Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.     

人工湿地不同季节与单元之间根际微生物多样性

采用选择性平板培养基法和PCR-DGGE法比较研究了三级垂直潜流人工湿地污水处理系统中不同季节与单元之间根际微生物数量与多样性,结果表明,随着“秋-冬-春-夏”的季节的变化,2种方法测定的微生物数量都呈现“高-低-高”季节性变化现象,但选择性平板培养基法显示秋季数量最多,PCR-DGGE法显示夏季数量最多.从三级处理单元的1-3级,两种方法测定的微生物数量都呈现“低-高-低”的规律.除土样11外,其余相同季节的土样均出现聚集在同一个分支下.得到的4个差异序列(YP1-YP 4),经BLAST比较,分别找到同源性最近的已知类群,分别为肠杆菌属、弧菌、欧文氏菌、产酸克雷伯菌.BLASTX比较发现YP1与细菌代谢分解有机物有关;YP2蜡样芽胞杆菌生命活动相关,具有解磷的作用;YP3与APOE载脂蛋白e功能相似,参与细菌的分解代谢;YP4功能可能与细菌去氮有关.     

Influences of Nano-TiO2 on the chloroplast aging of spinach under light

The aim of this study was to investigate the biochemical effects of niacin and chromium(III)-chloride on serum lipid peroxidation, uric and sialic acids, and the extent of lipid peroxidation and glutathione levels in skin and lung tissues of hyperlipidemic rats. In this study, female Swiss albino rats, 12 mo old, were used. They were randomly divided into four groups. Group I animals were fed with a standard pellet diet and water ad libitium. Group II rats were fed with a standard pellet diet and were treated with a dose of 250 μg/kg body weight CrCI₃·6H₂O and 100 mg/kg body weight niacin, for 45 d, by the gavage technique. Group III rats were fed a lipogenic diet in which 2% cholesterol, 0.5% cholic acid, and 20% sunflower oil were added to the pellet chow. In addition, the animals in this group drank water containing 3% ethanol. This regime was maintained for 60 d. The rats in group IV were maintained in the same food and drink regime as the animals in group III. After 2 wk, the animals showed symptoms of hyperlipemia and they were treated with 250 μg/kg body weight CrCI₃·6H₂O and 100 mg/kg body weight niacin, by gavage, for 45 d. On d 60, the blood and the skin and lungs samples were taken from animals. In the hyperlipemic groups, a reduction of the lung glutathione level and an increase in serum, lung, and skin lipid peroxidation levels and in serum sialic and uric acid were observed. In rats treated with a combination of niacin and Cr(III), the skin and serum lipid peroxidation and the sialic and uric acid levels decreased while showing an increase of lung glutathione activity. These results suggest that niacin and Cr(III), when administered in combination, have a protective effect against skin and lung tissues damage as a result of hyperlipidemia.     

未培养环境微生物培养方法的研究进展

自然界中微生物种类繁多、功能多样、分布广泛,对人类健康安全、生态稳定和物种进化等发挥不可替代的作用。尽管微生物培养技术至今已有一百多年,然而由于各种限制因素的制约,目前成功分离培养的微生物仅占0.1%-1.0%,自然界中仍有十分丰富的微生物资源有待挖掘和开发利用。如何理解难培养微生物的制约因素并探索作用机制,同时借此开发新型微生物培养方法则尤为必要。本文首先分析了典型环境微生物生长的限制因素,然后介绍了目前利用传统培养改进措施进行分离筛选的研究成果,进一步综述了原位培养、共培养、微流控培养技术、细胞分选技术等新型培养技术在难培养微生物分离培养中的应用,最后对目前培养法存在的瓶颈问题进行深入分析,提出今后可在多技术联合使用、难培养微生物复苏机制、微生物互作机制、代谢途径与调控机制等方面进行研究,以期为今后微生物资源开发利用提供借鉴。     

MicroRNA-449b-5p targets HMGB1 to attenuate hepatocyte injury in liver ischemia and reperfusion

MicroRNAs (miRNAs) participate in the pathological process of liver ischemia/reperfusion (I/R) injury. MiR-449b-5p is the target miRNA of high mobility group box 1 (HMGB1). Its role and molecular mechanism in liver I/R injury remain unidentified. In this study, we found a protective effect of miR-449b-5p against hepatic I/R injury. HMGB1 expression significantly increased, whereas miR-449b-5p dramatically decreased in patients after liver transplant and in L02 cells exposed to hypoxia/reoxygenation (H/R). A dual-luciferase reporter assay confirmed the direct interaction between miR-449b-5p and the 3′ untranslated region of HMGB1 messenger RNA. We also found that overexpression of miR-449b-5p significantly promoted cell viability and inhibited cell apoptosis of L02 cells exposed to H/R. Moreover, miR-449b-5p repressed HMGB1 protein expression and nuclear factor-κB (NF-κB) pathway activation in these L02 cells. In an in vivo rat model of hepatic I/R injury, overexpression of miR-449b-5p significantly decreased alanine aminotransferase and aspartate aminotransferase and inhibited the HMGB1/NF-κB pathway. Our study thus suggests that miR-449b-5p alleviated hepatic I/R injury by targeting HMGB1 and deactivating the NF-κB pathway, which may provide a novel and promising therapeutic target for hepatic I/R injury.     

Performance improvement of cephalosporin C fermentation by Acremonium chrysogenum with DO-Stat based strategy of co-feeding soybean oil and glucose

Cephalosporin C (CPC) fermentation by Acremonium chrysogenum featured with two major problems: (1) high raw materials cost (low CPC yield from soybean oil) and (2) low oxygen transfer rate between gaseous/aqueous phases leading to low CPC productivity and quality instability of CPC fermentation product due to the accumulation of deacetoxycephalosporin C (DAOC). To solve the problems, in this study, we proposed a novel DO-Stat based co-substrates feeding strategy by simultaneously supplementing soybean oil and glucose, and testified the effectiveness of the strategy in a 7 L bioreactor. The CPC fermentation performance were significantly improved when co-feeding soybean oil and glucose at a weight ratio of 1:0.7, as compared with those when feeding pure soybean oil: (1) final CPC concentration and yield reached higher levels of 37 g/L and 23.5%, the increments were 46% and 82%, respectively; (2) oxygen transfer rate was largely improved, oil consumption rate and CPC productivity were enhanced by 31% and 136%, respectively; and (3) DO could be controlled at adequately high levels so that DAOC accumulation could be minimized and the quality of CPC fermentation product be ensured. The proposed strategy showed application potential in improving the economics of industrial CPC productions.     

Immunosuppressive Drugs Modulate the Replication of Hepatitis B Virus (HBV) in a Hydrodynamic Injection Mouse Model

Hepatitis B virus (HBV) reactivation and recurrence are common in patients under immunosuppression and can be controlled by hepatitis B immunoglobulin, antivirals, and hepatitis B vaccine. However, the detailed analysis of HBV infection under immunosuppression is essential for the prophylaxis and therapy for HBV reactivation and recurrence. In this study, HBV replication and T cell responses were analyzed in a HBV-transfected mouse model under immunosuppressive therapy. During the treatment, HBV replication was at a high level in mice treated with dexamethasone, cyclosporine, and cyclophosphamide, whereas was terminated in mice treated with mycophenolate mofetil. After the withdrawal, HBV replication was at low or high levels in the dexamethasone-treated mice or in both cyclosporine- and cyclophosphamide-treated mice. The early withdrawal of cyclosporine allowed the recovery of suppressed T cell responses and led to subsequent HBV clearance, while the adoptive immune transfer to the mice with HBV persistence led to HBV suppression. Taken together, long-term HBV persistence under immunosuppression depends on the immunosuppressive drugs used and on the treatment duration and is mediated by the suppressed intrahepatic CD8 T cell response. These data may be helpful for individualized immunosuppressive therapy in patients with high risk of HBV reactivation and recurrence, and the mouse system is suitable for studying HBV reactivation and recurrence under immunosuppression.     

DGGE技术在环境微生物多样性研究中的应用

微生物是污水净化的主要作用者之一.采用变性梯度凝胶电泳(denaturing gradient gel electrophresis,DGGE)方法培育和鉴定土壤微生物具有可靠性强、重复性好、方便快捷等优点,已被广泛应用于环境科学和污染防治研究领域.综述了基于PCR-DGGE技术的基本原理、关键环节及其在微生物多样性研究中的应用,同时就其自身存在的不足进行了评价并提出了解决方案.