Regeneration of non-chimeric plants from DNA-free edited grapevine protoplasts

The application of New Breeding Techniques (NBTs) in Vitis vinifera is highly desirable to introduce valuable traits while preserving the genotype of the elite cultivars. However, a broad application of NBTs through standard DNA-based transformation is poorly accepted by public opinion and law regulations in Europe and other countries due to the stable integration of exogenous DNA, which leads to transgenic plants possibly affected by chimerism. A single-cell based approach, coupled with a DNA-free transfection of the CRISPR/Cas editing machinery, constitutes a powerful tool to overcome these problems and maintain the original genetic make-up in the whole organism. We here describe a successful single-cell based, DNA-free methodology to obtain edited grapevine plants, regenerated from protoplasts isolated from embryogenic callus of two table grapevine varieties (V. vinifera cv. Crimson seedless and Sugraone). The regenerated, non-chimeric plants were edited on the downy- and powdery-mildew susceptibility genes, VviDMR6 and VviMlo6 respectively, either as single or double mutant

UV-light-driven prebiotic synthesis of iron–sulfur clusters

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

Simultaneous editing of two DMR6 genes in grapevine results in reduced susceptibility to downy mildew

The reduction of pesticides’ treatments is of paramount importance for the sustainability of viticulture, and it can be achieved by a combination of strategies including the cultivation of vines (Vitis vinifera) that are resistant or tolerant to diseases such as downy mildew (DM). In many crops, the knock-out of Downy Mildew Resistant 6 (DMR6) proved successful in controlling DM-resistance, but the effect of mutations in DMR6 genes in not yet known in grapevine.Today, gene editing serves crop improvement with small and specific mutations while maintaining the genetic background of commercially important clones. Moreover, recent technological advances allowed to produce non-transgenic grapevine clones by regeneration of protoplasts 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 on the impact of editing on plant phenotype and fitness in different cultivars. In this work we generated single and double knock-out mutants by editing DMR6 susceptibility (S) genes using CRISPR/Cas9, and showed that only the combined mutations in VviDMR6-1 and VviDMR6-2 are effective in reducing susceptibility to DM in two table-grape cultivars by increasing the levels of endogenous salicylic acid. Therefore, editing both genes may be necessary for effective DM control in real-world agricultural settings, which could potentially lead to unwanted phenotypes. Additional research, including trials conducted in experimental vineyards, is required to gain a deeper understanding of DMR6-based resistance

Copper-Triggered Aggregation of Ubiquitin

Neurodegenerative disorders share common features comprising aggregation of misfolded proteins, failure of the ubiquitin-proteasome system, and increased levels of metal ions in the brain. Protein aggregates within affected cells often contain ubiquitin, however no report has focused on the aggregation propensity of this protein. Recently it was shown that copper, differently from zinc, nickel, aluminum, or cadmium, compromises ubiquitin stability and binds to the N-terminus with 0.1 micromolar affinity. This paper addresses the role of copper upon ubiquitin aggregation. In water, incubation with Cu(II) leads to formation of spherical particles that can progress from dimers to larger conglomerates. These spherical oligomers are SDS-resistant and are destroyed upon Cu(II) chelation or reduction to Cu(I). In water/trifluoroethanol (80∶20, v/v), a mimic of the local decrease in dielectric constant experienced in proximity to a membrane surface, ubiquitin incubation with Cu(II) causes time-dependent changes in circular dichroism and Fourier-transform infrared spectra, indicative of increasing β-sheet content. Analysis by atomic force and transmission electron microscopy reveals, in the given order, formation of spherical particles consistent with the size of early oligomers detected by gel electrophoresis, clustering of these particles in straight and curved chains, formation of ring structures, growth of trigonal branches from the rings, coalescence of the trigonal branched structures in a network. Notably, none of these ubiquitin aggregates was positive to tests for amyloid and Cu(II) chelation or reduction produced aggregate disassembly. The early formed Cu(II)-stabilized spherical oligomers, when reconstituted in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes and in POPC planar bilayers, form annular and pore-like structures, respectively, which are common to several neurodegenerative disorders including Parkinson's, Alzheimer's, amyotrophic lateral sclerosis, and prion diseases, and have been proposed to be the primary toxic species. Susceptibility to aggregation of ubiquitin, as it emerges from the present study, may represent a potential risk factor for disease onset or progression while cells attempt to tag and process toxic substrates

Ru(III) anticancer agents with aromatic and non-aromatic dithiocarbamates as ligands: Loading into nanocarriers and preliminary biological studies

Since the discovery of cisplatin in the 1960s, other metal complexes have been investigated as potential antitumor agents to overcome the side-effects associated with the administration of the Pt-based drug. In line with our previous research, in this work we report the synthesis and characterization of mono- and dinuclear Ru(III) complexes with the pyrrolidinedithiocarbamate (PDT) ligand and the more sterically-hindered carbazole-dithiocarbamato ligand (CDT), to compare their properties (both at the chemical and antiproliferative level), in an attempt to assess a structure-activity rationale. Moreover, to overcome the scarce solubility under physiological conditions of the Ru(III)-dithiocarbamato compounds, the biocompatible copolymer Pluronic\uae F127 has been used, to encapsulate the metal derivatives in water-soluble micellar carriers. Finally, preliminary biological evaluations on CDT and PDT compounds along with their nanoformulations, open intriguing perspectives in anticancer chemotherapy. In particular, comparing the structure of the Ru(III) derivatives, the ionic dinuclear PDT complex shows an important cytotoxic action in comparison to its neutral counterparts. Moreover, the micellar carrier improves the overall activity of the encapsulated Ru(III)-PDT chemotherapeutics. On the other hand, the nanoformulation of the CDT derivatives allows us to solubilize both the 1:3 and the 2:5 complexes and to state their inactivity

Structural Determinants of Cisplatin and Transplatin Binding to the Met-Rich Motif of Ctr1: A Computational Spectroscopy Approach

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

Methods to identify and characterize iron-sulfur oligopeptides in water

Iron-sulfur clusters are ubiquitous cofactors that mediate central biological processes. However, despite their long history, these metallocofactors remain challenging to investigate when coordinated to small (≤ six amino acids) oligopeptides in aqueous solution. In addition to being often unstable in vitro, iron-sulfur clusters can be found in a wide variety of forms with varied characteristics, which makes it difficult to easily discern what is in solution. This difficulty is compounded by the dynamics of iron-sulfur peptides, which frequently coordinate multiple types of clusters simultaneously. To aid investigations of such complex samples, a summary of data from multiple techniques used to characterize both iron-sulfur proteins and peptides is provided. Although not all spectroscopic techniques are equally insightful, it is possible to use several, readily available methods to gain insight into the complex composition of aqueous solutions of iron-sulfur peptides.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Structural Determinants of Cisplatin and Transplatin Binding to the Met-Rich Motif of Ctr1: A Computational Spectroscopy Approach

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

Duplications of an iron-sulphur tripeptide leads to the formation of a protoferredoxin

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. Duplications of the tripeptide sequence led to a protoferredoxin with increased stability. Duplications of primitive peptides may have contributed to the formation of contemporary ferredoxins

Novel Antitumor Cisplatin and Transplatin Derivatives Containing 1‑Methyl-7-Azaindole: Synthesis, Characterization, and Cellular Responses

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 <b>1</b> and <b>2</b>, 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 <b>1</b> and <b>2</b> 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