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Current Protocols in Chemical Biology

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https://www.readbyqxmd.com/read/28910858/overview-of-methods-and-strategies-for-conducting-virtual-small-molecule-screening
#1
Xavier Fradera, Kerim Babaoglu
Virtual screening (VS) in the context of drug discovery is the use of computational methods to discover novel ligands with a desired biological activity from within a larger collection of molecules. These techniques have been in use for many years, there is a wide range of methodologies available, and many successful applications have been reported in the literature. VS is often used as an alternative or a complement to High-throughput screening (HTS) or other methods to identify ligands for target validation or medicinal chemistry projects...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910857/a-guide-to-quantitative-biomarker-assay-development-using-whispering-gallery-mode-biosensors
#2
Heather M Robison, Ryan C Bailey
Whispering gallery mode (WGM) sensors are a class of powerful analytical techniques defined by the measurement of changes in the local refractive index at or near the sensor surface. When functionalized with target-specific capture agents, analyte binding can be measured with very low limits of detection. There are many geometric manifestations of WGM sensors, with chip-integrated silicon photonic devices first commercialized because of the robust, wafer-scale device fabrication, facile optical interrogation, and amenability to the creation of multiplexed sensor arrays...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910856/high-throughput-screening-of-hect-e3-ubiquitin-ligases-using-ubfluor
#3
Peter K Foote, David T Krist, Alexander V Statsyuk
HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910855/optimized-pei-based-transfection-method-for-transient-transfection-and-lentiviral-production
#4
Shaozhe Yang, Xiaoling Zhou, Rongxiang Li, Xiuhong Fu, Pingnan Sun
Polyethyleneimine (PEI), a cationic polymer vehicle, forms a complex with DNA which then can carry anionic nucleic acids into eukaryotic cells. PEI-based transfection is widely used for transient transfection of plasmid DNA. The efficiency of PEI-based transfection is affected by numerous factors, including the way the PEI/DNA complex is prepared, the ratio of PEI to DNA, the concentration of DNA, the storage conditions of PEI solutions, and more. Considering the major influencing factors, PEI-based transfection has been optimized to improve its efficiency, reproducibility, and consistency...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28910854/characterizing-the-nano-bio-interface-using-microscopic-techniques-imaging-the-cell-system-is-just-as-important-as-imaging-the-nanoparticle-system
#5
Christie M Sayes, Henry Lujan
The rapid growth of nanotechnology and its industries has elevated the need to understand the risks associated with handling, using, and disposing of nanomaterials. These risks can be assessed through exposure measurement and hazard identification. One of the common challenges associated with quantifying nanomaterials in products, waste, humans, or the environment is the lack of tools available to measure concentration. The ability of refined tools and techniques to qualitatively detect nanoparticles in complex matrices has been demonstrated...
September 14, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628203/photoactivated-in-vivo-proximity-labeling
#6
David B Beck, Roberto Bonasio
Identification of molecular interactions is paramount to understanding how cells function. Most available technologies rely on co-purification of a protein of interest and its binding partners. Therefore, they are limited in their ability to detect low-affinity interactions and cannot be applied to proteins that localize to difficult-to-solubilize cellular compartments. In vivo proximity labeling (IPL) overcomes these obstacles by covalently tagging proteins and RNAs based on their proximity in vivo to a protein of interest...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628202/paper-based-invasion-assays-for-quantifying-cellular-movement-in-three-dimensional-tissue-like-structures
#7
C Chad Lloyd, Matthew W Boyce, Matthew R Lockett
To elucidate the chemical and environmental conditions that promote invasion of cancer cells, an assay is needed in which the chemical landscape of a tumor-like environment can be experimentally manipulated and probed. The three-dimensional paper-based invasion assays described here simulate poorly vascularized tissue and allow the invasion of cancerous cells to be visualized and quantified. These cultures are easy to assemble and allow multiple invasion assays to be performed in parallel. By using different materials to control gradients formed across the culture, the chemotactic potential of small molecules can be evaluated in a more representative tissue microenvironment...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628201/designing-drug-response-experiments-and-quantifying-their-results
#8
Marc Hafner, Mario Niepel, Kartik Subramanian, Peter K Sorger
We developed a Python package to help in performing drug-response experiments at medium and high throughput and evaluating sensitivity metrics from the resulting data. In this article, we describe the steps involved in (1) generating files necessary for treating cells with the HP D300 drug dispenser, by pin transfer or by manual pipetting; (2) merging the data generated by high-throughput slide scanners, such as the Perkin Elmer Operetta, with treatment annotations; and (3) analyzing the results to obtain data normalized to untreated controls and sensitivity metrics such as IC50 or GR50 ...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628200/local-generation-and-imaging-of-hydrogen-peroxide-in-living-cells
#9
Yulia A Bogdanova, Carsten Schultz, Vsevolod V Belousov
Described here is a localized H2 O2 generation-detection system consisting of a yeast D-amino acid oxidase (DAAO) and two spectrally distinct variants of biosensor, HyPer2 and HyPerRed based on circularly permutated yellow and red fluorescent proteins, respectively, which enables spatiotemporal production and examination of the intracellular H2 O2 dynamics. The protocol describes using this system in a simple cell culture model. We provide detailed instructions on imaging of H2 O2 generated by the activated DAAO...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28628199/measuring-cancer-drug-sensitivity-and-resistance-in-cultured-cells
#10
Mario Niepel, Marc Hafner, Mirra Chung, Peter K Sorger
Measuring the potencies of small-molecule drugs in cell lines is a critical aspect of preclinical pharmacology. Such experiments are also prototypical of high-throughput experiments in multi-well plates. The procedure is simple in principle, but many unrecognized factors can affect the results, potentially making data unreliable. The procedures for measuring drug response described here were developed by the NIH LINCS program to improve reproducibility. Key features include maximizing uniform cell growth during the assay period, accounting for the effects of cell density on response, and correcting sensitivity measures for differences in proliferation rates...
June 19, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253435/detecting-membrane-protein-protein-interactions-using-the-mammalian-membrane-two-hybrid-mamth-assay
#11
Punit Saraon, Ingrid Grozavu, Sang Hyun Lim, Jamie Snider, Zhong Yao, Igor Stagljar
Protein-protein interactions (PPIs) play an integral role in numerous cellular processes. Membrane protein interactions, in particular, are critical in cellular responses to stresses and stimuli, with dysfunction of these PPIs (e.g., due to aberrant expression and/or mutation of interaction partners) leading to a diverse array of pathological states. Exploration of the interaction space and dynamics of membrane proteins is difficult due to the limitations of current techniques used to study proteins in the biochemically complex environment of biological membranes...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253434/a-general-non-radioactive-atpase-assay-for-chromatin-remodeling-complexes
#12
Benjamin Z Stanton, Courtney Hodges, Gerald R Crabtree, Keji Zhao
Chromatin remodeling complexes couple the energy released from ATP hydrolysis to facilitate transcription, recombination, and repair mechanisms essential for a wide variety of biologic responses. While recombinant expression of the regulatory subunits of these enzymes is possible, measuring catalytic (ATPase) activity of the intact complexes recovered from normal or mutant cells is critical for understanding their mechanisms. SWI/SNF-like remodeling complexes can be megadaltons in size and include many regulatory subunits, making reconstitution of purified subunits challenging for recapitulating in vivo function...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/28253433/ubfluor-a-fluorescent-thioester-to-monitor-hect-e3-ligase-catalysis
#13
David T Krist, Peter K Foote, Alexander V Statsyuk
HECT E3 ubiquitin ligases (∼28 are known) are associated with many phenotypes in eukaryotes and are important drug targets. However, assays used to screen for small molecule inhibitors of HECT E3s are complex and require ATP, Ub, E1, E2, and HECT E3 enzymes, producing three covalent thioester enzyme intermediates E1∼Ub, E2∼Ub, and HECT E3∼Ub (where ∼ indicates a thioester bond), and mixtures of polyubiquitin chains. To reduce the complexity of the assay, we developed a novel class of fluorescent probes, UbFluor, that act as mechanistically relevant pseudosubstrates of HECT E3s...
March 2, 2017: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925669/biochemical-biophysical-and-cellular-techniques-to-study-the-guanine-nucleotide-exchange-factor-giv-girdin
#14
Pradipta Ghosh, Nicolas Aznar, Lee Swanson, I-Chung Lo, Inmaculada Lopez-Sanchez, Jason Ear, Cristina Rohena, Nicholas Kalogriopoulos, Linda Joosen, Ying Dunkel, Nina Sun, Peter Nguyen, Deepali Bhandari
Canonical signal transduction via heterotrimeric G proteins is spatiotemporally restricted, i.e., triggered exclusively at the plasma membrane, only by agonist activation of G protein-coupled receptors via a finite process that is terminated within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a noncanonical pathway for activation of heterotrimeric G proteins via the nonreceptor guanidine-nucleotide exchange factor, GIV/Girdin. Biochemical, biophysical, and functional studies evaluating this pathway have unraveled its unique properties and distinctive spatiotemporal features...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925668/cyclic-immunofluorescence-cycif-a-highly-multiplexed-method-for-single-cell-imaging
#15
Jia-Ren Lin, Mohammad Fallahi-Sichani, Jia-Yun Chen, Peter K Sorger
Cyclic Immunofluorescence (CycIF) is a public-domain method for performing highly multiplexed immunofluorescence imaging using a conventional epifluorescence microscope. It uses simple reagents and existing antibodies to construct images with up to 30 channels by sequential 4- to 6-channel imaging followed by fluorophore inactivation. Three variant methods are described, the most generally useful of which involves staining fixed cells with antibodies directly conjugated to Alexa Fluor dyes and imaging in four colors, inactivating fluorophores using a mild base in the presence of hydrogen peroxide and light, and then performing another round of staining and imaging...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925667/tracking-the-activity-of-mtorc1-in-living-cells-using-genetically-encoded-fret-based-biosensor-torcar
#16
Xin Zhou, Simin Li, Jin Zhang
Mechanistic target of rapamycin complex 1 (mTORC1) is a highly conserved serine/threonine protein kinase that responds to multiple distinct signals (e.g., growth factors, amino acids, stress, and energy level) and coordinates cell growth and proliferation. The underlying molecular mechanisms by which these stimuli regulate the activity of mTORC1 are still not fully understood. The spatial compartmentalization of mTORC1 signaling has been suggested as an important mechanism for mTORC1 to achieve the signal specificity and efficiency...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27925666/enriching-s-4-u-rna-using-methane-thiosulfonate-mts-chemistry
#17
Erin E Duffy, Matthew D Simon
Metabolic labeling of cellular RNA is a useful approach to study RNA biology. 4-Thiouridine (s(4) U) is a convenient nucleoside for metabolic labeling because it is cell permeable and is incorporated into newly transcribed RNA, and the sulfur moiety provides a handle for biochemical purification. However, a critical step in the purification of s(4) U-RNA is the efficiency of the chemistry used to enrich s(4) U-RNA. Here, we present a protocol for s(4) U-RNA enrichment that includes efficient and reversible covalent chemistry to biotinylate s(4) U-RNA using the activated disulfide methane thiosulfonate conjugated to biotin (MTS-biotin), followed by enrichment on streptavidin beads...
December 7, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622569/nanoparticle-templated-molecular-recognition-platforms-for-detection-of-biological-analytes
#18
Abraham G Beyene, Gozde S Demirer, Markita P Landry
Molecular recognition of biological analytes with optical nanosensors provides both spatial and temporal biochemical information. A recently developed sensing platform exploits near-infrared fluorescent single-wall carbon nanotubes combined with electrostatically pinned heteropolymers to yield a synthetic molecular recognition technique that is maximally transparent through biological matter. This molecular recognition technique is known as corona phase molecular recognition (CoPhMoRe). In CoPhMoRe, the specificity of a folded polymer toward an analyte does not arise from a pre-existing polymer-analyte chemical affinity...
September 13, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622568/reverse-phase-protein-arrays-for-compound-profiling
#19
Nathan Moerke, Mohammad Fallahi-Sichani
Reverse phase protein arrays (RPPAs), also called reverse phase lysate arrays (RPLAs), involve immobilizing cell or tissue lysates, in small spots, onto solid supports which are then probed with primary antibodies specific for proteins or post-translational modifications of interest. RPPA assays are well suited for large-scale, high-throughput measurement of protein and PTM levels in cells and tissues. RPPAs are affordable and highly multiplexable, as a large number of arrays can readily be produced in parallel and then probed separately with distinct primary antibodies...
September 13, 2016: Current Protocols in Chemical Biology
https://www.readbyqxmd.com/read/27622567/fabrication-of-3-d-reconstituted-organoid-arrays-by-dna-programmed-assembly-of-cells-dpac
#20
Michael E Todhunter, Robert J Weber, Justin Farlow, Noel Y Jee, Alec E Cerchiari, Zev J Gartner
Tissues are the organizational units of function in metazoan organisms. Tissues comprise an assortment of cellular building blocks, soluble factors, and extracellular matrix (ECM) composed into specific three-dimensional (3-D) structures. The capacity to reconstitute tissues in vitro with the structural complexity observed in vivo is key to understanding processes such as morphogenesis, homeostasis, and disease. In this article, we describe DNA-programmed assembly of cells (DPAC), a method to fabricate viable, functional arrays of organoid-like tissues within 3-D ECM gels...
September 13, 2016: Current Protocols in Chemical Biology
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