Simone Scintilla

Greater concentrations of hydrosulfide lead to the prebiotic formation of higher nuclearity Fe–S peptides, culminating in a putative nitrogenase-like [6Fe–9S] cluster. Higher nuclearity clusters are more stable with lower reduction potential.

The production and cultivation of vines ( Vitis vinifera ) tolerant or resistant to diseases such as downy mildew (DM) is a promising strategy to reduce fungicides and help viticulture sustainability. In many crops, generation of knock-out mutants in host genes controlling susceptibility to DM, such as Downy Mildew Resistant 6 ( DMR6 ) is a strategy of proven success to obtain resistant plants, while the effect of mutations in DMR6 genes has yet to be demonstrated in grapevine. In addition, small mutations in genes governing important traits can be obtained by gene-editing while maintaining the genetic background of commercially important clones. Moreover, very recent advances in the technology of gene-editing allowed to produce non-transgenic grapevine mutants, by regeneration of protoplasts previously edited with the CRISPR-Cas9 ribonucleoprotein. This approach may revolutionize the production of new grapevine varieties and clones, but it requires knowledge on the targets, and an extensive evaluation of the impact of their mutation on plant phenotype and fitness. In this work we generated single and double knock-out mutants in DMR6 susceptibility (S) genes in multiple grapevine cultivars with improved resistance to DM.

CRISPR-Cas technology has widely extended the application fields of genome editing in plant breeding, making possible specific and minimal mutations within a genetic pool. With respect to standard genome editing technologies, CRISPR-Cas machinery can be introduced in the form of ribonucleoproteins (RNPs), thus avoiding the introduction of exogenous DNA into cells. The interest on the application of DNA-free delivery into plant cells is constantly increasing, especially in the case of valuable woody plants elite varieties where CRISPR-Cas9 technology would preserve their genotype, while still resulting into targeted genetic modifications. The use of single cells fits well the requirements of New Breeding Technologies, by ensuring both the CRISPR-Cas DNA-free delivery as RNPs and, since every plant will be regenerated from a single edited cell, the absence of chimerism. However, the use of protoplasts cell culture from woody plants is generally hampered by low editing efficiencies and an unsuccessful regenerative process. We here describe a successful DNA-free methodology to obtain fully edited grapevine plants, regenerated from protoplasts obtained from V. vinifera cv. Crimson seedless L. embryogenic callus. The transfected protoplasts were edited on the Downy Mildew susceptibility gene VvDMR6-2. The regenerated edited plants exhibited homozygous deletions of 1bp or 2bp, and homozygous insertion of 1bp.

Iron–sulfur clusters are ancient cofactors that play a fundamental role in metabolism and may have impacted the prebiotic chemistry that led to life. However, it is unclear whether iron–sulfur clusters could have been synthesized on prebiotic Earth. Dissolved iron on early Earth was predominantly in the reduced ferrous state, but ferrous ions alone cannot form polynuclear iron–sulfur clusters. Similarly, free sulfide may not have been readily available. Here we show that UV light drives the synthesis of [2Fe–2S] and [4Fe–4S] clusters through the photooxidation of ferrous ions and the photolysis of organic thiols. Iron–sulfur clusters coordinate to and are stabilized by a wide range of cysteine-containing peptides and the assembly of iron–sulfur cluster-peptide complexes can take place within model protocells in a process that parallels extant pathways. Our experiments suggest that iron–sulfur clusters may have formed easily on early Earth, facilitating the emergence of an iron–sulfur-cluster-dependent metabolism.

Based on UV-Vis, NMR, and EPR spectroscopies and DFT and molecular dynamics calculations, a model prebiotic [2Fe–2S] tripeptide was shown to accept and donate electrons.

Cysteine containing dipeptide metal affinities do not follow the Irving–Williams series but rather follow the concentration trends found in seawater.

Four novel neutral copper(I) compounds were prepared, based on the chelating phosphine bis(2-(diphenylphosphino)phenyl)ether, (DPEPhos), and a series of nitrogen-containing heterocycles as ancillary ligands. The optical properties can be tuned according to the ancillary ligand and their emissions at room temperature in aprotic solvent (CH₂Cl₂) go from greenish blue to orange. Their photoluminescence in solid state is particularly attractive and make their employment in opto-electronic devices very appealing. In particular, the low-cost preparation method of solution-processed light emitting devices is suitable for copper(I) complexes. In order to choose a suitable device architecture, the electrochemical characteristics were investigated and the frontier orbitals values were determined. Preliminary results on solution-processed OLED are presented.

The current work investigates the effect of new bifunctional and mononuclear Pt(II) compounds, the cis- and trans-isomers of [PtCl₂(NH₃)(L)] (L = 1-methyl-7-azaindole, compounds 1 and 2, respectively), on growth and viability of human carcinoma cells as well as their putative mechanism(s) of cytotoxicity. The results show that substitution of 1-methyl-7-azaindole for ammine in cisplatin or transplatin results in an increase of the toxic efficiency, selectivity for tumor cells in cisplatin-resistant cancer cells, and activation of the trans geometry. The differences in the cytotoxic activities of 1 and 2 were suggested to be due to their different DNA binding mode, different capability to induce cell cycle perturbations, and fundamentally different role of transcription factor p53 in their mechanism of action. Interestingly, both isomers make it possible to detect their cellular uptake and distribution in living cells by confocal microscopy without their modification with an optically active tag.

The cellular uptake of cisplatin and of other platinum-based drugs is mediated by the high-affinity copper transporter Ctr1. The eight-residue long peptide called Mets7 (MTGMKGMS) mimics one of extracellular methionine (Met)-rich motifs of Ctr1. It is an excellent model for investigating the interaction of platinum drugs with Ctr1 under in vitro and in vivo conditions. Some of us have shown that (i) Cisplatin loses all of its ligands upon reaction with Mets7 and the metal ion binds to the three Met residues and completes its coordination shell with a fourth ligand that can be a chloride or a water/hydroxyl oxygen. (ii) Transplatin loses only the chlorido ligands, which are replaced by Met residues. Here, we provide information on the structural determinants of cisplatin/Mets7 and transplatin/Mets7 adducts by computational methods. The predictions are validated against EXAFS, NMR, and CD spectra. While EXAFS gives information restricted to the metal coordination shell, NMR provides information extended to residue atoms around the coordination shell, and finally, CD provides information about the overall conformation of the peptide. This allows us to elucidate the different reaction modes of cisplatin and transplatin toward the peptide, as well as to propose the platinated peptides [PtX]⁺–(M*TGM*KGM*S) (X = Cl^–, OH^–) and trans[Pt(NH₃)₂]²⁺–(M*TGM*KGMS) as the most relevant species occurring in water solution.