正廿面体病毒衣壳参数的研究

本项研究从多学科的不同角度出发,对正廿面体病毒衣壳参数进行了深入研究.整理和开发出衣壳参数50多个,对其每一个参数的概念、含义、来源、参数值和参数间的关系作了明确的认定,并阐述了衣壳参数的理论意义及其应用价值.     

黄瓜花叶病毒衣壳蛋白存在于被其侵染的烟草叶绿体中

用原生质体法制备出高纯度的完整叶绿体经SDS-PAGE电泳,银染后,发现黄瓜花叶病毒(CMV)侵染的烟草病叶叶绿体蛋白质图谱和健叶叶绿体相比,多出一条染色较弱的迁移率与CMV衣壳蛋白质相同的带,经Western转移,用CMV游离衣壳蛋白亚基抗血清进行斑点酶联(Immunoblot)检测,证明这条带就是CMV衣壳蛋白质。健康叶绿体中加入去掉叶绿体的病叶汁液而制备出的叶绿体中无CMV衣壳蛋白质,说明这不是在叶绿体提纯过程中得到的假象,即衣壳蛋白质存在于被CMV侵染的完整叶片叶绿体中。这个结果否认了以往报道的CMV衣壳蛋白质不存在于烟草叶绿体中的结论。另外还发现,叶绿体中的衣壳蛋白质浓度与叶片症状严重程度呈正相关。     

杆状病毒核衣壳组装相关蛋白研究进展

杆状病毒生命周期中会产生包埋型和芽生型两种病毒粒子,这两种病毒粒子的包膜组成存在明显的差异,但拥有相同的核衣壳结构.杆状病毒核衣壳是由衣壳蛋白和杆状病毒基因组两部分组成,核衣壳的正常组装对两种病毒粒子的形成都是不可或缺的,因此核衣壳的正常组装在病毒的整个感染传播过程中发挥着重要作用.尽管越来越多参与核衣壳组装的蛋白被鉴定出来,目前还有许多核衣壳组装细节不明了,例如这些衣壳蛋白之间的互作关系是怎样的,宿主通过何种方式参与到病毒核衣壳组装过程等.本文主要以杆状病毒模式物种苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)为例综述了参与杆状病毒核衣壳组装的相关蛋白,并对一些参与核衣壳运输有关的核衣壳蛋白也做了阐述.     

水痘—带状疱疹病毒分离株核衣壳的形态特征

本文用超萍切片电镜技术对水痘－带状疱疹病毒（VZV）分离株J1的核衣壳进行了形态学研究。结果表明,在病毒感染后5小时即可观察到细胞核内大量的病毒核心相关颗粒和少量核衣壳。在细胞核内和细胞浆内均可见到病毒基质或毒浆结构。VZVJ1株具有三种类型的核衣壳,命名为A型、B型、C型核衣壳。A型具有电子致密核心,B型的核心呈颗粒状,C型具有电子透明核心。三种核衣壳大小一致,直径75－100nm,核心为35－55nm。将VZV的核衣壳与疱疹病毒科其它成员作了比较分析,并对各种核衣壳在病毒成熟过程中的作用进行了探讨。     

水痘一带状疱疹病毒分离株核衣壳的形态特征

本文用超薄切片电镜技术对水痘一带状疱疹病毒(VZV)分离株J_1的核衣壳进行了形态学研究。结果表明,在病毒感染后5小时即可观察到细胞核内大量的病毒核心相关颗粒和少量核衣壳。在细胞核内和细胞浆内均可见到病毒基质或毒浆结构。VZVJ_1株具有三种类型的核衣壳,命名为A型、B型、C型核衣壳。A型具有电子致密核心,B型的核心呈颗粒状,C型具有电子透明核心。三种核衣壳大小一致,直径75—100nm,核心为35—55nm。将VZV的核衣壳与疱疹病毒科其它成员作了比较分析,并对各种核衣壳在病毒成熟过程中的作用进行了探讨。     

替换HIV-1衣壳蛋白基因SHIV的构建及其活性测定

将猴免疫缺陷病毒(Simianimmunodeficiencyvirus,SIVmm239)中gag基因的衣壳蛋白部分置换成人免疫缺陷病毒(Humanimmunodeficiencyvirustype1,HIV-1HXBc2)的相应部分,构建出替换了衣壳蛋白基因的人/猿嵌合免疫缺陷病毒(SHIV)原病毒DNA。用此SHIV原病毒DNA转染293T细胞,细胞中能够检测到嵌合病毒基因的转录与翻译;在细胞培养液上清中亦可检测到装配出的病毒颗粒。病毒颗粒形态正常,含有基因组RNA,具有反转录酶活性,嵌合的外源衣壳蛋白能够正确剪切,形成棒状的核心。将此嵌合SHIV病毒感染MT4细胞,病毒能够吸附并进入细胞,能完成反转录过程,但不能增殖。     

替换HIV-1衣壳蛋白基因SHIV的构建及其活性测定

将猴免疫缺陷病毒(Simian immunodeficiency virus,SIVmm239)中gag基因的衣壳蛋白部分置换成人免疫缺陷病毒(Human immunodeficiency virus type1,HIV-1 HXBc2)的相应部分,构建出替换了衣壳蛋白基因的人/猿嵌合免疫缺陷病毒(SHIV)原病毒DNA.用此SHIV原病毒DNA转染293T细胞,细胞中能够检测到嵌合病毒基因的转录与翻译;在细胞培养液上清中亦可检测到装配出的病毒颗粒.病毒颗粒形态正常,含有基因组RNA,具有反转录酶活性,嵌合的外源衣壳蛋白能够正确剪切,形成棒状的核心.将此嵌合SHIV病毒感染MT4细胞,病毒能够吸附并进入细胞,能完成反转录过程,但不能增殖.     

AcMNPV核衣壳的形态发生

报道了缺失多角体蛋白基因的AcMNPV在sf9细胞内核衣壳的形态发生过程。病毒衣壳蛋白首先装配成许多呈束状排列的直径为34nm中空长管状结构,然后是病毒DNA进入管内,装有DNA的长管按一定的长度间隔断开,形成成束的核衣壳,每个核衣壳的大小约34×260nm,最后成束的核衣壳被囊膜包被形成完整的多粒包埋型病毒粒子。     

衣壳蛋白靶向灭活策略应用于抗登革病毒感染的研究

衣壳蛋白靶向病毒灭活是近年来新兴的抗病毒策略。为探索该策略在抗登革病毒感染中的应用 ,首先建立了稳定表达登革 2型病毒衣壳蛋白 (D2C)与葡萄球菌核酸酶 (SN)融合蛋白D2C_SN的哺乳动物细胞系 ,然后以登革病毒攻击上述细胞系 ,研究表达的融合蛋白D2C_SN对产生的子代病毒颗粒感染性的影响。结果表明融合蛋白D2C_SN能够在病毒装配过程中与野生型衣壳蛋白共组装入子代病毒颗粒内部 ,并导致病毒基因组的降解。与正常BHK细胞相比较 ,融合蛋白D2C_SN可导致产生的子代病毒感染性滴度降低 10 3～ 10 4 ,显示出很强的抗病毒效果     

人巨细胞病毒病毒体的组装研究新进展

阐明人巨细胞病毒（HCMV）病毒体的组装过程对研究HCMV致病分子机制有重要意义,同时可为抗病毒药物的设计与运用提供新的思路。HCMV组装可概括为两大阶段：初期为入核阶段,主要为核衣壳的组装。在胞质中表达的病毒蛋白形成多种形式的多聚体进入细胞核,在核内相互作用形成衣壳并将病毒DNA装入衣壳中,核衣壳初步形成。第二阶段为出核阶段,主要涉及被膜与包膜的组装。在核中形成的原始核衣壳出核移至胞质,最终在胞质中组装完成,此过程极其复杂,涉及众多蛋白间相互作用及宿主细胞的参与。值得一提的是,组装过程中多种蛋白的变异会导致病毒复制失败。组装完成的病毒体经修饰成熟释放出细胞后,再感染新的宿主细胞。本文对HCMV病毒体组装机制的最新研究作一综述。     

Development of functional variability during the motor learning process of a complex cyclic movement

Traditionally, movement variability is considered an indicator for sensorimotor malfunctioning. However, functional movement variability is also a result of compensation mechanisms e.g. to account for prior movement deviations and is, therefore, crucial for stable movements. The aim of this study was to analyze functional variability during motor learning of a complex cyclic task.Thirteen young participants practised riding a Pedalo® slalom until they were able to complete the task without errors. Since trunk movements are controlled with high priority, we analyzed trunk kinematics as a result parameter. Since lower extremities affect the result parameter, foot, thigh and pelvis kinematics are considered execution parameters. The movement variability for result and execution parameters was determined for the first (poor performance), an intermediate (medium performance) and the last (good performance) training sessions. Furthermore, the variability ratio (execution/result parameter) was calculated as a measure of functional variability.Movement variability of the result parameter decreased significantly with increasing expertise. In contrast, movement variability of all execution parameters increased significantly from measurements representing poor to medium performance. No change from medium to good performance was found. Functional variability increased over time in all execution parameters.Since the movement variability of all execution parameters did not decrease with increasing Pedalo performance, applying a traditional interpretation approach of movement variability would have led to completely wrong conclusions. Possible mechanisms explaining the increased movement variability are discussed. The variability ratio seems to be the only parameter that can reveal improved sensorimotor functioning during all analyzed stages of motor learning.     

A sensitivity analysis of the volumetric spatial decomposition algorithm

Recently, we proposed a new computational approach for the volumetric spatial decomposition of a three-dimensional bone structure into its basic rod and plate elements. This method was based on an image skeletonization approach, where two model parameters were used to identify an ideal skeleton. The goal of this study was to estimate the sensitivity of local morphometric indices to these two model parameters. Our results showed that the rod derived indices behaved more smoothly than plate derived indices, which suggests that rod derived indices are more trustworthy. The results also demonstrated that it was reasonable to reduce the model to only one parameter by setting the noise parameter n to twice the value of the slenderness parameter s. In conclusion, we found that local morphometric indices are reliable measures showing large differences between samples and thus may shed new light on structural differences of trabecular bone in a local fashion by adequately choosing one single optimization parameter.     

Systematic search for the rate constants that control the exocytotic process from chromaffin cells by a genetic algorithm

We have recently created a kinetic model that reproduces the dynamics of exocytosis with high accuracy. The reconstruction necessitated a search, in a multi-dimensional parameter space, for 37 parameters that described the system, with no assurance that the parameters, which reconstructed the observations, are a unique set. In the present study, a Genetic Algorithm (GA) was used for a thorough search in the unknown parameter space, using a strategy of gradual increase of the complexity of the analyzed input data. Upon systematic incorporation of one to four measurable parameters, used as input signals for the analysis, the constraint set on the GA search imposed the convergence of the free parameters into a single narrow range. The mean values for each adjustable parameter represent a minimum for the fitness function in the multi-dimensional parameter space. The GA search demonstrates that the parameters that control the kinetics of exocytosis are the rate constants of the steps downstream to synaptotagmin binding, and that the equilibrium constant of the binding of calcium to Munc13 controls the calcium-dependent priming process. Thus, the systematic use of the GA creates a link between specific reactions in the process of exocytosis and experimental phenotypes.     

On the possibilities of diagnosis of the state of the phytoplankton photosynthetic apparatus by the method of nonlinear laser fluorimetry

The possibilities of diagnosis of the state of the phytoplankton photosynthetic apparatus by the method of nonlinear laser fluorimetry (saturation fluorimetry), which can be realized in remote sensing mode, were investigated. A procedure for the determination of the nonsaturated fluorescence parameter phi0, which is proportional to the concentration of chlorophyll a molecules, and the parameter A, which is a product of three photophysical parameters of chlorophyll a molecule in native chloroplast, was elaborated. Laboratory experiments with the axenic culture of eurihaline Thalassiosira weissflogii showed that the parameter A depends on the state of the photosynthetic apparatus of the alga, which was varied by either DCMU treatment or exposure to actinic light.     

Identifiability and retrievability of unique parameters describing intrinsic Andrews kinetics

A key factor contributing to the variability in the microbial kinetic parameters reported from batch assays is parameter identifiability, i.e., the ability of the mathematical routine used for parameter estimation to provide unique estimates of the individual parameter values. This work encompassed a three-part evaluation of the parameter identifiability of intrinsic kinetic parameters describing the Andrews growth model that are obtained from batch assays. First, a parameter identifiability analysis was conducted by visually inspecting the sensitivity equations for the Andrews growth model. Second, the practical retrievability of the parameters in the presence of experimental error was evaluated for the parameter estimation routine used. Third, the results of these analyses were tested using an example data set from the literature for a self-inhibitory substrate. The general trends from these analyses were consistent and indicated that it is very difficult, if not impossible, to simultaneously obtain a unique set of estimates of intrinsic kinetic parameters for the Andrews growth model using data from a single batch experiment.     

Electrical stimulation of smooth muscle strips from the urinary bladder of the pig

Strips of smooth muscle from pig urinary bladders were electrically stimulated to contract. Stimulation parameters and conditions were optimized so as to obtain a maximum number of isometric contractions with maximal force. It was found that the contractions could be described mathematically by a simple model. In the model there is a constant probability for cells to pass from the non-contractile to the contractile state during stimulation; this leads to a linearly decreasing phase plot (a plot of the rate of rise of a variable as a function of the variable) for the force. ‘Activation’ of the cells is described by a physical step function. Isometric contractions were thus characterized by a set of three parameters: U, the time derivative of the force, extrapolated to zero force, F_iso the value of the isometric force which is approached asymptotically after infinitely long periods of stimulation and t₁, the activation time. The sensitivity of these three parameters to variation of the stimulus parameters was investigated. It was found that the parameter U was consistently correlated with the stimulus parameters, suggesting that this parameter can be used to describe the effectiveness of electrical stimulation of such strips.     

Study of the stability of long-range-ordered lamellar structures for directed self-assembly lithography,performed using dissipative particle dynamics

We performed dissipative particle dynamics (DPD) simulations to obtain long-range-ordered lamellar structures for directed self-assembly lithography. The self-assembled structure of diblock copolymers (DBCs) depends on the length of the different blocks and the difference in their solubility parameters. In the DPD simulations, the DBCs were formed from coarse-grained particles, and the difference between the solubility parameters was represented by a repulsion parameter. We examined the phase separation morphology of the DBCs, which were confined using a trench model system. The repulsion parameter for the assembly of the lamellar structures from the DBC particles was chosen from six types of parameters. The orientation of the lamellar structure was controlled by the repulsion parameter that described the repulsion between the particles and the wall of the system. We changed the width of the trench, and examined the probability for the formation of the lamellar structure. The lamellar structure could not be obtained by increasing the width. To increase the probability, we placed a ridge at the centre of the bottom wall. It was found that the presence of the ridge increased the probability for the formation of the long-range-ordered lamellar structures.     

Effect of various freezing parameters on the immediate post-thaw membrane integrity of adipose tissue derived adult stem cells

The effect of four thermal parameters on post-thaw membrane integrity of adipose tissue derived adult stem (ADAS) cells after controlled-rate freezing was investigated with the help of a two-level four-parameter (2(4)) experimental design. The four thermal parameters studied were cooling rate (CR), end temperature (ET), hold time (HT), and thawing rate (TR). Several passages, including Passage-0 (P0), Passage-1 (P1), Passage-2 (P2), Passage-3 (P3), and Passage-4 (P4), obtained from the suspended culture of stromal vascular fraction (SVF) of the ADAS cells were used for this study. The two levels (low and high) of the four parameters [CR (1 and 40 degrees C/min); ET (-80 and -20 degrees C); HT (1 and 15 min); and TR (10 and 200 degrees C/min)] are chosen in such a way that they enclosed all parameter values possible using commercially available controlled-rate freezing equipment. Individual effect of each parameter on the immediate post-thaw membrane integrity was determined through the calculation of parameter effect values (E), and any synergy among the parameters on post-thaw membrane integrity was assessed through the calculation of two or more parameter interaction effect values (I). Nonlinearity in the experimental results was represented through the calculation of curvature value (CV). The results suggest that for 99% confidence level the parameters CR and ET have considerable effect on post-thaw membrane integrity of all passages of ADAS cells. A significant individual effect of TR was observed with P3 and P4 cells and a significant two-parameter interaction was observed between CR-ET for all passages. These observed results will be used as a basis to further develop freezing storage protocols of ADAS cells.     

Steady state analysis of influx and transbilayer distribution of ergosterol in the yeast plasma membrane

Mathematical modeling is now frequently used in outbreak investigations to understand underlying mechanisms of infectious disease dynamics, assess patterns in epidemiological data, and forecast the trajectory of epidemics. However, the successful application of mathematical models to guide public health interventions lies in the ability to reliably estimate model parameters and their corresponding uncertainty. Here, we present and illustrate a simple computational method for assessing parameter identifiability in compartmental epidemic models. We describe a parametric bootstrap approach to generate simulated data from dynamical systems to quantify parameter uncertainty and identifiability. We calculate confidence intervals and mean squared error of estimated parameter distributions to assess parameter identifiability. To demonstrate this approach, we begin with a low-complexity SEIR model and work through examples of increasingly more complex compartmental models that correspond with applications to pandemic influenza, Ebola, and Zika. Overall, parameter identifiability issues are more likely to arise with more complex models (based on number of equations/states and parameters). As the number of parameters being jointly estimated increases, the uncertainty surrounding estimated parameters tends to increase, on average, as well. We found that, in most cases, R0 is often robust to parameter identifiability issues affecting individual parameters in the model. Despite large confidence intervals and higher mean squared error of other individual model parameters, R0 can still be estimated with precision and accuracy. Because public health policies can be influenced by results of mathematical modeling studies, it is important to conduct parameter identifiability analyses prior to fitting the models to available data and to report parameter estimates with quantified uncertainty. The method described is helpful in these regards and enhances the essential toolkit for conducting model-based inferences using compartmental dynamic models.