Project description:Polyethylene glycol (PEG) is one of the most common polymer contaminations in MS samples. At present, the detection of PEG and other polymers relies largely on manual inspection of raw data which is laborious and frequently difficult due to sample complexity and retention characteristics of polymer species in reversed phase chromatography. We developed a new strategy for the automated identification of PEG molecules from MSMS data using protein identification algorithms in combination with a database containing “PEG-proteins”. Through definition of variable modifications, we extend the approach for the identification of commonly used PEG-based detergents. We exemplify the identification of different types of polymers by static nanoESI-MSMS analysis of pure detergent solutions and data analysis using Mascot. Analysis of LC-MSMS runs of a PEG contaminated sample by Mascot identified 806 PEG-spectra originating from four PEG species using a defined set of modifications covering PEG and common PEG-based detergents. Further characterization of the sample for unidentified PEG species using error tolerant and mass tolerant searches resulted in identification of 3409 and 3187 PEG related MSMS spectra, respectively. We further demonstrate the applicability of the strategy for Protein Pilot and MaxQuant.
Project description:Transcriptome comparison to assess the responses of human monocytic cells (THP-1) to gold-nanoparticles (Au-NPs) of two different diameter sizes (5 or 20 nm) with different surface functional groups, i.e., alkylammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated thiolate Au-NPs.
Project description:Poly-ethylene-glycol (PEG)-based nanoparticles (NPs) - including cylindrical micelles (CNPs), spherical micelles (SNPs), and PEGylated liposomes (PLs) - are hypothesized to be cleared in vivo by opsonization followed by liver macrophage phagocytosis. This hypothesis has been used to explain the rapid and significant localization of NPs to the liver after administration into the mammalian vasculature. Here, we show that the opsonization-phagocytosis nexus is not the major factor driving PEG-NP – macrophage interactions. First, mouse and human blood proteins had insignificant affinity for PEG-NPs. Second, PEG-NPs bound macrophages in the absence of serum proteins. Third, lipoproteins blocked PEG-NP binding to macrophages. Because of these findings, we tested the postulate that PEG-NPs bind (apo)lipoprotein receptors. Indeed, PEG-NPs triggered an in vitro macrophage transcription program that was similar to that triggered by lipoproteins and different from that triggered by lipopolysaccharide (LPS) and group A Streptococcus. Unlike LPS and pathogens, PLs did not increase transcripts involved in phagocytosis or inflammation. High-density lipoprotein (HDL) and SNPs triggered remarkably similar mouse bone-marrow-derived macrophage transcription programs. Unlike opsonized pathogens, CNPs, SNPs, and PLs lowered macrophage autophagosome levels and either reduced or did not increase the secretion of key macrophage pro-inflammatory cytokines and chemokines. Thus, the sequential opsonization and phagocytosis process is likely a minor aspect of PEG-NP – macrophage interactions. Instead, PEG-NP interactions with (apo)lipoprotein and scavenger receptors appear to be a strong driving force for PEG-NP – macrophage binding, entry, and downstream effects. We hypothesize that the high presence of these receptors on liver macrophages and on liver sinusoidal endothelial cells is the reason PEG-NPs localize rapidly and strongly to the liver. To investigate the response of macrophages to nanoparticles, we incubated PEGylated liposomes (PLs), spherical nanoparticles (SNPs), high-density lipoproteins (HDL), low-density lipoproteins (LDL), Group A Streptococcus pathogens (JRS4), and lipopolysaccharide (LPS) with RAW264.7 and primary bone-marrow derived mouse macrophages
Project description:ORganically MOdified SILica (ORMOSIL) nanoparticles (NPs) appear promising carriers for the delivery of drugs to target tissues and cells but some concerns on possible cytotoxic effects still exist. We therefore studied the in vitro responses to ORMOSIL NPs in different types of human lung cells (i.e. CCD-34Lu normal fibroblasts, carcinoma alveolar type II A549 cells, NCI-H2347 adenocarcinoma cells) to determine the effects of polyethylene glycol (PEG) coating on NP cytotoxicity. Our results show that non-PEG NPs caused a concentration-dependent decrease of viability of all types of cells. On the contrary, PEG-coated NPs increased plasma membrane permeability and induced cell death only in carcinoma alveolar type II A549 cells, while did not produce deleterious effects on CCD-34Lu and NCI-H2347 cells. PEG-coated NPs promoted the formation of reactive oxygen species (ROS) in A549 and CCD-34Lu cells; nevertheless, the decrease of ROS levels in the presence of superoxide dismutase and catalase did not protect A549 cells from death, suggesting that the oxidative stress was not the main determinant of cytotoxicity. Analysis of gene expression in A549 cells exposed to PEG-coated NPs showed alterations in genes involved in inflammation, signal transduction and cell death, while the transcriptional response was not significantly affected in CCD-34Lu fibroblasts. Our transmission electron microscopy analysis evidenced a unique intracellular localization of PEG NPs in the lamellar bodies of A549 cells, which could be the most relevant factor leading to cytotoxicity by reducing the production of surfactant proteins and by interfering with the pulmonary surfactant system.
Project description:A proteome comparison was performed to assess the responses of human monocytic cells (THP-1) to gold-nanoparticles (Au-NPs) of two different diameter sizes (5 or 20 nm) with different surface functional groups, i.e., alkylammonium bromide, alkyl sodium carboxylate, or poly(ethylene glycol) (PEG)-terminated thiolate Au-NPs.
Project description:To understand the extent that Heat shock protein 90 (Hsp90) regulated its target proteins at the transcription level, transcriptomic change was profiled in yeast cells upon Hsp90 compromising. We genetically modified the R1158 strain (resulting genotype of mutant strain: TETp-HSC82 hsp82Δ arg4Δ lys5Δ car2Δ::URA3) and then reduced the Hsp90 amount with doxycycline treatment. Fold change of mRNA from untreated to treated cells indicated the transcriptomic change. Totally, we identified 1104 genes mis-regulated with a fold change of no less than 1.5 (P <0.05) upon Hsp90 compromising. Two-condition experiment, treated vs. untreated cells. Biological duplicates, independently grown and harvested. Technical triplicates for RNA isolation.
Project description:The study was conducted to identify differentially expressed polyethylene glycol (PEG) induced water stress responsive genes in E. grandis. Forty day old rooted cutting of E. grandis was subjected to -0.225 MPa PEG treatment and total RNA was isolated from leaves of water treated control and PEG treated samples after three hours of treatment. The differential expression of water stress responsive genes was analyzed using microarray technique. Agilent two-color experiment, Organism: Eucalyptus,Custom Agilent Eucalyptus 8x60k Microarray Gene expression (AMADID: 59849 ) designed by Genotypic Technology Pvt.Ltd.
Project description:Sour passion fruit (Passiflora edulis Sims) has economic and social relevance and is an alternative crop mainly for family farming agriculture. The use of micropropagation by somatic embryogenesis offers scale-up clonal propagation with high phytosanitary quality. The aim of this work was to evaluate the influence of polyethylene glycol (PEG) on the maturation of somatic embryos associated with differential accumulation of proteins and changes in the endogenous polyamines (PAs) content during somatic embryogenesis of P. edulis ‘UENF Rio Dourado’. Maturation of somatic embryos was performed using embryogenic callus in MS culture medium with PEG 6% or without PEG (control). PEG 6% promoted the maturation of a significantly higher number of somatic embryos at globular and cotyledonary stages when compared to control treatment. The higher somatic embryo formation induced by PEG 6% was associated with an increase in endogenous contents of free spermine, a PA with an important role in the maturation process of somatic embryogenesis cultures. PEG also promoted a significantly higher content of putrescine and total free PAs at the end of maturation, which was relevant for the increase in the number of somatic embryos. Comparative proteomic analyses of PEG 6%/control revealed that PEG 6% treatment induced the up-accumulation of proteins related to the glycolytic process, generation of precursor metabolites energy, and response to light stimulus, we can highlight the enolase ENO1, triosephosphate isomerase (TPI), ribulose bisphophate carboxylase/oxygenase activase chloroplastic (RCA), glyceraldehyde-3-phosphate dehydrogenase GAPCP-1, chloroplastic (GAPCP-1), succinate dehydrogenase [ubiquinone] flavoprotein subunit1, mitochondrial (SDH1-1), pyruvate dehydrogenase E1 component subunit alpha, mitochondrial (IAR4), ATP synthase CF1 beta subunit (PB), malate dehydrogenase [NADP], chloroplastic (AT5G58330) and chlorophyll a-b binding protein 21, chloroplastic (LHB1B1), and the down-accumulated proteins related mainly to the cellular metabolic process with proteins such as NADP-dependent malic enzyme (NADP-ME4), phosphoenolpyruvate carboxylase 2 (PPC1), UDP-glucose 6-dehydrogenase 1 and 4 (UGD2) and sucrose synthase 2 isoform X2 (SSA) and cell division cycle protein 48 homolog (ATCDC48B). The use of PEG induced thematuration and development of somatic embryos of P. edulis Sims ‘UENF Rio Dourado’ by the differential accumulation of proteins and modulation of endogenous contents of PAs
Project description:Drought and salinity reduce plant growth and grain yield in wheat. To explain the role of Cu and Fe NPs in the improvement of growth and yield of wheat varieties, gel-free proteomic technique was used. Spike length, number of grains per spike, and 100 grain weight were increased at 25 ppm Cu and Fe NPs in high yielding galaxy-13, drought tolerant Pakistan-13, and salinity tolerant NARC-11. A total of 86, 131, and 30 proteins were significantly changed in abundance under Cu and Fe NPs exposure. The number of proteins related to stress, proteins, and glycolysis were mainly changed by Cu and Fe NPs exposure. By Cu NPs, in galaxy-13 starch degradation and glycolysis pathway were increased, while decreased in Pakistan-13 and NARC-11. Furthermore, by Fe NPs, only in galaxy-13 tricarboxylic acid cycle was increased while did not change glycolysis and starch degradation. Uptake of Cu NPs increased at 25 ppm in galaxy-13, Pakistan-13 and NARC-11 while Fe NPs uptake increased only at 35 and 40 ppm in NARC-11.
Project description:Our goal is to find new genes regulated by p21 in human primary cells . To get it we carried out a gene expression profiling in two different models, human myeloid leukemia K562 cells and human keratinocytes both of them with conditional expression of p21. In order to find genes regulated by p21 in human primary cells we carried out a gene expression profiling in human myeloid leukemia K562 cells with conditional expression of p21. We previously described a K562 derivative, termed Kp21-4, that carries a zinc-inducible p21 gene (Munoz-Alonso MJ et at., 2005). We performed a kinetic study to identify the expression peak of p21 in this system. This transient induction of p21 was accompanied by proliferation arrest and an increase in polyploid cells after 48-72 h (Munoz-Alonso MJ et at., 2005). Actually, 6-12 h of p21 induction with ZnSO4 is sufficient to irreversibly trigger proliferation arrest. Therefore, we chose 12 h as the induction time to analyse p21 effects on the transcriptome of these cells, as gene expression changes later on may be indirect due to other phenotypic effects. We next carried out the gene expression profiling of Kp21-4 cells upon p21 induction by ZnSO4. In order to identify genes specifically modulated by p21 we compared with the cell line Kp27-5, which carries a Zn2+-inducible p27 allele (Munoz-Alonso MJ et at., 2005). p27 is a close relative to p21 that also inhibits CDKs and induce cell cycle arrest . Thus, the comparison serves to identify genes specifically regulated by p21 in our analysis. We subtracted the gene expression changes occurring at 72 h in Kp21-4 cells those genes regulated by p27 in the Kp27-5 cells and genes changed by ZnSO4 treatment in parental K562 cells. So, our intention is to identify only genes regulated at short time of induction by p21 and not by p27. In order to confirm these results we studied the p21-dependent repression of mitotic genes in a different cellular system. A dramatic increase in p21 and p27 in Kp21 and Kp27 were demonstrated by RT-qPCR and immunoblot (as we show in the manuscript). We prepared RNA 12 h and 72h after induction with ZnSO4 and performed large-scale expression assay using the Afftymetrix platform. The clustering analysis revealed that p21 provoked the down-regulation of a number genes involved in cell cycle control not shared by cells expressing p27 (as we show in the manuscript). Our goal, has been getting genes regulated more strongly by p21 and not by p27, in cell cycle and itosis. Our result are supported because we have found the same genes in two different models and also we have validated (by RT-qPCR) more than 20 cell cycle and mitotic genes, found in our affymetrix arrays. Also we have found the region of p21 that is sufficient for gene regulation and for one gene we have described as p21 bind to the promoter. Finally, we have discussed in our manuscript how p21 can do this regulation by bioinformatic analysis of p21-target genes. The sucess of this study is describe a new role of p21 as a transcriptional co-repressor in some systems.