Biology subjects

Hirata, H.

Publications and source records attributed to Hirata, H..

4 recordsLinked to original sources

bioRxiv2021

RNA-Seq data analysis for Planarian with tensor decomposition-based unsupervised feature extraction

RNA-Seq data analysis of non-model organisms is often difficult because of the lack of a well-annotated genome. However, in non-model organisms, contigs can be generated by de novo assembling. This can result in a large number of transcripts, making it difficult to easily remove redundancy. A large number of transcripts can also lead to difficulty in the recognition of differentially expressed transcripts (DETs) between more than two experimental conditions, because P-values must be corrected by considering multiple comparison corrections whose effect is enhanced as the number of transcripts increases. Heavily corrected P-values often fail to take sufficiently small P-values as significant. In this study, we applied a recently proposed tensor decomposition (TD)-based unsupervised feature extraction (FE) to the RNA-seq data obtained for a non-model organism, planarian Dugesia japonica; Although we used de novo assembled transcriptome reference with high redundancy, we successfully obtained a larger number of transcripts whose expression was altered between normal and defective samples as well as during time development than those identified by a conventional method. TD-based unsupervised FE is expected to be an effective tool that can identify a substantial number of DETs, even when a poorly annotated genome is available.

bioinformatics↗

bioRxiv2021

Intracellular and intercellular gene regulatory networks inference from time-course individual RNA-Seq

Gene regulatory network (GRN) inference is an effective approach to understand the molecular mechanisms underlying biological events. Generally, GRN inference mainly targets intracellular regulatory relationships such as transcription factors and their associated targets. In multicellular organisms, there are both intracellular and intercellular regulatory mechanisms. Thus, we hypothesize that GRNs inferred from time-course individual (whole embryo) RNA-Seq during development can reveal intercellular regulatory relationships (signaling pathways) underlying the development. Here, we conducted time-course bulk RNA-Seq of individual mouse embryos during early development, followed by pseudo-time analysis and GRN inference. The results demonstrated that GRN inference from RNA-Seq with pseudo-time can be applied for individual bulk RNA-Seq similar to scRNA-Seq. Validation using an experimental-source-based database showed that our approach could significantly infer GRN for all transcription factors in the database. Furthermore, the inferred ligand-related and receptor-related downstream genes were significantly overlapped. Thus, the inferred GRN based on whole organism could include intercellular regulatory relationships, which cannot be inferred from scRNA-Seq based only on gene expression data. Overall, inferring GRN from time-course bulk RNA-Seq is an effective approach for understanding the regulatory relationships underlying biological events in multicellular organisms.

bioinformatics↗

bioRxiv2021

Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) {beta} treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

physiology↗

bioRxiv2020

DeLTa-Seq: direct-lysate targeted RNA-Seq from crude tissue lysate.

Using current mRNA quantification methods such as RT-qPCR and RNA-Seq, it is very difficult to examine thousands of tissue samples due to cost and labor of RNA extraction and quantification steps. Here, we developed Direct-RT buffer in which homogenization of tissue samples and direct-lysate reverse transcription can be conducted without RNA purification. We showed that appreciate concentration of DTT prevented RNA degradation but not RT in the lysates of several plants tissues, yeast, and zebrafish larvae. Using the buffer, direct reverse transcription on the lysates could produce comparable amount of cDNA with that synthesized from purified RNA. Furthermore, we established DeLTa-Seq (Direct-Lysate reverse transcription and Targeted RNA-Seq) method. DeLTa-Seq is a cost-effective, high-throughput and highly-precise quantification method for the expressions of hundreds of genes. It enables us to conduct large-scale studies using thousands of samples such as chemical screening, field experiments and studies focusing on individual variability.

molecular biology↗