scholarly journals EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

2020 ◽  
Author(s):  
T.A. Hartjes ◽  
J.A. Slotman ◽  
M.S. Vredenbregt ◽  
N. Dits ◽  
R. Van der Meel ◽  
...  
Keyword(s):  
Protein Content ◽  
Size Distribution ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Ideal Liquid ◽  
Extracellular Vesicle ◽  
Cell Of Origin ◽  
Clinical Implementation ◽  
High Throughput Assay

AbstractExtracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

scholarly journals Semi-automated high-throughput substrate screening assay for nucleoside kinases

2021 ◽  
Author(s):  
Katja Hellendahl ◽  
Maryke Fehlau ◽  
Sebastian Hans ◽  
Peter Neubauer ◽  
Anke Kurreck
Keyword(s):  
High Throughput ◽  
Viral Infections ◽  
Screening Assay ◽  
Liquid Handling ◽  
Wide Range ◽  
High Throughput Assay ◽  
Liquid Handling Robot

Nucleoside kinases (NKs) are key enzymes involved in the in vivo phosphorylation of nucleoside analogues used as drugs to treat cancer or viral infections. Having different specificities, the characterization of NKs is essential for drug design and the production of nucleotide analogues in an in vitro enzymatic process. Therefore, a fast and reliable substrate screening assay for NKs is of great importance. Here, we report the validation of a well-known luciferase-based assay for the detection of NK activity in 96-well plate format. The assay was semi-automated using a liquid handling robot. A good linearity was demonstrated (r² >0.98) in the range of 0 to 500 µM ATP, and it was shown that also alternative phosphate donors like dATP or CTP were accepted by the luciferase. The developed high-throughput assay revealed comparable results to HPLC analysis. The assay was exemplary used for the comparison of the substrate spectra of four nucleoside kinases using 20 (8 natural and 12 modified) substrates. The screening results correlated well with literature data and, additionally, previously unknown substrates were identified for three of the NKs studied. Our results demonstrate that the developed semi-automated high-throughput assay is suitable to identify best performing NKs for a wide range of substrates.

A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

2009 ◽  
Vol 386 (2) ◽  
pp. 244-250 ◽  
Author(s):  
Thomas J. McQuade ◽  
Abbie D. Shallop ◽  
Anita Sheoran ◽  
James E. DelProposto ◽  
Oleg V. Tsodikov ◽  
...  
Keyword(s):  
High Throughput ◽  
Combinatorial Biosynthesis ◽  
Nonribosomal Peptide ◽  
High Throughput Assay

scholarly journals High-Throughput Single Extracellular Vesicle Profiling

2021 ◽  
Author(s):  
Yanling Cai ◽  
Di Wu
Keyword(s):  
Extracellular Vesicles ◽  
High Throughput ◽  
Liquid Biopsy ◽  
Trace Amount ◽  
Extracellular Vesicle ◽  
Body Fluids ◽  
Imaging Systems

Extracellular vesicles (EVs) are heterogeneous due to their cell of origins, biogenesis, stimuli in the microenvironment and so on. Single EV analysis is required for the study of EV heterogeneity. Besides the investigation of EV biology, single EV analysis technologies are promising approach for liquid biopsy, which relies on the detection of biomarker EVs readily available in body fluids but in trace amount. However, EVs are nano-scaled structures, which beyond the resolution of conventional technologies like optical microscopes, flow cytometers and so on. In this chapter, we will discuss advanced strategies for studying single EVs, including single EV imaging systems, flow cytometers, nano-sensing technologies and single EV barcoding assay.

scholarly journals Characterization of fluorescent probe substrates to develop an efficient high-throughput assay for neonatal hepatic CYP3A7 inhibition screening

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hannah M. Work ◽  
Sylvie E. Kandel ◽  
Jed N. Lampe
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Antiviral Drug ◽  
Dehydroepiandrosterone Sulfate ◽  
Fluorescent Compound ◽  
Critical Function ◽  
Drug Candidates ◽  
High Throughput Assay ◽  
Fetus And Neonate

AbstractCYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub-family that is expressed in the fetus and neonate. In addition to its role metabolizing retinoic acid and the endogenous steroid dehydroepiandrosterone sulfate (DHEA-S), it also has a critical function in drug metabolism and disposition during the first few weeks of life. Despite this, it is generally ignored in the preclinical testing of new drug candidates. This increases the risk for drug-drug interactions (DDI) and toxicities occurring in the neonate. Therefore, screening drug candidates for CYP3A7 inhibition is essential to identify chemical entities with potential toxicity risks for neonates. Currently, there is no efficient high-throughput screening (HTS) assay to assess CYP3A7 inhibition. Here, we report our testing of various fluorescent probes to assess CYP3A7 activity in a high-throughput manner. We determined that the fluorescent compound dibenzylfluorescein (DBF) is superior to other compounds in meeting the criteria considered for an efficient HTS assay. Furthermore, a preliminary screen of an HIV/HCV antiviral drug mini-library demonstrated the utility of DBF in a HTS assay system. We anticipate that this tool will be of great benefit in screening drugs that may be used in the neonatal population in the future.

scholarly journals High throughput imaging of nanoscale extracellular vesicles by scanning electron microscopy for accurate size-based profiling and morphological analysis

2021 ◽  
Author(s):  
Sara Cavallaro ◽  
Petra Hååg ◽  
Kristina Viktorsson ◽  
Anatol Krozer ◽  
Kristina Fogel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Throughput ◽  
Extracellular Vesicle ◽  
Physical Characterization ◽  
Nsclc Cell Line ◽  
Visual Approach ◽  
High Throughput Imaging ◽  
Scanning Electron

ABSTRACTNanoscale extracellular vesicle (EVs) have been found to play a key role in intercellular communication, offering opportunities for both diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for their physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The investigation was performed with small EVs (sEVs) isolated from a non-small cell lung cancer (NSCLC) cell line H1975 as well as from a human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter >70 nm), the microscopy based approach showed a better capacity for analyses on smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.

scholarly journals Colorimetric LPMO assay with direct implication for cellulolytic activity

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Søren Brander ◽  
Stine Lausten ◽  
Johan Ø. Ipsen ◽  
Kristoffer B. Falkenberg ◽  
Andreas B. Bertelsen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Biochemical Characterization ◽  
Cellulose Degradation ◽  
Microtiter Plate ◽  
Enzyme Characterization ◽  
Cellulolytic Activity ◽  
Polysaccharide Monooxygenases ◽  
High Throughput Assay

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization. Results A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis–Menten kinetics with kcat = 0.09 s−1 and KM = 244 µM, and the assay was used to confirm stoichiometric copper–enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation. Conclusions This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.

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