SLC7A11 and the glutathione pathway as novel prognostic markers in resectable pancreatic ductal adenocarcinoma: A metabolomics study of clinical specimens.

BACKGROUND/OBJECTIVES: Despite the poor prognosis associated with pancreatic ductal adenocarcinoma (PDAC), there remains a lack of clarity regarding the metabolic pathways and their significant impact on its phenotype. Therefore, we aimed to utilize metabolomics to capture changes in clinical PDAC tissues and elucidate the significant metabolic pathways close to its phenotypes. METHODS: This basic research was retrospectively validated using database research, immunohistochemistry, and protein analysis based on the findings obtained from metabolomics using clinical tissues collected from prospectively registered patients with PDAC. mRNA expression analysis using a database and protein analysis using archived clinical specimens was performed to validate the candidate pathways identified using metabolomics. Between-group comparisons were analyzed using paired t-tests and log-rank test, and Kaplan-Meier curves illustrated survival times. RESULTS: Patients subjected to metabolomics revealed a significant increase in glutathione disulfide levels in PDAC tissues when compared to normal pancreatic tissues. The Cancer Genome Atlas database analysis revealed significant changes in glutathione pathway-related mRNAs in PDAC compared to that in the normal pancreas. Protein analysis of previously resected specimens demonstrated a significant increase in SLC7A11 expression in PDAC tissues. The abundance ratio of SLC7A11 isoforms was associated with the post-operative prognosis in resectable PDAC. CONCLUSION: Glutathione disulfide levels were significantly increased in clinical PDAC metabolomics. Additionally, increased mRNA and protein expression in SLC7A11 was observed in PDAC. Furthermore, the SLC7A11 isoform abundance ratio may be a valuable prognostic marker in patients with resectable PDAC.

Total Biosynthesis of Antiangiogenic Agent (-)-Terpestacin by Artificial Reconstitution of the Biosynthetic Machinery in Aspergillus oryzae.

The total biosynthesis of (-)-terpestacin was achieved by heterologous expression of four biosynthetic enzyme genes ( tpcA- D) in Aspergillus oryzae. After construction of preterpestacin I by the action of bifunctional terpene synthase (TpcA), two cytochrome P450s (TpcBC) activate inert C-H bond to install three hydroxyl groups on the A-ring in stereo- and regioselective manners. Subsequently, a flavin-dependent oxidase (TpcD) catalyzes oxidation of the vicinal diol moiety to give a α-diketone, which undergoes an enolization to furnish terpestacin. The successful synthesis of structurally elaborated terpestacin showed that a reconstitution approach that harnesses several biosynthetic enzyme genes in A. oryzae could be a promising alternative to the current chemical synthesis of natural terpenoids.

Bacterial exposure risk to the endoscopist's face while performing endoscopy.

Objectives: Gastrointestinal endoscopy increases the risk of bacterial exposure to endoscopists. However, before 2019, most endoscopists did not pay attention to microorganism transmission from patients. This study aimed to investigate the incidence of bacterial exposure to endoscopists' faces during gastrointestinal endoscopic procedures using the bacterial culture method. Methods: This was a single-centered, retrospective study including endoscopists who performed various gastrointestinal endoscopy procedures at the Division of Endoscopy, Hirosaki University Hospital between August 31 and October 6, 2020. Endoscopists wore surgical masks and affixed pre-sterilized films over them. Following the gastrointestinal endoscopic procedures, attached microbes were collected from the endoscopists' surface films using sterilized swabs. Collected microorganisms were cultured on tryptic soy agar and 5% sheep blood agar, and the incidence of bacterial exposure was determined by bacterial culture positivity. Cultured bacteria were identified by gram staining and 16S rRNA gene sequencing. Results: Bacterial culture positivity was 12.6%, and it was significantly higher in therapeutic than in diagnostic endoscopy. Notably, therapeutic endoscopy increased bacterial culture positivity in colonoscopy, but not in esophagogastroduodenoscopy. Staphylococci, including Staphylococcus epidermidis and Staphylococcus capitis, were the most commonly found bacteria in samples identified through 16S rRNA gene sequencing. Conclusions: The risk of bacterial exposure to the endoscopist's face was increased in colonoscopy treatment procedures. Therefore, endoscopists should be aware of the significant risk of microbial infection from scattering fluid that comes from the endoscopy's working channel.

Characterization and Demonstration of Mock Communities as Control Reagents for Accurate Human Microbiome Community Measurements.

Standardization and quality assurance of microbiome community analysis by high-throughput DNA sequencing require widely accessible and well-characterized reference materials. Here, we report on newly developed DNA and whole-cell mock communities to serve as control reagents for human gut microbiota measurements by shotgun metagenomics and 16S rRNA gene amplicon sequencing. The mock communities were formulated as near-even blends of up to 20 bacterial species prevalent in the human gut, span a wide range of genomic guanine-cytosine (GC) contents, and include multiple strains with Gram-positive type cell walls. Through a collaborative study, we carefully characterized the mock communities by shotgun metagenomics, using previously developed standardized protocols for DNA extraction and sequencing library construction. Further, we validated fitness of the mock communities for revealing technically meaningful differences among protocols for DNA extraction and metagenome/16S rRNA gene amplicon library construction. Finally, we used the mock communities to reveal varying performance of metagenome-based taxonomic profilers and the impact of trimming and filtering of sequencing reads on observed species profiles. The latter showed that aggressive preprocessing of reads may result in substantial GC-dependent bias and should thus be carefully evaluated to minimize unintended effects on species abundances. Taken together, the mock communities are expected to support a myriad of applications that rely on well-characterized control reagents, ranging from evaluation and optimization of methods to assessment of reproducibility in interlaboratory studies and routine quality control. IMPORTANCE Application of high-throughput DNA sequencing has greatly accelerated human microbiome research and its translation into new therapeutic and diagnostic capabilities. Microbiome community analyses results can, however, vary considerably across studies or laboratories, and establishment of measurement standards to improve accuracy and reproducibility has become a priority. The here-developed mock communities, which are available from the NITE Biological Resource Center (NBRC) at the National Institute of Technology and Evaluation (NITE, Japan), provide well-characterized control reagents that allow users to judge the accuracy of their measurement results. Widespread and consistent adoption of the mock communities will improve reproducibility and comparability of microbiome community analyses, thereby supporting and accelerating human microbiome research and development.

Sulphur metabolism in colon cancer tissues: a case report and literature review.

Sulphur-containing compounds have been linked to colorectal cancer by factors such as the presence of methyl mercaptan in intestinal gas and long-term dietary intake associated with sulphur-metabolizing microbiota. Therefore, this current case report hypothesized that active sulphur metabolism in colorectal cancer results in the formation of sulphur compounds in the intestine and, thus, examined sulphur metabolites possibly associated with sulphur respiration in colon cancer tissues. The patient was a 73-year-old female that underwent laparoscopic right hemicolectomy for ascending colon cancer. During the surgery, colon cancer tissues and normal intestinal mucosa samples were collected. After optimizing the sample concentrations for homogenization (pre-treatment), the samples were stabilized using a hydroxyphenyl-containing derivative and the relevant metabolites were quantified using liquid chromatography with tandem mass spectrometry. The results showed that cysteine persulfide and cysteine trisulfide levels were higher in colon cancer tissues than in normal mucosal tissues. Thus, sulphur metabolism, possibly sulphur respiration, is enhanced in colon cancer tissues.

Validation and standardization of DNA extraction and library construction methods for metagenomics-based human fecal microbiome measurements.

BACKGROUND: Validation and standardization of methodologies for microbial community measurements by high-throughput sequencing are needed to support human microbiome research and its industrialization. This study set out to establish standards-based solutions to improve the accuracy and reproducibility of metagenomics-based microbiome profiling of human fecal samples. RESULTS: In the first phase, we performed a head-to-head comparison of a wide range of protocols for DNA extraction and sequencing library construction using defined mock communities, to identify performant protocols and pinpoint sources of inaccuracy in quantification. In the second phase, we validated performant protocols with respect to their variability of measurement results within a single laboratory (that is, intermediate precision) as well as interlaboratory transferability and reproducibility through an industry-based collaborative study. We further ascertained the performance of our recommended protocols in the context of a community-wide interlaboratory study (that is, the MOSAIC Standards Challenge). Finally, we defined performance metrics to provide best practice guidance for improving measurement consistency across methods and laboratories. CONCLUSIONS: The validated protocols and methodological guidance for DNA extraction and library construction provided in this study expand current best practices for metagenomic analyses of human fecal microbiota. Uptake of our protocols and guidelines will improve the accuracy and comparability of metagenomics-based studies of the human microbiome, thereby facilitating development and commercialization of human microbiome-based products. Video Abstract.

Complete Genome Sequence of Flavonifractor plautii JCM 32125T.

We report the complete genome sequence of Flavonifractor plautii JCM 32125T (=VPI 0310T). The genome consists of a single circular chromosome of 3,985,392 bp (G+C content, 60.9%) and was predicted to contain 3 complete sets of rRNA genes, 63 tRNA genes, and 3,764 protein-coding sequences.

Complete Genome Sequence of Blautia producta JCM 1471T.

We report a complete genome sequence of Blautia producta JCM 1471T The genome consists of a single circular chromosome of 6,197,116 bp with a G+C content of 45.7%. The genome was annotated as containing 5 complete sets of rRNA genes, 70 tRNA genes, and 5,516 protein-coding sequences.

Complete Genome Sequence of Collinsella aerofaciens JCM 10188T.

We report a complete genome sequence of Collinsella aerofaciens JCM 10188T (=VPI 1003T). The genome consists of a circular chromosome (2,428,218 bp with 60.6% G+C content) and two extrachromosomal elements. The genome was predicted to contain 5 sets of rRNA genes, 58 tRNA genes, and 2,079 protein-encoding sequences.

Complete Genome Sequence of Megamonas funiformis JCM 14723T.

We announce the complete genome sequence of Megamonas funiformis JCM 14723T (YIT 11815T). The genome consists of a circular chromosome (2,522,577 bp, 31.5% G+C content) and a plasmid of 46,189 bp (29.4% G+C content). The genome was predicted to contain 6 rRNA operons, 53 tRNA genes, and 2,440 protein-coding sequences.

Theoretical Study of Sesterfisherol Biosynthesis: Computational Prediction of Key Amino Acid Residue in Terpene Synthase.

The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods. Density functional theory calculations revealed unexpected intermediates and transition states, and implied that C-H···π interaction between a carbocation intermediate and an aromatic residue of sesterfisherol synthase (NfSS) plays a critical role, serving to accelerate the 1,2-H shift (thereby preventing triquinane carbocation formation) and to protect reactive carbocation intermediates from bases such as pyrophosphate or water in the active site. Site-directed mutagenesis of NfSS guided by docking simulations confirmed that phenylalanine F191 is a critical amino acid residue for sesterfisherol synthase, as the F191A mutant of NfSS produces novel sesterterpenes, but not sesterfisherol. Although both pathways are energetically viable, on the basis of our computational and experimental results, NfSS-mediated sesterfisherol biosynthesis appears to proceed via Path a. These findings may also provide new insight into the cyclization mechanisms in related sesterterpene synthases.

Focused Genome Mining of Structurally Related Sesterterpenes: Enzymatic Formation of Enantiomeric and Diastereomeric Products.

Heterologous expression of four clade-A bifunctional terpene synthases (BFTSs), giving di/sesterterpenes with unique polycyclic carbon skeletons such as sesterfisherol, enabled the isolation of the sesterterpenes Bm1, Bm2, Bm3, and Pb1. Their structures suggested that formation of the products occurs via various diastereomeric carbocation intermediates, allowing the proposal that clade-A BFTSs catalyze three-step cyclizations using several stereofacial combinations of allylic cation-olefin pairs in the corresponding intermediates to generate various stereoisomers.

Multiple Oxidative Modifications in the Ophiobolin Biosynthesis: P450 Oxidations Found in Genome Mining.

Heterologous expression of four candidate genes found in ophiobolin gene clusters from three fungal strains was employed to elucidate the late-stage biosynthetic pathway of phytotoxin ophiobolin. Expression of oblBAc (cytochrome P450) from the cryptic gene cluster gave unexpected products, and that of oblBBm/oblBEv from the gene cluster of ophiobolin producers, with oblDBm as the transporter, yielded intermediate ophiobolin C through an unusual four-step oxidation process. The observation made in this study may provide a useful guideline for the elucidation of genuine biosynthetic pathways of natural products.