Optimization of a fluorogenic assay to determine caspase 3/7 activity in meat extracts

2021 ◽  
pp. 108201322199357
Author(s):  
Claudia Fuente-Garcia ◽  
Enrique Sentandreu ◽  
Noelia Aldai ◽  
Miguel A Sentandreu
Keyword(s):  
Caspase 3 ◽  
Extraction Procedure ◽  
Chemical Compounds ◽  
Microtiter Plate ◽  
Enzyme Concentration ◽  
General Purpose ◽  
Experimental Conditions ◽  
High Enzyme ◽  
Biological Extracts ◽  
Environment Sustainability

Usefulness of general-purpose fluorogenic assay kits to determine caspase 3/7 activity of biological extracts is highly compromised in meat-based samples due to their scarce enzyme concentration. In the present work, a straightforward protocol is presented with two main purposes: 1) to enhance sensitivity of the fluorogenic approach addressing caspase 3/7 activity in tissues showing scarce enzyme concentration such as skeletal muscle, and 2) to reduce/economize the volume of employed reagents. The enzyme extraction procedure, peptide substrate, dithiothreitol concentration and detection settings were appropriately optimized for use in microtiter-plate fluorometers. As a result, low to high enzyme activity extracts (from 10,000 to 260,000 relative fluorescence units) can be measured under developed sampling and experimental conditions. The fact that enzyme reactions took place in 96-microtiter well plates reduces the consumption of chemical compounds when analysing a high number of samples, thus contributing to environment sustainability.

scholarly journals High Voltage Electric Discharge for Recovery of Chlorogenic Acid from Tobacco Waste

2021 ◽  
Vol 13 (8) ◽  
pp. 4481
Author(s):  
Marija Banožić ◽  
Antun Jozinović ◽  
Jovana Grgić ◽  
Borislav Miličević ◽  
Stela Jokić
Keyword(s):  
Chlorogenic Acid ◽  
Process Parameters ◽  
Electric Discharge ◽  
High Voltage ◽  
Treatment Time ◽  
Strong Dependence ◽  
Extraction Procedure ◽  
Polynomial Equation ◽  
Experimental Conditions ◽  
Tobacco Waste

Three fractions of tobacco waste (scrap, dust and midrib) were subjected to a high voltage electric discharge (HVED) assisted extraction procedure under different experimental conditions: solvent:solid ratio (300, 500, 700 mL/g), frequency (40, 70, 100 Hz) and treatment time (15, 30, 45 min), in order to study the influence of these conditions on the content of chlorogenic acid. The content of chlorogenic acid ranged from 1.54 to 3.66 mg/100 g for scrap, from 1.90 to 2.97 mg/100 g for dust, and from 2.30 to 3.38 mg/100 g for midrib extract, showing a strong dependence on the applied process parameters. The temperature change and the change in pH and electrical conductivity of the extracts after high voltage discharge treatment were also observed. The studied process parameters showed a statistically significant effect on the chemical and physical properties of the extracts from tobacco waste as well as on the content of chlorogenic acid, indicating the potential of HVED assisted processes in the separation of chlorogenic acid from tobacco industry waste. Multiple regression analysis was used to fit the results for the chlorogenic acid to a second order polynomial equation and the optimum conditions were determined.

waveRAPID—A Robust Assay for High-Throughput Kinetic Screens with the Creoptix WAVEsystem

2021 ◽  
pp. 247255522110138
Author(s):  
Önder Kartal ◽  
Fabio Andres ◽  
May Poh Lai ◽  
Rony Nehme ◽  
Kaspar Cottier
Keyword(s):  
Drug Discovery ◽  
Fold Increase ◽  
Microtiter Plate ◽  
Experimental Conditions ◽  
Short Pulses ◽  
Cycle Times ◽  
Fragment Screening ◽  
New Instrument ◽  
Single Well ◽  
Compound Screening

Surface-based biophysical methods for measuring binding kinetics of molecular interactions, such as surface plasmon resonance (SPR) or grating-coupled interferometry (GCI), are now well established and widely used in drug discovery. Increasing throughput is an often-cited need in the drug discovery process and this has been achieved with new instrument generations where multiple interactions are measured in parallel, shortening the total measurement times and enabling new application areas within the field. Here, we present the development of a novel technology called waveRAPID for a further—up to 10-fold—increase in throughput, consisting of an injection method using a single sample. Instead of sequentially injecting increasing analyte concentrations for constant durations, the analyte is injected at a single concentration in short pulses of increasing durations. A major advantage of the new method is its ability to determine kinetics from a single well of a microtiter plate, making it uniquely suitable for kinetic screening. We present the fundamentals of this approach using a small-molecule model system for experimental validation and comparing kinetic parameters to traditional methods. By varying experimental conditions, we furthermore assess the robustness of this new technique. Finally, we discuss its potential for improving hit quality and shortening cycle times in the areas of fragment screening, low-molecular-weight compound screening, and hit-to-lead optimization.

scholarly journals Improved Sugar Production by Optimizing Planetary Mill Pretreatment and Enzyme Hydrolysis Process

2015 ◽  
Vol 2015 ◽  
pp. 1-5
Author(s):  
Jeong Heo Kwon ◽  
Siseon Lee ◽  
Jae-Won Lee ◽  
Youn-Woo Hong ◽  
Jeong Ho Chang ◽  
...  
Keyword(s):  
Glucose Production ◽  
Process Variable ◽  
Enzyme Concentration ◽  
Planetary Mill ◽  
Enzyme Hydrolysis ◽  
Experimental Conditions ◽  
Citrate Buffer ◽  
Pinus Rigida ◽  
Hydrolysis Process ◽  
Optimal Values

This paper describes an optimization of planetary mill pretreatment and saccharification processes for improving biosugar production. Pitch pine (Pinus rigida) wood sawdust waste was used as biomass feedstock and the process parameters optimized in this study were the buffering media, the milling time, the enzyme quantity, and the incubation time. Glucose yields were improved when acetate buffer was used rather than citrate buffer. Initially, with each process variable tests, the optimal values were 100 minutes of milling, an enzyme concentration of 16 FPU/g-biomass, and a 12-hour enzymatic hydrolysis. Typically, interactions between these experimental conditions and their effects on glucose production were next investigated using RSM. Glucose yields from thePinus rigidawaste exceeded 80% with several of the conditions tested, demonstrating that milling can be used to obtain high levels of glucose bioconversion from woody biomass for biorefinery purposes.

Scalable Microfluidic Platform for Flexible Configuration of and Experiments with Microtissue Multiorgan Models

2018 ◽  
Vol 24 (1) ◽  
pp. 79-95 ◽  
Author(s):  
Christian Lohasz ◽  
Nassim Rousset ◽  
Kasper Renggli ◽  
Andreas Hierlemann ◽  
Olivier Frey
Keyword(s):  
Large Scale ◽  
Plastic Material ◽  
Microtiter Plate ◽  
Scale Production ◽  
Experimental Conditions ◽  
Precise Control ◽  
Open Design ◽  
Gravity Driven ◽  
Gravity Driven Flow

Microphysiological systems hold the promise to increase the predictive and translational power of in vitro substance testing owing to their faithful recapitulation of human physiology. However, the implementation of academic developments in industrial settings remains challenging. We present an injection-molded microfluidic microtissue (MT) culture chip that features two channels with 10 MT compartments each and that was designed in compliance with microtiter plate standard formats. Polystyrene as a chip material enables reliable, large-scale production and precise control over experimental conditions due to low adsorption or absorption of small, hydrophobic molecules at or into the plastic material in comparison with predecessor chips made of polydimethylsiloxane. The chip is operated by tilting, which actuates gravity-driven flow between reservoirs at both ends of every channel, so that the system does not require external tubing or pumps. The flow rate can be modulated by adjusting the tilting angle on demand. The top-open design of the MT compartment enables efficient MT loading using standard or advanced pipetting equipment, ensures oxygen availability in the chip, and allows for high-resolution imaging. Every channel can be loaded with up to 10 identical or different MTs, as demonstrated by culturing liver and tumor MTs in the same medium channel on the chip.

scholarly journals MMHub, a database for the mulberry metabolome

Database ◽  
2020 ◽  
Vol 2020 ◽  
Author(s):  
Dong Li ◽  
Bi Ma ◽  
Xiaofei Xu ◽  
Guo Chen ◽  
Tian Li ◽  
...  
Keyword(s):  
Mass Spectra ◽  
Chemical Compounds ◽  
Bioactive Metabolites ◽  
Molecular Formula ◽  
Experimental Conditions ◽  
Chemical Structures ◽  
Pubchem Compound ◽  
Specific Distribution ◽  
Small Chemical ◽  
Metabolome Data

Abstract Mulberry is an important economic crop plant and traditional medicine. It contains a huge array of bioactive metabolites such as flavonoids, amino acids, alkaloids and vitamins. Consequently, mulberry has received increasing attention in recent years. MMHub (version 1.0) is the first open public repository of mass spectra of small chemical compounds (<1000 Da) in mulberry leaves. The database contains 936 electrospray ionization tandem mass spectrometry (ESI-MS2) data and lists the specific distribution of compounds in 91 mulberry resources with two biological duplicates. ESI-MS2 data were obtained under non-standardized and independent experimental conditions. In total, 124 metabolites were identified or tentatively annotated and details of 90 metabolites with associated chemical structures have been deposited in the database. Supporting information such as PubChem compound information, molecular formula and metabolite classification are also provided in the MS2 spectral tag library. The MMHub provides important and comprehensive metabolome data for scientists working with mulberry. This information will be useful for the screening of quality resources and specific metabolites of mulberry. Database URL: https://biodb.swu.edu.cn/mmdb/

scholarly journals Challenges and Advances in Genome Editing Technologies in Streptomyces

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 734 ◽  
Author(s):  
Yawei Zhao ◽  
Guoquan Li ◽  
Yunliang Chen ◽  
Yinhua Lu
Keyword(s):  
Natural Product ◽  
Genome Editing ◽  
Genetic Manipulation ◽  
Chemical Compounds ◽  
Gene Clusters ◽  
Future Research ◽  
Biosynthetic Gene ◽  
Biosynthetic Gene Clusters ◽  
Research Focus ◽  
Experimental Conditions

The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

1987 ◽  
Vol 65 (7) ◽  
pp. 1416-1420 ◽  
Author(s):  
Jean-Pierre Caillé ◽  
Marie Pigeon-Gosselin ◽  
Michel Pézolet
Keyword(s):  
Raman Spectra ◽  
Sea Water ◽  
Muscle Fibers ◽  
Extraction Procedure ◽  
Organic Solutes ◽  
Experimental Conditions ◽  
Scattering Intensity ◽  
Barnacle Muscle ◽  
Α Helix ◽  
Barnacle Muscle Fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023–1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the α-helix conformation (1645–1650 cm−1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660–1670 cm−1 region where the amide I bands of either β-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm−1 is assigned to a change of orientation of α-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.

scholarly journals Switches induced by quorum sensing in a model of enzyme-loaded microparticles

2018 ◽  
Vol 15 (140) ◽  
pp. 20170945 ◽  
Author(s):  
Tamás Bánsági ◽  
Annette F. Taylor
Keyword(s):  
Drug Delivery ◽  
Quorum Sensing ◽  
Cell Density ◽  
Group Size ◽  
Enzyme Concentration ◽  
Autocatalytic Reaction ◽  
Enzyme Loading ◽  
High Enzyme ◽  
Different Types ◽  
Hydrolysis Of

Quorum sensing refers to the ability of bacteria and other single-celled organisms to respond to changes in cell density or number with population-wide changes in behaviour. Here, simulations were performed to investigate quorum sensing in groups of diffusively coupled enzyme microparticles using a well-characterized autocatalytic reaction which raises the pH of the medium: hydrolysis of urea by urease. The enzyme urease is found in both plants and microorganisms, and has been widely exploited in engineering processes. We demonstrate how increases in group size can be used to achieve a sigmoidal switch in pH at high enzyme loading, oscillations in pH at intermediate enzyme loading and a bistable, hysteretic switch at low enzyme loading. Thus, quorum sensing can be exploited to obtain different types of response in the same system, depending on the enzyme concentration. The implications for microorganisms in colonies are discussed, and the results could help in the design of synthetic quorum sensing for biotechnology applications such as drug delivery.

scholarly journals Be Aware of Aggregators in the Search for Potential Human ecto-5′-Nucleotidase Inhibitors

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 1876 ◽  
Author(s):  
Lucas Viviani ◽  
Erika Piccirillo ◽  
Arquimedes Cheffer ◽  
Leandro de Rezende ◽  
Henning Ulrich ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Kinetic Studies ◽  
Enzyme Concentration ◽  
Experimental Conditions ◽  
Time Aggregation ◽  
Compound C ◽  
Promising Target ◽  
Ic50 Values ◽  
Potential Inhibitors ◽  
Triton X

Promiscuous inhibition due to aggregate formation has been recognized as a major concern in drug discovery campaigns. Here, we report some aggregators identified in a virtual screening (VS) protocol to search for inhibitors of human ecto-5′-nucleotidase (ecto-5′-NT/CD73), a promising target for several diseases and pathophysiological events, including cancer, inflammation and autoimmune diseases. Four compounds (A, B, C and D), selected from the ZINC-11 database, showed IC50 values in the micromolar range, being at the same time computationally predicted as potential aggregators. To confirm if they inhibit human ecto-5′-NT via promiscuous mechanism, forming aggregates, enzymatic assays were done in the presence of 0.01% (v/v) Triton X-100 and an increase in the enzyme concentration by 10-fold. Under both experimental conditions, these four compounds showed a significant decrease in their inhibitory activities. To corroborate these findings, turbidimetric assays were performed, confirming that they form aggregate species. Additionally, aggregation kinetic studies were done by dynamic light scattering (DLS) for compound C. None of the identified aggregators has been previously reported in the literature. For the first time, aggregation and promiscuous inhibition issues were systematically studied and evaluated for compounds selected by VS as potential inhibitors for human ecto-5′-NT. Together, our results reinforce the importance of accounting for potential false-positive hits acting by aggregation in drug discovery campaigns to avoid misleading assay results.

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