A workbench for multiple alignment construction and analysis

Multiple sequence alignment can be a useful technique for studying molecular evolution, as well as for analyzing relationships between structure or function and primary sequence. We have developed for this purpose an interactive program, MACAW (Multiple Alignment Construction and Analysis Workbench), that allows the user to construct multiple alignments by locating, analyzing, editing, and combining "blocks" of aligned sequence segments. MACAW incorporates several novel features. (1) Regions of local similarity are located by a new search algorithm that avoids many of the limitations of previous techniques. (2) The statistical significance of blocks of similarity is evaluated using a recently developed mathematical theory. (3) Candidate blocks may be evaluated for potential inclusion in a multiple alignment using a variety of visualization tools. (4) A user interface permits each block to be edited by moving its boundaries or by eliminating particular segments, and blocks may be linked to form a composite multiple alignment. No completely automatic program is likely to deal effectively with all the complexities of the multiple alignment problem; by combining a powerful similarity search algorithm with flexible editing, analysis and display tools, MACAW allows the alignment strategy to be tailored to the problem at hand.     

Informing climate models with rapid chamber measurements of forest carbon uptake

Models predicting ecosystem carbon dioxide (CO₂) exchange under future climate change rely on relatively few real‐world tests of their assumptions and outputs. Here, we demonstrate a rapid and cost‐effective method to estimate CO₂ exchange from intact vegetation patches under varying atmospheric CO₂ concentrations_. We find that net ecosystem CO₂ uptake (NEE) in a boreal forest rose linearly by 4.7 ± 0.2% of the current ambient rate for every 10 ppm CO₂ increase, with no detectable influence of foliar biomass, season, or nitrogen (N) fertilization. The lack of any clear short‐term NEE response to fertilization in such an N‐limited system is inconsistent with the instantaneous downregulation of photosynthesis formalized in many global models. Incorporating an alternative mechanism with considerable empirical support – diversion of excess carbon to storage compounds – into an existing earth system model brings the model output into closer agreement with our field measurements. A global simulation incorporating this modified model reduces a long‐standing mismatch between the modeled and observed seasonal amplitude of atmospheric CO₂. Wider application of this chamber approach would provide critical data needed to further improve modeled projections of biosphere–atmosphere CO₂ exchange in a changing climate.     

Transcriptome‐based target‐enrichment baits for stony corals (Cnidaria: Anthozoa: Scleractinia)

Despite the ecological and economic significance of stony corals (Scleractinia), a robust understanding of their phylogeny remains elusive due to patchy taxonomic and genetic sampling, as well as the limited availability of informative markers. To increase the number of genetic loci available for phylogenomic analyses in Scleractinia, we designed 15,919 DNA enrichment baits targeting 605 orthogroups (mean 565 ± SD 366 bp) over 1,139 exon regions. A further 236 and 62 barcoding baits were designed for COI and histone H3 genes respectively for quality and contamination checks. Hybrid capture using these baits was performed on 18 coral species spanning the presently understood scleractinian phylogeny, with two corallimorpharians as outgroup. On average, 74% of all loci targeted were successfully captured for each species. Barcoding baits were matched unambiguously to their respective samples and revealed low levels of cross‐contamination in accordance with expectation. We put the data through a series of stringent filtering steps to ensure only scleractinian and phylogenetically informative loci were retained, and the final probe set comprised 13,479 baits, targeting 452 loci (mean 531 ± SD 307 bp) across 865 exon regions. Maximum likelihood, Bayesian and species tree analyses recovered maximally supported, topologically congruent trees consistent with previous phylogenomic reconstructions. The phylogenomic method presented here allows for consistent capture of orthologous loci among divergent coral taxa, facilitating the pooling of data from different studies and increasing the phylogenetic sampling of scleractinians in the future.     

A proposed bioactive form of peptide T and the design of peptidomimetics

Peptide T is a non-natural octapeptide of sequence Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr, taken from the sequence of the protein gp120 of HIV. The peptide has been shown to bind competitively to the CD4 receptors of the helper/inducer lymphocytes T. The peptide is presently used for the treatment of AIDS-associated dementia and has been proven useful for the treatment of psoriasis. Using molecular modeling procedures, we studied the conformational profile of this peptide as well as those of several active and inactive analogs. The analysis of these results gave rise to the proposal of a bioactive conformation of the peptide, which can be described as a pseudo -turn structure, involving the last four residues at the C-terminus of the peptide. The secondary structure is stabilized by a hydrogen bond between the hydroxyl hydrogen of the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷. From the bioactive form and different structure–activity relationship studies, a pharmacophore was proposed. This hypothesis was used to search on several 3D data bases. One of the hits obtained was the natural compound amigdalin, which was tested and exhibited moderate activity.     

Impact of adolescent obesity on middle-age health of women given data MAR

We analyze adolescent BMI and middle-age systolic blood pressure (SBP) repeatedly measured on women enrolled in the Fels Longitudinal Study (FLS) between 1929 and 2010 to address three questions: Do adolescent-specific growth rates in body mass index (BMI) and menarche affect middle-age SBP? Do they moderate the aging effect on middle-age SBP? Have the effects changed over historical time? To address the questions, we propose analyzing a growth curve model (GCM) that controls for age, birth-year cohort, and historical time. However, several complications in the data make the GCM analysis nonstandard. First, the person-specific adolescent BMI and middle-age SBP trajectories are unobservable. Second, missing data are substantial on BMI, SBP, and menarche. Finally, modeling the latent trajectories for BMI and SBP, repeatedly measured on two distinct sets of unbalanced time points, are computationally intensive. We adopt a bivariate GCM for BMI and SBP with correlated random coefficients. To efficiently handle missing values of BMI, SBP, and menarche assumed missing at random, we estimate their joint distribution by maximum likelihood via the EM algorithm where the correlated random coefficients and menarche are multivariate normal. The estimated distribution will be transformed to the desired GCM for SBP that includes the random coefficients of BMI and menarche as covariates. We demonstrate unbiased estimation by simulation. We find that adolescent growth rates in BMI and menarche are positively associated with, and moderate, the aging effect on SBP in middle age, controlling for age, cohort, and historical time, but the effect sizes are at most modest. The aging effect is significant on SBP, controlling for cohort and historical time, but not vice versa.     

Segmentation by classification: A novel and reliable approach for semi-automatic selection of HIV/SIV envelope spikes

We describe a semiautomated approach to segment Env spikes from the membrane envelope of Simian Immunodeficiency Virus visualized by cryoelectron tomography of frozen-hydrated specimens. Multivariate data analysis is applied to a large set of overlapping subvolumes extracted semiautomatically from the viral envelope and does not utilize a template of the target structure. The major manual step used in the method involves determination of six points that define an ellipsoid approximating the virion shape. The approach is robust to departures of the actual virion from this starting ellipsoid. A point cage of sufficient density is generated to ensure that any spike-like protein is identified multiple times. Subsequently translational alignment of class averages to a cylindrical reference on a curved surface separates subvolumes with spikes from those without. Spike containing subvolumes identified multiple times are removed by proximity analysis. Slightly different procedures segment spikes in the equatorial and the polar regions. Once all spikes are segmented, further alignment of class averages using separately the polar and spin angles produces recognizable spike images. Our approach localized 96% of the equatorial spikes and 85% of all spikes identified manually; it identifies a significant number of additional spikes missed by manual selection. Two types of spike shapes were segmented, one with near 3-fold symmetry resembling the conventional spike, the other had a T-shape resembling the spike structure obtained when antibodies such as PG9 bind to HIV Env. The approach should be applicable to segmentation of any protein spikes extending from a cellular or virion envelope.     

SimpleDSFviewer: A tool to analyze and view differential scanning fluorimetry data for characterizing protein thermal stability and interactions

Differential scanning fluorimetry (DSF) is a widely used thermal shift assay for measuring protein stability and protein–ligand interactions that are simple, cheap, and amenable to high throughput. However, data analysis remains a challenge, requiring improved methods. Here, the program SimpleDSFviewer, a user‐friendly interface, is described to help the researchers who apply DSF technique in their studies. SimpleDSFviewer integrates melting curve (MC) normalization, smoothing, and melting temperature (Tm) analysis and directly previews analyzed data, providing an efficient analysis tool for DSF. SimpleDSFviewer is developed in Matlab, and it is freely available for all users to use in Matlab workspace or with Matlab Runtime. It is easy to use and an efficient tool for researchers to preview and analyze their data in a very short time.     

Estimating evolutionary parameters when viability selection is operating

Some individuals die before a trait is measured or expressed (the invisible fraction), and some relevant traits are not measured in any individual (missing traits). This paper discusses how these concepts can be cast in terms of missing data problems from statistics. Using missing data theory, I show formally the conditions under which a valid evolutionary inference is possible when the invisible fraction and/or missing traits are ignored. These conditions are restrictive and unlikely to be met in even the most comprehensive long-term studies. When these conditions are not met, many selection and quantitative genetic parameters cannot be estimated accurately unless the missing data process is explicitly modelled. Surprisingly, this does not seem to have been attempted in evolutionary biology. In the case of the invisible fraction, viability selection and the missing data process are often intimately linked. In such cases, models used in survival analysis can be extended to provide a flexible and justified model of the missing data mechanism. Although missing traits pose a more difficult problem, important biological parameters can still be estimated without bias when appropriate techniques are used. This is in contrast to current methods which have large biases and poor precision. Generally, the quantitative genetic approach is shown to be superior to phenotypic studies of selection when invisible fractions or missing traits exist because part of the missing information can be recovered from relatives.     

Passive multivariable temperature and conductivity RFID sensors for single-use biopharmaceutical manufacturing components

Single-use biopharmaceutical manufacturing requires monitoring of critical manufacturing parameters. We have developed an approach for passive radio-frequency identification (RFID)-based sensing that converts ubiquitous passive 13.56 MHz RFID tags into inductively coupled sensors. We combine several measured parameters from the resonant sensor antenna with multivariate data analysis and deliver unique capability of multiparameter sensing and rejection of environmental interferences with a single sensor. We demonstrate here the integration of these RFID sensors into single-use biopharmaceutical manufacturing components. We have tested these sensors for over 500 h for measurements of temperature and solution conductivity with the accuracy of 0.1°C (32-48°C range) and accuracy of 0.3-2.9 mS/cm (0.5-230 mS/cm range). We further demonstrate simultaneous temperature and conductivity measurements with an individual RFID sensor with the accuracy of 0.2°C (5-60°C range) and accuracy of 0.9 mS/cm (0.5-183 mS/cm range). Developed RFID sensors provide several important features previously unavailable from other single-use sensing technologies such as the same sensor platform for measurements of physical, chemical, and biological parameters; multi-parameter monitoring with individual sensors; and simultaneous digital identification.     

Structure–Function Analysis of the ADAM Family of Disintegrin-Like and Metalloproteinase-Containing Proteins (Review)

The ADAMs belong to a disintegrin-like and metalloproteinase-containing protein family that are zinc-dependent metalloproteinases. These proteins share all or some of the following domain structure: a signal peptide, a propeptide, a metalloproteinase, a disintegrin, a cysteine-rich, and an epidermal growth factor (EGF)-like domains, a transmembrane region, and a cytoplasmic tail. ADAMs are widely distributed in many organs, tissues, and cells, such as brain, testis, epididymis, ovary, breast, placenta, liver, heart, lung, bone, and muscle. These proteins are capable of four potential functions: proteolysis, adhesion, fusion, and intracellular signaling. Because the number of ADAM genes has grown rapidly and the biological functions of most members are unclear, this review analyzes the protein structures and functions, their activation and processing, their known and potential activities, and their evolutionary relationships. A sequence alignment of human ADAMs is compiled and their homology and physical data are calculated. The conceivable functions of ADAMs in reproduction, development, and diseases are also discussed.