Size correction: comparing morphological traits among populations and environments

Morphological relationships change with overall body size and body size often varies among populations. Therefore, quantitative analyses of individual traits from organisms in different populations or environments (e.g., in studies of phenotypic plasticity) often adjust for differences in body size to isolate changes in allometry. Most studies of among population variation in morphology either (1) use analysis of covariance (ANCOVA) with a univariate measure of body size as the covariate, or (2) compare residuals from ordinary least squares regression of each trait against body size or the first principal component of the pooled data (shearing). However, both approaches are problematic. ANCOVA depends on assumptions (small variance in the covariate) that are frequently violated in this context. Residuals analysis assumes that scaling relationships within groups are equal, but this assumption is rarely tested. Furthermore, scaling relationships obtained from pooled data typically mischaracterize within-group scaling relationships. We discuss potential biases imposed by the application of ANCOVA and residuals analysis for quantifying morphological differences, and elaborate and demonstrate a more effective alternative: common principal components analysis combined with Burnaby’s back-projection method.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Amphisbaenian paleobiogeography: Evidence of vicariance and geodispersal patterns

Paleobiogeographic patterns within the Amphisbaenia were evaluated using the modified Brooks Parsimony Analysis (BPA) and recently published morphological and molecular phylogenies. Extant amphisbaenians are present in Africa, South America, North America, Europe, and the Middle East. The modified BPA was used to determine the relative effects of Pangean breakup, sea-level change, and climate change on evolutionary and distributional patterns within the Amphisbaenia. The modified BPA also tested the biogeographic effect of the Rhineuridae's phylogenetic position as either most basal in the morphologic phylogeny or most derived in the molecular phylogeny. The morphological and molecular analyses resulted in two different biogeographic hypotheses. The morphological analysis indicated three major biogeographic regions for the Amphisbaenia: 1) Africa, South America, and the Caribbean, 2) western Asia, and 3) North America. The molecular analysis indicated two major biogeographic regions: 1) Africa, western Asia, and North America, and 2) South America. The morphological biogeographic pattern corresponds with the known timing of the breakup of Pangea and the resulting paleogeographic reconstructions of the Mesozoic and Early Cenozoic. While the molecular pattern is similar to patterns recovered from dinosaurian biogeographic studies, the closer connection of Africa with North America rather than South America does not match well-constrained geologic evidence for the sequence of Pangean breakup. Both paleobiogeographic analyses, however, resulted in congruent patterns of speciation through vicariance and geodispersal. This suggests that in addition to the breakup of Pangea, such cyclical Earth history processes as sea-level and climate changes played an important role in the biogeographic patterns of the Amphisbaenia.     

Correlation of RNA Secondary Structure Statistics with Thermodynamic Stability and Applications to Folding

The accurate prediction of the secondary and tertiary structure of an RNA with different folding algorithms is dependent on several factors, including the energy functions. However, an RNA higher-order structure cannot be predicted accurately from its sequence based on a limited set of energy parameters. The inter- and intramolecular forces between this RNA and other small molecules and macromolecules, in addition to other factors in the cell such as pH, ionic strength, and temperature, influence the complex dynamics associated with transition of a single stranded RNA to its secondary and tertiary structure. Since all of the factors that affect the formation of an RNAs 3D structure cannot be determined experimentally, statistically derived potential energy has been used in the prediction of protein structure. In the current work, we evaluate the statistical free energy of various secondary structure motifs, including base-pair stacks, hairpin loops, and internal loops, using their statistical frequency obtained from the comparative analysis of more than 50,000 RNA sequences stored in the RNA Comparative Analysis Database (rCAD) at the Comparative RNA Web (CRW) Site. Statistical energy was computed from the structural statistics for several datasets. While the statistical energy for a base-pair stack correlates with experimentally derived free energy values, suggesting a Boltzmann-like distribution, variation is observed between different molecules and their location on the phylogenetic tree of life. Our statistical energy values calculated for several structural elements were utilized in the Mfold RNA-folding algorithm. The combined statistical energy values for base-pair stacks, hairpins and internal loop flanks result in a significant improvement in the accuracy of secondary structure prediction; the hairpin flanks contribute the most.     

Reduced PDZ Interactions of Rescued ΔF508CFTR Increases Its Cell Surface Mobility

Deletion of phenylalanine 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) plasma membrane chloride channel is the most common cause of cystic fibrosis (CF). Though several maneuvers can rescue endoplasmic reticulum-retained ΔF508CFTR and promote its trafficking to the plasma membrane, rescued ΔF508CFTR remains susceptible to quality control mechanisms that lead to accelerated endocytosis, ubiquitination, and lysosomal degradation. To investigate the role of scaffold protein interactions in rescued ΔF508CFTR surface instability, the plasma membrane mobility of ΔF508CFTR was measured in live cells by quantum dot single particle tracking. Following rescue by low temperature, chemical correctors, thapsigargin, or overexpression of GRASP55, ΔF508CFTR diffusion was more rapid than that of wild-type CFTR because of reduced interactions with PDZ domain-containing scaffold proteins. Knock-down of the plasma membrane quality control proteins CHIP and Hsc70 partially restored ΔF508CFTR-scaffold association. Quantitative comparisons of CFTR cell surface diffusion and endocytosis kinetics suggested an association between reduced scaffold binding and CFTR internalization. Our surface diffusion measurements in live cells indicate defective scaffold interactions of rescued ΔF508CFTR at the cell surface, which may contribute to its defective peripheral processing.     

Different sample treatment approaches for the analysis of T-2 and HT-2 toxins from oats-based media

A LC-DAD method is proposed for the determination of the T-2 and HT-2 toxins in cultures of Fusarium langsethiae in oat-based and other in vitro media. Test media consisted of freshly prepared milled oats to which T-2 and HT-2 toxin stock solutions were added. Different mixtures of extraction solvent (acetonitrile:water and methanol:water), extraction times (30′, 60′ or 90′) and drying methods were investigated. Results showed that extraction with methanol:water (80:20, v/v) for 90 min, drying with N₂ and subsequent analysis by LC-DAD was the fastest and most user friendly method for detecting HT-2 and T-2 toxins production by F. langsethiae strains grown on oat-based media at levels of 0.459 and 0.508 mg of toxin/kg of agar, respectively. The proposed method was used to investigate toxin production of 6 F. langsethiae strains from northern Europe and provided clear chromatograms with no interfering peaks in media with and without glycerol as water activity modifier.     

Magnetizable antibody-like proteins

Use of paramagnetic particles to isolate molecules or cells from complex media is well established. Typically, particles are manufactured and coated with a biological molecule that confers specific biorecognition. Incubation of particles with sample and exposure to magnetic fields isolates the species of interest. We have designed, produced and assessed magnetized fusion proteins consisting of the antigen-binding portion of an antibody (single chain variable fraction; scFv) fused to the heavy chain of the iron-binding protein ferritin. The fusion protein subunits expressed in E. coli assemble to form a fusion protein consisting of a ferritin sphere with scFvs on the surface. The fusion proteins were chemically magnetized by introducing a paramagnetic iron core. The resultant fusion protein was shown to be magnetizable and capable of binding target antigens. These “organic” magnetizable particles possess a number of theoretical advantages over traditional inorganic particles.     

Proteomics data repositories: Providing a safe haven for your data and acting as a springboard for further research

Despite the fact that data deposition is not a generalised fact yet in the field of proteomics, several mass spectrometry (MS) based proteomics repositories are publicly available for the scientific community. The main existing resources are: the Global Proteome Machine Database (GPMDB), PeptideAtlas, the PRoteomics IDEntifications database (PRIDE), Tranche, and NCBI Peptidome. In this review the capabilities of each of these will be described, paying special attention to four key properties: data types stored, applicable data submission strategies, supported formats, and available data mining and visualization tools. Additionally, the data contents from model organisms will be enumerated for each resource. There are other valuable smaller and/or more specialized repositories but they will not be covered in this review. Finally, the concept behind the ProteomeXchange consortium, a collaborative effort among the main resources in the field, will be introduced.     

Sequence signatures of allosteric proteins towards rational design

Allostery is the phenomenon of changes in the structure and activity of proteins that appear as a consequence of ligand binding at sites other than the active site. Studying mechanistic basis of allostery leading to protein design with predetermined functional endpoints is an important unmet need of synthetic biology. Here, we screened the amino acid sequence landscape in search of sequence-signatures of allostery using Recurrence Quantitative Analysis (RQA) method. A characteristic vector, comprised of 10 features extracted from RQA was defined for amino acid sequences. Using Principal Component Analysis, four factors were found to be important determinants of allosteric behavior. Our sequence–based predictor method shows 82.6% accuracy, 85.7% sensitivity and 77.9% specificity with the current dataset. Further, we show that Laminarity-Mean-hydrophobicity representing repeated hydrophobic patches is the most crucial indicator of allostery. To our best knowledge this is the first report that describes sequence determinants of allostery based on hydrophobicity. As an outcome of these findings, we plan to explore possibility of inducing allostery in proteins.     

Analysis conditions for proteomic profiling of mammalian tissue and cell extracts with antibody microarrays

Antibody microarrays are a developing tool for global proteomic profiling. A protocol was established that permits robust analyses of protein extracts from mammalian tissues and cells rather than body fluids. The factors optimized were buffer composition for surface blocking, blocking duration, protein handling and processing, labeling parameters like type of dye, molar ratio of label versus protein, and dye removal, as well as incubation parameters such as duration, temperature, buffer, and sample agitation.     

重组萝卜磷脂氢谷胱甘肽过氧化物酶在毕赤酵母优化表达初步纯化与鉴定

将萝卜磷脂氢谷胱甘肽过氧化物酶（RsPHGPx）基因插入到分泌表达载体pPIC9K中,转化巴斯德毕赤酵母GS115细胞,筛选具有G418抗性的单拷贝转化子。经过优化表达条件,RsPHGPx在1%甲醇、pH6.0、28℃条件下诱导60h后得到最大表达量,产率约为102 mg/L。通过硫酸铵分级沉淀、脱盐柱脱盐、凝胶过滤等纯化步骤,得到了90%以上纯度的RsPHGPx．活性分析显示纯化获得的RsPHGPx具有依赖于GSH的还原活性, 比活性为4.2μmol/min·mg,为获得大量RsPHGPx而用于应用开发研究奠定了基础。