8、IL=8RA和IL-15各基因的含量,并发现WHBE兔IL-15基因的相对表达量显著高于JW兔（P〈0．05）,IL-1R1基因的相对表达量显著高于JW兔（P〈0．01）,IL-8RA基因在WHBE兔中的相对表达量也高于JW兔（P〈0．05）。结论建立了实验兔白介素基因的液相悬浮芯片检测方法,WHBE兔的IL-15、IL-1R1和IL-8RA基因表达量较高,可能与WHBE兔独特的免疫学特性有关。