Molecular and structural characterization of the domain 2 of hepatitis C virus non-structural protein 5A

Hepatitis C virus (HCV) non-structural protein 5A protein (NS5A), which consists of three functional domains, is involved in regulating viral replication, interferon resistance, and apoptosis. Recently, the three-dimensional structure of the domain 1 was determined. However, currently the molecular basis for the domains 2 and 3 of HCV NS5A is yet to be defined. Toward this end, we expressed, purified the domain 2 of the NS5A (NS5A-D2), and then performed biochemical and structural studies. The purified domain 2 was active and was able to bind NS5B and PKR, biological partners of NS5A. The results from gel filtration, CD analysis, 1D 1H NMR and 2D 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy indicate that the domain 2 of NS5A appears to be flexible and disordered.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

Dominant-negative effects in prion diseases: insights from molecular dynamics simulations on mouse prion protein chimeras

Mutations in the prion protein (PrP) can cause spontaneous prion diseases in humans (Hu) and animals. In transgenic mice, mutations can determine the susceptibility to the infection of different prion strains. Some of these mutations also show a dominant-negative effect, thus halting the replication process by which wild type mouse (Mo) PrP is converted into Mo scrapie. Using all-atom molecular dynamics (MD) simulations, here we studied the structure of HuPrP, MoPrP, 10?Hu/MoPrP chimeras, and 1 Mo/sheepPrP chimera in explicit solvent. Overall, ～2?μs of MD were collected. Our findings suggest that the interactions between α1 helix and N-terminal of α3 helix are critical in prion propagation, whereas the β2–α2 loop conformation plays a role in the dominant-negative effect.

An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:4.     

Heterologous expression of an aspartic protease gene from biocontrol fungus Trichoderma asperellum in Pichia pastoris

Trichoderma asperellum parasitizes a large variety of phytopathogenic fungi. The mycoparasitic activity of T. asperellum depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall and proteases which are a group of enzymes capable of degrading proteins from host. In this study, a full-length cDNA clone of aspartic protease gene, TaAsp, from T. asperellum was obtained and sequenced. The 1,185 bp long cDNA sequence was predicted to encode a 395 amino acid polypeptide with molecular mass of 42.3 kDa. The cDNA of TaAsp was inserted into the pPIC9K vector and transformed into yeast Pichia pastoris GS115 for heterologous expression. A clearly visible band with molecular mass about 42 kDa in the SDS-PAGE gel indicated that the transformant harboring the gene TaAsp had been successfully translated in P. pastoris and produced a recombinant protein. Enzyme characterization test showed that the optimum fermentation time for P. pastoris GS115 transformant was 72 h. Enzyme activity of the recombinant aspartic proteinase remained relatively stable at 25–60 °C and pH 3.0–9.0, which indicated its good prospect of application in biocontrol. The optimal pH value and temperature of the enzyme activity were pH 4.0 and 40 °C, and under this condition, with casein as the substrate, the recombinant protease activity was 18.5 U mL^?1. In order to evaluate antagonistic activity of the recombinant protease against pathogenic fungi, five pathogenic fungi, Fusarium oxysporum, Alternaria alternata, Cytospora chrysosperma, Sclerotinia sclerotiorum and Rhizoctonia solani, were applied to the test of in vitro inhibition of their mycelial growth by culture supernatant of P. pastoris GS115 transformant.     

Getting on the right track: Interactions between viruses and the cytoskeletal motor proteins

The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments and their accessory proteins. Motor proteins, which comprise the dynein, kinesin and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along with the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as rabies disease and acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, that is, rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins.     

Quantitative analysis of mitral valve morphology in atrial functional mitral regurgitation using real-time 3-dimensional echocardiography atrial functional mitral regurgitation

Background

Atrial fibrillation (AF) can result in atrial functional mitral regurgitation (MR), but the mechanism remains controversial. Few data about the relationship between the 3-dimensional morphology of the MV and the degree of MR in AF exist.

Methods

Real-time 3-dimensional transesophageal echocardiography (3D-TEE) of the MV was acquired in 168 patients with AF (57.7% persistent AF), including 25 (14.9%) patients with moderate to severe MR (the MR+ group) and 25 patients without AF as controls. The 3-dimensional geometry of the MV apparatus was acquired using dedicated quantification software.

Results

Compared with the group of patients with no or mild MR (the MR- group) and the controls, the MR+ group had a larger left atrium (LA), a more dilated mitral annulus (MA), a reduced annular height to commissural width ratio (AHCWR), indicating flattening of the annular saddle shape, and greater leaflet surfaces and tethering. MR severity was correlated with the MA area (r²?=?0.43, P?<?0.01) and the annulus circumference (r²?=?0.38, P?<?0.01). A logistic regression analysis indicated that the MA area (OR: 1.02, 95% CI: 1.01–1.03, P?<?0.01), AHCWR (OR: 0.24, 95% CI: 0.14–0.35, P?=?0.04) and MV tenting volume (OR: 3.24, 95% CI: 1.16–9.08, P?=?0.03) were independent predictors of MR severity in AF patients.

Conclusions

The mechanisms of “atrial functional MR” are complex and include dilation of the MA, flattening of the annular saddle shape and greater leaflet tethering.

     

云南中度退化的喀斯特天坑草地植物群落优势种种间关系

喀斯特天坑作为独特的宏大地表负地形,其坑底生境与外界环境相互独立,呈现出独特的植物群落格局及特征.本研究选取云南沾益天坑群中可进入性较强的中度退化天坑--巴家陷塘为对象,筛选出重要值大于1的优势种,结合总体关联性分析、χ²检验、AC联结系数及Spearman秩相关系数对巴家陷塘退化天坑植物群落中11种优势种的种间关联性和相关性进行研究.结果表明: 巴家陷塘退化天坑植物群落种间联结指数VR=1.377>1,总体呈显著正关联,但正负关联差异较大,阳生型植物和阴生型植物呈极显著负关联,现阶段仍具有独立的分布格局,但呈逐渐趋于稳定的趋势;Spearman分析中种间正相关对数(26对)少于种间负相关对数(29对),结合天坑微环境的差异,将退化天坑的优势物种划分为3个生态种组,群落总体呈现出一定的圈层分布规律.     

Adaptive Tensor-Based Principal Component Analysis for Low-Dose CT Image Denoising

Computed tomography (CT) has a revolutionized diagnostic radiology but involves large radiation doses that directly impact image quality. In this paper, we propose adaptive tensor-based principal component analysis (AT-PCA) algorithm for low-dose CT image denoising. Pixels in the image are presented by their nearby neighbors, and are modeled as a patch. Adaptive searching windows are calculated to find similar patches as training groups for further processing. Tensor-based PCA is used to obtain transformation matrices, and coefficients are sequentially shrunk by the linear minimum mean square error. Reconstructed patches are obtained, and a denoised image is finally achieved by aggregating all of these patches. The experimental results of the standard test image show that the best results are obtained with two denoising rounds according to six quantitative measures. For the experiment on the clinical images, the proposed AT-PCA method can suppress the noise, enhance the edge, and improve the image quality more effectively than NLM and KSVD denoising methods.     

黄土高寒区典型植被类型土壤入渗特征及其影响因素

为了研究黄土高寒区典型植被在不同坡位下土壤入渗性能差异及其影响因素,采用恒定水头法测定了不同植被类型的土壤入渗过程,并分析了土壤孔隙状况、机械组成、水稳性团聚体等与渗透速率的相关性。结果表明:(1)初渗速率和稳渗速率均表现为青海云杉>祁连圆柏>华北落叶松>荒草地,且差异性显著;同一植被类型的土壤入渗速率沿坡面向下逐渐增大,但差异性不显著;(2)对不同植被类型的土壤入渗过程模拟发现Horton模型拟合效果最好,决定系数均在0.8以上,通用经验模型拟合精度较差,决定系数在0.614—0.982之间;(3)土壤入渗性能与孔隙状况、水稳性团聚体质量分数、有机质含量均有极显著相关性;>0.25 mm团聚体质量分数是影响稳渗速率的主要决策因素;总孔隙度是影响初渗速率的最主要的决策因素,而毛管孔隙度是主要的限制因子。为高寒地区土壤入渗模拟以及植被配置等提供了科学依据。     

A C-terminal region of RAG1 contacts the coding DNA during V(D)J recombination

The site-specific DNA rearrangement process, called V(D)J recombination, creates much of the diversity of immune receptor molecules in the adaptive immune system. Central to this reaction is the organization of the protein-DNA complex containing the proteins RAG1 and RAG2 and their DNA targets. A long-term goal is to appreciate the three-dimensional relationships between the proteins and DNA that allow the assembly of the appropriate reaction intermediates, resulting in concerted cleavage and directed rejoining of the DNA ends. Previous cross-linking approaches have mapped RAG1 contacts on the DNA. RAG1 protein contacts the DNA at the conserved heptamer and nonamer sequences as well as at the coding DNA adjacent to the heptamer. Here we subject RAG1, covalently cross-linked to DNA substrates, to partial cyanogen bromide degradation or trypsin proteolysis in order to map contacts on the protein. We find that coding-sequence contacts occur near the C terminus of RAG1, while contacts made within the recombination signal sequence occur nearer the N terminus of the core region of RAG1. A deletion protein lacking the C-terminal DNA-contacting region is still capable of making the N-terminal contacts. This suggests that the two binding interactions may exist on two separate domains of the protein. A trypsin cleavage pattern of the native protein supports this conclusion. A two-domain model for RAG1 is evaluated with respect to the larger recombination complex.