Combinations of β-lactam or aminoglycoside antibiotics with plectasin are synergistic against methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as β-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with β-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with β-lactams (penicillin, amoxicillin or flucloxacillin) in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD

Development of a versatile laboratory experiment to teach the metabolic transformation of hydrolysis

In this paper we describe an easy, reliable, versatile and inexpensive laboratory experiment to teach the metabolic transformation of hydrolysis to Pharmacy students. The experiment does not require the sacrifice of any experimental animal, or any work with organs or tissues, and so can be implemented in a typical university chemistry laboratory. We used acetylsalicylic acid (ASA), hexyl salicylate (HS) and two enzymes, a lipase and an esterase. Since both ASS and HS liberate salicylic acid (SA) upon hydrolysis, students can evaluate the different enzymatic transformations by monitoring the amount of SA liberated. The learning outcomes are an enhanced student understanding of: (1) the process of hydrolysis; (2) the application of enzymatic transformations of molecules from food to xenobiotics; (3) the differences between the general specificity of substrate of both enzymes; (4) the concepts of the lipophilic pocket; (5) the catalytic triad and its regioselectivity in relation to the ester bond. A questionnaire was administered to participating students at three points in time: at the beginning of the module, after enzymatic hydrolysis was taught in class, and after the laboratory experiment. From an analysis of the questionnaire data we conclude that this practical helped Pharmacy students to understand these concepts

A threading receptor for polysaccharides

Cellulose, chitin and related polysaccharides are key renewable sources of organic molecules and materials. However, poor solubility tends to hamper their exploitation. Synthetic receptors could aid dissolution provided they are capable of cooperative action, for example by multiple threading on a single polysaccharide molecule. Here we report a synthetic receptor designed to form threaded complexes (polypseudorotaxanes) with these natural polymers. The receptor binds fragments of the polysaccharides in aqueous solution with high affinities (Ka up to 19,000 M−1), and is shown—by nuclear Overhauser effect spectroscopy—to adopt the threading geometry. Evidence from induced circular dichroism and atomic force microscopy implies that the receptor also forms polypseudorotaxanes with cellulose and its polycationic analogue chitosan. The results hold promise for polysaccharide solubilization under mild conditions, as well as for new approaches to the design of biologically active molecules

Genomic Binding Profiling of the Fission Yeast Stress-Activated MAPK Sty1 and the bZIP Transcriptional Activator Atf1 in Response to H2O2

10.1371/journal.pone.0011620PLoS ONE57

Warfarin therapy and incidence of cerebrovascular complications in left-sided native valve endocarditis

International audienceAnticoagulant therapy has been anticipated to increase the risk of cerebrovascular complications (CVC) in native valve endocarditis (NVE). This study investigates the relationship between ongoing oral anticoagulant therapy and the incidence of symptomatic CVC in left-sided NVE. In a prospective cohort study, the CVC incidence was compared between NVE patients with and without ongoing warfarin. Among 587 NVE episodes, 48 (8%) occurred in patients on warfarin. A symptomatic CVC was seen in 144 (25%) patients, with only three on warfarin. CVC were significantly less frequent in patients on warfarin (6% vs. 26%, odds ratio [OR] 0.20, 95% confidence interval [CI] 0.06-0.6,  = 0.006). No increase in haemorrhagic lesions was detected in patients on warfarin. aetiology (adjusted OR [aOR] 6.3, 95% CI 3.8-10.4) and vegetation length (aOR 1.04, 96% CI 1.01-1.07) were risk factors for CVC, while warfarin on admission (aOR 0.26, 95% CI 0.07-0.94), history of congestive heart failure (adjusted OR 0.22, 95% CI 0.1-0.52) and previous endocarditis (aOR 0.1, 95% CI 0.01-0.79) correlated with lower CVC frequency

Unraveling incompatibility between wheat and the fungal pathogen Zymoseptoria tritici through apoplastic proteomics

Background: Hemibiotrophic fungal pathogen Zymoseptoria tritici causes severe foliar disease in wheat. However, current knowledge of molecular mechanisms involved in plant resistance to Z. tritici and Z. tritici virulence factors is far from being complete. The present work investigated the proteome of leaf apoplastic fluid with emphasis on both host wheat and Z. tritici during the compatible and incompatible interactions. Results: The proteomics analysis revealed rapid host responses to the biotrophic growth, including enhanced carbohydrate metabolism, apoplastic defenses and stress, and cell wall reinforcement, might contribute to resistance. Compatibility between the host and the pathogen was associated with inactivated plant apoplastic responses as well as fungal defenses to oxidative stress and perturbation of plant cell wall during the initial biotrophic stage, followed by the strong induction of plant defenses during the necrotrophic stage. To study the role of anti-oxidative stress in Z. tritici pathogenicity in depth, a YAP1 transcription factor regulating antioxidant expression was deleted and showed the contribution to anti-oxidative stress in Z. tritici ,but was not required for pathogenicity. This result suggests the functional redundancy of antioxidants in the fungus. Conclusions: The data demonstrate that incompatibility is probably resulted from the proteome-level activation of host apoplastic defenses as well as fungal incapability to adapt to stress and interfere with host cell at the biotrophic stage of the interaction

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design

Identification of molecular pathways affected by pterostilbene, a natural dimethylether analog of resveratrol

<p>Abstract</p> <p>Background</p> <p>Pterostilbene, a naturally occurring phenolic compound produced by agronomically important plant genera such as <it>Vitis </it>and <it>Vacciunium</it>, is a phytoalexin exhibiting potent antifungal activity. Additionally, recent studies have demonstrated several important pharmacological properties associated with pterostilbene. Despite this, a systematic study of the effects of pterostilbene on eukaryotic cells at the molecular level has not been previously reported. Thus, the aim of the present study was to identify the cellular pathways affected by pterostilbene by performing transcript profiling studies, employing the model yeast <it>Saccharomyces cerevisiae</it>.</p> <p>Methods</p> <p><it>S. cerevisiae </it>strain S288C was exposed to pterostilbene at the IC₅₀concentration (70 μM) for one generation (3 h). Transcript profiling experiments were performed on three biological replicate samples using the Affymetrix GeneChip Yeast Genome S98 Array. The data were analyzed using the statistical methods available in the GeneSifter microarray data analysis system. To validate the results, eleven differentially expressed genes were further examined by quantitative real-time RT-PCR, and <it>S. cerevisiae </it>mutant strains with deletions in these genes were analyzed for altered sensitivity to pterostilbene.</p> <p>Results</p> <p>Transcript profiling studies revealed that pterostilbene exposure significantly down-regulated the expression of genes involved in methionine metabolism, while the expression of genes involved in mitochondrial functions, drug detoxification, and transcription factor activity were significantly up-regulated. Additional analyses revealed that a large number of genes involved in lipid metabolism were also affected by pterostilbene treatment.</p> <p>Conclusion</p> <p>Using transcript profiling, we have identified the cellular pathways targeted by pterostilbene, an analog of resveratrol. The observed response in lipid metabolism genes is consistent with its known hypolipidemic properties, and the induction of mitochondrial genes is consistent with its demonstrated role in apoptosis in human cancer cell lines. Furthermore, our data show that pterostilbene has a significant effect on methionine metabolism, a previously unreported effect for this compound.</p