Distinct transcriptional response of Caenorhabditis elegans to different exposure routes of perfluorooctane sulfonic acid.

Although people are exposed daily to per- and polyfluorinated alkyl substances (PFASs), the biological consequences are poorly explored. The health risks associated with PFAS exposure are currently based on chemical analysis with a weak correlation to potential harmful effects in man and animals. In this study, we show that perfluorooctane sulfonic acid (PFOS), often the most enriched PFAS in the environment, can be transferred via bacteria to higher organisms such as Caenorhabditis elegans. C. elegans nematodes were exposed to PFOS directly in buffer or by feeding on bacteria pretreated with PFOS, and this led to distinct gene expression profiles. Specifically, heavy metal and heat shock associated genes were significantly, although inversely, expressed following the different PFOS exposures. The innate immunity receptor for microbial pathogens, clec-60, was shown for the first time to be down-regulated by PFOS. This is in line with a previous study indicating that PFOS is associated with children's susceptibility to certain infectious diseases. Furthermore, bar-1, a gene associated with various cancers was highly up-regulated only when C. elegans were exposed to PFOS pretreated live bacteria. Furthermore, dead bacterial biomass had higher binding capacity for linear and isomeric PFOS than live bacteria, which correlated to the higher levels of PFOS detected in C. elegans when fed the treated E. coli, respectively. These results reveal new aspects concerning trophic chain transport of PFOS.

Pseudomonas aeruginosa Regulated Intramembrane Proteolysis: Protease MucP Can Overcome Mutations in the AlgO Periplasmic Protease To Restore Alginate Production in Nonmucoid Revertants.

The progression of cystic fibrosis (CF) from an acute to a chronic disease is often associated with the conversion of the opportunistic pathogen Pseudomonas aeruginosa from a nonmucoid form to a mucoid form in the lung. This conversion involves the constitutive synthesis of the exopolysaccharide alginate, whose production is under the control of the AlgT/U sigma factor. This factor is regulated posttranslationally by an extremely unstable process and has been commonly attributed to mutations in the algT (algU) gene. By exploiting this unstable phenotype, we isolated 34 spontaneous nonmucoid variants arising from the mucoid strain PDO300, a PAO1 derivative containing the mucA22 allele commonly found in mucoid CF isolates. Complementation analysis using a minimal tiling path cosmid library revealed that most of these mutants mapped to two protease-encoding genes, algO, also known as prc or PA3257, and mucP Interestingly, our algO mutations were complemented by both mucP and algO, leading us to delete, clone, and overexpress mucP, algO, mucE, and mucD in both wild-type PAO1 and PDO300 backgrounds to better understand the regulation of this complex regulatory mechanism. Our findings suggest that the regulatory proteases follow two pathways for regulated intramembrane proteolysis (RIP), where both the AlgO/MucP pathway and MucE/AlgW pathway are required in the wild-type strain but where the AlgO/MucP pathway can bypass the MucE/AlgW pathway in mucoid strains with membrane-associated forms of MucA with shortened C termini, such as the MucA22 variant. This work gives us a better understanding of how alginate production is regulated in the clinically important mucoid variants of Pseudomonas aeruginosaIMPORTANCE Infection by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality seen in CF patients. Poor patient prognosis correlates with the genotypic and phenotypic change of the bacteria from a typical nonmucoid to a mucoid form in the CF lung, characterized by the overproduction of alginate. The expression of this exopolysaccharide is under the control an alternate sigma factor, AlgT/U, that is regulated posttranslationally by a series of proteases. A better understanding of this regulatory phenomenon will help in the development of therapies targeting alginate production, ultimately leading to an increase in the length and quality of life for those suffering from CF.

Aerial Oxidation of a V(IV)-Iminopyridine Hydroquinonate Complex: A Trap for the V(IV)-Semiquinonate Radical Intermediate.

The reaction of 2,5-bis[N,N'-bis(2-pyridyl-aminomethyl)aminomethyl]-p-hydroquinone (H2bpymah) with VO(2+) salts in acetonitrile or water at a low pH (2.2-3.5) results in the isolation of [{V(IV)(O)(Cl)}2(µ-bpymah)], the p-semiquinonate complex [{V(IV)(O)(Cl)}2(µ-bpymas)](OH), the cyclic mixed-valent hexanuclear compound [{V(V)(O)(µ-O)V(IV)(O)}(µ-bpymah)]3, and [(V(V)O2)2(µ-bpymah)]. [{V(IV)(O)(Cl)}2(µ-bpymas)](OH) is an intermediate of the radical-mediated oxidation of [{V(IV)(O)(Cl)}2(µ-bpymah)] from O2. At lower pH values (2.2), a reversible intramolecular electron transfer from the metal to the ligand of [{V(IV)(O)(Cl)}2(µ-bpymas)](OH) is induced with the concurrent substitution of chlorine atoms by the oxygen-bridging atoms, resulting in the formation of [{V(V)(O)(µ-O)V(IV)(O)}(µ-bpymah)]3. The metal complexes were fully characterized by X-ray crystallography, infrared (IR) spectroscopy, and magnetic measurements in the solid state, as well as by conductivity measurements, UV-vis spectroscopy, and electrochemical measurements in solution. The oxidation states of the metal ions and ligands were determined by the crystallographic data. The [{V(IV)(O)(Cl)}2(µ-bpymah)]-[{V(IV)(O)(Cl)}2(µ-bpymas)](OH) redox process is electrochemically reversible. The V(IV) ion in the semiquinonate compound exhibits a surprisingly low oxophilicity, resulting in the stabilization of OH(-) counterions at acidic pH values. An investigation of the mechanism of this reaction reveals that these complexes induce the reduction of O2 to H2O2, mimicking the activity of enzymes incorporating two redox-active centers (metal-organic) in the active site.

Antifungal application of nonantifungal drugs.

Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to ∼150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.

High levels of short-chain fatty acids secreted by Candida albicans hyphae induce neutrophil chemotaxis via free fatty acid receptor 2.

Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.

Molybdenum(VI) coordination chemistry of the N,N-disubstituted bis(hydroxylamido)-1,3,5-triazine ligand, H2bihyat. Water-assisted activation of the Mo(VI)═O bond and reversible dimerization of cis-[Mo(VI)O2(bihyat)] to [Mo(VI)2O4(bihyat)2(H2O)2].

Reaction of the N,N-disubstituted bis(hydroxylamino) ligand 2,6-bis[hydroxy(methyl)amino]-4-morpholino-1,3,5-triazine (H(2)bihyat) with cis-[Mo(VI)O(2)(acac)(2)] in tetrahydrofuran resulted in isolation of the mononuclear compound cis-[Mo(VI)O(2)(bihyat)] (1). The treatment of Na(2)Mo(VI)O(4)·2H(2)O with the ligand H(2)bihyat in aqueous solution gave the dinuclear compounds cis-[Mo(VI)(2)O(4)(bihyat)(2)(H(2)O)(2)] (2) and trans-[Mo(VI)(2)O(4)(bihyat)(2)(H(2)O)(2)] (3) at pH values of 3.5 and 5.5, respectively. The structures for the three molybdenum(VI) compounds were determined by X-ray crystallography. Compound 1 has a square-pyramidal arrangement around molybdenum, while in the two dinuclear compounds, each molybdenum atom is in a distorted pentagonal-bipyramidal environment of two bridging and one terminal oxido groups, a tridentate (O,N,O) bihyat(2-) ligand that forms two five-membered chelate rings, and a water molecule trans to the terminal oxido group. The dinuclear compounds constitute rare examples containing the {Mo(2)(VI)O(2)(µ(2)-O(2))}(4+) moiety. The potentiometry revealed that the Mo(VI)bihyat(2-) species exhibit high hydrolytic stability in aqueous solution at a narrow range of pH values, 3-5. A subtle change in the coordination environment of the five-coordinate compound 1 with ligation of a weakly bound water molecule trans to the oxido ligand (1w) renders the equatorial oxido group in 1w more nucleophilic than that in 1, and this oxido group attacks a molybdenum atom and thus the dinuclear compounds 2 and 3 are formed. This process might be considered as the first step of the oxido group nucleophilic attack on organic substrates, resulting in oxidation of the substrate, in the active site of molybdenum enzymes such as xanthine oxidase. Theoretical calculations in the gas phase were performed to examine the influence of water on the dimerization process (1 → 2/3). In addition, the molecular structures, cis/trans geometrical isomerism for the dinuclear molybdenum(VI) species, vibrational spectra, and energetics of the metal-ligand interaction for the three molybdenum(VI) compounds 1-3 have been studied by means of density functional theory calculations.

Per- and polyfluoroalkyl substances enhance Staphylococcus aureus pathogenicity and impair host immune response.

Per- and Poly-fluoroalkyl substances (PFAS) are major persistent environmental contaminants. Epidemiological studies have linked PFAS exposures to altered immunity and increased occurrence of infections in children. However, the mechanisms leading to immune susceptibility to bacterial infections remains unclear. To elucidate the mechanism, transcriptional alteration in the Caenorhabditis elegans model caused by a PFAS contaminated environmental water and two reconstituted PFAS solutions were evaluated using RNA-sequencing. PFAS affected the expression of several genes involved in C. elegans immune surveillance to Gram-positive bacteria (cpr-2, tag-38, spp-1, spp-5, clec-7, clec-172). The combined exposure to PFAS and Staphylococcus aureus significantly reduced C. elegans survival and increased intestinal membrane permeability. Furthermore, the growth of S. aureus in the presence of PFAS increased the expression of virulence genes, specifically, the virulence gene regulator saeR and α-hemolysin, hla, which resulted in increased hemolytic activity. The present study demonstrated that PFAS exposure not only increased C. elegans susceptibility to pathogens by reducing host immunity and increasing intestinal membrane permeability, but also increased bacteria virulence. This presents a broader implication for humans and other animals, where environmental contaminants simultaneously reduce host resilience, while, increasing microbial pathogenicity.

pH-Potentiometric Investigation towards Chelating Tendencies of p-Hydroquinone and Phenol Iminodiacetate Copper(II) Complexes.

Copper ions in the active sites of several proteins/enzymes interact with phenols and quinones, and this interaction is associated to the reactivity of the enzymes. In this study the speciation of the Cu(2+) with iminodiacetic phenolate/hydroquinonate ligands has been examined by pH-potentiometry. The results reveal that the iminodiacetic phenol ligand forms mononuclear complexes with Cu(2+) at acidic and alkaline pHs, and a binuclear O(phenolate)-bridged complex at pH range from 7 to 8.5. The binucleating hydroquinone ligand forms only 2 : 1 metal to ligand complexes in solution. The pK values of the protonation of the phenolate oxygen of the two ligands are reduced about 2 units after complexation with the metal ion and are close to the pK values for the copper-interacting tyrosine phenol oxygen in copper enzymes.

Synthesis, solution, and structural characterization of tetrahydrofuranyl-2,2-bisphosphonic acid disodium salt.

Bisphosphonates are biologically relevant therapeutics for bone disorders and cancer. Reaction of gamma-chlorobutyric acid, phosphorus acid, and phosphorus trichloride without the use of solvent gave the tetrahydrofuranyl-2,2-bisphosphonate sodium salt (Na(2)H(2)L). The Na(2)H(2)L was isolated, characterized in solution by (1)H, (13)C, and (31)P NMR spectroscopy and in solid state by single X-Ray crystallography. The crystal structure showed that the Na(2)H(2)L forms in the crystal infinite two-dimensional sheets stacked one parallel to the other. A comparison of the chelating properties of H(2)L(2-) with similar hydroxyl bisphosphonate ligands shows that the strength of the Na-O(furanyl/hydroxyl) bond is directly related to the total charge of the ligand anion.

Cryptococcus neoformans Induces MCP-1 Release and Delays the Death of Human Mast Cells.

Cryptococcosis, caused by the basidiomycete Cryptococcus neoformans, is a life-threatening disease affecting approximately one million people per year worldwide. Infection can occur when C. neoformans cells are inhaled by immunocompromised people. In order to establish infection, the yeast must bypass recognition and clearance by immune cells guarding the tissue. Using in vitro infections, we characterized the role of mast cells (MCs) in cryptococcosis. We found that MCs recognize C. neoformans and release inflammatory mediators such as tryptase and cytokines. From the latter group MCs released mainly CCL-2/MCP-1, a strong chemoattractant for monocytic cells. We demonstrated that supernatants of infected MCs recruit monocytes but not neutrophils. During infection with C. neoformans, MCs have a limited ability to kill the yeast depending on the serotype. C. neoformans, in turn, modulates the lifespan of MCs both, by presence of its polysaccharide capsule and by secreting soluble modulators. Taken together, MCs might have important contributions to fungal clearance during early stages of cryptocococis where these cells regulate recruitment of monocytes to mucosal tissues.

Stable Redox-Cycling Nitroxide Tempol Has Antifungal and Immune-Modulatory Properties.

Invasive mycoses remain underdiagnosed and difficult to treat. Hospitalized individuals with compromised immunity increase in number and constitute the main risk group for severe fungal infections. Current antifungal therapy is hampered by slow and insensitive diagnostics and frequent toxic side effects of standard antifungal drugs. Identification of new antifungal compounds with high efficacy and low toxicity is therefore urgently required. We investigated the antifungal activity of tempol, a cell-permeable nitroxide. To narrow down possible mode of action we used RNA-seq technology and metabolomics to probe for pathways specifically disrupted in the human fungal pathogen Candida albicans due to tempol administration. We found genes upregulated which are involved in iron homeostasis, mitochondrial stress, steroid synthesis, and amino acid metabolism. In an ex vivo whole blood infection, tempol treatment reduced C. albicans colony forming units and at the same time increased the release of pro-inflammatory cytokines, such as interleukin 8 (IL-8, monocyte chemoattractant protein-1, and macrophage migration inhibitory factor). In a systemic mouse model, tempol was partially protective with a significant reduction of fungal burden in the kidneys of infected animals during infection onset. The results obtained propose tempol as a promising new antifungal compound and open new opportunities for the future development of novel therapies.

Vanadium(V) compounds with the bis-(hydroxylamino)-1,3,5-triazine ligand, H2bihyat: synthetic, structural, and physical studies of [V2(V)O3(bihyat)2] and of the enhanced hydrolytic stability species cis-[V(V)O2(bihyat)]-.

Reaction of the ligand 2,6-bis[hydroxy(methyl)amino]-4-morpholino-1,3,5-triazine (H(2)bihyat) with NaV(V)O(3) in aqueous solution followed by addition of either Ph(4)PCl or C(NH(2))(3)Cl, respectively, gave the mononuclear vanadium(V) compounds Ph(4)P[V(V)O(2)(bihyat)].1.5H(2)O (1) and C(NH(2))(3)[V(V)O(2)(bihyat)] (2). Treatment of V(IV)OSO(4).5H(2)O with the ligand H(2)bihyat in methyl alcohol under specific conditions gave the oxo-bridged dimer [V(V)(2)O(2)(mu(2)-O)(bihyat)(2)] (3). The structures for 1 and 3 were determined by X-ray crystallography and indicate that these compounds have distorted square-pyramidal arrangement around vanadium. The ligand bihyat(2-) is bonded to vanadium atom in a tridentate fashion at the pyridine-like nitrogen atom and the two deprotonated hydroxylamino oxygen atoms. The high electron density of the triazine ring nitrogen atoms, which results from the resonative contribution of electrons of exocyclic nitrogen atoms (Scheme 4 ), leads to very strong V-N bonds. The cis-[V(V)O(2)(bihyat)](-) species exhibits high hydrolytic stability in aqueous solution over a wide pH range, 3.3-11.0, as it was evidenced by (1)H and (51)V NMR spectroscopy and potentiometry. The high affinity of the H(2)bihyat ligand for the V(V)O(2)(+) unit, its tridentate character, as well as its small size, paves the way for potential applications in medicine, analysis, and catalysis for the C(NH(2))(3)[V(V)O(2)(bihyat)] compound. The molecular structures, vibrational and electronic spectra, and the energetics of the metal-ligand interaction for compounds 1 and 3 have been studied by means of density functional calculations.

Evasion of Immune Surveillance in Low Oxygen Environments Enhances Candida albicans Virulence.

Microbial colonizers of humans have evolved to adapt to environmental cues and to sense nutrient availability. Oxygen is a constantly changing environmental parameter in different host tissues and in different types of infection. We describe how Candida albicans, an opportunistic fungal pathogen, can modulate the host response under hypoxia and anoxia. We found that high infiltration of polymorphonuclear leukocytes (PMNs) to the site of infection contributes to a low oxygen milieu in a murine subdermal abscess. A persistent hypoxic environment did not affect viability or metabolism of PMNs. Under oxygen deprivation, however, infection with C. albicans disturbed specific PMN responses. PMNs were not able to efficiently phagocytose, produce ROS, or release extracellular DNA traps. Failure to launch an adequate response was caused by C. albicans cell wall masking of ß-glucan upon exposure to low oxygen levels which hindered PAMP sensing by Dectin-1 on the surfaces of PMNs. This in turn contributed to immune evasion and enhanced fungal survival. The cell wall masking effect is prolonged by the accumulation of lactate produced by PMNs under low oxygen conditions. Finally, adaptation to oxygen deprivation increased virulence of C. albicans which we demonstrated using a Caenorhabditis elegans infection model.IMPORTANCE Successful human colonizers have evolved mechanisms to bypass immune surveillance. Infiltration of PMNs to the site of infection led to the generation of a low oxygen niche. Exposure to low oxygen levels induced fungal cell wall masking, which in turn hindered pathogen sensing and antifungal responses by PMNs. The cell wall masking effect was prolonged by increasing lactate amounts produced by neutrophil metabolism under oxygen deprivation. In an invertebrate infection model, C. albicans was able to kill infected C. elegans nematodes within 2 days under low oxygen conditions, whereas the majority of uninfected controls and infected worms under normoxic conditions survived. These results suggest that C. albicans benefited from low oxygen niches to increase virulence. The interplay of C. albicans with innate immune cells under these conditions contributed to the overall outcome of infection. Adaption to low oxygen levels was in addition beneficial for C. albicans by reducing susceptibility to selected antifungal drugs. Hence, immunomodulation of host cells under low oxygen conditions could provide a valuable approach to improve current antifungal therapies.

Assessment of Neutrophil Chemotaxis Upon G-CSF Treatment of Healthy Stem Cell Donors and in Allogeneic Transplant Recipients.

Neutrophils are crucial for the human innate immunity and constitute the majority of leukocytes in circulation. Thus, blood neutrophil counts serve as a measure for the immune system's functionality. Hematological patients often have low neutrophil counts due to disease or chemotherapy. To increase neutrophil counts and thereby preventing infections in high-risk patients, recombinant G-CSF is widely used as adjunct therapy to stimulate the maturation of neutrophils. In addition, G-CSF is utilized to recruit stem cells (SCs) into the peripheral blood of SC donors. Still, the actual functionality of neutrophils resulting from G-CSF treatment remains insufficiently understood. We tested the ex vivo functionality of neutrophils isolated from blood of G-CSF-treated healthy SC donors. We quantified chemotaxis, oxidative burst, and phagocytosis before and after treatment and detected significantly reduced chemotactic activity upon G-CSF treatment. Similarly, in vitro treatment of previously untreated neutrophils with G-CSF led to reduced chemotactic activity. In addition, we revealed that this effect persists in the allogeneic SC recipients up to 4 weeks after neutrophil engraftment. Our data indicates that neutrophil quantity, as a sole measure of immunocompetence in high-risk patients should be considered cautiously as neutrophil functionality might be affected by the primary treatment.

Investigation of Phenols Activity in Early Stage Oxidation of Edible Oils by Electron Paramagnetic Resonance and 19F NMR Spectroscopies Using Novel Lipid Vanadium Complexes As Radical Initiators.

A novel dynamic method for the investigation of the phenols activity in early stage oxidation of edible oils based on the formation of α-tocopheryl radicals initiated by oil-soluble vanadium complexes is developed. Two new vanadium complexes in oxidation states V and IV were synthesized by reacting 2,2'-((2-hydroxyoctadecyl)azanediyl)bis(ethan-1-ol) (C18DEA) with [VO(acac)2] and 1-(bis(pyridin-2-ylmethyl)amino)octadecan-2-ol (C18DPA) with VOCl2. Addition of a solution of either complex in edible oils resulted in the formation of α-tocopheryl radical, which was monitored by electron paramagnetic resonance (EPR) spectroscopy. The intensity of the α-tocopheryl signal in the EPR spectra was measured versus time. It was found that the profile of the intensity of the α-tocopheryl signal versus time depends on the type of oil, the phenolic content, and the storage time of the oil. The time interval until the occurrence of maximum peak intensity be reached (tm), the height of the maximum intensity, and the rate of the quenching of the α-tocopheryl radical were used for the investigation of the mechanism of the edible oils oxidation. 19F NMR of the 19F labeled phenolic compounds (through trifluoroacetate esters) and radical trap experiments showed that the vanadium complexes in edible oil activate the one electron reduction of dioxygen to superperoxide radical. Superperoxide reacts with the lipids to form alkoperoxyl and alkoxyl lipid radicals, and all these radicals react with the phenols contained in oils.

Phenol-Soluble Modulin α Peptide Toxins from Aggressive Staphylococcus aureus Induce Rapid Formation of Neutrophil Extracellular Traps through a Reactive Oxygen Species-Independent Pathway.

Neutrophils have the ability to capture and kill microbes extracellularly through the formation of neutrophil extracellular traps (NETs). These are DNA and protein structures that neutrophils release extracellularly and are believed to function as a defense mechanism against microbes. The classic NET formation process, triggered by, e.g., bacteria, fungi, or by direct stimulation of protein kinase C through phorbol myristate acetate, is an active process that takes several hours and relies on the production of reactive oxygen species (ROS) that are further modified by myeloperoxidase (MPO). We show here that NET-like structures can also be formed by neutrophils after interaction with phenol-soluble modulin α (PSMα) that are cytotoxic membrane-disturbing peptides, secreted from community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA). The PSMα-induced NETs contained the typical protein markers and were able to capture microbes. The PSMα-induced NET structures were disintegrated upon prolonged exposure to DNase-positive S. aureus but not on exposure to DNase-negative Candida albicans. Opposed to classic NETosis, PSMα-triggered NET formation occurred very rapidly, independently of ROS or MPO, and was also manifest at 4°C. These data indicate that rapid NETs release may result from cytotoxic membrane disturbance by PSMα peptides, a process that may be of importance for CA-MRSA virulence.

Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells.

Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal, colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast cell responses included the launch of initial, intermediate and late phase components determined by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans infection. Consequently, these findings open new doors for understanding fungal pathogenicity.

Novel high-throughput screening method for identification of fungal dimorphism blockers.

Invasive mycoses have been increasing worldwide, with Candida spp. being the most prevalent fungal pathogen causing high morbidity and mortality in immunocompromised individuals. Only few antimycotics exist, often with severe side effects. Therefore, new antifungal drugs are urgently needed. Because the identification of antifungal compounds depends on fast and reliable assays, we present a new approach based on high-throughput image analysis to define cell morphology. Candida albicans and other fungi of the Candida clade switch between different growth morphologies, from budding yeast to filamentous hyphae. Yeasts are considered proliferative, whereas hyphae are required for invasion and dissemination. Thus, morphotype switching in many Candida spp. is connected to virulence and pathogenesis. It is, consequently, reasonable to presume that morphotype blockers interfere with the virulence, thereby preventing hazardous colonization. Our method efficiently differentiates yeast from hyphal cells using a combination of automated microscopy and image analysis. We selected the parameters length/width ratio and mean object shape to quantitatively discriminate yeasts and hyphae. Notably, Z' factor calculations for these parameters confirmed the suitability of our method for high-throughput screening. As a second stage, we determined cell viability to discriminate morphotype-switching inhibitors from those that are fungicidal. Thus, our method serves as a basis for the identification of candidates for next-generation antimycotics.

Synthesis, structure, magnetic properties and aqueous solution characterization of p-hydroquinone and phenol iminodiacetate copper(II) complexes.

The reaction of Cu2+ acetate monohydrate with 2-[N,N'-bis(carboxymethyl)aminomethyl]-4-carboxyphenol (H4cacp), 2-[N,N-bis(carboxymethyl)aminomethyl]hydroquinone (H4cah) and the dinucleating 2,5-bis[N,N-bis(carboxymethyl)aminomethyl]hydroquinone (H6bicah) in water results in the formation of several Cu2+ species, which are in dynamic equilibrium in aqueous solution and their stability is pH dependent. A systematic crystallographic study of these species was pursued, resulting in the characterization of most of the species. Additional techniques were employed to characterize the molecules in the solid state (infrared spectroscopy) and in solution (UV-vis spectroscopy and electrochemistry). These measurements show that the Cu2+ ions are ligated mainly to the iminodiacetate at pH < 6, exhibiting only weak interactions with the phenol oxygen. At pH > 6, the phenol oxygen was deprotonated and dinuclear-bridged species, from the phenolate oxygen complexes exhibiting a Cu2+ 2O2 core, were isolated. The coordination environment around the copper ions varies between trigonal bipyramidal, tetragonal pyramidal and distorted octahedral geometries. The two unpaired electrons of the Cu2+ ions are found to be antiferromagnetically coupled. A survey of the magnetic and structural properties of the dinuclear phenoxide bridged Cu2+ complexes shows that the strength of the antiferromagnetic coupling is linearly dependent on the Cu-Ophenolate bond lengths, at bond distances below 1.98 angstroms. The effect of the Cu-O-Cu angles on the magnetic properties of the complexes is also discussed.

Polynuclear cobalt(II/III) sulfites: synthesis, structure, and magnetic properties of the octanuclear cluster (NH4)11(Li subset[Co4IICo4III(SO3)16(NH3)8]).10H2O encapsulating a lithium cation.

Partial oxidation of an aqueous solution of CoIICl(2).6H2O with (NH4)6[Mo7VIO24].4H2O in the presence of (NH4)2SO3.H2O and LiCl, at pH approximately 5.3, leads to isolation of the octanuclear cluster (NH4)11(Li subset[Co4IICo4III(SO3)16(NH3)8].10H2O), 1. The structure of the anion of 1 consists of a central [Co4II], almost ideal square planar unit, and a pair of symmetry-related CoIII dimers above and below the Co4II plane grafting onto the tetramer by 16 bridging sulfite groups. The [Co8(SO3)16(NH3)8]12- cluster encapsulates a lithium cation which lies at the center of the Co4II square.