Possible explanations for different surface quality in laser cutting with 1 micron and 10 microns beams

In laser　cutting　of　thick　steel　sheets,　quality　difference　is　observed　between　cut　surfaces　obtained　with 1 micron and 10 micron laser beams. This paper investigates physical mechanisms for this interesting and important problem of the wavelength dependence. First, striation generation process is described, based on a 3D structure of melt flow on a kerf front, which was revealed for the first time by our recent experimental observations. Two fundamental processes are suggested to explain the difference in the cut surface quality: destabilization of the melt flow in the central part of the kerf front and downward displacement of discrete melt accumulations along the side parts of the front. Then each of the processes is analyzed using a simplified analytical model. The results show that in both processes, different angular dependence of the absorptivity of the laser beam can result in the quality difference. Finally we propose use of radial polarization to improve the quality with the 1 micron wavelength

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

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

Breeding for grapevine downy mildew resistance via gene editing

Downy mildew (DM) caused by the oomycete Plasmopara viticola ranks in the top diseases affecting grapevine (Vitis vinifera L.) cultivation and its control requires every year a large use of fungicides. The Farm to Fork strategy newly promoted by the EU aims to accelerate the transition to a sustainable food system and has set very ambitious targets including the reduction by 50% of the use and risk of pesticides by 2030. The introduction of disease-tolerant grapevine varieties or clones clearly represents a step forward to reach this goal. The recent advent of new breeding tools such as genome editing and cis-genesis offers a great opportunity to obtain resistant plants with higher precision and speed than by conventional breeding, either by knocking down susceptibility genes or by introducing known resistance-genes in commercial cultivars. Based on reports in other crops, the family of Downy Mildew Resistant 6 (DMR6) and DMR6-like oxygenases (DLOs) are candidate susceptibility genes for the control of DM resistance in V. vinifera. Deep-sequencing the putative susceptibility genes in 190 genetically diverse grapevine genotypes identified several Single Nucleotide Polymorphisms then screened for their impact on protein structure/function and association with DM resistant genotypes. Gene expression and gene network analysis suggested that grapevine DMR6 and DLO genes have distinct functions, and that VviDMR6-1 is co-regulated with several Pathogenesis-related genes. Based on this evidence, we generated a large collection of DMR6-1 and DMR6-2 single and double knock-out mutants in multiple grapevine cultivars and evaluated their resistance to DM. Phenotypic resistance data upon artificial infection are being collected and will be presented here. In parallel, we also developed a new DNA-free gene editing methodology and obtained non-transgenic and non-chimeric edited grapevine plants regenerated from a single cell

A DNA-free editing approach for viticulture sustainability: dual editing of DMR6-1 and DMR6-2 enhances resistance to downy mildew

The sustainability of viticulture hinges on maintaining quality and yield while reducing pesticide use. Promising strides in this direction involve the development of clones with enhanced disease tolerance, particularly through the knockout of plant susceptibility genes. Knocking out of Downy Mildew Resistant 6 (DMR6) led to increased levels of endogenous salicylic acid (SA), a regulator of immunity, resulting in enhanced tolerance to Downy Mildew (DM) and other diseases in various crops. Mutations in both DMR6-1 and DMR6-2 genes were introduced into two grapevine cultivars using CRISPR-Cas9 using two methods. In the first case, transgene delivery mediated by A. tumefaciens was employed, while in the second case, we developed a 'single-cell technology' for gene editing, creating non-transgenic grapevine mutants through the regeneration of protoplasts previously edited with the CRISPR/Cas9 ribonucleoprotein. We tested the susceptibility of single and double mutants to DM through artificial inoculation assays on detached leaves and whole plants. Our findings indicate that a simultaneous mutation in both DMR6-1 and DMR6-2 is needed to significantly enhance resistance to DM, with the double mutant (dmr6-1-dmr6-2) outperforming either single mutant in both cultivars. Elevated levels of endogenous SA were only observed in the double mutant, while single mutation in DMR6-1 or DMR6-2 proved ineffective. Collectively, our data highlight the need for a double knockout to achieve appreciable results against DM-susceptibility. Currenlty, we are adapting the 'single-cell technology' to generate edited vines from various agronomically relevant cultivars. In parallel, we are assessing the performance of plants edited in different susceptibility genes

The effect of minimum quantity lubrication in the intermittent turning of magnesium based on vibration signals

The present work shows an experimental investigation on intermittent turning based on vibration signals. The dependence of vibrations on the feed rate, minimum quantity lubrication (MQL) flow rate and the type of the interruption of the workpiece is evaluated. The results indicate that a part of the vibrations depends on the flow rate of the MQL system and its interaction with the feed rate, finding no dependency on the type of interruption. The influence of the MQL system is greater when machining at the lower feed rate. In addition, a strong relation between surface roughness and vibrations is identified. However, this relation is quite different depending on the environment used. In general, under dry conditions, the higher the vibrations the higher the surface roughness, while the opposite occurs when the MQL system is used.publishe

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

Telegram, 1932, Sidney, NY, to Amelia Earhart Putnam, London

Congratulatory telegram sent to Amelia Earhart from Scintilla Magneto Co., Inc., for Earhart's solo Atlantic flight, 193