Search results for "cycle"
showing 10 items of 3119 documents
Cell Proliferation High-Content Screening on Adherent Cell Cultures
2019
Pulse-chase experiments using 5-bromo-2'-deoxyuridine (BrdU), or the more recent EdU (5-etynil-2'-deoxyuridine), enable the identification of cells going through S phase. This chapter describes a high-content proliferation assay pipeline for adherent cell cultures. High-throughput imaging is followed by high-content data analysis using a non-supervised ImageJ macroinstruction that segments the individual nuclei, determines the nucleoside analogue absence/presence, and measures the signal of up to two additional nuclear markers. Based upon the specific combination with proliferation-specific protein immunostaining, the percentage of cells undergoing different phases of the cell cycle (G0, G1…
Cooperation between CRISPR-Cas types enables adaptation in an RNA-targeting system
2020
AbstractCRISPR-Cas immune systems adapt to new threats by acquiring spacers from invading nucleic acids such as phage genomes. However, some CRISPR-Cas loci lack genes necessary for spacer acquisition, despite apparent variation in spacer content between strains. It has been suggested that such loci may use acquisition machinery from co-occurring CRISPR-Cas systems. Here, using a lytic dsDNA phage, we observe spacer acquisition in the native host Flavobacterium columnare that carries an acquisition-deficient subtype VI-B locus and a complete subtype II-C locus. We characterize acquisition events in both loci and show that the RNA-targeting VI-B locus acquires spacers in trans using acquisit…
Comparison of CRISPR and marker based methods for the engineering of phage T7
2020
With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genescmkortrxhave been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple …
Nuclear Pore Complex Acetylation Regulates mRNA Export and Cell Cycle Commitment in Budding Yeast
2021
AbstractNuclear pore complexes (NPCs) mediate communication between the nucleus and the cytoplasm and regulate gene expression by interacting with transcription and mRNA export factors. Lysine acetyl-transferases (KATs) promote transcription through acetylation of chromatin-associated proteins. We find that Esa1, the KAT subunit of the yeast NuA4 complex, also acetylates the nuclear pore basket component Nup60 to promote mRNA export. Acetylation of Nup60 recruits to the nuclear basket the mRNA export factor Sac3, the scaffolding subunit of the Transcription and Export 2 (TREX-2) complex. Esa1-dependent nuclear export of mRNAs promotes entry into S phase, and is inhibited by the Hos3 deacety…
2020
We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with β, β'-iminodipropionitrile (IDPN) did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the nonstructural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, leading to prolonged cell surviva…
SMAC mimetics promote NIK-dependent inhibition of CD4 + T H 17 cell differentiation
2019
Second mitochondria-derived activator of caspase (SMAC) mimetics (SMs) are selective antagonists of the inhibitor of apoptosis proteins (IAPs), which activate noncanonical NF-κB signaling and promote tumor cell death. Through gene expression analysis, we found that treatment of CD4+ T cells with SMs during T helper 17 (TH17) cell differentiation disrupted the balance between two antagonistic transcription factor modules. Moreover, proteomics analysis revealed that SMs altered the abundance of proteins associated with cell cycle, mitochondrial activity, and the balance between canonical and noncanonical NF-κB signaling. Whereas SMs inhibited interleukin-17 (IL-17) production and ameliorated …
Arctic aerosols and the ‘Divergence Problem’ in dendroclimatology
2021
Considering the importance and complexity of natural (e.g., volcanic eruptions and wildfires) and anthropogenic (e.g., mining, oil and shipping industries) aerosol emissions to Arctic warming is particularly timely given the recent temperature extremes recorded at high-northern latitudes (Cohen et al., 2020; Overland and Wang, 2021). Despite our knowledge about the observed and modelled climatic effects of rising Arctic aerosol concentrations (Schmale et al., 2021), which may exhibit regional and seasonal differences and call for diverse research priorities from local to circumpolar scales, we feel that the ecological consequences of an aerosol-induced reduction in surface irradiance (i.e.,…
Viral entry, lipid rafts and caveosomes.
2005
Lipid rafts and caveolae are detergent-insoluble plasma membrane microdomains, involved in cellular endocytic processes and signalling. Several viruses, including a human pathogen, echovirus 1, and an extensively studied simian virus 40 utilize these domains for internalization into the host cells. Interaction of viruses with receptors on the cell surface triggers specific conformational changes of the virus particle and can give rise to signalling events, which determine the mechanisms of virus entry. After internalization via cell surface lipid rafts or caveolae, virus-containing vesicles can fuse with caveosomes, pre-existing cytoplasmic organelles, or dock on other intracellular organel…
Antiproliferative activity of green, black tea and olive leaves polyphenols subjected to biosorption and in vitro gastrointestinal digestion in Caco-…
2020
Olive (Olea europaea L.) leaves and tea (Camellia sinensis) are rich sources of bioactive compounds, especially polyphenols. Our previous studies have evidenced the potential use of Saccharomyces cerevisiae as a natural delivery system for these antioxidants and a means to improve their bioaccessibility in the human gut. In the present work, the antiproliferative effect of green tea (GT), black tea (BT) and olive leaves (OL) infusions and suspensions of S. cerevisiae were evaluated, for the first time, in human colon cancer cells (Caco-2) after biosorption and in vitro gastrointestinal digestion. The bioaccessible fractions (BF) were not overtly cytotoxic, not affecting cell viability. ROS …
A reactive nitrogen budget for forest land and wetlands in Latvia and Estonia
2020
Human intervention in global nitrogen cycling has led to excess reactive nitrogen (Nr) flows to the environment, impacting terrestrial ecosystems as well as the quality of the atmosphere and waterb...