ALBERT

Description: The predicted effect of effective population size on the distribution of fitness effects and substitution rate is critically dependent on the relationship between sequence and fitness. This highlights the importance of using models that are informed by the molecular biology, biochemistry, and biophysics of the evolving systems. We describe a computational model based on fundamental aspects of biophysics, the requirement for (most) proteins to be thermodynamically stable. Using this model, we find that differences in population size have minimal impact on the distribution of population-scaled fitness effects, as well as on the rate of molecular evolution. This is because larger populations result in selection for more stable proteins that are less affected by mutations. This reduction in the magnitude of the fitness effects almost exactly cancels the greater selective pressure resulting from the larger population size. Conversely, changes in the population size in either direction cause transient increases in the substitution rate. As differences in population size often correspond to changes in population size, this makes comparisons of substitution rates in different lineages difficult to interpret.

Description: O -GlcNAcylation is an inducible, highly dynamic and reversible post-translational modification, mediated by a unique enzyme named O -linked N -acetyl- d -glucosamine ( O -GlcNAc) transferase (OGT). In response to nutrients, O -GlcNAc levels are differentially regulated on many cellular proteins involved in gene expression, translation, immune reactions, protein degradation, protein–protein interaction, apoptosis and signal transduction. In contrast to eukaryotic cells, little is known about the role of O -GlcNAcylation in the viral life cycle. Here, we show that the overexpression of the OGT reduces the replication efficiency of Kaposi's sarcoma-associated herpesvirus (KSHV) in a dose-dependent manner. In order to investigate the global impact of O -GlcNAcylation in the KSHV life cycle, we systematically analyzed the 85 annotated KSHV-encoded open reading frames for O -GlcNAc modification. For this purpose, an immunoprecipitation (IP) strategy with three different approaches was carried out and the O -GlcNAc signal of the identified proteins was properly controlled for specificity. Out of the 85 KSHV-encoded proteins, 18 proteins were found to be direct targets for O -GlcNAcylation. Selected proteins were further confirmed by mass spectrometry for O -GlcNAc modification. Correlation of the functional annotation and the O -GlcNAc status of KSHV proteins showed that the predominant targets were proteins involved in viral DNA synthesis and replication. These results indicate that O -GlcNAcylation plays a major role in the regulation of KSHV propagation.

Description: Galectins are potent adhesion/growth-regulatory effectors with characteristic expression profiles. Understanding the molecular basis of gene regulation in each case requires detailed information on copy number of genes and sequence(s) of their promoter(s). Our report reveals plasticity in this respect between galectins and species. We here describe occurrence of a two-gene constellation for human galectin (Gal)-7 and define current extent of promoter-sequence divergence. Interestingly, cross-species genome analyses also detected single-copy display. Because the regulatory potential will then be different, extrapolations of expression profiles are precluded between respective species pairs. Gal-4 coding in chromosomal vicinity was found to be confined to one gene, whereas copy-number variation also applied to Gal-9. The example of rat Gal-9 teaches the lesson that the presence of multiple bands in Southern blotting despite a single-copy gene constellation is attributable to two pseudogenes. The documented copy-number variability should thus be taken into consideration when studying regulation of galectin genes, in a species and in comparison between species.

Description: In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous -〉 alpha-1 (AI-1) -〉 alpha-2 (AI-2) -〉 gamma (GI) -〉 delta (DI) -〉 zeta (ZI) -〉 epsilon (EI) -〉 omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using 1 H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

Description: The methylotrophic yeast, Pichia pastoris , is an important organism used for the production of therapeutic proteins. Previously, we have reported the glycoengineering of this organism to produce human-like N -linked glycans but up to now no one has addressed engineering the O -linked glycosylation pathway. Typically, O -linked glycans produced by wild-type P. pastoris are linear chains of four to five α-linked mannose residues, which may be capped with β- or phospho-mannose. Previous genetic engineering of the N-linked glycosylation pathway of P. pastoris has eliminated both of these two latter modifications, resulting in O -linked glycans which are linear α-linked mannose structures. Here, we describe a method for the co-expression of an α-1,2-mannosidase, which reduces these glycans to primarily a single O -linked mannose residue. In doing so, we have reduced the potential of these glycans to interact with carbohydrate-binding proteins, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin. Furthermore, the introduction of the enzyme protein- O -linked-mannose β-1,2- N -acetylglucosaminyltransferase 1, resulted in the capping of the single O -linked mannose residues with N -acetylglucosamine. Subsequently, this glycoform was extended into human-like sialylated glycans, similar in structure to α-dystroglycan-type glycoforms. As such, this represents the first example of sialylated O -linked glycans being produced in yeast and extends the utility of the P. pastoris production platform beyond N -linked glycosylated biotherapeutics to include molecules possessing O -linked glycans.

Description: Neurons and other cells require intracellular transport of essential components for viability and function. Previous work has shown that while net amyloid precursor protein (APP) transport is generally anterograde, individual vesicles containing APP move bi-directionally. This discrepancy highlights our poor understanding of the in vivo regulation of APP-vesicle transport. Here, we show that reduction of presenilin (PS) or suppression of gamma-secretase activity substantially increases anterograde and retrograde velocities for APP vesicles. Strikingly, PS deficiency has no effect on an unrelated cargo vesicle class containing synaptotagmin, which is powered by a different kinesin motor. Increased velocities caused by PS or gamma-secretase reduction require functional kinesin-1 and dynein motors. Together, our findings suggest that a normal function of PS is to repress kinesin-1 and dynein motor activity during axonal transport of APP vesicles. Furthermore, our data suggest that axonal transport defects induced by loss of PS-mediated regulatory effects on APP-vesicle motility could be a major cause of neuronal and synaptic defects observed in Alzheimer's Disease (AD) pathogenesis. Thus, perturbations of APP/PS transport could contribute to early neuropathology observed in AD, and highlight a potential novel therapeutic pathway for early intervention, prior to neuronal loss and clinical manifestation of disease.

Description: With age, muscle mass and integrity are progressively lost leaving the elderly frail, weak and unable to independently care for themselves. Defined as sarcopenia, this age-related muscle atrophy appears to be multifactorial but its definite cause is still unknown. Mitochondrial dysfunction has been implicated in this process. Using a novel transgenic mouse model of mitochondrial DNA (mtDNA) double-strand breaks (DSBs) that presents a premature aging-like phenotype, we studied the role of mtDNA damage in muscle wasting. We caused DSBs in mtDNA of adult mice using a ubiquitously expressed mitochondrial-targeted endonuclease, mito- Pst I. We found that a short, transient systemic mtDNA damage led to muscle wasting and a decline in locomotor activity later in life. We found a significant decline in muscle satellite cells, which decreases the muscle's capacity to regenerate and repair during aging. This phenotype was associated with impairment in acetylcholinesterase (AChE) activity and assembly at the neuromuscular junction (NMJ), also associated with muscle aging. Our data suggests that systemic mitochondrial dysfunction plays important roles in age-related muscle wasting by preferentially affecting the myosatellite cell pool.

Description: Populations of widely distributed species encounter and must adapt to local environmental conditions. However, comprehensive characterization of the genetic basis of adaptation is demanding, requiring genome-wide genotype data, multiple sampled populations, and an understanding of population structure and potential selection pressures. Here, we used single-nucleotide polymorphism genotyping and data on numerous environmental variables to describe the genetic basis of local adaptation in 21 populations of teosinte, the wild ancestor of maize. We found complex hierarchical genetic structure created by altitude, dispersal events, and admixture among subspecies, which complicated identification of locally beneficial alleles. Patterns of linkage disequilibrium revealed four large putative inversion polymorphisms showing clinal patterns of frequency. Population differentiation and environmental correlations suggest that both inversions and intergenic polymorphisms are involved in local adaptation.

Description: Not all calcite ballast is created equal: differing effects of foraminiferan and coccolith calcite on the formation and sinking of aggregates Biogeosciences Discussions, 10, 14861-14885, 2013 Author(s): K. Schmidt, C. L. De La Rocha, M. Gallinari, and G. Cortese Correlation between particulate organic carbon (POC) and calcium carbonate sinking through the deep ocean has led to the idea that ballast provided by calcium carbonate is important for the export of POC from the surface ocean. While this idea is certainly to some extent true, it is worth considering in more nuance, for example, examining the different effects on the aggregation and sinking of POC of small, non-sinking calcite particles like coccoliths and large, rapidly sinking calcite like planktonic foraminiferan tests. We have done that here in a simple experiment carried out in roller tanks that allow particles to sink continuously without being impeded by container walls. Coccoliths were efficiently incorporated into aggregates that formed during the experiment, increasing their sinking speed compared to similarly sized aggregates lacking added calcite ballast. The foraminiferan tests, which sank as fast as 700 m d −1 , became associated with only very minor amounts of POC. In addition, when they collided with other, larger, foraminferan-less aggregates, they fragmented them into two smaller, more slowly sinking aggregates. While these effects were certainly exaggerated within the confines of the roller tanks, they clearly demonstrate that calcium carbonate ballast is not just calcium carbonate ballast- different forms of calcium carbonate ballast have notably different effects on POC aggregation, sinking, and export.

Description: There is much interest in using high-throughput DNA sequencing methodology to monitor microorganisms, complex plant and animal communities. However, there are experimental and analytical issues to consider before applying a sequencing technology, which was originally developed for genome projects, to ecological projects. Many of these issues have been highlighted by recent microbial studies. Understanding how high-throughput sequencing is best implemented is important for the interpretation of recent results and the success of future applications. Addressing complex biological questions with metagenomics requires the interaction of researchers who bring different skill sets to problem solving. Educators can help by nurturing a collaborative interdisciplinary approach to genome science, which is essential for effective problem solving. Educators are in a position to help students, teachers, the public and policy makers interpret the new knowledge that metagenomics brings. To do this, they need to understand, not only the excitement of the science but also the pitfalls and shortcomings of methodology and research designs. We review these issues and some of the research directions that are helping to move the field forward.

Description: We believe that undergraduate biology students must acquire a foundational background in computing including how to formulate a computational problem; develop an algorithmic solution; implement their solution in software and then test, document and use their code to explore biological phenomena. Moreover, by learning these skills in the first year, students acquire a powerful tool set that they can use and build on throughout their studies. To address this need, we have developed a first-year undergraduate course that teaches students the foundations of computational thinking and programming in the context of problems in biology. This article describes the structure and content of the course and summarizes assessment data on both affective and learning outcomes.

Description: Bioinformatics is an integral part of modern life sciences. It has revolutionized and redefined how research is carried out and has had an enormous impact on biotechnology, medicine, agriculture and related areas. Yet, it is only rarely integrated into high school teaching and learning programs, playing almost no role in preparing the next generation of information-oriented citizens. Here, we describe the design principles of bioinformatics learning environments, including our own, that are aimed at introducing bioinformatics into senior high school curricula through engaging learners in scientifically authentic inquiry activities. We discuss the bioinformatics-related benefits and challenges that high school teachers and students face in the course of the implementation process, in light of previous studies and our own experience. Based on these lessons, we present a new approach for characterizing the questions embedded in bioinformatics teaching and learning units, based on three criteria: the type of domain-specific knowledge required to answer each question (declarative knowledge, procedural knowledge, strategic knowledge, situational knowledge), the scientific approach from which each question stems (biological, bioinformatics, a combination of the two) and the associated cognitive process dimension (remember, understand, apply, analyze, evaluate, create). We demonstrate the feasibility of this approach using a learning environment, which we developed for the high school level, and suggest some of its implications. This review sheds light on unique and critical characteristics related to broader integration of bioinformatics in secondary education, which are also relevant to the undergraduate level, and especially on curriculum design, development of suitable learning environments and teaching and learning processes.

Description: Next-generation sequencing (NGS) is increasingly being adopted as the backbone of biomedical research. With the commercialization of various affordable desktop sequencers, NGS will be reached by increasing numbers of cellular and molecular biologists, necessitating community consensus on bioinformatics protocols to tackle the exponential increase in quantity of sequence data. The current resources for NGS informatics are extremely fragmented. Finding a centralized synthesis is difficult. A multitude of tools exist for NGS data analysis; however, none of these satisfies all possible uses and needs. This gap in functionality could be filled by integrating different methods in customized pipelines, an approach helped by the open-source nature of many NGS programmes. Drawing from community spirit and with the use of the Wikipedia framework, we have initiated a collaborative NGS resource: The NGS WikiBook. We have collected a sufficient amount of text to incentivize a broader community to contribute to it. Users can search, browse, edit and create new content, so as to facilitate self-learning and feedback to the community. The overall structure and style for this dynamic material is designed for the bench biologists and non-bioinformaticians. The flexibility of online material allows the readers to ignore details in a first read, yet have immediate access to the information they need. Each chapter comes with practical exercises so readers may familiarize themselves with each step. The NGS WikiBook aims to create a collective laboratory book and protocol that explains the key concepts and describes best practices in this fast-evolving field.

Description: Phenology as a strategy for carbon optimality: a global model Biogeosciences Discussions, 10, 15107-15152, 2013 Author(s): S. Caldararu, D. W. Purves, and P. I. Palmer Phenology is essential to our understanding of biogeochemical cycles and the climate system. We develop a global mechanistic model of leaf phenology based on the hypothesis that phenology is a strategy for optimal carbon gain at the canopy level so that trees adjust leaf gains and losses in response to environmental factors such as light, temperature and soil moisture, to achieve maximum carbon assimilation. We fit this model to five years of satellite observations of leaf area index (LAI) using a Bayesian fitting algorithm. We show that our model is able to reproduce phenological patterns for all vegetation types and use it to explore variations in growing season length and the climate factors that limit leaf growth for different biomes. Phenology in wet tropical areas is limited by leaf age physiological constraints while at higher latitude leaf seasonality is limited by low temperature and light availability. Leaf growth in grassland regions is limited by water availability but often in combination with other factors. This model will advance the current understanding of phenology for ecosystem carbon models and our ability to predict future phenological behaviour.

Description: The genomes of related species contain valuable information on the history of the considered taxa. Great apes in particular exhibit variation of evolutionary patterns along their genomes. However, the great ape data also bring new challenges, such as the presence of incomplete lineage sorting and ancestral shared polymorphisms. Previous methods for genome-scale analysis are restricted to very few individuals or cannot disentangle the contribution of mutation rates and fixation biases. This represents a limitation both for the understanding of these forces as well as for the detection of regions affected by selection. Here, we present a new model designed to estimate mutation rates and fixation biases from genetic variation within and between species. We relax the assumption of instantaneous substitutions, modeling substitutions as mutational events followed by a gradual fixation. Hence, we straightforwardly account for shared ancestral polymorphisms and incomplete lineage sorting. We analyze genome-wide synonymous site alignments of human, chimpanzee, and two orangutan species. From each taxon, we include data from several individuals. We estimate mutation rates and GC-biased gene conversion intensity. We find that both mutation rates and biased gene conversion vary with GC content. We also find lineage-specific differences, with weaker fixation biases in orangutan species, suggesting a reduced historical effective population size. Finally, our results are consistent with directional selection acting on coding sequences in relation to exonic splicing enhancers.

Description: Genetic control of male or female gonad development displays between different groups of organisms a remarkable diversity of "master sex-determining genes" at the top of the genetic hierarchies, whereas downstream components surprisingly appear to be evolutionarily more conserved. Without much further studies, conservation of sequence has been equalized to conservation of function. We have used the medaka fish to investigate the generality of this paradigm. In medaka, the master male sex-determining gene is dmrt1bY , a highly conserved downstream regulator of sex determination in vertebrates. To understand its function in orchestrating the complex gene regulatory network, we have identified targets genes and regulated pathways of Dmrt1bY. Monitoring gene expression and interactions by transgenic fluorescent reporter fish lines, in vivo tissue-chromatin immunoprecipitation and in vitro gene regulation assays revealed concordance but also major discrepancies between mammals and medaka, notably amongst spatial, temporal expression patterns and regulations of the canonical Hedgehog and R-spondin/Wnt/Follistatin signaling pathways. Examination of Foxl2 protein distribution in the medaka ovary defined a new subpopulation of theca cells, where ovarian-type aromatase transcriptional regulation appears to be independent of Foxl2. In summary, these data show that the regulation of the downstream regulatory network of sex determination is less conserved than previously thought.

Description: Timing of sea ice retreat can alter phytoplankton community structure in the western Arctic Ocean Biogeosciences Discussions, 10, 15153-15180, 2013 Author(s): given_name prefix surname suffix, A. Fujiwara, T. Hirawake, K. Suzuki, I. Imai, and S.-I. Saitoh This study assesses the response of phytoplankton assemblages to recent climate change, especially with regard to the shrinking of sea ice in the northern Chukchi Sea of the western Arctic Ocean. Distribution patterns of phytoplankton groups in the late summers of 2008–2010 were analyzed based on HPLC pigment signatures and, the following four major algal groups were inferred via multiple regression and cluster analyses: prasinophytes, diatoms, haptophytes and dinoflagellates. A remarkable interannual difference in the distribution pattern of the groups was found in the northern basin area. Haptophytes dominated and dispersed widely in warm surface waters in 2008, whereas prasinophytes dominated in cold water in 2009 and 2010. A difference in the onset date of sea ice retreat was evident among years – the sea ice retreat in 2008 was 1–2 months earlier than in 2009 and 2010. The spatial distribution of early sea ice retreat matched the areas in which a shift in algal community composition was observed. Steel-Dwass's multiple comparison tests were used to assess the physical, chemical and biological parameters of the four clusters. We found a statistically significant difference in temperature between the haptophyte-dominated cluster and the other clusters, suggesting that the change in the phytoplankton communities was related to the earlier sea ice retreat in 2008 and the corollary increase in sea surface temperatures. Longer periods of open water during the summer, which are expected in the future, may affect food webs and biogeochemical cycles in the western Arctic due to shifts in phytoplankton community structure.

Description: Motivation: Pyrosequencing technology provides an important new approach to more extensively characterize diverse sequence populations and detect low frequency variants. However, the promise of this technology has been difficult to realize, as careful correction of sequencing errors is crucial to distinguish rare variants (~1%) in an infected host with high sensitivity and specificity. Results: We developed a new approach, referred to as Indel and Carryforward Correction (ICC), to cluster sequences without substitutions and locally correct only indel and carryforward sequencing errors within clusters to ensure that no rare variants are lost. ICC performs sequence clustering in the order of (i) homopolymer indel patterns only, (ii) indel patterns only and (iii) carryforward errors only, without the requirement of a distance cutoff value. Overall, ICC removed 93–95% of sequencing errors found in control datasets. On pyrosequencing data from a PCR fragment derived from 15 HIV-1 plasmid clones mixed at various frequencies as low as 0.1%, ICC achieved the highest sensitivity and similar specificity compared with other commonly used error correction and variant calling algorithms. Availability and implementation: Source code is freely available for download at http://indra.mullins.microbiol.washington.edu/ICC . It is implemented in Perl and supported on Linux, Mac OS X and MS Windows. Contact: jmullins@uw.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Tiki proteins appear to antagonize Wnt signalling pathway by acting as Wnt proteases, thereby affecting Wnt solubility by its amino-terminal cleavage. Tiki1 protease activity was shown to be metal ion-dependent and was inhibited by chelating agents and thus was tentatively proposed to be a metalloprotease. Nevertheless, Tiki proteins exhibit no detectable sequence similarity to previously described metalloproteases, but instead have been reported as being homologues of TraB proteins (Pfam ID: PF01963), a widely distributed family of unknown function and structure. Here, we show that Tiki proteins are members of a new superfamily of domains contained not just in TraB proteins, but also in erythromycin esterase (Pfam ID: PF05139), DUF399 (domain of unknown function 399; Pfam ID: PF04187) and MARTX toxins that contribute to host invasion and pathogenesis by bacteria. We establish the core fold of this enzymatic domain and its catalytic residues. Contact: luis.sanchezpulido@dpag.ox.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The research area metabolomics achieved tremendous popularity and development in the last couple of years. Owing to its unique interdisciplinarity, it requires to combine knowledge from various scientific disciplines. Advances in the high-throughput technology and the consequently growing quality and quantity of data put new demands on applied analytical and computational methods. Exploration of finally generated and analyzed datasets furthermore relies on powerful tools for data mining and visualization. Results: To cover and keep up with these requirements, we have created MeltDB 2.0, a next-generation web application addressing storage, sharing, standardization, integration and analysis of metabolomics experiments. New features improve both efficiency and effectivity of the entire processing pipeline of chromatographic raw data from pre-processing to the derivation of new biological knowledge. First, the generation of high-quality metabolic datasets has been vastly simplified. Second, the new statistics tool box allows to investigate these datasets according to a wide spectrum of scientific and explorative questions. Availability: The system is publicly available at https://meltdb.cebitec.uni-bielefeld.de . A login is required but freely available. Contact: nkessler@cebitec.uni-bielefeld.de

Description: Motivation: High - throughput next - generation sequencing technologies enable increasingly fast and affordable sequencing of genomes and transcriptomes, with a broad range of applications. The quality of the sequencing data is crucial for all applications. A significant portion of the data produced contains errors, and ever more efficient error correction programs are needed. Results: We propose RACER (Rapid and Accurate Correction of Errors in Reads), a new software program for correcting errors in sequencing data. RACER has better error-correcting performance than existing programs, is faster and requires less memory. To support our claims, we performed extensive comparison with the existing leading programs on a variety of real datasets. Availability: RACER is freely available for non-commercial use at www.csd.uwo.ca/~ilie/RACER/ . Contact: ilie@csd.uwo.ca Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Interactions between various types of molecules that regulate crucial cellular processes are extensively investigated by high-throughput experiments and require dedicated computational methods for the analysis of the resulting data. In many cases, these data can be represented as a bipartite graph because it describes interactions between elements of two different types such as the influence of different experimental conditions on cellular variables or the direct interaction between receptors and their activators/inhibitors. One of the major challenges in the analysis of such noisy datasets is the statistical evaluation of the relationship between any two elements of the same type. Here, we present SICOP (significant co-interaction patterns), an implementation of a method that provides such an evaluation based on the number of their common interaction partners, their so-called co-interaction. This general network analytic method, proved successful in diverse fields, provides a framework for assessing the significance of this relationship by comparison with the expected co-interaction in a suitable null model of the same bipartite graph. SICOP takes into consideration up to two distinct types of interactions such as up- or downregulation. The tool is written in Java and accepts several common input formats and supports different output formats, facilitating further analysis and visualization. Its key features include a user-friendly interface, easy installation and platform independence. Availability: The software is open source and available at cna.cs.uni-kl.de/SICOP under the terms of the GNU General Public Licence (version 3 or later). Contact: agnes.horvat@iwr.uni-heidelberg.de or zweig@cs.uni-kl.de

Description: : Molecular recognition features (MoRFs) are small, intrinsically disordered regions in proteins that undergo a disorder-to-order transition on binding to their partners. MoRFs are involved in protein–protein interactions and may function as the initial step in molecular recognition. The aim of this work was to collect, organize and store all membrane proteins that contain MoRFs. Membrane proteins constitute ~30% of fully sequenced proteomes and are responsible for a wide variety of cellular functions. MoRFs were classified according to their secondary structure, after interacting with their partners. We identified MoRFs in transmembrane and peripheral membrane proteins. The position of transmembrane protein MoRFs was determined in relation to a protein’s topology. All information was stored in a publicly available mySQL database with a user-friendly web interface. A Jmol applet is integrated for visualization of the structures. mpMoRFsDB provides valuable information related to disorder-based protein–protein interactions in membrane proteins. Availability: http://bioinformatics.biol.uoa.gr/mpMoRFsDB Contact: shamodr@biol.uoa.gr

Description: : The signaling Petri net (SPN) simulator, designed to provide insights into the trends of molecules’ activity levels in response to an external stimulus, contributes to the systems biology necessity of analyzing the dynamics of large-scale cellular networks. Implemented into the freely available software, BioLayout Express 3D , the simulator is publicly available and easy to use, provided the input files are prepared in the GraphML format, typically using the network editing software, yEd, and standards specific to the software. However, analysis of complex networks represented using other systems biology formatting languages (on which popular software, such as CellDesigner and Cytoscape, are based) requires manual manipulation, a step that is prone to error and limits the use of the SPN simulator in BioLayout Express 3D . To overcome this, we present a Cytoscape plug-in that enables users to automatically convert networks for analysis with the SPN simulator from the standard systems biology markup language. The automation of this step opens the SPN simulator to a far larger user group than has previously been possible. Availability and implementation: Distributed under the GNU General Public License Version 3 at http://apps.cytoscape.org/apps/spnconverter . Contact: christine@picb.ac.cn

Description: Motivation: Conformational diversity is a key concept in the understanding of different issues related with protein function such as the study of catalytic processes in enzymes, protein-protein recognition, protein evolution and the origins of new biological functions. Here, we present a database of proteins with different degrees of conformational diversity. Conformational Diversity of Native State (CoDNaS) is a redundant collection of three-dimensional structures for the same protein derived from protein data bank. Structures for the same protein obtained under different crystallographic conditions have been associated with snapshots of protein dynamism and consequently could characterize protein conformers. CoDNaS allows the user to explore global and local structural differences among conformers as a function of different parameters such as presence of ligand, post-translational modifications, changes in oligomeric states and differences in pH and temperature. Additionally, CoDNaS contains information about protein taxonomy and function, disorder level and structural classification offering useful information to explore the underlying mechanism of conformational diversity and its close relationship with protein function. Currently, CoDNaS has 122 122 structures integrating 12 684 entries, with an average of 9.63 conformers per protein. Availability: The database is freely available at http://www.codnas.com.ar/ . Contact: gusparisi@gmail.com

Description: Motivation: Recent experimental advancements allow determining positions of nucleosomes for complete genomes. However, the resulting nucleosome occupancy maps are averages of heterogeneous cell populations. Accordingly, they represent a snapshot of a dynamic ensemble at a single time point with an overlay of many configurations from different cells. To study the organization of nucleosomes along the genome and to understand the mechanisms of nucleosome translocation, it is necessary to retrieve features of specific conformations from the population average. Results: Here, we present a method for identifying non-overlapping nucleosome configurations that combines binary-variable analysis and a Monte Carlo approach with a simulated annealing scheme. In this manner, we obtain specific nucleosome configurations and optimized solutions for the complex positioning patterns from experimental data. We apply the method to compare nucleosome positioning at transcription factor binding sites in different mouse cell types. Our method can model nucleosome translocations at regulatory genomic elements and generate configurations for simulations of the spatial folding of the nucleosome chain. Availability: Source code, precompiled binaries, test data and a web-based test installation are freely available at http://bioinformatics.fh-stralsund.de/nucpos/ Contact: gero.wedemann@fh-stralsund.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Wolbachia , endosymbiotic bacteria of the order Rickettsiales, are widespread in arthropods but also present in nematodes. In arthropods, A and B supergroup Wolbachia are generally associated with distortion of host reproduction. In filarial nematodes, including some human parasites, multiple lines of experimental evidence indicate that C and D supergroup Wolbachia are essential for the survival of the host, and here the symbiotic relationship is considered mutualistic. The origin of this mutualistic endosymbiosis is of interest for both basic and applied reasons: How does a parasite become a mutualist? Could intervention in the mutualism aid in treatment of human disease? Correct rooting and high-quality resolution of Wolbachia relationships are required to resolve this question. However, because of the large genetic distance between Wolbachia and the nearest outgroups, and the limited number of genomes so far available for large-scale analyses, current phylogenies do not provide robust answers. We therefore sequenced the genome of the D supergroup Wolbachia endosymbiont of Litomosoides sigmodontis , revisited the selection of loci for phylogenomic analyses, and performed a phylogenomic analysis including available complete genomes (from isolates in supergroups A, B, C, and D). Using 90 orthologous genes with reliable phylogenetic signals, we obtained a robust phylogenetic reconstruction, including a highly supported root to the Wolbachia phylogeny between a (A + B) clade and a (C + D) clade. Although we currently lack data from several Wolbachia supergroups, notably F, our analysis supports a model wherein the putatively mutualist endosymbiotic relationship between Wolbachia and nematodes originated from a single transition event.

Description: Many insects rely on bacterial symbionts with tiny genomes specialized for provisioning nutrients lacking in host diets. Xylem sap and phloem sap are both deficient as insect diets, but differ dramatically in nutrient content, potentially affecting symbiont genome evolution. For sap-feeding insects, sequenced symbiont genomes are available only for phloem-feeding examples from the suborder Sternorrhyncha and xylem-feeding examples from the suborder Auchenorrhyncha, confounding comparisons. We sequenced genomes of the obligate symbionts, Sulcia muelleri and Nasuia deltocephalinicola , of the phloem-feeding pest insect, Macrosteles quadrilineatus (Auchenorrhyncha: Cicadellidae). Our results reveal that Nasuia- ALF has the smallest bacterial genome yet sequenced (112 kb), and that the Sulcia- ALF genome (190 kb) is smaller than that of Sulcia in other insect lineages. Together, these symbionts retain the capability to synthesize the 10 essential amino acids, as observed for several symbiont pairs from xylem-feeding Auchenorrhyncha. Nasuia retains genes enabling synthesis of two amino acids, DNA replication, transcription, and translation. Both symbionts have lost genes underlying ATP synthesis through oxidative phosphorylation, possibly as a consequence of the enriched sugar content of phloem. Shared genomic features, including reassignment of the UGA codon from Stop to tryptophan, and phylogenetic results suggest that Nasuia -ALF is most closely related to Zinderia , the betaproteobacterial symbiont of spittlebugs. Thus, Nasuia / Zinderia and Sulcia likely represent ancient associates that have co-resided in hosts since the divergence of leafhoppers and spittlebugs 〉200 Ma, and possibly since the origin of the Auchenorrhyncha, 〉260 Ma.

Description: It has been argued that scale is the central problem in ecology ( Levin, 1992 ). Studies on carbon cycles and global climate change, the current major themes in modern ecology, require the interfacing of phenomena that occur on different scales of space, time, and ecological organization. For several decades, tremendous efforts have been made to reveal the general patterns of, and the mechanisms for the global carbon cycles. However, many uncertainties remain, particularly on local to regional scales. To reduce these uncertainties, regional collaborations across the board of nations are required.

Description: Aims Boreal forest is the largest and contains the most soil carbon among global terrestrial biomes. Soil respiration during the prolonged winter period may play an important role in the carbon cycles in boreal forests. This study aims to explore the characteristics of winter soil respiration in the boreal forest and to show how it is regulated by environmental factors, such as soil temperature, soil moisture and snowpack. Methods Soil respiration in an old-growth larch forest ( Larix gmelinii Ruppr.) in Northeast China was intensively measured during the winter soil-freezing process in 2011 using an automated soil CO 2 flux system. The effects of soil temperature, soil moisture and thin snowpack on soil respiration and its temperature sensitivity were investigated. Important Findings Total soil respiration and heterotrophic respiration both showed a declining trend during the observation period, and no significant difference was found between soil respiration and heterotrophic respiration until the snowpack exceeded 20cm. Soil respiration was exponentially correlated with soil temperature and its temperature sensitivity (Q 10 value) for the entire measurement duration was 10.5. Snow depth and soil moisture both showed positive effects on the temperature sensitivity of soil respiration. Based on the change in the Q 10 value, we proposed a ‘freeze–thaw critical point’ hypothesis, which states that the Q 10 value above freeze–thaw critical point is much higher than that below it (16.0 vs . 3.5), and this was probably regulated by the abrupt change in soil water availability during the soil-freezing process. Our findings suggest interactive effects of multiple environmental factors on winter soil respiration and recommend adopting the freeze–thaw critical point to model soil respiration in a changing winter climate.

Description: Aims Clear-cutting is a common forest management practice, especially in subtropical China. However, the potential ecological consequences of clear-cutting remain unclear. In particular, the effect of clear-cutting on soil processes, such as the carbon cycle, has not been quantified in subtropical forests. Here, we investigated the response of soil respiration (Rs) to clear-cutting during a 12-month period in a subtropical forest in eastern China. Methods We randomly selected four clear-cut (CC) plots and four corresponding undisturbed forest (UF) plots. Measurements of Rs were made at monthly time points and were combined with continuous climatic measurements in both CC and UF. Daily Rs was estimated by interpolating data with an exponential model dependent on soil temperature. Daily Rs was cumulated to annual Rs estimates. Important Findings In the first year after clear-cutting, annual estimates of Rs in CC (508±23g C m –2 yr –1 ) showed no significant difference to UF plots (480±12g C m –2 yr –1 ). During the summer, soil temperatures were usually higher, whereas the soil volumetric water content was lower in CC than in UF plots. The long-term effects of clear-cutting on Rs are not significant, although there might be effects during the first several months after clear-cutting. Compared with previous work, this pattern was more pronounced in our subtropical forest than in the temperate and boreal forests that have been studied by others. With aboveground residuals off-site after clear-cutting, our results indicate that the stimulation of increasing root debris, as well as environmental changes, will not lead to a significant increase in Rs. In addition, long-term Rs will not show a significant decrease from the termination of root respiration, and this observation might be because of the influence of fast-growing vegetation after clear-cutting in situ .

Description: Aims Understanding carbon (C) and nitrogen (N) dynamics and their dependence on the stand density of an even-aged, mature forest provides knowledge that is important for forest management. This study investigated the differences in ecosystem total C and N storage and flux between a low-density stand (LD) and a high-density stand (HD) and examined the effects of stand density on aboveground net primary productivity (ANPP), total belowground C allocation (TBCA) and net ecosystem production (NEP) in a naturally regenerated, 65- to 75-year-old Pinus densiflora S. et Z. forest. Methods LD (450 trees ha –1 ) and HD (842 trees ha –1 ) were established in an even-aged, mature P. densiflora forest in September 2006. The forest had been naturally regenerated following harvesting, and the stand density was naturally maintained without any artificial management such as thinning. The diameter at breast height (DBH ≥ 5.0cm) of all live stems within the stands was measured yearly from 2007 to 2011. To compare C and N storage and fluxes in LD and HD, C and N pools in aboveground and belowground biomass, the forest floor, coarse woody debris (CWD) and soil; soil CO 2 efflux ( R S ); autotrophic respiration ( R A ); litter production; and soil N availability were measured. Further, ANPP, TBCA and NEP were estimated from plot-based measurement data. Important Findings Ecosystem C (Mg C ha –1 ) and N (Mg N ha –1 ) storage was, respectively, 173.0±7.3 (mean ± SE) and 4.69±0.30 for LD and 162±11.8 and 4.08±0.18 for HD. There were no significant differences in C and N storage in the ecosystem components, except for soils, between the two stands. In contrast, there were significant differences in aboveground ANPP and TBCA between the two stands ( P 〈 0.05). Litterfall, biomass increment and R S were major C flux components with values of, respectively, 3.89, 3.74 and 9.07 Mg C ha –1 year –1 in LD and 3.15, 2.94 and 7.06 Mg C ha –1 year –1 in HD. Biometric-based NEP (Mg C ha –1 year –1 ) was 4.18 in LD and 5.50 in HD. Although the even-aged, mature P. densiflora forest had similar C and N allocation patterns, it showed different C and N dynamics depending on stand density. The results of the current study will be useful for elucidating the effects of stand density on C and N storage and fluxes, which are important issues in managing natural mature forest ecosystems.

Description: Aims Tropical forest plays a key role in global C cycle; however, there are few studies on the C budget in the tropical rainforests in Asia. This study aims to (i) reveal the seasonal patterns of total soil respiration ( R T ), litter respiration ( R L ) and soil respiration without surface organic litter ( R NL ) in the primary and secondary Asian tropical mountain rainforests and (ii) quantify the effects of soil temperature, soil moisture and substrate availability on soil respiration. Methods The seasonal dynamics of soil CO 2 efflux was measured by an automatic chamber system (Li-8100), within the primary and secondary tropical mountain rainforests located at the Jianfengling National Reserve in Hainan Island, China. The litter removal treatment was used to assess the contribution of litter to belowground CO 2 production. Important Findings The annual R T was higher in the primary forest (16.73±0.87 Mg C ha –1 ) than in the secondary forest (15.10±0.26 Mg C ha –1 ). The rates of R T , R NL and R L were all significantly higher in the hot and wet season (May–October) than those in the cool and dry season (November–April). Soil temperature at 5cm depth could explain 55–61% of the seasonal variation in R T , and the temperature sensitivity index ( Q 10 ) ranked by R L ( Q 10 = 3.39) 〉 R T (2.17) 〉 R NL (1.76) in the primary forest and by R L (4.31) 〉 R T (1.86) 〉 R NL (1.58) in the secondary forest. The contribution of R L to R T was 22–23%, while litter input and R T had 1 month time lag. In addition, the seasonal variation of R T was mainly determined by soil temperature and substrate availability. Our findings suggested that global warming and increased substrate availability are likely to cause considerable losses of soil C in the tropical forests.

Description: Aims Understanding of the ecophysiological dynamics of forest canopy photosynthesis and its spatial and temporal scaling is crucial for revealing ecological response to climate change. Combined observations and analyses of plant ecophysiology and optical remote sensing would enable us to achieve these studies. In order to examine the utility of spectral vegetation indices (VIs) for assessing ecosystem-level photosynthesis, we investigated the relationships between canopy-scale photosynthetic productivity and canopy spectral reflectance over seasons for 5 years in a cool, temperate deciduous broadleaf forest at ‘Takayama’ super site in central Japan. Methods Daily photosynthetic capacity was assessed by in situ canopy leaf area index (LAI), (LAI x V cmax [single-leaf photosynthetic capacity]), and the daily maximum rate of gross primary production (GPP max ) was estimated by an ecosystem carbon cycle model. We examined five VIs: normalized difference vegetation index (NDVI), enhanced vegetation index (EVI), green–red vegetation index (GRVI), chlorophyll index (CI) and canopy chlorophyll index (CCI), which were obtained by the in situ measurements of canopy spectral reflectance. Important Findings Our in situ observation of leaf and canopy characteristics, which were analyzed by an ecosystem carbon cycling model, revealed that their phenological changes are responsible for seasonal and interannual variations in canopy photosynthesis. Significant correlations were found between the five VIs and canopy photosynthetic capacity over the seasons and years; four of the VIs showed hysteresis-type relationships and only CCI showed rather linear relationship. Among the VIs examined, we applied EVI–GPP max relationship to EVI data obtained by Moderate Resolution Imaging Spectroradiometer to estimate the temporal and spatial variation in GPP max over central Japan. Our findings would improve the accuracy of satellite-based estimate of forest photosynthetic productivity in fine spatial and temporal resolutions, which are necessary for detecting any response of terrestrial ecosystem to meteorological fluctuations.

Description: Aims This study aimed to develop radial growth models and to predict the potential spatial distribution of Pinus densiflora (Japanese red pine) and Quercus spp. (Oaks) in South Korea, considering topographic and climatic factors. Methods We used a dataset of diameter at breast height and radial growth estimates of individual trees, topographic and climatic factors in systematic sample plots distributed over the whole of South Korea. On the basis that radial growth is attributed primarily to tree age, we developed a radial growth model employing tree age as an explanatory variable. We estimated standard growth (SG), defined as radial growth of the tree at age 30, to eliminate the influence of tree age on radial growth. In addition, SG estimates including the Topographic Wetness Index, temperature and precipitation were calculated by the Generalized Additive Model. Important Findings As a result of variogram analysis of SG, we found spatial autocorrelation between SG, topographic and climatic factors. Incremental temperature had negative impacts on radial growth of P. densiflora and positive impacts on that of Quercus spp. Precipitation was associated with positive effects on both tree species. Based on the model, we found that radial growth of P. densiflora would be more vulnerable than that of Quercus spp. to climatic factors. Through simulation with the radial growth model, it was predicted that P. densiflora stands would be gradually replaced with Quercus spp. stands in eastern coastal and southern regions of South Korea in the future. The models developed in this study will be helpful for understanding the impact of climatic factors on tree growth and for predicting changes in distribution of P. densiflora and Quercus spp. due to climate change in South Korea.

Description: Aims Vast grasslands on the Tibetan Plateau are almost all under livestock grazing. It is unclear, however, what is the role that the grazing will play in carbon cycle of the grassland under future climate warming. We found in our previous study that experimental warming can shift the optimum temperature of saturated photosynthetic rate into higher temperature in alpine plants. In this study, we proposed and tested the hypothesis that livestock grazing would alter the warming effect on photosynthetic and respiration through changing physical environments of grassland plants. Methods Experimental warming was carried by using an infrared heating system to increase the air temperature by 1.2 and 1.7°C during the day and night, respectively. The warming and ambient temperature treatments were crossed over to the two grazing treatments, grazing and un-grazed treatments, respectively. To assess the effects of grazing and warming, we examined photosynthesis, dark respiration, maximum rates of the photosynthetic electron transport ( J max ), RuBP carboxylation ( V cmax ) and temperature sensitivity of respiration Q 10 in Gentiana straminea , an alpine species widely distributed on the Tibetan grassland. Leaf morphological and chemical properties were also examined to understand the physiological responses. Important findings 1) Light-saturated photosynthetic rate ( A max ) of G. straminea showed similar temperature optimum at around 16°C in plants from all experimental conditions. Experimental warming increased A max at all measuring temperatures from 10 to 25°C, but the positive effect of the warming occurred only in plants grown under the un-grazed conditions. Under the same measuring temperature, A max was significantly higher in plants from the grazed than the un-grazed condition. 2) There was significant crossing effect of warming and grazing on the temperature sensitivity ( Q 10 ) of leaf dark respiration. Under the un-grazed condition, plants from the warming treatment showed lower respiration rate but similar Q 10 in comparison with plants from the ambient temperature treatment. However, under the grazed condition Q 10 was significantly lower in plants from the warming than the ambient treatment. 3) The results indicate that livestock grazing can alter the warming effects on leaf photosynthesis and temperature sensitivity of leaf dark respiration through changing physical environment of the grassland plants. The study suggests for the first time that grazing effects should be taken into account in predicting global warming effects on photosynthesis and respiration of plants in those grasslands with livestock grazing.

Description: Uptake of phytodetritus by benthic foraminifera under oxygen depletion at the Indian Margin (Arabian Sea) Biogeosciences Discussions, 10, 15305-15335, 2013 Author(s): A. J. Enge, U. Witte, M. Kucera, and P. Heinz Benthic foraminifera in sediments on the Indian margin of the Arabian Sea where the oxygen minimum zone (OMZ) impinges on the continental slope are exposed to particularly severe levels of oxygen depletion. Food supply for the benthic community is high but delivered in distinct pulses during upwelling and water mixing events associated with summer and winter monsoon periods. In order to investigate the response by benthic foraminifera to such pulsed food delivery under oxygen concentrations of less than 0.1 mL L −1 (4.5 μmol L −1 ), an in situ isotope labeling experiment ( 13 C, 15 N) was performed at the western continental slope of India at 540 m water depth (OMZ core region). The assemblage of living foraminifera (〉125 μm) in the uppermost centimeter at this depth is characterized by an unexpectedly high population density of 3982 ind. 10 cm −2 and a strong dominance by few calcareous species. For the experiment, we concentrated on the nine most abundant taxa, which constitute 93% of the entire foraminifera population at 0–1 cm sediment depth. Increased concentrations of 13 C and 15 N in the cytoplasm indicate that all investigated taxa took up the labeled phytodetritus during the 4 day experimental phase. In total, these nine species had assimilated 113.8 mg C m −2 (17.5% of the total added carbon). The uptake of nitrogen by the three most abundant taxa ( Bolivina aff. B. dilatata , Cassidulina sp., Bulimina gibba ) was 2.7 mg N m −2 (2% of the total added nitrogen) and showed the successful application of 15 N as tracer in foraminiferal studies. The short-term response to the offered phytodetritus varied largely among foraminiferal species with Uvigerina schwageri being by far the most important species in short-term processing whereas the most abundant taxa Bolivina aff. B. dilatata and Cassidulina sp. showed comparably low uptake of the offered food. We suggest that the observed species-specific differences are related to individual biomass of species and to specific feeding preferences. The high numbers of living foraminifera and their rapid response to deposited fresh phytodetritus demonstrate the importance of foraminifera in short-term carbon cycling under oxygen-depleted conditions. We propose that foraminifera at the studied site benefit from unique adaptations in their metabolisms to nearly anoxic conditions as well as from the exclusion of macrofauna and the resulting relaxation of competition for food and low predation pressure.

Description: Summertime canopy albedo is sensitive to forest thinning Biogeosciences Discussions, 10, 15373-15414, 2013 Author(s): J. Otto, D. Berveiller, F.-M. Bréon, N. Delpierre, G. Geppert, A. Granier, W. Jans, A. Knohl, A. Kuusk, B. Longdoz, E. Moors, M. Mund, B. Pinty, M.-J. Schelhaas, and S. Luyssaert Despite an emerging body of literature linking canopy albedo to forest management, understanding of the process is still fragmented. We combined a stand-level forest gap model with a canopy radiation transfer model and satellite-derived model parameters to quantify the effects of forest thinning, that is removing trees at a certain time during the forest rotation, on summertime canopy albedo. The effects of different forest species (pine, beech, oak) and four thinning strategies (light to intense thinning regimes) were examined. During stand establishment, summertime canopy albedo is driven by tree species. In the later stages of stand development, the effect of tree species on summertime canopy albedo decreases in favour of an increasing influence of forest thinning on summertime canopy albedo. These trends continue until the end of the rotation where thinning explains up to 50% of the variance in near-infrared canopy albedo and up to 70% of the variance in visible canopy albedo. More intense thinning lowers the summertime shortwave albedo in the canopy by as much as 0.02 compared to unthinned forest. The structural changes associated with forest thinning can be described by the change in LAI in combination with crown volume. However, forests with identical canopy structure can have different summertime albedo values due to their location: the further north a forest is situated, the more the solar zenith angle increases and thus the higher is the summertime canopy albedo, independent of the wavelength. Despite the increase of absolute summertime canopy albedo values with latitude, the difference in canopy albedo between managed and unmanaged forest decreases with increasing latitude. Forest management thus strongly altered summertime forest albedo.

Description: We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs , encoding the acetyl coenzyme A synthetase, in Escherichia coli . We confirm most of the known genetic regulators, including CRP–cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA , sucB and lpdA , encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC . The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.

Description: Tandem repeats (TRs) are often present in proteins with crucial functions, responsible for resistance, pathogenicity and associated with infectious or neurodegenerative diseases. This motivates numerous studies of TRs and their evolution, requiring accurate multiple sequence alignment. TRs may be lost or inserted at any position of a TR region by replication slippage or recombination, but current methods assume fixed unit boundaries, and yet are of high complexity. We present a new global graph-based alignment method that does not restrict TR unit indels by unit boundaries. TR indels are modeled separately and penalized using the phylogeny-aware alignment algorithm. This ensures enhanced accuracy of reconstructed alignments, disentangling TRs and measuring indel events and rates in a biologically meaningful way. Our method detects not only duplication events but also all changes in TR regions owing to recombination, strand slippage and other events inserting or deleting TR units. We evaluate our method by simulation incorporating TR evolution, by either sampling TRs from a profile hidden Markov model or by mimicking strand slippage with duplications. The new method is illustrated on a family of type III effectors, a pathogenicity determinant in agriculturally important bacteria Ralstonia solanacearum. We show that TR indel rate variation contributes to the diversification of this protein family.

Description: A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.

Description: The synthesis of protein from messenger RNA during translation is a highly dynamic process that plays a key role in controlling the efficiency and fidelity of genome-wide protein expression. The availability of aminoacylated transfer RNA (tRNA) is a major factor influencing the speed of ribosomal movement, which depending on codon choices, varies considerably along a transcript. Furthermore, it has been shown experimentally that tRNA availability can vary significantly under different growth and stress conditions, offering the cell a way to adapt translational dynamics across the genome. Existing models of translation have neglected fluctuations of tRNA pools, instead assuming fixed tRNA availabilities over time. This has lead to an incomplete understanding of this process. Here, we show for the entire Escherichia coli genome how and to what extent translational speed profiles, which capture local aspects of translational elongation, respond to measured shifts in tRNA availability. We find that translational profiles across the genome are affected to differing degrees, with genes that are essential or related to fundamental processes such as translation, being more robust than those linked to regulation. Furthermore, we reveal how fluctuating tRNA availability influences profiles of specific sequences known to play a significant role in translational control of gene expression.

Description: Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap .

Description: Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.

Description: One of the obstacles hindering a better understanding of cancer is its heterogeneity. However, computational approaches to model cancer heterogeneity have lagged behind. To bridge this gap, we have developed a new probabilistic approach that models individual cancer cases as mixtures of subtypes. Our approach can be seen as a meta-model that summarizes the results of a large number of alternative models. It does not assume predefined subtypes nor does it assume that such subtypes have to be sharply defined. Instead given a measure of phenotypic similarity between patients and a list of potential explanatory features, such as mutations, copy number variation, microRNA levels, etc., it explains phenotypic similarities with the help of these features. We applied our approach to Glioblastoma Multiforme (GBM). The resulting model Prob_GBM, not only correctly inferred known relationships but also identified new properties underlining phenotypic similarities. The proposed probabilistic framework can be applied to model relations between similarity of gene expression and a broad spectrum of potential genetic causes.

Description: Estrogen regulates over a thousand genes, with an equal number of them being induced or repressed. The distinct mechanisms underlying these dual transcriptional effects remain largely unknown. We derived comprehensive views of the transcription machineries assembled at estrogen-responsive genes through integrating multiple types of genomic data. In the absence of estrogen, the majority of genes formed higher-order chromatin structures, including DNA loops tethered to protein complexes involving RNA polymerase II (Pol II), estrogen receptor alpha (ERα) and ERα-pioneer factors. Genes to be ‘repressed’ by estrogen showed active transcription at promoters and throughout the gene bodies; genes to be ‘induced’ exhibited active transcription initiation at promoters, but with transcription paused in gene bodies. In the presence of estrogen, the majority of estrogen-induced genes retained the original higher-order chromatin structures, whereas most estrogen-repressed genes underwent a chromatin reconfiguration. For estrogen-induced genes, estrogen enhances transcription elongation, potentially through recruitment of co-activators or release of co-repressors with unique roles in elongation. For estrogen-repressed genes, estrogen treatment leads to chromatin structure reconfiguration, thereby disrupting the originally transcription-efficient chromatin structures. Our in silico studies have shown that estrogen regulates gene expression, at least in part, through modifying previously assembled higher-order complexes, rather than by facilitating de novo assembly of machineries.

Description: The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1–MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.

Description: Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene ( CYP1A1 ) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene ( CYP1B1 ) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

Description: TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB–TFIIIC complexes in Saccharomyces cerevisiae , obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed ‘bootprints’. Global analysis reveals that the TFIIIB–TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site ( ETC10 ). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.

Description: The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.

Description: Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo , whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo . These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.

Description: Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe . Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.

Description: In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1 . Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.

Description: Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies 〉4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5–22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1 . This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes.

Description: The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

Description: RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans -splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre– trans -splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans -splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv , illustrating that a given PTM can be suitable for a variety of mutations.

Description: Oxygen minimum zone of the open Arabian Sea: variability of oxygen and nitrite from daily to decadal time scales Biogeosciences Discussions, 10, 15455-15517, 2013 Author(s): K. Banse, S. W. A. Naqvi, P. V. Narvekar, J. R. Postel, and D. A. Jayakumar The oxygen minimum zone (OMZ) of the Arabian Sea is the thickest of the three oceanic OMZs, which is of global biogeochemical significance because of denitrification in the upper part leading to N 2 and N 2 O production. The residence time of the OMZ water is believed to be less than a decade. The upper few hundred meters of this zone are nearly anoxic but non-sulfidic and still support animal (metazoan) pelagic life, possibly as a result of episodic injections of O 2 by physical processes. The very low O 2 values obtained with the new STOX sensor in the eastern tropical South Pacific probably also characterize the Arabian Sea OMZ, but there is no apparent reason as to why the temporal trends of the historic data should not hold. We report on discrete measurements of dissolved O 2 and NO 2 - , besides temperature and salinity, made between 1959 and 2004 well below the tops of the sharp pycno- and oxyclines near 150, 200, 300, 400, and 500 m depth. We assemble nearly all O 2 determinations (originally, 849 values, 695 in the OMZ) by the visual endpoint detection of the iodometric Winkler procedure, which in our data base yields about 0.04 mL L −1 (∼2 μM) O 2 above the endpoint from modern automated titration methods. We find 632 values acceptable (480 from 150 stations in the OMZ). The data are grouped in zonally-paired boxes of 1° lat. and 2° long. centered at 8°, 10°, 12°, 15°, 18°, 20°, and 21° N along 65° E and 67° E. The latitudes of 8–12° N, outside the OMZ, are only treated in passing. The principal results are as follows: (1) an O 2 climatology for the upper OMZ reveals a marked seasonality at 200 to 500 m depth with O 2 levels during the northeast monsoon and spring intermonsoon season elevated over those during the southwest monsoon season (median difference, 0.08 mL L −1 [3.5 μM]). The medians of the slopes of the seasonal regressions of O 2 on year for the NE and SW monsoon seasons are −0.0043 and −0.0019 mL L −1 a −1 , respectively (−0.19 and −0.08 μM a −1 ; n = 10 and 12, differing at p = 0.01); (2) four decades of statistically significant decreases of O 2 between 15° and 20° N but a trend to a similar increase near 21° N are observed. The balance of the mechanisms that more or less annually maintain the O 2 levels are still uncertain. At least between 300 and 500 m the annual reconstitution of the decrease is inferred to be due to lateral, isopycnal re-supply of O 2 , while at 200 (250?) m it is diapycnal, most likely by eddies. Similarly, recent models show large vertical advection of O 2 well below the pycno- cum -oxycline. The spatial (within drift stations) and temporal (daily) variability in hydrography and chemistry is large also below the principal pycnocline. The seasonal change of hydrography is considerable even at 500 m. There is no trend in the redox environment for a quarter of a century at a GEOSECS station near 20° N. In the entire OMZ the slopes on year within seasons for the quite variable NO 2 - (taken as an indicator of active denitrification) do not show a clear pattern. Also, future O 2 or nutrient budgets for the OMZ should not be based on single cruises or sections obtained during one season only. Steady state cannot be assumed any longer for the intermediate layers of the central Arabian Sea.

Description: Thanks to the microarray technology, our understanding of transcriptome evolution at the genome level has been considerably advanced in the past decade. Yet, further investigation was challenged by several technical limitations of this technology. Recent innovation of next-generation sequencing, particularly the invention of RNA-seq technology, has shed insightful lights on resolving this problem. Though a number of statistical and computational methods have been developed to analyze RNA-seq data, the analytical framework specifically designed for evolutionary genomics remains an open question. In this article we develop a new method for estimating the genome expression distance from the RNA-seq data, which has explicit interpretations under the model of gene expression evolution. Moreover, this distance measure takes the data overdispersion, gene length variation, and sequencing depth variation into account so that it can be applied to multiple genomes from different species. Using mammalian RNA-seq data as example, we demonstrated that this expression distance is useful in phylogenomic analysis.

Description: The control of RNA splicing is often modulated by exonic motifs near splice sites. Chief among these are exonic splice enhancers (ESEs). Well-described ESEs in mammals are purine rich and cause predictable skews in codon and amino acid usage toward exonic ends. Looking across species, those with relatively abundant intronic sequence are those with the more profound end of exon skews, indicative of exonization of splice site recognition. To date, the only intron-rich species that have been analyzed are mammals, precluding any conclusions about the likely ancestral condition. Here, we examine the patterns of codon and amino acid usage in the vicinity of exon–intron junctions in the brown alga Ectocarpus siliculosus , a species with abundant large introns, known SR proteins, and classical splice sites. We find that amino acids and codons preferred/avoided at both 3' and 5' ends in Ectocarpus , of which there are many, tend, on average, to also be preferred/avoided at the same exon ends in humans. Moreover, the preferences observed at the 5' ends of exons are largely the same as those at the 3' ends, a symmetry trend only previously observed in animals. We predict putative hexameric ESEs in Ectocarpus and show that these are purine rich and that there are many more of these identified as functional ESEs in humans than expected by chance. These results are consistent with deep phylogenetic conservation of SR protein binding motifs. Assuming codons preferred near boundaries are "splice optimal" codons, in Ectocarpus , unlike Drosophila, splice optimal and translationally optimal codons are not mutually exclusive. The exclusivity of translationally optimal and splice optimal codon sets is thus not universal.

Description: Autonomous retrotransposons lacking long terminal repeats (LTR) account for much of the variation in genome size and structure among vertebrates. Mammalian genomes contain hundreds of thousands of non-LTR retrotransposon copies, mostly resulting from the amplification of a single clade known as L1. The genomes of teleost fish and squamate reptiles contain a much more diverse array of non-LTR retrotransposon families, whereas copy number is relatively low. The majority of non-LTR retrotransposon insertions in nonmammalian vertebrates also appear to be very recent, suggesting strong purifying selection limits the accumulation of non-LTR retrotransposon copies. It is however unclear whether this turnover model, originally proposed in Drosophila , applies to nonmammalian vertebrates. Here, we studied the population dynamics of L1 in the green anole lizard ( Anolis carolinensis ). We found that although most L1 elements are recent in this genome, truncated insertions accumulate readily, and many are fixed at both the population and species level. In contrast, full-length L1 insertions are found at lower population frequencies, suggesting that the turnover model only applies to longer L1 elements in Anolis . We also found that full-length L1 inserts are more likely to be fixed in populations of small effective size, suggesting that the strength of purifying selection against deleterious alleles is highly dependent on host demographic history. Similar mechanisms seem to be controlling the fate of non-LTR retrotransposons in both Anolis and teleostean fish, which suggests that mammals have considerably diverged from the ancestral vertebrate in terms of how they interact with their intragenomic parasites.

Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.

Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.

Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.

Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.

Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.

Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.

Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.

Description: Motivation: Residue–residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize. Results: We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score 〉0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity 〉30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins. Availability: www.csbio.sjtu.edu.cn/bioinf/MemBrain/ Contact: hbshen@sjtu.edu.cn or zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Identification of protein–ligand binding sites is critical to protein function annotation and drug discovery. However, there is no method that could generate optimal binding site prediction for different protein types. Combination of complementary predictions is probably the most reliable solution to the problem. Results: We develop two new methods, one based on binding-specific substructure comparison (TM-SITE) and another on sequence profile alignment (S-SITE), for complementary binding site predictions. The methods are tested on a set of 500 non-redundant proteins harboring 814 natural, drug-like and metal ion molecules. Starting from low-resolution protein structure predictions, the methods successfully recognize 〉51% of binding residues with average Matthews correlation coefficient (MCC) significantly higher (with P -value 〈10 –9 in student t -test) than other state-of-the-art methods, including COFACTOR, FINDSITE and ConCavity. When combining TM-SITE and S-SITE with other structure-based programs, a consensus approach (COACH) can increase MCC by 15% over the best individual predictions. COACH was examined in the recent community-wide COMEO experiment and consistently ranked as the best method in last 22 individual datasets with the Area Under the Curve score 22.5% higher than the second best method. These data demonstrate a new robust approach to protein–ligand binding site recognition, which is ready for genome-wide structure-based function annotations. Availability: http://zhanglab.ccmb.med.umich.edu/COACH/ Contact: zhng@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The nucleosome is the basic repeating unit of chromatin. It contains two copies each of the four core histones H2A, H2B, H3 and H4 and about 147 bp of DNA. The residues of the histone proteins are subject to numerous post-translational modifications, such as methylation or acetylation. Chromatin immunoprecipitiation followed by sequencing (ChIP-seq) is a technique that provides genome-wide occupancy data of these modified histone proteins, and it requires appropriate computational methods. Results: We present NucHunter, an algorithm that uses the data from ChIP-seq experiments directed against many histone modifications to infer positioned nucleosomes. NucHunter annotates each of these nucleosomes with the intensities of the histone modifications. We demonstrate that these annotations can be used to infer nucleosomal states with distinct correlations to underlying genomic features and chromatin-related processes, such as transcriptional start sites, enhancers, elongation by RNA polymerase II and chromatin-mediated repression. Thus, NucHunter is a versatile tool that can be used to predict positioned nucleosomes from a panel of histone modification ChIP-seq experiments and infer distinct histone modification patterns associated to different chromatin states. Availability: The software is available at http://epigen.molgen.mpg.de/nuchunter/ . Contact: chung@molgen.mpg.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: In biomedical research a growing number of platforms and technologies are used to measure diverse but related information, and the task of clustering a set of objects based on multiple sources of data arises in several applications. Most current approaches to multisource clustering either independently determine a separate clustering for each data source or determine a single ‘joint’ clustering for all data sources. There is a need for more flexible approaches that simultaneously model the dependence and the heterogeneity of the data sources. Results: We propose an integrative statistical model that permits a separate clustering of the objects for each data source. These separate clusterings adhere loosely to an overall consensus clustering, and hence they are not independent. We describe a computationally scalable Bayesian framework for simultaneous estimation of both the consensus clustering and the source-specific clusterings. We demonstrate that this flexible approach is more robust than joint clustering of all data sources, and is more powerful than clustering each data source independently. We present an application to subtype identification of breast cancer tumor samples using publicly available data from The Cancer Genome Atlas. Availability: R code with instructions and examples is available at http://people.duke.edu/%7Eel113/software.html . Contact: Eric.Lock@duke.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: More and more evidences have indicated that long–non-coding RNAs (lncRNAs) play critical roles in many important biological processes. Therefore, mutations and dysregulations of these lncRNAs would contribute to the development of various complex diseases. Developing powerful computational models for potential disease-related lncRNAs identification would benefit biomarker identification and drug discovery for human disease diagnosis, treatment, prognosis and prevention. Results : In this article, we proposed the assumption that similar diseases tend to be associated with functionally similar lncRNAs. Then, we further developed the method of Laplacian Regularized Least Squares for LncRNA–Disease Association (LRLSLDA) in the semisupervised learning framework. Although known disease–lncRNA associations in the database are rare, LRLSLDA still obtained an AUC of 0.7760 in the leave-one-out cross validation, significantly improving the performance of previous methods. We also illustrated the performance of LRLSLDA is not sensitive (even robust) to the parameters selection and it can obtain a reliable performance in all the test classes. Plenty of potential disease–lncRNA associations were publicly released and some of them have been confirmed by recent results in biological experiments. It is anticipated that LRLSLDA could be an effective and important biological tool for biomedical research. Availability: The code of LRLSLDA is freely available at http://asdcd.amss.ac.cn/Software/Details/2 . Contact: xingchen@amss.ac.cn or yangy@amt.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Genomic repositories are rapidly growing, as witnessed by the 1000 Genomes or the UK10K projects. Hence, compression of multiple genomes of the same species has become an active research area in the past years. The well-known large redundancy in human sequences is not easy to exploit because of huge memory requirements from traditional compression algorithms. Results: We show how to obtain several times higher compression ratio than of the best reported results, on two large genome collections (1092 human and 775 plant genomes). Our inputs are variant call format files restricted to their essential fields. More precisely, our novel Ziv-Lempel-style compression algorithm squeezes a single human genome to ~400 KB. The key to high compression is to look for similarities across the whole collection, not just against one reference sequence, what is typical for existing solutions. Availability: http://sun.aei.polsl.pl/tgc (also as Supplementary Material) under a free license. Supplementary data: Supplementary data are available at Bioinformatics online. Contact: sebastian.deorowicz@polsl.pl

Description: Motivation:  Biological systems are understood through iterations of modeling and experimentation. Not all experiments, however, are equally valuable for predictive modeling. This study introduces an efficient method for experimental design aimed at selecting dynamical models from data. Motivated by biological applications, the method enables the design of crucial experiments: it determines a highly informative selection of measurement readouts and time points. Results:  We demonstrate formal guarantees of design efficiency on the basis of previous results. By reducing our task to the setting of graphical models, we prove that the method finds a near-optimal design selection with a polynomial number of evaluations. Moreover, the method exhibits the best polynomial-complexity constant approximation factor, unless P = NP. We measure the performance of the method in comparison with established alternatives, such as ensemble non-centrality, on example models of different complexity. Efficient design accelerates the loop between modeling and experimentation: it enables the inference of complex mechanisms, such as those controlling central metabolic operation. Availability:  Toolbox ‘NearOED’ available with source code under GPL on the Machine Learning Open Source Software Web site (mloss.org). Contact:   busettoa@inf.ethz.ch Supplementary information:   Supplementary data are available at Bioinformatics online.

Description: : Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA–mRNA interaction databases. Availability and Implementation: The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/ . Contact: rotter@genxpro.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: : We present PARSEC (PAtteRn Search and Contextualization), a new open source platform for guided discovery, allowing localization and biological characterization of short genomic sites in entire eukaryotic genomes. PARSEC can search for a sequence or a degenerated pattern. The retrieved set of genomic sites can be characterized in terms of (i) conservation in model organisms, (ii) genomic context (proximity to genes) and (iii) function of neighboring genes. These modules allow the user to explore, visualize, filter and extract biological knowledge from a set of short genomic regions such as transcription factor binding sites. Availability: Web site implemented in Java, JavaScript and C++, with all major browsers supported. Freely available at lbgi.fr/parsec. Source code is freely available at sourceforge.net/projects/genomicparsec. Contact: odile.lecompte@unistra.fr Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : At the rate that prokaryotic genomes can now be generated, comparative genomics studies require a flexible method for quickly and accurately predicting orthologs among the rapidly changing set of genomes available. SPOCS implements a graph-based ortholog prediction method to generate a simple tab-delimited table of orthologs and in addition, html files that provide a visualization of the predicted ortholog/paralog relationships to which gene/protein expression metadata may be overlaid. Availability and Implementation: A SPOCS web application is freely available at http://cbb.pnnl.gov/portal/tools/spocs.html . Source code for Linux systems is also freely available under an open source license at http://cbb.pnnl.gov/portal/software/spocs.html ; the Boost C++ libraries and BLAST are required. Contact: leeann.mccue@pnnl.gov

Description: : Automated image processing has allowed cell migration research to evolve to a high-throughput research field. As a consequence, there is now an unmet need for data management in this domain. The absence of a generic management system for the quantitative data generated in cell migration assays results in each dataset being treated in isolation, making data comparison across experiments difficult. Moreover, by integrating quality control and analysis capabilities into such a data management system, the common practice of having to manually transfer data across different downstream analysis tools will be markedly sped up and made more robust. In addition, access to a data management solution creates gateways for data standardization, meta-analysis and structured public data dissemination. We here present CellMissy, a cross-platform data management system for cell migration data with a focus on wound healing data. CellMissy simplifies and automates data management, storage and analysis from the initial experimental set-up to data exploration. Availability and implementation: CellMissy is a cross-platform open-source software developed in Java. Source code and cross-platform binaries are freely available under the Apache2 open source license at http://cellmissy.googlecode.com . Contact: lennart.martens@ugent.be Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Motivation: With the expansion of high-throughput technologies, understanding different kinds of genome-level data is a common task. MicroRNA (miRNA) is increasingly profiled using high-throughput technologies (microarrays or next-generation sequencing). The downstream analysis of miRNA targets can be difficult. Although there are many databases and algorithms to predict miRNA targets, there are few tools to integrate miRNA–gene interaction data into high-throughput genomic analyses. Results: We present targetHub, a CouchDB database of miRNA–gene interactions. TargetHub provides a programmer-friendly interface to access miRNA targets. The Web site provides RESTful access to miRNA–gene interactions with an assortment of gene and miRNA identifiers. It can be a useful tool to integrate miRNA target interaction data directly into high-throughput bioinformatics analyses. Availability: TargetHub is available on the web at http://app1.bioinformatics.mdanderson.org/tarhub/_design/basic/index.html . Contact: coombes.3@osu.edu

Description: Increasing evidence from sequence data from various environments, including the human gut, suggests the existence of a previously unknown putative seventh order of methanogens. The first genomic data from members of this lineage, Methanomassiliicoccus luminyensis and " Candidatus Methanomethylophilus alvus," provide insights into its evolutionary history and metabolic features. Phylogenetic analysis of ribosomal proteins robustly indicates a monophyletic group independent of any previously known methanogenic order, which shares ancestry with the Marine Benthic Group D, the Marine Group II, the DHVE2 group, and the Thermoplasmatales. This phylogenetic position, along with the analysis of enzymes involved in core methanogenesis, strengthens a single ancient origin of methanogenesis in the Euryarchaeota and indicates further multiple independent losses of this metabolism in nonmethanogenic lineages than previously suggested. Genomic analysis revealed an unprecedented loss of the genes coding for the first six steps of methanogenesis from H 2 /CO 2 and the oxidative part of methylotrophic methanogenesis, consistent with the fact that M. luminyensis and " Ca. M. alvus" are obligate H 2 -dependent methylotrophic methanogens. Genomic data also suggest that these methanogens may use a large panel of methylated compounds. Phylogenetic analysis including homologs retrieved from environmental samples indicates that methylotrophic methanogenesis (regardless of dependency on H 2 ) is not restricted to gut representatives but may be an ancestral characteristic of the whole order, and possibly also of ancient origin in the Euryarchaeota. 16S rRNA and McrA trees show that this new order of methanogens is very diverse and occupies environments highly relevant for methane production, therefore representing a key lineage to fully understand the diversity and evolution of methanogenesis.

Description: Stable isotopes dissect food webs from top to the bottom Biogeosciences Discussions, 10, 14923-14952, 2013 Author(s): J. J. Middelburg Stable isotopes have been used extensively to study food web functioning, i.e. the flow of energy and matter among organisms. Traditional food-web studies are based on the natural variability of carbon and nitrogen isotopes and are limited to larger organisms that can be physically separated from their environment. Recent developments allow isotope ratio measurements of microbes and this in turn allows then measurement of entire food webs, i.e. from small producers at the bottom to large consumers at the top. Here, I provide a concise review on the use and potential of stable isotope to reconstruct end-to-end food webs. I will first discuss food web reconstruction based on natural abundances isotope data and will then show that the use of stable isotopes as deliberately added tracers provides complementary information. Finally, challenges and opportunities for end-to-end food web reconstructions in a changing world are discussed.

Description: With the advent of YouTube channels in bioinformatics, open platforms for problem solving in bioinformatics, active web forums in computing analyses and online resources for learning to code or use a bioinformatics tool, the more traditional continuing education bioinformatics training programs have had to adapt. Bioinformatics training programs that solely rely on traditional didactic methods are being superseded by these newer resources. Yet such face-to-face instruction is still invaluable in the learning continuum. Bioinformatics.ca, which hosts the Canadian Bioinformatics Workshops, has blended more traditional learning styles with current online and social learning styles. Here we share our growing experiences over the past 12 years and look toward what the future holds for bioinformatics training programs.

Description: Teaching students with very diverse backgrounds can be extremely challenging. This article uses the Bioinformatics and Systems Biology MSc in Amsterdam as a case study to describe how the knowledge gap for students with heterogeneous backgrounds can be bridged. We show that a mix in backgrounds can be turned into an advantage by creating a stimulating learning environment for the students. In the MSc Programme, conversion classes help to bridge differences between students, by mending initial knowledge and skill gaps. Mixing students from different backgrounds in a group to solve a complex task creates an opportunity for the students to reflect on their own abilities. We explain how a truly interdisciplinary approach to teaching helps students of all backgrounds to achieve the MSc end terms. Moreover, transferable skills obtained by the students in such a mixed study environment are invaluable for their later careers.

Description: The number of bioinformatics tools and resources that support molecular and cell biology approaches is continuously expanding. Moreover, systems and network biology analyses are accompanied more and more by integrated bioinformatics methods. Traditional information-centered university teaching methods often fail, as (1) it is impossible to cover all existing approaches in the frame of a single course, and (2) a large segment of the current bioinformation can become obsolete in a few years. Signaling network offers an excellent example for teaching bioinformatics resources and tools, as it is both focused and complex at the same time. Here, we present an outline of a university bioinformatics course with four sample practices to demonstrate how signaling network studies can integrate biochemistry, genetics, cell biology and network sciences. We show that several bioinformatics resources and tools, as well as important concepts and current trends, can also be integrated to signaling network studies. The research-type hands-on experiences we show enable the students to improve key competences such as teamworking, creative and critical thinking and problem solving. Our classroom course curriculum can be re-formulated as an e-learning material or applied as a part of a specific training course. The multi-disciplinary approach and the mosaic setup of the course have the additional benefit to support the advanced teaching of talented students.

Description: Today, Bioinformatics has become a scientific discipline with great relevance for the Molecular Biosciences and for the Omics sciences in general. Although developed countries have progressed with large strides in Bioinformatics education and research, in other regions, such as Central America, the advances have occurred in a gradual way and with little support from the Academia, either at the undergraduate or graduate level. To address this problem, the University of Costa Rica’s Medical School, a regional leader in Bioinformatics in Central America, has been conducting a series of Bioinformatics workshops, seminars and courses, leading to the creation of the region’s first Bioinformatics Master’s Degree. The recent creation of the Central American Bioinformatics Network (BioCANET), associated to the deployment of a supporting computational infrastructure (HPC Cluster) devoted to provide computing support for Molecular Biology in the region, is providing a foundational stone for the development of Bioinformatics in the area. Central American bioinformaticians have participated in the creation of as well as co-founded the Iberoamerican Bioinformatics Society (SOIBIO). In this article, we review the most recent activities in education and research in Bioinformatics from several regional institutions. These activities have resulted in further advances for Molecular Medicine, Agriculture and Biodiversity research in Costa Rica and the rest of the Central American countries. Finally, we provide summary information on the first Central America Bioinformatics International Congress, as well as the creation of the first Bioinformatics company (Indromics Bioinformatics), spin-off the Academy in Central America and the Caribbean.

Description: Pattern recognition is concerned with the development of systems that learn to solve a given problem using a set of example instances, each represented by a number of features. These problems include clustering, the grouping of similar instances; classification, the task of assigning a discrete label to a given instance; and dimensionality reduction, combining or selecting features to arrive at a more useful representation. The use of statistical pattern recognition algorithms in bioinformatics is pervasive. Classification and clustering are often applied to high-throughput measurement data arising from microarray, mass spectrometry and next-generation sequencing experiments for selecting markers, predicting phenotype and grouping objects or genes. Less explicitly, classification is at the core of a wide range of tools such as predictors of genes, protein function, functional or genetic interactions, etc., and used extensively in systems biology. A course on pattern recognition (or machine learning) should therefore be at the core of any bioinformatics education program. In this review, we discuss the main elements of a pattern recognition course, based on material developed for courses taught at the BSc, MSc and PhD levels to an audience of bioinformaticians, computer scientists and life scientists. We pay attention to common problems and pitfalls encountered in applications and in interpretation of the results obtained.

Description: Responses of carbon dioxide flux and plant biomass to drought in a treed peatland in northern Alberta: a climate change perspective Biogeosciences Discussions, 10, 14999-15031, 2013 Author(s): T. M. Munir, B. Xu, M. Perkins, and M. Strack Northern peatland ecosystems represent large carbon stocks that are susceptible to changes such as accelerated mineralization due to water table lowering expected under a climate change scenario. During the growing seasons of 2011 and 2012 we monitored CO 2 fluxes and plant biomass along a microtopographic gradient (hummocks-hollows) in an undisturbed dry continental boreal treed bog (control) and a nearby site that was drained (drained) in 2001. Ten years of drainage in the bog significantly increased coverage of shrubs at hummocks and lichens at hollows. Considering measured hummock coverage and including tree incremental growth, we estimate that the control site was a larger sink in 2011 of −40 than that of −13 g C m −2 in 2012 while the drained site was a source of 144 and 140 g C m −2 over the same years. We infer that, drainage induced changes in vegetation growth led to increased biomass to counteract a portion of soil carbon losses. These results suggest that spatial variability (microtopography) and changes in vegetation community in boreal peatlands will affect how these ecosystems respond to lowered water table potentially induced by climate change.

Description: The coccolithophores Emiliania huxleyi and Coccolithus pelagicus : extant populations from the Norwegian-Iceland Sea and Fram Strait Biogeosciences Discussions, 10, 15077-15106, 2013 Author(s): C. V. Dylmer, J. Giraudeau, V. Hanquiez, and K. Husum Extant coccolithophores and their relation to the governing oceanographic features in the northern North Atlantic were investigated along two zonal transects of surface water sampling, both conducted during summer 2011 and fall 2007. The northern transects crossed Fram Strait and its two opposing boundary currents (West Spitsbergen Current and East Greenland Current), while the southern transects sampled the Norwegian and Iceland Seas (passing the island Jan Mayen) from the Lofoten Islands to the continental margin off Eastern Greenland. The distribution of the dominant coccolithophore species Emiliania huxleyi and Coccolithus pelagicus is discussed in view of both the surface hydrology at the time of sampling and the structure of the surface mixed layer. Remote-sensing images as well as CTD and ARGO profiles are used to constrain the physico-chemical state of the surface water at the time of sampling. Both transects were characterized by strong seasonal differences in bulk coccolithophore standing stocks with maximum values of 53 × 10 3 cells L −1 for the northern transect and 72 × 10 3 cells L −1 for the southern transect in fall and summer, respectively. The highest recorded bulk cell densities are essentially explained by E. huxleyi . This species shows a zonal shift in peak abundance in the Norwegian-Iceland Seas from a summer maximum in the Lofoten gyre to peak cell densities around the island Jan Mayen in fall. Vertical mixing of Atlantic waters west of Lofoten Island, a phenomenom related to pervasive summer large scale atmospheric changes in the eastern Nordic Seas, on one hand, and strengthened influence of melt-water and related surface water stratification around the island Jan Mayen during fall, on the other hand, explains the observed seasonal migration of the E. huxleyi peak production area, as well as the seasonal change in dominating species within the Iceland Sea. In addition our datasets are indicative of a well-defined maximum boundary temperature of 6 °C for the production of C. pelagicus in the northern North Atlantic. The Fram Strait transects provides, to our knowledge, a first view of the zonal distribution of extant coccolithophores in this remote setting during summer and fall. Our datasets are indicative of a seasonal change in the species community from an E. huxleyi -dominated assemblage during summer to a C. pelagicus -rich population during fall. Here, higher irradiance and increased Atlantic water influence during summer favored the production of the opportunistic species E. huxleyi close to the Arctic Front, whereas the peak production area during fall, with high concentrations of C. pelagicus , lays in true Arctic/Polar waters.

Description: Synonymous codon usage patterns are shaped by a balance between mutation, drift, and natural selection. To date, detection of translational selection in vertebrates has proven to be a challenging task, obscured by small long-term effective population sizes in larger animals and the existence of isochores in some species. The consensus is that, in such species, natural selection is either completely ineffective at overcoming mutational pressures and genetic drift or perhaps is effective but so weak that it is not detectable. The aim of this research is to understand the interplay between mutation, selection, and genetic drift in vertebrates. We observe that although variation in mutational bias is undoubtedly the dominant force influencing codon usage, translational selection acts as a weak additional factor influencing synonymous codon usage. These observations indicate that translational selection is a widespread phenomenon in vertebrates and is not limited to a few species.