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: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:Background: RNA interference (RNAi) is an indispensable regulatory mechanism governing developmental processes and stress responses via sequence-specific control of target RNAs mediated by the action of small, 20–24-nt-long, non-coding regulatory (s)RNAs such as micro (mi) and small interfering (si) RNAs. Biogenesis and sorting of miRNAs into ARGONAUTE (AGO) proteins are intensively investigated, however very few information is available about the presence and distribution of distinct sRNA pools in plant cells. Results: High-throughput sequencing of size-separated sRNA pools of plant crude extracts revealed that the majority of the canonical miRNAs were associated with high molecular weight RNA-induced silencing complexes co-migrating with AGO1 (HMW RISC). In contrast, the majority of 24-nt-long siRNAs were found in association with low molecular weight complexes co-migrating with AGO4 (LMW RISC). Intriguingly, we identified a large set of sRNAs in the cytoplasm, including mature miRNA sequences, in the low molecular size range corresponding to protein-unbound sRNAs. By comparing the RISC-loaded and protein-unbound pools of miRNAs, we identified miRNAs with highly different loading efficiencies. Investigation of some selected miRNAs in a transient expression system validated this finding. We also showed that the availability of RISCs is a limiting factor determining the loading efficiency of miRNAs. Conclusion: Our data reveal the existence of a regulatory checkpoint, likely controlled by information carried by the diverse miRNA precursors, determining the RISC-loading efficiencies of various miRNAs by sorting only a subset of the produced miRNAs into the biologically active RISCs.
Project description:HRMS guided study of 42 marine Streptomyces in the MAR4 clade. Metabolomics data collected from Ethyl Acetate crude extracts of 50 ml liquid cultures.
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: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.