Redoxisome Update: TRX and TXNIP/TBP2-Dependent Regulation of NLRP-1/NLRP-3 Inflammasome.

Recent studies have provided evidence for the direct binding of thioredoxin-1 (TRX1) to a component of inflammasome complex NLR family pyrin domain containing 1 (NLRP-1). This interaction suggests a potential role for TRX1 in the regulation of the NLRP-1 inflammasome. Furthermore, the NLRP-3 inflammasome is known to bind TRX1 and its inhibitor, TRX-binding protein-2/TRX-interacting protein/vitamin D3 upregulated protein-1 (TBP2/TXNIP/VDUP-1). This binding forms a redox-sensitive complex, termed the "Redoxisome," as described previously. However, the specific functions of NLRP-1 within the redoxisome complex remain undefined. Antioxid. Redox Signal. 40, 595-597.

The nociceptin/orphanin FQ receptor partial agonist sunobinop promotes non-REM sleep in rodents and patients with insomnia.

The potent and partial agonist sunobinop activates the nociceptin/orphanin-FQ peptide receptor and promotes non-REM sleep in rodents and patients with insomnia.

Using nanopore sequencing to identify fungi from clinical samples with high phylogenetic resolution.

The study of microbiota has been revolutionized by the development of DNA metabarcoding. This sequence-based approach enables the direct detection of microorganisms without the need for culture and isolation, which significantly reduces analysis time and offers more comprehensive taxonomic profiles across broad phylogenetic lineages. While there has been an accumulating number of researches on bacteria, molecular phylogenetic analysis of fungi still remains challenging due to the lack of standardized tools and the incompleteness of reference databases limiting the accurate and precise identification of fungal taxa. Here, we present a DNA metabarcoding workflow for characterizing fungal microbiota with high taxonomic resolution. This method involves amplifying longer stretches of ribosomal RNA operons and sequencing them using nanopore long-read sequencing technology. The resulting reads were error-polished to generate consensus sequences with 99.5-100% accuracy, which were then aligned against reference genome assemblies. The efficacy of this method was explored using a polymicrobial mock community and patient-derived specimens, demonstrating the marked potential of long-read sequencing combined with consensus calling for accurate taxonomic classification. Our approach offers a powerful tool for the rapid identification of pathogenic fungi and has the promise to significantly improve our understanding of the role of fungi in health and disease.

Impact of neoadjuvant therapy on gut microbiome in patients with resectable/borderline resectable pancreatic ductal adenocarcinoma.

BACKGROUND: /Objectives: Effects of chemotherapy on gut microbiota have been reported in various carcinomas. The current study aimed to evaluate the changes in the gut microbiota before and after neoadjuvant chemotherapy (NAC) in patients with resectable (R) and borderline resectable (BR) pancreatic ductal adenocarcinoma (PDAC) and understand their clinical implications. METHODS: Twenty patients diagnosed with R/BR-PDAC were included in this study. Stool samples were collected at two points, before and after NAC, for microbiota analysis using 16S ribosomal RNA (16S rRNA) gene sequences. RESULTS: Of the 20 patients, 18 (90%) were treated with gemcitabine plus S-1 as NAC, and the remaining patients received gemcitabine plus nab-paclitaxel and a fluorouracil, leucovorin, irinotecan, and oxaliplatin combination. No significant differences were observed in the α- and ß-diversity before and after NAC. Bacterial diversity was not associated with Evans classification (histological grade of tumor destruction by NAC) or postoperative complications. The relative abundance of Actinobacteria phylum after NAC was significantly lower than that before NAC (P = 0.02). At the genus level, the relative abundance of Bifidobacterium before NAC in patients with Evans grade 2 disease was significantly higher than that in patients with Evans grade 1 disease (P = 0.03). Patients with Evans grade 2 lost significantly more Bifidobacterium than patients with Evans grade 1 (P = 0.01). CONCLUSIONS: The diversity of gut microbiota was neither decreased by NAC for R/BR-PDAC nor associated with postoperative complications. Lower incidence of Bifidobacterium genus before NAC may be associated with a lower pathological response to NAC.

Full-Length 16S rRNA Gene Analysis Using Long-Read Nanopore Sequencing for Rapid Identification of Bacteria from Clinical Specimens.

Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene is a practical and reliable measure for taxonomic profiling of bacterial communities. This chapter describes the detailed workflow for full-length 16S rRNA gene amplicon analysis using nanopore sequencing and bioinformatics pipelines to analyze nanopore sequencing data for taxonomic assignment. This approach offers a higher taxonomic resolution for bacterial identification from clinical specimens with a markedly reduced timeframe and improved versatility.

Inflammatory Cytokine-Induced HIF-1 Activation Promotes Epithelial-Mesenchymal Transition in Endometrial Epithelial Cells.

The endometrium undergoes repeated proliferation and shedding during the menstrual cycle. Significant changes to this environment include fluctuations in the partial pressure of oxygen, exposure to a high-cytokine environment associated with intrauterine infection, and inflammation. Chronic endometritis is a condition wherein mild inflammation persists in the endometrium and is one of the causes of implantation failure and miscarriage in early pregnancy. It is thought that the invasion of embryos into the endometrium requires epithelial-mesenchymal transition (EMT)-associated changes in the endometrial epithelium. However, the effects of inflammation on the endometrium remain poorly understood. In this study, we investigated the effects of the intrauterine oxygen environment, hypoxia-inducible factor (HIF), and inflammation on the differentiation and function of endometrial epithelial cells. We elucidated the ways in which inflammatory cytokines affect HIF activity and EMT in an immortalized cell line (EM-E6/E7/TERT) derived from endometrial epithelium. Pro-inflammatory cytokines caused significant accumulation of HIF-1α protein, increased HIF-1α mRNA levels, and enhanced hypoxia-induced accumulation of HIF-1α protein. The combined effect of inflammatory cytokines and hypoxia increased the expression of EMT-inducing factors and upregulated cell migration. Our findings indicate that pro-inflammatory factors, including cytokines and LPS, work synergistically with hypoxia to activate HIF-1 and promote EMT in endometrial epithelial cells.

Sevoflurane Does Not Promote the Colony-Forming Ability of Human Mesenchymal Glioblastoma Stem Cells In Vitro.

Background and Objectives: Clinically used concentrations of sevoflurane, an inhaled anesthetic, have been reported to significantly inhibit tumor growth. We investigated the effects of sevoflurane on sphere formation and the proliferation of human glioblastoma stem cells (GSCs) to determine whether sevoflurane exerts short- and long-term effects on human tumor cells. Materials and Methods: High-grade patient-derived GSCs (MD13 and Me83) were exposed to 2% sevoflurane. To evaluate the effect of sevoflurane on viability, proliferation, and stemness, we performed a caspase-3/7 essay, cell proliferation assay, and limiting dilution sphere formation assays. The expression of CD44, a cell surface marker of cancer stem-like cells in epithelial tumors, was evaluated using quantitative reverse transcription PCR. Differences between groups were evaluated with a one-way analysis of variance (ANOVA). Results: Sevoflurane exposure for 4 days did not significantly promote caspase 3/7 activity in MD13 and Me83, and cell proliferation was not observed after 5 days of exposure. Furthermore, prolonged exposure to sevoflurane for 6 days did not promote the sphere-forming and proliferative potential of MD13 and Me83 cells. These results suggest that sevoflurane does not promote either apoptosis, proliferative capacity, or the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. Conclusions: Sevoflurane at clinically used concentrations does not promote the colony-forming ability of human mesenchymal glioblastoma stem cells in vitro. It is very important for neurosurgeons and anesthesiologists to know that sevoflurane, a volatile anesthetic used in surgical anesthesia, would not exacerbate the disease course of GSCs.

16S rRNA nanopore sequencing for the diagnosis of ocular infection: a feasibility study.

OBJECTIVE: We conducted a feasibility study to verify the effectiveness of 16S ribosomal RNA (rRNA) gene analysis using the nanopore sequencer MinION for identifying causative bacteria in several types of ocular infections. METHODS AND ANALYSIS: Four cases of corneal ulcers, one case of endophthalmitis and one case of a conjunctival abscess were included in this study. DNA was extracted from corneal scraping, vitreous samples and secretions from the conjunctival abscess. We conducted 16S rRNA gene amplicon sequencing using MinION and metagenomic DNA analysis. The efficacy of bacterial identification was verified by comparing the conventional culture method with smear observations. RESULTS: 16S rRNA gene sequencing analysis with MinION identified the causative organisms promptly with high accuracy in approximately 4 hours, from ophthalmic specimens. The results of the conventional culture method and 16S rRNA gene sequencing were consistent in all cases. In four of the six cases, a greater variety of organisms was found in the 16S rRNA gene analysis than in bacterial culture. CONCLUSION: Using our workflow, 16S rRNA gene analysis using MinION enabled rapid and accurate identification possible in various kinds of bacterial ocular infections.

AGE/RAGE axis regulates reversible transition to quiescent states of ALK-rearranged NSCLC and pancreatic cancer cells in monolayer cultures.

Cancer recurrence due to tumor cell quiescence after therapy and long-term remission is associated with cancer-related death. Previous studies have used cell models that are unable to return to a proliferative state; thus, the transition between quiescent and proliferative states is not well understood. Here, we report monolayer cancer cell models wherein the human non-small cell lung carcinoma cell line H2228 and pancreatic cancer cell line AsPC-1 can be reversibly induced to a quiescent state under hypoxic and serum-starved (HSS) conditions. Transcriptome and metabolome dual-omics profiles of these cells were compared with those of the human lung adenocarcinoma cell line A549, which was unable to enter a quiescent state under HSS conditions. The quiescence-inducible cells had substantially lower intracellular pyruvate and ATP levels in the quiescent state than in the proliferative state, and their response to sudden demand for energy was dramatically reduced. Furthermore, in quiescence-inducible cells, the transition between quiescent and proliferative states of these cells was regulated by the balance between the proliferation-promoting Ras and Rap1 signaling and the suppressive AGE/RAGE signaling. These cell models elucidate the transition between quiescent and proliferative states, allowing the development of drug-screening systems for quiescent tumor cells.

Successful identification of Granulicatella adiacens in postoperative acute infectious endophthalmitis using a bacterial 16S ribosomal RNA gene-sequencing platform with MinION™: A case report.

Purpose: To evaluate the efficacy of identifying the bacteria by aqueous sampling and vitreous sampling in postoperative infectious endophthalmitis using 16S ribosomal RNA (rRNA) gene analysis with a nanopore sequencer (MinION™). Observation: A 55-year-old woman who underwent cataract surgery at an ophthalmology clinic 18 days ago was referred to our hospital for suspected endophthalmitis. She had light perception visual acuity in her right eye; however, the eye was severely inflamed, with a hypopyon and a fibrinous membrane in the anterior chamber. The fundus was not visible because of vitreous opacity on a B-scan image. Based on the diagnosis of postoperative acute infectious endophthalmitis, we performed a vitrectomy, intraocular lens extraction, and silicone oil tamponade. On postoperative day 14, the inflammation resolved. An aqueous sample was collected before surgical treatment, and the vitreous sample was collected during the operation. Both samples underwent 16S rRNA gene analysis with a nanopore sequencer MinION™ to identify the causative organism. Conclusions and Importance: In the aqueous humor, Granulicatella adiacens and Cutibacterium acnes were identified before the operation, while only Granulicatella adiacens was detected in the vitreous sample after the operation. Although the aqueous humor sample might contain commensal bacteria, it could provide a predictable result before the operation. It can also provide a substitute for a vitreous sample to allow earlier identification of the causative organism.

Activation of transcription factor HIF inhibits IL-1ß-induced NO production in primary cultured rat hepatocytes.

Roxadustat and other hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) have recently been approved for the treatment of chronic renal anemia. In macrophages and monocytes, the activation of HIF-1 by pro-inflammatory cytokines induces iNOS expression and activity through the NF-κB pathway to produce nitric oxide (NO), which causes liver injury when excessively produced. Few studies have reported a relationship between HIF activity and iNOS induction in hepatocytes. We investigated the effect of drug- and hypoxia-induced HIF activations on NO production in primary cultured rat hepatocytes. Roxadustat treatment and hypoxic conditions activated HIF. Contrary to expectations, HIF-PHI treatment and hypoxia inhibited IL-1ß-induced NO production. RNA-Seq analysis of mRNA expression in rat hepatocytes showed that roxadustat treatment decreased the expression of genes related to inflammation, and genes in the NF-κB signaling pathway were induced by IL-1ß. Moreover, roxadustat suppressed IL-1ß-activated signaling pathways in an HIF-dependent manner. GalN/LPS-treated rats were used as in vivo models of hepatic injury, and roxadustat treatment showed a tendency to suppress the death of rats. Therefore, exogenous HIF-1 activation, including HIF-PHI and hypoxia exposures, suppressed IL-1ß-induced iNOS mRNA expression and subsequent NO production in hepatocytes, by suppressing the NF-κB signaling pathway. Roxadustat treatment suppresses the expression of pro-inflammatory genes by activating HIF, and thus may exhibit hepatoprotective effects.

Establishment of a novel assessment of the quality of human spermatozoa measuring mitochondrial oxygen metabolism.

OBJECTIVE: We aimed to establish a novel sperm quality evaluation technology by measuring mitochondrial oxygen metabolism in human spermatozoa. RESULTS: Normozoospermic human sperm samples were used. After establishing the optimal parameters for measuring the oxygen metabolism of human sperm cells using the extracellular flux analyzer, we measured the oxygen consumption rate (OCR) of human spermatozoa exposed to different storage temperatures. Although sperm motility was significantly lower at 4 °C when compared with sperm motility at 37 °C, there were no significant differences in sperm vitality and the OCR under both conditions. The present study established a methodology for human sperm quality evaluation using extracellular flux analysis technology. The OCR evaluation could be a reliable measurement tool for assessing human sperm quality.

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

BACKGROUND: It has been suggested that the local microbiota in the reproductive organs is relevant to women's 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 rapid bacterial identification from a small specimen amount in a clinical context. METHODS: We developed 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. RESULTS: The principal coordinate analysis showed no differences in the bacterial compositions regardless of the sample collection method. The analysis of vaginal microbiota could be completed with a total analysis time of approximately four hours, allowing same-day results. Taxonomic profiling by MinION sequencing revealed relatively low diversity of the vaginal bacterial community, identifying the prevailing Lactobacillus species and several causative agents of bacterial vaginosis. CONCLUSIONS: 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.

Rapid detection of single nucleotide polymorphisms using the MinION nanopore sequencer: a feasibility study for perioperative precision medicine.

INTRODUCTION: Precision medicine is a phrase used to describe personalized medical care tailored to specific patients based on their clinical presentation and genetic makeup. However, despite the fact that several single nucleotide polymorphisms (SNPs) have been reported to be associated with increased susceptibility to particular anesthetic agents and the occurrence of perioperative complications, genomic profiling and thus precision medicine has not been widely applied in perioperative management. METHODS: We validated six SNP loci known to affect perioperative outcomes in Japanese patients using genomic DNA from saliva specimens and nanopore sequencing of each SNP loci to facilitate allele frequency calculations and then compared the nanopore results to those produced using the conventional dideoxy sequencing method. RESULTS: Nanopore sequencing reads clustered into the expected genotypes in both homozygous and heterozygous cases. In addition, the nanopore sequencing results were consistent with those obtained using conventional dideoxy sequencing and the workflow provided reliable allele frequency estimation, with a total analysis time of less than 4 h. CONCLUSION: Thus, our results suggest that nanopore sequencing is a promising and versatile tool for SNP genotyping, allowing for rapid and feasible risk prediction of perioperative outcomes.

Introducing Thioredoxin-Related Transmembrane Proteins: Emerging Roles of Human TMX and Clinical Implications.

Significance: The presence of a large number of thioredoxin superfamily members suggests a complex mechanism of redox-based regulation in mammalian cells. However, whether these members are functionally redundant or play separate and distinct roles in each cellular compartment remains to be elucidated. Recent Advances: In the mammalian endoplasmic reticulum (ER), ∼20 thioredoxin-like proteins have been identified. Most ER oxidoreductases are soluble proteins located in the luminal compartment, whereas a small family of five thioredoxin-related transmembrane proteins (TMX) also reside in the ER membrane and play crucial roles with specialized functions. Critical Issues: In addition to the predicted function of ER protein quality control, several independent studies have suggested the diverse roles of TMX family proteins in the regulation of cellular processes, including calcium homeostasis, bioenergetics, and thiol-disulfide exchange in the extracellular space. Moreover, recent studies have provided evidence of their involvement in the pathogenesis of various diseases. Future Directions: Extensive research is required to unravel the physiological roles of TMX family proteins. Given that membrane-associated proteins are prime targets for drug discovery in a variety of human diseases, expanding our knowledge on the mechanistic details of TMX action on the cell membrane will provide the molecular basis for developing novel diagnostic and therapeutic approaches as a potent molecular target in a clinical setting. Antioxid. Redox Signal. 36, 984-1000.

Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal.

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

Polysulfide inhibits hypoxia-elicited hypoxia-inducible factor activation in a mitochondria-dependent manner.

In cellular signaling, the diverse physiological actions of biological gases, including O2, CO, NO, and H2S, have attracted much interest. Hypoxia-inducible factors (HIFs), including HIF-1 and HIF-2, are transcription factors that respond to reduced intracellular O2 availability. Polysulfides are substances containing varying numbers of sulfur atoms (H2Sn) that are generated endogenously from H2S by 3-mercaptopyruvate sulfurtransferase in the presence of O2, and regulate ion channels, specific tumor suppressors, and protein kinases. However, the effect of polysulfides on HIF activation in hypoxic mammalian cells is largely unknown. Here, we have investigated the effect of polysulfide on cells in vitro. In established cell lines, polysulfide donors reversibly reduced cellular O2 consumption and inhibited hypoxia-induced HIF-1α protein accumulation and the expression of genes downstream of HIFs; however, these effects were not observed in anoxia. In Von Hippel-Lindau tumor suppressor (VHL)- and mitochondria-deficient cells, polysulfides did not affect HIF-1α protein synthesis but destabilized it in a VHL- and mitochondria-dependent manner. For the first time, we show that polysulfides modulate intracellular O2 homeostasis and regulate HIF activation and subsequent hypoxia-induced gene expression in a VHL- and mitochondria-dependent manner.

Development of antitumor biguanides targeting energy metabolism and stress responses in the tumor microenvironment.

To develop antitumor drugs capable of targeting energy metabolism in the tumor microenvironment, we produced a series of potent new biguanide derivatives via structural modification of the arylbiguanide scaffold. We then conducted biological screening using hypoxia inducible factor (HIF)-1- and unfolded protein response (UPR)-dependent reporter assays and selective cytotoxicity assay under low glucose conditions. Homologation studies of aryl-(CH2)n-biguanides (n = 0-6) yielded highly potent derivatives with an appropriate alkylene linker length (n = 5, 6). The o-chlorophenyl derivative 7l (n = 5) indicated the most potent inhibitory effects on HIF-1- and UPR-mediated transcriptional activation (IC50; 1.0 ± 0.1 µM, 7.5 ± 0.1 µM, respectively) and exhibited selective cytotoxicity toward HT29 cells under low glucose condition (IC50; 1.9 ± 0.1 µM). Additionally, the protein expression of HIF-1α induced by hypoxia and of GRP78 and GRP94 induced by glucose starvation was markedly suppressed by the biguanides, thereby inhibiting angiogenesis. Metabolic flux and fluorescence-activated cell sorting analyses of tumor cells revealed that the biguanides strongly inhibited oxidative phosphorylation and activated compensative glycolysis in the presence of glucose, whereas both were strongly suppressed in the absence of glucose, resulting in cellular energy depletion and apoptosis. These findings suggest that the pleiotropic effects of these biguanides may contribute to more selective and effective killing of cancer cells due to the suppression of various stress adaptation systems in the tumor microenvironment.

Cigarette Smoke Extract Activates Hypoxia-Inducible Factors in a Reactive Oxygen Species-Dependent Manner in Stroma Cells from Human Endometrium.

Cigarette smoking (CS) is a major contributing factor in the development of a large number of fatal and debilitating disorders, including degenerative diseases and cancers. Smoking and passive smoking also affect the establishment and maintenance of pregnancy. However, to the best of our knowledge, the effects of smoking on the human endometrium remain poorly understood. In this study, we investigated the regulatory mechanism underlying CS-induced hypoxia-inducible factor (HIF)-1α activation using primary human endometrial stromal cells and an immortalized cell line (KC02-44D). We found that the CS extract (CSE) increased reactive oxygen species levels and stimulated HIF-1α protein stabilization in endometrial stromal cells, and that CS-induced HIF-1α-dependent gene expression under non-hypoxic conditions in a concentration- and time-dependent manner. Additionally, we revealed the upregulated expression of a hypoxia-induced gene set following the CSE treatment, even under normoxic conditions. These results indicated that HIF-1α might play an important role in CS-exposure-induced cellular stress, inflammation, and endometrial remodeling.

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

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.