Project description:Slime proteins were separated into high, middle, and low MW regions by SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) . The bands from different MW regions were carefully cut off, in-gel digested, subjected to LC-MS/MS, and searched against the transcriptome library of the slime gland.
Project description:We did bulk RNA sequencing in newborn cystic fibrosis (CF) and non-CF pig kidney. We compared kidney gene expression profiling between non-CF and CF pigs. RNA sequencing results showed that there is not significant difference between non-CF and CF in terms of gene expression, suggesting that CFTR knockout does not affect kidney development in newborn pigs.
Project description:The Human Tracheal Gland (HTG) cell line MM39 and the CF HTG cell line CF-KM4 were incubated with or without a P. aeruginosa supernatant for 3 hours.
Project description:A small-scale whole genome microarray study of gene expression in human native nasal epithelial cells from F508del-CFTR homozygous CF patients and non-CF controls. We used the custom designed Affymetrix HsAirwaya520108F Arrays to compare gene expression in 5 CF and 5 non CF nasal epithelial cell samples. We analysed a total of 10 samples (5 CF and 5 non CF). The CF group contained 2 males and 3 females, with an average age of 14 years and an average of 6% inflammatory cells per sample, and the non CF group contained 3 males and 2 females with an average age of 14.8 years and an average of 4.7% inflammatory cells.
Project description:A small-scale whole genome microarray study of gene expression in human native nasal epithelial cells from F508del-CFTR homozygous CF patients and non-CF controls. We used the custom designed Affymetrix HsAirwaya520108F Arrays to compare gene expression in 5 CF and 5 non CF nasal epithelial cell samples.
Project description:To provide a more detailed survey of adaptive changes in the physiology of P. aeruginosa (PA) during long-term infection of the cystic fibrosis (CF) lung, we performed a comparative proteome and transcriptome analysis of a set of isogenic sequential non-mutator and mutator isolates from three selected CF patients. Recently, we showed that during CF lung persistence PA mutators converge to a virulence-attenuated phenotype. In this study, we demonstrate that besides virulence-associated traits (VATs) the adaptation process of PA predominantly comprises metabolic pathways. In end-stage mutator strains, transcripts of genes encoding VATs, chemotaxis, degradation of aromatic compounds and several two-component regulatory systems were decreased. In contrast, several transcripts of genes or proteins involved in metabolism of fatty acids, nucleotides, amino acids and the generation of energy were increased. Of particular interest is the increased expression level of genes involved in (i) the anaerobic arginine-deiminase pathway, (ii) the anaerobic respiration such as nitrate-uptake protein OprF, redox-active azurin and cytchrome c551 peroxidase, (iii) the micro-aerobic respiration such as high oxygen-affinity cytochrome oxidase cbb3 (iv) the tricarboxylic acid cycle (TCA), glyoxylate shunt and anaplerotic carboxylation reactions to oxaloacetate. Strikingly, an increased transcription of the anaerobic regulator gene anr correlates with the up-regulation of ANR-dependent genes. In conclusion, these changes in transcriptome and proteome indicate an adaptive shift towards constitutive expression of genes of metabolic pathways obviously required for growth under micro-aerobic and nutritional conditions of suppurative CF lung tissue. Finally, these results provide us with new targets for antimicrobial agents and biomarkers reflecting adaptation of PA towards progressive CF lung disease. Experiment Overall Design: P. aeruginosa isolates recovered from different time points of chronic cystic fibrosis lung disease were cultered in vitro, harvested for RNA extraction and hybridization on Affymetrix microarrays. We compared the transcriptome (triplicate microarrays) of early non-mutator P. aeruginosa isolates with late mutator isolates with high mutation frequency probably the driving force of an efficient adaptation to changing environements to conclude from differences in gene expression to the requirements of CF lung environment. Experiment Overall Design: Second publication of array data to be added later
Project description:Mutations in CFTR have been shown to alter the immune response of macrophages, for example, by reducing the ability of macrophages to phagocytose and kill bacteria. This contributes to chronic bacterial infection and inflammation in the lungs, which leads to significant morbidity and mortality in cystic fibrosis (CF). Extracellular vesicles (EVs) are secreted by a variety of cell types in the lungs and participate in the host immune response to bacterial infection. However, nothing is known about the effect of EVs secreted by CF airway epithelial cells (AEC) on CF macrophages. Therefore, we examined the effect of EVs secreted by primary CF AEC on CF monocyte derived macrophages (MDM) and compared it with the effect of EVs secreted by wild type (WT) AEC on WT MDM. EVs increased pro-inflammatory cytokine secretion and enhanced the expression of numerous innate immune genes in WT MDM. However, the response of CF MDM to EVs was significantly attenuated compared to WT MDM, a difference that was also observed when EVs were isolated from WT and CF AEC exposed to Pseudomonas aeruginosa. Attenuated responses by CF MDM can be attributed to defects in the CF macrophages themselves rather than differences between CF and WT EVs, because EVs secreted by CF AEC or WT AEC elicited similar cytokine secretion by CF MDM. EVs secreted by P. aeruginosa exposed AEC resulted in the upregulation of immune response genes and increased secretion of pro-inflammatory cytokines, chemoattractants and chemokines involved in tissue repair by WT MDM, whereas the response of CF MDM was attenuated by comparison. To our knowledge, this is the first study examining the effect of EVs secreted by CF AEC on CF MDM, and it demonstrates that the Phe508del mutation in CFTR attenuates the innate immune response of MDM to EVs.
Project description:Cystic Fibrosis (CF) is associated with pathology in multiple tissues including the lung, digestive tract and reproductive system. Lung disease is primarily a post-natal event but other organs are affected before birth. Here we use the CF sheep model to investigate the initiation and progression of CF disease through gestation.