Eradication of Candida albicans persister cell biofilm by the membranotropic peptide gH625

Biofilm formation poses an important clinical trouble due to resistance to antimicrobial agents; therefore, there is an urgent demand for new antibiofilm strategies that focus on the use of alternative compounds also in combination with conventional drugs. Drug-tolerant persisters are present in Candida albicans biofilms and are detected following treatment with high doses of amphotericin B. In this study, persisters were found in biofilms treated with amphotericin B of two clinical isolate strains, and were capable to form a new biofilm in situ. We investigated the possibility of eradicating persister-derived biofilms from these two Candida albicans strains, using the peptide gH625 analogue (gH625-M). Confocal microscopy studies allowed us to characterize the persister-derived biofilm and understand the mechanism of interaction of gH625-M with the biofilm. These findings confirm that persisters may be responsible for Candida biofilm survival, and prove that gH625-M was very effective in eradicating persister-derived biofilms both alone and in combination with conventional antifungals, mainly strengthening the antibiofilm activity of fluconazole and 5-flucytosine. Our strategy advances our insights into the development of effective antibiofilm therapeutic approaches

Review of a viral peptide nanosystem for intracellular delivery

The internalization of bioactive molecules is one of the most critical problems to overcome in theranostics. In order to improve pharmacokinetic and pharmacodynamic proper- ties, synthetic transporters are widely investigated. A new nanotechnological transporter, gH625, is based on a viral peptide sequence derived from the herpes simplex virus type 1 glycoprotein H (gH) that has proved to be a useful delivery vehicle, due to its intrinsic properties of inducing membrane perturbation. The peptide functionalization with several kinds of nanoparticles like quantum dots, dendrimers, and liposomes could be of particular interest in biomedical applica- tions to improve drug release within cells, to increase site-specific action, and eventually to reduce related cytotoxicity. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI. (DOI: 10.1117/1.JNP.7.071599

GENETICS AND ENVIRONMENT: IMPACT ON MAMMALIAN SPERMATOZOA

Aim of this doctorate has been to explore the links between environment, genetics and characteristics of spermatozoa in mammals and in particular three species have been studied: humans, dogs and horses. This study was in fact conceived following the collaboration of two important research areas: human reproduction and the genetic improvement of livestock species. We started from the premise of wanting to fill gap of information concerning semen quality of men living in the high environmental pressure area of the "Land of Fires" (LF), and to investigate the potential association withenvironmental pollutants, such as cadmium (Cd). Specific aims included the assessment of the potential epigenetic changes induced by heavy metals exposure, particularly Cd, and to correlate these changes to semen quality and spermatozoa charachteristics (in humans and dogs). The study included two different cohorts of subjects comprising 730 (C1) and 512 (C2) participants belonging to the LF. All subjects in C1 were offered a diagnostic clinical examination in their domestic dogs. The dogs recruited (N=30) for the study had to be clinically healthy and with at least one litter in the last 12 months. In C2 subjects a strict correlation was demonstrated between seminal parameters, particularly sperm total count, and Cd burden in semen samples, testifying a potential harmful effect on reproductive health in humans. Moreover, reduced SDGM was associated to reduced semen quality, demonstrating an overall similar relationship between epigenetic changes and seminal parameters in men and their dogs. This is the first report demonstrating a correlation between SGDM percentage and conventional seminal parameters in dogs; this epigenetic finding could be of considerable interest also in the zootechnical field, due to the possible reproductive and economic repercussions. Lastly, a further specific aim was to assess the effects of healthy lifestyle, particularly the supplementation with nutraceuticals, on semen quality, in order to identify natural substances as alternative treatments for seminal impairment in stallions. Four stallions were food supplemented with maca (Lepidium meyenii) during the breeding season. Maca food supplementation in stallions during breeding season reduced the percentage of spermatozoa with fragmented DNA, significantly increased sperm concentration and lengthened spermatozoa head, suggesting that food supplementation of maca could be useful in horse breeding

Septic Shock by Gram-Negative Infections: Role of Outer Membrane Proteins

The magnitude of septic shock as a clinical problem is often understated. Despite advances in our ability to diagnose and treat infectious diseases, severe sepsis leading to shock due to gram-negative infections remains one of the leading causes of mortality worldwide. Septic shock develops because of a disregulation in the host response, and the mechanisms initially recruited to fight infection produce life-threatening tissue damage and death. Recent research has witnessed a significant increase in our understanding of host-pathogen interactions, particularly in the area of innate immunity and the molecular recognition of gram-positive and gram-negative bacteria. Important new mediators of sepsis and novel mechanisms of host-cell toxicity have been identified and, together with clinical trials targeting pathways considered central to sepsis pathogenesis, provide new insight into the molecular and cellular basis of sepsis for the formulation of new strategies of intervention. Research on septic shock pathogenesis by gram-negative bacteria is mainly focused on the understanding of the molecular and cellular role played by lipopolysaccharide (LPS). Strong experimental evidence and clinical observations suggest that the release of proinflammatory cytokine mediators by LPS-responsive cells (mainly macrophages, endothelial cells and neutrophils) in response to toxic products sets in motion the genetic and physiologic program that manifests as shock. The best characterized of these toxic components is LPS, which is considered as a paradigm for other less well-characterized toxic microbial molecules. The immune protection stimulated by highly purified LPS in animals does not resolve the symptomatology of septic shock, while LPS mixed to outer membrane proteins shows a better protective activity. Several studies evidence the major role played by outer membrane proteins in the molecular interaction between the host cell and the gram-negative bacteria. Endotoxin-associated proteins consist of a complex of several major proteins that are intimately associated with the LPS. Very little is known about release of non-LPS gramnegative outer membrane components such as OMPs in sepsis. Among the OMPs, porins have been shown to play an important role in pathogenesis of bacterial infections. Porins were pyrogenic in rabbits and elicited a localized reaction when used as the sensiting and eliciting agent. Porins were also shown to kill D-galactosamine sensitized LPS-responsive and LPS-unresponsive mice. Treatment of Human Umbilical Vein Endothelial Cells: (HUVEC) with porins increased the transmigration of different leukocyte populations, inparticular of neutrophils. Porins by several gram-negative bacteria induce cytokine release by human leukocytes as well as enhancement of cytokine gene expression. Also, other components of the bacterial envelope are important in the induction and pathogenesis of septic shock such as bacterial lipoproteins (LP). As anti-LPS therapies does not seem to improve by the addition of proteins from the outer membrane or small fragments of these proteins, a great alternative to existing strategies will involve the blockage of signal transduction pathways, cytokine and inflammatory mechanisms

Viral fusion peptides induce several signal transduction pathway activations that are essential for interleukin-10 and beta-interferon production

Objectives: The deciphering of intracellular signaling pathways that are activated by the interaction between viral fusion peptides and cellular membranes are important for the understanding of both viral replication strategies and host defense mechanisms. Methods: Fusion peptides of several enveloped viruses belonging to different virus families were prepared by standard 9-fluorenylmethoxycarbonyl polyamine solid-phase synthesis and used to stimulate U937 cells in vitro to analyze the phosphorylation patterns of the signaling pathways (PKC, Src, Akt, and MAPK pathways). Immunoprecipitation and Western blotting were carried out by using phosphospecific antibodies. All samples were also assayed for the presence of IL-10 and IFN-beta by ELISA and activation of nuclear factors (AP-1 and NF-kappa B). Results: We have demonstrated that hydrophobic domains of fusion proteins are able to induce several transduction pathways that lead to cytokine (IFN-beta and IL-10) production, an event that appears to be dependent on early activation of AP-1 and NF-kappa B. Conclusions: The results obtained on the signaling activity of fusion peptides from different viruses enabled us to shed some light on the complex mechanism of viral entry and more precisely we focused on the exact signaling event induced by hydrophobic domains characteristic of fusion peptides interacting with the cell membrane. Copyright (C) 2010 S. Karger AG, Base

Public Private Partnership in Italian Health Care Management An Organizational Maturity Assessment Model

This work aims to analyze a specific phenomenon of innovation in health management: public private partnerships within the Italian healthcare sector. The object of the study is to measure the degree of organizational maturity (OM) of the forms of public-private partnerships (PPP) analyzing and measuring key managerial processes, in terms of innovation in meeting the partnership‘s goals/targets. The analysis is based on the identification of key processes relevant to the management of partnerships, to check which systems of governance are able to meet different stakeholder interests. We therefore built a conceptual standard for analysis of the OM through a field survey based on visits, participant observation, analysis of documents and semi-structured interviews with the management

Fusogenic domains in herpes simplex virus type 1 glycoprotein H.

Infection of eukaryotic cells by enveloped viruses requires fusion between the viral envelope and the cellular plasma or endosomal membrane. The actual merging of the two membranes is mediated by viral envelope glycoproteins, which generally contain a highly hydrophobic region termed the fusion peptide. The entry of herpesviruses is mediated by three conserved proteins: glycoproteins B, H (gH), and L. However, how fusion is executed remains unknown. Herpes simplex virus type 1 gH exhibits features typical of viral fusion glycoproteins, and its ectodomain seems to contain a putative internal fusion peptide. Here, we have identified additional internal segments able to interact with membranes and to induce membrane fusion of large unilamellar vesicles. We have applied the hydrophobicity-at-interface scale proposed by Wimley and White (Wimley, W. C., and White, S. H. (1996) Nat. Struct. Biol. 3, 842-848) to identify six hydrophobic stretches within gH with a tendency to partition into the membrane interface, and four of them were able to induce membrane fusion. Experiments in which equimolar mixtures of gH peptides were used indicated that different fusogenic regions may act in a synergistic way. The functional and structural characterization of these segments suggests that herpes simplex virus type 1 gH possesses several fusogenic internal peptides that could participate in the actual fusion event