Characteristics of the Serum Microbiota and Circulating Metabolites in Cynomolgus Monkeys With Spontaneous Type 2 Diabetes Mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is widely recognized as a serious global public health concern with a substantial economic burden on patients, their families, and society. Accumulating evidence suggests that an etiologic role for serum microbiota and circulating metabolites in the pathogenesis of T2DM. This study aims to characterize the serum microbiota and circulating metabolites in cynomolgus monkeys with spontaneous T2DM, and provide a reference for the diagnosis and treatment of clinical T2DM. METHODS: We collected serum samples of the 14 cynomolgus monkeys (15-20 years old, male) for serum microbiota analysis by 16S rRNA gene V3-V4 region amplicon sequencing and circulating metabolites analysis by ultra-high-performance liquid chromatography-tandem mass spectrometry, of which seven were spontaneous T2DM cynomolgus monkeys and seven were healthy controls. RESULTS: Our results showed that the serum microbiota abundance and diversity were significantly increased in cynomolgus monkeys with spontaneous T2DM compared to healthy controls, the phyla of Cyanobacteria and Chloroflexi and the genera of Lactobacillus, rhodobacter and collinsella were also significantly increased. A total of 114 serum differentially expressed metabolites (DEMs) were identified, of which 22 were selected as potential biomarkers candidates related to spontaneous T2DM in cynomolgus monkeys by multivariate data analysis. In addition, serum levels of total SCFAs, acetate, butyrate, caproate, isobutyrate, and isovalerate were also significantly increased in the present study. The correlation network analyses have selected certain key DEMs, such as D-Psicose, 4-Oxoproline, D-Glutamine, and Hydroxyphenyllactic acid, which may serve as potential biomarkers for distinguishing between T2DM and healthy controls. CONCLUSION: Our results provide preliminary insights on perturbed serum microbiota and circulating metabolites of cynomolgus monkeys with spontaneous T2DM. These findings would be useful to develop microbiota-based strategies for T2DM prevention and control.

Combined untargeted and targeted lipidomics approaches reveal potential biomarkers in type 2 diabetes mellitus cynomolgus monkeys.

BACKGROUND: The significantly increasing incidence of type 2 diabetes mellitus (T2DM) over the last few decades triggers the demands of T2DM animal models to explore the pathogenesis, prevention, and therapy of the disease. The altered lipid metabolism may play an important role in the pathogenesis and progression of T2DM. However, the characterization of molecular lipid species in fasting serum related to T2DM cynomolgus monkeys is still underrecognized. METHODS: Untargeted and targeted LC-mass spectrometry (MS)/MS-based lipidomics approaches were applied to characterize and compare the fasting serum lipidomic profiles of T2DM cynomolgus monkeys and the healthy controls. RESULTS: Multivariate analysis revealed that 196 and 64 lipid molecules differentially expressed in serum samples using untargeted and targeted lipidomics as the comparison between the disease group and healthy group, respectively. Furthermore, the comparative analysis of differential serum lipid metabolites obtained by untargeted and targeted lipidomics approaches, four common serum lipid species (phosphatidylcholine [18:0_22:4], lysophosphatidylcholine [14:0], phosphatidylethanolamine [PE] [16:1_18:2], and PE [18:0_22:4]) were identified as potential biomarkers and all of which were found to be downregulated. By analyzing the metabolic pathway, glycerophospholipid metabolism was associated with the pathogenesis of T2DM cynomolgus monkeys. CONCLUSION: The study found that four downregulated serum lipid species could serve as novel potential biomarkers of T2DM cynomolgus monkeys. Glycerophospholipid metabolism was filtered out as the potential therapeutic target pathway of T2DM progression. Our results showed that the identified biomarkers may offer a novel tool for tracking disease progression and response to therapeutic interventions.

Preliminary serum and fecal metabolomics study of spontaneously diabetic cynomolgus monkeys based on LC-MS/MS.

BACKGROUND: Using untargeted metabolomics techniques, the goal of the study is to differentially screen serum and feces metabolite profiles of spontaneously diabetic and healthy cynomolgus monkeys, to explore potential serum and fecal biomarkers and analyze affected metabolic pathways. METHODS: We adopted the diagnostic criteria for T2DM recommended by ADA for humans: FSG ≥7.0 mmol/L (126 mg/dl) and HbA1c ≥ 6.5%. The serum and feces samples from three diagnosed spontaneously T2DM cynomolgus monkeys and 11 age-matched healthy controls were enrolled in the study. We employed LC-MS/MS-based untargeted metabolomic methods to reveal the differential metabolite profiles of serum and feces samples between the two groups and to analyze the affected metabolic pathways in MetaboAnalyst 5.0 based on KEGG library. RESULTS: Six and 44 differential metabolites were identified in serum and feces samples, respectively, and the corresponding affected commonly metabolic pathways involved several metabolic ways, such as arginine biosynthesis, pantothenate and CoA biosynthesis, alanine, aspartate and glutamate metabolism, valine, leucine and isoleucine biosynthesis, and histidine metabolism. CONCLUSION: The differential potential serum and feces biomarkers obtained from the LC-MS/MS based untargeted metabolomic may help to explain the potential pathophysiological mechanisms of T2DM and offer pivotal information for the early diagnosis and treatment of DM.

[Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase（PcHDR1）gene in Phlegmarirus carinatus].

The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.

Strategies on biosynthesis and production of bioactive compounds in medicinal plants.

Medicinal plants are a valuable source of essential medicines and herbal products for healthcare and disease therapy. Compared with chemical synthesis and extraction, the biosynthesis of natural products is a very promising alternative for the successful conservation of medicinal plants, and its rapid development will greatly facilitate the conservation and sustainable utilization of medicinal plants. Here, we summarize the advances in strategies and methods concerning the biosynthesis and production of natural products of medicinal plants. The strategies and methods mainly include genetic engineering, plant cell culture engineering, metabolic engineering, and synthetic biology based on multiple "OMICS" technologies, with paradigms for the biosynthesis of terpenoids and alkaloids. We also highlight the biosynthetic approaches and discuss progress in the production of some valuable natural products, exemplifying compounds such as vindoline (alkaloid), artemisinin and paclitaxel (terpenoids), to illustrate the power of biotechnology in medicinal plants.

Quantitative Proteomic Analysis of Serum Reveals MST1 as a Potential Candidate Biomarker in Spontaneously Diabetic Cynomolgus Monkeys.

The prevalence of type 2 diabetes (T2DM) is increasing globally, creating essential demands for T2DM animal models for the study of disease pathogenesis, prevention, and therapy. A non-human primate model such as cynomolgus monkeys can develop T2DM spontaneously in an age-dependent way similar to humans. In this study, a data-independent acquisition-based quantitative proteomics strategy was employed to investigate the serum proteomic profiles of spontaneously diabetic cynomolgus monkeys compared with healthy controls. The results revealed significant differences in protein abundances. A total of 95 differentially expressed proteins (DEPs) were quantitatively identified in the current study, among which 31 and 64 proteins were significantly upregulated and downregulated, respectively. Bioinformatic analysis revealed that carbohydrate digestion and absorption was the top enriched pathway by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Protein-protein interaction network analysis demonstrated that MST1 was identified as the most connected protein in the network and could be considered as the hub protein. MST1 was significantly and inversely associated with FSG and HbA1c. Furthermore, recent lines of evidence also indicate that MST1 acts as a crucial regulator in regulating hepatic gluconeogenesis to maintain metabolic homeostasis while simultaneously suppressing the inflammatory processes. In conclusion, our study provides novel insights into serum proteome changes in spontaneously diabetic cynomolgus monkeys and points out that the dysregulation of several DEPs may play an important role in the pathogenesis of T2DM.

bHLH transcription factor SmbHLH92 negatively regulates biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Objective: Salvia miltiorrhiza is a valuable herbal medicine with tanshinone and phenolic acid as the main biological active ingredients. The biosynthetic regulation of these bioactive compounds is controlled by a set of transcription factors (TFs). The basic helix-loop-helix (bHLH) transcription factor plays an important role in various physiological and biochemical processes in plants. However, research on bHLH TFs regulating phenolic acid or tanshinone biosynthesis in S. miltiorrhiza is limited. Methods: qRT-PCR was used for gene expression analysis. The subcellular localization of SmbHLH92 was detected by SmbHLH92-GFP transient transformation into tobacco leaves, and its fluorescence was observed using a confocal laser scanning microscope. The transcriptional activity of SmbHLH92 was confirmed in the AH109 yeast strain. RNA interference hairy roots of SmbHLH92-RNAi transgenic lines were obtained through Agrobacterium-mediated genetic transformation. Ultra performance liquid chromatography (UPLC) was used to detect the changes of phenolic acids and tanshinones. Results: SmbHLH92 is a bHLH transcription factor that is highly expressed in the root and phloem of S. miltiorrhiza. The subcellular localization and transcriptional activity of SmbHLH92 indicated that SmbHLH92 was located in the nucleus and may be a transcription factor. RNA interference (RNAi) of SmbHLH92 in hairy roots of S. miltiorrhiza significantly increased the accumulation of phenolic acid and tanshinone. Quantitative RT-PCR (RT-qPCR) analysis showed the transcription level of genes encoding the key enzymes involved in the phenolic acid and tanshinone biosynthetic pathways was increased in the hairy roots of the SmbHLH92-RNAi transgenic line, comparing with the control line. Conclusion: These data indicate that SmbHLH92 is a negative regulator involved in the regulation of phenolic acid and tanshinone biosynthesis in S. miltiorrhiza.

Cloning and analysis of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase genes HsHDR1 and HsHDR2 in Huperzia serrate.

We cloned and analyzed the two genes of the 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) gene family from Huperzia serrate. The two transcripts coding HDR, named HsHDR1 and HsHDR2, were discovered in the transcriptome dataset of H. serrate and were cloned by reverse transcription-polymerase chain reaction (RT-PCR). The physicochemical properties, protein domains, protein secondary structure, and 3D structure of the putative HsHDR1 and HsHDR2 proteins were analyzed. The full-length cDNA of the HsHDR1 gene contained 1431 bp encoding a putative protein with 476 amino acids, whereas the HsHDR2 gene contained 1428 bp encoding a putative protein of 475 amino acids. These two proteins contained the conserved domain of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (PF02401), but without the transmembrane region and signal peptide. The most abundant expression of HsHDR1 and HsHDR2 was detected in H. serrate roots, followed by the stems and leaves. Our results provide a foundation for exploring the function of HsHDR1 and HsHDR2 in terpenoid and sterol biosynthesis in Huperziaceae plants.