Gene Deletion Algorithms for Minimum Reaction Network Design by Mixed-Integer Linear Programming for Metabolite Production in Constraint-Based Models: gDel_minRN.

Genome-scale constraint-based metabolic networks play an important role in the simulation of growth-coupled production, which means that cell growth and target metabolite production are simultaneously achieved. For growth-coupled production, a minimal reaction-network-based design is known to be effective. However, the obtained reaction networks often fail to be realized by gene deletions due to conflicts with gene-protein-reaction (GPR) relations. Here, we developed gDel_minRN that determines gene deletion strategies using mixed-integer linear programming to achieve growth-coupled production by repressing the maximum number of reactions via GPR relations. The results of computational experiments showed that gDel_minRN could determine the core parts, which include only 30% to 55% of whole genes, for stoichiometrically feasible growth-coupled production for many target metabolites, which include useful vitamins such as biotin (vitamin B7), riboflavin (vitamin B2), and pantothenate (vitamin B5). Since gDel_minRN calculates a constraint-based model of the minimum number of gene-associated reactions without conflict with GPR relations, it helps biological analysis of the core parts essential for growth-coupled production for each target metabolite. The source codes, implemented in MATLAB using CPLEX and COBRA Toolbox, are available on https://github.com/MetNetComp/gDel-minRN.

NeuroGT: A brain atlas of neurogenic tagging CreER drivers for birthdate-based classification and manipulation of mouse neurons.

Neuronal birthdate is one of the major determinants of neuronal phenotypes. However, most birthdating methods are retrospective in nature, allowing very little experimental access to the classified neuronal subsets. Here, we introduce four neurogenic tagging mouse lines, which can assign CreER-loxP recombination to neuron subsets that share the same differentiation timing in living animals and enable various experimental manipulations of the classified subsets. We constructed a brain atlas of the neurogenic tagging mouse lines (NeuroGT), which includes holistic image data of the loxP-recombined neurons and their processes across the entire brain that were tagged on each single day during the neurodevelopmental period. This image database, which is open to the public, offers investigators the opportunity to find specific neurogenic tagging driver lines and the stages of tagging appropriate for their own research purposes.

BD5: An open HDF5-based data format to represent quantitative biological dynamics data.

BD5 is a new binary data format based on HDF5 (hierarchical data format version 5). It can be used for representing quantitative biological dynamics data obtained from bioimage informatics techniques and mechanobiological simulations. Biological Dynamics Markup Language (BDML) is an XML (Extensible Markup Language)-based open format that is also used to represent such data; however, it becomes difficult to access quantitative data in BDML files when the file size is large because parsing XML-based files requires large computational resources to first read the whole file sequentially into computer memory. BD5 enables fast random (i.e., direct) access to quantitative data on disk without parsing the entire file. Therefore, it allows practical reuse of data for understanding biological mechanisms underlying the dynamics.

Phenotype Analysis Method for Identification of Gene Functions Involved in Asymmetric Division of Caenorhabditis elegans.

In gene function analysis, it is arduous to identify gene function individually, and the way to screen out all involved genes according to a particular phenotype or disease usually shows us little information for a specific problem. We present a data-driven analysis system based on wild type (WT) embryos to study the concrete function of each gene associated with certain category of abnormal phenotypes. It can be applied to genes with very few RNAi embryos. Instead of presupposing the particular function of a gene, its function is confirmed by the statistical testing of built models. The scheme includes the following five: first, verify the to be detected genes and determine related recognized features according to the given category; second, compute the value of each feature based on WT embryos and merge them by principal component analysis (PCA); third, for each of the selected components of PCA, build a normal distribution and verify its normality; fourth, project the RNAi embryos to each component and probe them; and finally, analyze the more detailed functions of each gene based on the physical or biological meaning of each component. Choosing the first-round asymmetric division process of Caenorhabditis elegans as the phenotype, experimental results show that on the different aspects of the asymmetric division process, par-2, par-3, and let-754 are related to scalar differences; dcn-1 and mcm-5 are associated with the divergences of scalar variation, which may reflect the disaccord in development; and dcn-1, par-2, and par-3 are involved with morphological discrepancies.

SSBD: a database of quantitative data of spatiotemporal dynamics of biological phenomena.

MOTIVATION: Rapid advances in live-cell imaging analysis and mathematical modeling have produced a large amount of quantitative data on spatiotemporal dynamics of biological objects ranging from molecules to organisms. There is now a crucial need to bring these large amounts of quantitative biological dynamics data together centrally in a coherent and systematic manner. This will facilitate the reuse of this data for further analysis. RESULTS: We have developed the Systems Science of Biological Dynamics database (SSBD) to store and share quantitative biological dynamics data. SSBD currently provides 311 sets of quantitative data for single molecules, nuclei and whole organisms in a wide variety of model organisms from Escherichia coli to Mus musculus The data are provided in Biological Dynamics Markup Language format and also through a REST API. In addition, SSBD provides 188 sets of time-lapse microscopy images from which the quantitative data were obtained and software tools for data visualization and analysis. AVAILABILITY AND IMPLEMENTATION: SSBD is accessible at http://ssbd.qbic.riken.jp CONTACT: sonami@riken.jp.

Investigation of glycan evolution based on a comprehensive analysis of glycosyltransferases using phylogenetic profiling.

Glycans play important roles in such cell-cell interactions as signaling and adhesion, including processes involved in pathogenic infections, cancers, and neurological diseases. Glycans are biosynthesized by multiple glycosyltransferases (GTs), which function sequentially. Excluding mucin-type O-glycosylation, the non-reducing terminus of glycans is biosynthesized in the Golgi apparatus after the reducing terminus is biosynthesized in the ER. In the present study, we performed genome-wide analyses of human GTs by investigating the degree of conservation of homologues in other organisms, as well as by elucidating the phylogenetic relationship between cephalochordates and urochordates, which has long been controversial in deuterostome phylogeny. We analyzed 173 human GTs and functionally linked glycan synthesis enzymes by phylogenetic profiling and clustering, compiled orthologous genes from the genomes of other organisms, and converted them into a binary sequence based on the presence (1) or absence (0) of orthologous genes in the genomes. Our results suggest that the non-reducing terminus of glycans is biosynthesized by newly evolved GTs. According to our analysis, the phylogenetic profiles of GTs resemble the phylogenetic tree of life, where deuterostomes, metazoans, and eukaryotes are resolved into separate branches. Lineage-specific GTs appear to play essential roles in the divergence of these particular lineages. We suggest that urochordates lose several genes that are conserved among metazoans, such as those expressing sialyltransferases, and that the Golgi apparatus acquires the ability to synthesize glycans after the ER acquires this function.

Biological Dynamics Markup Language (BDML): an open format for representing quantitative biological dynamics data.

MOTIVATION: Recent progress in live-cell imaging and modeling techniques has resulted in generation of a large amount of quantitative data (from experimental measurements and computer simulations) on spatiotemporal dynamics of biological objects such as molecules, cells and organisms. Although many research groups have independently dedicated their efforts to developing software tools for visualizing and analyzing these data, these tools are often not compatible with each other because of different data formats. RESULTS: We developed an open unified format, Biological Dynamics Markup Language (BDML; current version: 0.2), which provides a basic framework for representing quantitative biological dynamics data for objects ranging from molecules to cells to organisms. BDML is based on Extensible Markup Language (XML). Its advantages are machine and human readability and extensibility. BDML will improve the efficiency of development and evaluation of software tools for data visualization and analysis. AVAILABILITY AND IMPLEMENTATION: A specification and a schema file for BDML are freely available online at http://ssbd.qbic.riken.jp/bdml/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

GenoBase: comprehensive resource database of Escherichia coli K-12.

Comprehensive experimental resources, such as ORFeome clone libraries and deletion mutant collections, are fundamental tools for elucidation of gene function. Data sets by omics analysis using these resources provide key information for functional analysis, modeling and simulation both in individual and systematic approaches. With the long-term goal of complete understanding of a cell, we have over the past decade created a variety of clone and mutant sets for functional genomics studies of Escherichia coli K-12. We have made these experimental resources freely available to the academic community worldwide. Accordingly, these resources have now been used in numerous investigations of a multitude of cell processes. Quality control is extremely important for evaluating results generated by these resources. Because the annotation has been changed since 2005, which we originally used for the construction, we have updated these genomic resources accordingly. Here, we describe GenoBase (http://ecoli.naist.jp/GB/), which contains key information about comprehensive experimental resources of E. coli K-12, their quality control and several omics data sets generated using these resources.

Parameter optimization and sensitivity analysis for large kinetic models using a real-coded genetic algorithm.

Dynamic modeling is a powerful tool for predicting changes in metabolic regulation. However, a large number of input parameters, including kinetic constants and initial metabolite concentrations, are required to construct a kinetic model. Therefore, it is important not only to optimize the kinetic parameters, but also to investigate the effects of their perturbations on the overall system. We investigated the efficiency of the use of a real-coded genetic algorithm (RCGA) for parameter optimization and sensitivity analysis in the case of a large kinetic model involving glycolysis and the pentose phosphate pathway in Escherichia coli K-12. Sensitivity analysis of the kinetic model using an RCGA demonstrated that the input parameter values had different effects on model outputs. The results showed highly influential parameters in the model and their allowable ranges for maintaining metabolite-level stability. Furthermore, it was revealed that changes in these influential parameters may complement one another. This study presents an efficient approach based on the use of an RCGA for optimizing and analyzing parameters in large kinetic models.

Biochemical studies on sphingolipids of Artemia franciscana: complex neutral glycosphingolipids.

Brine shrimp are primitive crustacean arthropodal model organisms, second to daphnia, which can survive in high-salinity environments. Their oviposited cysts, cuticle-covered diapausing eggs, are highly resistant to dryness. To elucidate specialties of brine shrimp, this study characterized glycosphingolipids, which are signal transduction-associated material. A group of novel and complex fucosyl glycosphingolipids were separated and identified from cysts of the brine shrimp Artemia franciscana by repeated lipid extraction, alkaline methanolysis, acid treatment, successive column chromatography, and post-source decay measurements by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Structures of the glycosphingolipids were elucidated by conventional structural characterization and mass spectrometry, and the compounds were identified as GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, GalNAcß1-4(Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer, and GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3GalNAcß1-4(GlcNAcα1-2Fucα1-3)GlcNAcß1-3Manß1-4Glcß1-Cer. These compounds also contained a branching, non-arthro-series disaccharide with an α-GlcNAc terminus, similar to that found in a previously reported ceramide hexasaccharide (III(3)(GlcNAcα2Fucα)-At4Cer). The glycans within these complex GSLs are longer than reported glycans of the animal kingdom containing α-GlcNAc terminus. These complex GSLs as well as the longest GSL with ten sugar residues, ceramide decasaccharide (CDeS), contain the fucosylated LacdiNAc sequence reported to associate with parasitism/immunosuppression and the α-GlcNAc terminus reported to show a certain antibacterial effect in other reports. CDeS, the longest GSL of this species, was found in the highest amount, which indicates that CDeS may be functionally important.

ECOH: an enzyme commission number predictor using mutual information and a support vector machine.

MOTIVATION: The enzyme nomenclature system, commonly known as the enzyme commission (EC) number, plays a key role in classifying and predicting enzymatic reactions. However, numerous reactions have been described in various pathways that do not have an official EC number, and the reactions are not expected to have an EC number assigned because of a lack of articles published on enzyme assays. To predict the EC number of a non-classified enzymatic reaction, we focus on the structural similarity of its substrate and product to the substrate and product of reactions that have been classified. RESULTS: We propose a new method to assign EC numbers using a maximum common substructure algorithm, mutual information and a support vector machine, termed the Enzyme COmmission numbers Handler (ECOH). A jack-knife test shows that the sensitivity, precision and accuracy of the method in predicting the first three digits of the official EC number (i.e. the EC sub-subclass) are 86.1%, 87.4% and 99.8%, respectively. We furthermore demonstrate that, by examining the ranking in the candidate lists of EC sub-subclasses generated by the algorithm, the method can successfully predict the classification of 85 enzymatic reactions that fall into multiple EC sub-subclasses. The better performance of the ECOH as compared with existing methods and its flexibility in predicting EC numbers make it useful for predicting enzyme function. AVAILABILITY: ECOH is freely available via the Internet at http://www.bioinfo.sk.ritsumei.ac.jp/apps/ecoh/. This program only works on 32-bit Windows.

Intrinsically disordered proteins in human mitochondria.

Intrinsically disordered (ID) proteins (IDPs) are abundant in eukaryotes but are scarce in prokaryotes. Mitochondria, cellular organelles that descended from Rickettsia-like α-proteobacteria, are at the intersection between prokaryotes and eukaryotes. Although IDPs are reportedly as rare in mitochondria as in bacteria, these details remained to be clarified. Human mitochondrial proteins (n = 706) were obtained from the UniProt database, and information on ID regions of all human proteins was extracted from the DICHOT database. A BLAST search carried out against all α-proteobacterial proteins identified two types of mitochondrial proteins: those with (B) and without (E) bacterial homologues. The B-type proteins (n = 387) descended from a bacterial ancestor, whereas the E-type proteins (n = 319) were more recently added to the mitochondria via the host cell during the early evolution of eukaryotes. The average ID ratios of B-type/E-type proteins are 10.3% and 21.4%, respectively. The 706 proteins were further classified into four groups based on the mitochondrial subcompartment, namely, the matrix, intermembrane space, inner membrane, or outer membrane. The ID ratios in these different locations suggest that the frequency of IDPs in mitochondria might be due to the evolutionary origin (B-type/E-type) of the protein, rather than differences in its functional environment.

Comparative proteomic analysis reveals differentially expressed proteins in Caenorhabditis elegans pgl-1 mutants grown at 20°C and 25°C.

PGL-1 is an RNA-binding protein component of germ granules and essential for fertility in Caenorhabditis elegans. To clarify the molecular function of PGL-1, we performed comparative proteomic analysis using 2-D DIGE and LC-MS/MS. Five groups of synchronized adult hermaphrodites were analyzed: (1) wild-type N2 grown at 20°C, (2) pgl-1(bn101) mutants grown at 20°C, (3) pgl-1(bn101) mutants grown at 20°C then upshifted to 25°C after the L1 stage, (4) pgl-1(ct131) mutants grown at 20°C, and (5) pgl-1(ct131) mutants grown at 20°C then upshifted to 25°C after the L1 stage. The five groups were divided into two experimental sets for 2-D DIGE: set A included N2 and pgl-1(bn101) mutants, and set B included N2 and pgl-1(ct131) mutants. Dunnett's test indicated 90 and 100 specific spots, respectively, with significantly different expression levels from the rest of the experimental set (q≤0.1). Among them, 69 and 58 spots, respectively, were analyzed by LC-MS/MS. Finally, we identified 19 proteins from 24 specific spots common to both the experimental sets. RNAi analysis indicated that decreased eef-1G expression is strongly associated with the temperature-sensitive sterile phenotype of pgl-1. Our results suggest that PGL-1 is closely involved in translational processes during C. elegans germline development.

Environmental dependency of gene knockouts on phenotype microarray analysis in Escherichia coli.

Systematic studies have revealed that single gene deletions often display little phenotypic effects under laboratory conditions and that in many cases gene dispensability depends on the experimental conditions. To elucidate the environmental dependency of genes, we analyzed the effects of gene deletions by Phenotype MicroArray™ (PM), a system for quantitative screening of thousands of phenotypes in a high-throughput manner. Here, we proposed a new statistical approach to minimize error inherent in measurements of low respiration rates and find which mutants showed significant phenotypic changes in comparison to the wild-type. We show analyzing results from comprehensive PM assays of 298 single-gene knockout mutants in the Keio collection and two additional mutants under 1,920 different conditions. We focused on isozymes of these genes as simple duplications and analyzed correlations between phenotype changes and protein expression levels. Our results revealed divergence of the environmental dependency of the gene among the knockout genes and have also given some insights into possibilities of alternative pathways and availabilities of information on protein synthesis patterns to classify or predict functions of target genes from systematic phenotype screening.

Phenotype profiling of single gene deletion mutants of E. coli using Biolog technology.

Phenotype MicroArray (PM) technology is high-throughput phenotyping system and is directly applicable to assay the effects of genetic changes in cells. In this study, we performed comprehensive PM analysis using single gene deletion mutants of central metabolic pathway and related genes. To elucidate the structure of central metabolic networks in Escherichia coli K-12, we focused 288 different PM conditions of carbon and nitrogen sources and performed bioinformatic analysis. For data processing, we employed noise reduction procedures. The distance between each of the mutants was defined by Manhattan distance and agglomerative Ward's hierarchical method was applied for clustering analysis. As a result, five clusters were revealed which represented to activate or repress cellular respiratory activities. Furthermore, the results might suggest that Glyceraldehyde-3P plays a key role as a molecular switch of central metabolic network.