Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation,Oxidative and Nitrosative Stress

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO^•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.     

Manufacture of stable palladium and gold nanoparticles on native and genetically engineered flagella scaffolds

The use of bacterial flagella as templates for the immobilization of Pd and Au nanoparticles is described. Complete coverage of D. desulfuricans flagellar filaments by Pd(0) nanoparticles was obtained via the H(2)-mediated reduction of Pd(NH3)4]Cl2 but similar results were not obtained using HAuCl4. The introduction of additional cysteine-derived thiol residues in the E. coli FliC protein increased Au(III) sorption and reduction onto the surface of the flagellar filament and resulted in the production of stabilized Au(0) nanoparticles of approximately 20-50 nm diameter. We demonstrate the application of molecular engineering techniques to manufacture biologically passivated Au(0) nanoparticles of a size suitable for catalytic applications.     

Peptidomics of Cpefat/fat mouse brain regions: implications for neuropeptide processing

Quantitative peptidomics was used to compare levels of peptides in wild type (WT) and Cpe^fat/fat mice, which lack carboxypeptidase E (CPE) activity because of a point mutation. Six different brain regions were analyzed: amygdala, hippocampus, hypothalamus, prefrontal cortex, striatum, and thalamus. Altogether, 111 neuropeptides or other peptides derived from secretory pathway proteins were identified in WT mouse brain extracts by tandem mass spectrometry, and another 47 peptides were tentatively identified based on mass and other criteria. Most secretory pathway peptides were much lower in Cpe^fat/fat mouse brain, relative to WT mouse brain, indicating that CPE plays a major role in their biosynthesis. Other peptides were only partially reduced in the Cpe^fat/fat mice, indicating that another enzyme (presumably carboxypeptidase D) contributes to their biosynthesis. Approximately 10% of the secretory pathway peptides were present in the Cpe^fat/fat mouse brain at levels similar to those in WT mouse brain. Many peptides were greatly elevated in the Cpe^fat/fat mice; these peptide processing intermediates with C‐terminal Lys and/or Arg were generally not detectable in WT mice. Taken together, these results indicate that CPE contributes, either directly or indirectly, to the production of the majority of neuropeptides.     

NCAM induces CaMKIIalpha-mediated RPTPalpha phosphorylation to enhance its catalytic activity and neurite outgrowth

Receptor protein tyrosine phosphatase α (RPTPα) phosphatase activity is required for intracellular signaling cascades that are activated in motile cells and growing neurites. Little is known, however, about mechanisms that coordinate RPTPα activity with cell behavior. We show that clustering of neural cell adhesion molecule (NCAM) at the cell surface is coupled to an increase in serine phosphorylation and phosphatase activity of RPTPα. NCAM associates with T- and L-type voltage-dependent Ca²⁺ channels, and NCAM clustering at the cell surface results in Ca²⁺ influx via these channels and activation of NCAM-associated calmodulin-dependent protein kinase IIα (CaMKIIα). Clustering of NCAM promotes its redistribution to lipid rafts and the formation of a NCAM–RPTPα–CaMKIIα complex, resulting in serine phosphorylation of RPTPα by CaMKIIα. Overexpression of RPTPα with mutated Ser180 and Ser204 interferes with NCAM-induced neurite outgrowth, which indicates that neurite extension depends on NCAM-induced up-regulation of RPTPα activity. Thus, we reveal a novel function for a cell adhesion molecule in coordination of cell behavior with intracellular phosphatase activity.     

Autophagy in organelle homeostasis: peroxisome turnover

When cells are confronted with an insufficient supply of nutrients in their extracellular fluid, they may begin to cannibalize some of their internal proteins as well as whole organelles for reuse in the synthesis of new components. This process is termed autophagy and it involves the formation of a double-membrane structure within the cell, which encloses the material to be degraded into a vesicle called an autophagosome. The autophagosome subsequently fuses with a lysosome/vacuole whose hydrolytic enzymes degrade the sequestered organelle. Degradation of peroxisomes is a specific type of autophagy, which occurs in a selective manner and has been mostly studied in yeast. Recently, it was reported that a similar selective process of autophagy occurs in mammalian cells with proliferated peroxisomes. Here we discuss characteristics of the autophagy of peroxisomes in mammalian cells and present a comprehensive model of their likely mechanism of degradation on the basis of known and common elements from other systems.     

Real-time optical detection of methicillin-resistant Staphylococcus aureus using lytic phage probes

Staphylococcus aureus (S. aureus)-specific bacteriophage was used as a probe for detection of methicillin-resistant S. aureus (MRSA) in aqueous solution using a novel optical method. Biorecognition phage monolayers transferred to glass substrates using Langmuir-Blodgett (LB) technique were exposed individually to MRSA in solution at logarithmic concentrations ranging from 10(6) to 10(9)cfu/ml, and observed for real-time binding using a CytoVivatrade mark optical light microscope system. Results indicate that LB monolayers possessed high levels of elasticity (K), measuring 22 and 29mN/m for 10(9) and 10(11)pfu/ml phage concentrations, respectively. Near-instantaneous MRSA-phage binding produced 33+/-5%, 10+/-1%, 1.1+/-0.1%, and 0.09+/-0.01% coverage of the substrate that directly correlated to a decrease in MRSA concentrations of 10(9), 10(8), 10(7), and 10(6)cfu/ml. The exclusive selectivity of phage monolayers was verified with Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Bacillus subtilis.     

Contribution of inducible and neuronal nitric oxide synthases to mitochondrial damage and melatonin rescue in LPS-treated mice

NOS isoform activation is related to liver failure during sepsis, but the mechanisms driving mitochondrial impairment remain unclear. We induced sepsis by LPS administration to inducible nitric oxide synthase (iNOS^?/?) and neuronal nitric oxide synthase (nNOS^?/?) mice and their respective wild-type controls to examine the contribution of iNOS to mitochondrial failure in the absence of nNOS. To achieve this goal, the determination of messenger RNA (mRNA) expression and protein content of iNOS in cytosol and mitochondria, the mitochondrial respiratory complex content, and the levels of nitrosative and oxidative stress (by measuring 3-nitrotyrosine residues and carbonyl groups, respectively) were examined in the liver of control and septic mice. We detected strongly elevated iNOS mRNA expression and protein levels in liver cytosol and mitochondria of septic mice, which were related to enhanced oxidative and nitrosative stress, and with fewer changes in respiratory complexes. The absence of the iNOS, but not nNOS, gene absolutely prevented mitochondrial impairment during sepsis. Moreover, the nNOS gene did not modify the expression and the effects of iNOS here shown. Melatonin administration counteracted iNOS activation and mitochondrial damage and enhanced the expression of the respiratory complexes above the control values. These effects were unrelated to the presence or absence of nNOS. iNOS is a main target to prevent liver mitochondrial impairment during sepsis, and melatonin represents an efficient antagonist of these iNOS-dependent effects whereas it may boost mitochondrial respiration to enhance liver survival.     

Interleukin-6, Age,and Corpus Callosum Integrity

The contribution of inflammation to deleterious aging outcomes is increasingly recognized; however, little is known about the complex relationship between interleukin-6 (IL-6) and brain structure, or how this association might change with increasing age. We examined the association between IL-6, white matter integrity, and cognition in 151 community dwelling older adults, and tested whether age moderated these associations. Blood levels of IL-6 and vascular risk (e.g., homocysteine), as well as health history information, were collected. Processing speed assessments were administered to assess cognitive functioning, and we employed tract-based spatial statistics to examine whole brain white matter and regions of interest. Given the association between inflammation, vascular risk, and corpus callosum (CC) integrity, fractional anisotropy (FA) of the genu, body, and splenium represented our primary dependent variables. Whole brain analysis revealed an inverse association between IL-6 and CC fractional anisotropy. Subsequent ROI linear regression and ridge regression analyses indicated that the magnitude of this effect increased with age; thus, older individuals with higher IL-6 levels displayed lower white matter integrity. Finally, higher IL-6 levels were related to worse processing speed; this association was moderated by age, and was not fully accounted for by CC volume. This study highlights that at older ages, the association between higher IL-6 levels and lower white matter integrity is more pronounced; furthermore, it underscores the important, albeit burgeoning role of inflammatory processes in cognitive aging trajectories.