An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.     

仁用杏SRAP-PCR体系的正交设计优化

改良CTAB法提取仁用杏基因组DNA,用于建立SRAP-PCR体系。采用L16（44）正交设计试验,对影响仁用杏SRAP-PCR反应体系的4因素（dNTPs浓度、Mg^2＋浓度、引物浓度、Taq聚合酶量）4水平进行筛选,PCR结果经统计软件SPSS V13.0分析,并对退火温度进行了摸索,确立了仁用杏SRAP-PCR的优化体系：总体积为20 L,dNTPs浓度为0.2 mmol/L,Mg^2＋浓度为2.5mmol/L,引物浓度为0.9μmol/L,Taq聚合酶量为1.5 U,退火温度为52℃。本优化体系为仁用杏资源遗传分析奠定了技术基础。     

GaAs MMIC开关MESFET电路建模技术研究

基于Agilent公司的IC—CAP软件及GaAs圆片工艺加工线,研究了砷化镓微波毫米波单片集成电路（GaAsM—MMIC）开关MESFET电路建模技术及其应用。讨论了MMIC设计中电路建模技术的重要性,提出了高精度GaAs MMIC开关MESF田简化电路模型及不同栅宽模型参数的比例缩放方案,扩展了电路模型的应用频率范围,解决了电路模型参数提取中的关键问题,如：开关MESFET模型的版图设计、微波探针校准图形的设计、电路模型参数的提取、统计和确认。采用该技术提取的电路模型参数,成功研制出GaAs MMIC控制电路系列产品,验证了该方法的有效性。     

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.     

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-β. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β are strongly associated with Alzheimer's disease, are more toxic than amyloid-β, residues 1-42 (Aβ(1-42)) and Aβ(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aβ may trigger Alzheimer's disease. Aβ(3(pE)-42) co-oligomerizes with excess Aβ(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ(3(pE)-42) plus 95% Aβ(1-42) (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ(1-42) monomers in the absence of additional Aβ(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aβ(3(pE)-42), and enhanced Aβ(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aβ(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.     

Bone marrow cells regenerate infarcted myocardium

Myocardial infarction leads to loss of tissue and impairment of cardiac performance. The remaining myocytes are unable to reconstitute the necrotic tissue, and the post-infarcted heart deteriorates with time. Injury to a target organ is sensed by distant stem cells, which migrate to the site of damage and undergo alternate stem cell differentiation; these events promote structural and functional repair. This high degree of stem cell plasticity prompted us to test whether dead myocardium could be restored by transplanting bone marrow cells in infarcted mice. We sorted lineage-negative (Lin-) bone marrow cells from transgenic mice expressing enhanced green fluorescent protein by fluorescence-activated cell sorting on the basis of c-kit expression. Shortly after coronary ligation, Lin- c-kitPOS cells were injected in the contracting wall bordering the infarct. Here we report that newly formed myocardium occupied 68% of the infarcted portion of the ventricle 9 days after transplanting the bone marrow cells. The developing tissue comprised proliferating myocytes and vascular structures. Our studies indicate that locally delivered bone marrow cells can generate de novo myocardium, ameliorating the outcome of coronary artery disease.     

Genome sequencing in microfabricated high-density picolitre reactors

The proliferation of large-scale DNA-sequencing projects in recent years has driven a search for alternative methods to reduce time and cost. Here we describe a scalable, highly parallel sequencing system with raw throughput significantly greater than that of state-of-the-art capillary electrophoresis instruments. The apparatus uses a novel fibre-optic slide of individual wells and is able to sequence 25 million bases, at 99% or better accuracy, in one four-hour run. To achieve an approximately 100-fold increase in throughput over current Sanger sequencing technology, we have developed an emulsion method for DNA amplification and an instrument for sequencing by synthesis using a pyrosequencing protocol optimized for solid support and picolitre-scale volumes. Here we show the utility, throughput, accuracy and robustness of this system by shotgun sequencing and de novo assembly of the Mycoplasma genitalium genome with 96% coverage at 99.96% accuracy in one run of the machine.     

超临界CO2气溶胶溶剂萃取法制备尤特奇S100纳米颗粒

采用超临界CO2气溶胶溶剂萃取法(ASES)制备了尤特奇S100(Eudragit S100)纳米粒,考察了有机溶剂的选择、压力、温度、溶液浓度及流速等主要工艺参数对Eudragit S100纳米粒形貌及粒径大小的影响,并采用扫描电镜、傅立叶红外光谱分析对结果进行了表征。结果表明,在以丙酮作为溶剂、压力为16 MPa、温度为45℃、溶液浓度为1 mg/m L的工艺条件下,可制备出有机溶剂残留少、分布均匀、平均粒径为30~60 nm的Eudragit S100纳米颗粒。纳米粒子的平均粒径随温度、压力的升高而减小,随溶液浓度的减小而减小。     

The genome sequence of the rice blast fungus Magnaporthe grisea

Magnaporthe grisea is the most destructive pathogen of rice worldwide and the principal model organism for elucidating the molecular basis of fungal disease of plants. Here, we report the draft sequence of the M. grisea genome. Analysis of the gene set provides an insight into the adaptations required by a fungus to cause disease. The genome encodes a large and diverse set of secreted proteins, including those defined by unusual carbohydrate-binding domains. This fungus also possesses an expanded family of G-protein-coupled receptors, several new virulence-associated genes and large suites of enzymes involved in secondary metabolism. Consistent with a role in fungal pathogenesis, the expression of several of these genes is upregulated during the early stages of infection-related development. The M. grisea genome has been subject to invasion and proliferation of active transposable elements, reflecting the clonal nature of this fungus imposed by widespread rice cultivation.