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Frontiers in Toxicology 2022The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM...
The methods outlined here are part of a series of papers designed specifically for genotoxicity assessment of nanomaterials (NM). Common Considerations such as NM characterization, sample preparation and dose selection, relevant to all genotoxicity assays, are found in an accompanying paper. The present paper describes methods for evaluation of mutagenicity in the mammalian (mouse) () gene occurring in L5178Y mouse lymphoma (ML) cells and in the designated gene in human lymphoblastoid TK6 cells. Mutations change the functional genotype from TK to TK, detectable as cells surviving on media selective for the lack of thymidine kinase (TK) function. Unlike cells with TK enzyme function, the TK cells are unable to integrate the toxic selection agent, allowing these cells to survive as rare mutant colonies. The ML assay has been shown to detect a broad spectrum of genetic damage, including both small scale (point) mutations and chromosomal alterations. This assay is a widely used mammalian cell gene mutation assay for regulatory purposes and is included in the core battery of genotoxicity tests for regulatory decision-making. The TK6 assay is an assay using a human cell line derived similarly via mutagenic manipulations and optimal selection. Details are provided on the materials required, cell culture methods, selection of test chemical concentrations, cytotoxicity, treatment time, mutation expression, cloning, and data calculation and interpretation. The methods describe the microwell plate version of the assays without metabolic activation.
PubMed: 35757197
DOI: 10.3389/ftox.2022.864753 -
PloS One 2020Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of...
Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available-particularly for common model organisms like E. coli-fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we exchanged the original fluorescent reporter in a previously developed plasmid (pBSU101) with one of eight fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different phases of aerobic growth. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations under aerobic growth conditions. Finally, we incorporated codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.
Topics: Biofilms; Enterococcus faecalis; Fluorescence; Luminescent Proteins; Plasmids; Single-Cell Analysis
PubMed: 32369497
DOI: 10.1371/journal.pone.0232539 -
Analytical Chemistry Jan 2021Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several...
Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1-500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.
Topics: Automation; Chromatography, Liquid; HeLa Cells; Humans; Proteome; Proteomics; Tandem Mass Spectrometry; Tumor Cells, Cultured
PubMed: 33352054
DOI: 10.1021/acs.analchem.0c04240 -
Frontiers in Microbiology 2022In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is...
In order to exploit the microbes present in the environment for their beneficial resources, effective selection and isolation of microbes from environmental samples is essential. In this study, we fabricated a gel-filled microwell array device using resin for microbial culture. The device has an integrated sealing mechanism that enables high-density isolation based on the culture of microorganisms; the device is easily manageable, facilitating observation using bright-field microscopy. This low-cost device made from polymethyl methacrylate (PMMA)/polyethylene terephthalate (PET) has 900 microwells (600 μm × 600 μm × 700 μm) filled with a microbial culture gel medium in glass slide-sized plates. It also has grooves for maintaining the moisture content in the micro-gel. The partition wall between the wells has a highly hydrophobic coating to inhibit microbial migration to neighboring wells and to prevent exchange of liquid substances. After being hermetically sealed, the device can maintain moisture in the agarose gels for 7 days. In the bacterial culture experiment using this device, environmental bacteria were isolated and cultured in individual wells after 3 days. Moreover, the isolated bacteria were then picked up from wells and re-cultured. This device is effective for the first screening of microorganisms from marine environmental samples.
PubMed: 36590440
DOI: 10.3389/fmicb.2022.1031439 -
Journal of Virological Methods Dec 2009This study describes a colorimetric method for detecting and genotyping hepatitis C virus (HCV) in which four different oligonucleotide probes are fixed onto microwell...
This study describes a colorimetric method for detecting and genotyping hepatitis C virus (HCV) in which four different oligonucleotide probes are fixed onto microwell plates and hybridized separately with biotinylated PCR amplification products derived from clinical samples. The first probe capable of hybridizing with all seven known HCV genotypes was used for overall detection, and the remaining probes were used to recognize specifically genotypes 1-3. When combined with an improved silica-based RNA extraction method, the sensitivity of the test was 50 IU/mL. Eighty-five of the 86 samples analyzed (98.8%) yielded results in agreement with reference detection methods. The remaining sample was HCV-RNA positive in the COBAS Amplicor qualitative assay, but was negative using the reverse-hybridization method. The usefulness of the new genotyping test was confirmed by comparison with direct sequencing of PCR products: 98% of samples tested (54/55) were in agreement using the two methods (21, 7 and 27 from genotypes 1-3, respectively). The single discrepancy might have been due to a mixed HCV infection. The new method is an alternative to the use of commercially available genotyping kits and should be particularly convenient in developing countries where genotypes 1-3 represent a high proportion of HCV isolates.
Topics: Biotinylation; Colorimetry; Genotype; Hepacivirus; Humans; Nucleic Acid Hybridization; Oligonucleotide Probes; Polymerase Chain Reaction; RNA, Viral; Reagent Kits, Diagnostic; Species Specificity
PubMed: 19643142
DOI: 10.1016/j.jviromet.2009.07.017 -
F1000Research 2022Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires...
Animal models have provided many insights into ocular development and disease, but they remain suboptimal for understanding human oculogenesis. Eye development requires spatiotemporal gene expression patterns and disease phenotypes can differ significantly between humans and animal models, with patient-associated mutations causing embryonic lethality reported in some animal models. The emergence of human induced pluripotent stem cell (hiPSC) technology has provided a new resource for dissecting the complex nature of early eye morphogenesis through the generation of three-dimensional (3D) cellular models. By using patient-specific hiPSCs to generate optic vesicle-like models, we can enhance the understanding of early developmental eye disorders and provide a pre-clinical platform for disease modelling and therapeutics testing. A major challenge of optic vesicle generation is the low efficiency of differentiation in 3D cultures. To address this, we adapted a previously published protocol of retinal organoid differentiation to improve embryoid body formation using a microwell plate. Established morphology, upregulated transcript levels of known early eye-field transcription factors and protein expression of standard retinal progenitor markers confirmed the optic vesicle/presumptive optic cup identity of models between day 20 and 50 of culture. This adapted protocol is relevant to researchers seeking a physiologically relevant model of early human ocular development and disease with a view to replacing animal models.
Topics: Animals; Cell Differentiation; Embryoid Bodies; Humans; Induced Pluripotent Stem Cells; Retina; Transcription Factors
PubMed: 35811797
DOI: 10.12688/f1000research.108829.1 -
Applied and Environmental Microbiology May 2022The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment....
The capacity to defluorinate polyfluorinated organic compounds is a rare phenotype in microbes but is increasingly considered important for maintaining the environment. New discoveries will be greatly facilitated by the ability to screen many natural and engineered microbes in a combinatorial manner against large numbers of fluorinated compounds simultaneously. Here, we describe a low-volume, high-throughput screening method to determine defluorination capacity of microbes and their enzymes. The method is based on selective binding of fluoride to a lanthanum chelate complex that gives a purple-colored product. It was miniaturized to determine biodefluorination in 96-well microtiter plates by visual inspection or robotic handling and spectrophotometry. Chemicals commonly used in microbiological studies were examined to define usable buffers and reagents. Base-catalyzed, purified enzyme and whole-cell defluorination reactions were demonstrated with fluoroatrazine and showed correspondence between the microtiter assay and a fluoride electrode. For discovering new defluorination reactions and mechanisms, a chemical library of 63 fluorinated compounds was screened with Pseudomonas putida F1 in microtiter well plates. These data were also calibrated against a fluoride electrode. Our new method revealed 21 new compounds undergoing defluorination. A compound with four fluorine substituents, 4-fluorobenzotrifluoride, was shown to undergo defluorination to the greatest extent. The mechanism of its defluorination was studied to reveal a latent microbial propensity to defluorinate trifluoromethylphenyl groups, a moiety that is commonly incorporated into numerous pharmaceutical and agricultural chemicals. Thousands of organofluorine chemicals are known, and a number are considered to be persistent and toxic environmental pollutants. Environmental bioremediation methods are avidly being sought, but few bacteria biodegrade fluorinated chemicals. To find new organofluoride biodegradation, a rapid screening method was developed. The method is versatile, monitoring chemical, enzymatic, and whole-cell biodegradation. Biodegradation of organofluorine compounds invariably releases fluoride anions, which was sensitively detected. Our method uncovered 21 new microbial defluorination reactions. A general mechanism was delineated for the biodegradation of trifluoromethylphenyl groups that are increasingly being used in drugs and pesticides.
Topics: Biodegradation, Environmental; Fluorides; Fluorine; Pseudomonas putida
PubMed: 35435713
DOI: 10.1128/aem.00288-22 -
Nature Communications Jan 2023Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with...
Sensing small biomolecules in biofluids remains challenging for many optical chemosensors based on supramolecular host-guest interactions due to adverse interplays with salts, proteins, and other biofluid components. Instead of following the established strategy of developing alternative synthetic binders with improved affinities and selectivity, we report a molecular engineering approach that addresses this biofluid challenge. Here we introduce a cucurbit[8]uril-based rotaxane chemosensor feasible for sensing the health-relevant biomarker tryptophan at physiologically relevant concentrations, even in protein- and lipid-containing human blood serum and urine. Moreover, this chemosensor enables emission-based high-throughput screening in a microwell plate format and can be used for label-free enzymatic reaction monitoring and chirality sensing. Printed sensor chips with surface-immobilized rotaxane-microarrays are used for fluorescence microscopy imaging of tryptophan. Our system overcomes the limitations of current supramolecular host-guest chemosensors and will foster future applications of supramolecular sensors for molecular diagnostics.
Topics: Humans; Serum; Rotaxanes; Tryptophan; Body Fluids
PubMed: 36720875
DOI: 10.1038/s41467-023-36057-3 -
The Analyst Apr 2020The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was...
The majority of bioassays are cell-lethal and thus cannot be used for cell assay and selection prior to live-cell sorting. A quad microraft array-based platform was developed to perform semi-automated cell sampling, bioassay, and banking on ultra-small sample sizes. The system biopsies and collects colony fragments, quantifies intracellular protein levels via immunostaining, and then retrieves the living mother colonies based on the fragments' immunoassay outcome. To accomplish this, a magnetic, microwell-based plate was developed to mate directly above the microraft array and capture colony fragments with a one-to-one spatial correspondence to their mother colonies. Using the Signal Transducer and Activator of Transcription 3 (STAT3) model pathway in basophilic leukemia cells, the system was used to sort cells based on the amount of intracellular STAT3 protein phosphorylation (pSTAT3). Colonies were detected on quad arrays using bright field microscopy with 96 ± 20% accuracy (true-positive rate), 49 ± 3% of the colonies were identified as originating from a single cell, and the majority (95 ± 3%) of biopsied clonal fragments were successfully collected into the microwell plate for immunostaining. After assay, biopsied fragments were matched back to their mother colonies and mother colonies with fragments possessing the greatest and least pSTAT3/STAT3 were resampled for expansion and downstream biological assays for pSTAT3/STAT3 and immune granule exocytosis. This approach has the potential to enable colony screening and sorting based on assays not compatible with cell viability, greatly expanding the cell selection criteria available to identify cells with unique phenotypes for subsequent biomedical research.
Topics: Animals; Cell Line, Tumor; Humans; Image Processing, Computer-Assisted; Immunoassay; Leukemia, Basophilic, Acute; Magnetics; Microarray Analysis; Microscopy, Fluorescence; Phosphorylation; Rats; STAT3 Transcription Factor
PubMed: 32048684
DOI: 10.1039/d0an00030b -
RSC Advances Oct 2023This study describes the development of two highly sensitive and selective sensor-assisted fluorescence immunoassays for the trace determination of copper ions, Cu(ii)...
This study describes the development of two highly sensitive and selective sensor-assisted fluorescence immunoassays for the trace determination of copper ions, Cu(ii) residues, in food samples. These assays were the microwell-based fluoroimmuoassay (FIA) and the kinetic exclusion assay (KinExA). FIA and KinExA were assisted by a microplate reader and a KinExA™ 3200 immunosensor, respectively. Both FIA and KinExA were developed utilizing the same antibody, capturing reagent, and fluorescence signal-generating reagent. The antibody was a mouse monoclonal antibody, designated as 8D66, that specifically recognized the Cu(ii)-ethylenediaminetetraacetic acid complex (Cu(ii)-EDTA) but did not recognize Cu(ii)-free EDTA. The capturing reagent was Cu(ii)-EDTA covalently linked to bovine serum albumin protein (Cu(ii)-EDTA-BSA). The fluorescence-generating reagent was an anti-mouse IgG conjugated with fluorescein isothiocyanate (IgG-FITC). Both FIA and KinExA involved competitive binding reactions between Cu(ii)-EDTA complexes, formed in the sample solution, and Cu(ii)-EDTA-BSA conjugate which has been immobilized onto microwell fluorescence assay plates (in FIA) or polymethylmethacrylate beads (in KinExA) for a limited quantity of binding sites of 8D66 antibody. The conditions of both FIA and KinExA were investigated, and the optimum procedures were established. Both FIA and KinExA were validated, and all validation parameters were acceptable. Many different metal ions that are commonly encountered in food samples did not interfere with Cu(ii) analysis by both FIA and KinExA. Both assays were applied to the determination of Cu(ii) in food samples with satisfactory accuracy and precision. Both assays were compared favorably with inductively coupled plasma atomic emission spectroscopy. Comparative evaluation of FIA and KinExA revealed that KinExA had higher sensitivity and better precision than FIA, whereas, both assays had comparable accuracy. Both FIA and KinExA were superior to the existing atomic spectrometric methods for Cu(ii). The proposed FIA and KinExA are anticipated to effectively contribute to assessing Cu(ii) concentrations and controlling the exposure of humans to its potential toxicities.
PubMed: 37818275
DOI: 10.1039/d3ra04415g