A high-throughput method for the quantification of iron saturation in lactoferrin preparations.
Ontology highlight
ABSTRACT:
SUBMITTER: Majka G
PROVIDER: S-EPMC3656221 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Calcium carbonate which is extracted from the Earth in combination with other mineral impurities, is largely used in preparations for pediatric supplements. Elemental impurities in drug products pose toxicological concerns without therapeutic benefits. Thus, it is very urgent to assess the safety of chronic exposure to elements that may be present in trace amounts. In the present study, we developed high throughput ICP-MS method for the quantitative determination of 62 elemental impurities in high matric calcium carbonate samples and validated according to USP 233. Calcium carbonate preparations which state clearly used for child (including neonates, infants, toddlers and children) from 9 manufactures and two types of raw materials (light calcium carbonate and ground calcium carbonate) were investigated in terms of the content and variability of 62 elemental impurities. According to the results, ground calcium carbonate was more suitable to be used in pediatric preparations concerning elemental impurities. Parts of elemental impurities in CaCO3 preparations which are derived from the raw materials and the preparation process, may cause potential risks for children. These results indicate that it is necessary to establish a modern instrumental analysis method to evaluate and control elemental impurities in CaCO3 raw materials and preparations.
| S-EPMC8173443 | biostudies-literature
Project description:Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.
| S-EPMC5159830 | biostudies-literature
Project description:Introduction:Global metabolomics analyses using body fluids provide valuable results for the understanding and prediction of diseases. However, the mechanism of a disease is often tissue-based and it is advantageous to analyze metabolomic changes directly in the tissue. Metabolomics from tissue samples faces many challenges like tissue collection, homogenization, and metabolite extraction. Objectives:We aimed to establish a metabolite extraction protocol optimized for tissue metabolite quantification by the targeted metabolomics AbsoluteIDQ™ p180 Kit (Biocrates). The extraction method should be non-selective, applicable to different kinds and amounts of tissues, monophasic, reproducible, and amenable to high throughput. Methods:We quantified metabolites in samples of eleven murine tissues after extraction with three solvents (methanol, phosphate buffer, ethanol/phosphate buffer mixture) in two tissue to solvent ratios and analyzed the extraction yield, ionization efficiency, and reproducibility. Results:We found methanol and ethanol/phosphate buffer to be superior to phosphate buffer in regard to extraction yield, reproducibility, and ionization efficiency for all metabolites measured. Phosphate buffer, however, outperformed both organic solvents for amino acids and biogenic amines but yielded unsatisfactory results for lipids. The observed matrix effects of tissue extracts were smaller or in a similar range compared to those of human plasma. Conclusion:We provide for each murine tissue type an optimized high-throughput metabolite extraction protocol, which yields the best results for extraction, reproducibility, and quantification of metabolites in the p180 kit. Although the performance of the extraction protocol was monitored by the p180 kit, the protocol can be applicable to other targeted metabolomics assays.
| S-EPMC5748028 | biostudies-literature
Project description:Colony forming unit (CFU) determination by agar plating is still regarded as the gold standard for biofilm quantification despite being time- and resource-consuming. Here, we propose an adaption of the high-throughput Start-Growth-Time (SGT) method from planktonic to biofilm analysis, which indirectly quantifies CFU/mL numbers by evaluating regrowth curves of detached biofilms. For validation, the effect of dalbavancin, rifampicin and gentamicin against mature biofilms of Staphylococcus aureus and Enterococcus faecium was measured by accessing different features of the viability status of the cell, i.e., the cultivability (conventional agar plating), growth behavior (SGT) and metabolic activity (resazurin assay). SGT correlated well with the resazurin assay for all tested antibiotics, but only for gentamicin and rifampicin with conventional agar plating. Dalbavancin treatment-derived growth curves showed a compared to untreated controls significantly slower increase with reduced cell doubling times and reduced metabolic rate, but no change in CFU numbers was observed by conventional agar plating. Here, unspecific binding of dalbavancin to the biofilm interfered with the SGT methodology since the renewed release of dalbavancin during detachment of the biofilms led to an unintended antimicrobial effect. The application of the SGT method for anti-biofilm testing is therefore not suited for antibiotics which stick to the biofilm and/or to the bacterial cell wall. Importantly, the same applies for the well-established resazurin method for anti-biofilm testing. However, for antibiotics which do not bind to the biofilm as seen for gentamicin and rifampicin, the SGT method presents a much less labor-intensive method suited for high-throughput screening of anti-biofilm compounds.
| S-EPMC8428173 | biostudies-literature
Project description:Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.
| S-EPMC7376206 | biostudies-literature
Project description:The accurate determination of allele frequencies is crucially important across a wide range of problems in genetics, such as developing population genetic models, making inferences from genome-wide association studies, determining genetic risk for diseases, as well as other scientific and medical applications. Furthermore, understanding how allele frequencies change over time in populations is central to ascertaining their evolutionary dynamics. We present a precise, efficient, and economical method (FREQ-Seq2) for quantifying the relative frequencies of different alleles at loci of interest in mixed population samples. Through the creative use of paired barcode sequences, we exponentially increased the throughput of the original FREQ-Seq method from 48 to 2,304 samples. FREQ-Seq2 can be targeted to specific genomic regions of interest, which are amplified using universal barcoded adapters to generate Illumina sequencing libraries. Our enhanced method, available as a kit along with open-source software for analyzing sequenced libraries, enables the detection and removal of errors that are undetectable in the original FREQ-Seq method as well as other conventional methods for allele frequency quantification. Finally, we validated the performance of our sequencing-based approach with a highly multiplexed set of control samples as well as a competitive evolution experiment in Escherichia coli and compare the latter to estimates derived from manual colony counting. Our analyses demonstrate that FREQ-Seq2 is flexible, inexpensive, and produces large amounts of data with low error, low noise, and desirable statistical properties. In summary, FREQ-Seq2 is a powerful method for quantifying allele frequency that provides a versatile approach for profiling mixed populations.
| S-EPMC10542570 | biostudies-literature
Project description:Reporter-based assays underlie many high-throughput screening (HTS) platforms, but most are limited to in vitro applications. Here, we report a simple whole-organism HTS method for quantifying changes in reporter intensity in individual zebrafish over time termed, Automated Reporter Quantification in vivo (ARQiv). ARQiv differs from current "high-content" (e.g., confocal imaging-based) whole-organism screening technologies by providing a purely quantitative data acquisition approach that affords marked improvements in throughput. ARQiv uses a fluorescence microplate reader with specific detection functionalities necessary for robust quantification of reporter signals in vivo. This approach is: 1) Rapid; achieving true HTS capacities (i.e., >50,000 units per day), 2) Reproducible; attaining HTS-compatible assay quality (i.e., Z'-factors of ≥0.5), and 3) Flexible; amenable to nearly any reporter-based assay in zebrafish embryos, larvae, or juveniles. ARQiv is used here to quantify changes in: 1) Cell number; loss and regeneration of two different fluorescently tagged cell types (pancreatic beta cells and rod photoreceptors), 2) Cell signaling; relative activity of a transgenic Notch-signaling reporter, and 3) Cell metabolism; accumulation of reactive oxygen species. In summary, ARQiv is a versatile and readily accessible approach facilitating evaluation of genetic and/or chemical manipulations in living zebrafish that complements current "high-content" whole-organism screening methods by providing a first-tier in vivo HTS drug discovery platform.
| S-EPMC3251595 | biostudies-literature
Project description:High throughput screening (HTS) projects like the U.S. Environmental Protection Agency's ToxCast program are required to address the large and rapidly increasing number of chemicals for which we have little to no toxicity measurements. Concentration-response parameters such as potency and efficacy are extracted from HTS data using nonlinear regression, and models and analyses built from these parameters are used to predict in vivo and in vitro toxicity of thousands of chemicals. How these predictions are impacted by uncertainties that stem from parameter estimation and propagated through the models and analyses has not been well explored. While data size and complexity makes uncertainty quantification computationally expensive for HTS datasets, continued advancements in computational resources have allowed these computational challenges to be met. This study uses nonparametric bootstrap resampling to calculate uncertainties in concentration-response parameters from a variety of HTS assays. Using the ToxCast estrogen receptor model for bioactivity as a case study, we highlight how these uncertainties can be propagated through models to quantify the uncertainty in model outputs. Uncertainty quantification in model outputs is used to identify potential false positives and false negatives and to determine the distribution of model values around semi-arbitrary activity cutoffs, increasing confidence in model predictions. At the individual chemical-assay level, curves with high variability are flagged for manual inspection or retesting, focusing subject-matter-expert time on results that need further input. This work improves the confidence of predictions made using HTS data, increasing the ability to use this data in risk assessment.
| S-EPMC6059398 | biostudies-other
Project description:Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2×10(8) TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ≈1.3 days) or subjected to multiple freeze-thaw cycles (τ=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.
| S-EPMC4562269 | biostudies-literature
Project description:BACKGROUND: Protein quantification is an essential step in many proteomics experiments. A number of labeling approaches have been proposed and adopted in mass spectrometry (MS) based relative quantification. The mTRAQ, one of the stable isotope labeling methods, is amine-specific and available in triplex format, so that the sample throughput could be doubled when compared with duplex reagents. METHODS AND RESULTS: Here we propose a novel data analysis algorithm for peptide quantification in triplex mTRAQ experiments. It improved the accuracy of quantification in two features. First, it identified and separated triplex isotopic clusters of a peptide in each full MS scan. We designed a schematic model of triplex overlapping isotopic clusters, and separated triplex isotopic clusters by solving cubic equations, which are deduced from the schematic model. Second, it automatically determined the elution areas of peptides. Some peptides have similar atomic masses and elution times, so their elution areas can have overlaps. Our algorithm successfully identified the overlaps and found accurate elution areas. We validated our algorithm using standard protein mixture experiments. CONCLUSIONS: We showed that our algorithm was able to accurately quantify peptides in triplex mTRAQ experiments. Its software implementation is compatible with Trans-Proteomic Pipeline (TPP), and thus enables high-throughput analysis of proteomics data.
| S-EPMC3044303 | biostudies-other