Analysis of conformational motions and related key residue interactions responsible for a specific function of proteins with elastic network model

Protein collective motions play a critical role in many biochemical processes. How to predict the functional motions and the related key residue interactions in proteins is important for our understanding in the mechanism of the biochemical processes. Normal mode analysis (NMA) of the elastic network model (ENM) is one of the effective approaches to investigate the structure-encoded motions in proteins. However, the motion modes revealed by the conventional NMA approach do not necessarily correspond to a specific function of protein. In the present work, a new analysis method was proposed to identify the motion modes responsible for a specific function of proteins and then predict the key residue interactions involved in the functional motions by using a perturbation approach. In our method, an internal coordinate that accounts for the specific function was introduced, and the Cartesian coordinate space was transformed into the internal/Cartesian space by using linear approximation, where the introduced internal coordinate serves as one of the axes of the coordinate space. NMA of ENM in this internal/Cartesian space was performed and the function-relevant motion modes were identified according to their contributions to the specific function of proteins. Then the key residue interactions important for the functional motions of the protein were predicted as the interactions whose perturbation largely influences the fluctuation along the internal coordinate. Using our proposed methods, the maltose transporter (MalFGK₂) from E. Coli was studied. The functional motions and the key residue interactions that are related to the channel-gating function of this protein were successfully identified.     

一种新型免疫层析技术在临床乳腺癌Her-2蛋白定量检测中的应用

建立一种靶点蛋白质快速定量检测方法。在原有侧向流动免疫层析技术的基础上,通过优化层析材料和纳米微球的均一性、改进检测区的检测方法,经逐点扫描技术,建立标准浓度曲线,以达到对临床靶点蛋白质的定量检测。以乳腺癌组织中的Her2表达为例,通过对已知浓度样品的检测,验证本技术方法的准确度大于96%。另外,以蛋白质免疫印迹作为组织中特定蛋白质检测金标准,分析临床肿瘤组织中Her2蛋白的含量,其准确率也达到95.5%,而免疫组织化学方法检测准确率仅为69.58%。新型免疫层析法检测结果与靶向治疗患者的愈后密切相关(P<0.01)。改进后的新型免疫层析方法能够准确地对临床靶点蛋白质进行定量检测,而且结合侧向流动技术的简单、快速和易用性,这种新型检测方法可以广泛应用于临床组织标本、血液标本和体液标本中靶点蛋白质的临场定量检测,在一定程度上可以替代免疫组化技术。     

Photocurrent Enhancement in Si-Ge Photodetectors by Utilizing Surface Plasmons

A circular slit-groove surface plasmon polaritons (SPPs) launcher surrounding a photodetector is employed theoretically to enhance the photocurrent of atypical Si-Ge photodetectors. The slit and grooves are designed such that the SPPs are focused at the center of the absorption layer of the photodetector to result in additional electric current. Fabry–Perot resonance condition accurately calculates the period of the groove, slit-groove distance, photodetector radius, and slit-photodetector distance. The manipulation leads to constructive interference between the incident light impinging from the top and the SPPs propagating toward the photodetector. Simulation result shows that photocurrent increases by approximately 13-fold when the SPPs are introduced.     

Soluble expression and purification of Brucella cell surface protein (BCSP31) of Brucella melitensis and preparation of anti-BCSP31 monoclonal antibodies

Brucella cell surface protein (BCSP31) is potentially useful for diagnosing brucellosis. We aimed to establish a monoclonal antibody (MAb) against Brucella melitensis BCSP31 and to investigate its distribution in diagnosis. Soluble recombinant BCSP31 was successfully expressed and purified. Two MAbs (1F1 and 1E5) against B. melitensis BCSP31, effective in detecting both recombinant and cellular proteins, were obtained and characterized. The MAbs did not react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus aeruginosus, but strongly reacted with BCSP31 and B. melitensis by ELISA and Western blot analysis. We also tested different Brucella species and brucellosis using the prepared anti-BCSP31 MAbs. BCSP31 and anti-BCSP31 MAbs may play important roles in future research in diagnosing brucellosis.     

Identifying a Kinase Network Regulating FGF14:Nav1.6 Complex Assembly Using Split-Luciferase Complementation

Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.     

Modelling Infectious Hematopoietic Necrosis Virus Dispersion from Marine Salmon Farms in the Discovery Islands,British Columbia,Canada

Finite volume ocean circulation and particle tracking models are used to simulate water-borne transmission of infectious hematopoietic necrosis virus (IHNV) among Atlantic salmon (Salmo salar) farms in the Discovery Islands region of British Columbia, Canada. Historical simulations for April and July 2010 are carried out to demonstrate the seasonal impact of river discharge, wind, ultra-violet (UV) radiation, and heat flux conditions on near-surface currents, viral dispersion and survival. Numerical particles released from infected farm fish in accordance with IHNV shedding rates estimated through laboratory experiments are dispersed by model oceanic flows. Viral particles are inactivated by ambient UV radiation levels and by the natural microbial community at rates derived through laboratory studies. Viral concentration maps showing temporal and spatial changes are produced and combined with lab-determined minimum infectious dosages to estimate the infective connectivity among farms. Results demonstrate that neighbouring naïve farms can become exposed to IHNV via water-borne transport from an IHNV diseased farm, with a higher risk in April than July, and that many events in the sequence of farm outbreaks in 2001-2002 are consistent with higher risks in our farm connectivity matrix. Applications to other diseases, transfers between farmed and wild fish, and the effect of vaccinations are also discussed.     

Simplified Aeroelastic Model for Fluid Structure Interaction between Microcantilever Sensors and Fluid Surroundings

Fluid-structural coupling occurs when microcantilever sensors vibrate in a fluid. Due to the complexity of the mechanical characteristics of microcantilevers and lack of high-precision microscopic mechanical testing instruments, effective methods for studying the fluid-structural coupling of microcantilevers are lacking, especially for non-rectangular microcantilevers. Here, we report fluid-structure interactions (FSI) of the cable-membrane structure via a macroscopic study. The simplified aeroelastic model was introduced into the microscopic field to establish a fluid-structure coupling vibration model for microcantilever sensors. We used the finite element method to solve the coupled FSI system. Based on the simplified aeroelastic model, simulation analysis of the effects of the air environment on the vibration of the commonly used rectangular microcantilever was also performed. The obtained results are consistent with the literature. The proposed model can also be applied to the auxiliary design of rectangular and non-rectangular sensors used in fluid environments.     

Survival rates of mutant genes under artificial selection using individual and family information

We have used diffusion and branching process methods to investigate fixation rates, probabilities of survival per generation, and times to fixation of mutant genes under different selection methods incorporating individual and family information. Diffusion approximations fit well to simulated results even for large selection coefficients. Methods that give much weight to family information, such as BLUP evaluation which is widely used in animal breeding, reduce fixation rates of mutant genes because of the reduced effective population sizes. In general, it is observed that even mutants with relatively small heterozygous effects (say 0.1 phenotypic standard deviation) are practically ‘safe’ (i.e. their probability of loss from one generation to the next is smaller than, say, 10%) after just a few generations, typically less than 10. For methods of selection with larger effective size, such as within-family selection, the mutant is ‘safe’ in the population somewhat earlier but eventual fixation takes a longer time. Finally we evaluate the amount by which the use of marker assisted selection reduces the fixation probability of newly arisen mutants.     

厚壳贻贝whirlin类贝壳基质蛋白的重组表达与功能分析

贝壳历来是生物工程和材料学研究的重要对象。贝壳中的贝壳基质蛋白质在贝壳的形成与发育过程中具有重要的调控作用。Whirlin类蛋白质(Whirlin-like protein,WLP)是一种从厚壳贻贝(Mytilus coruscus)中鉴定的新型贝壳基质蛋白质。序列分析结果显示,该蛋白质含有PDZ(postsynaptic density/Discs large/Zonula occludens)结构域,而该结构域对贝壳生物矿化的影响目前尚无报道。为深入了解WLP在贝壳形成中对碳酸钙晶体的影响,在序列分析基础上,采用密码子优化结合原核重组表达,获得其重组表达产物后,开展了重组WLP对碳酸钙晶体形貌及晶型的影响研究,结晶速度抑制以及碳酸钙晶体结合分析。分析结果表明,重组WLP能诱导文石型碳酸钙晶体的形貌和方解石型碳酸钙晶体的晶型发生改变;同时重组WLP对碳酸钙晶体具有结合作用,且能抑制碳酸钙晶体的结晶速度。上述结果表明,WLP对贝壳的形成及发育具有重要影响,并可能在贝壳肌棱柱层的形成中发挥了重要作用。