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Regioselective C-H Functionalization in the Six-Membered Ring from the Some,5-Fused Heterocyclic Techniques: An understanding.
This one-step method generates, for any locus, a conditional knockout allele in Drosophila. The allele carries a bright fluorescent marker for easy screening and an attP landing site for subsequent genetic manipulations. After removing the selectable marker with Cre, the attP site can be used to insert DNA fragments expressing tagged or mutant alleles to determine protein localization and function in a tissue- and stage-specific manner. Only a single round of CRISPR-Cas9-mediated editing is required. For complete details on the use and execution of this protocol, please refer to the DWnt4[cKO] example in Yu et al. (2020).Mitochondrial dysfunction and metabolic reprogramming are implicated in a variety of neurological disorders. Here, we present a protocol that enables complex profiling of brain metabolic function using acute mouse brain slices ex vivo. Utilizing differential metabolic conditions, substrates, and inhibitors, this protocol can be broadly applied to determine metabolic shift or reprogramming upon genetic manipulations, pathological insults, or therapeutic interventions and could thus further the understanding of the dynamic role of energy metabolism in brain physiological function and diseases. For complete details on the use and execution of this protocol, please refer to Qi et al. (2021).Tracking the inheritance patterns of proteins (TrIPP) is a live-cell imaging technique used for tracking maternal protein segregation patterns between mother and daughter cells during asymmetric divisions of budding yeast. We use the photo-convertible fluorescent protein Dendra2 fused to a protein of interest (POI). Irreversible conversion from green to red fluorescence allows for parallel monitoring of old and new proteins for several generations. Single-cell quantitative image analysis of time-lapse microscopy gives synthesis and decay rates, as well as segregation patterns of the POI. For complete details on the use and execution of this protocol, please refer to Auboiron et al. (2021).Mechanical forces are important in (patho)physiological processes, including how host epithelial cells interact with intracellular bacterial pathogens. As these pathogens disseminate within host epithelial monolayers, large mounds of infected cells are formed due to the forceful action of surrounding uninfected cells, limiting bacterial spread across the basal cell monolayer. Here, we present a protocol for mound volume measurement and biophysical characterization of mound formation. Modifications to this protocol may be necessary for studying different host cell types or pathogenic organisms. For complete details on the use and execution of this protocol, please refer to Bastounis et al. (2021).The SPrime program detects the variants in current-day populations that were introgressed from an archaic source in the past. It is optimized for detecting introgression from Neanderthals and Denisovans in modern humans. We provide a protocol for detecting Neanderthal and Denisovan introgression in 1000 Genomes Project data, specifically focusing on the CHB (Han Chinese in Beijing) population. For complete details on the use and execution of this protocol, please refer to Browning et al. (2018).CD8+ T cells are key effector cells in adaptive immune responses against intracellular pathogens and cancer cells. Systemic drug treatments, like chemotherapy, may positively or negatively affect CD8+ T cell function. In this protocol, we describe robust and optimized ex vivo polyclonal activation and cell culture conditions to measure drug treatments' effects on primary human CD8+ T cell activation and cytolytic potential. We provide streamlined methods for measuring effector cytokines and activation markers of CD8+ T cells via flow cytometry. For complete details on the use and execution of this protocol, please refer to Loo Yau et al. (2021).PAX6 is a key determinant of human neuroectoderm cell fate. Here, we describe a protocol for genome-scale CRISPR screening for use in genetically engineered human pluripotent stem cells (hPSCs). Using the germ layer reporter PAX6 and an inducible CRISPR/Cas9 knockout system, we describe how to identify lineage-specific preventing genes. This protocol can be applied for use with other reporter genes to study cell fate determination in hPSCs. For complete details on the use and execution of this protocol, please refer to Xu et al. (2021).Post-translational modification by S-nitrosylation regulates numerous cellular functions and impacts most proteins across phylogeny. We describe a protocol for isolating S-nitrosylated proteins (SNO-proteins) from C. elegans, suitable for assessing SNO levels of individual proteins and of the global proteome. This protocol features efficient nematode lysis and SNO capture, while protection of SNO proteins from degradation is the major challenge. This protocol can be adapted to mammalian tissues. For complete information on the generation and use of this protocol, please refer to Seth et al. (2019).Here, we describe a protocol to construct, calibrate, and operate a versatile tiling light sheet microscope for imaging cleared tissues. The microscope uses adjustable tiling light sheets to achieve higher spatial resolution and better optical sectioning ability than conventional light sheet microscopes and to image cleared tissues with the cellular to the subcellular spatial resolution. It is compatible with all tissue clearing methods and aligned semiautomatically through the phase modulation of the illumination light. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).This protocol is a comprehensive guide to phage display-based selection of virus neutralizing VH antibody domains. It details three optimized parts including (1) construction of a large-sized (theoretically > 1011) naïve human antibody heavy chain domain library, (2) SARS-CoV-2 antigen expression and stable cell line construction, and (3) library panning for selection of SARS-CoV-2-specific antibody domains. Using this protocol, we identified a high-affinity neutralizing human VH antibody domain, VH ab8, which exhibits high prophylactic and therapeutic efficacy. RHPS 4 chemical structure For complete details on the use and execution of this protocol, please refer to Li et al. (2020).
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