Biology subjects

Hirota, K.

Publications and source records attributed to Hirota, K..

26 records · Page 2Linked to original sources

bioRxiv2021

MinION™, a portable long-read sequencer, enables rapid vaginal microbiota analysis in a clinical setting

It has been suggested that the local microbiota in the reproductive organs is relevant to womens health and may also affect pregnancy outcomes. Analysis of partial 16S ribosomal RNA (rRNA) gene sequences generated by short-read sequencers has been used to identify vaginal and endometrial microbiota, but it requires a long time to obtain the results, making it unsuitable for the rapid analysis of small samples in a clinical context. We demonstrated a simple workflow using the nanopore sequencer MinION that allows high-resolution and rapid differentiation of vaginal microbiota. Vaginal samples collected from 18 participants were subjected to DNA extraction and full-length 16S rRNA gene sequencing with MinION. The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The vaginal microbiota results could be reported within 2 days of specimen receipt. Although bacterial vaginosis (BV) was not diagnosed by the Nugent score in any cases, groups with both healthy and BV-like vaginal microbiota were clearly characterized by MinION sequencing. We conclude that full-length 16S rRNA gene sequencing analysis with MinION provides a rapid means for identifying vaginal bacteria with higher resolution. Species-level profiling of human vaginal microbiota by MinION sequencing can allow the analysis of associations with conditions such as genital infections, endometritis, and threatened miscarriage.

microbiology↗

bioRxiv2020

Hydrophilic Shell Matrix Proteins of Nautilus pompilius and The Identification of a Core Set of Conchiferan Domains

Despite being a member of the shelled mollusks (Conchiferans), most members of extant cephalopods have lost their external biomineralized shells, except for the Nautiloids. Here, we report the result of our study to identify major Shell Matrix Proteins and their domains in the Nautiloid Nautilus pompilius, in order to gain a general insight into the evolution of Conchiferan Shell Matrix Proteins. In order to do so, we conducted transcriptomics of the mantle, and proteomics of the shell of N. pompilius simultaneously. Analyses of obtained data identified 61 distinct shell-specific sequences. Of the successfully annotated 27 sequences, protein domains were predicted in 19. Comparative analysis of Nautilus sequences with four Conchiferans for which Shell Matrix Protein data were available (the pacific oyster, the pearl oyster, the limpet, and the Euhadra snail) revealed that three proteins and six domains of the shell proteins are conserved in all Conchiferans. Interestingly, when the terrestrial Euhadra snail was excluded, another five proteins and six domains were found to be shared among the four marine Conchiferans. Phylogenetic analyses indicated that most of these proteins and domains were present in the ancestral Conchiferan, but employed in shell formation later and independently in most clades. Although further studies utilizing deeper sequencing techniques to obtain genome and full-length sequences, and functional analyses, must be done in the future, our results here provide important pieces of information for the elucidation of the evolution of Conchiferan shells at the molecular level.

zoology↗

bioRxiv2020

A dual prokaryotic (E. coli) expression system (pdMAX)

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. This pdMAX system is a manageable size at 5811 bp. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-{beta}-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. Interestingly, the dual induction plasmid system shows limited protein expression. To analyze the expression of inserted genes, we performed an -complementation assay, in which the -peptide of the lacZ ({beta}-galactosidase) gene is inserted in XL-10 E. coli cells. E. coli expressing the recombinant plasmid form blue colonies when plated on ampicillin/IPTG- or arabinose/X-gal-containing plates if the -peptide was correctly inserted in-frame to produce the tagged protein. This assay system assesses whether the -peptide was inserted at the restriction sites (EcoRV or SmaI), whether the inserted peptide was expressed, and whether the inserted -peptide sequence was ligated in-frame to produce a tagged protein. Frameshifts result in no -complementation and white colonies. With the dual promoter plasmid (pdMAX) system, expressed lacZ activity was significantly decreased comparing with the solo expression (pgMAX) system. Despite this disadvantage, we believe the pdMAX system is still useful for the analysis of distinct genes in E. coli, which will enable different types of expression analysis. Overall, the novel pdMAX system allows for efficient subcloning of two different genes. Furthermore, the pdMAX system could be used to induce and analyze the expression of two distinct genes and adopted to various types of prokaryotic gene expression analyses.

molecular biology↗

bioRxiv2020

A new dual prokaryotic (E. coli) and mammalian expression system (pgMAXs)

We introduce an efficient subcloning and expression plasmid system with two different modes (prokaryotic for expression in Escherichia coli with lac promoter and mammalian modes with cytomegalovirus promoter). The efficient subcloning (DNA insertion) is based upon a DNA topoisomerase II toxin-originated gene for effective selection with isopropyl-{beta}-D-thiogalactoside (IPTG) induction. The new pgMAXs system is manageable size (4452 bp) and has also various types of protein tags (flag, myc, poly-histidine, Human influenza hemagglutinin, strep, and v5) for expression analysis. With pgMAXs system, various types of fluorescent proteins were subcloned and prtein expressions were confirmed. We also tried to identify epitope amino acid sequences for anti-calcium channel {beta}2 antibody, by constructing epitope-library with DNaseI-partial digestion and subcloning into EcoRV site in pgMAXs. The new pgMAXs plasmid system enables highly efficient subcloning, simple expression in E. coli and that it has a simple deletion step of rare 8-nucleotide rare-cutter blunt-end enzymes for mammalian expression plasmid construction. Taken together, the pgMAXs system simplifies prokaryotic and mammalian gene expression analyses.

molecular biology↗

bioRxiv2020

Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution

BackgroundSpecies-level genetic characterization of complex bacterial communities has important clinical applications in both diagnosis and treatment. Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene has proven to be a powerful strategy for the taxonomic classification of bacteria. This study aims to improve the method for full-length 16S rRNA gene analysis using the nanopore long-read sequencer MinION. We compared it to the conventional short-read sequencing method in both a mock bacterial community and human fecal samples. ResultsWe modified our existing protocol for full-length 16S amplicon sequencing by MinION. A new strategy for library construction with an optimized primer set overcame PCR-associated bias and enabled taxonomic classification across a broad range of bacterial species. We compared the performance of full-length and short-read 16S amplicon sequencing for the characterization of human gut microbiota with a complex bacterial composition. The relative abundance of dominant bacterial genera was highly similar between full-length and short-read sequencing. At the species level, MinION long-read sequencing had better resolution for discriminating between members of particular taxa such as Bifidobacterium, allowing an accurate representation of the sample bacterial composition. ConclusionsOur present microbiome study, comparing the discriminatory power of full-length and short-read sequencing, clearly illustrated the analytical advantage of sequencing the full-length 16S rRNA gene, which provided the requisite species-level resolution and accuracy in clinical settings.

microbiology↗

bioRxiv2018

Direct PCR amplification of 16S rRNA genes offers accelerated bacterial identification using the MinION™ nanopore sequencer

Rapid identification of bacterial pathogens is crucial for appropriate and adequate antibiotic treatment, which significantly improves patient outcomes. 16S ribosomal RNA (rRNA) gene amplicon sequencing has proven to be a powerful strategy for diagnosing bacterial infections. We have recently established a sequencing method and bioinformatics pipeline for 16S rRNA gene analysis utilizing the Oxford Nanopore Technologies MinION sequencer. In combination with our taxonomy annotation analysis pipeline, the system enabled the molecular detection of bacterial DNA in a reasonable timeframe for diagnostic purposes. However, purification of bacterial DNA from specimens remains a rate-limiting step in the workflow. To further accelerate the process of sample preparation, we adopted a direct PCR strategy that amplifies 16S rRNA genes from bacterial cell suspensions without DNA purification. Our results indicate that differences in cell wall morphology significantly affect direct PCR efficiency and sequencing data. Notably, mechanical cell disruption preceding direct PCR was indispensable for obtaining an accurate representation of the specimen bacterial composition. Furthermore, 16S rRNA gene analysis of mock polymicrobial samples indicated that primer sequence optimization is required to avoid preferential detection of particular taxa and to cover a broad range of bacterial species. This study establishes a relatively simple workflow for rapid bacterial identification via MinIONTM sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

microbiology↗

bioRxiv2018

In renal cell carcinoma, cancerous phenotypes linked to hypoxia-inducible factors are insensitive to the volatile anesthetic isoflurane

The possibility that anesthesia during cancer surgery may affect cancer recurrence, metastasis, and patient prognosis has become one of the most important topics of interest in cancer treatment. For example, the volatile anesthetic isoflurane was reported in several studies to induce hypoxia-inducible factors, and thereby enhance malignant phenotypes in vitro. Indeed, these transcription factors are considered critical regulators of cancer-related hallmarks, including "sustained proliferative signaling, evasion of growth suppressors, resistance to cell death, replicative immortality, angiogenesis, invasion, and metastasis." This study aimed to investigate the impact of isoflurane on the growth and migration of derivatives of the renal cell line RCC4. We indicated that isoflurane treatment did not positively influence cancer cell phenotypes, and that hypoxia-inducible factors (HIFs) maintain hallmark cancer cell phenotypes including gene expressions signature, metabolism, cell proliferation and cell motility. The present results indicate that HIF activity is not influenced by the volatile anesthetic isoflurane.

cancer biology↗

bioRxiv2017

Propofol induces metabolic reprogramming and cell death in a mitochondrial electron transport chain-dependent manner

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia in operating rooms and for sedation in intensive care units. Although there is no widely accepted definition of propofol infusion syndrome (PRIS), PRIS is defined as the development of metabolic acidosis, rhabdomyolysis, hyperkalemia, hepatomegaly, renal failure, arrhythmia, and progressive cardiac failure. In vitro evidence suggests that PRIS is related to the impaired mitochondrial function. There are indications that preexisting mitochondrial disorders predispose to PRIS. However, the precise molecular mechanisms, including mitochondrial defects and a metabolic conversion by propofol, are largely unknown as yet. To elucidate the underlying cellular and molecular mechanisms of PRIS, we investigated the effects of propofol on the cellular metabolic mode and cell death. We demonstrated that clinically relevant concentrations of propofol, used within a clinically relevant exposure time, suppressed the mitochondrial function, caused the generation of reactive oxygen species, and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting complexes I and III of mitochondria. The data also indicated that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted the cell death and caspase activation induced by propofol.

pharmacology and toxicology↗