Synthetic prions with novel strain-specified properties

Prions are infectious proteins that possess multiple self-propagating structures. The information for strains and structural specific barriers appears to be contained exclusively in the folding of the pathological isoform, PrP(Sc). Many recent studies determined that de novo prion strains could be generated in vitro from the structural conversion of recombinant (rec) prion protein (PrP) into amyloidal structures. Our aim was to elucidate the conformational diversity of pathological recPrP amyloids and their biological activities, as well as to gain novel insights in characterizing molecular events involved in mammalian prion conversion and propagation. To this end we generated infectious materials that possess different conformational structures. Our methodology for the prion conversion of recPrP required only purified rec full-length mouse (Mo) PrP and common chemicals. Neither infected brain extracts nor amplified PrP(Sc) were used. Following two different in vitro protocols recMoPrP converted to amyloid fibrils without any seeding factor. Mouse hypothalamic GT1 and neuroblastoma N2a cell lines were infected with these amyloid preparations as fast screening methodology to characterize the infectious materials. Remarkably, a large number of amyloid preparations were able to induce the conformational change of endogenous PrPC to harbor several distinctive proteinase-resistant PrP forms. One such preparation was characterized in vivo habouring a synthetic prion with novel strain specified neuropathological and biochemical properties

Antifungal activity of the frog skin peptide Temporin G and its effect on Candida albicans virulence factors

The increasing resistance to conventional antifungal drugs is a widespread concern, and a search for new compounds, active against different species of fungi, is demanded. Antimicrobial peptides (AMPs) hold promises in this context. Here we investigated the activity of the frog skin AMP Temporin G (TG) against a panel of fungal strains, by following the Clinical and Laboratory Standards Institute protocols. TG resulted to be active against (i) Candida species and Cryptococcus neoformans, with MIC50 between 4 μM and 64 μM after 24 h of incubation; (ii) dermatophytes with MIC80 ranging from 4 to 32 μM, and (iii) Aspergillus strains with MIC80 of 128 μM. In addition, our tests revealed that TG reduced the metabolic activity of Candida albicans cells, with moderate membrane perturbation, as proven by XTT and Sytox Green assays, respectively. Furthermore, TG was found to be effective against some C. albicans virulence factors; indeed, at 64 μM it was able to inhibit ~90% of yeast–mycelial switching, strongly prevented biofilm formation, and led to a 50% reduction of metabolic activity in mature biofilm cells, and ~30–35% eradication of mature biofilm biomass. Even though further studies are needed to deepen our knowledge of the mechanisms of TG antifungal activity, our results suggest this AMP as an attractive lead compound for treatment of fungal diseases

Antifungal Activity of the Frog Skin Peptide Temporin G and Its Effect on Candida albicans Virulence Factors

The increasing resistance to conventional antifungal drugs is a widespread concern, and a search for new compounds, active against different species of fungi, is demanded. Antimicrobial peptides (AMPs) hold promises in this context. Here we investigated the activity of the frog skin AMP Temporin G (TG) against a panel of fungal strains, by following the Clinical and Laboratory Standards Institute protocols. TG resulted to be active against (i) Candida species and Cryptococcus neoformans, with MIC50 between 4 µM and 64 µM after 24 h of incubation; (ii) dermatophytes with MIC80 ranging from 4 to 32 µM, and (iii) Aspergillus strains with MIC80 of 128 µM. In addition, our tests revealed that TG reduced the metabolic activity of Candida albicans cells, with moderate membrane perturbation, as proven by XTT and Sytox Green assays, respectively. Furthermore, TG was found to be effective against some C. albicans virulence factors; indeed, at 64 µM it was able to inhibit ~90% of yeast–mycelial switching, strongly prevented biofilm formation, and led to a 50% reduction of metabolic activity in mature biofilm cells, and ~30–35% eradication of mature biofilm biomass. Even though further studies are needed to deepen our knowledge of the mechanisms of TG antifungal activity, our results suggest this AMP as an attractive lead compound for treatment of fungal diseases.</jats:p

Distinct disease phenotypes produced by a de novo generated synthetic prion strain: Conformational instability before adaptation

International audienc

The Inhibition of DNA Viruses by the Amphibian Antimicrobial Peptide Temporin G: A Virological Study Addressing HSV-1 and JPCyV

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV.</jats:p

AFM characterization of amyloid fibrils.

<p>Atomic Force Microscopy (AFM) imaging analysis was performed at the end of the fibrillization reactions after 72 hours (amyloids #4, #19, #28, #32). AFM scan topographical images of prion protein (PrP) deposited on mica surface, large-scale images (<b>A</b>). AFM height profiles along the numbered lines in topographical images. The profile reflects the lines as numbered in the images. Higher resolution scan images belonging to the area are marked by a white dashed square in part A (<b>B</b>). Three-dimensional representation of AFM topography images and height distribution data obtained from the AFM images in part B (<b>C</b>).</p

The inhibition of DNA viruses by the amphibian antimicrobial peptide temporin G. A virological study addressing HSV-1 and JPCyV

Herpes simplex virus type-1 (HSV-1) and John Cunningham polyomavirus (JCPyV) are widely distributed DNA viruses causing mainly asymptomatic infection, but also mild to very severe diseases, especially when these viruses reach the brain. Some drugs have been developed to inhibit HSV-1 replication in host cells, but their prolonged use may induce resistance phenomena. In contrast, to date, there is no cure for JCPyV. The search for alternative drugs that can reduce viral infections without undermining the host cell is moving toward antimicrobial peptides (AMPs) of natural occurrence. These include amphibian AMPs belonging to the temporin family. Herein, we focus on temporin G (TG), showing that it strongly affects HSV-1 replication by acting either during the earliest stages of its life cycle or directly on the virion. Computational studies have revealed the ability of TG to interact with HSV-1 glycoprotein B. We also found that TG reduced JCPyV infection, probably affecting both the earliest phases of its life cycle and the viral particle, likely through an interaction with the viral capsid protein VP1. Overall, our results are promising for the development of short naturally occurring peptides as antiviral agents used to counteract diseases related to HSV-1 and JCPyV

Detection of PMCA-#4 in brain and blood of infected animals and related immunohistochemical/biochemical characterization.

<p>Neuropathological analysis of PMCA-#4 and PMCA-PBS injected animals and comparison to that of RML infected mice. Animals injected with PMCA-#4 showed widespread deposition of PrP^Res in the hippocampus with focal plaque-like deposits found in the cerebral cortex. Cerebellum is completely spared by PrP^Res accumulation. RML injected animals showed the typical pattern of widespread, synaptic and diffuse PrP^Res accumulation in the whole brain with major involvement of thalamus and hippocampus. Spongiform changes were mainly found in the hippocampus of PMCA-#4 injected animals. Few vacuoles were detected in the cerebral cortex, while cerebellum did not show any vacuolation. RML injected animals showed severe vacuolation in the thalamus and hippocampus. Mild alterations were found in cerebellum and septum. Brain of PMCA-PBS injected animal was used as control (A). Higher magnification of the red square in panel A (see asterisk) and Thioflavin-S staining showing the lack of amyloid properties of the deposits found in the submeningeal level of the cerebral cortex (B). PMCA of blood collected at 140 day post infection (d.p.i.) from a symptomatic animal injected with PMCA-#4. RML dilutions (10⁻¹⁰ and 10⁻¹¹) were used in PMCA to estimate the concentration of circulating infectious PrP (C). Biochemical analysis of the brains harvested from the first two animals injected with PMCA-#4 (sacrificed at terminal stage of the disease) were performed and compared to that of RML injected mice (D). Scale bar in A is 10 μm; scale bar in B is 5 μm. Western blots were performed using 6D11 monoclonal antibody to PrP (0.2 μg/mL, Covance). Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized using a G:BOX Chemi Syngene system.</p

Different height of clusters of recMoPrP (23–231) aggregates.

Different height of clusters of recMoPrP (23–231) aggregates.</p