Comprehensive Profiling of Phosphomonoester Metabolites in Saccharomyces cerevisiae by the Chemical Isotope Labeling-LC-MS Method.

Phosphomonoesters are important biosynthetic and energy metabolism intermediates in microorganisms. A comprehensive analysis of phosphomonoester metabolites is of great significance for the understanding of their metabolic phosphorylation process and inner mechanism. In this study, we established a pair of isotope reagent d0/d5-2-diazomethyl-N-methyl-phenyl benzamide-labeling-based LC-MS method for the comprehensive analysis of phosphomonoester metabolites. By this method, the labeled phosphomonoester metabolites specifically produced characteristic isotope paired peaks with an m/z difference of 5.0314 in the MS1 spectra and a pair of diagnostic ions (m/z 320.0693/325.1077) in the MS2 spectra. Based on this, a diagnostic ion-based strategy was established for the rapid screening, identification, and relative quantification of phosphomonoester metabolites. Using this strategy, 42 phosphomonoester metabolites were highly accurately identified fromSaccharomyces cerevisiae (S. cerevisiae). Notably, two phosphomonoesters were first detected fromS. cerevisiae. The relative quantification results indicated that the contents of nine phosphomonoester metabolites including two intermediates (Ru5P and S7P) in the pentose phosphate pathway (PPP) were significantly different between lycopene-producible and wild-type S. cerevisiae. A further enzyme assay indicated that the activity of the PPP was closely related to the production of lycopene. Our findings provide new perspectives for the related mechanism study and valuable references for making informed microbial engineering decisions.

New Insights of Corynebacterium kroppenstedtii in Granulomatous Lobular Mastitis based on Nanopore Sequencing.

BACKGROUND: The etiology of granulomatous lobular mastitis (GLM) remains unknown. This study aimed to detect bacteria in GLM using Nanopore sequencing and identify the relationship between GLM and Corynebacterium kroppenstedtii. METHODS AND MATERIALS: The bacterial detection on fresh samples (including breast pus and tissue) of 50 GLM patients using nanopore sequencing and culture methods. The bacterial detection rate of participants with different stages were compared and analyzed. Formalin-fixed and paraffin-embedded (FFPE) tissues from 39 patients were performed on Gram staining to identify Gram-positive bacilli (GPB) within lipid vacuoles. Moreover, the clinicopathological characteristics of GLM patients in different bacterial subgroups were also conducted. RESULTS: In 50 GLM patients, the detection rate of bacteria was 78% using nanopore sequencing method, especially in the early stage of GLM (over 80%), which was significantly higher than that using culture methods (24%, p < 0.001). The dominant bacteria were Corynebacterium species (64%), especially for the Corynebacterium kroppenstedtii. The detection rate of C. kroppenstedtii in nanopore sequencing method (56%) was higher than that in culture methods (16%, p < 0.001). Gram staining positive of bacteria in 7 patients, and 5 of them were C. kroppenstedtii. Thirty-one patients (31/39, 79.5%) exhibited typical histological structure of cystic neutrophilic granulomatous mastitis (CNGM), and eighteen patients detected with C. kroppenstedtii. CONCLUSION: Nanopore sequencing showed rapid and accurate bacteria detection over culture method in GLM patients. GLM is not sterile inflammation and closely related to C. kroppenstedtii. CNGM was associated with Corynebacterium infection, especially for C. kroppenstedtii.

Bacteria Associated with Granulomatous Lobular Mastitis and the Potential for Personalized Therapy.

Granulomatous lobular mastitis (GLM), also known as idiopathic granulomatous mastitis (IGM), is a chronic inflammatory lesion of the breast. The incidence of GLM has been increasing in recent years, especially among young women. The etiologies of GLM have not been fully elucidated but are associated with autoimmunity and bacterial infection. Bacteria, especially Corynebacterium species, play important roles in GLM. In this article, we review research progress regarding the bacteriology of GLM attained with the application of several new high-throughput detection techniques. Accurate detection might be important for deepening our understanding of the pathogenesis of GLM and hold promise for personalized GLM therapy.

[The third-generation sequencing combined with targeted capture technology for high-resolution HLA typing and MHC region haplotype identification].

The high-resolution and accurate typing of human leukocyte antigen (HLA) is of great significance for the study of tissue matching in organ transplantation and the correlation between HLA and disease. In this study, the peripheral blood of 12 patients with primary hepatocellular carcinoma was used to compare the advantages and disadvantages of the next- and third-generation sequencing technology for high-resolution HLA typing. In addition, probe capture technology was used to capture the MHC region of YH and HeLa standard cell lines, and a primary hepatocellular carcinoma patient. The captured products were sequenced using PacBio platform to assess the potential of ultra-long reads sequencing technology for analysis of the entire MHC region. Our results showed that: (1) the next- and third-generation sequencing technology can both achieve 6-8 digit high resolution in HLA typing. However, the coverage of the third-generation is significantly better than the next-generation sequencing technology. (2) The ultra-long reads of the third generation sequencing can directly span the entire amplicon region, which has obvious advantages for haplotype phasing, with 92.79% of the HLA genes having accurate phasing results, which is much higher than the 75.65% from the next-generation data. (3) The long-reads from the third generating sequencing can not only be used to assemble the MHC region but also the ability to phase the entire MHC region of 3.6 Mb, thereby helping to clarify the localization information of the mutation sites, alleles and non-coding regions on each MHC haplotype, and providing a theoretical basis for the study of immune and other related diseases.

[Genetic engineering of microbial metabolic pathway for production of advanced biodiesel].

Biodiesel is a renewable biofuel and alternative diesel, but the first generation of biodiesel, which has many defects in properties and in production methods, mainly comes from the chemical transesterification of triglyceride from plant oil. With the fast development in the field of synthetic biology and metabolic engineer-ing, the researchers can choose suitable microbes and engineer its metabolic pathways, such as fatty acid bio-synthesis pathway and isoprenoid biosynthesis pathway, to directly produce the second generation of advanced biodiesel---long chain hydrocarbons, which have better properties and quality using the newest biotechnology techniques. In this review, we summarized the research progress about microbial production of advanced bio-diesel and also pointed the deficiencies and future direction in this new field.