ALBERT

Description: Telomeres, the ends of linear chromosomes, safeguard against genome instability. The enzyme responsible for extension of the telomere 3' terminus is the ribonucleoprotein telomerase. Whereas telomerase activity can be reconstituted in vitro with only the telomerase RNA (hTR) and telomerase reverse transcriptase (TERT), additional components are required in vivo for enzyme assembly, stability and telomere extension activity. One such associated protein, dyskerin, promotes hTR stability in vivo and is the only component to co-purify with active, endogenous human telomerase. We used oligonucleotide-based affinity purification of hTR followed by native gel electrophoresis and in-gel telomerase activity detection to query the composition of telomerase at different purification stringencies. At low salt concentrations (0.1 M NaCl), affinity-purified telomerase was ‘supershifted’ with an anti-dyskerin antibody, however the association with dyskerin was lost after purification at 0.6 M NaCl, despite the retention of telomerase activity and a comparable yield of hTR. The interaction of purified hTR and dyskerin in vitro displayed a similar salt-sensitive interaction. These results demonstrate that endogenous human telomerase, once assembled and active, does not require dyskerin for catalytic activity. Native gel electrophoresis may prove useful in the characterization of telomerase complexes under various physiological conditions.

Description: We describe an inexpensive and efficient method for generating functional pools of Dicer-substrate small interfering RNAs (siRNAs) in a single reaction tube. The method exploits a highly active form of the enzyme Dicer from Giardia lamblia , which is capable of accurately processing double-stranded RNA (dsRNA) into 25–27 nt RNA pools during in vitro transcription. The small RNAs produced function as substrates of human Dicer in vitro and induce gene silencing with potency equivalent to traditional siRNAs when introduced into mammalian cells. The overall reaction is simple, can be carried out in any laboratory with access to a PCR machine, and is amenable to high-throughput processes.

Description: Microglial cells are the main HIV-1 targets in the central nervous system (CNS) and constitute an important reservoir of latently infected cells. Establishment and persistence of these reservoirs rely on the chromatin structure of the integrated proviruses. We have previously demonstrated that the cellular cofactor CTIP2 forces heterochromatin formation and HIV-1 gene silencing by recruiting HDAC and HMT activities at the integrated viral promoter. In the present work, we report that the histone demethylase LSD1 represses HIV-1 transcription and viral expression in a synergistic manner with CTIP2. We show that recruitment of LSD1 at the HIV-1 proximal promoter is associated with both H3K4me3 and H3K9me3 epigenetic marks. Finally, our data suggest that LSD1-induced H3K4 trimethylation is linked to hSET1 recruitment at the integrated provirus.

Description: This short review aims at presenting some recent illustrative examples of spontaneous nucleolipids self-assembly. High-resolution structural investigations reveal the diversity and complexity of assemblies formed by these bioinspired amphiphiles, resulting from the interplay between aggregation of the lipid chains and base–base interactions. Nucleolipids supramolecular assemblies are promising soft drug delivery systems, particularly for nucleic acids. Regarding prodrugs, squalenoylation is an innovative concept for improving efficacy and delivery of nucleosidic drugs.

Description: Determining the underlying haplotypes of individual human genomes is an essential, but currently difficult, step toward a complete understanding of genome function. Fosmid pool-based next-generation sequencing allows genome-wide generation of 40-kb haploid DNA segments, which can be phased into contiguous molecular haplotypes computationally by Single Individual Haplotyping (SIH). Many SIH algorithms have been proposed, but the accuracy of such methods has been difficult to assess due to the lack of real benchmark data. To address this problem, we generated whole genome fosmid sequence data from a HapMap trio child, NA12878, for which reliable haplotypes have already been produced. We assembled haplotypes using eight algorithms for SIH and carried out direct comparisons of their accuracy, completeness and efficiency. Our comparisons indicate that fosmid-based haplotyping can deliver highly accurate results even at low coverage and that our SIH algorithm, ReFHap, is able to efficiently produce high-quality haplotypes. We expanded the haplotypes for NA12878 by combining the current haplotypes with our fosmid-based haplotypes, producing near-to-complete new gold-standard haplotypes containing almost 98% of heterozygous SNPs. This improvement includes notable fractions of disease-related and GWA SNPs. Integrated with other molecular biological data sets, this phase information will advance the emerging field of diploid genomics.

Description: RNA tetraloops can recognize receptors to mediate long-range interactions in stable natural RNAs. In vitro selected GNRA tetraloop/receptor interactions are usually more ‘G/C-rich’ than their ‘A/U-rich’ natural counterparts. They are not as widespread in nature despite comparable biophysical and chemical properties. Moreover, while AA, AC and GU dinucleotide platforms occur in natural GAAA/11 nt receptors, the AA platform is somewhat preferred to the others. The apparent preference for ‘A/U-rich’ GNRA/receptor interactions in nature might stem from an evolutionary adaptation to avoid folding traps at the level of the larger molecular context. To provide evidences in favor of this hypothesis, several riboswitches based on natural and artificial GNRA receptors were investigated in vitro for their ability to prevent inter-molecular GNRA/receptor interactions by trapping the receptor sequence into an alternative intra-molecular pseudoknot. Extent of attenuation determined by native gel-shift assays and co-transcriptional assembly is correlated to the G/C content of the GNRA receptor. Our results shed light on the structural evolution of natural long-range interactions and provide design principles for RNA-based attenuator devices to be used in synthetic biology and RNA nanobiotechnology.

Description: Many types of DNA structures are generated in response to DNA damage, repair and recombination that require processing via specialized nucleases. DNA hairpins represent one such class of structures formed during V(D)J recombination, palindrome extrusion, DNA transposition and some types of double-strand breaks. Here we present biochemical and genetic evidence to suggest that Pso2 is a robust DNA hairpin opening nuclease in budding yeast. Pso2 (SNM1A in mammals) belongs to a small group of proteins thought to function predominantly during interstrand crosslink (ICL) repair. In this study, we characterized the nuclease activity of Pso2 toward a variety of DNA substrates. Unexpectedly, Pso2 was found to be an efficient, structure-specific DNA hairpin opening endonuclease. This activity was further shown to be required in vivo for repair of chromosomal breaks harboring closed hairpin ends. These findings provide the first evidence that Pso2 may function outside ICL repair and open the possibility that Pso2 may function at least in part during ICL repair by processing DNA intermediates including DNA hairpins or hairpin-like structures.

Description: The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N 6 -(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N 6 -methyltransferases ( hin1523 , nma1821 , hia5 , respectively). Products of the hin1523 , hia5 and nma1821 genes modify adenine residues to N 6 -methyladenine, both in vitro and in vivo . All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential ‘sequence specificity’ could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.

Description: The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common ‘transition zone’ located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro , all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5' region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

Description: Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis- o -(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.

Description: Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo . Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1 G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

Description: The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using ‘click’ chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

Description: Rosen, S., Engås, A., Fernö, A., and Jörgensen, T. 2012. The reactions of shoaling adult cod to a pelagic trawl: implications for commercial trawling. – ICES Journal of Marine Science, 69: 303–312. The reactions of shoaling adult Atlantic cod to a pelagic trawl were measured during fishing off the north coast of Norway. Cod remaining in the trawl track dived at rates as fast as 0.35 m s –1 following vessel passage and swam away from the vessel, in the direction of the approaching trawl, at an average rate of 0.6 m s –1 . They did not attempt to swim ahead of the trawl as documented previously, but passed into the lower half of the trawl entrance and swam slowly in the direction of trawling at a rate of 0.2–0.5 m s –1 as the trawl's greater speed through water carried them deeper into the trawl. Shoals compressed vertically once inside the trawl, suggesting that packing density increased at least fourfold. Fish remained in the lower part of the trawl as they moved through its tapered section towards the codend, with little to no clearance above the bottom panel, but significant clearance beneath the top panel. Catches were sufficient to support commercial harvest, and the behaviour observed suggests that changes in trawl design and fishing strategy might improve fuel economy and species selectivity.

Description: Shackell, N. L., Bundy, A., Nye, J. A., and Link, J. S. 2012. Common large-scale responses to climate and fishing across Northwest Atlantic ecosystems. – ICES Journal of Marine Science, 69: 151–152. Investigating whether there were common biological responses to climate and fishing across seven Northwest Atlantic ecosystems, a minimum/maximum autocorrelation factor analysis of biological indicators for each region revealed a common primary multivariate trend of a rapid change during the 1980s and early 1990s. There was a strong common pattern in the biological indicators responsible for the primary multivariate temporal trend in the five more northerly regions: an increase in the abundance of phytoplankton, an increase in biomass at mid-trophic levels, and a decline in predatory groundfish size. The common associations between patterns and drivers were fishing indices and the Atlantic Multidecadal Oscillation, but all associations weakened when co-varying drivers were held constant. The results are consistent with known long-term effects of intense fishing, such as a decline in average fish size and changes in species composition. Less fishing pressure has allowed some regions to recover to former predatory biomass levels since the late 1990s, but the bulk of the biomass consists of fewer species. However, fishing was not the only driver, and a more mechanistic understanding of how the climate affects lower trophic levels is needed to contextualize climate effects in heavily fished ecosystems.

Description: Deporte, N., Ulrich, C., Mahévas, S., Demanèche, S., and Bastardie, F. 2012. Regional métier definition: a comparative investigation of statistical methods using a workflow applied to international otter trawl fisheries in the North Sea. – ICES Journal of Marine Science, 69: 331–342. The European Common Fisheries Policy recognizes the importance of accounting for heterogeneity in fishing practices, and métier-based sampling is now at the core of the EU Data Collection Framework. The implementation of such an approach would require Member States to agree on the standard regional métier definitions and on practical rules to categorize logbook records into métiers. Several alternative approaches have been used in the past to categorize landings profiles, but no consensus has yet emerged. A generic open-source workflow is developed to test and compare a selection of methods, including principal components analysis (PCA), hierarchical agglomerative clustering (HAC), K -means, and Clustering LARge Applications (CLARA), and to provide simple allocation rules. This workflow is applied to a unique regional dataset consisting of bottom-trawl logbooks of five North Sea countries. No method proved to be infallible, but combining PCA with either CLARA or HAC performed best. For 2008, a hierarchical classification with 14 species assemblages is proposed. Discriminant analysis proved more robust than simple ordination methods for allocating a new logbook record into an existing métier. The whole approach is directly operational and could contribute to defining more objective and consistent métiers across European fisheries.

Description: Greenstreet, S. P. R., Rogers, S. I., Rice, J. C., Piet, G. J., Guirey, E. J., Fraser, H. M., and Fryer, R. J. 2012. A reassessment of trends in the North Sea Large Fish Indicator and a re-evaluation of earlier conclusions. – ICES Journal of Marine Science, 69: 343–345. Previous analysis of the Large Fish Indicator, the basis for the North Sea "fish communities" EcoQO, suggested a strong recovery between 2001 and 2008. Discovery of a mistake in this earlier analysis now suggests that this recovery is not as strong as first thought, but reanalysis of the corrected data still supports the majority of conclusions drawn in the original paper.

Description: Xie, Y., Michielsens, C. G. J., and Martens, F. J. 2012. Classification of fish and non-fish acoustic tracks using discriminant function analysis. – ICES Journal of Marine Science, 69: 313–322. Hydroacoustic data acquired for estimating fish populations contain information on both fish and non-fish targets, so sonar technicians traditionally rely on their knowledge of fish behaviour and experience with hydroacoustics to remove non-fish targets from the hydroacoustic data. This process is often labour-intensive and time-consuming, making real-time assessment of fish populations difficult. Simple solutions are not always available for all circumstances. However, the split-beam sonar data collected in the lower Fraser River, British Columbia, Canada, showed distinct signatures between actively swimming fish and non-fish objects such as drifting debris, surface bubbles, and stationary objects in the water column and off the river bottom. Acoustic tracks of fish and non-fish targets were characterized by differentiable statistical patterns that were amenable to discriminant function analysis (DFA). An application of DFA to segregate fish and non-fish targets detected by a split-beam sonar system in the lower Fraser River is presented, characteristics of user-identified fish and non-fish acoustic tracks being utilized as learning samples for the DFA. Also, a method to rank the discriminating power of individual variables is presented, providing guidance for constructing efficient and effective discriminant functions with variables that offer high discriminating power. The DFA yielded classification accuracies of 96% for fish and 91% for non-fish tracks and reduced the manual sorting time by 50–75%.

Description: Rey, F. 2012. Declining silicate concentrations in the Norwegian and Barents Seas. – ICES Journal of Marine Science, 69: 208–212. Since 1990, a decline in silicate concentrations together with increasing salinities has been observed in the Atlantic water of the Norwegian and Barents Seas. This decline in silicate has been found to be related to the relative proportion in which eastern and western source water masses from the northeastern North Atlantic enter the Norwegian Sea.

Description: Zhou, S., Milton, D. A., and Fry, G. C. 2012. Integrated risk analysis for rare marine species impacted by fishing: sustainability assessment and population trend modelling. – ICES Journal of Marine Science, 69: 271–280. Risk assessment and conservation management of rare species are challenging due to a lack of data. We developed an integrated risk assessment approach to assess human impact on population sustainability of rare species. The approach involved two components: a quantitative sustainability assessment coupled with modelling trends in relative abundance. Both components took nil catches into account through zero-inflated statistical distributions that simultaneously modelled the zero and non-zero catches separately in submodels. The sustainability assessment used detection–non-detection data for population estimation and linked sustainability to easily collected life-history traits. This component provides an assessment of population sustainability at one point in time. The trend modelling applied zero-inflated negative binomial models to temporal trends in density and dispersion of species. It provided a complement to the static sustainability assessment. We applied this integrated approach to assess the risk to 14 species of rare, protected sea snakes incidentally caught in the Australian Northern Prawn Fishery. This approach can be applicable for risk assessment of many species with limited abundance data, a large number of absences and some presence–absence information only.

Description: Frid, C. L. J., and Paramor, O. A. L. 2012. Feeding the world: what role for fisheries? – ICES Journal of Marine Science, 69: 145–150. Fisheries (wild capture and aquaculture) deliver more than 110 million tonnes of food and around 15% of the dietary protein to the 7 billion people currently living on the planet. With the global population expected to peak at 9 billion by 2050, and 〉80% of global fish stocks currently fully or overexploited (and aquaculture is at least in part dependent on capture fisheries), the contribution of fisheries looks set to decline. The challenge is therefore determining how better management, an ecosystem perspective, and more efficient utilization of fisheries waste can support fisheries products continuing to contribute significantly to "feeding the world" up to and beyond the population peak.

Description: Boström, M. K., Östman, Ö., Bergenius, M. A. J., and Lunneryd, S-G. 2012. Cormorant diet in relation to temporal changes in fish communities. – ICES Journal of Marine Science, 69: 175–183. The stomach contents of 229 great cormorants ( Phalacrocorax carbo sinensis ) shot between March and October 2009 along the Swedish east coast were analysed for differences in diet between gender, age, and breeding phase. Sticklebacks ( Gasterosteus aculeatus and Pungitius pungitius ) were the most common prey, followed by eelpout ( Zoarces viviparus ), herring ( Clupea harengus membras ), and cyprinids (Cyprinidae). Diet did not differ between age and gender, but changed over the breeding season. The different phases explained around 10% of the total variation in stomach content between cormorants, suggesting no major shift in diet over the breeding season. The diet of cormorants in 2009 was compared with the results of a study conducted in the same area in 1992. There were evident changes in the diet between 1992 and 2009, with less perch ( Perca fluviatilis ) and cyprinids and more eelpout and herring in 2009. This change in diet could partly be related to changes in the fish community. The seasonal changes in diet composition of whole stomachs were less notable than in many previous studies, but long-term changes in the fish communities may induce changes in cormorant diet. It is clearly important to use stomach contents in areas with many small fish species for a comprehensive assessment of cormorant diet.

Description: Cañás, L., Stransky, C., Schlickeisen, J., Sampedro, M. P., and Fariña, A. C. 2012. Use of the otolith shape analysis in stock identification of anglerfish ( Lophius piscatorius ) in the Northeast Atlantic. – ICES Journal of Marine Science, 69: 250–256. The geographical variability in the anglerfish Lophius piscatorius otolith shape was investigated to examine biological evidence for the current separation into Northern and Southern stocks of L. piscatorius established in the Southwest European waters (Southern Shelf area) by the International Council for the Exploration of the Sea (ICES). In all, 629 otoliths of L. piscatorius (12–90 cm total length) from commercial fishing and research surveys were collected in two consecutive years, 2008 and 2009. The otolith shape was compared between stocks for differences in univariate descriptors using non-parametric analysis of covariance, as well as elliptical Fourier shape descriptors, through a metric multidimensional scaling and a linear discriminant analysis (LDA). The differences found in the otolith shape between Northern and Southern stocks were not large enough to hold the current status of L. piscatorius regarding stock definition in the Southern Shelf area, although homogeneous mixing between both stocks is not expected as there were significant differences between three univariate descriptors and the LDA was significant. These results are in agreement with the previous L. piscatorius stock identification studies (genetics, body morphology, and migratory behaviour), which do not support the current separation of these stocks but found substructures within the area.

Description: Motivation: High-throughput sequencing (HTS) technologies have made low-cost sequencing of large numbers of samples commonplace. An explosion in the type, not just number, of sequencing experiments has also taken place including genome re-sequencing, population-scale variation detection, whole transcriptome sequencing and genome-wide analysis of protein-bound nucleic acids. Results: We present Artemis as a tool for integrated visualization and computational analysis of different types of HTS datasets in the context of a reference genome and its corresponding annotation. Availability: Artemis is freely available (under a GPL licence) for download (for MacOSX, UNIX and Windows) at the Wellcome Trust Sanger Institute websites: http://www.sanger.ac.uk/resources/software/artemis/ . Contact: artemis@sanger.ac.uk ; tjc@sanger.ac.uk

Description: : microRibonucleic acid (miRNAs) are small regulatory molecules that act by mRNA degradation or via translational repression. Although many miRNAs are ubiquitously expressed, a small subset have differential expression patterns that may give rise to tissue-specific complexes. Motivation: This work studies gene targeting patterns amongst miRNAs with differential expression profiles, and links this to control and regulation of protein complexes. Results: We find that, when a pair of miRNAs are not expressed in the same tissues, there is a higher tendency for them to target the direct partners of the same hub proteins. At the same time, they also avoid targeting the same set of hub-spokes. Moreover, the complexes corresponding to these hub-spokes tend to be specific and nonoverlapping. This suggests that the effect of miRNAs on the formation of complexes is specific. Contact: wongls@comp.nus.edu.sg Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Small interfering RNAs (siRNAs) are produced from much longer sequences of double-stranded RNA precursors through cleavage by Dicer or a Dicer-like protein. These small RNAs play a key role in genetic and epigenetic regulation; however, a full understanding of the mechanisms by which they operate depends on the characterization of the precursors from which they are derived. Results: High-throughput sequencing of small RNA populations allows the locations of the double-stranded RNA precursors to be inferred. We have developed methods to analyse small RNA sequencing data from multiple biological sources, taking into account replicate information, to identify robust sets of siRNA precursors. Our methods show good performance on both a set of small RNA sequencing data in Arabidopsis thaliana and simulated datasets. Availability: Our methods are available as the Bioconductor ( www.bioconductor.org ) package segmentSeq (version 1.5.6 and above). Contact: tjh48@cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Intrinsically disordered regions are key for the function of numerous proteins, and the scant available experimental annotations suggest the existence of different disorder flavors. While efficient predictions are required to annotate entire genomes, most existing methods require sequence profiles for disorder prediction, making them cumbersome for high-throughput applications. Results: In this work, we present an ensemble of protein disorder predictors called ESpritz. These are based on bidirectional recursive neural networks and trained on three different flavors of disorder, including a novel NMR flexibility predictor. ESpritz can produce fast and accurate sequence-only predictions, annotating entire genomes in the order of hours on a single processor core. Alternatively, a slower but slightly more accurate ESpritz variant using sequence profiles can be used for applications requiring maximum performance. Two levels of prediction confidence allow either to maximize reasonable disorder detection or to limit expected false positives to 5%. ESpritz performs consistently well on the recent CASP9 data, reaching a S w measure of 54.82 and area under the receiver operator curve of 0.856. The fast predictor is four orders of magnitude faster and remains better than most publicly available CASP9 methods, making it ideal for genomic scale predictions. Conclusions: ESpritz predicts three flavors of disorder at two distinct false positive rates, either with a fast or slower and slightly more accurate approach. Given its state-of-the-art performance, it can be especially useful for high-throughput applications. Availability: Both a web server for high-throughput analysis and a Linux executable version of ESpritz are available from: http://protein.bio.unipd.it/espritz/ Contact: silvio.tosatto@unipd.it Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Clustering protein structures is an important task in structural bioinformatics. De novo structure prediction, for example, often involves a clustering step for finding the best prediction. Other applications include assigning proteins to fold families and analyzing molecular dynamics trajectories. Results: We present Pleiades, a novel approach to clustering protein structures with a rigorous mathematical underpinning. The method approximates clustering based on the root mean square deviation by first mapping structures to Gauss integral vectors—which were introduced by Røgen and co-workers—and subsequently performing K-means clustering. Conclusions: Compared to current methods, Pleiades dramatically improves on the time needed to perform clustering, and can cluster a significantly larger number of structures, while providing state-of-the-art results. The number of low energy structures generated in a typical folding study, which is in the order of 50 000 structures, can be clustered within seconds to minutes. Contact: thamelry@binf.ku.dk ; harder@binf.ku.dk Supplementary Information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Transmembrane β barrel proteins (TMBs) are found in the outer membrane of Gram-negative bacteria, chloroplast and mitochondria. They play a major role in the translocation machinery, pore formation, membrane anchoring and ion exchange. TMBs are also promising targets for antimicrobial drugs and vaccines. Given the difficulty in membrane protein structure determination, computational methods to identify TMBs and predict the topology of TMBs are important. Results: Here, we present BOCTOPUS; an improved method for the topology prediction of TMBs by employing a combination of support vector machines (SVMs) and Hidden Markov Models (HMMs). The SVMs and HMMs account for local and global residue preferences, respectively. Based on a 10-fold cross-validation test, BOCTOPUS performs better than all existing methods, reaching a Q3 accuracy of 87%. Further, BOCTOPUS predicted the correct number of strands for 83% proteins in the dataset. BOCTOPUS might also help in reliable identification of TMBs by using it as an additional filter to methods specialized in this task. Availability: BOCTOPUS is freely available as a web server at: http://boctopus.cbr.su.se/ . The datasets used for training and evaluations are also available from this site. Contact: arne@bioinfo.se Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: High-dimensional data such as microarrays have created new challenges to traditional statistical methods. One such example is on class prediction with high-dimension, low-sample size data. Due to the small sample size, the sample mean estimates are usually unreliable. As a consequence, the performance of the class prediction methods using the sample mean may also be unsatisfactory. To obtain more accurate estimation of parameters some statistical methods, such as regularizations through shrinkage, are often desired. Results: In this article, we investigate the family of shrinkage estimators for the mean value under the quadratic loss function. The optimal shrinkage parameter is proposed under the scenario when the sample size is fixed and the dimension is large. We then construct a shrinkage-based diagonal discriminant rule by replacing the sample mean by the proposed shrinkage mean. Finally, we demonstrate via simulation studies and real data analysis that the proposed shrinkage-based rule outperforms its original competitor in a wide range of settings. Contact: tongt@hkbu.edu.hk

Description: Motivation: The advent of high-throughput sequencing technologies is revolutionizing our ability in discovering and genotyping DNA copy number variants (CNVs). Read count-based approaches are able to detect CNV regions with an unprecedented resolution. Although this computational strategy has been recently introduced in literature, much work has been already done for the preparation, normalization and analysis of this kind of data. Results: Here we face the many aspects that cover the detection of CNVs by using read count approach. We first study the characteristics and systematic biases of read count distributions, focusing on the normalization methods designed for removing these biases. Subsequently, we compare the algorithms designed to detect the boundaries of CNVs and we investigate the ability of read count data to predict the exact number of DNA copy. Finally, we review the tools publicly available for analysing read count data. To better understand the state of the art of read count approaches, we compare the performance of the three most widely used sequencing technologies (Illumina Genome Analyzer, Roche 454 and Life Technologies SOLiD) in all the analyses that we perform. Contact: albertomagi@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: We investigate and quantify the generalizability of the white blood cell (WBC) transcriptome to the general, multiorgan transcriptome. We use data from the NCBI's Gene Expression Omnibus (GEO) public repository to define two datasets for comparison, WBC and OO (Other Organ) sets. Results: Comprehensive pair-wise correlation and expression level profiles are calculated for both datasets (with sizes of 81 and 1463, respectively). We have used mapping and ranking across the Gene Ontology (GO) categories to quantify similarity between the two sets. GO mappings of the most correlated and highly expressed genes from the two datasets tightly match, with the notable exceptions of components of the ribosome, cell adhesion and immune response. That is, 10 877 or 48.8% of all measured genes do not change 〉10% of rank range between WBC and OO; only 878 (3.9%) change rank 〉50%. Two trans -tissue gene lists are defined, the most changing and the least changing genes in expression rank. We also provide a general, quantitative measure of the probability of expression rank and correlation profile in the OO system given the expression rank and correlation profile in the WBC dataset. Contact: vvaltchinov@partners.org Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The understanding of the molecular sources for diseases like cancer can be significantly improved by computational models. Recently, Boolean networks have become very popular for modeling signaling and regulatory networks. However, such models rely on a set of Boolean functions that are in general not known. Unfortunately, while detailed information on the molecular interactions becomes available in large scale through electronic databases, the information on the Boolean functions does not become available simultaneously and has to be included manually into the models, if at all known. Results: We propose a new Boolean approach which can directly utilize the mechanistic network information available through modern databases. The Boolean function is implicitly defined by the reaction mechanisms. Special care has been taken for the treatment of kinetic features like inhibition. The method has been applied to a signaling model combining the Wnt and MAPK pathway. Availability: A sample C++ implementation of the proposed method is available for Linux and compatible systems through http://code.google.com/p/libscopes/wiki/Paper2011 Contact: handorf@physik.hu-berlin.de Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: Multiple sequence alignment (MSA) is a core method in bioinformatics. The accuracy of such alignments may influence the success of downstream analyses such as phylogenetic inference, protein structure prediction, and functional prediction. The importance of MSA has lead to the proliferation of MSA methods, with different objective functions and heuristics to search for the optimal MSA. Different methods of inferring MSAs produce different results in all but the most trivial cases. By measuring the differences between inferred alignments, we may be able to develop an understanding of how these differences (i) relate to the objective functions and heuristics used in MSA methods, and (ii) affect downstream analyses. Results: We introduce four metrics to compare MSAs, which include the position in a sequence where a gap occurs or the location on a phylogenetic tree where an insertion or deletion (indel) event occurs. We use both real and synthetic data to explore the information given by these metrics and demonstrate how the different metrics in combination can yield more information about MSA methods and the differences between them. Availability: MetAl is a free software implementation of these metrics in Haskell. Source and binaries for Windows, Linux and Mac OS X are available from http://kumiho.smith.man.ac.uk/whelan/software/metal/ . Contact: simon.whelan@manchester.ac.uk

Description: Motivation: Peptide detection is a crucial step in mass spectrometry (MS) based proteomics. Most existing algorithms are based upon greedy isotope template matching and thus may be prone to error propagation and ineffective to detect overlapping peptides. In addition, existing algorithms usually work at different charge states separately, isolating useful information that can be drawn from other charge states, which may lead to poor detection of low abundance peptides. Results: BPDA2d models spectra as a mixture of candidate peptide signals and systematically evaluates all possible combinations of possible peptide candidates to interpret the given spectra. For each candidate, BPDA2d takes into account its elution profile, charge state distribution and isotope pattern, and it combines all evidence to infer the candidate's signal and existence probability. By piecing all evidence together—especially by deriving information across charge states—low abundance peptides can be better identified and peptide detection rates can be improved. Instead of local template matching, BPDA2d performs global optimization for all candidates and systematically optimizes their signals. Since BPDA2d looks for the optimal among all possible interpretations of the given spectra, it has the capability in handling complex spectra where features overlap. BPDA2d estimates the posterior existence probability of detected peptides, which can be directly used for probability-based evaluation in subsequent processing steps. Our experiments indicate that BPDA2d outperforms state-of-the-art detection methods on both simulated data and real liquid chromatography–mass spectrometry data, according to sensitivity and detection accuracy. Availability: The BPDA2d software package is available at http://gsp.tamu.edu/Publications/supplementary/sun11a/ Contact: Michelle.Zhang@utsa.edu ; edward@ece.tamu.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The continued progress in developing technological platforms, availability of many published experimental datasets, as well as different statistical methods to analyze those data have allowed approaching the same research question using various methods simultaneously. To get the best out of all these alternatives, we need to integrate their results in an unbiased manner. Prioritized gene lists are a common result presentation method in genomic data analysis applications. Thus, the rank aggregation methods can become a useful and general solution for the integration task. Results: Standard rank aggregation methods are often ill-suited for biological settings where the gene lists are inherently noisy. As a remedy, we propose a novel robust rank aggregation (RRA) method. Our method detects genes that are ranked consistently better than expected under null hypothesis of uncorrelated inputs and assigns a significance score for each gene. The underlying probabilistic model makes the algorithm parameter free and robust to outliers, noise and errors. Significance scores also provide a rigorous way to keep only the statistically relevant genes in the final list. These properties make our approach robust and compelling for many settings. Availability: All the methods are implemented as a GNU R package R obust R ank A ggreg , freely available at the Comprehensive R Archive Network http://cran.r-project.org/ . Contact: vilo@ut.ee Supplementary information Supplementary data are available at Bioinformatics online.

Description: : CLARE is a computational method designed to reveal sequence encryption of tissue-specific regulatory elements. Starting with a set of regulatory elements known to be active in a particular tissue/process, it learns the sequence code of the input set and builds a predictive model from features specific to those elements. The resulting model can then be applied to user-supplied genomic regions to identify novel candidate regulatory elements. CLARE's model also provides a detailed analysis of transcription factors that most likely bind to the elements, making it an invaluable tool for understanding mechanisms of tissue-specific gene regulation. Availability: CLARE is freely accessible at http://clare.dcode.org/ . Contact: taherl@ncbi.nlm.nih.gov ; ovcharen@nih.gov Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: We present a pipeline for the pre-processing, quality assessment, read distribution and methylation estimation for methylated DNA immunoprecipitation (MeDIP)-sequence datasets. This is the first MeDIP-seq-specific analytic pipeline that starts at the output of the sequencers. This pipeline will reduce the data analysis load on staff and allows the easy and straightforward analysis of sequencing data for DNA methylation. The pipeline integrates customized scripting and several existing tools, which can deal with both paired and single end data. Availability: The package and extensive documentation, and comparison to public data is available at http://life.tongji.edu.cn/meqa/ Contact: jhuang@cephb.fr

Description: Motivation: A plethora of bioinformatics analysis has led to the discovery of numerous gene sets, which can be interpreted as discrete measurements emitted from latent signaling pathways. Their potential to infer signaling pathway structures, however, has not been sufficiently exploited. Existing methods accommodating discrete data do not explicitly consider signal cascading mechanisms that characterize a signaling pathway. Novel computational methods are thus needed to fully utilize gene sets and broaden the scope from focusing only on pairwise interactions to the more general cascading events in the inference of signaling pathway structures. Results: We propose a gene set based simulated annealing (SA) algorithm for the reconstruction of signaling pathway structures. A signaling pathway structure is a directed graph containing up to a few hundred nodes and many overlapping signal cascades, where each cascade represents a chain of molecular interactions from the cell surface to the nucleus. Gene sets in our context refer to discrete sets of genes participating in signal cascades, the basic building blocks of a signaling pathway, with no prior information about gene orderings in the cascades. From a compendium of gene sets related to a pathway, SA aims to search for signal cascades that characterize the optimal signaling pathway structure. In the search process, the extent of overlap among signal cascades is used to measure the optimality of a structure. Throughout, we treat gene sets as random samples from a first-order Markov chain model. We evaluated the performance of SA in three case studies. In the first study conducted on 83 KEGG pathways, SA demonstrated a significantly better performance than Bayesian network methods. Since both SA and Bayesian network methods accommodate discrete data, use a ‘search and score’ network learning strategy and output a directed network, they can be compared in terms of performance and computational time. In the second study, we compared SA and Bayesian network methods using four benchmark datasets from DREAM. In our final study, we showcased two context-specific signaling pathways activated in breast cancer. Availibility: Source codes are available from http://dl.dropbox.com/u/16000775/sa_sc.zip Contact: dzhu@wayne.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : We provide a Bioconductor package with quality assessment, processing and visualization tools for high-throughput sequencing data, with emphasis in ChIP-seq and RNA-seq studies. It includes detection of outliers and biases, inefficient immuno-precipitation and overamplification artifacts, de novo identification of read-rich genomic regions and visualization of the location and coverage of genomic region lists. Availability: www.bioconductor.org Contact: david.rossell@irbbarcelona.org Supplementary information: Supplementary data available at Bioinformatics online.

Description: Motivation: We study a stochastic method for approximating the set of local minima in partial RNA folding landscapes associated with a bounded-distance neighbourhood of folding conformations. The conformations are limited to RNA secondary structures without pseudoknots. The method aims at exploring partial energy landscapes p L induced by folding simulations and their underlying neighbourhood relations. It combines an approximation of the number of local optima devised by Garnier and Kallel (2002) with a run-time estimation for identifying sets of local optima established by Reeves and Eremeev (2004). Results: The method is tested on nine sequences of length between 50 nt and 400 nt, which allows us to compare the results with data generated by RNAsubopt and subsequent barrier tree calculations. On the nine sequences, the method captures on average 92% of local minima with settings designed for a target of 95%. The run-time of the heuristic can be estimated by O ( n 2 D ln), where n is the sequence length, is the number of local minima in the partial landscape p L under consideration and D is the maximum number of steepest descent steps in attraction basins associated with p L . Contact: a.albrecht@qub.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: RNA-seq is a powerful technology for the study of transcriptome profiles that uses deep-sequencing technologies. Moreover, it may be used for cellular phenotyping and help establishing the etiology of diseases characterized by abnormal splicing patterns. In RNA-Seq, the exact nature of splicing events is buried in the reads that span exon–exon boundaries. The accurate and efficient mapping of these reads to the reference genome is a major challenge. Results: We developed PASSion, a pattern growth algorithm-based pipeline for splice site detection in paired-end RNA-Seq reads. Comparing the performance of PASSion to three existing RNA-Seq analysis pipelines, TopHat, MapSplice and HMMSplicer, revealed that PASSion is competitive with these packages. Moreover, the performance of PASSion is not affected by read length and coverage. It performs better than the other three approaches when detecting junctions in highly abundant transcripts. PASSion has the ability to detect junctions that do not have known splicing motifs, which cannot be found by the other tools. Of the two public RNA-Seq datasets, PASSion predicted ~ 137 000 and 173 000 splicing events, of which on average 82 are known junctions annotated in the Ensembl transcript database and 18% are novel. In addition, our package can discover differential and shared splicing patterns among multiple samples. Availability: The code and utilities can be freely downloaded from https://trac.nbic.nl/passion and ftp://ftp.sanger.ac.uk/pub/zn1/passion Contact: y.zhang@lumc.nl ; k.ye@lumc.nl Supplementary information: Supplementary data are available at Bioinformatics online.

Description: Motivation: The completion of 168 genome sequences from a single population of Drosophila melanogaster provides a global view of genomic variation and an understanding of the evolutionary forces shaping the patterns of DNA polymorphism and divergence along the genome. Results: We present the ‘Population Drosophila Browser’ (PopDrowser), a new genome browser specially designed for the automatic analysis and representation of genetic variation across the D. melanogaster genome sequence. PopDrowser allows estimating and visualizing the values of a number of DNA polymorphism and divergence summary statistics, linkage disequilibrium parameters and several neutrality tests. PopDrowser also allows performing custom analyses on-the-fly using user-selected parameters. Availability: PopDrowser is freely available from http://PopDrowser.uab.cat . Contact: miquel.ramia@uab.cat

Description: Motivation: Probabilistic approaches for inferring transcription factor binding sites (TFBSs) and regulatory motifs from DNA sequences have been developed for over two decades. Previous work has shown that prediction accuracy can be significantly improved by incorporating features such as the competition of multiple transcription factors (TFs) for binding to nearby sites, the tendency of TFBSs for co-regulated TFs to cluster and form cis -regulatory modules and explicit evolutionary modeling of conservation of TFBSs across orthologous sequences. However, currently available tools only incorporate some of these features, and significant methodological hurdles hampered their synthesis into a single consistent probabilistic framework. Results: We present MotEvo, a integrated suite of Bayesian probabilistic methods for the prediction of TFBSs and inference of regulatory motifs from multiple alignments of phylogenetically related DNA sequences, which incorporates all features just mentioned. In addition, MotEvo incorporates a novel model for detecting unknown functional elements that are under evolutionary constraint, and a new robust model for treating gain and loss of TFBSs along a phylogeny. Rigorous benchmarking tests on ChIP-seq datasets show that MotEvo's novel features significantly improve the accuracy of TFBS prediction, motif inference and enhancer prediction. Availability: Source code, a user manual and files with several example applications are available at www.swissregulon.unibas.ch . Contact: erik.vannimwegen@unibas.ch Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : We present LaTcOm, a new web tool, which offers several alternative methods for ‘rare codon cluster’ (RCC) identification from a single and simple graphical user interface. In the current version, three RCC detection schemes are implemented: the recently described %MinMax algorithm and a simplified sliding window approach, along with a novel modification of a linear-time algorithm for the detection of maximally scoring subsequences tailored to the RCC detection problem. Among a number of user tunable parameters, several codon-based scales relevant for RCC detection are available, including tRNA abundance values from Escherichia coli and several codon usage tables from a selection of genomes. Furthermore, useful scale transformations may be performed upon user request (e.g. linear, sigmoid). Users may choose to visualize RCC positions within the submitted sequences either with graphical representations or in textual form for further processing. Availability: LaTcOm is freely available online at the URL http://troodos.biol.ucy.ac.cy/latcom.html . Contact: vprobon@ucy.ac.cy Supplementary information: Supplementary data are available at Bioinformatics online.

Description: : Many existing databases annotate experimentally characterized single nucleotide polymorphisms (SNPs). Each non-synonymous SNP (nsSNP) changes one amino acid in the gene product (single amino acid substitution;SAAS). This change can either affect protein function or be neutral in that respect. Most polymorphisms lack experimental annotation of their functional impact. Here, we introduce SNPdbe—SNP database of effects, with predictions of computationally annotated functional impacts of SNPs. Database entries represent nsSNPs in dbSNP and 1000 Genomes collection, as well as variants from UniProt and PMD. SAASs come from 〉2600 organisms; ‘human’ being the most prevalent. The impact of each SAAS on protein function is predicted using the SNAP and SIFT algorithms and augmented with experimentally derived function/structure information and disease associations from PMD, OMIM and UniProt. SNPdbe is consistently updated and easily augmented with new sources of information. The database is available as an MySQL dump and via a web front end that allows searches with any combination of organism names, sequences and mutation IDs. Availability: http://www.rostlab.org/services/snpdbe Contact: schaefer@rostlab.org ; snpdbe@rostlab.org

Description: : We have implemented in a single package all the features required for extracting, visualizing and manipulating fully conserved positions as well as those with a family-dependent conservation pattern in multiple sequence alignments. The program allows, among other things, to run different methods for extracting these positions, combine the results and visualize them in protein 3D structures and sequence spaces. Availability and implementation: JDet is a multiplatform application written in Java. It is freely available, including the source code, at http://csbg.cnb.csic.es/JDet . The package includes two of our recently developed programs for detecting functional positions in protein alignments ( Xdet and S3Det ), and support for other methods can be added as plug-ins. A help file and a guided tutorial for JDet are also available. Contact: pazos@cnb.csic.es

Description: : VarSifter is a graphical software tool for desktop computers that allows investigators of varying computational skills to easily and quickly sort, filter, and sift through sequence variation data. A variety of filters and a custom query framework allow filtering based on any combination of sample and annotation information. By simplifying visualization and analyses of exome-scale sequence variation data, this program will help bring the power and promise of massively-parallel DNA sequencing to a broader group of researchers. Availability and Implementation: VarSifter is written in Java, and is freely available in source and binary versions, along with a User Guide, at http://research.nhgri.nih.gov/software/VarSifter/ . Contact: mullikin@mail.nih.gov Supplementary Information: Additional figures and methods available online at the journal's website.

Description: Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades mRNAs containing premature termination codons (PTCs). SMG-1-mediated Upf1 phosphorylation takes place in the decay inducing complex (DECID), which contains a ribosome, release factors, Upf1, SMG-1, an exon junction complex (EJC) and a PTC-mRNA. However, the significance and the consequence of Upf1 phosphorylation remain to be clarified. Here, we demonstrate that SMG-6 binds to a newly identified phosphorylation site in Upf1 at N-terminal threonine 28, whereas the SMG-5:SMG-7 complex binds to phosphorylated serine 1096 of Upf1. In addition, the binding of the SMG-5:SMG-7 complex to Upf1 resulted in the dissociation of the ribosome and release factors from the DECID complex. Importantly, the simultaneous binding of both the SMG-5:SMG-7 complex and SMG-6 to phospho-Upf1 are required for both NMD and Upf1 dissociation from mRNA. Thus, the SMG-1-mediated phosphorylation of Upf1 creates a binding platforms for the SMG-5:SMG-7 complex and for SMG-6, and triggers sequential remodeling of the mRNA surveillance complex for NMD induction and recycling of the ribosome, release factors and NMD factors.

Description: The ciliate Tetrahymena thermophila is an important eukaryotic model organism that has been used in pioneering studies of general phenomena, such as ribozymes, telomeres, chromatin structure and genome reorganization. Recent work has shown that Tetrahymena has many classes of small RNA molecules expressed during vegetative growth or sexual reorganization. In order to get an overview of medium-sized (40–500 nt) RNAs expressed from the Tetrahymena genome, we created a size-fractionated cDNA library from macronuclear RNA and analyzed 80 RNAs, most of which were previously unknown. The most abundant class was small nucleolar RNAs (snoRNAs), many of which are formed by an unusual maturation pathway. The modifications guided by the snoRNAs were analyzed bioinformatically and experimentally and many Tetrahymena -specific modifications were found, including several in an essential, but not conserved domain of ribosomal RNA. Of particular interest, we detected two methylations in the 5'-end of U6 small nuclear RNA (snRNA) that has an unusual structure in Tetrahymena . Further, we found a candidate for the first U8 outside metazoans, and an unusual U14 candidate. In addition, a number of candidates for new non-coding RNAs were characterized by expression analysis at different growth conditions.

Description: DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.

Description: The discovery of a plethora of small non-coding RNAs (ncRNAs) has fundamentally changed our understanding of how genes are regulated. In this study, we employed the power of deep sequencing of RNA (RNA-seq) to examine the repertoire of ncRNAs present in small ribonucleoprotein particles (RNPs) of Trypanosoma brucei , an important protozoan parasite. We identified new C/D and H/ACA small nucleolar RNAs (snoRNAs), as well as tens of putative novel non-coding RNAs; several of these are processed from trans -spliced and polyadenylated transcripts. The RNA-seq analysis provided information on the relative abundance of the RNAs, and their 5'- and 3'-termini. The study demonstrated that three highly abundant snoRNAs are involved in rRNA processing and highlight the unique trypanosome-specific repertoire of these RNAs. Novel RNAs were studied using in situ hybridization, association in RNP complexes, and ‘RNA walk’ to detect interaction with their target RNAs. Finally, we showed that the abundance of certain ncRNAs varies between the two stages of the parasite, suggesting that ncRNAs may contribute to gene regulation during the complex parasite’s life cycle. This is the first study to provide a whole-genome analysis of the large repertoire of small RNPs in trypanosomes.

Description: The FokI restriction endonuclease is a monomeric protein that recognizes an asymmetric sequence and cleaves both DNA strands at fixed loci downstream of the site. Its single active site is positioned initially near the recognition sequence, distant from its downstream target 13 nucleotides away. Moreover, to cut both strands, it has to recruit a second monomer to give an assembly with two active sites. Here, the individual steps in the FokI reaction pathway were examined by fluorescence resonance energy transfer (FRET). To monitor DNA binding and domain motion, a fluorescence donor was attached to the DNA, either downstream or upstream of the recognition site, and an acceptor placed on the catalytic domain of the protein. A FokI variant incapable of dimerization was also employed, to disentangle the signal due to domain motion from that due to protein association. Dimerization was monitored separately by using two samples of FokI labelled with donor and acceptor, respectively. The stopped-flow studies revealed a complete reaction pathway for FokI, both the sequence of events and the kinetics of each individual step.

Description: Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and rapidly degrades mRNAs containing premature termination codons (PTC). The strength of the NMD response appears to reflect multiple determinants on a target mRNA. We have previously reported that mRNAs containing PTCs in close proximity to the translation initiation codon (AUG-proximal PTCs) can substantially evade NMD. Here, we explore the mechanistic basis for this NMD resistance. We demonstrate that translation termination at an AUG-proximal PTC lacks the ribosome stalling that is evident in an NMD-sensitive PTC. This difference is associated with demonstrated interactions of the cytoplasmic poly(A)-binding protein 1, PABPC1, with the cap-binding complex subunit, eIF4G and the 40S recruitment factor eIF3 as well as the ribosome release factor, eRF3. These interactions, in combination, underlie critical 3'–5' linkage of translation initiation with efficient termination at the AUG-proximal PTC and contribute to an NMD-resistant PTC definition at an early phase of translation elongation.

Description: The yeast RNA helicase Dhh1p has been shown to associate with components of mRNA decay and is involved in mRNA decapping and degradation. An RNA-binding protein, Rbp1p, is known to bind to the 3'-UTR of porin ( POR1 ) mRNA, and induces mRNA decay by an uncharacterized mechanism. Here, we show that Dhh1p can associate with POR1 mRNA and specifically promote POR1 mRNA decay via its interaction with Rbp1p. As compared to its mammalian homolog RCK/p54/DDX6, Dhh1p has a unique and long extension at its C-terminus. Interestingly, this non-conserved C-terminal region of Dhh1p is required for interaction with Rbp1p and modulating Rbp1p-mediated POR1 mRNA decay. Notably, expression of a C-terminal 81-residue deleted Dhh1p can fully complement the growth defect of a dhh1 strain and retains its function in regulating the mRNA level of an RNA-binding protein Edc1p. Moreover, mammalian DDX6 became capable of interacting with Rbp1p and could confer Rbp1p-mediated POR1 mRNA decay in the dhh1 strain upon fusion to the C-terminal unique region of Dhh1p. Thus, we propose that the non-conserved C-terminus of Dhh1p plays a role in defining specific interactions with mRNA regulatory factors that promote distinct mRNA decay.

Description: DNA replication is strictly regulated through a sequence of steps that involve many macromolecular protein complexes. One of them is the replicative helicase, which is required for initiation and elongation phases. A MCM helicase found as a prophage in the genome of Bacillus cereus is fused with a primase domain constituting an integrative arrangement of two essential activities for replication. We have isolated this helicase–primase complex (BcMCM) showing that it can bind DNA and displays not only helicase and primase but also DNA polymerase activity. Using single-particle electron microscopy and 3D reconstruction, we obtained structures of BcMCM using ATPS or ADP in the absence and presence of DNA. The complex depicts the typical hexameric ring shape. The dissection of the unwinding mechanism using site-directed mutagenesis in the Walker A, Walker B, arginine finger and the helicase channels, suggests that the BcMCM complex unwinds DNA following the extrusion model similarly to the E1 helicase from papillomavirus.

Description: Single-stranded RNAs (ssRNAs) are ubiquitous RNA elements that serve diverse functional roles. Much of our understanding of ssRNA conformational behavior is limited to structures in which ssRNA directly engages in tertiary interactions or is recognized by proteins. Little is known about the structural and dynamic behavior of free ssRNAs at atomic resolution. Here, we report the collaborative application of nuclear magnetic resonance (NMR) and replica exchange molecular dynamics (REMD) simulations to characterize the 12 nt ssRNA tail derived from the prequeuosine riboswitch. NMR carbon spin relaxation data and residual dipolar coupling measurements reveal a flexible yet stacked core adopting an A-form-like conformation, with the level of order decreasing toward the terminal ends. An A-to-C mutation within the polyadenine tract alters the observed dynamics consistent with the introduction of a dynamic kink. Pre-ordering of the tail may increase the efficacy of ligand binding above that achieved by a random-coil ssRNA. The REMD simulations recapitulate important trends in the NMR data, but suggest more internal motions than inferred from the NMR analysis. Our study unmasks a previously unappreciated level of complexity in ssRNA, which we believe will also serve as an excellent model system for testing and developing computational force fields.

Description: The kallikrein ( KLK ) gene family comprises the largest uninterrupted locus of serine proteases in the human genome and represents a notable case for studying the evolutionary fate of duplicated genes. In primates, a recent duplication event gave rise to KLK2 and KLK3 , both encoding essential proteins for the cascade of seminal plasma liquefaction. We reconstructed the evolutionary history of KLK2 and KLK3 by comparative analysis of the orthologous sequences from 22 primate species, calculated d N / d S ratios, and addressed the hypothesis of coevolution with their substrates, the semenogelins (SEMG1 and SEMG2). Our findings support the placement of the KLK2–KLK3 duplication in the Catarrhini ancestor and unveil the frequent loss of KLK2 throughout primate evolution by different genomic mechanisms, including unequal crossing-over, deletions, and pseudogenization. We provide evidences for an adaptive evolution of KLK3 toward an expanded enzymatic spectrum, with an effect on the hydrolysis of semen coagulum. Furthermore, we found associations between mating system, the number of SEMG repeat units, and the number of functional KLK2 and KLK3 , suggesting complex evolutionary dynamics shaped by reproductive biology.

Description: Insect cells are widely used for recombinant glycoprotein production, but they cannot provide the glycosylation patterns required for some biotechnological applications. This problem has been addressed by genetically engineering insect cells to express mammalian genes encoding various glycoprotein glycan processing functions. However, for various reasons, the impact of a mammalian cytosine-5'-monophospho (CMP)-sialic acid transporter has not yet been examined. Thus, we transformed Spodoptera frugiperda (Sf9) cells with six mammalian genes to generate a new cell line, SfSWT-4, that can produce sialylated glycoproteins when cultured with the sialic acid precursor, N -acetylmannosamine. We then super-transformed SfSWT-4 with a human CMP-sialic acid transporter (hCSAT) gene to isolate a daughter cell line, SfSWT-6, which expressed the hCSAT gene in addition to the other mammalian glycogenes. SfSWT-6 cells had higher levels of cell surface sialylation and also supported higher levels of recombinant glycoprotein sialylation, particularly when cultured with low concentrations of N -acetylmannosamine. Thus, hCSAT expression has an impact on glycoprotein sialylation, can reduce the cost of recombinant glycoprotein production and therefore should be included in ongoing efforts to glycoengineer the baculovirus-insect cell system. The results of this study also contributed new insights into the endogenous mechanism and potential mechanisms of CMP-sialic acid accumulation in the Golgi apparatus of lepidopteran insect cells.

Description: Chondroitin sulfate (CS) chains regulate the development of the central nervous system in vertebrates and are linear polysaccharides consisting of variously sulfated repeating disaccharides, [–4GlcUAβ1-3GalNAcβ1–] n , where GlcUA and GalNAc represent d -glucuronic acid and N -acetyl- d -galactosamine, respectively. CS chains containing D-disaccharide units [GlcUA(2- O -sulfate)-GalNAc(6- O -sulfate)] are involved in the development of cerebellar Purkinje cells and neurite outgrowth-promoting activity through interaction with a neurotrophic factor, pleiotrophin, resulting in the regulation of signaling. In this study, to obtain further structural information on the CS chains containing d -disaccharide units involved in brain development, oligosaccharides containing D-units were isolated from a shark fin cartilage. Seven novel hexasaccharide sequences, O-D-D, A-D-D, C-D-D, E-A-D, D-D-C, E-D-D and A-B-D, in addition to three previously reported sequences, C-A-D, C-D-C and A-D-A, were isolated from a CS preparation of shark fin cartilage after exhaustive digestion with chondroitinase AC-I, which cannot act on the galactosaminidic linkages bound to D-units. The symbol stands for a 4,5-unsaturated bond of uronic acids, whereas A, B, C, D, E and O represent [GlcUA-GalNAc(4- O -sulfate)], [GlcUA(2- O -sulfate)-GalNAc(4- O -sulfate)], [GlcUA-GalNAc(6- O -sulfate)], [GlcUA(2- O -sulfate)-GalNAc(6- O -sulfate)], [GlcUA-GalNAc(4- O -, 6- O -sulfate)] and [GlcUA-GalNAc], respectively. In binding studies using an anti-CS monoclonal antibody, MO-225, the epitopes of which are involved in cerebellar development in mammals, novel epitope structures, A-D-A, A-D-D and A-B-D, were revealed. Hexasaccharides containing two consecutive D-units or a B-unit will be useful for the structural and functional analyses of CS chains particularly in the neuroglycobiological fields.

Description: Protein O -fucosyltransferase 1 (Pofut1) and protein O -fucosyltransferase 2 (Pofut2) add O -linked fucose at distinct consensus sequences in properly folded epidermal growth factor (EGF)-like repeats and thrombospondin type-1 (TSR) repeats, respectively. Glycan chain elongation past O -fucose can occur to yield a tetrasaccharide on EGF repeats and a disaccharide on TSRs. Elimination of Pofut1 in mice causes embryonic lethality with Notch-like phenotypes demonstrating that O -fucosylation of Notch is essential for its function. Similarly, elimination of Pofut2 results in an early embryonic lethal phenotype in mice, although the molecular mechanism for the lethality is unknown. The recent development of sugar analogs has revolutionized the study of glycans by providing a convenient method for labeling and tracking glycosylation. In order to study O -fucosylation, we took advantage of the recently developed reporter, 6-alkynyl fucose. Using the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), or "click" reaction, azido-biotin allows tagging and detection of 6AF-modified proteins. Here we examine whether proteins containing EGF repeats or TSRs with O -fucose consensus sequences are specifically modified with 6AF in cell culture. Using mass spectrometry (MS), we demonstrate that 6AF is efficiently incorporated onto the appropriate consensus sequences on EGF repeats and TSRs. Furthermore, the elongation of the O -fucose monosaccharide on EGF repeats and TSRs is not hampered when 6AF is used. These results show that 6AF is efficiently utilized in a truly bioorthogonal manner by Pofut1, Pofut2 and the enzymes that elongate O -fucose, providing evidence that 6AF is a significant new tool in the study of protein O -fucosylation.

Description: Xanthan is a polysaccharide secreted by Xanthomonas campestris that contains pentameric repeat units. The biosynthesis of xanthan involves an operon composed of 12 genes ( gumB to gumM ). In this study, we analyzed the proteins encoded by gumB and gumC . Membrane fractionation showed that GumB was mainly associated with the outer membrane, whereas GumC was an inner membrane protein. By in silico analysis and specific globomycin inhibition, GumB was characterized as a lipoprotein. By reporter enzyme assays, GumC was shown to contain two transmembrane segments flanking a large periplasmic domain. We confirmed that gumB and gumC mutant strains uncoupled the synthesis of the lipid-linked repeat unit from the polymerization process. We studied the effects of gumB and gumC gene amplification on the production, composition and viscosity of xanthan. Overexpression of GumB, GumC or GumB and GumC simultaneously did not affect the total amount or the chemical composition of the polymer. GumB overexpression did not affect xanthan viscosity; however, a moderate increase in xanthan viscosity was achieved when GumC protein levels were increased 5-fold. Partial degradation of GumC was observed when only that protein was overexpressed; but co-expression of GumB and GumC diminished GumC degradation and resulted in higher xanthan viscosity than individual GumB or GumC overexpression. Compared with xanthan from the wild-type strain, longer polymer chains from the strain that simultaneously overexpressed GumB and GumC were observed by atomic force microscopy. Our results suggest that GumB–GumC protein levels modulate xanthan chain length, which results in altered polymer viscosity.

Description: Bifidobacterium bifidum is one of the most frequently found bifidobacteria in the intestines of newborn infants. We previously reported that B. bifidum possesses unique metabolic pathways for O -linked glycans on gastrointestinal mucin (Yoshida E, Sakurama H, Kiyohara M, Nakajima M, Kitaoka M, Ashida H, Hirose J, Katayama T, Yamamoto K, Kumagai H. 2012. Bifidobacterium longum subsp. infantis uses two different β-galactosidases for selectively degrading type-1 and type-2 human milk oligosaccharides. Glycobiology . 22:361–368). The nonreducing termini of O -linked glycans on mucin are frequently covered with histo-blood group antigens. Here, we identified a gene agabb from B. bifidum JCM 1254, which encodes glycoside hydrolase (GH) family 110 α-galactosidase. AgaBb is a 1289-amino acid polypeptide containing an N-terminal signal sequence, a GH110 domain, a carbohydrate-binding module (CBM) 51 domain, a bacterial Ig-like (Big) 2 domain and a C-terminal transmembrane region, in this order. The recombinant enzyme expressed in Escherichia coli hydrolyzed α1,3-linked Gal in branched blood group B antigen [Galα1-3(Fucα1-2)Galβ1-R], but not in a linear xenotransplantation antigen (Galα1-3Galβ1-R). The enzyme also acted on group B human salivary mucin and erythrocytes. We also revealed that CBM51 specifically bound blood group B antigen using both isothermal titration calorimetry and a solid-phase binding assay, and it enhanced the affinity of the enzyme toward substrates with multivalent B antigens. We suggest that this enzyme plays an important role in degrading B antigens to acquire nutrients from mucin oligosaccharides in the gastrointestinal tracts.

Description: Biovolume is commonly used as a size descriptor in the study of phytoplankton ecology. Usually, biovolume is not measured directly but is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. This commonly used method allows visualization and measurement in two dimensions (2D) yielding no information at all on the third dimension of phytoplankton cells. Inaccurate biovolume assessment resulting from geometric approximation leads to erroneous interpretation of eco-physiological processes and morpho-functional traits. Here, we use confocal microscopy coupled with an image analysis system (NIS Elements AR software, Nikon) to determine directly shape and biovolume by means a 3D reconstruction of Coscinodiscus cf . granii specimens. We evaluate the accuracy of current methods by comparing the results obtained using geometric models with direct biovolume and shape. We find that calculation of biovolume by approximation to geometric models (cylindrical and complex shapes) leads to a significant overestimation with respect to direct volume. We also propose a data-driven formula for calculating the biovolume of Coscinodiscus cf. granii specimens based on diameter or other linear measurements.

Description: The phytoplankton colour index (PCI) of the Continuous Plankton Recorder (CPR) survey is an in situ measure of ocean colour, which is considered a proxy of the phytoplankton biomass. PCI has been extensively used to describe the major spatiotemporal patterns of phytoplankton in the North Atlantic Ocean and North Sea since 1931. Regardless of its wide application, the lack of an adequate evaluation to test the PCI's quantitative nature is an important limitation. To address this concern, a field trial over the main production season has been undertaken to assess the numerical values assigned by previous investigations for each category of the greenness of the PCI. CPRs were towed across the English Channel from Roscoff to Plymouth consecutively for each of 8 months producing 76 standard CPR samples, each representing 10 nautical miles of tow. The results of this experiment test and update the PCI methodology, and confirm the validity of this long-term in situ ocean colour data set. In addition, using a 60-year time series of the PCI of the western English Channel, a comparison is made between the previous and the current revised experimental calculations of PCI.

Description: We investigated the selective predation of mixotrophic and heterotrophic flagellates (MF and HF) on different heterotrophic prokaryote phylotypes (HPP; Bacteria + Archaea ) living in natural assemblages from oligotrophic Antarctic lakes. In situ prey preference was analyzed for the first time on different mixotrophic taxa ( Pseudopedinella sp., Ochromonas -like cells, Chrysophyceae 〉5 µm). The relative abundances of seven different HPP hybridized by CARD-FISH (catalyzed reporter deposition-fluorescent in situ hybridization) in natural community were compared with the proportions of hybridized cells inside digestive vacuoles. Our results showed some general trends to selectivity over some HPP. Alphaproteobacteria and Betaproteobacteria were the most abundant groups, and strikingly, a negative selection trend was detected in most samples by all bacterivorous protists. In contrast, for Actinobacteria a positive selection trend was observed in most samples, whereas Bacteroidetes seemed to be randomly preyed upon. Interestingly, similar prey preferences were observed in all bacterivorous flagellates. Our results suggest that phylogenetic affiliation determines part of the process of prey selection by protists in these lakes. Nevertheless, other features, such as cell size, morphology and the presence of the S-layer, might also significantly contribute to prey selectivity on the HPP.

Description: The invasive ctenophore Mnemiopsis leidyi , which now may be established in the NW Mediterranean, is a voracious predator of zooplankton and ichthyoplankton. Pelagia noctiluca , an abundant scyphomedusa there, eats other gelatinous species. We measured predation, digestion and escape when different sizes of medusae fed on ctenophores. Clearance rates increased with predator size and ingestion rates increased with prey concentration. Digestion times were longer when medusae were smaller than the ctenophores. All large ctenophores escaped from the medusae, but small ctenophores were ingested completely. Pelagia noctiluca is potentially an important predator of M. leidyi that may help control populations of this ctenophore.

Description: Allometric scaling rules have been classically used in plankton ecology to describe how the maximum ingestion rate ( I max ) under steady-state conditions changes with the body size of the consumer. Empirical and theoretical concerns, however, motivate a more accurate and mechanistic description of size–ingestion relations. Here, I propose to relate I max to the digestive surface area, which expresses the capacity in preprocessing and digesting food items. This surface area depends on both the body size and the optimal prey size of the consumer. The allometry in I max , hence, includes a second major variable which describes different feeding modes within a consumer size class. Species with a small optimal-prey-to-predator-size ratio and, thus, a small "internal" surface-to-volume ratio, as is typical for filter feeders, have large intra-body transport lengths and lower I max than raptorial-feeding species of the same body size. Digestive surface scaling tries to mechanistically accommodate feeding ecology, physiology and geometry. It does not explicitly resolve further possible factors affecting maximal ingestion such as nutritional quality. Still, digestive surface scaling explains the variability in published data compilations better than classical approaches when applied to the entire plankton size range. This is corroborated in further applications where the theory precisely fits anomalously steep scaling relations reported for heterotrophic nanoflagellates, ctenophores and a scyphomedusa. By introducing feeding mode and related morphological diversity into the size-dependency in ingestion rates, digestive surface scaling can be expected to improve the accuracy of size-based plankton models.

Description: The photosynthetic capacity of phytoplankton is frequently limited by the availability of nutrients. To explore the mechanism of the effect of nutrient availability on photosynthesis, batch cultures of a marine diatom Skeletonema costatum (Greville) Cleve were carried out under different nitrate (N: 0–220.75 µM) and phosphate (P: 0–9.075 µM) concentrations. Changes of photosynthetic characters, including the photosynthetic rate (measured by O 2 evolution) and the rbcL mRNA content (encoding the large subunit of the Calvin cycle enzyme, RuBisCO), were both studied. Within the range tested, both the photosynthetic rate and rbcL transcript levels correlated significantly with N and P concentrations in the medium. Additionally, the photosynthetic rates and rbcL transcript levels were both growth dependent. Significant correlations were also found between the abundance of rbcL mRNA and the photosynthetic rate ( R 2 = 0.800), rbcL and growth rate ( R 2 = 0.855), the photosynthetic rate and growth rate ( R 2 = 0.815). These results are consistent with N and P nutrition-regulating photosynthesis from the transcriptional levels of rbcL . The rbcL transcript level has the potential to be a good marker of algal growth and primary productivity.

Description: Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man 5 GlcNAc 2 -PP-Dol resulting in the formation of Man 6 GlcNAc 2 -PP-Dol, which is then further processed to the final precursor oligosaccharide Glc 3 Man 9 GlcNAc 2 for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the alg3 strain of S. cerevisiae . Inactivation of alg3 in S. commune resulted in the production of predominantly Man 3 GlcNAc 2 protein-linked N -glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.

Description: We previously demonstrated that Siglec-15, a member of the Siglec family of glycan-recognition proteins, is expressed on a subset of macrophages and preferentially recognizes the sialyl-Tn (sTn) antigen, a tumor-associated glycan structure. In this study, we report on the biological significance of the Siglec-15-mediated interaction between monocytes/macrophages and cancer cells. Siglec-15 is expressed on tumor-associated macrophages (TAMs) in various human tumor tissues. We further demonstrated that its expression is substantially elevated in macrophage colony-stimulating factor-induced M2-like macrophages, which produced more transforming growth factor-β (TGF-β) in response to sTn-positive cells than to negative cells. We designed a co-culture model of THP-1 (human monocytic leukemia) cells and H157 (human lung carcinoma) cells mimicking the interaction between monocytes/macrophages and cancer cells that recapitulated the enhanced TGF-β production in Siglec-15 expressing THP-1 cells by the cellular interaction with sTn expressing H157 cells. The enhanced TGF-β production required a direct interaction between the two cell lines through sialic acids. Siglec-15 associates with adaptor protein DNAX activation protein of 12 kDa (DAP12) at the binding determinant Lys 274 in the transmembrane domain and transduces a signal to spleen tyrosine kinase (Syk). The enhanced TGF-β secretion was significantly attenuated by Syk inhibitor treatment of THP-1 cells or by substitution of the Siglec-15 Lys 274 to Ala, which disrupts the molecular interaction between Siglec15 and DAP12. These findings indicate that Siglec-15 recognizes the tumoral sTn antigen and transduces a signal for enhanced TGF-β secretion in TAMs and further suggest that Siglec-15 on macrophages may contribute to tumor progression by the TGF-β-mediated modulation of intratumoral microenvironments.

Description: We suggest that podocysts are responsible for bloom versus non-bloom years of the giant jellyfish Nemopilema nomurai . They were capable of dormancy for at least 6 years. Their excystment was very rare (ca. 1%) in well-aerated seawater at constant temperature (19°C), but increased significantly under exposure to abnormally high temperatures (≥27°C), low salinities (8–24), hypoxia (1.0 mg O 2 L –1 ) and burial in organic-rich mud. Their behavior (e.g. maintaining dormancy or mass excystment) may influence medusa population sizes in any given year.

Description: Molecular probes were developed for the dinoflagellate genus Azadinium to discriminate among three taxa difficult to differentiate by light microscopy. This genus contains azaspiracid toxin-producing Azadinium spinosum , but also non-toxigenic species. Quantitative polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were applied to cultured isolates and Azadinium -spiked field plankton. Molecular methods were highly specific and sensitive in the unambiguous detection of Azadinium , and thus are valuable for routine plankton, biogeographic and phylogenetic investigations.

Description: The in situ reproductive activity of the copepods Centropages ponticus and Temora stylifera was investigated in autumn 2006 in the Bizerte Channel in relation to abiotic factors and phytoplankton composition. Egg production rate (EPR), hatching success (HS) and nauplii survival rate of both species were measured from samples taken daily over 45 days from September 28 to November 11 and then weekly over 8 supplementary weeks. Over this period, C. ponticus and T. stylifera had mean EPRs of 13.7 ± 0.3 and 35.2 ± 3.8 eggs f –1 day –1 , and mean HS of 24.4 ± 1.6 and 50.4 ± 5.5%, respectively. EPR and HS remained fairly stable for C. ponticus throughout the study period compared with T. stylifera in which reproductive traits fluctuated considerably. Changes in EPR and HS for both species resulted from environmental parameter variations and in particular, from phytoplankton composition, with slight decreasing EPR and HS observed after the important phytoplankton blooms. Centropages ponticus , a lagoon species, was able to withstand high turbidity, whereas T. stylifera , a marine species, had low EPR and HS under turbid conditions. The low HS recorded for C. ponticus , in particular in December, may be due to production of resting eggs.

Description: The 20th annual Database Issue of Nucleic Acids Research includes 176 articles, half of which describe new online molecular biology databases and the other half provide updates on the databases previously featured in NAR and other journals. This year’s highlights include two databases of DNA repeat elements; several databases of transcriptional factors and transcriptional factor-binding sites; databases on various aspects of protein structure and protein–protein interactions; databases for metagenomic and rRNA sequence analysis; and four databases specifically dedicated to Escherichia coli . The increased emphasis on using the genome data to improve human health is reflected in the development of the databases of genomic structural variation (NCBI’s dbVar and EBI’s DGVa), the NIH Genetic Testing Registry and several other databases centered on the genetic basis of human disease, potential drugs, their targets and the mechanisms of protein–ligand binding. Two new databases present genomic and RNAseq data for monkeys, providing wealth of data on our closest relatives for comparative genomics purposes. The NAR online Molecular Biology Database Collection, available at http://www.oxfordjournals.org/nar/database/a/ , has been updated and currently lists 1512 online databases. The full content of the Database Issue is freely available online on the Nucleic Acids Research website ( http://nar.oxfordjournals.org/ ).

Description: The International Nucleotide Sequence Database Collaboration (INSDC; http://www.insdc.org ), one of the longest-standing global alliances of biological data archives, captures, preserves and provides comprehensive public domain nucleotide sequence information. Three partners of the INSDC work in cooperation to establish formats for data and metadata and protocols that facilitate reliable data submission to their databases and support continual data exchange around the world. In this article, the INSDC current status and update for the year of 2012 are presented. Among discussed items of international collaboration meeting in 2012, BioSample database and changes in submission are described as topics.

Description: The European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/ ) collects, maintains and presents comprehensive nucleic acid sequence and related information as part of the permanent public scientific record. Here, we provide brief updates on ENA content developments and major service enhancements in 2012 and describe in more detail two important areas of development and policy that are driven by ongoing growth in sequencing technologies. First, we describe the ENA data warehouse, a resource for which we provide a programmatic entry point to integrated content across the breadth of ENA. Second, we detail our plans for the deployment of CRAM data compression technology in ENA.

Description: The University of California Santa Cruz (UCSC) Genome Browser ( http://genome.ucsc.edu ) offers online public access to a growing database of genomic sequence and annotations for a wide variety of organisms. The Browser is an integrated tool set for visualizing, comparing, analysing and sharing both publicly available and user-generated genomic datasets. As of September 2012, genomic sequence and a basic set of annotation ‘tracks’ are provided for 63 organisms, including 26 mammals, 13 non-mammal vertebrates, 3 invertebrate deuterostomes, 13 insects, 6 worms, yeast and sea hare. In the past year 19 new genome assemblies have been added, and we anticipate releasing another 28 in early 2013. Further, a large number of annotation tracks have been either added, updated by contributors or remapped to the latest human reference genome. Among these are an updated UCSC Genes track for human and mouse assemblies. We have also introduced several features to improve usability, including new navigation menus. This article provides an update to the UCSC Genome Browser database, which has been previously featured in the Database issue of this journal.

Description: HEXEvent ( http://hexevent.mmg.uci.edu ) is a new database that permits the user to compile genome-wide exon data sets of human internal exons showing selected splicing events. User queries can be customized based on the type and the frequency of alternative splicing events. For each splicing version of an exon, an ESTs count is given, specifying the frequency of the event. A user-specific definition of constitutive exons can be entered to designate an exon exclusion level still acceptable for an exon to be considered as constitutive. Similarly, the user has the option to define a maximum inclusion level for an exon to be called an alternatively spliced exon. Unlike other existing splicing databases, HEXEvent permits the user to easily extract alternative splicing information for individual, multiple or genome-wide human internal exons. Importantly, the generated data sets are downloadable for further analysis.

Description: The non-B DB, available at http://nonb.abcc.ncifcrf.gov , catalogs predicted non-B DNA-forming sequence motifs, including Z-DNA, G-quadruplex, A-phased repeats, inverted repeats, mirror repeats, direct repeats and their corresponding subsets: cruciforms, triplexes and slipped structures, in several genomes. Version 2.0 of the database revises and re-implements the motif discovery algorithms to better align with accepted definitions and thresholds for motifs, expands the non-B DNA-forming motifs coverage by including short tandem repeats and adds key visualization tools to compare motif locations relative to other genomic annotations. Non-B DB v2.0 extends the ability for comparative genomics by including re-annotation of the five organisms reported in non-B DB v1.0, human, chimpanzee, dog, macaque and mouse, and adds seven additional organisms: orangutan, rat, cow, pig, horse, platypus and Arabidopsis thaliana . Additionally, the non-B DB v2.0 provides an overall improved graphical user interface and faster query performance.

Description: The spliceosome is the extremely complex macromolecular machine responsible for pre-mRNA splicing. It assembles from five U-rich small nuclear RNAs (snRNAs) and over 200 proteins in a highly dynamic fashion. One important challenge to studying the spliceosome is simply keeping track of all these proteins, a situation further complicated by the variety of names and identifiers that exist in the literature for them. To facilitate studies of the spliceosome and its components, we created a database of spliceosome-associated proteins and snRNAs, which is available at http://spliceosomedb.ucsc.edu and can be queried through a simple browser interface. In the database, we cataloged the various names, orthologs and gene identifiers of spliceosome proteins to navigate the complex nomenclature of spliceosome proteins. We also provide links to gene and protein records for the spliceosome components in other databases. To navigate spliceosome assembly dynamics, we created tools to compare the association of spliceosome proteins with complexes that form at specific stages of spliceosome assembly based on a compendium of mass spectrometry experiments that identified proteins in purified splicing complexes. Together, the information in the database provides an easy reference for spliceosome components and will support future modeling of spliceosome structure and dynamics.

Description: The Eukaryotic Promoter Database (EPD), available online at http://epd.vital-it.ch , is a collection of experimentally defined eukaryotic POL II promoters which has been maintained for more than 25 years. A promoter is represented by a single position in the genome, typically the major transcription start site (TSS). EPD primarily serves biologists interested in analysing the motif content, chromatin structure or DNA methylation status of co-regulated promoter subsets. Initially, promoter evidence came from TSS mapping experiments targeted at single genes and published in journal articles. Today, the TSS positions provided by EPD are inferred from next-generation sequencing data distributed in electronic form. Traditionally, EPD has been a high-quality database with low coverage. The focus of recent efforts has been to reach complete gene coverage for important model organisms. To this end, we introduced a new section called EPDnew, which is automatically assembled from multiple, carefully selected input datasets. As another novelty, we started to use chromatin signatures in addition to mRNA 5'tags to locate promoters of weekly expressed genes. Regarding user interfaces, we introduced a new promoter viewer which enables users to explore promoter-defining experimental evidence in a UCSC genome browser window.

Description: CTCF is a highly conserved transcriptional regulator protein that performs diverse functions such as regulating gene expression and organizing the 3D structure of the genome. Here, we describe recent updates to a database of CTCF-binding sites, CTCFBSDB ( http://insulatordb.uthsc.edu/ ), which now contains almost 15 million CTCF-binding sequences in 10 species. Since the original publication of the database, studies of the 3D structure of the genome, such as those provided by Hi-C experiments, have suggested that CTCF plays an important role in mediating intra- and inter-chromosomal interactions. To reflect this important progress, we have integrated CTCF-binding sites with genomic topological domains defined using Hi-C data. Additionally, the updated database includes new features enabled by new CTCF-binding site data, including binding site occupancy and the ability to visualize overlapping CTCF-binding sites determined in separate experiments.

Description: Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM ( http://dbPTM.mbc.nctu.edu.tw/ ) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, 〉60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein–protein interaction and domain–domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Description: We here present The Online Protein Processing Resource (TOPPR; http://iomics.ugent.be/toppr/ ), an online database that contains thousands of published proteolytically processed sites in human and mouse proteins. These cleavage events were identified with COmbinded FRActional DIagonal Chromatography proteomics technologies, and the resulting database is provided with full data provenance. Indeed, TOPPR provides an interactive visual display of the actual fragmentation mass spectrum that led to each identification of a reported processed site, complete with fragment ion annotations and search engine scores. Apart from warehousing and disseminating these data in an intuitive manner, TOPPR also provides an online analysis platform, including methods to analyze protease specificity and substrate-centric analyses. Concretely, TOPPR supports three ways to retrieve data: (i) the retrieval of all substrates for one or more cellular stimuli or assays; (ii) a substrate search by UniProtKB/Swiss-Prot accession number, entry name or description; and (iii) a motif search that retrieves substrates matching a user-defined protease specificity profile. The analysis of the substrates is supported through the presence of a variety of annotations, including predicted secondary structure, known domains and experimentally obtained 3D structure where available. Across substrates, substrate orthologs and conserved sequence stretches can also be shown, with iceLogo visualization provided for the latter.

Description: We present the Database of Disordered Protein Prediction (D 2 P 2 ), available at http://d2p2.pro (including website source code). A battery of disorder predictors and their variants, VL-XT, VSL2b, PrDOS, PV2, Espritz and IUPred, were run on all protein sequences from 1765 complete proteomes (to be updated as more genomes are completed). Integrated with these results are all of the predicted (mostly structured) SCOP domains using the SUPERFAMILY predictor. These disorder/structure annotations together enable comparison of the disorder predictors with each other and examination of the overlap between disordered predictions and SCOP domains on a large scale. D 2 P 2 will increase our understanding of the interplay between disorder and structure, the genomic distribution of disorder, and its evolutionary history. The parsed data are made available in a unified format for download as flat files or SQL tables either by genome, by predictor, or for the complete set. An interactive website provides a graphical view of each protein annotated with the SCOP domains and disordered regions from all predictors overlaid (or shown as a consensus). There are statistics and tools for browsing and comparing genomes and their disorder within the context of their position on the tree of life.

Description: The goal of the Papillomavirus Episteme (PaVE) is to provide an integrated resource for the analysis of papillomavirus (PV) genome sequences and related information. The PaVE is a freely accessible, web-based tool ( http://pave.niaid.nih.gov ) created around a relational database, which enables storage, analysis and exchange of sequence information. From a design perspective, the PaVE adopts an Open Source software approach and stresses the integration and reuse of existing tools. Reference PV genome sequences have been extracted from publicly available databases and reannotated using a custom-created tool. To date, the PaVE contains 241 annotated PV genomes, 2245 genes and regions, 2004 protein sequences and 47 protein structures, which users can explore, analyze or download. The PaVE provides scientists with the data and tools needed to accelerate scientific progress for the study and treatment of diseases caused by PVs.

Description: The Nucleic acid—Protein Interaction DataBase ( http://npidb.belozersky.msu.ru/ ) contains information derived from structures of DNA–protein and RNA–protein complexes extracted from the Protein Data Bank (3846 complexes in October 2012). It provides a web interface and a set of tools for extracting biologically meaningful characteristics of nucleoprotein complexes. The content of the database is updated weekly. The current version of the Nucleic acid—Protein Interaction DataBase is an upgrade of the version published in 2007. The improvements include a new web interface, new tools for calculation of intermolecular interactions, a classification of SCOP families that contains DNA–binding protein domains and data on conserved water molecules on the DNA–protein interface.

Description: Disease and Gene Annotations database (DGA, http://dga.nubic.northwestern.edu ) is a collaborative effort aiming to provide a comprehensive and integrative annotation of the human genes in disease network context by integrating computable controlled vocabulary of the Disease Ontology (DO version 3 revision 2510, which has 8043 inherited, developmental and acquired human diseases), NCBI Gene Reference Into Function (GeneRIF) and molecular interaction network (MIN). DGA integrates these resources together using semantic mappings to build an integrative set of disease-to-gene and gene-to-gene relationships with excellent coverage based on current knowledge. DGA is kept current by periodically reparsing DO, GeneRIF, and MINs. DGA provides a user-friendly and interactive web interface system enabling users to efficiently query, download and visualize the DO tree structure and annotations as a tree, a network graph or a tabular list. To facilitate integrative analysis, DGA provides a web service Application Programming Interface for integration with external analytic tools.

Description: EcoCyc ( http://EcoCyc.org ) is a model organism database built on the genome sequence of Escherichia coli K-12 MG1655. Expert manual curation of the functions of individual E. coli gene products in EcoCyc has been based on information found in the experimental literature for E. coli K-12-derived strains. Updates to EcoCyc content continue to improve the comprehensive picture of E. coli biology. The utility of EcoCyc is enhanced by new tools available on the EcoCyc web site, and the development of EcoCyc as a teaching tool is increasing the impact of the knowledge collected in EcoCyc.

Description: HAMAP (High-quality Automated and Manual Annotation of Proteins—available at http://hamap.expasy.org/ ) is a system for the classification and annotation of protein sequences. It consists of a collection of manually curated family profiles for protein classification, and associated annotation rules that specify annotations that apply to family members. HAMAP was originally developed to support the manual curation of UniProtKB/Swiss-Prot records describing microbial proteins. Here we describe new developments in HAMAP, including the extension of HAMAP to eukaryotic proteins, the use of HAMAP in the automated annotation of UniProtKB/TrEMBL, providing high-quality annotation for millions of protein sequences, and the future integration of HAMAP into a unified system for UniProtKB annotation, UniRule. HAMAP is continuously updated by expert curators with new family profiles and annotation rules as new protein families are characterized. The collection of HAMAP family classification profiles and annotation rules can be browsed and viewed on the HAMAP website, which also provides an interface to scan user sequences against HAMAP profiles.

Description: SILVA (from Latin silva, forest, http://www.arb-silva.de ) is a comprehensive web resource for up to date, quality-controlled databases of aligned ribosomal RNA (rRNA) gene sequences from the Bacteria , Archaea and Eukaryota domains and supplementary online services. The referred database release 111 (July 2012) contains 3 194 778 small subunit and 288 717 large subunit rRNA gene sequences. Since the initial description of the project, substantial new features have been introduced, including advanced quality control procedures, an improved rRNA gene aligner, online tools for probe and primer evaluation and optimized browsing, searching and downloading on the website. Furthermore, the extensively curated SILVA taxonomy and the new non-redundant SILVA datasets provide an ideal reference for high-throughput classification of data from next-generation sequencing approaches.

Description: Quorum-sensing (QS) peptides are biologically attractive molecules, with a wide diversity of structures and prone to modifications altering or presenting new functionalities. Therefore, the Quorumpeps database ( http://quorumpeps.ugent.be ) is developed to give a structured overview of the QS oligopeptides, describing their microbial origin (species), functionality (method, result and receptor), peptide links and chemical characteristics (3D-structure-derived physicochemical properties). The chemical diversity observed within this group of QS signalling molecules can be used to develop new synthetic bio-active compounds.

Description: SecReT4 ( http://db-mml.sjtu.edu.cn/SecReT4/ ) is an integrated database providing comprehensive information of type IV secretion systems (T4SSs) in bacteria. T4SSs are versatile assemblages that promote genetic exchange and/or effector translocation with consequent impacts on pathogenesis and genome plasticity. T4SSs have been implicated in conjugation, DNA uptake and release and effector translocation. The effectors injected into eukaryotic target cells can lead to alteration of host cellular processes during infection. SecReT4 offers a unique, highly organized, readily exploreable archive of known and putative T4SSs and cognate effectors in bacteria. It currently contains details of 10 752 core components mapping to 808 T4SSs and 1884 T4SS effectors found in representatives of 289 bacterial species, as well as a collection of more than 900 directly related references. A broad range of similarity search, sequence alignment, phylogenetic, primer design and other functional analysis tools are readily accessible via SecReT4. We propose that SecReT4 will facilitate efficient investigation of large numbers of these systems, recognition of diverse patterns of sequence-, gene- and/or functional conservation and an improved understanding of the biological roles and significance of these versatile molecular machines. SecReT4 will be regularly updated to ensure its ongoing maximum utility to the research community.

Description: EuPathDB ( http://eupathdb.org ) resources include 11 databases supporting eukaryotic pathogen genomic and functional genomic data, isolate data and phylogenomics. EuPathDB resources are built using the same infrastructure and provide a sophisticated search strategy system enabling complex interrogations of underlying data. Recent advances in EuPathDB resources include the design and implementation of a new data loading workflow, a new database supporting Piroplasmida (i.e. Babesia and Theileria ), the addition of large amounts of new data and data types and the incorporation of new analysis tools. New data include genome sequences and annotation, strand-specific RNA-seq data, splice junction predictions (based on RNA-seq), phosphoproteomic data, high-throughput phenotyping data, single nucleotide polymorphism data based on high-throughput sequencing (HTS) and expression quantitative trait loci data. New analysis tools enable users to search for DNA motifs and define genes based on their genomic colocation, view results from searches graphically (i.e. genes mapped to chromosomes or isolates displayed on a map) and analyze data from columns in result tables (word cloud and histogram summaries of column content). The manuscript herein describes updates to EuPathDB since the previous report published in NAR in 2010.

Description: The microbial genome database for comparative analysis (MBGD, available at http://mbgd.genome.ad.jp/ ) is a platform for microbial genome comparison based on orthology analysis. As its unique feature, MBGD allows users to conduct orthology analysis among any specified set of organisms; this flexibility allows MBGD to adapt to a variety of microbial genomic study. Reflecting the huge diversity of microbial world, the number of microbial genome projects now becomes several thousands. To efficiently explore the diversity of the entire microbial genomic data, MBGD now provides summary pages for pre-calculated ortholog tables among various taxonomic groups. For some closely related taxa, MBGD also provides the conserved synteny information (core genome alignment) pre-calculated using the CoreAligner program. In addition, efficient incremental updating procedure can create extended ortholog table by adding additional genomes to the default ortholog table generated from the representative set of genomes. Combining with the functionalities of the dynamic orthology calculation of any specified set of organisms, MBGD is an efficient and flexible tool for exploring the microbial genome diversity.