Expansion of EasyClone-MarkerFree toolkit for Saccharomyces cerevisiae genome with new integration sites.

Biotechnological production requires genetically stable recombinant strains. To ensure genomic stability, recombinant DNA is commonly integrated into the genome of the host strain. Multiple genetic tools have been developed for genomic integration into baker's yeast Saccharomyces cerevisiae. Previously, we had developed a vector toolkit EasyClone-MarkerFree for stable integration into eleven sites on chromosomes X, XI, and XII of S. cerevisiae. The markerless integration was enabled by CRISPR-Cas9 system. In this study, we have expanded the kit with eight additional intergenic integration sites located on different chromosomes. The integration efficiency into the new sites was above 80%. The expression level of green fluorescence protein (gfp) for all eight sites was similar or above XI-2 site from the original EasyClone-MarkerFree toolkit. The cellular growth was not affected by the integration into any of the new eight locations. The eight-vector expansion kit is available from AddGene.

Preparation for the next COVID-19 wave: The European Hip Society and European Knee Associates recommendations.

PURPOSE: To plan for the continuance of elective hip and knee arthroplasty during a resurgence or new wave of COVID-19 infections. METHOD: A systematic review was conducted using the terms "COVID-19" or "SARS-Cov-2" and "second wave". No relevant citations were found to inform on recommendations the plan. Therefore, an expert panel of the European Hip Society and the European Knee Associates was formed to provide the recommendations. RESULTS: Overall, the recommendations consider three phases; review of the first wave, preparation for the next wave, and during the next wave. International and national policies will drive most of the management. The recommendations focus on the preparation phase and, in particular, the actions that the individual surgeon needs to undertake to continue with, and practice, elective arthroplasty during the next wave, as well as planning their personal and their family's lives. The recommendations expect rigorous data collection during the next wave, so that a cycle of continuous improvement is created to take account of any future waves. CONCLUSIONS: The recommendations for planning to continue elective hip and knee arthroplasty during a new phase of the SARS-Cov-2 pandemic provide a framework to reduce the risk of a complete shutdown of elective surgery. This involves engaging with hospital managers and other specialities in the planning process. Individuals have responsibilities to themselves, their colleagues, and their families, beyond the actual delivery of elective arthroplasty.

Acceleration of CRISPR/Cas9-Mediated Editing at Multiple Sites in the Saccharomyces cerevisiae Genome.

The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments.

Tree-ring data set for dendroclimatic reconstructions and dendrochronological dating in European Russia.

The data set presented represents 15 years of collection. It contains tree-ring width measurements from 64 sites of living trees and ten historical chronologies based on archaeological and construction wood up to year 572 CE, altogether 2909 tree-ring series and more than 450000 measured and cross-dated tree rings. It covers the vast territory of European Russia, including its forested northern and central parts, and the Northern Caucasus mountains. The potential use of these data include climatic reconstructions of regional and hemispheric scale, dendrochronological dating of historical and cultural wood, ecological and remote sensing studies.

Effect of detonation nanodiamonds on phagocyte activity.

Detonation ND (nanodiamond) holds much promise for biological studies and medical applications. Properties like size of particles, inclination for modification of their surface and unambiguous biocompatibility are crucial. Of prime importance is interaction between ND and immune cells, which supervise foreign intrusion into an organism and eliminate it. Neutrophils are more reactive in inflammatory response implementing cytotoxical arsenal including ROS (reactive oxygen species). The aim of the work was to estimate the ability of two ND samples (produced by Diamond Center and PlasmaChem) to keep the vitality of neutrophils from the inflammatory site. The ability of cells to generate ROS in the presence of ND particles is considered as indicating their biocompatibility. IR spectra and size of particles in the samples were characterized. Acid modification of ND was carried out to get the luminescent form. In the biological aspect, ND demonstrated up or down action, depending on the concentration, time and conditions of activation of cells. Weak action of ND in whole blood was obtained possibly owing to the ND adsorbed plasma proteins, which mask active functional groups to interact with the cell membrane. ND did not influence the viability of isolated inflammatory neutrophils in low and moderate concentrations and suppressed it in high concentrations (≥1 g/l). Addition of ND to the cell suspension initiated concentration-dependent reaction to produce ROS similar to respiratory burst. ND up-regulated response to bacterial formylpeptide, but up- and down-modified (low or high concentrations, accordingly) response to such bacterial agents as OZ (opsonized zymosan), which neutrophils swallow up by oxygen-dependent phagocytosis. Localization of the particles on the cell surface as into the cells was identified by monitoring the intrinsic fluorescence of oxidized ND. The various mechanisms that could account for penetration of ND particles into the cell are discussed. Common conclusion concerns compatibility of ND with living neutrophils from inflammatory site and their normal functioning for infection safeguard.