An in vitro fluorescence screen to identify antivirals that disrupt hepatitis B virus capsid assembly

Virus assembly has not been routinely targeted in the development of antiviral drugs, in part because of the lack of tractable methods for screening in vitro. We have developed an in vitro assay of hepatitis B virus (HBV) capsid assembly, based on fluorescence quenching of dye-labeled capsid protein, for testing potential inhibitors. This assay is adaptable to high-throughput screening and can identify small-molecule inhibitors of virus assembly that prevent, inappropriately accelerate and/or misdirect capsid formation to yield aberrant particles. An in vitro primary screen has the advantage of identifying promising lead compounds affecting assembly without the requirement that they be taken up by cells in culture and be nontoxic. Our approach may facilitate the identification of antivirals targeting viruses other than HBV, such as avian influenza and HIV.     

A new dynamic null model for phylogenetic community structure

Phylogenies are increasingly applied to identify the mechanisms structuring ecological communities but progress has been hindered by a reliance on statistical null models that ignore the historical process of community assembly. Here, we address this, and develop a dynamic null model of assembly by allopatric speciation, colonisation and local extinction. Incorporating these processes fundamentally alters the structure of communities expected due to chance, with speciation leading to phylogenetic overdispersion compared to a classical statistical null model assuming equal probabilities of community membership. Applying this method to bird and primate communities in South America we show that patterns of phylogenetic overdispersion – often attributed to negative biotic interactions – are instead consistent with a species neutral model of allopatric speciation, colonisation and local extinction. Our findings provide a new null expectation for phylogenetic community patterns and highlight the importance of explicitly accounting for the dynamic history of assembly when testing the mechanisms governing community structure.     

Differentiating between niche and neutral assembly in metacommunities using null models of β‐diversity

The β‐null deviation measure, developed as a null model for β‐diversity, is increasingly used in empirical studies to detect the underlying structuring mechanisms in communities (e.g. niche versus neutral and stochastic versus deterministic). Despite growing use, the ecological interpretation of the presence/absence and abundance‐based versions of the β‐null diversity measure have not been tested against communities with known assembly mechanisms, and thus have not been validated as an appropriate tool for inferring assembly mechanisms. Using a mechanistic model with known assembly mechanisms, we simulated replicate metacommunities and examined β‐null deviation values 1) across a gradient of niche (species‐sorting) to neutrally structured metacommunities, 2) through time, and 3) we compared the effect of changes in assembly mechanism on the performance of the β‐null deviation measures. The impact of stochasticity on assembly outcomes was also considered. β‐null deviation measures proved to be interpretable as a measure of niche or neutral assembly. However, the presence/absence version of the β‐null deviation measure could not differentiate between niche and neutral metacommunities if demographic stochasticity were present. The abundance‐based β‐null deviation measure was successful in distinguishing between niche and neutral metacommunities and was robust to the presence of stochasticity, changes through time, and changing assembly mechanisms. However, we suggest that it is not robust to changing abundance evenness distributions or sampling of communities, and so its interpretation still requires some care. We encourage the testing of the assumptions behind null models for ecology and care in their application.     

Evaluation of Membrane Filter Field Monitors for Microbiological Air Sampling

Due to area constraints encountered in assembly and testing areas of spacecraft, the membrane filter field monitor (MF) and the National Aeronautics and Space Administration-accepted Reyniers slit air sampler were compared for recovery of airborne microbial contamination. The intramural air in a microbiological laboratory area and a clean room environment used for the assembly and testing of the Apollo spacecraft was studied. A significantly higher number of microorganisms was recovered by the Reyniers sampler. A high degree of consistency between the two sampling methods was shown by a regression analysis, with a correlation coefficient of 0.93. The MF samplers detected 79% of the concentration measured by the Reyniers slit samplers. The types of microorganisms identified from both sampling methods were similar. Variables in the MF samplers, such as pore size, relative humidity, and flow rates, have been studied, but no effect was noted on recovery. The results show that the MF method could be used to estimate the number and types of microorganisms found in the air.     

Mapping human chromosomes by walking with sequence-tagged sites from end fragments of yeast artificial chromosome inserts.

Sequence-tagged sites (STSs) derived from end fragments of chromosome-specific yeast artificial chromosomes (YACs) can facilitate the assembly of an overlapping YAC/STS map. Contigs form rapidly by iteratively screening YAC collections with end-fragment STSs from YACs that have not yet been detected by any previous STS. The map is rendered rapidly useful during its assembly by incorporating supplementary STSs from genes and genetic linkage probes with known locations. Methods for the systematic development and testing of the end-fragments STSs are given here, and a group of 100 STSs is presented for the X chromosome. The mapping strategy is shown to be successful in simulations with portions of the X chromosome already largely mapped into overlapping YACs by other means.     

Design factors influencing performance of constrained acetabular liners: finite element characterization

Constrained acetabular liners are utilized to deal with the infrequent but devastating problem of recurrent dislocation. While an encouraging treatment of last resort, the clinical performance of contemporary constrained liners has been somewhat mixed. There are multiple factors contributing to this variability, one of which is the limited understanding of the intrinsic mechanical characteristics of these specialty devices. To address this issue, a three-dimensional, materially nonlinear, multi-surface contact finite element model of a representative constrained liner was created. The model was physically validated, and then used for parametric testing to explore the effects of individual design features. The model was exercised for both intra-operative assembly and lever-out dislocation. It was found that the coefficient of friction between the femoral head and the liner substantially affected both the force required to seat the femoral head into the liner during assembly, and the peak moment resisting dislocation (226% increase in assembly force for friction coefficients of 0.2 versus 0.0; 49% reduction in dislocation moment for friction coefficients of 0.013 versus 0.135). As expected, the cup opening radius also had a dominant effect on both maneuvers: decreasing the opening radius from 13.9 to 13.6 mm increased assembly force by 506 N and increased the dislocation moment by over 3.5 N-m, whereas the influence of other design parameters was much more modest.     

Three biodiversity facets and assembly mechanism of the oligochaete community in the karst spring environment

Hydrobiologia - Springs as ecotones represent a suitable environment for testing hypotheses regarding the principles of a benthic community assembly. In this study, we aimed to uncover the...     

Cholesterol and phospholipid efflux from cultured cells

The removal of phospholipids and cholesterol from tissues is the major mechanism mediating the initial assembly of high density lipoproteins (HDL), as well as being the main reason HDL are thought to protect against atherosclerosis. Investigations of the mechanisms of HDL assembly and testing of novel HDL-raising agents typically involve assays to determine phospholipid and/or cholesterol removal or "efflux" from cultured cells. The purpose of this chapter is to describe experimental protocols that can be used in the determination of cholesterol and phospholipid efflux from cultured cells by HDL apolipoproteins for the formation of new HDL particles, and the testing of novel HDL-raising therapies in vitro. A protocol is also provided for determining the size and nature of HDL particles formed in cell-conditioned medium using two-dimensional gel electrophoresis.     

A review of the effectiveness of vaccine potency control testing

The use of potency control testing is a valuable tool for testing the actual relative strength of manufactured assembly lots of vaccine. Biological-based manufacturing methods are inherently variable and potency testing is a tool to ensure lot-to-lot consistency of commercial vaccines. A strong historical link to clinical efficacy has been established where correlation to efficacy and adequate test validation have been achieved. The link to immunogenicity and efficacy has traditionally been strongest with attenuated vaccines and toxoids. Control potency test failure does predict that a serial or batch of vaccine would most likely provide insufficient immunogenicity in typical field applications. Because of the complexity of pathogenic processes and associated immune responses, potency tests may not always directly predict the effectiveness of a vaccine. Thus, vaccines that pass control potency testing may not always provide adequate efficacy. This is particularly true of adjuvanted, inactivated vaccines. In the development of vaccine formulations and control tests for vaccines, the nature of the desired protective immune responses to the targeted pathogen (when known) should be considered. These considerations could provide better alternatives in the assays chosen as correlates of immunity and may more accurately predict efficacy and assure batch-to-batch consistency. Also, the effects of the dose and duration of antigen exposure as well as the nature of antigen presentation and generation of extrinsic cytokines could be characterised and correlated to vaccine potency as additional indicators of vaccine efficacy.     

Functional traits,spatial patterns and species associations: what is their combined role in the assembly of wetland plant communities?

Studies of community assembly focus on finding rules that predict which species can become member of a plant community. Within a community, species can be categorized in two ways: functional groups classify species according to their functional traits, whereas generalized guilds group species based on their (co-)occurrence, spatial distribution and abundance patterns. This study searches for community assembly rules by testing for coherence among functional groups and generalized guilds, as well as for correlations between the individual functional traits and assembly features, in two wetland plant communities in South Africa. The classifications of functional groups and generalized guilds were not consistent. However, rhizome internode length was related to fine-scale spatial pattern, suggesting that in systems dominated by clonal species (including wetlands, where recruitment sites are strongly limited) community assembly may be strongly linked to colonization ability. Functional groups do not predict guilds in wetland plant communities, precluding their use as the basis for assembly rules. However, an explicit consideration of clonal strategies and their effect on species’ spatial patterns appears to be important for understanding community assembly in systems dominated by clonal plants.     

植物群落构建机制研究进展

群落构建研究对于解释物种共存和物种多样性的维持是至关重要的,因此一直是生态学研究的中心论题。尽管近年来关于生态位和中性理论的验证研究已经取得了显著的成果,但对于局域群落构建机制的认识仍存在很大争议。随着统计和理论上的进步使得用功能性状和群落谱系结构解释群落构建机制变为可能,主要是通过验证共存物种的性状和谱系距离分布模式来实现。然而,谱系和功能性状不能相互替代,多种生物和非生物因子同时控制着群落构建,基于中性理论的扩散限制、基于生态位的环境过滤和竞争排斥等多个过程可能同时影响着群落的构建。所以,综合考虑多种方法和影响因素探讨植物群落的构建机制,对于预测和解释植被对干扰的响应,理解生物多样性维持机制有重要意义。试图在简要回顾群落构建理论及研究方法发展的基础上,梳理其最新研究进展,并探讨整合功能性状及群落谱系结构的研究方法,解释群落构建和物种多样性维持机制的可能途径。在结合功能性状和谱系结构研究群落构建时,除了考虑空间尺度、环境因子、植被类型外,还应该关注时间尺度、选择性状的种类和数量、性状的种内变异、以及人为干扰等因素对群落构建的影响。     

Integrated network reconstruction,visualization and analysis using YANAsquare

Background  

Modeling of metabolic networks includes tasks such as network assembly, network overview, calculation of metabolic fluxes and testing the robustness of the network.     

Synthesis of Anti-Sense Phosphorothioate Analogues for Pharmacokinetic and Pre-Clinical Studies

Abstract

Nudease-resistant oligonucleotides (11 to 28-mers) containing stereorandom phosphorothioate linkages have been recently reported to exhibit potent anti-HIV III effects and sequence-specific inhibition of protein synthesis. Relatively large amounts (100 mg 1g) of these analogues. which are needed for further biological testing and initial pharmacokinetic and pre-clinical studies, were readily obtained by automated hydrogen phosphonate chemistry followed by reversed-phase HPLC and further processing. This chemistry features 1 -adamanetanecarbonyl chloride as the activator, capping with isopropyl phosphite, and more complete sulfurization in only one-step following chain assembly. An automated, quantitative. picomole method for analysis of the analogues in blood samples has been developed.     

Test of Fox's assembly rule for functional groups in lemur communities in Madagascar

This paper examines to what extent an assembly rule designed by Fox for small terrestrial mammal communities is also applicable to communities of arboreal lemurs in Madagascar. This approach extends the testing of Fox's assembly rule to a new biogeographic region and a different phylogenetic radiation. The rule operates on a functional rather than on the taxonomic level. It specifies that: "There is a much higher probability that each species entering a community will be drawn from a different functional group, until each group is represented before the cycle repeats" (Fox, 1987: 201). This rule was tested with lemur communities from 14 sites in evergreen rain forests and nine sites in dry deciduous forests of Madagascar. Lemur species were assigned to one of three different functional groups based on dietary preferences: omnivores, frugivores, folivores. The rule applies almost perfectly to extant lemur communities in evergreen rain forest. Present communities in dry deciduous forests are not ordered as perfectly as communities of the rain forest sites, but they also deviate from random assembly as generated by the null model. Including extinct species of the dry forest ecosystem indicates that the at present unfavoured composition of the lemur community has been derived from a favoured state through extinction of the large folivorous and frugivorous lemur species. In the lemur communities of the eastern Malagasy rain forest, the assembly went through at least four, and in the Holocene dry forest ecosystem through even five, cycles without failing. This assigns considerable significance to the underlying mechanisms (most likely interspecific competition) and indicates that they are still effective in the forests of Madagascar. The dry deciduous forests might have been subject to recent natural and anthropogenic changes which interfere with the operation of these deterministic processes.     

Identifying models of trait‐mediated community assembly using random forests and approximate Bayesian computation

Ecologists often use dispersion metrics and statistical hypothesis testing to infer processes of community formation such as environmental filtering, competitive exclusion, and neutral species assembly. These metrics have limited power in inferring assembly models because they rely on often‐violated assumptions. Here, we adapt a model of phenotypic similarity and repulsion to simulate the process of community assembly via environmental filtering and competitive exclusion, all while parameterizing the strength of the respective ecological processes. We then use random forests and approximate Bayesian computation to distinguish between these models given the simulated data. We find that our approach is more accurate than using dispersion metrics and accounts for uncertainty in model selection. We also demonstrate that the parameter determining the strength of the assembly processes can be accurately estimated. This approach is available in the R package CAMI; Community Assembly Model Inference. We demonstrate the effectiveness of CAMI using an example of plant communities living on lava flow islands.     

The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation?

Amyloidosis is a class of diseases caused by protein aggregation and deposition in various tissues and organs. In this paper, a yeast amyloid-forming protein Sup35 was used as a model for understanding amyloid fiber formation. The dynamics of amyloid formation by Sup35 were studied with scanning force microscopy. We found that: 1) the assembly of Sup35 fibers begins with individual NM peptides that aggregate to form large beads or nucleation units which, in turn, form dimers, trimers, tetramers and longer linear assemblies appearing as a string of beads; 2) the morphology of the linear assemblies differ; and 3) fiber assembly suggests an analogy to the aggregation of colloidal particles. A dipole assembly model is proposed based on this analogy that will allow further experimental testing.     

四种常用高通量测序拼接软件的应用比较

新一代测序平台的诞生推动了对全基因组鸟枪法测序数据的拼接算法和软件的研究,自2005年以来多种用于高通量测序的序列拼接软件已经被开发出来,并且在不断地进行改进以提高拼接效果.本文利用目前广泛使用的高通量测序拼接软件Velvet、AbySS、SOAPdenovo和CLC Genomic Workbench分别对本试验室分离的一株噬菌体IME08的高通量测序结果进行拼接,介绍这几种拼接软件的安装使用及参数优化,并对不同软件的拼接结果进行比较,针对不同的拼接软件得到优化的拼接参数,可为其他研究人员使用上述软件提供参考借鉴.     

Utilizing mapping targets of sequences underrepresented in the reference assembly to reduce false positive alignments

The human reference assembly remains incomplete due to the underrepresentation of repeat-rich sequences that are found within centromeric regions and acrocentric short arms. Although these sequences are marginally represented in the assembly, they are often fully represented in whole-genome short-read datasets and contribute to inappropriate alignments and high read-depth signals that localize to a small number of assembled homologous regions. As a consequence, these regions often provide artifactual peak calls that confound hypothesis testing and large-scale genomic studies. To address this problem, we have constructed mapping targets that represent roughly 8% of the human genome generally omitted from the human reference assembly. By integrating these data into standard mapping and peak-calling pipelines we demonstrate a 10-fold reduction in signals in regions common to the blacklisted region and identify a comprehensive set of regions that exhibit mapping sensitivity with the presence of the repeat-rich targets.     

Self-assembly of plant cell walls

The object of this paper is to define criteria for distinguishing between self-assembly and template-based assembly in plant cell walls. The example of cellulose shows that cell wall polymers biosynthesized at a membrane may retain parallel chain packing arrangements that are thermodynamically unstable and cannot be reproduced in vitro, making the experimental testing of the self-assembly hypothesis difficult. Also, natural cellulose is ordered on a number of scales of pattern, each of which may be constructed by either self- or template-based assembly independently of the rest. These conceptual problems apply equally to the self-assembly of complete cell walls and other cell wall polymers. It is suggested that the self-assembly concept should be applied only to one stage or level in the synthesis of a cell wall, and that an additional concept of parallel assembly may be useful for understanding the synthesis of some polysaccharides.     

Community assembly,natural selection and maximum entropy models

The papers in this Forum discussion debate various aspects of my maximum entropy model of community assembly. The questions raised centre around (1) the possible mechanisms generating the patterns predicted by my maxent model of community assembly, and (2) the appropriate statistical methods for testing the patterns. Here I briefly explain the proposed mechanistic basis of the model: natural selection occurring between individuals of different species. If trait differences are linked to differential demographic probabilities (i.e. fitness differences) then natural selection will constrain the average trait values found in the community and such average (‘community‐aggregated’) traits will then possess information that is translated into the maximum entropy probabilities. If community assembly is strictly neutral then the maxent model will have no predictive ability. This also justifies the null model, and the permutation test, proposed by Roxburgh and Mokany.