Evaluation of Methods To Assess the Biofilm-Forming Ability of Listeria monocytogenes

2012 ◽  
Vol 75 (8) ◽  
pp. 1411-1417 ◽  
Author(s):  
ANTÓNIO LOURENÇO ◽  
FRANCISCO REGO ◽  
LUISA BRITO ◽  
JOSEPH F. FRANK
Keyword(s):  
Stainless Steel ◽  
Listeria Monocytogenes ◽  
High Throughput ◽  
Biofilm Formation ◽  
High Throughput Screening ◽  
Screening Methods ◽  
Genetic Lineage ◽  
Production Lines ◽  
Genetic Characteristics

The contamination of ready-to-eat products with Listeria monocytogenes has been related to the presence of biofilms in production lines, as biofilms protect cells from chemical sanitizers. The ability of L. monocytogenes to produce biofilms is often evaluated using in vitro methodologies. This work aims to compare the most frequently used methodologies, including high-throughput screening methods based on microplates (crystal violet and the Calgary Biofilm Device) and methods based on CFU enumeration and microscopy after growth on stainless steel. Thirty isolates with diverse origins and genetic characteristics were evaluated. No (or low) correlations between methods were observed. The only significant correlation was found between the methods using stainless steel. No statistically significant correlation (P > 0.05) was detected among genetic lineage, serovar, and biofilm-forming ability. Because results indicate that biofilm formation is influenced by the surface material, the extrapolation of results from high-throughput methods using microplates to more industrially relevant surfaces should be undertaken with caution.

scholarly journals Recent Advances in In-Vitro Assays for Type 2 Diabetes Mellitus: An Overview

2020 ◽  
Vol 16 (1) ◽  
pp. 13-23
Author(s):  
Nazmina Vhora ◽  
Ujjal Naskar ◽  
Aishwarya Hiray ◽  
Abhijeet S. Kate ◽  
Alok Jain
Keyword(s):  
Type 2 Diabetes ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antidiabetic Activity ◽  
Screening Methods ◽  
Selection Of

BACKGROUND: A higher rate of attenuation of molecules in drug discovery has enabled pharmaceutical companies to enhance the efficiency of their hit identification and lead optimization. Selection and development of appropriate in-vitro and in-vivo strategies may improve this process as primary and secondary screening utilize both strategies. In-vivo approaches are too relentless and expensive for assessing hits. Therefore, it has become indispensable to develop and implement suitable in-vitro screening methods to execute the required activities and meet the respective targets. However, the selection of an appropriate in-vitro assay for specific evaluation of cellular activity is no trivial task. It requires thorough investigation of the various parameters involved. AIM: In this review, we aim to discuss in-vitro assays for type 2 diabetes (T2D), which have been utilized extensively by researchers over the last five years, including target-based, non-target based, low-throughput, and high-throughput screening assays. METHODS: The literature search was conducted using databases including Scifinder, PubMed, ScienceDirect, and Google Scholar to find the significant published articles. DISCUSSION and CONCLUSION: The accuracy and relevance of in-vitro assays have a significant impact on the drug discovery process for T2D, especially in assessing the antidiabetic activity of compounds and identifying the site of effect in high-throughput screening. The report reviews the advantages, limitations, quality parameters, and applications of the probed invitro assays, and compares them with one another to enable the selection of the optimal method for any purpose. The information on these assays will accelerate numerous procedures in the drug development process with consistent quality and accuracy.

scholarly journals High-throughput screening of biomolecules using cell-free gene expression systems

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Luis E Contreras-Llano ◽  
Cheemeng Tan
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Screening Methods ◽  
Free Gene ◽  
On Chip ◽  
Translation Systems ◽  
Selection Of

Abstract The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

Development and assessment of a high-throughput biofilm and biomass testing platform

2021 ◽  
Vol 30 (Sup7) ◽  
pp. S36-S46
Author(s):  
Hosan Kim ◽  
Matthew Aquino ◽  
Mina Izadjoo
Keyword(s):  
Growth Rate ◽  
High Throughput ◽  
Bacterial Growth ◽  
Biofilm Formation ◽  
High Throughput Screening ◽  
Bacterial Strains ◽  
Bacterial Growth Rate ◽  
Testing Platform ◽  
Biofilm Development

Objective: To develop and evaluate a simple platform technology for developing static biofilms in a 96-well microtitre plate for various downstream applications. The technology allows monitoring of growth rate, biofilm formation and quantifying biofilm biomass by using crystal violet (CV) and safranin O (SO) staining over seven-day time periods for pathogens including clinical isolates most commonly associated with hard-to-treat wound infections. Method: A total of 157 bacteria including Acinetobacter, Enterobacter, Klebsiella, Pseudomonas and Staphylococcus spp. were used in the study. Bacterial growth was measured at 600nm optical density (OD). Biofilm formation was monitored and assessed quantitatively with CV at 570nm and SO staining at 492nm for one-, two-, three- and seven-day incubation periods. Results: Bacterial growth rate and static biofilm biomass in the 96-well plates varied for various strains tested. Both CV and SO staining showed similar results in the biomass, with SO assay displaying more reproducible data throughout the study. Most of the strains were metabolically active even at the seven-day incubation period. Microbial adherences of all bacterial strains on the plastic surface was assessed with CV staining: 28 Acinetobacter, 17 Staphylococcus, 12 Pseudomonas and four Enterobacter strains were strong biofilm producers. Moderate biofilm-producing strains included 27 Staphylococcus, 14 Acinetobacter, eight Pseudomonas and three Enterobacter. Weak biofilm-producing strains included: 33 Staphylococcus, six Enterobacter, two Pseudomonas and one Acinetobacter. Only one Pseudomonas aeruginosa strain did not develop biofilm. Conclusion: Our results demonstrate the feasibility of using 96-well microtitre plates as a high-throughput platform for quantitative measurement and assessment of biofilm development over time. Studying microbial adherence or biofilm biomass generated on various surfaces using a high-throughput system could provide valuable information for in vitro testing and developing therapeutics for biofilm infections. Employing the biofilm testing platform described in this study makes it possible to simultaneously develop different biofilms formed by specific pathogens, and study potential association between the quantity of bacterial biomass and strength of a biofilm formed by specific wound pathogens. In addition, the described testing approach could provide an optimal model for standardised and high-throughput screening of candidate antibiofilm therapeutics.

From Classical to High Throughput Screening Methods for Feruloyl Esterases: A Review

2016 ◽  
Vol 19 (8) ◽  
pp. 616-626 ◽  
Author(s):  
Lorena Ramírez-Velasco ◽  
Mariana Armendáriz-Ruiz ◽  
Jorge Alberto Rodríguez-González ◽  
Marcelo Müller-Santos ◽  
Ali Asaff-Torres ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Methods ◽  
Feruloyl Esterases

scholarly journals A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities

2020 ◽  
Author(s):  
Yuru Wang ◽  
Christopher D Katanski ◽  
Christopher Watkins ◽  
Jessica N Pan ◽  
Qing Dai ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Targeted Mutagenesis ◽  
Rna Repair ◽  
Dna And Rna ◽  
Modified Dna ◽  
Dna Substrates ◽  
Trna Sequencing

Abstract AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhou Fang ◽  
Junjian Chen ◽  
Ye Zhu ◽  
Guansong Hu ◽  
Haoqian Xin ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rational Design ◽  
Click Reaction ◽  
Reaction Times ◽  
Great Promise ◽  
Biomaterial Surfaces

AbstractPeptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

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