Published papers

Background: There has been a lot of interest in recent years focusing on the modeling and simulation of Gene Regulatory Networks (GRNs). However, the evolutionary mechanisms that give rise to GRNs in the first place are still largely unknown. In an earlier work, we developed a framework to analyze the effect of objective functions, input types and starting populations on the evolution of GRNs with a specific emphasis on the robustness of evolved GRNs. Results: In this work, we present a parallel software package, GeNESiS for the modeling and simulation of the evolution of gene regulatory networks (GRNs). The software models the process of gene regulation through a combination of finite-state and stochastic models. The evolution of GRNs is then simulated by means of a genetic algorithm with the network connections represented as binary strings. The software allows users to simulate the evolution under varying selective pressures and starting conditions. We believe that the software provides a way for researchers to understand the evolutionary behavior of populations of GRNs. Conclusion: We believe that GeNESiS will serve as a useful tool for scientists interested in understanding the evolution of gene regulatory networks under a range of different conditions and selective pressures. Such modeling efforts can lead to a greater understanding of the network characteristics of GRNs. © 2008 Kratz et al; licensee BioMed Central Ltd.

Background: The reduced cost of sequencing has made de novo sequencing and the assembly of draft microbial genomes feasible in any ordinary biology lab. However, the process of finishing and completing the genome remains labor-intensive and computationally challenging in some cases, such as in the study of complete genome sequences, genomic rearrangements, long-range syntenic relationships, and structural variations. Methods: Here, we show a contig reordering strategy based on experimental replication profiling (eRP) to recapitulate the bacterial genome structure within draft genomes. During the exponential growth phase, the majority of bacteria show a global genomic copy number gradient that is enriched near the replication origin and gradually declines toward the terminus. Therefore, if genome sequencing is performed with appropriate timing, the short-read coverage reflects this copy number gradient, providing information about the contig positions relative to the replication origin and terminus. Results: We therefore investigated the appropriate timing for genomic DNA sampling and developed an algorithm for the reordering of the contigs based on eRP. As a result, this strategy successfully recapitulates the genomic structure of various structural mutants with draft genome sequencing. Conclusions: Our strategy was successful for contig rearrangement with intracellular DNA replication behavior mechanisms and can be applied to almost all bacteria because the DNA replication system is highly conserved. Therefore, eRP makes it possible to understand genomic structural information and long-range syntenic relationships using a draft genome that is based on short reads.

Cancer cells alter their metabolism for the production of precursors of macromolecules. However, the control mechanisms underlying this reprogramming are poorly understood. Here we show that metabolic reprogramming of colorectal cancer is caused chiefly by aberrant MYC expression. Multiomics-based analyses of paired normal and tumor tissues from 275 patients with colorectal cancer revealed that metabolic alterations occur at the adenoma stage of carcinogenesis, in a manner not associated with specific gene mutations involved in colorectal carcinogenesis. MYC expression induced at least 215 metabolic reactions by changing the expression levels of 121 metabolic genes and 39 transporter genes. Further, MYC negatively regulated the expression of genes involved in mitochondrial biogenesis and maintenance but positively regulated genes involved in DNA and histone methylation. Knockdown of MYC in colorectal cancer cells reset the altered metabolism and suppressed cell growth. Moreover, inhibition of MYC target pyrimidine synthesis genes such as CAD, UMPS, and CTPS blocked cell growth, and thus are potential targets for colorectal cancer therapy.

Gut microbiota is an essential factor in the shaping of intestinal immune system development and driving inflammation in inflammatory bowel disease (IBD). We report the effects and microbe-host interactions underlying an intervention using fine powder of eggshell membrane (ESM) against IBD. ESM attenuated lipopolysaccharide-induced inflammatory cytokine production and promoted the Caco-2 cell proliferation by up-regulating growth factors in vitro. In a murine model of dextran sodium sulphate-induced colitis, ESM significantly suppressed the disease activity index and colon shortening. These effects were associated with significant ameliorations of gene expressions of inflammatory mediators, intestinal epithelial cell proliferation, restitution-related factors and antimicrobial peptides. Multifaceted integrated omics analyses revealed improved levels of energy metabolism-related genes, proteins and metabolites. Concomitantly, cecal metagenomic information established an essential role of ESM in improving dysbiosis characterized by increasing the diversity of bacteria and decreasing absolute numbers of pathogenic bacteria such as Enterobacteriaceae and E. coli, as well as in the regulation of the expansion of Th17 cells by suppressing the overgrowth of segmented filamentous bacteria. Such modulations have functional effects on the host; i.e., repairing the epithelium, regulating energy requirements and eventually alleviating mucosal inflammation. These findings are first insights into ESM's modulation of microbiota and IBD suppression, providing new perspectives on the prevention/treatment of IBD.

With advances in high-throughput sequencing technologies, de novo transcriptome sequencing and assembly has become a cost-effective method to obtain comprehensive genetic information of a species of interest, especially in nonmodel species with large genomes such as spiders. However, high-quality RNA is essential for successful sequencing, and sample preservation conditions require careful consideration for the effective storage of field-collected samples. To this end, we report a streamlined feasibility study of various storage conditions and their effects on de novo transcriptome assembly results. The storage parameters considered include temperatures ranging from room temperature to -80°C; preservatives, including ethanol, RNAlater, TRIzol and RNAlater-ICE; and sample submersion states. As a result, intact RNA was extracted and assembly was successful when samples were preserved at low temperatures regardless of the type of preservative used. The assemblies as well as the gene expression profiles were shown to be robust to RNA degradation, when 30 million 150-bp paired-end reads are obtained. The parameters for sample storage, RNA extraction, library preparation, sequencing and in silico assembly considered in this work provide a guideline for the study of field-collected samples of spiders.

Transfer RNA (tRNA) is essential for the translation of genetic information into proteins, and understanding its molecular evolution is important if we are to understand the genetic code. In general, long variable-arm (V-arm) structures form in tRNALeu, tRNASer, and bacterial and organellar tRNATyr. However, as we have previously reported, noncanonical V-arms occur in nematode tRNAGly and tRNAIle, and potentially affect translational fidelity. Here, we comprehensively analyzed 69 eukaryotic genome sequences and examined the evolutionary divergence of the V-arm-containing tRNAs. In total, 253 V-arm-containing tRNAs, with neither leucine nor serine anticodons, were identified in organisms ranging from nematodes to fungi, plants, and vertebrates. We defined them as "noncanonical V-arm-containing tRNAs" (nov-tRNAs). Moreover, 2,415 nov-tRNA-like sequences lacking some of the conserved features of tRNAs were also identified, largely in vertebrate genomes. These nov-tRNA/nov-tRNA-like sequences can be categorized into three types, based on differences in their possible evolutionary origins. The type A nov-tRNAs in nematodes probably evolved not only from tRNALeu but also from tRNASer and other isotypes on several independent occasions. The type B nov-tRNAs are dispersed abundantly throughout vertebrate genomes, and seem to have originated from retrotransposable elements. The type C nov-tRNAs may have been acquired from plant chloroplasts or from bacteria through horizontal transfer. Our findings provide unexpected insight into the evolution of the tRNA molecule, which was diverse and occurred independently in nematodes, vertebrates, and plants.

© 2018 The Author(s). Bacillus subtilis offers a platform for giant DNA synthesis, which is mediated by the connection of overlapping DNA segments called domino DNA, in the cloning locus of the host. The domino method was successfully used to produce DNA fragments as large as 3500 kbp. However, domino DNA is limited to <100 kbp because of size restrictions regarding the transformation (TF) of B. subtilis competent cells. A novel conjugal transfer (CT) method was designed to eliminate the TF size limit. The CT method enables rapid and efficient domino reactions in addition to the transfer of giant DNA molecules of up to 875 kbp to another B. subtilis genome within 4 hours. The combined use of the TF and CT should enable significantly rapid giant DNA production.

Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.

Copyright © 2019 Harada et al. Strain SCT is an iodate-reducing bacterium isolated from marine sediment in Kanagawa Prefecture, Japan. In this study, we determined the draft genome sequence of strain SCT and compared it to complete genome sequences of other closely related bacteria, including Pseudomonas stutzeri. A phylogeny inferred from concatenation of core genes revealed that strain SCT was closely related to marine isolates of P. stutzeri. Genes present in the SCT genome but absent from the other analyzed P. stutzeri genomes comprised clusters corresponding to putative prophage regions and possible operons. They included pil genes, which encode type IV pili for natural transformation; the mer operon, which encodes resistance systems for mercury; and the pst operon, which encodes a Pi-specific transport system for phosphate uptake. We found that strain SCT had more prophage-like genes than the other P. stutzeri strains and that the majority (70%) of them were SCT strain-specific. These genes, encoded on distinct prophage regions, may have been acquired after branching from a common ancestor following independent phage transfer events. Thus, the genome sequence of Pseudomonas sp. strain SCT can provide detailed insights into its metabolic potential and the evolution of genetic elements associated with its unique phenotype.

With the availability of numerous curated databases, researchers are now able to efficiently use the multitude of biological data by integrating these resources via hyperlinks and cross-references. A large proportion of bioinformatics research tasks, however, may include labor-intensive tasks such as fetching, parsing, and merging datasets and functional annotations from distributed multi-domain databases. This data integration issue is one of the key challenges in bioinformatics. We aim to provide an identifier conversion and data aggregation system as a part of solution to solve this problem with a service named G-Links, 1) by gathering resource URI information from 130 databases and 30 web services in a gene-centric manner so that users can retrieve all available links about a given gene, 2) by providing RESTful API for easy retrieval of links including facet searching based on keywords and/or predicate types, and 3) by producing a variety of outputs as visual HTML page, tab-delimited text, and in Semantic Web formats such as Notation3 and RDF. G-Links as well as other relevant documentation are available at http://link.g-language.org/.

Metabolite concentrations reflect the physiological states of tissues and cells. However, the role of metabolic changes in species evolution is currently unknown. Here, we present a study of metabolome evolution conducted in three brain regions and two non-neural tissues from humans, chimpanzees, macaque monkeys, and mice based on over 10,000 hydrophilic compounds. While chimpanzee, macaque, and mouse metabolomes diverge following the genetic distances among species, we detect remarkable acceleration of metabolome evolution in human prefrontal cortex and skeletal muscle affecting neural and energy metabolism pathways. These metabolic changes could not be attributed to environmental conditions and were confirmed against the expression of their corresponding enzymes. We further conducted muscle strength tests in humans, chimpanzees, and macaques. The results suggest that, while humans are characterized by superior cognition, their muscular performance might be markedly inferior to that of chimpanzees and macaque monkeys. © 2014 Bozek et al.

Background: There is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism. Results: To resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency. Conclusions: These findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.

The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy [PALM]) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.

The developmental program of the heart requires accurate regulation to ensure continuous circulation and simultaneous cardiac morphogenesis, because any functional abnormalities may progress to congenital heart malformation. Notably, energy metabolism in fetal ventricular cells is regulated in a manner that differs from adult ventricular cells: fetal cardiomyocytes generally have immature mitochondria and fetal ventricular cells show greater dependence on glycolytic ATP production. However, although various characteristics of energy metabolism in fetal ventricular cells have been reported, to our knowledge, a quantitative description of the contributions of these factors to fetal ventricular cell functions has not yet been established. Here, we constructed a mathematical model to integrate various characteristics of fetal ventricular cells and predicted the contribution of each characteristic to the maintenance of intracellular ATP concentration and sarcomere contraction under anoxic conditions. Our simulation results demonstrated that higher glycogen content, higher hexokinase activity, and lower creatine concentration helped prolong the time for which ventricular cell contraction was maintained under anoxic conditions. The integrated model also enabled us to quantitatively assess the contributions of factors related to energy metabolism in ventricular cells. Because fetal cardiomyocytes exhibit similar energy metabolic profiles to stem cell-derived cardiomyocytes and those in the failing heart, an improved understanding of these fetal ventricular cells will contribute to a better comprehension of the processes in stem cell-derived cardiomyocytes or under pathological conditions.

Background: This study characterized the changes in quality and quantity of saliva, and changes in the salivary metabolomic profile, to understand the effects of masticatory stimulation. Methods: Stimulated and unstimulated saliva samples were collected from 55 subjects and salivary hydrophilic metabolites were comprehensively quantified using capillary electrophoresis-time-of-flight mass spectrometry. Results: In total, 137 metabolites were identified and quantified. The concentrations of 44 metabolites in stimulated saliva were significantly higher than those in unstimulated saliva. Pathway analysis identified the upregulation of the urea cycle and synthesis and degradation pathways of glycine, serine, cysteine and threonine in stimulated saliva. A principal component analysis revealed that the effect of masticatory stimulation on salivary metabolomic profiles was less dependent on sample population sex, age, and smoking. The concentrations of only 1 metabolite in unstimulated saliva, and of 3 metabolites stimulated saliva, showed significant correlation with salivary secretion volume, indicating that the salivary metabolomic profile and salivary secretion volume were independent factors. Conclusions: Masticatory stimulation affected not only salivary secretion volume, but also metabolite concentration patterns. A low correlation between the secretion volume and these patterns supports the conclusion that the salivary metabolomic profile may be a new indicator to characterize masticatory stimulation.

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

The development of high-throughput metabolite measurement technologies has enabled the use of metabolomics for epidemiologic studies by profiling metabolite concentrations in large cohorts of human blood samples. Standard protocols are necessary to obtain unbiased profiles through multiple runs over long periods of time and to allow reliable statistical analyses. This study assessed the effects of sampling procedures and storage conditions on the stability of metabolomic profiles in plasma and serum. Charged metabolomic profiles were determined by capillary electrophoresis-mass spectrometry (CE-MS) and compared by multivariate analyses. The effects of pre-analytical procedures, including times for clotting and incubation of serum and plasma, respectively; incubation temperatures; and number of freeze-thaw cycles, were assessed. Overall, inter-individual differences in profiles were larger than intra-individual differences, and profiles in plasma showed better stability than those in serum. These quantified datasets of metabolites, along with their stability and variation, may help in interpreting data from long-term cohort studies.

© 2018, The Author(s). Salivary polyamines are potential non-invasive tools for screening various types of cancers. For clinical use, the reproducibility of these metabolites should be evaluated under various storage conditions, including duration and temperature, to establish standard operating protocols. Polyamines and amino acids in unstimulated whole saliva were quantified via liquid chromatography-mass spectrometry. Concentrations of time course samples were analysed after short-term storage for up to 240 min and long-term storage for up to 8 days under various storage conditions. As expected, storage at the lowest temperature (−18 °C) exerted the least pronounced effects on the quantified values in both tests. At a higher temperature, polyamines were more stable than amino acids, as evident from polyamine profiling. Addition of ethanol significantly stabilized polyamine profiles even at a higher temperature. Comparative processing of saliva revealed a minor effect of the solvent, whereas drying had a more prominent effect on polyamine profiles. Computational analyses evaluated the ability of polyamines to discriminate pancreatic cancer from controls. Repeated noise added tests were designed on the basis of the results of the storage tests; these analyses confirmed that the discriminative abilities were robust. These data contribute to the standardization of salivary storage conditions, thereby highlighting the clinical utility of saliva.

© 2018 Elsevier B.V. Background: Salivary secretion is an important parameter reflecting the health status of an individual and has been used clinically for the diagnosis of various oral diseases, such as xerostomia. Salivary metabolomic profiling is considered an emerging potential tool for the detection of various systemic diseases. To our knowledge, this is the first study to investigate the quantitative relationship between salivary secretion volume and salivary metabolomic profile. Methods: To evaluate inter- and intra-day variations in salivary secretion, 234 saliva samples were collected three times per day for three days from 13 subjects and analyzed. Capillary electrophoresis-mass spectrometry was used for non-targeted quantification of water-soluble metabolites. Results: No significant inter- or intra-day variations were observed in salivary secretion volume. No significant inter-day variations were observed in metabolomic patterns. In contrast, significant intra-day variations were observed in salivary metabolomic profiles. The difference was more obvious for stimulated saliva than for unstimulated saliva. These profile changes were independent of salivary secretion volume. Conclusions: Our results indicated that diurnal change had a greater effect on salivary metabolomic profiles than the other factors. Hence, sampling time should be tightly controlled to minimize unexpected bias in the clinical use of salivary metabolomics.

© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. Synechococcus elongatus PCC7942, a cyanobacterium that uses light and carbon dioxide to grow, has a high ability to incorporate DNA by transformation. To assess the effective delivery of large DNA in plasmid form, we cloned the endogenous plasmid pANL (46.4 kbp) into a BAC vector of Escherichia coli. The plasmid p38ANL (54.3 kbp) replaced the native plasmid. To assess the delivery of larger DNA into PCC7942, p38ANL was fused to the broad-host-range conjugal transfer plasmid pUB307IP (53.5 kbp). The resulting plasmid pUB307IP501 (107.9 kbp) was transmitted from E. coli to PCC7942 by simple mixing of donor and recipient cultures. PCC7942 transcipients possessed only pUB307IP501, replacing the preexisting pANL. In contrast, the pUB307IP501 plasmid was unable to transform PCC7942, indicating that natural transformation of DNA may be restricted by size limitations. The ability to deliver large DNA by conjugation may lead to genetic engineering in PCC7942.