Background: Species-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. Results: We modified our existing protocol for full-length 16S rRNA gene 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 rRNA gene 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. Conclusions: Our 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.

During the past few decades, it has been widely recognized that reducing-oxidizing (Redox) responses occurring at the intra- and extra-cellular levels are one of most important biological phenomena and dysregulated redox responses are involved in the initiation and progression of multiple diseases. Thioredoxin 1 (Trx1) and Thioredoxin 2 (Trx2), mainly located in the cytoplasm and mitochondria, respectively, are ubiquitously expressed in variety of cells and control cellular reactive oxygen species (ROS) by reducing the disulfides into thiol groups. Thioredoxin interacting protein (Txnip/TBP-2/VDUP1) directly binds to Trx1 & Trx2 (Trx) and inhibit the reducing activity of Trx through their disulfide exchange. Recent studies have revealed that Trx1 and Txnip are involved in some critical redox-dependent signal pathways including NLRP3 inflammasome activation in a redox-dependent manner. Therefore, Trx/Txnip, a redox-sensitive signaling complex is a regulator of cellular redox status and has emerged as a key component in the link between redox-regulation and the pathogenesis of diseases. Here, we review the novel functional concept of the redox-related protein complex, named “Redoxisome”, consisting of Trx/Txnip, as a critical regulator for intra- and extra-cellular redox signaling, involved in the pathogenesis of various diseases such as cancer, autoimmune disease, and diabetes.

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 time frame 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 MinION™ sequencing, which reduces the turnaround time from sample to result, and provides a reliable method that may be applicable to clinical settings.

Thioredoxin-binding protein-2 (TBP-2)/vitamin D₃ up-regulated protein 1 is an endogenous molecule interacting with thioredoxin (TRX), negatively regulating TRX function, and being implicated in the suppression of tumor development and metastasis. We found that TBP-2 ectopically expressed in the breast cancer cell line MCF-7 was localized predominantly in the nucleus exhibiting growth suppressive activity. The nuclear accumulation of endogenous TBP-2 protein was also demonstrated when the cells were treated with an anti-cancer drug, suberoylanilide hydroxamic acid. To investigate the mechanism underlying the nuclear localization, we performed a yeast two-hybrid screening and identified importin α₁ (Rch1) as a protein interacting with TBP-2. The physical interaction between TBP-2 and Rch1 was confirmed with a glutathione S-transferase pull-down assay. The interaction of TBP-2 was specific to Rch1 among other importin α subfamilies (Qip1 and NPI-1), and amino acids 1–227 of TBP-2 were sufficient for both the interaction with Rch1 and the nuclear localization, although there is no typical nuclear localization signal in this sequence. The expression of short interfering RNA of Rch1 suppressed suberoylanilide hydroxamic acid-induced nuclear accumulation of TBP-2. Collectively, our results strongly suggest that an interaction with importin system is required for TBP-2 nuclear translocation and growth control tightly associated with TRX-dependent redox regulation of transcription factors.

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia and sedation in critical patient care. However, the rare but severe complication propofol infusion syndrome (PRIS) can occur, especially in patients receiving high doses of propofol for prolonged periods. In vivo and in vitro evidence suggests that the propofol toxicity is related to the impaired mitochondrial function. However, underlying molecular mechanisms remain unknown. Therefore, we investigated effects of propofol on cell metabolism and death using a series of established cell lines of various origins, including neurons, myocytes, and trans-mitochondrial cybrids, with defined mitochondrial DNA deficits. We demonstrated that supraclinical concentrations of propofol in not less than 50 μM disturbed the mitochondrial function and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting mitochondrial complexes I, II and III. This disturbance in mitochondrial electron transport caused the generation of reactive oxygen species, resulting in apoptosis. We also found 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 propofol-induced caspase activation and cell death induced by clinical relevant concentrations of propofol in not more than 25 μM. With further experiments with appropriate in vivo model, it is possible that the processes to constitute the molecular basis of PRIS are identified.