Gene transfer and expression in human neutrophils. The phox homology domain of p47(phox )translocates to the plasma membrane but not to the membrane of mature phagosomes

BACKGROUND: Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. RESULTS: Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX) domain of p47(phox )localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47(phox), is translocated to the membrane of mature phagosomes. CONCLUSION: We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils

The role of open-air inhalatoria in the air quality improvement in spa towns

Objectives: The present study was aimed at evaluating microbiological contamination of air in Ciechocinek and Ino­wro­cław – Polish lowland spa towns. Additionally, the impact of open-air inhalatoria on the quality of air was evaluated. Material and Methods: Air samples were collected seasonally in the urban areas, in the recreation areas and in the vicinity of inhalatoria in both towns using impaction. The numbers of mesophilic bacteria, staphylococci, hemolytic bacteria and actinomycetes were determined on media according to the Polish Standard PN-86/Z-04111/02. The number of moulds was determined on media according to the Polish Standard PN-86/Z-04111/03. Results: While the highest numbers of microorganisms were noted at the sites located in the urban areas, the lowest numbers were noted in the vicinity of the open-air inhalatoria. In all the investigated air samples the values of bioaerosol concentrations were below the recommended TLVs (≤ 5000 CFU×m–3 for both bacteria and fungi in outdoor environments). Location of the sampling site was invariably a decisive factor in determining the number of microorganisms in the air. Conclusions: The aerosol which is formed in the open-air inhalatoria has a positive influence on microbiological air quality. Owing to a unique microclimate and low air contamination, Ciechocinek and Inowrocław comply with all necessary requirements set for health resorts specializing in treating upper respiratory tract infections

Three-dimensional organization of pyrrolo[3,2-b]pyrrole-based triazine framework using nanostructural spherical carbon: enhancing electrochemical performance of materials for supercapacitors

Abstract Covalent triazine-based frameworks have attracted much interest recently due to their high surface area and excellent thermal and electrochemical stabilities. This study shows that covalently immobilizing triazine-based structures on spherical carbon nanostructures results in the organization of micro- and mesopores in a three-dimensional manner. We selected the nitrile-functionalized pyrrolo[3,2-b]pyrrole unit to form triazine rings to construct a covalent organic framework. Combining spherical carbon nanostructures with the triazine framework produced a material with unique physicochemical properties, exhibiting the highest specific capacitance value of 638 F g−1 in aqueous acidic solutions. This phenomenon is attributed to many factors. The material exhibits a large surface area, a high content of micropores, a high content of graphitic N, and N-sites with basicity and semi-crystalline character. Thanks to the high structural organization and reproducibility, and remarkably high specific capacitance, these systems are promising materials for use in electrochemistry. For the first time, hybrid systems containing triazine-based frameworks and carbon nano-onions were used as electrodes for supercapacitors

Cross-talk between IRAK-4 and the NADPH oxidase1

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation