Combining in silico prediction and ribosome profiling in a genome-wide search for novel putatively coding sORFs

Background: It was long assumed that proteins are at least 100 amino acids (AAs) long. Moreover, the detection of short translation products (e. g. coded from small Open Reading Frames, sORFs) is very difficult as the short length makes it hard to distinguish true coding ORFs from ORFs occurring by chance. Nevertheless, over the past few years many such non-canonical genes (with ORFs < 100 AAs) have been discovered in different organisms like Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster. Thanks to advances in sequencing, bioinformatics and computing power, it is now possible to scan the genome in unprecedented scrutiny, for example in a search of this type of small ORFs. Results: Using bioinformatics methods, we performed a systematic search for putatively functional sORFs in the Mus musculus genome. A genome-wide scan detected all sORFs which were subsequently analyzed for their coding potential, based on evolutionary conservation at the AA level, and ranked using a Support Vector Machine (SVM) learning model. The ranked sORFs are finally overlapped with ribosome profiling data, hinting to sORF translation. All candidates are visually inspected using an in-house developed genome browser. In this way dozens of highly conserved sORFs, targeted by ribosomes were identified in the mouse genome, putatively encoding micropeptides. Conclusion: Our combined genome-wide approach leads to the prediction of a comprehensive but manageable set of putatively coding sORFs, a very important first step towards the identification of a new class of bioactive peptides, called micropeptides

Spatially resolved metabolic distribution for unraveling the physiological change and responses in tomato fruit using matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI)

Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI–MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI–MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI–MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI–MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations

A Chemometrics-driven Strategy for the Bioactivity Evaluation of Complex Multicomponent Systems and the Effective Selection of Bioactivity-predictive Chemical Combinations

Although understanding their chemical composition is vital for accurately predicting the bioactivity of multicomponent drugs, nutraceuticals, and foods, no analytical approach exists to easily predict the bioactivity of multicomponent systems from complex behaviors of multiple coexisting factors. We herein represent a metabolic profiling (MP) strategy for evaluating bioactivity in systems containing various small molecules. Composition profiles of diverse bioactive herbal samples from 21 green tea extract (GTE) panels were obtained by a high-throughput, non-targeted analytical procedure. This employed the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) technique, using 1,5-diaminonaphthalene (1,5-DAN) as the optical matrix for detecting GTE-derived components. Multivariate statistical analyses revealed differences among the GTEs in their antioxidant activity, oxygen radical absorbance capacity (ORAC). A reliable bioactivity-prediction model was constructed to predict the ORAC of diverse GTEs from their compositional balance. This chemometric procedure allowed the evaluation of GTE bioactivity by multicomponent rather than single-component information. The bioactivity could be easily evaluated by calculating the summed abundance of a few selected components that contributed most to constructing the prediction model. 1,5-DAN-MALDI-MS-MP, using diverse bioactive sample panels, represents a promising strategy for screening bioactivity-predictive multicomponent factors and selecting effective bioactivity-predictive chemical combinations for crude multicomponent systems

Mass spectrometry of short peptides reveals common features of metazoan peptidergic neurons

The evolutionary origins of neurons remain unknown. Although recent genome data of extant early-branching animals have shown that neural genes existed in the common ancestor of animals, the physiological and genetic properties of neurons in the early evolutionary phase are still unclear. Here, we performed a mass spectrometry-based comprehensive survey of short peptides from early-branching lineages Cnidaria, Porifera and Ctenophora. We identified a number of mature ctenophore neuropeptides that are expressed in neurons associated with sensory, muscular and digestive systems. The ctenophore peptides are stored in vesicles in cell bodies and neurites, suggesting volume transmission similar to that of cnidarian and bilaterian peptidergic systems. A comparison of genetic characteristics revealed that the peptide-expressing cells of Cnidaria and Ctenophora express the vast majority of genes that have pivotal roles in maturation, secretion and degradation of neuropeptides in Bilateria. Functional analysis of neuropeptides and prediction of receptors with machine learning demonstrated peptide regulation of a wide range of target effector cells, including cells of muscular systems. The striking parallels between the peptidergic neuronal properties of Cnidaria and Bilateria and those of Ctenophora, the most basal neuron-bearing animals, suggest a common evolutionary origin of metazoan peptidergic nervous systems

Identification of neuropeptides and evolutionarily conserved peptide receptors in Hydra

Neuropeptides are utilized as transmitters and hormones in higher metazoans. In lower metazoans, the information of neuropeptides are still limited. Hydra, a member of Cnidaria, is one of the most basal metazoans that have a definite body plan and nervous system. It has been implicated that developmental processes and physiology of Hydra are highly regulated by various signaling peptides. Furthermore, neurotransmission in Cnidaria are thought to be performed exclusively with peptides. Thus, identification and characterization of neuropeptides are of particular importance to understand both development and physiology of Hydra. The information obtained from Hydra should also shed light on evolution of peptides and their receptors.Chapter Ⅰ Identification of evolutionarily conserved neuropeptides and prediction of their receptors in Hydra.Some neuropeptide families are conserved among Chordata and Arthropoda. This suggests that neuropeptides are conserved throughout animal evolution and that evolutionarily conserved neuropeptides may be identified in Hydra. Thus, I attempted to identify such neuropeptides in Hydra by in silico data mining. First, I developed an algorithm to detect possible neuropeptides in the Hydra EST database. I discovered a novel precursor gene that possibly encodes neuropeptide Y (NPY)-related peptides. The deduced precursor contained a N-terminal signal peptide, 3 putative NPY-related peptides, each flanked by an amidation motif "G(K/R)" at the Cterminal side. The sequence of C-terminal 5 amino acids in one of the deduced peptides was quite similar to neuropeptide F, a member of NPY family, of Helix aspersa (Brown garden snail). In situ hybridization revealed that the gene was expressed in a subpopulation of neurons distributed in tentacles and foot region of Hydra. Thus, the peptides were designated as Hydra neruropeptide Fs. Next, I looked for homologs of Hydra neuropeptides, Hym-355 and GLWamides in the Caenorhabditis elegans EST database as a midpoint between primitive and higher organisms. Genes that possibly encode Hym355-like and GLWamide-like peptides were found. The peptides similar to these C. elegans peptides were searched in the insect DNA databases. C.elegans Hym-355-like peptides showed similarity to insect PRXamides (Ecdysis triggering hormone, CAP, Pyrokinin), and C.elegans GLWamide-like peptides showed similarity to insect Allatostatin type B. These findings suggest that some of the neuropeptides are evolutionarily conserved throughout animal evolution. Neuropeptides and their receptors (GPCRs) evolve together. In fact, GPCRs form clusters in a phylogenetic tree and those in a cluster often use the same or similar ligands. In this study, sets of novel GPCR genes were identified from the Hydra whole genome shotgun database. Phylogenetic analysis showed that some of the Hydra GPCRs were highly related to neuropeptide receptors known in higher metazoans. One of them (HGR001) was related to neuropeptide Y and cholecystokinin receptors. The gene was expressed in dividing nematoblasts (sting cell precursors) suggesting its involvement in proliferation and/or early differentiation of nematocytes. These results suggest that some of the neuropeptides and their receptors are evolutionarily conserved and based on the conservation, novel peptides and GPCRs can be identified not only in Hydra but also in other animals.Chapter Ⅱ Identification of novel neuropeptide family, FRamide-1 and FRamide-2 In the course of systematic identification of peptide signaling molecules combined with EST database analysis in Hydra, we have identified a novel neuropeptide family that consists of two members with a C-terminal motif of FRamide; FRamide-1 (IPTGTLIFRamide) and FRamide-2 (APGSLLFRamide). The precursor sequence deduced from cDNA contained a single copy each of FRamide-1 and FRamide-2 precursor. The expression analysis by whole-mount in situ hybridization showed that the peptide encoding gene was specifically expressed in a subpopulation of neurons that were distributed throughout the body from tentacles to basal disc. In order to analyze the subpopulation of FRamide-1 and FRamide-2 expressing neuron in Hydra, the expression pattern was compared with other neuropeptides (Hym-176, Hym-355, GLWamides) expressing neurons. Double in situ hybridization analysis showed that the population was further subdivided into two; one consisted of neurons expressing both FRamide-1/FRamide-2 and Hym-176 encoding genes and the other consisted of neurons expressing only the FRamide-1/FRamide-2 encoding gene. The neuron population did not overlap with that expressing the Hym-355 or GLWamdie neuropeptide gene. FRamide-1 evoked elongation of the body column of epithelial Hydra that is composed of epithelial cells and gland cells but lacks essentially all the cells in the interstitial stem cell lineage including neurons. In contrast, FRamide-2 evoked the body column contraction. These results suggest that both of the neuropeptides directly act on epithelial (muscle) cells as neurotransmitters and are involved in the body movement in a longitudinal direction

Nondestructive detection of hidden chemical compounds with laser Compton-scattering gamma rays

金属で厳重に遮へいされた爆発物の非破壊測定法を発明. 京都大学プレスリリース. 2009-04-29.A nondestructive assay method for measuring a shielded chemical compound has been proposed. The chemical compound is measured by using a nuclear resonance fluorescence (NRF) measurement technique with an energy tunable laser Compton-scattering (LCS) -ray source. This method has an advantage that hidden materials can be detected through heavy shields such as iron plates of a thickness of several centimeters. A detection of a chemical compound of melamine, C3H6N6, shielded by 15-mm-thick iron and 4-mm-thick lead plates is demonstrated. The NRF -rays of 12C and 14N of the melamine are measured by using the LCS -rays of the energies of up to 5.0 MeV. The observed ratio (12C/14N)exp=0.39±0.12 is consistent with (C/N)melamine=0.5