Project description:Background: Dendrobium officinale, an endangered Chinese herb, has extensive therapeutic effects and contains bioactive ingredients including a large number of polysaccharides and alkaloids, and minimal flavonoids. Firstly, this study attempts to obtain the protocorm-like bodies of this plant through tissue culture to produce the main secondary metabolites whose distribution in each organelle and protocorm like bodies is analyzed. Then, analysis of the correlation between comparative transcriptome sequence and the metabolite content in different organs enables the discovery of putative genes encoding enzymes involved in the biosynthesis of polysaccharides and alkaloids, and flavonoids. Results: The optimum condition for protocorm-like bodies (PLBs) induction and propagation of D. officinale is established. For protocorm induction, we use the seed as the explant, and the optimum medium formula for PLBs propagation is 1/2 MS + α-NAA 0.5 mg·L-1 +6-BA 1.0 mg·L-1 + 2, 4-D 1.5-2.0 mg·L-1 + potato juice 100 g·L-1. The distribution of polysaccharides, alkaloids and flavonoids in D. officinale organs was clarified. Stems, PLBs and leaves have the highest content of polysaccharides, alkaloids and flavonoids, respectively. PLBs replace organs to produce alkaloids in D. officinale, and naringenin was only produced in stem. Hot water extraction (HWE) method was found outperforming the ultrasound-assisted extraction (UAE) method for polysaccharides from D. officinale. A comparative transcriptome analysis of the protocorm-like bodies and leaves of D. officinale showed genes encoding enzymes involved in polysaccharides, alkaloids and flavonoids biosynthetic pathway were differentially expressed. Putative genes encoding enzymes involved in polysaccharides, alkaloids and flavonoids synthetic pathway were identified. Notably, genes encoding enzymes of strictosidine beta-glucosidase, geissoschizine synthase and vinorine synthase in alkaloids biosynthesis of D. officinale are first reported. Conclusions: Our works, especially the identification of candidate genes encoding enzymes involved in metabolites biosynthesis will help to explore and protect the endangered genetic resources and will also facilitate further analysis of the molecular mechanism of secondary metabolites’ biosynthesis in D. officinale.
Project description:Recent advances in (meta)genomic methods have provided new opportunities to examine host-microbe-environment interactions in the human gut. While opportunities exist to extract DNA from freshly sourced colonic tissue there are potentially valuable sources of DNA from historical studies that might also be examined. We examined how four different tissue DNA extraction methods employed in past clinical trials might impact the recovery of microbial DNA from a colonic tissue sample as assessed using a custom designed phylogenetic microarray for human gut bacteria and archaebacteria. While all methods of DNA extraction produced similar phylogenetic profiles some extraction specific biases were also observed. Real time PCR analysis targeting several bacterial groups substantiated this observation. These data suggest that while the efficacy of different DNA extraction methods differs somewhat all the methods tested produce an accurate representation of microbial diversity. This suggests that DNA samples archived in biobanks should be suitable for retrospective analyses.
Project description:Proteomics research has increasingly focused on human cells, tissues, and fluids; however, comprehensive data regarding dental tissues remain limited. Dentin, a mineralized component of the tooth, contains structural proteins and bioactive molecules that, upon release, can modulate pulp cell activity and facilitate regeneration. Therefore, an understanding of the protein composition of dentin is of considerable importance. Previous studies have reported a limited number of proteins, and technical difficulties have often hindered replication. Furthermore, traditional extraction methods rely on strong acids, which are not clinically relevant. In this technical note, we introduce a workflow that combines EDTA-based dentin extraction under clinically relevant conditions with peptide-level fractionation using high-pH reverse-phase chromatography. This approach was compared against unfractionated samples (control), protein-level fractionation via SDS-PAGE, and peptide-level fractionation utilizing strong cation exchange (SCX), all followed by LC-MS/MS analysis. This workflow facilitated the identification of 514 proteins, compared with 238 (unfractionated control), 428 (SDS-PAGE), and 193 (SCX). High-pH reverse-phase chromatography contributed 217 unique identifications, surpassing the number of unique fractions identified through the other techniques. Although this methodology has been utilized in other protein systems, it has not previously been applied to dentin matrix extracts and represents a promising approach for enhancing the comprehensiveness of protein discovery.
Project description:Comparison of two solid-phase extraction (SPE) methods for the identification and quantification of porcine retinal protein markers by LC MS/MS