Microplate Format Research Articles

Affordable infectious pathogen detection using a dual-mode biosensor integrating exonuclease III-assisted target recycling amplification with high-throughput 96-well microplate format

The ongoing challenge of infectious pathogens highlights the need for accurate and accessible methods to discern their genetic signatures, especially in resource-limited settings. In response to this crucial requirement, we introduce an affordable large-scale screening platform for infectious pathogen detection, using Hepatitis B virus (HBV) as a fundamental model. This proposed biosensor integrates an exonuclease III-assisted target recycling amplification strategy within a high-throughput 96-well microplate format. The HBV DNA target binds to a capture probe DNA and exonuclease III digests the probe to release the target. This mechanism enables the target to engage in binding cycles with new probes, each digested in turn, increasing detection sensitivity for even small quantities of HBV DNA. The implemented approach incorporates a biotin-streptavidin interaction allowing the undigested capture probe DNA to bind to a 5′-biotin-modified detection probe for effective HBV DNA quantification. This interaction generates a signal that, following the enzyme-substrate reaction, can be detected on-site using a smartphone, offering either optical or electrochemical readouts. The developed biosensor was capable of detecting HBV DNA with a detection limit of 5.62 fM and provided a considerable linear range covering concentrations from 100 fM to 100 nM. The determination of HBV DNA quantities in spiked human serum was achieved with a recovery of 90.0 % – 107.4 % as well. The results suggest that the developed dual-mode biosensor offers an adaptable and cost-effective approach for detecting infectious diseases, with promising applications in medical diagnostics and environmental monitoring to support public health efforts.

6651 A Traceable Clinical Free Thyroxine Assay Based on Equilibrium Dialysis ID-LC/MS/MS

Abstract Disclosure: D. Spector: None. L. Zhang: None. A. Ribera: None. A. Doty: None. C. Tse: None. O. Sugahara: None. N. Vazquez: None. U. Danilenko: None. H.W. Vesper: None. Accurate diagnosis and effective treatment of thyroid diseases rely on reliable thyroid function tests. Serum free thyroxine (FT4) immunoassays (IAs) are most commonly used for FT4 measurements in clinical practice. However, FT4 IAs have not been standardized with large variations between different assay platforms. A reference measurement procedure (RMP) for FT4 based on well-defined equilibrium dialysis (ED) of serum has been established for CDC standardization program to support the standardization of clinical assays for patient care. In the current study, we aim to develop a routine clinical FT4 assay based on ED-LC/MS/MS that is traceable to the RMP. ED-LC/MS/MS analysis was performed using a commercially available micro-ED plate. FT4 in dialysate was extracted with a 96-well C18 SPE plate. FT4 in the samples was quantitated by using LC/MS/MS in positive ion mode. The assay was calibrated with the certified primary reference material. By using RMP as a reference, we compared the analysis of 40 single donor sera between the ED-LC/MS/MS assay and FT4 IA on Roche e401platform. ED-LC/MS/MS assay could resolve T4, T3, reverse triiodothyronine (rT3), and other interferences in the samples with C18 chromatography in 4 min. The linear range of the routine FT4 assay was from 0.5-100 pg/mL covering clinically relevant FT4 concentrations including hypo-, hyperthyroid patient samples. The intra and inter-run CV% of the assay in 20 days was 3.72 and 5.25%, respectively. The effect of minor changes to the method conditions was studied. It was determined that serum/buffer ratios from 1:1 to 1:2 in the ED inserts, speeds of shaker for ED, and ED incubation time, did not influence the FT4 measurement. However, elevated ED temperature at 38°C could result in a more than 10% increase in FT4 values. A microplate format for the procedures of ED and SPE is amiable to the application of an automation system which significantly improves the throughput of the procedure. Method comparison showed good agreement between ED-LC/MS/MS assay and RMP in Deming regression analysis with a slope close to 1, while there was significant bias of IA from RMP. ED-LC/MS/MS assay was also applied for pregnancy serum samples with minimal difference on the FT4 measurement from RMP. We have developed a routine clinical FT4 assay that is traceable to RMP. The accuracy, precision, and sensitivity of the assay are suitable for high-throughput FT4 measurements in clinical laboratories and large epidemiologic studies. We demonstrate that the developed method can provide accurate FT4 measurements in patients including pregnant women. Presentation: 6/3/2024

A-299 Assessment of a new miniaturized line blot system for fully automated serodiagnosis of Borrelia infections

Abstract Background Serodiagnosis of Lyme disease caused by Borrelia burgdorferi commonly affords the combination of a sensitive antibody screening test followed by confirmatory immunoblot analysis. We developed and evaluated a new immunoblot system enabling the multiparametric analysis of anti-Borrelia antibodies by means of miniaturized line blots in a fully automated microplate format. Methods The EUROMicroblots Anti-Borrelia (IgG and IgM, EUROIMMUN) are based on diagnostically relevant Borrelia-specific antigens. Diagnostic sensitivity was determined by analyzing 203 pre-characterized samples from patients with Lyme disease stratified by clinical picture and antibiotic treatment. Specificity was assessed using 168 sera from patients clinically diagnosed with rheumatic diseases. The reference range was analyzed using a control panel of 211 (IgG) and 210 (IgM) samples from healthy individuals, comprising 151 (IgG) and 150 (IgM) blood donors as well as 60 pregnant women (IgG, IgM). The Microwell Imager and EUROLineScan software (EUROIMMUN) were used for image acquisition and evaluation. Results Depending on the clinical category, the diagnostic sensitivity of the EUROMicroblots ranged from 68.0% in the early phase of infection to 100% (IgG/IgM combined) at a specificity of 86.9% (IgG) and 94.0% (IgM). The analysis of the panel of healthy control subjects revealed a prevalence of 10.9% (IgG) and 2.8% (IgM). Conclusions The new EUROMicroblot system enables high-throughput serodiagnosis of Borrelia infections due to the microplate format and automated processing and evaluation. Depending on clinical symptoms and treatment, the assays provide excellent diagnostic sensitivity and specificity for the detection of anti-Borrelia IgG and IgM.

Simultaneous spectral illumination of microplates for high-throughput optogenetics and photobiology.

The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis ofaction spectra. Current methods to determine action spectraonly allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination. The RainbowCap provides equal photon flux for each wavelength, with the output of the LEDs narrowed by optical bandpass filters. We validatedthe RainbowCap for photoactivatable G protein-coupled receptors (opto-GPCRs) and enzymes for the control of intracellular downstream signaling. The simultaneous, spectrally defined illumination provides minimal interruption during time-series measurements, while resolving 10 nm differences in the action spectra of optogenetic proteins under identical experimental conditions. The RainbowCap is also suitable for studying the spectral dependence of light-regulated gene expression in bacteria, which requires illumination over several hours. In summary, the RainbowCap provides high-throughput spectral illumination of microplates, while its modular, customizable design allows easy adaptation to a wide range of optogenetic and photobiological applications.

Loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles for colorimetric detection of Trichoderma spp. in Agaricus bisporus cultivation substrates

One of the significant challenges in organic cultivation of edible mushrooms is the control of invasive Trichoderma species that can hinder the mushroom production and lead to economic losses. Here, we present a novel loop-mediated isothermal amplification (LAMP) assay coupled with gold nanoparticles (AuNPs) for rapid colorimetric detection of Trichoderma spp. The specificity of LAMP primers designed on the tef1 gene was validated in silico and through gel-electrophoresis on Trichoderma harzianum and non-target soil-borne fungal and bacterial strains. LAMP amplification of genomic DNA templates was performed at 65 °C for only 30 min. The results were rapidly visualized in a microplate format within less than 5 min. The assay is based on salt-induced aggregation of AuNPs that is being prevented by the amplicons produced in case of positive LAMP reaction. As the solution color changes from red to violet upon nanoparticle aggregation can be observed with the naked eye, the developed LAMP-AuNPs assay can be easily operated to provide a simple initial screening for the rapid detection of Trichoderma in button mushroom cultivation substrate.

High-Throughput Fluorometric Assay For Quantifying Polysorbate In Biopharmaceutical Products Using Micelle Activated Fluorescence Probe N-Phenyl-1-Naphthylamine.

Polysorbates are among the most used surfactants in biopharmaceutical products containing proteins. Our work aims to develop a high-throughput fluorometric assay to further diversify the analytical toolbox for quantification of PSs. The assay leverages the micelle activated fluorescence signal from N-Phenyl-1-Naphthylamine (NPN). The development and optimization of assay parameters were guided by the pre-defined analytical target profile. Furthermore, NMR was used to probe the interaction between protein, PS80 and NPN in the measurement system and understand protein interference. All assay parameters including excitation and emission wavelengths, standard curve, NPN concentration, and incubation time have been optimized and adapted to a microplate format, making it compatible with automated solutions that will be pursued in the near future to drive consistency and efficiency in our workflows. The specificity, accuracy, and precision of the assay have been demonstrated through a case study. Furthermore, NMR results provided additional insight into the change of the interaction dynamics between PS80 and NPN as the protein concentration increases. The results indicate minimal interaction between the protein and PS80 at lower concentration. However, when the concentration exceeds 75mg/mL, there is a significant interaction between the protein and PS-80 micelle and monomer. A high-throughput fluorometric assay has been developed for quantification of polysorbates in biopharmaceutical samples including in-process samples, drug substance and drug product. The assay reported herein could serve as a powerful analytical tool for polysorbate quantification and control, complementing the widely used liquid chromatography with charged aerosol detection method.

Smart Biointerfaces via Click Chemistry-Enabled Nanopatterning of Multiple Bioligands and DNA Force Sensors.

Nanoscale biomolecular placement is crucial for advancing cellular signaling, sensor technology, and molecular interaction studies. Despite this, current methods fall short in enabling large-area nanopatterning of multiple biomolecules while minimizing nonspecific interactions. Using bioorthogonal tags at a submicron scale, we introduce a novel hole-mask colloidal lithography method for arranging up to three distinct proteins, DNA, or peptides on large, fully passivated surfaces. The surfaces are compatible with single-molecule fluorescence microscopy and microplate formats, facilitating versatile applications in cellular and single-molecule assays. We utilize fully passivated and transparent substrates devoid of metals and nanotopographical features to ensure accurate patterning and minimize nonspecific interactions. Surface patterning is achieved using bioorthogonal TCO-tetrazine (inverse electron-demand Diels-Alder, IEDDA) ligation, DBCO-azide (strain-promoted azide-alkyne cycloaddition, SPAAC) click chemistry, and biotin-avidin interactions. These are arranged on surfaces passivated with dense poly(ethylene glycol) PEG brushes crafted through the selective and stepwise removal of sacrificial metallic and polymeric layers, enabling the directed attachment of biospecific tags with nanometric precision. In a proof-of-concept experiment, DNA tension gauge tether (TGT) force sensors, conjugated to cRGD (arginylglycylaspartic acid) in nanoclusters, measured fibroblast integrin tension. This novel application enables the quantification of forces in the piconewton range, which is restricted within the nanopatterned clusters. A second demonstration of the platform to study integrin and epidermal growth factor (EGF) proximal signaling reveals clear mechanotransduction and changes in the cellular morphology. The findings illustrate the platform's potential as a powerful tool for probing complex biochemical pathways involving several molecules arranged with nanometer precision and cellular interactions at the nanoscale.

Abstract 6790: Breaking barriers: A high-throughput 3D drug discovery platform for ovarian cancer

Abstract Background: Ovarian Cancer (OVCA) is a formidable challenge in oncology and remains the most lethal gynecologic malignancy with many cases (~80%) diagnosed at a late stage with an equally high rate of recurrence. Yet, no established OVCA models exist that faithfully recapitulate the biological complexity of metastatic OVCA. This multidisciplinary team developed a new in vitro high-throughput model that maintains the characteristics of the patient’s tumor including resident immune cells. Key breakthroughs include long term (>21 day) culture of patient-derived microtumors, a functional and perfusable microgel medium (Liquid Like Solids - LLS™), microplates with controlled “pumpless” perfusion of culture media (Darcy Plates™), and integrated in situ imaging. Methods: We designed, fabricated, and validated a new 3D culture device in the standard SBS microplate format (Darcy Plate) that integrates in situ microscopy and perfusion through a bio-conjugated microgel bed (LLS). Patient tumors were collected during debulking surgery, micronized into 1 µl microtumors, suspended in LLS, and plated into the 48-well Darcy Plate. Viability and proliferation were assessed over 21 days in situ using a standard LIVE/DEAD™ kit and EDU staining, respectively. Epithelial OVCA cells and immune cells were identified with immunofluorescence microscopy. Results: Optimal media conditions (HPLM media with 10% FBS, 1% Pen/Strep, 2.5 mg L−1 Amphotericin B, 10 mM HEPES, 0.5 µM A83-01 -TGF-β inhibitor-, and ITS) and perfusion flow rates (100 µl/day per well) were identified that resulted in outstanding cell viability after 21 days (~100% - no detectable cell death using LIVE/DEAD™). PAX8 nuclear expression, a marker for epithelial metastatic OVCA, was retained. EDU staining confirmed cell proliferation and CD45 staining revealed retention of viable immune cells on and within the microtumor at 21 days. Drug testing to evaluate tumor response to chemotherapeutic agents, carboplatin, and emerging targeted therapies was performed by dosing the feed-wells at different concentrations and evaluating cell death through LIVE/DEAD imaging after 5 days of treatment. Conclusions: OVCA is a challenging disease with understudied biological complexity and extensive metastatic involvement. This study demonstrates that drug screening with 3D microtumors has important clinical implications and provides a unique opportunity to evaluate drug responses and personalized treatment strategies. These patient-derived microtumors retain crosstalk between diverse cell populations and the tumor microenvironment, which increases the likelihood of identifying effective therapies and drug candidates. This system retains patient-specific tumor heterogeneity and microenvironmental complexity of OVCA, offering a promising platform for drug screening, high-throughput assays, biomarker discovery, and ultimately improving treatment outcomes. Citation Format: Erin George, Diego I. Pedro, Alfonso Pepe, Angie M. Rivera, Ryan A. Smolchek, Jack E. Famiglietti, W. Gregory Sawyer. Breaking barriers: A high-throughput 3D drug discovery platform for ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6790.

MicroMPN: methods and software for high-throughput screening of microbe suppression in mixed populations.

We created a unified set of software and laboratory protocols for screening microbe libraries to assess the suppression of a pathogen in a mixed microbial community. Existing methods of fluorescent labeling were combined with the most probable number (MPN) assay in a microplate format to enumerate the reduction of a pathogenic soil microbe from complex soil matrices. This work provides a fluorescent expression vector available from Addgene, step-by-step laboratory protocols hosted by protocols.io, and MicroMPN, a command-line software for processing plate reader outputs. MicroMPN simplifies MPN estimation from 96- and 384-well microplates. The microplate screening assay is amenable to robotic automation with standard liquid handling robots, further reducing the hands-on processing time. This tool was designed to evaluate synthetic microbial communities for use as microbial inoculates or probiotics. The fluorMPN method is also useful for screening chemical and antimicrobial libraries for pathogen suppression in complex bacterial communities like soil.

Abstract LB_A21: NCI60 HTS384: The next phase of the NCI60 screen

Abstract The NCI60 human tumor cell line screen, which has operated as a free service to the cancer research community for over 20 years, has proven to be an important drug discovery tool. The screen has operated using cutting-edge technology of the 1990s including a 96-well microplate format, a 2-day drug exposure period and following cell fixation, and a visible absorbance endpoint by the protein-staining dye sulforhodamine B (SRB). The SRB endpoint determines cell density based on protein content with a lower resolution limit of 1,000 – 2,000 cells. This report describes the next phase of this important cancer research tool, the NCI60 HTS384 screen format. While the cell lines remain the same, the screen is performed using 384-well microplates with a 3-day compound exposure period and a CellTiter-Glo luminescence endpoint. The CellTiter-Glo endpoint determines cell number based upon ATP content with a lower resolution limit of <10 cells. To develop a baseline comparison, data were generated from a library of 1,000 FDA-approved and investigational small molecule anticancer agents by the two assay formats. Although the mean-graph patterns and COMPARE analyses are very similar for many compounds, there is a divergence between the two screens for some molecular targets. Within subsets of approved and investigational agents directed toward the same target (e.g., EGFR, BRAF/MEK/ERK, and PI3K inhibitors), the clustering of compound data from the NCI60 HTS384 screen is qualitatively similar to the clustering of compound data from the classic NCI60 SRB screen and there is a strong COMPARE correlation between the two screens. However, for some drug targets there is very little or no clustering between data from the two screens, although the clustering within each screen is moderate-to-good. Examples include mTOR rapalogs, BET bromodomain and NAMPRTase inhibitors. Differences in data between the two screens might be partially attributed to increases in the assay endpoint sensitivity and compound exposure time of the NCI60 HTS384 assay. Compounds submitted for NCI60 screening after September 30, 2023, will be tested in the modernized NCI60 HTS384 format. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I and by the Intramural Research Program of the NIH, National Cancer Institute. Citation Format: Beverly A Teicher, Mark W Kunkel, Joel Morris, Ronald C Taylor, Thomas S Dexheimer, Nathan P Coussens, James H Doroshow. NCI60 HTS384: The next phase of the NCI60 screen [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A21.

Genotoxic and Mutagenic Effects of the Alternaria Mycotoxin Alternariol in Combination with the Process Contaminant Acrylamide.

Humans are constantly exposed to mixtures of different xenobiotics through their diet. One emerging concern is the Alternaria mycotoxin alternariol (AOH), which can occur in foods typically contaminated by the process contaminant acrylamide (AA). AA is a byproduct of the Maillard reaction produced in carbohydrate-rich foods during thermal processing. Given the genotoxic properties of AOH and AA as single compounds, as well as their potential co-occurrence in food, this study aimed to assess the cytotoxic, genotoxic, and mutagenic effects of these compounds in combination. Genotoxicity was assessed in HepG2 cells by quantifying the phosphorylation of the histone γ-H2AX, induced as a response to DNA double-strand breaks (DSBs). Mutagenicity was tested in Salmonella typhimurium strains TA98 and TA100 by applying the Ames microplate format test. Our results showed the ability of AOH and AA to induce DSBs and increase revertant numbers in S. typhimurium TA100, with AOH being more potent than AA. However, no synergistic effects were observed during the combined treatments. Notably, the results of the study suggest that the compounds exert mutagenic effects primarily through base pair substitutions. In summary, the data indicate no immediate cause for concern regarding synergistic health risks associated with the consumption of foods co-contaminated with AOH and AA.

Development of a high-throughput fluorescence resonance energy transfer assay for biotin in food

A new fluorescence resonance energy transfer (FRET) biosensor to evaluate the presence of biotin has been developed and optimized. The biosensor is based on the direct evaluation of BirA enzyme from Escherichia coli homodimerization as a result of biotin binding. BirA monomer was genetically fused either to fluorescent protein mTurquoise2 (mTur) or to cp173Venus (Venus). In the presence of biotin, BirA dimerization brings mTur and Venus molecules close enough for an energy transfer. The analytical performances of the biosensor in the presence biotin using the purified fusion proteins which expressed in Escherichia coli have been evaluated and optimized. The biotin assay can be performed in 96-well microplate format with a 10 min incubation. The FRET signal increased with the biotin concentration in the range of 0.4 nmol/L - 4 nmol/L, and the linear equation was Y = 0.235 + 0.333X (R2 = 0.991), which was slightly affected by other water-soluble vitamins and trace metal elements. The FRET biosensor was used to detect biotin in infant formula samples, and the results were in good agreement with those obtained from the microbial method, indicating that the developed FRET biosensor was applicable to the biotin detection in food.

The Analytical Application of Quenching Phenomena of CdTe Quantum Dot Nanoparticles

This paper is devoted to the synthesis and application of CdTe quantum dot (QD) nanoparticles covered with organic ligands containing a thiol group, mostly mercaptopropionic acid (MPA) and glutathione (GSH). The simple one-step synthetic procedure was optimized to prepare greater quantities of nanoparticles for analytical purposes. The prepared CdTe QD nanoparticles were characterized by various analytical techniques, and their interaction with some metal ions (Cu(II), Pb(II), and Hg(II)) was studied by using luminescence spectroscopy in both steady-state and time-resolved modes. The mathematical analysis of the quenching effect of Cu(II) ions on the luminescence of CdTe QD nanoparticles shows that the static contribution is mostly responsible for the overall effect, but experimental conditions, such as pH, ionic strength, or the concentration of nanoparticles in aqueous solution, could also be important. The presence of metal ions in the form of a metal complex species could play an important role, and this phenomenon could be used to tune the selectivity of the quenching process. These findings have been utilized for the development of an analytical procedure for the detection and quantitative analysis of Cu(II) and Pb(II) ions in environmental water samples. In practice, this procedure could be easily implemented in a microplate format to increase throughput.

Post-Traumatic Stress Response and Appendicitis in Children-Clinical Usefulness of Selected Biomarkers.

Acute appendicitis is an inflammatory process which is one of the most frequent global causes of surgical interventions in children. The goal of the study was to determine whether acute phase proteins, that is, C-reactive protein (CRP), procalcitonin (PCT) and neutrophil gelatinase-associated lipocalin (NGAL), interleukin 6 (IL-6), transforming growth factor-beta1 (TGF-β1) and cortisol (HC) play a role in the pathomechanism of post-trauma stress response of the organism and to establish the impact of the applied surgical procedure and/or of inflammation on their concentrations. An additional purpose was to establish the clinical usefulness of the studied biomarkers in the diagnostics of appendicitis. CRP concentrations were quantified via the immunoturbidimetric method, while the levels of IL-6 and PCT were assessed using a bead-based multiplexed immunoassay system in a microplate format (Luminex xMAP technology); NGAL, TGF-β1 and cortisol concentrations were determined via the enzyme-linked immunosorbent assay (ELISA) technique. All the investigated biomarkers were assayed twice, i.e., immediately before the surgery and 12-24 h after its completion. Significant increases in CRP, IL-6 and PCT concentrations were found in all children subjected to laparoscopic surgeries (p = 0.001, p = 0.006, and p = 0.009, respectively) and open (classic) surgeries (p = 0.001, p = 0.016, and p = 0.044, respectively) compared to the initial concentrations. The patients undergoing classical surgery moreover presented with significant (p = 0.002, and p = 0.022, respectively) increases in NGAL and TGF-β1 levels after the procedures. In a group of children undergoing laparoscopic surgery, the appendicitis induced an increase in cortisol concentration, whereas in patients undergoing classical surgery the increase in the levels of this biomarker was caused by the type of performed surgical procedure. Simultaneously assaying the levels of CRP, NGAL and IL-6 (p = 0.008, p = 0.022, and p = 0.000, respectively) may prove useful in clinical practice, enabling the diagnosis of appendicitis in paediatric patients reporting to a hospital with abdominal pains, in addition to data from anamnesis and from clinical or ultrasound examination. The performed study confirms the participation of examined biomarkers in the pathomechanism of post-injury stress reaction of the organism to surgical trauma.

Enzymatic assay for UDP-GlcNAc and its application in the parallel assessment of substrate availability and protein O-GlcNAcylation

O-linked N-acetylglucosaminylation (O-GlcNAcylation) is a ubiquitous and dynamic non-canonical glycosylation of intracellular proteins. Several branches of metabolism converge at the hexosamine biosynthetic pathway (HBP) to produce the substrate for protein O-GlcNAcylation, the uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Availability of UDP-GlcNAc is considered a key regulator of O-GlcNAcylation. Yet UDP-GlcNAc concentrations are rarely reported in studies exploring the HBP and O-GlcNAcylation, most likely because the methods to measure it are restricted to specialized chromatographic procedures. Here, we introduce an enzymatic method to quantify cellular and tissue UDP-GlcNAc. The method is based on O-GlcNAcylation of a substrate peptide by O-linked N-acetylglucosamine transferase (OGT) and subsequent immunodetection of the modification. The assay can be performed in dot-blot or microplate format. We apply it to quantify UDP-GlcNAc concentrations in several mouse tissues and cell lines. Furthermore, we show how changes in UDP-GlcNAc levels correlate with O-GlcNAcylation and the expression of OGT and O-GlcNAcase (OGA).

Application of human iPSC-derived macrophages in a miniaturized high-content-imaging-based efferocytosis assay.

Macrophages play a pivotal role in drug discovery due to their key regulatory functions in health and disease. Overcoming the limited availability and donor variability of human monocyte-derived macrophages (MDMs), human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) could provide a promising tool for both disease modeling and drug discovery. To access large numbers of model cells for medium- to high-throughput application purposes, an upscaled protocol was established for differentiation of iPSCs into progenitor cells and subsequent maturation into functional macrophages. These IDM cells resembled MDMs both with respect to surface marker expression and phago- as well as efferocytotic function. A statistically robust high-content-imaging assay was developed to quantify the efferocytosis rate of IDMs and MDMs allowing for measurements both in the 384- and 1536-well microplate format. Validating the applicability of the assay, inhibitors of spleen tyrosine kinase (Syk) were shown to modulate efferocytosis in IDMs and MDMs with comparable pharmacology. The miniaturized cellular assay with the upscaled provision of macrophages opens new routes to pharmaceutical drug discovery in the context of efferocytosis-modulating substances.

Evaluation of 96-well high-throughput DNA extraction methods for 16S rRNA gene metabarcoding.

Gaining meaningful insights into bacterial communities associated with animal hosts requires the provision of high-quality nucleic acids. Although many studies have compared DNA extraction methods for samples with low bacterial biomass (e.g. water) or specific PCR inhibitors (e.g. plants), DNA extraction bias in samples without inherent technical constraint (e.g. animal samples) has received little attention. Furthermore, there is an urgent need to identify a DNA extraction methods in a high-throughput format that decreases the cost and time for processing large numbers of samples. We here evaluated five DNA extraction protocols, using silica membrane-based spin columns and a 96-well microplate format and based on either mechanical or enzymatic lysis or a combination of both, using three bacterial mock communities and Illumina sequencing of the V4 region of the 16SrRNA gene. Our results showed that none of the DNA extraction methods fully eliminated bias associated with unequal lysis efficiencies. However, we identified a DNA extraction method with a lower bias for each mock community standard. Of these methods, those including an enzymatic lysis showed biases specific to some bacteria. Altogether, these results again demonstrate the importance of DNA extraction standardization to be able to compare the microbiome results of different samples. In this attempt, we advise for the use of the 96-well DNeasy Blood and Tissue kit (Qiagen) with a zirconia bead-beating procedure, which optimizes altogether the cost, handling time and bacteria-specific effects associated with enzymatic lysis.

Rapid and cost-efficient microplate assay for the accurate quantification of total phenolics in seaweeds

Brown seaweeds (Phaeophyceae) are a rich source of polyphenols (up to 20% dry weight) with a structure based on phloroglucinol (1,3,5-trihydroxybenzene). To-date the determination of total phenolics content (TPC) involves a redox reaction with the Folin-Ciocalteu (FC) reagent. However, side reactions with other reducing substances preclude accurate, direct measurement of TPC. This research reports a novel microplate assay involving a coupling reaction between phloroglucinol with Fast Blue BB (FBBB) diazonium salt, at basic pH, to form a stable tri-azo complex with maximum absorbance at 450 nm. Linear regression correlation values (R2) were ≥0.99 with phloroglucinol as standard. Direct quantification of TPCs (phloroglucinol equivalents, PGEs) in crude aqueous and ethanolic extracts from A. nodosum demonstrated that the new FBBB assay is not subject to side-redox interference and provides a more accurate estimate of TPC (1.2–3.9-fold lower than with the FC assay) in a relatively rapid (30 min), cost-effective (0.24€/test) microplate format.

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis.

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

Biochemical factors in the blood serum of neuroendocrine tumor patients with carcinoid syndrome

Introduction. Carcinoid syndrome is the most common functional syndrome in patients with neuroendocrine tumors. More than 40 biochemical factors are responsible for the manifestation of carcinoid syndrome, among which serotonin is the most important. The study of biochemical markers of carcinoid syndrome and associated carcinoid heart disease is an important aim of laboratory examination in neuroendocrine tumors patients.Aim. Analysis of levels and diagnostic efficiency evaluation of chromogranin A (CgA), serotonin, pro-brain natriuretic peptide (proBNP) and platelet-derived growth factor (PDGF-BB) in the blood serum of neuroendocrine tumors patients with various clinical manifestations, including carcinoid syndrome and carcinoid heart disease.Materials and methods. 66 patients with neuroendocrine tumors of various localizations were examined (pancreas – 24 cases, small intestine – 21, large intestine – 6, lungs – 10, unkown primary focus – 5). 38 patients had liver metastases. In 43 patients, a clinic of carcinoid syndrome was observed, 16 had signs of carcinoid heart disease. The control group consisted of 30 practically healthy people. Serum levels of CgA, serotonin, and PDGF-BB were determined by enzyme immunoassay in microplate format: Chromogranin A NEOLISA (Eurodiagnostica, Sweden), Serotonin ELISA (IBL, German), and PDGF-BB ELISA Kit (Invitrogen, USA). The proBNP analysis was performed on a Cobas e601 automated analyzer (Roche, Switzerland).Results. In carcinoid syndrome, the medians of CgA, serotonin, and proBNP were the highest, differing statistically significantly from the control group. In patients with G3 tumors, the median PDGF-BB was statistically significantly higher than in controls, in contrast to G1 and G2. The highest diagnostic sensitivity in the general neuroendocrine tumors group was in CgA – 63.6 %, with a specificity of 100 %. In patients with carcinoid syndrome, the highest diagnostic sensitivity was characteristic of serotonin and chromogranin A (79 %), while in patients with CAD clinic, proBNP had the highest sensitivity – 93.8 %.Conclusion. The study revealed the high efficiency of СgA, with the highest sensitivity in common forms and tumors with high biological activity. Serotonin can be used in the diagnosis of carcinoid syndrome, associated with cardiofibrosis development. Pro-brain natriuretic peptide is a highly sensitive and specific marker of carcinoid heart disease. The highest levels of PDGF-BB are associated with a high grade of neuroendocrine tumors malignancy.