Project description:The success of bottom-up proteomic analysis frequently depends on the efficient removal of contaminants from protein or peptide samples before LC-MS/MS. For a peptide clean-up workflow, the single-pot solid-phase-enhanced peptide sample preparation on carboxylate-modified paramagnetic beads (termed SP2) was evaluated for sodium dodecyl sulfate or polyethylene glycol removal from Arabidopsis thaliana tryptic peptides. The robust and efficient 40-min SP2 protocol, tested for a 10 ng, 250ng and 10µg peptide sample, was proposed and benchmarked thoroughly against the ethyl acetate extraction protocol. The SP2 protocol on carboxylated magnetic beads proved to be the robust approach even for simultaneous removal of massive sodium dodecyl sulfate (SDS) and polyethylene glycol (PEG) contaminations from AT peptide samples in respect of the LC-MS/MS data outperforming ethyl acetate extraction.
Project description:Purpose: Antenoron Filiforme is a traditionally used herb medicine in China. Our findings indicate that the ethyl acetate extract of Antenoron Filiforme (AF-EAE) has the ability to inhibit the proliferation of triple negative breast cancer cells (TNBC). However, little is known about the underlying mechanism of AF-EAE's action on TNBC. Methods: Here, we performed RNA sequencing on MDA-MB-231 treated by AF-EAE with two concentrations for 24 hours. The sequence reads that passed quality filters were analyzed at the transcript isoform level with hisat2 followed by HTSeq. Results: Using an optimised data analysis workflow, RNA-seq data showed 1,392 differentially expressed genes (DEGs) after low concentration drug treatment and 2,847 DEGs in the high concentration drug treatment group, with a fold change of ≥1.5 and p-value <0.05. Expression changes in six genes were confirmed by qRT-PCR, demonstrating the high sensitivity of the RNA-seq method. Based on the KEGG pathway enrichment analysis results, cell growth relevant pathways such as "p53 signaling pathway" and "cell cycle" were greatly enriched. In addition, GSEA analysis indicated that DEGs in AF-EAE treated MDA-MB-231 cells were also well enriched in "cell cycle", "DNA replication" and "G2/M cell cycle". Conclusion: Our study indicated that AF-EAE could inhibit the proliferation of TNBC by inducing cell cycle arrest. Meanwhile, we provide a new potential drug treating breast cancer.
Project description:Inflammatory Bowel Disease (IBD) is a term describing a collection of conditions characterised by chronic inflammatory disorder of the gastrointestinal tract involving an inappropriate immune response to commensal microorganisms in a genetically susceptible host. Four kiwifruit extracts, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa), have previously demonstrated anti-inflammatory activity using in vitro models of IBD. This study examined whether these kiwifruit extracts had immune modulating effects in vivo against inflammatory processes known to be increased in patients with IBD. KFEs were used as a dietary intervention in Il10-/- mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 x 2 factorial design. While all Il10-/- mice developed significant colonic inflammation compared to the C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. Whole genome gene and protein expression level profiling indicated that KFEs influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, adaptive immune pathways were reduced by three out of four kiwifruit extracts, with greater reduction seen in the C57BL/6J mice. This suggests that while immune-modulating activity was present in vivo, KFEs did not reduce inflammatory processes relevant to IBD. Experimental design. Two experiments were conducted, one using extracts from gold kiwifruit and one using extracts from green kiwifruit. Within each experiment, both Il10-/- and C57 mice were randomly divided into three diet treatment groups, to ive the following 12 treatments: 1) Gold Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 2) Gold Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 3) Gold Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 4) Gold Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 5) Gold Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 6) Gold Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 7) Green Kiwifruit Experiment, C57BL/6J mice, Control diet (AIN-76A), 8) Green Kiwifruit Experiment, C57BL/6J mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 9) Green Kiwifruit Experiment, C57BL/6J mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) 10) Green Kiwifruit Experiment, Il10-/- mice, Control diet (AIN-76A), 11) Green Kiwifruit Experiment, Il10-/- mice, Aqueous Kiwifruit Extract Supplemented Diet (AIN-76A + 5% Aqueous KFE) 12) Green Kiwifruit Experiment, Il10-/- mice, Ethyl Acetate Kiwifruit Extract Supplemented Diet (AIN-76A + 0.11% Ethyl Acetate KFE) Six biological replicates were analysed from each treatment group, except for groups 3 and 7 where five replicates were analysed due to array quality issues with the sixth replicate. Each replicate contained mRNA from one mouse. A reference design was used, where each slide was hybridised with mRNA from one sample (green channel) and mRNA from a common reference pool (red channel).
Project description:Pulmonary fibrosis (PF) is a terminal lung disease characterized by fibroblast proliferation, accumulation of extracellular matrix accumulation, inflammatory damage, and tissue structure destruction. The pathogenesis of this disease, especiallyparticularly idiopathic pulmonary fibrosis (IPF), is still remains unknown. Macrophages play a significant rolemajor roles in organ fibrosis diseases, including pulmonary fibrosis. The phenotype and polarization of macrophages are closely associated with the process of pulmonary fibrosis. A new direction in drug research on for antipulmonary fibrosis is focuseds on developing drugs that maintain the stability of the pulmonary microenvironment. Here, tThrough bioinformatics analysis and experiments involving bleomycininduced pulmonary fibrosis in mice, we confirmed the importance of macrophage polarization in IPF. The analysis revealed that macrophage polarization in IPF involves a change in the phenotypice spectrum. Furthermore, the experiments demonstrated showed high expression of M2-type macrophage-related-associated biomarkers and inducible nitric oxide synthase, thus indicating an imbalance in M1/M2 polarization of pulmonary macrophages in mice with pulmonary fibrosis. Our investigation revealed that the ethyl acetate extract (HG2) obtained from the roots of Prismatomeris connataPrismatomeris connata Y. Z. Ruan exhibits therapeutic efficacy against bleomycin-induced pulmonary fibrosis. HG2 demonstrates the ability to modulates macrophage polarization, alterations in the TGF‐β/Smads pSmad pathway, and downstream protein expression in the context of pulmonary fibrosis. Drawing upon On the basis of our findings, we believe that HG2 exhibits has potential as a novel component of traditional Chinese medicine component for treating pulmonary fibrosis.
Project description:Ethyl Acetate Extract of Antenoron Filiforme Inhibits the Proliferation of Triple Negative Breast Cancer Cell via Suppressing SKP2/P21 Signaling Axis
Project description:This experiment aims to ascertain a profile of secondary metabolites produced by Ilyonectria species capable of causing disappearing root rot in ginseng. Ilyonectria isolates were grown on potato dextrose agar for 20 days, then plugs were taken from the cultures and extracted with ethyl acetate. Extracts were analyzed by LC-HRMS and tandem HRMS. Data were analyzed by Principal component analysis and molecular networking with GNPS.
Project description:Ethyl carbamate is a common food contaminant prevalent in fermented food with probable carcinogenic effects in animals. To date, other toxicological properties of ethyl carbamate are not well characterized. Using the genetic model Caenorhabditis elegans, we found that chronic exposure to ethyl carbamate during larval development inhibits growth while exposure at adulthood inhibits reproduction, shortens lifespan, and promotes degeneration of dopaminergic neurons. Through whole-transcriptome RNA-sequencing, we found that ethyl carbamate invokes a widespread transcriptomic response inducing the differential expression of > 4,000 genes by at least 2-fold. Functional analysis of RNA-sequencing data revealed that genes up-regulated enrich to various neuron regulatory processes and xenobiotic defense. Gene expression analysis confirms that various genes functioning within phase 1 and 2 detoxification responses along with ABC transporters are highly up-regulated in response to ethyl carbamate exposure, suggesting the induction of oxidative stress. Overall, these findings demonstrates new toxicological properties of chronic ethyl carbamate exposure and provide new insights into the effects in has on transcriptome regulation in the C. elegans model.
Project description:The Ethyl Acetate phase (EP) obtained from the liquid-liquid partition of methanolic extract of Casearia aborea leaves was phytochemicaly investigated about the flavonoid-3-O-glycosides.