ABSTRACT: We performed immunoprecipitation analysis with leaves from 35S::NusG:MYC transgenic lines throung the MYCantibody, and the product was subjected to LC-MS analysis.
Project description:A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNA associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP and an RNAse III domain-containing protein MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs. One small RNA library was generated using QDE-2 immunoprecipitate from Neurospora crassa.
Project description:Impact of CDK4-deficiency on EM-BM-5-myc driven B-lymphoma By crossing mating CDK4 Knockout mice with EM-BM-5-myc mice,We found CDK4-deficiency enhances the EM-BM-5-myc induced B-lymphoma.We further to discover the molecular mechanism B-lymphoma cells form the cdk4+/+ EM-BM-5-myc and cdk4-/-EM-BM-5-myc mice and then culturing for RNA extraction and hybridization on Affymetrix microarrays.
Project description:Salinity causes osmotic stress to crops and limits their productivity. To understand the mechanism underlying soybean salt tolerance, quantitative phosphoproteomics approach was used to identify phosphoproteins that were altered by NaCl treatment.
Project description:Mg alloy is one of the lightest metals on earth and has the most similar mechanical properties to human bones. However, the active chemical properties and fast in vivo degrade speed significantly hinder its further employment as human implant. In this study, three different Mg alloys are employed to investigate the in vitro corrosion behavior including weight loss, pH value evolution and surface hardness and in vivo degradable speed and tissue compatibility. All three Mg alloys are well tolerated when implanted in SD rats, with minimal influence on hepato-and renal functions and vital organs. Compared to cold extruded AZ31 (CE AZ31) and pure Mg (PM) specimens, fully annealed AZ31 (FA AZ31) presents much stable surface asperity. Proteomics analysis of tissues near implant site showed that FA AZ31 activates few inflammation and immune associated signaling pathways; while the CE AZ31 and pure Mg led more significant inflammatory responses as confirmed by the cytokine array that pure Mg stimulate higher IL-1 production than FA AZ31. Further, FA AZ31 can activate pathways of cell organization and development that may improve the recovery of the injured tissues neighboring the implant sites. Besides, all three Mg alloys cannot inhibit a methicillin-resistant S. aureus growth if the implants are contaminated with this bacterium. FA AZ31 has a higher ratio of first-order pyramidal slips system (10-11) {10-1-2} than pure Mg and cold extruded AZ31 analysed by EBSD, suggesting the process of dynamic recovery may plays an important role in the improvement of mechanical properties, especially bio-properties including corrosion resistance and biocompatibility. In conclusion, FA AZ31 has better biocompatibilities and corrosion resistance, makes it a promising candidate of metal-based degradable implant and warrant further investigation.
Project description:FUSCA3 (FUS3) is a B3 domain transcription factor that is a member of the LEAFY COTYLEDON (LEC) group of genes. The LEC genes encode proteins that also include LEC2, a B3 domain factor related to FUS3, and LEC1, a CCAAT box binding factor. LEC1, LEC2 and FUS3 are essential for plant embryo development. We report ChIP-chip experiments using the Affymetrix tiling array to globally map binding sites for FUS3. Fangfang Wang and Sharyn E. Perry (2013) Plant Physiology preview FUSCA3 was expressed by the native promoter and included a C-terminal 10x-c-myc tag. This transgene was able to rescue the fus3-3 mutant. A second transgene (35S:AGL15) was present to allow establishment of stable somatic embryo cultures. ChIP was performed using anti-c-myc antibody (Myc-Tag (9B11) Mouse mAb; Cell Signaling 2276S) on tissue expressing FUS3-myc (three biological replicates) and on three controls of tissue expressing FUS3 (no epitope tag).
Project description:In this study, we demonstrate that, the topical application of a temperature-sensitive gel containing caerin 1.1 and 1.9 peptides significantly reduces the tumour weight of HPV 16 E6/E7 transformed TC-1 tumour bearing mice via improving the tumour microenvironment (TME) when compared with untreated tumour or a control peptide-containing gel. We use single cell transcriptomics and TMT10 plex-labelling proteomics to quantify changes in cellular activity across different cell types within the TME. We show that caerin 1.1/1.9 gel increased immune activating macrophages, modulated the heterogeneity of NK and dendritic cells to be more pro-inflammatory, and increased numbers of activated CD8+ T cells. Proteomic profiling demonstrated higher innate immune responses, especially the positive regulations of interferon-alpha/beta secretion and response to cytokine stimulus in the caerin gel groups. Further, computational integration of the proteome with the single cell transcriptome consistently suggested more activated T cells and NK cells with the treatment of caerin peptide gel.
Project description:Development of a vaccine formula that alters the tumour-infiltrating lymphocytes to be more immune active against a tumour is key to the improvement of clinical responses to immunotherapy. Here, we demonstrate that, in conjunction with E7 antigen specific immunotherapy, and IL-10 and PD-1 blockade, intra-tumoral administration of caerin 1.1 and 1.9 peptides further improves the tumour microenvironment (TME) when compared with injection of a control peptide. We used single cell transcriptomics and mass spectrometry-based proteomics to quantify changes in cellular activity across different cell types within the TME. We show that the injection of caerin 1.1/1.9 increases immune activating macrophages and NK cells, while reducing immunosuppressive macrophages with M2 phenotype, and increased numbers of activated CD8+ T cells with higher populations of memory and effector-memory CD8+ T subsets. Proteomic profiling demonstrated activation of Stat1 modulated apoptosis and production of nitric oxide. Further, computational integration of the proteome with the single cell transcriptome was consistent with deactivation of immune suppressive B cell function following caerin 1.1 and 1.9 treatment.
Project description:Caerin 1 is a family of host-defense peptides with antimicrobial property originally isolated from Australia tree frog. Caerin 1.1+1.9 has been shown to inhibit multiple antibiotic resistant bacteria infection both in vitro and in vivo. In current study, we compare the MICs of caerin 1.1/1.9 with commonly used antibiotics against S. aureus, Copper-Green Pseudomonas aeruginosa, Acinetobacter baumannii, and Streptococcus haemolyticus. We demonstrate that caerin 1.1/1.9 not only prevent the formation of biofilm by A. Baumann, but also have therapeutic effect on established biofilm. In addition, we find that caerin1.1/1.9 significantly inhibit the growth of methicillin-resistant Staphylococcus aureus (MRSA) strain in a murine skin infection model. The quantitative proteomic analysis suggested that caerin1.1/1.9 largely activate oxidative phosphorylation, as well as several pathways associated with tissue repair and growth, with respect to the untreated tissues infected with MRSA in mice. In summary, our results suggest that caerin 1.1/1.9 have the potential to be used as a drug candidate treating complicated antibiotic resistant bacterial infection in human.