Analysis of molecular mechanism for acceleration of polyembryony using gene functional annotation pipeline in Copidosoma floridanum.

BACKGROUND: Polyembryony is defined as the formation of several embryos from a single egg. This phenomenon can occur in humans, armadillo, and some endoparasitoid insects. However, the mechanism underlying polyembryogenesis in animals remains to be elucidated. The polyembryonic parasitoid wasp Copidosoma floridanum oviposits its egg into an egg of the host insect; eventually, over 2000 individuals will arise from one egg. Previously, we reported that polyembryogenesis is enhanced when the juvenile hormone (JH) added to the culture medium in the embryo culture. Hence, in the present study, we performed RNA sequencing (RNA-Seq) analysis to investigate the molecular mechanisms controlling polyembryogenesis of C. floridanum. Functional annotation of genes is not fully available for C.floridanum; however, whole genome assembly has been archived. Hence, we constructed a pipeline for gene functional annotation in C. floridanum and performed molecular network analysis. We analyzed differentially expressed genes between control and JH-treated molura after 48 h of culture, then used the tblastx program to assign whole C. floridanum transcripts to human gene. RESULTS: We obtained 11,117 transcripts in the JH treatment group and identified 217 differentially expressed genes compared with the control group. As a result, 76% of C. floridanum transcripts were assigned to human genes. Gene enrichment analysis revealed genes associated with platelet degranulation, fatty acid biosynthesis, cell morphogenesis in the differentiation and integrin signaling pathways were fluctuated following JH treatment. Furthermore, Cytoscape analysis revealed a molecular interaction that was possibly associated with polyembryogenesis . CONCLUSIONS: We have constructed a pipeline for gene functional annotation of C. floridanum, and identified transcripts with high similarity to human genes during early embryo developmental. Additionally, this study reveals new molecular interactions associated with polyembryogenesis; these interactions could indicate the molecular mechanisms underlying polyembryony. Our results highlight the potential utility of molecular interaction analysis in human twins.

Identification of functional enolase genes of the silkworm Bombyx mori from public databases with a combination of dry and wet bench processes.

BACKGROUND: Various insect species have been added to genomic databases over the years. Thus, researchers can easily obtain online genomic information on invertebrates and insects. However, many incorrectly annotated genes are included in these databases, which can prevent the correct interpretation of subsequent functional analyses. To address this problem, we used a combination of dry and wet bench processes to select functional genes from public databases. RESULTS: Enolase is an important glycolytic enzyme in all organisms. We used a combination of dry and wet bench processes to identify functional enolases in the silkworm Bombyx mori (BmEno). First, we detected five annotated enolases from public databases using a Hidden Markov Model (HMM) search, and then through cDNA cloning, Northern blotting, and RNA-seq analysis, we revealed three functional enolases in B. mori: BmEno1, BmEno2, and BmEnoC. BmEno1 contained a conserved key amino acid residue for metal binding and substrate binding in other species. However, BmEno2 and BmEnoC showed a change in this key amino acid. Phylogenetic analysis showed that BmEno2 and BmEnoC were distinct from BmEno1 and other enolases, and were distributed only in lepidopteran clusters. BmEno1 was expressed in all of the tissues used in our study. In contrast, BmEno2 was mainly expressed in the testis with some expression in the ovary and suboesophageal ganglion. BmEnoC was weakly expressed in the testis. Quantitative RT-PCR showed that the mRNA expression of BmEno2 and BmEnoC correlated with testis development; thus, BmEno2 and BmEnoC may be related to lepidopteran-specific spermiogenesis. CONCLUSIONS: We identified and characterized three functional enolases from public databases with a combination of dry and wet bench processes in the silkworm B. mori. In addition, we determined that BmEno2 and BmEnoC had species-specific functions. Our strategy could be helpful for the detection of minor genes and functional genes in non-model organisms from public databases.

MeSH ORA framework: R/Bioconductor packages to support MeSH over-representation analysis.

BACKGROUND: In genome-wide studies, over-representation analysis (ORA) against a set of genes is an essential step for biological interpretation. Many gene annotation resources and software platforms for ORA have been proposed. Recently, Medical Subject Headings (MeSH) terms, which are annotations of PubMed documents, have been used for ORA. MeSH enables the extraction of broader meaning from the gene lists and is expected to become an exhaustive annotation resource for ORA. However, the existing MeSH ORA software platforms are still not sufficient for several reasons. RESULTS: In this work, we developed an original MeSH ORA framework composed of six types of R packages, including MeSH.db, MeSH.AOR.db, MeSH.PCR.db, the org.MeSH.XXX.db-type packages, MeSHDbi, and meshr. CONCLUSIONS: Using our framework, users can easily conduct MeSH ORA. By utilizing the enriched MeSH terms, related PubMed documents can be retrieved and saved on local machines within this framework.

High expression of serine protease, Brachyurin in the posterior midgut of black soldier fly (Hermetia illucens) during horse dropping processing.

OBJECTIVE: Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. RESULTS: First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings.

A functional platform for remote use of electron microscopes using web conferencing systems.

We propose a useful system for remote sharing of transmission electron microscope (TEM) images by connecting three computers: a computer connected to a TEM, a computer distributing images and a computer receiving images. Then, we confirmed the performance of three web conferencing systems, Microsoft Teams, Zoom and Google Meet, to evaluate the usefulness of their remote use based on the clarity of images, smoothness of movement and time lag in images on each computer in the system. Display image of operation computer for TEM can be captured using the following two methods: a virtual camera of a video distribution software that provided a good reaction speed to transfer images and the screen sharing by conference system software that could share high-quality images.

Gendoo: functional profiling of gene and disease features using MeSH vocabulary.

Genome-wide data enables us to clarify the underlying molecular mechanisms of complex phenotypes. The Online Mendelian Inheritance in Man (OMIM) is a widely employed knowledge base of human genes and genetic disorders for biological researchers. However, OMIM has not been fully exploited for omics analysis because its bibliographic data structure is not suitable for computer automation. Here, we characterized diseases and genes by generating feature profiles of associated drugs, biological phenomena and anatomy with the MeSH (Medical Subject Headings) vocabulary. We obtained 1 760 054 pairs of OMIM entries and MeSH terms by utilizing the full set of MEDLINE articles. We developed a web-based application called Gendoo (gene, disease features ontology-based overview system) to visualize these profiles. By comparing feature profiles of types 1 and 2 diabetes, we clearly illustrated their differences: type 1 diabetes is an autoimmune disease (P-value = 4.55 x 10(-5)) and type 2 diabetes is related to obesity (P-value = 1.18 x 10(-15)). Gendoo and the developed feature profiles should be useful for omics analysis from molecular and clinical viewpoints. Gendoo is available at http://gendoo.dbcls.jp/.

Construction of TUATinsecta database that integrated plant and insect database for screening phytophagous insect metabolic products with medicinal potential.

Phytophagous insect larvae feed on plants containing secondary metabolic products with biological activity against other predatory organisms. Phytophagous insects can use their specialised metabolic systems to covert these secondary metabolic products into compounds with therapeutic properties useful to mankind. Some Asians drink tea decoctions made from phytophagous insect frass which is believed to be effective against inflammatory diseases. However, insects that can convert plant-derived secondary metabolic products into useful human therapeutic agents remain poorly studied. Here, we constructed the TUATinsecta database by integrating publicly plant/insect datasets for the purpose of selecting insect species. Using TUAT-insecta we selected the Asian swallowtail butterfly, Papilio xuthus larvae fed on several species of Rutaceous plants and examined whether the plant-derived secondary metabolites, especially those present in frass, were chemically altered or not. We extracted metabolic products from frass using three organic solvents with different polarities, and evaluated solvent fractions for their cytotoxic effects against several human cell lines. We found that chloroform frass extracts from P. xuthus larvae fed on Poncirus trifoliata leaves contained significant cytotoxic activity. Our findings demonstrate that screening of insect species using the 'TUATinsecta' database provides an important pipeline for discovering novel therapeutic agents that might be useful for mankind.

BioHackathon 2015: Semantics of data for life sciences and reproducible research.

We report on the activities of the 2015 edition of the BioHackathon, an annual event that brings together researchers and developers from around the world to develop tools and technologies that promote the reusability of biological data. We discuss issues surrounding the representation, publication, integration, mining and reuse of biological data and metadata across a wide range of biomedical data types of relevance for the life sciences, including chemistry, genotypes and phenotypes, orthology and phylogeny, proteomics, genomics, glycomics, and metabolomics. We describe our progress to address ongoing challenges to the reusability and reproducibility of research results, and identify outstanding issues that continue to impede the progress of bioinformatics research. We share our perspective on the state of the art, continued challenges, and goals for future research and development for the life sciences Semantic Web.

Identification of renal transporters involved in sulfate excretion in marine teleost fish.

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

Comparative analysis of seven types of superoxide dismutases for their ability to respond to oxidative stress in Bombyx mori.

Insects are well adapted to changing environmental conditions. They have unique systems for eliminating reactive oxygen species (ROS). Superoxide dismutase (SOD) is a key enzyme that plays a primary role in removing ROS. Bombyx mori is a lepidopteran insect, whose body size is larger than the model insect Drosophila melanogaster, which enabled us to more easily examine gene expression at the tissue level. We searched B. mori SOD (BmSOD) genes using genome database, and we analyzed their function under different type of oxidative stress. Consequently, we identified four new types of BmSODs in addition to the three types already known. Two of the seven types had a unique domain architecture that has not been discovered previously in the SOD family, and they were expressed in different tissues and developmental stages. Furthermore, these BmSODs responded differently to several kinds of stressors. Our results showed that the seven types of BmSODs are likely to play different roles in B. mori; therefore, B. mori could be used to distinguish the functions of each SOD for resistance to oxidative stress that changes with the environmental conditions.

Construction of a simple evaluation system for the intestinal absorption of an orally administered medicine using Bombyx mori larvae.

Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.

Calculating the quality of public high-throughput sequencing data to obtain a suitable subset for reanalysis from the Sequence Read Archive.

It is important for public data repositories to promote the reuse of archived data. In the growing field of omics science, however, the increasing number of submissions of high-throughput sequencing (HTSeq) data to public repositories prevents users from choosing a suitable data set from among the large number of search results. Repository users need to be able to set a threshold to reduce the number of results to obtain a suitable subset of high-quality data for reanalysis. We calculated the quality of sequencing data archived in a public data repository, the Sequence Read Archive (SRA), by using the quality control software FastQC. We obtained quality values for 1 171 313 experiments, which can be used to evaluate the suitability of data for reuse. We also visualized the data distribution in SRA by integrating the quality information and metadata of experiments and samples. We provide quality information of all of the archived sequencing data, which enable users to obtain sufficient quality sequencing data for reanalyses. The calculated quality data are available to the public in various formats. Our data also provide an example of enhancing the reuse of public data by adding metadata to published research data by a third party.

Data integration aids understanding of butterfly-host plant networks.

Although host-plant selection is a central topic in ecology, its general underpinnings are poorly understood. Here, we performed a case study focusing on the publicly available data on Japanese butterflies. A combined statistical analysis of plant-herbivore relationships and taxonomy revealed that some butterfly subfamilies in different families feed on the same plant families, and the occurrence of this phenomenon more than just by chance, thus indicating the independent acquisition of adaptive phenotypes to the same hosts. We consequently integrated plant-herbivore and plant-compound relationship data and conducted a statistical analysis to identify compounds unique to host plants of specific butterfly families. Some of the identified plant compounds are known to attract certain butterfly groups while repelling others. The additional incorporation of insect-compound relationship data revealed potential metabolic processes that are related to host plant selection. Our results demonstrate that data integration enables the computational detection of compounds putatively involved in particular interspecies interactions and that further data enrichment and integration of genomic and transcriptomic data facilitates the unveiling of the molecular mechanisms involved in host plant selection.

Superoxide dismutases, SOD1 and SOD2, play a distinct role in the fat body during pupation in silkworm Bombyx mori.

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation.

Experimental design-based functional mining and characterization of high-throughput sequencing data in the sequence read archive.

High-throughput sequencing technology, also called next-generation sequencing (NGS), has the potential to revolutionize the whole process of genome sequencing, transcriptomics, and epigenetics. Sequencing data is captured in a public primary data archive, the Sequence Read Archive (SRA). As of January 2013, data from more than 14,000 projects have been submitted to SRA, which is double that of the previous year. Researchers can download raw sequence data from SRA website to perform further analyses and to compare with their own data. However, it is extremely difficult to search entries and download raw sequences of interests with SRA because the data structure is complicated, and experimental conditions along with raw sequences are partly described in natural language. Additionally, some sequences are of inconsistent quality because anyone can submit sequencing data to SRA with no quality check. Therefore, as a criterion of data quality, we focused on SRA entries that were cited in journal articles. We extracted SRA IDs and PubMed IDs (PMIDs) from SRA and full-text versions of journal articles and retrieved 2748 SRA ID-PMID pairs. We constructed a publication list referring to SRA entries. Since, one of the main themes of -omics analyses is clarification of disease mechanisms, we also characterized SRA entries by disease keywords, according to the Medical Subject Headings (MeSH) extracted from articles assigned to each SRA entry. We obtained 989 SRA ID-MeSH disease term pairs, and constructed a disease list referring to SRA data. We previously developed feature profiles of diseases in a system called "Gendoo". We generated hyperlinks between diseases extracted from SRA and the feature profiles of it. The developed project, publication and disease lists resulting from this study are available at our web service, called "DBCLS SRA" (http://sra.dbcls.jp/). This service will improve accessibility to high-quality data from SRA.

BioCompass: a novel functional inference tool that utilizes MeSH hierarchy to analyze groups of genes.

Microarray technology has become employed widely for biological researchers to identify genes associated with conditions such as diseases and drugs. To date, many methods have been developed to analyze data covering a large number of genes, but they focus only on statistical significance and cannot decipher the data with biological concepts. Gene Ontology (GO) is utilized to understand the data with biological interpretation; however, it is restricted to specific ontology such as biological process, molecular function, and cellular component. Here, we attempted to apply MeSH (Medical Subject Headings) to interpret groups of genes from biological viewpoint. To assign MeSH terms to genes, in this study, contexts associated with genes are retrieved from full set of MEDLINE data using machine learning, and then extracted MeSH terms from retrieved articles. Utilizing the developed method, we implemented a software called BioCompass. It generates high-scoring lists and hierarchical lists for diseases MeSH terms associated with groups of genes to utilize MeSH and GO tree, and illustrated a wiring diagram by linking genes with extracted association from articles. Researchers can easily retrieve genes and keywords of interest, such as diseases and drugs, associated with groups of genes. Using retrieved MeSH terms and OMIM in conjunction with, we could obtain more disease information associated with target gene. BioCompass helps researchers to interpret groups of genes such as microarray data from a biological viewpoint.

Mechanism of acid adaptation of a fish living in a pH 3.5 lake.

Despite unfavorable conditions, a single species of fish, Osorezan dace, lives in an extremely acidic lake (pH 3.5) in Osorezan, Aomori, Japan. Physiological studies have established that this fish is able to prevent acidification of its plasma and loss of Na(+). Here we show that these abilities are mainly attributable to the chloride cells of the gill, which are arranged in a follicular structure and contain high concentrations of Na(+)-K(+)-ATPase, carbonic anhydrase II, type 3 Na(+)/H(+) exchanger (NHE3), type 1 Na(+)-HCO(3)(-) cotransporter, and aquaporin-3, all of which are upregulated on acidification. Immunohistochemistry established their chloride cell localization, with NHE3 at the apical surface and the others localized to the basolateral membrane. These results suggest a mechanism by which Osorezan dace adapts to its acidic environment. Most likely, NHE3 on the apical side excretes H(+) in exchange for Na(+), whereas the electrogenic type 1 Na(+)-HCO(3)(-) cotransporter in the basolateral membrane provides HCO(3)(-) for neutralization of plasma using the driving force generated by Na(+)-K(+)-ATPase and carbonic anhydrase II. Increased expression of glutamate dehydrogenase was also observed in various tissues of acid-adapted dace, suggesting a significant role of ammonia and bicarbonate generated by glutamine catabolism.