Silver nanoparticles induced heat shock protein 70, oxidative stress and apoptosis in Drosophila melanogaster.

Due to the intensive commercial application of silver nanoparticles (Ag NPs), risk assessment of this nanoparticle is of great importance. Our previous in vitro study demonstrated that Ag NPs caused DNA damage and apoptosis in mouse embryonic stem cells and fibroblasts. However, toxicity of Ag NPs in vivo is largely lacking. This study was undertaken to examine the toxic effects of well-characterized polysaccharide coated 10 nm Ag NPs on heat shock stress, oxidative stress, DNA damage and apoptosis in Drosophila melanogaster. Third instar larvae of D. melanogaster were fed a diet of standard cornmeal media mixed with Ag NPs at the concentrations of 50 and 100 microg/ml for 24 and 48 h. Ag NPs up-regulated the expression of heat shock protein 70 and induced oxidative stress in D. melanogaster. Malondialdehyde level, an end product of lipid peroxidation was significantly higher while antioxidant glutathione content was significantly lower in Ag NPs exposed organisms. Activities of antioxidant enzyme superoxide dismutase and catalase were also significantly higher in the organisms exposed to Ag NPs. Furthermore, Ag NPs up-regulated the cell cycle checkpoint p53 and cell signaling protein p38 that are involved in the DNA damage repair pathway. Moreover, activities of caspase-3 and caspase-9, markers of apoptosis were significantly higher in Ag NPs exposed organisms. The results indicate that Ag NPs in D. melanogaster induce heat shock stress, oxidative stress, DNA damage and apoptosis. This study suggests that the organism is stressed and thus warrants more careful assessment of Ag NPs using in vivo models to determine if chronic exposure presents developmental and reproductive toxicity.

Pseudomonas aeruginosa anaerobic respiration in biofilms: relationships to cystic fibrosis pathogenesis.

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Proteomic, microarray, and signature-tagged mutagenesis analyses of anaerobic Pseudomonas aeruginosa at pH 6.5, likely representing chronic, late-stage cystic fibrosis airway conditions.

Patients suffering from cystic fibrosis (CF) commonly harbor the important pathogen Pseudomonas aeruginosa in their airways. During chronic late-stage CF, P. aeruginosa is known to grow under reduced oxygen tension and is even capable of respiring anaerobically within the thickened airway mucus, at a pH of approximately 6.5. Therefore, proteins involved in anaerobic metabolism represent potentially important targets for therapeutic intervention. In this study, the clinically relevant "anaerobiome" or "proteogenome" of P. aeruginosa was assessed. First, two different proteomic approaches were used to identify proteins differentially expressed under anaerobic versus aerobic conditions. Microarray studies were also performed, and in general, the anaerobic transcriptome was in agreement with the proteomic results. However, we found that a major portion of the most upregulated genes in the presence of NO(3)(-) and NO(2)(-) are those encoding Pf1 bacteriophage. With anaerobic NO(2)(-), the most downregulated genes are those involved postglycolytically and include many tricarboxylic acid cycle genes and those involved in the electron transport chain, especially those encoding the NADH dehydrogenase I complex. Finally, a signature-tagged mutagenesis library of P. aeruginosa was constructed to further screen genes required for both NO(3)(-) and NO(2)(-) respiration. In addition to genes anticipated to play important roles in the anaerobiome (anr, dnr, nar, nir, and nuo), the cysG and dksA genes were found to be required for both anaerobic NO(3)(-) and NO(2)(-) respiration. This study represents a major step in unraveling the molecular machinery involved in anaerobic NO(3)(-) and NO(2)(-) respiration and offers clues as to how we might disrupt such pathways in P. aeruginosa to limit the growth of this important CF pathogen when it is either limited or completely restricted in its oxygen supply.

Anaerobic metabolism and quorum sensing by Pseudomonas aeruginosa biofilms in chronically infected cystic fibrosis airways: rethinking antibiotic treatment strategies and drug targets.

Recent evidence indicates that Pseudomonas aeruginosa residing as biofilms in airway mucus of cystic fibrosis (CF) patients is undergoing anaerobic metabolism, a form of growth requiring gene products that are not utilized during aerobic growth. The outer membrane protein, OprF, and the rhl quorum sensing circuit are two previously unrecognized cellular factors that are required for optimal anaerobic biofilm viability. Without OprF, bacteria grow extremely poorly because they lack nitrite reductase activity while lacking rhlR or rhlI forces bacteria to undergo metabolic suicide by overproduction of nitric oxide. Furthermore, anaerobic growth favors maintenance of the mucoid, alginate-overproducing phenotype. Thus, with increasing age of CF patients, mucoid populations predominate, indicating that anaerobic bacteria reside in the inspissated airway mucus. Because many frontline antibiotics used in the treatment of CF airway disease are either ineffective or show reduced efficacy during anaerobic conditions, we propose development of new drugs to combat anaerobic metabolism by P. aeruginosa for more effective treatment of chronic CF lung infections.

ZnO nanoparticles induce apoptosis in human dermal fibroblasts via p53 and p38 pathways.

The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continues to expand. Zinc oxide nanoparticles (ZnO NPs) are used in various applications, including catalysis, electronics, biosensors, medicine, paints, sunscreens and cosmetics, thus it is important to understand the biological effects and risks of ZnO NPs. This study was designed to investigate the apoptosis induction by ZnO NPs via mitogen-activated protein kinase p38 and cell cycle checkpoint protein p53 pathways in human dermal fibroblasts. MTT-based cell viability assay showed a significant decrease in cell survivorship after ZnO NP exposure, and phase contrast images revealed that ZnO NP treated cells had lower density and a rounded morphology. Apoptosis induction was confirmed by the annexin V assay and Western blot analysis showed the up-regulation of p53 and phospho-p38 proteins. Furthermore, in ZnO NP exposed cells, p53 protein was phosphorylated at Ser33 and Ser46 sites known to be phosphorylated by p38. Our results suggest that ZnO NPs have the potential to induce apoptosis in human dermal fibroblasts via p53-p38 pathways.

Differential toxicity of silver and titanium dioxide nanoparticles on Drosophila melanogaster development, reproductive effort, and viability: size, coatings and antioxidants matter.

Silver and titanium dioxide nanoparticles are known to induce oxidative stress in vitro and in vivo. Here we test if they impact development, mating success, and survivorship in Drosophila melanogaster, and if so, if these effects are reversible by antioxidants. Ingestion of nanotitanium dioxide during the larval stage of the life cycle showed no effects on development or survivorship, up to doses of 200 µg mL(-1). Conversely, ingestion of nanosilver had major dose, size, and coating-dependent effects on each of these aspects of life history. Each of these effects was partially or fully reversible by vitamin C. Larvae growing on nanosilver supplemented with vitamin C showed a greater than twofold increase in survivorship compared to flies reared on nanosilver alone, and a threefold increase in mating success. Vitamin C also rescued cuticular and pigmentation defects in nanosilver fed flies. Biochemical assays of superoxide dismutase and glutathione show these markers respond to nanotitanium dioxide and nanosilver induced oxidative stress, and this response is reduced by vitamin C. These results indicate that life history effects of nanosilver ingestion result from oxidative stress, and suggest antioxidants as a potential remediation for nanosilver toxicity. Conversely, the lack of nanotitanium dioxide life history toxicity shows that oxidative stress does not necessarily result in whole organism effects, and argues that nanoparticle toxicity needs to be examined at different levels of biological organization.

Inhalation method for delivery of nanoparticles to the Drosophila respiratory system for toxicity testing.

The growth of the nanotechnology industry and subsequent proliferation of nanoparticle types present the need to rapidly assess nanoparticle toxicity. We present a novel, simple and cost-effective nebulizer-based method to deliver nanoparticles to the Drosophila melanogaster respiratory system, for the purpose of toxicity testing. FluoSpheres, silver, and CdSe/ZnS nanoparticles of different sizes were effectively aerosolized, showing the system is capable of functioning with a wide range of nanoparticle types and sizes. Red fluorescent CdSe/ZnS nanoparticles were successfully delivered to the fly respiratory system, as visualized by fluorescent microscopy. Silver coated and uncoated nanoparticles were delivered in a toxicity test, and induced Hsp70 expression in flies, confirming the utility of this model in toxicity testing. This is the first method developed capable of such delivery, provides the advantage of the Drosophila health model, and can serve as a link between tissue culture and more expensive mammalian models in a tiered toxicity testing strategy.

Artificial control of nitrate respiration through the lac promoter permits the assessment of oxygen-mediated posttranslational regulation of the nar operon in Pseudomonas aeruginosa.

In this study, oxygen and nitrate regulation of transcription and subsequent protein expression of the unique narK1K2GHJI respiratory operon of Pseudomonas aeruginosa were investigated. Under the control of PLAC, P. aeruginosa was able to transcribe nar and subsequently express methyl viologen-linked nitrate reductase activity under aerobic conditions without nitrate. Modulation of PLAC through the LacI repressor enabled us to assess both transcriptional and posttranslational regulation by oxygen during physiological whole-cell nitrate reduction.

Involvement of NarK1 and NarK2 proteins in transport of nitrate and nitrite in the denitrifying bacterium Pseudomonas aeruginosa PAO1.

Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the DeltanarK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants were found to be severely restricted in nitrate-dependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the DeltanarK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the DeltanarK2::Gm and DeltanarK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions.

The mobA gene is required for assimilatory and respiratory nitrate reduction but not xanthine dehydrogenase activity in Pseudomonas aeruginosa.

The requirement for the mobA gene in key assimilatory and respiratory nitrogen metabolism of Pseudomonas aeruginosa PAO1 was investigated by mutational analysis of PA3030 (mobA; MoCo guanylating enzyme), PA1779 (nasA; assimilatory nitrate reductase), and PA3875 (narG; respiratory nitrate reductase). The mobA mutant was deficient in both assimilatory and respiratory nitrate reductase activities, whereas xanthine dehydrogenase activity remained unaffected. Thus, P. aeruginosa requires both the molybdopterin (MPT) and molybdopterin guanine dinucleotide (MGD) forms of the molybdenum cofactor for a complete spectrum of nitrogen metabolism, and one form cannot substitute for the other. Regulation studies using a Phi(PA3030-lacZGm) reporter strain suggest that expression of mobA is not influenced by the type of nitrogen source or by anaerobiosis, whereas assimilatory nitrate reductase activity was detected only in the presence of nitrate.