Project description:To identify those proteins interacting with the cytoplasmic dynein-2 using mammalian cell lines stably expressing HA-tagged intermediate chain subunits, WDR34 (DYNC2I2) or WDR60 (DYNC2I1).
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.
Project description:Melanoma cells are highly plastic and have the ability to switch to a dedifferentiated, invasive phenotype in response to multiple stimuli. Here, we show that exposure of melanoma cell lines and patient specimens to multiple stresses including BRAF-MEK inhibitor therapy, hypoxia and UV-irradiation leads to an increase in histone deacetylase 8 (HDAC8) expression/activity, and in turn, the adoption of a drug-resistant, invasive phenotype. Systems level analyses using mass spectrometry-based phosphoproteomics implicated HDAC8 in the regulation of MAPK and AP-1 signaling pathways. Introduction of HDAC8 into drug-naïve melanoma cells conveyed resistance both in vitro and in in vivo xenograft models. HDAC8-mediated BRAF inhibitor resistance was mediated via receptor tyrosine kinase (RTK) activation leading to Ras/CRAF/MEK/ERK signaling. Although HDACs primarily function at the histone level, they also regulate signaling through the modulation of non-histone substrates. In line with this, HDAC8 introduction decreased the acetylation of c-Jun, increasing its transcriptional activity and enriching for an AP-1 gene signature. Mutation of the putative c-Jun acetylation site at lysine residue 273 reduced the transcriptional activation of c-Jun in melanoma cells and conveyed resistance to BRAF inhibition through increased RTK expression and enhanced MAPK pathway activity. In vivo xenograft studies confirmed the key role of HDAC8 in therapeutic adaptation, with both non-selective and HDAC8-specific inhibitors enhancing the durability of response to BRAF inhibitor therapy. Our studies demonstrate that HDAC8-specific inhibitors could represent an excellent strategy to limit the adaptation of melanoma cells to multiple stresses and therapeutic interventions, including BRAF-MEK inhibitor combinations.
Project description:Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Pectins are made in the Golgi apparatus by the coordinated action of transporters and enzymes. Here, in this project, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. We did an immunoprecipitation-mass spectrometry (IP-MS) analysis to identify potential interactors of NKS1. And we also performed an IP-MS analysis of QUA1, which is one of the interactors of NKS1.
Project description:PURPOSE. Triamcinolone acetonide (TA) and dexamethasone (DEX) are corticosteroids commonly used for ocular inflammation but both can cause ocular hypertension. We investigated the differential gene expression profile of human trabecular meshwork (TM) cells in response to treatment by TA compared to that by DEX. METHODS. Total RNA was extracted from cultured human TM cells treated with TA or DEX and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by an empirical Bayes approach and confirmed by real-time quantitative PCR. RESULTS. 0.1 mg/ml TA treatment resulted in 15 genes up-regulated and 12 genes down-regulated while 1mg/ml TA treatment resulted in 36 genes up-regulated and 21 genes down-regulated. These genes were mainly associated with acute-phase response, cell adhesion, cell cycle and growth, growth factor, ion binding, metabolism, proteolysis and transcription factor. Two genes, MYOC and GAS1, were up-regulated and 3 genes, SENP1, ZNF343 and SOX30, were down-regulated by both TA and DEX treatment. Eight differentially expressed genes were located in known primary open angle glaucoma (POAG) loci, including MYOC, SOAT1, CYP27A1, SPOCK, SEMA6A, EGR1, GAS1 and ATP10A. CONCLUSIONS. Differential gene expression profiles of human TM cells treated by TA and DEX, and a dosage effect by TA, were revealed by microarray technology. TA and DEX treatment shared several differentially expressed genes, suggesting a common mechanism to cause ocular hypertension. Some differentially expressed genes located in the known POAG loci are potential candidates for glaucoma genes. A total of 9 samples were used for this study. There were 3 biological replicates for each of 3 treatments.
Project description:Aberrant regulation of angiogenesis involves in the growth and metastasis of tumors, but angiogenesis inhibitors fail to improve overall survival of pancreatic cancer patients in previous phase III clinical trials. A comprehensive knowledge of the mechanism of angiogenesis inhibitors against pancreatic cancer is helpful for clinical purpose and for the selection of patients who might benefit from the inhibitors. In this work, multi-omics analyses (transcriptomics, proteomics and phosphoproteomics profiling) were carried to delineate the mechanism of anlotinib, a novel angiogenesis inhibitor, against pancreatic cancer cells.
Project description:The present study was conducted to investigate the effect of graded levels of black soldier fly larvae (BSFL) (Hermetia illucens) meal and BSFL paste in extruded diets for Atlantic salmon (Salmo salar). A total of 1260 Atlantic salmon with 34 g of mean initial weight were randomly distributed into 21 fiberglass tanks and fed (n=3) with seven extruded isolipidic and isonitrogenous diets for seven weeks. The experimental diets consisted of a positive control diet based on fishmeal, soy protein concentrate, corn gluten, faba bean and fish oil (Control_1); three diets with increased levels of full lipid BSFL meal, substituting 6.25% (6.25_IM), 12.5% (12.5_IM) and 25% (25_IM) of the protein content of Control_1; two diets with increased levels of full lipid BSFL paste, substituting 3.7% (3.7_IP) and 6.7% (6.7_IP); and of protein from Control_1 and a negative a control with 0.84 % of formic acid (Control_2). We investigate the effect of diets on growth performance, mmune response and health.
Project description:Pseudomonas aeruginosa (P. aeruginosa) can cause severe acute infections, including pneumonia and sepsis, and also cause chronic infections commonly in patients with structural respiratory diseases. However, the molecular and pathophysiological mechanisms of P. aeruginosa respiratory infection are largely unknown. Here, we profiled performed to assay for transposase-accessible chromatin using sequencing (ATAC-seq), transcriptomics, and quantitative mass spectrometry-based proteomics and ubiquitin-proteomics in P. aeruginosa-infected lung tissues for multi-omics analysis, while ATAC-seq and transcriptomics were also examined in P. aeruginosa-infected mouse macrophages. To find the pivotal transcription factors that are likely involved in host immune defense, we integrally investigated systematic changes in chromatin accessibility and gene expression in P. aeruginosa-infected lung tissues combined with proteomics and ubiquitin-proteomics studies. We discovered that Stat1 and Stat3 were altered in various omics and found similar results in mouse alveolar macrophages. Taken together, these findings indicate that these crucial transcription factors and their downstream signaling molecules play a critical role in the mobilization of host immune response against P. aeruginosa infection and may serve as potential targets for bacterial infections and inflammatory diseases, as well as provide clear insights and resources for using integrative histological analyses.
Project description:Paracoccidioides spp. is the etiological agent of Paracoccidioidomycosis (PCM), a systemicinfection with wide distribution in Latin America. Macrophages are very important cells during the response to infection by Paracoccidoides brasiliensis and understanding the interaction between the fungus and immune cells is very relevant for understanding the disease. In this study, we performed a proteomic analysis to assess the consequences of P. brasiliensis yeast infection on the THP-1 macrophage proteome and to verify whether there are differences between the proteome of cells infected with the live fungus. We identified 443 upregulated and 2247 downregulated proteins in macrophages infected with live P. brasiliensis yeasts, compared to uninfected macrophages. Proteins differentially expressed in cells infected are related to metabolism and energy production, protein synthesis and processing, transcription, cell cycle, DNA processing, cell signaling, oxidative stress, immune response, among other processes . Proteomic analysis revealed that P. brasiliensis, causes metabolic alterations in infected cells, drastically affecting energy production pathways. In addition, macrophages showed many upregulated, mostly downregulated, immune system proteins. Thus, the present work contributes to elucidate the changes that occur in immune cells in response to infection by P. brasiliensis and may help to better understand PCM.