Herpes Simplex Virus ICP27 Protein Inhibits AIM 2-Dependent Inflammasome Influencing Pro-Inflammatory Cytokines Release in Human Pigment Epithelial Cells (hTert-RPE 1).

Although Herpes simplex virus type 1 (HSV-1) has been deeply studied, significant gaps remain in the fundamental understanding of HSV-host interactions: our work focused on studying the Infected Cell Protein 27 (ICP27) as an inhibitor of the Absent-in-melanoma-2 (AIM 2) inflammasome pathway, leading to reduced pro-inflammatory cytokines that influence the activation of a protective innate immune response to infection. To assess the inhibition of the inflammasome by the ICP27, hTert-immortalized Retinal Pigment Epithelial cells (hTert-RPE 1) infected with HSV-1 wild type were compared to HSV-1 lacking functional ICP27 (HSV-1∆ICP27) infected cells. The activation of the inflammasome by HSV-1∆ICP27 was demonstrated by quantifying the gene and protein expression of the inflammasome constituents using real-time PCR and Western blot. The detection of the cleavage of the pro-caspase-1 into the active form was performed by using a bioluminescent assay, while the quantification of interleukins 1ß (IL-1ß) and 18 (IL-18)released in the supernatant was quantified using an ELISA assay. The data showed that the presence of the ICP27 expressed by HSV-1 induces, in contrast to HSV-1∆ICP27 vector, a significant downregulation of AIM 2 inflammasome constituent proteins and, consequently, the release of pro-inflammatory interleukins into the extracellular environment reducing an effective response in counteracting infection.

Suppressing gain-of-function proteins via CRISPR/Cas9 system in SCA1 cells.

SCAs are autosomal dominant neurodegenerative disorders caused by a gain-of-function protein with toxic activities, containing an expanded polyQ tract in the coding region. There are no treatments available to delay the onset, stop or slow down the progression of these pathologies. In this work we focus our attention on SCA1 which is one of the most common genotypes circulating in Italy. Here, we develop a CRISPR/Cas9-based approach to reduce both forms of the ATXN1 protein, normal and mutated with expanded polyQ. We started with the screening of 10 different sgRNAs able to target Exon 8 of the ATXN1 gene. The two most promising sgRNAs were validated in fibroblasts isolated from SCA1 patients, following the identification of the best transfection method for this type of cell. Our silencing approach significantly downregulated the expression of ataxin1, due to large deletions and the introduction of small changes in the ATXN1 gene, evidenced by NGS analysis, without major effects on cell viability. Furthermore, very few significant guide RNA-dependent off-target effects were observed. These preliminary results not only allowed us to identify the best transfection method for SCA1 fibroblasts, but strongly support CRISPR/Cas9 as a promising approach for the treatment of expanded polyQ diseases. Further investigations will be needed to verify the efficacy of our silencing system in SCA1 neurons and animal models.

Control of Erwinia amylovora Growth by Moringa oleifera Leaf Extracts: In Vitro and in Planta Effects.

Erwinia amylovora (EA) is a phytopathogenic bacterium, the causative agent of bacterial fire blight, a disease that affects Rosaceaes. In order to replace antibiotics and copper, the antimicrobial activity of three extracts of Moringa oleifera Lam., methanolic (MeOH-MOE), hydroalcoholic (HA-MOE) and hydroalcoholic with maltodextrins (HAMD-MOE), was tested on eleven strains of EA isolated from apple trees by the Emilia-Romagna Phytosanitary Department. MIC and MBC have been evaluated; biofilm formation, swarming motility and amylovoran production were performed with the crystalviolet, soft-agar assay and the amylovoran method. All extracts demonstrated bacteriostatic activity at a concentration of 1 mg/mL, resulting in a 80% reduction in biofilm formation. HAMD-MOE, MeOH-MOE and HA-MOE caused an inhibition of motility of 60%, 65% and 30% after 6 days and a decrease in amylovoran synthesis of 84%, 63% and 93%, respectively. In planta results showed how the compounds were able to inhibit EA virulence on apple trees, mainly if they were applied as a preventive treatment, although the treatment showed a significant reduction in fire blight symptoms progression. The antibacterial activity of the extracts is mainly due to the high concentration of polyphenolic compounds detected in the extracts that was able to alter the permeability of bacterial membrane, resulting in slowing the synthesis of ATP and consequently of all ATP-dependent functions, such as motility and less selectivity towards harmful compounds, which can, thus, enter the cytoplasm and inhibit enzymes involved in replication and quorum sensing. The efficacy, eco-compatibility and low cost make such extracts a potential tool for the control of bacterial fire blight.

Effects of Moringa oleifera Leaf Extracts on Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a Gram-negative bacterium belonging to the Xanthomonodaceae family, causing black rot in crucifers. To control this pathogen, the study investigated the effect of different leaves extracts of Moringa oleifera Lam., a tropical plant, well known for its food properties and with countless applications in many different fields, from nutraceutical (hypoglycemic) to the cosmetic (sunscreen) properties. Nevertheless, several studies pointed to its antibacterial action against both Gram-negative and Gram-positive bacteria. Many bioactive compounds, including flavonoids, phenolic acids, alkaloids, isothiocyanates, tannins and saponins, contained in these extracts, are responsible for its countless activities. The analyses carried out in this study show that the methanolic, hydroalcoholic and hydroalcoholic maltodextrin extracts have both bacteriostatic and bactericidal effects at concentrations of 0.5, 0.5 and 0.1 mg/mL respectively. In particular, the study shows how all extracts can alter membrane permeability, to adversely affect swarming motility, and to alter biofilm formation in Xcc. The in planta experiments showed a reduction of the necrosis area in the infected radishes, although the ability of the extracts to be absorbed by root systems is yet to be understood, in order to reach the target point.

UPF1 silenced cellular model systems for screening of read-through agents active on ß039 thalassemia point mutation.

BACKGROUND: Nonsense mutations promote premature translational termination, introducing stop codons within the coding region of mRNAs and causing inherited diseases, including thalassemia. For instance, in ß039 thalassemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, ribosomal read-through molecules, such as aminoglycoside antibiotics, have been tested on mRNAs carrying premature stop codons. These findings have introduced new hopes for the development of a pharmacological approach to the ß039 thalassemia therapy. While several strategies, designed to enhance translational read-through, have been reported to inhibit NMD efficiency concomitantly, experimental tools for systematic analysis of mammalian NMD inhibition by translational read-through are lacking. RESULTS: We developed a human cellular model of the ß039 thalassemia mutation with UPF-1 suppressed and showing a partial NMD suppression. CONCLUSIONS: This novel cellular model could be used for the screening of molecules exhibiting preferential read-through activity allowing a great rescue of the mutated transcripts.