Structure-Based Design and Optimization of AIF/CypA peptide inhibitors with neuroprotective activity

In this research work, we described the importance of peptides as a therapeutic approach for the resolution of many diseases. In particular, we focused our attention on the lethal role of complex protein AIF/CypA, involved in neuronal cell death. The aim of this project was the structure-based design and optimization of AIF/CypA peptide inhibitors, using NMR studies and combinatorial chemistry. The peptides were designed from AIF(370-394) peptide, mimetic of amino acidic region 370-394 of AIF protein, able to block in vitro the proteins interaction, through its bond to CypA and able to induce neuroprotection. The identified peptides will be used to for the treatment of neurodegenerative disorders. Moreover a second project was focused on development of a simple and homogenous fluorescent HTS assays for the discovery of CypA cis-trans isomerase activity inhibitors, using a new FRET-based substrate probe useful for Chymotrypsin-coupled isomerase assays. For this purpose, we have designed a new fluorescent peptide substrate, useful to the use of in order to have a high proportion of cis conformers and to work by following fluorescence intensity increase or decrease, depending on enzyme activation or inhibition. The assay is helpful to screening set of large compound libraries

Structural and biochemical insights of CypA and AIF interaction

The Cyclophilin A (CypA)/Apoptosis Inducing Factor (AIF) complex is implicated in the DNA degradation in response to various cellular stress conditions, such as oxidative stress, cerebral hypoxia-ischemia and traumatic brain injury. The pro-apoptotic form of AIF (AIF(Δ1-121)) mainly interacts with CypA through the amino acid region 370-394. The AIF(370-394) synthetic peptide inhibits complex formation in vitro by binding to CypA and exerts neuroprotection in a model of glutamate-mediated oxidative stress. Here, the binding site of AIF(Δ1-121) and AIF(370-394) on CypA has been mapped by NMR spectroscopy and biochemical studies, and a molecular model of the complex has been proposed. We show that AIF(370-394) interacts with CypA on the same surface recognized by AIF(Δ1-121) protein and that the region is very close to the CypA catalytic pocket. Such region partially overlaps with the binding site of cyclosporin A (CsA), the strongest catalytic inhibitor of CypA. Our data point toward distinct CypA structural determinants governing the inhibitor selectivity and the differential biological effects of AIF and CsA, and provide new structural insights for designing CypA/AIF selective inhibitors with therapeutic relevance in neurodegenerative diseases

Target-site mutations and expression of als gene copies vary according to Echinochloa species

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target- site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance

Design, Optimization, and Structural Characterization of an Apoptosis-Inducing Factor Peptide Targeting Human Cyclophilin A to Inhibit Apoptosis Inducing Factor-Mediated Cell Death

Blocking the interaction between the apoptosis-inducing factor (AIF) and cyclophilin A (CypA) by the AIF fragment AIF(370-394) is protective against glutamate-induced neuronal cell death and brain injury in mice. Starting from AIF(370-394), we report the generation of the disulfide-bridged and shorter variant AIF(381-389) and its structural characterization by nuclear magnetic resonance (NMR) in the free and CypA-bound state. AIF(381-389) in both the free and bound states assumes a β-hairpin conformation similar to that of the fragment in the AIF protein and shows a highly reduced conformational flexibility. This peptide displays a similar in vitro affinity for CypA, an improved antiapoptotic activity in cells and an enhanced proteolytic stability compared to the parent peptide. The NMR-based 3D model of the AIF(381-389)/CypA complex provides a better understanding of the binding hot spots on both the peptide and the protein and can be exploited to design AIF/CypA inhibitors with improved pharmacokinetic and pharmacodynamics features

137Cs and 40K in Cortinarius caperatus mushrooms (1996–2016) in Poland - Bioconcentration and estimated intake: 137Cs in Cortinarius spp. from the Northern Hemisphere from 1974 to 2016

Cortinarius caperatus grows in the northern regions of Europe, North America and Asia and is widely collected by mushroom foragers across Europe. This study shows that in the last three decades since the Chernobyl nuclear accident, C. caperatus collected across much of Northern Poland exhibited high activity concentrations of radiocaesium (137Cs) - a long-lived radionuclide. The mushroom appears to efficiently bioconcentrate 137Cs from contaminated soil substrata followed by sequestration into its morphological parts such as the cap and stipe which are used as food. The gradual leaching of 137Cs into the lower strata of surface soils in exposed areas are likely to facilitate higher bioavailability to the mycelia of this species which penetrate to relatively greater depths and may account for the continuing high activity levels noticed in Polish samples (e.g. activity within caps in some locations was still at 11,000 Bq kg−1 dw in 2008 relative to a peak of 18,000 in 2002). The associated dietary intake levels of 137Cs have often exceeded the tolerance limits set by the European Union (370 and 600 Bq kg−1 ww for children and adults respectively) during the years 1996–2010. Human dietary exposure to 137Cs is influenced by the method of food preparation and may be mitigated by blanching followed by disposal of the water, rather than direct consumption after stir-frying or stewing. It may be prudent to provide precautionary advice and monitor activity levels, as this mushroom continues to be foraged by casual as well as experienced mushroom hunters

In margine all'edizione degli Annales Caesenates

Viene presentata, dal punto di vista storico e filologico, la nuova edizione, dopo quella settecentesca muratoriana, di Enrico Angiolini degli "Annales Caesenates", una fonte narrativa importantissima del Trecento romagnolo

Muzio Attendolo da Cotignola, capostipite degli Sforza

Biografia di uno dei pi\uf9 importanti capitani di ventura del Quattrocento; sono anche lumeggiati i suoi rapporti coi vari poteri politici da cui dipese, il costituirsi di un suo "esercito" personale, le modalit\ue0 della guerra in quell'epoc