CHAPTER 4. Best Practices for Design and Characterization of Covalent Chemical Probes

2020 ◽  
pp. 69-99
Author(s):  
Ronen Gabizon ◽  
Efrat Resnick ◽  
Nir London
Keyword(s):  
Best Practices ◽  
Chemical Probes

scholarly journals Characterization of Best Practices for Customer/Supplier Collaboration in Co-innovation Projects

2020 ◽  
Vol 15 (4) ◽  
pp. 5-18
Author(s):  
Carlos Abraham Moya ◽  
Vincent Boly ◽  
Laure Morel ◽  
Daniel Gálvez ◽  
Mauricio Camargo
Keyword(s):  
Best Practices ◽  
Innovation Projects ◽  
Supplier Collaboration

scholarly journals Best practices for bioinformatic characterization of neoantigens for clinical utility

2019 ◽  
Vol 11 (1) ◽  
Author(s):  
Megan M. Richters ◽  
Huiming Xia ◽  
Katie M. Campbell ◽  
William E. Gillanders ◽  
Obi L. Griffith ◽  
...  
Keyword(s):  
Best Practices ◽  
Clinical Utility

Covalent modification and inhibition of an epithelial sodium channel by tyrosine-reactive reagents

1980 ◽  
Vol 239 (3) ◽  
pp. F299-F306 ◽  
Author(s):  
C. S. Park ◽  
D. D. Fanestil
Keyword(s):  
Short Circuit ◽  
Short Circuit Current ◽  
Complete Recovery ◽  
Covalent Modification ◽  
Na Channel ◽  
Chemical Probes ◽  
Pharmacologic Activity ◽  
Identification And Characterization ◽  
Aerobic And Anaerobic

This study sought to elucidate the molecular mechanism involved in the Na+ entry across the apical membrane of the urinary bladder of the toad. Na+ transport, as measured by short-circuit current (SCC), was irreversibly inhibited by three tyrosine-specific reagents: N-acetylimidazole (ID50, 4.6 x 10(-2)M), tetranitromethane (1.8 x 10(-4) M), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl; 3.4 x 10(-5) M). The functional specificity of NBD-Cl to block Na+ entry via Na+ channels was attested by the following: 1) NBD-Cl produced comparable inhibition of SCC and Na+ influx under aerobic and anaerobic conditions; 2) amphotericin B produced complete recovery of inhibited SCC; 3) vasopressin increased SCC only in proportion to the uninhibited SCC; 4) Km for Na+ was not changed; and 5) the half time for the inhibition varied as a function of amiloride concentration or pharmacologic activity of its analogues. On the basis of the above findings, these tyrosine-specific reagents are believed to be useful chemical probes for the identification and characterization of Na+ channel protein.

scholarly journals Chemical probes to potently and selectively inhibit endocannabinoid cellular reuptake

2017 ◽  
Vol 114 (25) ◽  
pp. E5006-E5015 ◽  
Author(s):  
Andrea Chicca ◽  
Simon Nicolussi ◽  
Ruben Bartholomäus ◽  
Martina Blunder ◽  
Alejandro Aparisi Rey ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Cannabinoid Receptor ◽  
Facilitated Diffusion ◽  
Chemical Probes ◽  
Analgesic Effects ◽  
Complex Process ◽  
Arachidonoyl Glycerol ◽  
The Brain

The extracellular effects of the endocannabinoids anandamide and 2-arachidonoyl glycerol are terminated by enzymatic hydrolysis after crossing cellular membranes by facilitated diffusion. The lack of potent and selective inhibitors for endocannabinoid transport has prevented the molecular characterization of this process, thus hindering its biochemical investigation and pharmacological exploitation. Here, we report the design, chemical synthesis, and biological profiling of natural product-derivedN-substituted 2,4-dodecadienamides as a selective endocannabinoid uptake inhibitor. The highly potent (IC50= 10 nM) inhibitorN-(3,4-dimethoxyphenyl)ethyl amide (WOBE437) exerted pronounced cannabinoid receptor-dependent anxiolytic, antiinflammatory, and analgesic effects in mice by increasing endocannabinoid levels. A tailored WOBE437-derived diazirine-containing photoaffinity probe (RX-055) irreversibly blocked membrane transport of both endocannabinoids, providing mechanistic insights into this complex process. Moreover, RX-055 exerted site-specific anxiolytic effects on in situ photoactivation in the brain. This study describes suitable inhibitors to target endocannabinoid membrane trafficking and uncovers an alternative endocannabinoid pharmacology.

scholarly journals FplA FromFusobacterium nucleatumis a Type Vd autotransporter phospholipase with a proposed role in altered host signaling and evasion of autophagy

2017 ◽  
Author(s):  
Michael A. Casasanta ◽  
Christopher C. Yoo ◽  
Hans B. Smith ◽  
A. Jane Duncan ◽  
Kyla Cochrane ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Outer Membrane ◽  
Virulence Factors ◽  
Biochemical Characterization ◽  
Bacterial Virulence ◽  
Chemical Probes ◽  
Gram Negative ◽  
Enzyme Family ◽  
Host Signaling

ABSTRACTFusobacterium nucleatumis a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection,F. nucleatumlacks Type I, II, III, IV, and VI secretion. By contrast,F. nucleatumstrains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of aF. nucleatumType Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby renameFusobacteriumphospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined inF. nucleatumvirulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with anfplAgene deletion strain, encompass a powerful set of tools for the future analysis of FplAin vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.IMPORTANCEF. nucleatumis an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme fromF. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and forF. nucleatum, in colorectal carcinogenesis.

scholarly journals Optimal protocols for sequence-based characterization of the human vaginal microbiome

2020 ◽  
Author(s):  
Luisa W. Hugerth ◽  
Marcela Pereira ◽  
Yinghua Zha ◽  
Maike Seifert ◽  
Vilde Kaldhusdal ◽  
...  
Keyword(s):  
16S Rrna ◽  
Best Practices ◽  
Gene Sequencing ◽  
16S Rrna Gene Sequencing ◽  
Rrna Gene ◽  
Vaginal Microbiome ◽  
Wide Range ◽  
Rrna Gene Sequencing ◽  
Good Agreement

AbstractThe vaginal microbiome has been connected to a wide range of health outcomes. This has led to a thriving research environment, but also to the use of conflicting methodologies to study its microbial composition. Here we systematically assess best practices for the sequencing-based characterization of the human vaginal microbiome. As far as 16S rRNA gene sequencing is concerned, the V1-V3 region has the best theoretical properties, but limitations of current sequencing technologies mean that the V3-V4 region performs equally well. Both of these approaches present very good agreement with qPCR quantification of key taxa, provided an appropriate bioinformatic pipeline is used. Shotgun metagenomic sequencing presents an interesting alternative to 16S amplification and sequencing, but it is not without its challenges. We have assessed different tools for the removal of host reads and the taxonomic annotation of metagenomic reads, including a new, easy-to-build and – use, reference database of vaginal taxa. This strategy performed as well as the best performing previously published strategies. Despite the many advantages of shotgun sequencing none of the shotgun approaches assessed here had as good agreement with the qPCR data as 16S rRNA gene sequencing.ImportanceThe vaginal microbiome has been connected to a wide range of health outcomes, from susceptibility to sexually transmitted infections to gynecological cancers and pregnancy outcomes. This has led to a thriving research environment, but also to conflicting available methodologies, including many studies that do not report their molecular biological and bioinformatic methods in sufficient detail for them to be considered reproducible. This can lead to conflicting messages and delay progress from descriptive to intervention studies. By systematically assessing best practices for the characterization of the human vaginal microbiome, this study will enable past studies to be assessed more critically and assist future studies in the selection of appropriate methods for their specific research questions.

scholarly journals Development And Characterization Of Fluorescent Mannose- And Sialic Acid Based Chemical Probes For Lectin Discovery And Enzyme Studies

2021 ◽  
Author(s):  
Tasnim Abukar
Keyword(s):  
Sialic Acid ◽  
Enzymatic Synthesis ◽  
Biological Applications ◽  
Chemical Probes ◽  
Mannose Binding ◽  
Biological Labeling ◽  
Photochemical Properties ◽  
Enzymatic Detection ◽  
Fluorescent Molecules

BODIPY (4,4–Difluoro-4-boro-3a,4a-diaza-s-indacene) dyes are relatively nonpolar fluorescent molecules used as biological labeling reagents. The advantages of using BODIPY dyes as tags include their unique photochemical properties, allowing for an easy and sensitive monitoring of target components. This thesis will discuss the synthesis of novel fluorescent “BODIPY”-labeled carbohydrate-based chemical probes and highlight their uses in different biological applications. In chapter one, we have focused on the development of BODIPY-mannose-based chemical probes for the discovery and characterization of mannose-binding lectins on surfaces of Actinobacteria. The synthesis of these probes and their efficiency to detect mannose-binding lectins using different techniques such as Fluorescence microscopy and flow cytometry are discussed. In chapter two, we have focused on the chemo-enzymatic synthesis of BODIPY-sialic acid based chemical probes for enzymatic detection of specific glycoconjugates on proteins. The use of these probes as donor substrates to probe the enzymes involved in glycan sialylation are discussed

scholarly journals Study of Carbon Nanocones CNC k n via Connection Zagreb Indices

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Muhammad Asif ◽  
Muhammad Hussain ◽  
Hamad Almohamedh ◽  
Khalid M Alhamed ◽  
Rana Alabdan ◽  
...  
Keyword(s):  
Strain Energy ◽  
Chemical Properties ◽  
Graph Invariant ◽  
Practical Application ◽  
Chemical Probes ◽  
Topological Characterization ◽  
Mathematica Software ◽  
Physical And Chemical ◽  
And Chemical Properties

Topology of fullerenes, carbon nanotubes, and nanocones has considerable worth due to their effective applications in nanotechnology. These are emerging materials of practical application in gas storage devises, nanoelectronics devices, energy storage, biosensor, and chemical probes. The topological indices are graph invariant used to investigate the physical and chemical properties of the compounds such as boiling point, stability, and strain energy through associated chemical graph of the underlying compound. We computed recently modified Zagreb connection indices of nanocones CNC 4 n , CNC 5 n , and CNC 6 n and generalized our findings up to a large class of CNC k n . Topological characterization of nanocones via these indices is mathematically novel and assists to enable its emerging use in nanotechnology. For computation and verification of results, we use Mathematica software.

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