Screening Platform Research Articles

Acrolein scavengers and detoxification: From high-throughput screening of flavonoids to mechanistic study of epigallocatechin gallate

Acrolein (ACR) is a widespread, highly toxic substance that poses significant health risks. Flavonoids have been recognized as effective ACR scavengers, offering a possible way to reduce these risks. However, the lack of specific high-throughput screening methods has limited the identification of ACR scavengers, and their actual detoxifying capacity on ACR remains unknown. To address this, we developed a high-throughput screening platform to assess the ACR scavenging capacity of 322 flavonoids. Our results showed that 80.7 % of the flavonoids could scavenge ACR, but only 34.4 % exhibited detoxifying effects in an ACR-injured QSG7701 cell model. Some flavonoids even increased toxicity. Structure-activity relationship (SAR) analysis indicated that galloyl and pyrogallol units enhance scavenging but worsen ACR-induced cytotoxicity. Further investigation revealed that epigallocatechin gallate (EGCG) could exacerbate ACR-induced redox disorder, leading to cell apoptosis. Our findings provide crucial data on the scavenging and detoxifying capacities of 322 flavonoids, highlighting that ACR scavengers might not mitigate ACR-induced toxicity and could pose additional safety risks.

High-throughput screening of green amino acid and surfactant mixtures with high corrosion inhibition efficiency: Experimental and modelling perspectives

A high-throughput screening method is developed for rapid evaluation of corrosion inhibitors. By employing a fluorometric technique, this method quickly assesses the concentration of iron ions converted from corrosion products in the reaction pool to rapidly evaluate the corrosion inhibition performance of amino acid and surfactants. Utilizing this high-throughput screening platform, the corrosion inhibition effects of L-cysteine mixed with quaternary ammonium salts of different alkyl chain length were evaluated, and their optimal mixing ratios were determined. Through corrosion experiments and molecular simulations, the synergistic mechanisms of the inhibitor mixtures are revealed.

Streamlined screening platforms lead to the discovery of pachysiphine synthase from Tabernanthe iboga.

Plant-specialized metabolism is largely driven by the oxidative tailoring of key chemical scaffolds catalyzed by cytochrome P450 (CYP450s) enzymes. Monoterpene indole alkaloids (MIAs) tabersonine and pseudo-tabersonine, found in the medicinal plant Tabernanthe iboga (commonly known as iboga), are tailored with oxidations, and the enzymes involved remain unknown. Here, we developed a streamlined screening strategy to test the activity of T. iboga CYP450s in Nicotiana benthamiana. Using multigene constructs encoding the biosynthesis of tabersonine and pseudo-tabersonine scaffolds, we aimed to uncover the CYP450s responsible for oxidative transformations in these scaffolds. Our approach identified two T. iboga cytochrome P450 enzymes: pachysiphine synthase (PS) and 16-hydroxy-tabersonine synthase (T16H). These enzymes catalyze an epoxidation and site-specific hydroxylation of tabersonine to produce pachysiphine and 16-OH-tabersonine, respectively. This work provides new insights into the biosynthetic pathways of MIAs and underscores the utility of N. benthamiana and Catharanthus roseus as platforms for the functional characterization of plant enzymes.

Microdroplet-enhanced chip platform for high-throughput immunotherapy marker screening from extracellular vesicle RNAs and membrane proteins

Extracellular vesicles (EVs) are considered as promising candidates for predicting patients who respond to immunotherapy. Nevertheless, simultaneous detection of multiple EVs markers still presents significant technical challenges. In this work, we developed a high-throughput microdroplet-enhanced chip (MEC) platform, which utilizes thousands of individual microchambers (∼pL) as reactors, accelerating the detection efficiency of the CRISPR/Cas systems and increasing the sensitivity by up to 100-fold (aM level). Ten biomarkers (including 5 RNAs and 5 proteins) from patients’ EVs are successfully detected on one chip, and the comprehensive markers show increased accuracy (AUC 0.911) than the individual marker for the efficacy prediction of immunotherapy. This platform provides a high-throughput yet sensitive strategy for screening immunotherapy markers in clinical.

Real-time electro-mechanical profiling of dynamically beating human cardiac organoids by coupling resistive skins with microelectrode arrays

Cardiac organoids differentiated from induced pluripotent stem cells are emerging as a promising platform for pre-clinical drug screening, assessing cardiotoxicity, and disease modelling. However, it is challenging to simultaneously measure mechanical contractile forces and electrophysiological signals of cardiac organoids in real-time and in-situ with the existing methods. Here, we present a biting-inspired sensory system based on a resistive skin sensor and a microelectrode array. The bite-like contact can be established with a micromanipulator to precisely position the resistive skin sensor on the top of the cardiac organoid while the 3D microneedle electrode array probes from underneath. Such reliable contact is key to achieving simultaneous electro-mechanical measurements. We demonstrate the use of our system for modelling cardiotoxicity with the anti-cancer drug doxorubicin. The electro-mechanical parameters described here elucidate the acute cardiotoxic effects induced by doxorubicin. This integrated electro-mechanical system enables a suite of new diagnostic options for assessing cardiac organoids and tissues.

Screening of herbal extracts binding with vascular endothelial growth factor by applying HerboChip platform

BackgroundTraditional Chinese medicine (TCM) has been hailed as a rich source of medicine, but many types of herbs and their functions still need to be rapidly discovered and elucidated. HerboChip, a target-based drug screening platform, is an array of different fractions deriving from herbal extracts. This study was designed to identify effective components from TCM that interact with vascular endothelial growth factor (VEGF) as a target using HerboChip.MethodsSelected TCMs that are traditionally used as remedies for cancer prevention and wound healing were determined and extracted with 50% ethanol. Biotinylated-VEGF was hybridized with over 500 chips coated with different HPLC-separated fractions from TCM extracts and straptavidin-Cy5 was applied to identify plant extracts containing VEGF-binding fractions. Cytotoxicity of selected herbal extracts and their activities on VEGF-mediated angiogenic functions were evaluated.ResultsOver 500 chips were screened within a week, and ten positive hits were identified. The interaction of the identified herbal extracts with VEGF was confirmed in cultured endothelial cells. The identified herbs promoted or inhibited VEGF-mediated cell proliferation, migration and tube formation. Results from western blotting analysis demonstrated the identified herbal extracts significantly affected VEGF-triggered phosphorylations of eNOS, Akt and Erk. Five TCMs demonstrated potentiating activities on the VEGF response and five TCMs revealed suppressive activities.ConclusionsThe current results demonstrated the applicability of the HerboChip platform and systematically elucidated the activity of selected TCMs on angiogenesis and its related signal transduction mechanisms.

Evaluation of AI-enhanced non-mydriatic fundus photography for diabetic retinopathy screening

ObjectiveTo assess the feasibility of using non-mydriatic fundus photography in conjunction with an artificial intelligence (AI) reading platform for large-scale screening of diabetic retinopathy (DR). MethodsIn this study, we selected 120 patients with diabetes hospitalized in our institution from December 2019 to April 2021. Retinal imaging of 240 eyes was obtained using non-mydriatic fundus photography. The fundus images of these patients were divided into two groups based on different interpretation methods. In Experiment Group 1, the images were analyzed and graded for DR diagnosis using an AI reading platform. In Experiment Group 2, the images were analyzed and graded for DR diagnosis by an associate chief physician in ophthalmology, specializing in fundus diseases. Concurrently, all patients underwent the gold standard for DR diagnosis and grading—fundus fluorescein angiography (FFA)—with the outcomes serving as the Control Group. The diagnostic value of the two methods was assessed by comparing the results of Experiment Groups 1 and 2 with those of the Control Group. ResultsKeeping the control group (FFA results) as the gold standard, no significant differences were observed between the two experimental groups regarding diagnostic sensitivity, specificity, false positive rate, false negative rate, positive predictive value, negative predictive value, Youden's index, Kappa value, and diagnostic accuracy (X2 = 0.371, P > 0.05). ConclusionCompared with the manual reading group, the AI reading group revealed no significant differences across all diagnostic indicators, exhibiting high sensitivity and specificity, as well as a relatively high positive predictive value. Additionally, it demonstrated a high level of diagnostic consistency with the gold standard. This technology holds potential for suitability in large-scale screening of DR.

Multiplexed Microfluidic Platform for Parallel Bacterial Chemotaxis Assays.

The sensing of and response to ambient chemical gradients by microorganisms via chemotaxis regulates many microbial processes fundamental to ecosystem function, human health, and disease. Microfluidics has emerged as an indispensable tool for the study of microbial chemotaxis, enabling precise, robust, and reproducible control of spatiotemporal chemical conditions. Previous techniques include combining laminar flow patterning and stop-flow diffusion to produce quasi-steady chemical gradients to directly probe single-cell responses or loading micro-wells to entice and ensnare chemotactic bacteria in quasi-steady chemical conditions. Such microfluidic approaches exemplify a trade-off between high spatiotemporal resolution of cell behavior and high-throughput screening of concentration-specific chemotactic responses. However, both aspects are necessary to disentangle how a diverse range of chemical compounds and concentrations mediate microbial processes such as nutrient uptake, reproduction, and chemorepulsion from toxins. Here, we present a protocol for the multiplexed chemotaxis device (MCD), a parallelized microfluidic platform for efficient, high-throughput, and high-resolution chemotaxis screening of swimming microbes across a range of chemical concentrations. The first layer of the two-layer polydimethylsiloxane (PDMS) device comprises a serial dilution network designed to produce five logarithmically diluted chemostimulus concentrations plus a control from a single chemical solution input. Laminar flow in the second device layer brings a cell suspension and buffer solution into contact with the chemostimuli solutions in each of six separate chemotaxis assays, in which microbial responses are imaged simultaneously over time. The MCD is produced via standard photography and soft lithography techniques and provides robust,repeatable chemostimulus concentrations across each assay in the device. This microfluidic platform provides a chemotaxis assay that blends high-throughput screening approaches with single-cell resolution to achieve a more comprehensive understanding of chemotaxis-mediated microbial processes. Key features • Microchannel master molds are fabricated using photolithography techniques in a clean room with a mask aligner to fabricate multilevel feature heights. • The microfluidic device is fabricated from PDMS using standard soft lithography replica molding from the master molds. • The resulting microchannel requires a one-time calibration of the driving inlet pressures, after which devices from the same master molds have robust performance. • The microfluidic platform is optimized and tested for measuring chemotaxis of swimming prokaryotes.

An artificial intelligence accelerated virtual screening platform for drug discovery

Structure-based virtual screening is a key tool in early drug discovery, with growing interest in the screening of multi-billion chemical compound libraries. However, the success of virtual screening crucially depends on the accuracy of the binding pose and binding affinity predicted by computational docking. Here we develop a highly accurate structure-based virtual screen method, RosettaVS, for predicting docking poses and binding affinities. Our approach outperforms other state-of-the-art methods on a wide range of benchmarks, partially due to our ability to model receptor flexibility. We incorporate this into a new open-source artificial intelligence accelerated virtual screening platform for drug discovery. Using this platform, we screen multi-billion compound libraries against two unrelated targets, a ubiquitin ligase target KLHDC2 and the human voltage-gated sodium channel NaV1.7. For both targets, we discover hit compounds, including seven hits (14% hit rate) to KLHDC2 and four hits (44% hit rate) to NaV1.7, all with single digit micromolar binding affinities. Screening in both cases is completed in less than seven days. Finally, a high resolution X-ray crystallographic structure validates the predicted docking pose for the KLHDC2 ligand complex, demonstrating the effectiveness of our method in lead discovery.

Functional Aptamers In Vitro Evolution for Intranuclear Blockage of RNA-Protein Interaction.

Over the last 30 years, despite considerable research and endeavors aimed at harnessing aptamers as pharmaceutical molecules, the progress in developing aptamer-based drugs has been falling short of expectations. Sequential steps of affinity molecule acquisition and functional screening are typically required for discovering affinity-based macromolecule therapeutics, which can be time-consuming and limiting in candidate selection. Additionally, aptamers often necessitate tedious postselection modifications to overcome pharmacokinetic limitations, which usually impede the binding affinity. Herein, we propose a novel in vitro screening platform termed Functional Aptamers in vitro Evolution (FAIVE), which integrates affinity molecule acquisition with functional screening and introduces chemical diversity during the process. This platform aims to rapidly generate functional aptamers capable of binding to target proteins and regulating their functions. Illustrated by targeting intranuclear RNA-protein interactions involving HIV-1 Tat protein and TAR RNA, FAIVE demonstrates a selection of functional aptamers with significant intracellular blocking effects. The study also explores lipid nanoparticle delivery systems to enhance intracellular delivery efficiency, expanding aptamer targeting potential to broader intracellular and intranuclear domains. This study emphasizes the potential of FAIVE to expedite the development of aptamer-based drugs and facilitate the creation of more versatile and effective therapeutics.

Exploring the Effects of Optically Active Solvents in Chiral Chromatography on Polysaccharide-Based Columns.

Exploring the effectiveness of optically active solvents as mobile-phase modifiers in chiral liquid chromatography (LC) can offer an additional new tool to tune the chiral selectivity. Hence, the potential of l-ethyl lactate (LEL), a biobased solvent of this nature, was explored for its distinctive interactions with both the mobile phase and analytes, as anticipated from its chiral nature. The findings reveal that LEL provides distinct selectivity compared to commonly used modifiers in chiral LC. Reversed-phase LC (RPLC)-type chiral separations were therefore compared under various conditions whereby LEL was partially or completely replacing common achiral solvents such as acetonitrile (ACN) and methanol (MeOH). An increase in chiral resolution was obtained in 8 of 16 test compounds. For 5 of them a decrease was obtained, and 3 test solutes did not offer satisfactory results under any of the tested conditions on the polysaccharide columns. When LEL was combined with methanol instead of ACN, worse results were obtained, presumably due to its protic nature. Moreover, LEL demonstrates excellent compatibility with salt additives and is fully miscible with aqueous phases. Interestingly, a steeper increase in chiral resolution is observed for LEL, as compared to ACN at lower temperatures. While LEL is somewhat hindered by its higher UV absorbance, it paves the way toward more simplified chiral screening platforms, whereby chiral solutions can be found for fewer columns and greener solvents such as LEL are incorporated. Finally, to elucidate the impact of chiral interactions between the solvent and analytes, the influence of d-ethyl lactate (DEL) was compared with that of LEL. The results revealed different interactions between the stereoisomers of ethyl lactate (EL) and chiral analytes, demonstrating an influence of optically active solvents on enantioseparations.

Patient-derived organoids and mini-PDX for predicting METN375S-mutated lung cancer patient clinical therapeutic response

Lung cancer as a molecularly and histologically high heterogonous disease, there is an urgent need to predict lung cancer patients' responses to anti-cancer treatment, and patient-derived organoids (PDOs) have been recognized as a valuable platform for preclinical drug screening. In this study, we successfully established 26 PDO lines from various subtypes of lung cancers including benign tumor, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, large-cell carcinoma, and small-cell carcinoma. These PDOs were shown to retain the major genomic and histological characteristics of primary tumors and remain stable during long-term culture. With the help of targeted genomic sequencing, we found that lung cancer that harbors METN375S mutation is selectively sensitive to afatinib, and a combination of afatinib and gemcitabine induced synthetic lethality in PDO and mini-PDX models. In summary, our findings demonstrate the potential of PDO in predicting lung cancer drug response, and reveal a promising strategy for METN375S mutant lung cancer treatment.

P01-56 Primary human hepatocytes as screening platform for imaging-based assessment of DILI liabilities

P01-56 Primary human hepatocytes as screening platform for imaging-based assessment of DILI liabilities

Compressible Hollow Microlasers in Organoids for High-Throughput and Real-Time Mechanical Screening.

Mechanical stress within organoids is a pivotal indicator in disease modeling and pharmacokinetics, yet current tools lack the ability to rapidly and dynamically screen these mechanics. Here, we introduce biocompatible and compressible hollow microlasers that realize all-optical assessment of cellular stress within organoids. The laser spectroscopy yields identification of cellular deformation at the nanometer scale, corresponding to tens of pascals stress sensitivity. The compressibility enables the investigation of the isotropic component, which is the fundamental mechanics of multicellular models. By integrating with a microwell array, we demonstrate the high-throughput screening of mechanical cues in tumoroids, establishing a platform for mechano-responsive drug screening. Furthermore, we showcase the monitoring and mapping of dynamic contractile stress within human embryonic stem cell-derived cardiac organoids, revealing the internal mechanical inhomogeneity within a single organoid. This method eliminates time-consuming scanning and sample damage, providing insights into organoid mechanobiology.

Development of a Screening Platform for Optimizing Chemical Nanosensor Materials.

Chemical sensors, relying on changes in the electrical conductance of a gas-sensitive material due to the surrounding gas, typically react with multiple target gases and the resulting response is not specific for a certain analyte species. The purpose of this study was the development of a multi-sensor platform for systematic screening of gas-sensitive nanomaterials. We have developed a specific Si-based platform chip, which integrates a total of 16 sensor structures. Along with a newly developed measurement setup, this multi-sensor platform enables simultaneous performance characterization of up to 16 different sensor materials in parallel in an automated gas measurement setup. In this study, we chose the well-established ultrathin SnO2 films as base material. In order to screen the sensor performance towards type and areal density of nanoparticles on the SnO2 films, the films are functionalized by ESJET printing Au-, NiPt-, and Pd-nanoparticle solutions with five different concentrations. The functionalized sensors have been tested toward the target gases: carbon monoxide and a specific hydrogen carbon gas mixture of acetylene, ethane, ethne, and propene. The measurements have been performed in three different humidity conditions (25%, 50% and 75% r.h.). We have found that all investigated types of NPs (except Pd) increase the responses of the sensors towards CO and HCmix and reach a maximum for an NP type specific concentration.

Measuring the capacity of yeast for surface display of cell wall-anchored protein isoforms by using β-lactamase as a reporter enzyme.

Yeast surface display is a promising biotechnological tool that uses genetically modified yeast cell wall proteins as anchors for enzymes of interest, thereby transforming yeast cell wall into a living catalytic material. Here, we present a comprehensive protocol for quantifying surface-displayed β-lactamase on the cell wall of model yeast Saccharomyces cerevisiae. We use β-lactamase as a reporter enzyme, which we tagged to be anchored to the cell wall closer to its N or C terminus, through the portion of the Pir2 or Ccw12 cell wall proteins, respectively. The catalytic activity of surface-displayed β-lactamase is assessed by its ability to hydrolyze nitrocefin, which produces a colorimetric change that is quantitatively measured by spectrophotometric analysis at 482 nm. This system enables precise quantification of the potential of S. cerevisiae strains for surface display, continuous real-time monitoring of enzyme activity, and facilitates the study of enzyme kinetics and interactions with inhibitors within the cell's native environment. In addition, the system provides a platform for high-throughput screening of potential β-lactamase inhibitors and can be adapted for the visualization of other enzymes, making it a versatile tool for drug discovery and bioprocess development.

DNA-directed immobilization fluorescent immunoarray for multiplexed antibiotic residue determination in milk.

The presence of antibiotic residues in cow's milk entails high risk for consumers, the dairy industry, and the environment. Therefore, the development of highly specific and sensitive screening tools for the rapid and cost-effective identification of traces of these compounds is urgently needed. A multiplexed screening platform utilizing DNA-directed immobilization (DDI) was developed aiming to detect three classes of antibiotic residues (fluoroquinolones, sulfonamides, and tylosin) prevalently found in milk. Throughout this work, each oligonucleotide sequence was conjugated to a different hapten molecule, while the three complementary strands were immobilized in 24 independent microarray chips on a single glass slide. First, the array was incubated with the pool of hapten-oligonucleotide conjugate site encoded the signal through DNA hybridization. Next, commercial milk samples were incubated with the cocktail of monoclonal antibodies following a secondary fluorophore-labeled antibody which was required for fluorescent readout. Direct sample detection was achieved in milk diluting 20 times in assay buffer. The limits of detection (LODs) reached were 1.43µgkg-1, 1.67µgkg-1, and 0.89µgkg-1 for TYLA, STZ, and CIP, respectively, which represented in raw milk 7.15µgkg-1, 8.35µgkg-1, and 4.45µgkg-1 for TYLA, STZ, and CIP, respectively, that are below the EU regulatory limits. Cross-reactivity profiles were evaluated against the family of structurally related antibiotics in order to demonstrate the capability to detect antibiotics from the same family of compounds. A pre-validation study was performed by spiking 20 blind samples above and below the maximum residue limits established by the EU guidelines. The system was successfully implemented towards randomized sample classification as compliant or non-compliant. The proposed DDI-based immunoarray provides a fast and cost-effective alternative to obtain semi-quantitative information about the presence of three veterinary residues simultaneously in milk samples.

Free Radical Production Induced by Nitroimidazole Compounds Lead to Cell Death in Leishmania infantum Amastigotes.

Leishmania infantum is the vector-borne trypanosomatid parasite causing visceral leishmaniasis in the Mediterranean basin. This neglected tropical disease is treated with a limited number of obsolete drugs that are not exempt from adverse effects and whose overuse has promoted the emergence of resistant pathogens. In the search for novel antitrypanosomatid molecules that help overcome these drawbacks, drug repurposing has emerged as a good strategy. Nitroaromatic compounds have been found in drug discovery campaigns as promising antileishmanial molecules. Fexinidazole (recently introduced for the treatment of stages 1 and 2 of African trypanosomiasis), and pretomanid, which share the nitroimidazole nitroaromatic structure, have provided antileishmanial activity in different studies. In this work, we have tested the in vitro efficacy of these two nitroimidazoles to validate our 384-well high-throughput screening (HTS) platform consisting of L. infantum parasites emitting the near-infrared fluorescent protein (iRFP) as a biomarker of cell viability. These molecules showed good efficacy in both axenic and intramacrophage amastigotes and were poorly cytotoxic in RAW 264.7 and HepG2 cultures. Fexinidazole and pretomanid induced the production of ROS in axenic amastigotes but were not able to inhibit trypanothione reductase (TryR), thus suggesting that these compounds may target thiol metabolism through a different mechanism of action.

Expression levels of core spliceosomal proteins modulate the MBNL-mediated spliceopathy in DM1.

Myotonic dystrophy type 1 (DM1) is a heterogeneous multisystemic disease caused by a CTG repeat expansion in DMPK. Transcription of the expanded allele produces toxic CUG repeat RNA that sequesters the MBNL family of alternative splicing (AS) regulators into ribonuclear foci, leading to pathogenic mis-splicing. To identify genetic modifiers of toxic CUG RNA levels and the spliceopathy, we performed a genome-scale siRNA screen using an established HeLa DM1 repeat-selective screening platform. We unexpectedly identified core spliceosomal proteins as a new class of modifiers that rescue the spliceopathy in DM1. Modest knockdown of one of our top hits, SNRPD2, in DM1 fibroblasts and myoblasts, significantly reduces DMPK expression and partially rescues MBNL-regulated AS dysfunction. While the focus on the DM1 spliceopathy has centered around the MBNL proteins, our work reveals an unappreciated role for MBNL:spliceosomal protein stoichiometry in modulating the spliceopathy, revealing new biological and therapeutic avenues for DM1.

A Combined Computational and Experimental Approach to Studying Tropomyosin Kinase Receptor B Binders for Potential Treatment of Neurodegenerative Diseases.

Tropomyosin kinase receptor B (TrkB) has been explored as a therapeutic target for neurological and psychiatric disorders. However, the development of TrkB agonists was hindered by our poor understanding of the TrkB agonist binding location and affinity (both affect the regulation of disorder types). This motivated us to develop a combined computational and experimental approach to study TrkB binders. First, we developed a docking method to simulate the binding affinity of TrkB and binders identified by our magnetic drug screening platform from Gotu kola extracts. The Fred Docking scores from the docking computation showed strong agreement with the experimental results. Subsequently, using this screening platform, we identified a list of compounds from the NIH clinical collection library and applied the same docking studies. From the Fred Docking scores, we selected two compounds for TrkB activation tests. Interestingly, the ability of the compounds to increase dendritic arborization in hippocampal neurons matched well with the computational results. Finally, we performed a detailed binding analysis of the top candidates and compared them with the best-characterized TrkB agonist, 7,8-dyhydroxyflavon. The screening platform directly identifies TrkB binders, and the computational approach allows for the quick selection of top candidates with potential biological activities based on the docking scores.