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Competing Interests: Conflict of interest statement. None declared.

Commercially available glow luciferase assay kits are widely popular and convenient to use. However, concerning high-throughput screening, commercial kits are limited by huge running costs. As an alternative to commercial luciferase assay kits, this study presents a cost-effective and efficient methodology of performing a simple and rapid laboratory flash luciferase assay. The proposed luciferase assay method has a versatile use ranging from screening lysates in a microplate reader for quantitative assay as well as screening live cells qualitatively or quantitatively under an imaging system., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Diagnostics employing multiple modalities have been essential for controlling and managing COVID-19, caused by SARS-CoV-2. However, scaling up Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR), the gold standard for SARS-CoV-2 detection, remains challenging in low and middle-income countries. Cost-effective and high-throughput alternatives like enzyme-linked immunosorbent assay (ELISA) could address this issue. We developed an in-house SARS-CoV-2 nucleocapsid capture ELISA, and validated on 271 nasopharyngeal swab samples from humans (n = 252), bovines (n = 10), and dogs (n = 9). This ELISA has a detection limit of 195 pg/100 µL of nucleocapsid protein and does not cross-react with related coronaviruses, ensuring high specificity to SARS-CoV-2. Diagnostic performance was evaluated using receiver operating characteristic curve analysis, showing a diagnostic sensitivity of 67.78 % and specificity of 100 %. Sensitivity improved to 74.32 % when excluding positive clinical samples with RT-qPCR Ct values > 25. Furthermore, inter-rater reliability analysis demonstrated substantial agreement (κ values = 0.73-0.80) with the VIRALDTECT II Multiplex RT-qPCR kit and perfect agreement with the CoVeasy™ COVID-19 rapid antigen self-test (κ values = 0.89-0.93). Our findings demonstrated that the in-house nucleocapsid capture ELISA is suitable for SARS-CoV-2 testing in humans and animals, meeting the necessary sensitivity and specificity thresholds for cost-effective, large-scale screening., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Therapeutic drug monitoring of treatment with therapeutic antibodies is hampered by the application of a wide range of different methods in the quantification of serum levels. LC-MS based methods could significantly improve comparability of results from different laboratories, but such methods are often considered complicated and costly. We developed a method for LC-MS/MS based quantification of 11 therapeutic antibodies concomitantly measured in a single run, with emphasis on simplicity in sample preparation and low cost., Results: After a single-step sample purification using caprylic acid precipitation to remove interfering proteins, the sample underwent proteolysis followed by LC-MS/MS analysis. Infliximab is used as internal standard for sample preparation while isotope-labeled signature peptides identified for each analyte are internal standards for the LC-MS/MS normalization. The method was validated according to recognized guidelines, and pipetting steps can be performed by automated liquid handling systems. The total precision of the method ranged between 2.7 and 7.3 % (5.1 % average) across the quantification range of 4-256 μg/ml for all 11 drugs, with an average accuracy of 96.3 %. Matrix effects were xamined in 55 individual patient samples instead of the recommended 6, and 147 individual patient samples were screened for interfering compounds., Significance and Novelty: Our method for simultaneous quantification of 11 t-mAb in human serum allows an unprecedented integration of robustness, speed and reduced complexity, which could pave the way for uniform use in research projects and clinical settings alike. In addition, the first LC-MS protocol for signature peptide-based quantification of durvalumab is described. This high throughput method can be readily adapted to a drug panel of choice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences., (© 2024. The Author(s).)

The rapid identification and isolation of infected individuals remains a key strategy for controlling the spread of SARS-CoV-2. Frequent testing of populations to detect infection early in asymptomatic or presymptomatic individuals can be a powerful tool for intercepting transmission, especially when the viral prevalence is low. However, RT-PCR testing-the gold standard of SARS-CoV-2 diagnosis-is expensive, making regular testing of every individual unfeasible. Sample pooling is one approach to lowering costs. By combining samples and testing them in groups the number of tests required is reduced, substantially lowering costs. Here we report on the implementation of pooling strategies using 3-d and 4-d hypercubes to test a professional sports team in South Africa. We have shown that infected samples can be reliably detected in groups of 27 and 81, with minimal loss of assay sensitivity for samples with individual Ct values of up to 32. We report on the automation of sample pooling, using a liquid-handling robot and an automated web interface to identify positive samples. We conclude that hypercube pooling allows for the reliable RT-PCR detection of SARS-CoV-2 infection, at significantly lower costs than lateral flow antigen (LFA) tests., (© 2021. The Author(s).)

Novel technologies are needed to facilitate large-scale detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibodies in human blood samples. Such technologies are essential to support seroprevalence studies and vaccine clinical trials, and to monitor quality and duration of immunity. We developed a microfluidic nanoimmunoassay (NIA) for the detection of anti-SARS-CoV-2 IgG antibodies in 1,024 samples per device. The method achieved a specificity of 100% and a sensitivity of 98% based on the analysis of 289 human serum samples. To eliminate the need for venipuncture, we developed low-cost, ultralow-volume whole blood sampling methods based on two commercial devices and repurposed a blood glucose test strip. The glucose test strip permits the collection, shipment, and analysis of 0.6 μL of whole blood easily obtainable from a simple finger prick. The NIA platform achieves high throughput, high sensitivity, and specificity based on the analysis of 289 human serum samples, and negligible reagent consumption. We furthermore demonstrate the possibility to combine NIA with decentralized and simple approaches to blood sample collection. We expect this technology to be applicable to current and future SARS-CoV-2 related serological studies and to protein biomarker analysis in general., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often nonspecific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys., Methods: Here, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye., Results: We determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles., Conclusion: We demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Therapeutic drug development is a long, expensive, and complex process that usually takes 12-15 years. In the early phases of drug discovery, in particular, there is a growing need for animal models that ensure the reduction in both cost and time. Caenorhabditis elegans has been traditionally used to address fundamental aspects of key biological processes, such as apoptosis, aging, and gene expression regulation. During the last decade, with the advent of large-scale platforms for screenings, this invertebrate has also emerged as an essential tool in the pharmaceutical research industry to identify novel drugs and drug targets. In this review, we discuss the reasons why C. elegans has been positioned as an outstanding cost-effective option for drug discovery, highlighting both the advantages and drawbacks of this model. Particular attention is paid to the suitability of this nematode in large-scale genetic and pharmacological screenings. High-throughput screenings in C. elegans have indeed contributed to the breakthrough of a wide variety of candidate compounds involved in extensive fields including neurodegeneration, pathogen infections and metabolic disorders. The versatility of this nematode, which enables its instrumentation as a model of human diseases, is another attribute also herein underscored. As illustrative examples, we discuss the utility of C. elegans models of both human neurodegenerative diseases and parasitic nematodes in the drug discovery industry. Summing up, this review aims to demonstrate the impact of C. elegans models on the drug discovery pipeline., (© 2021 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Selective androgen receptor modulators (SARMs) are a group of anabolic enhancer drugs posing threats to the integrity of animal sports and the safety of animal-derived foods. The current research describes for the first time the development of a semi-quantitative assay for the monitoring of SARM family compounds in blood and establishes the relative stability of these analytes under various storage conditions prior to analysis. The presented screening method validation was performed in line with current EU legislation for the inspection of livestock and produce of animal origin, with detection capability (CCβ) values determined at 0.5 ng/mL (Ly2452473), 1 ng/mL (AC-262536 and PF-06260414), 2 ng/mL (bicalutamide, GLPG0492, LGD-2226, ostarine, S-1, S-6, and S-23), and 5 ng/mL (andarine, BMS-564929, LGD-4033, RAD140, and S-9), respectively. The applicability of the developed assay was demonstrated through the analysis of blood samples from racehorses and cattle. The developed method presents a high-throughput cost-effective tool for the routine screening for a range of SARM compounds in sport and livestock animals., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 μl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.

More than 800 G protein-coupled receptors (GPCRs) comprise the largest class of membrane receptors in humans. While there is ample biological understanding and many approved drugs for prototypic GPCRs, most GPCRs still lack well-defined biological ligands and drugs. Here, we report our efforts to tap the potential of understudied GPCRs by developing yeast-based technologies for high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) engineering and GPCR ligand discovery. We refer to these technologies collectively as Dynamic Cyan Induction by Functional Integrated Receptors, or DCyFIR. A major advantage of DCyFIR is that GPCRs and other assay components are CRISPR-integrated directly into the yeast genome, making it possible to decode ligand specificity by profiling mixtures of GPCR-barcoded yeast strains in a single tube. To demonstrate the capabilities of DCyFIR, we engineered a yeast strain library of 30 human GPCRs and their 300 possible GPCR-Gα coupling combinations. Profiling of these 300 strains, using parallel (DCyFIRscreen) and multiplex (DCyFIRplex) DCyFIR modes, recapitulated known GPCR agonism with 100% accuracy, and identified unexpected interactions for the receptors ADRA2B, HCAR3, MTNR1A, S1PR1, and S1PR2. To demonstrate DCyFIR scalability, we profiled a library of 320 human metabolites and discovered several GPCR-metabolite interactions. Remarkably, many of these findings pertained to understudied pharmacologically dark receptors GPR4, GPR65, GPR68, and HCAR3. Experiments on select receptors in mammalian cells confirmed our yeast-based observations, including our discovery that kynurenic acid activates HCAR3 in addition to GPR35, its known receptor. Taken together, these findings demonstrate the power of DCyFIR for identifying ligand interactions with prototypic and understudied GPCRs., Competing Interests: The authors declare no competing interest.

The traditional qPCR instrument is bulky, expensive, and inconvenient to carry, so we report a portable rotary real-time fluorescent PCR (polymerase chain reaction) that completes the PCR amplification of DNA in the field, and the reaction can be observed in real-time. Through the analysis of a target gene, namely pGEM-3Zf (+), the gradient amplification and melting curves are compared to commercial devices. The results confirm the stability of our device. This is the first use of a mechanical rotary structure to achieve gradient amplification curves and melting curves comparable to commercial instruments. The average power consumption of our system is about 7.6 W, which is the lowest energy consumption for real-time fluorescence quantification in shunting PCR and enables the use of our device in the field thanks to its self-contained power supply based on a lithium battery. In addition, all of the equipment costs only about 710 dollars, which is far lower than the cost of a commercial PCR instrument because the control system through mechanical displacement replaces the traditional TEC (thermoelectric cooler) temperature control. Moreover, the equipment has a low technical barrier, which can suit the needs of non-professional settings, with strong repeatability.

Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.

Epigenetic transformations may provide early indicators for cancer and other disease. Specifically, the amount of genomic 5-hydroxymethylcytosine (5-hmC) was shown to be globally reduced in a wide range of cancers. The integration of this global biomarker into diagnostic workflows is hampered by the limitations of current 5-hmC quantification methods. Here we present and validate a fluorescence-based platform for high-throughput and cost-effective quantification of global genomic 5-hmC levels. We utilized the assay to characterize cancerous tissues based on their 5-hmC content, and observed a pronounced reduction in 5-hmC level in various cancer types. We present data for glioblastoma, colorectal cancer, multiple myeloma, chronic lymphocytic leukemia and pancreatic cancer, compared to corresponding controls. Potentially, the technique could also be used to follow response to treatment for personalized treatment selection. We present initial proof-of-concept data for treatment of familial adenomatous polyposis., (© 2019 UICC.)

Therapeutic antibodies generation has to be faster with less development costs. This requires combination of in silico predictions associated with cutting edge screening and characterization technologies. Here, non-exhaustive examples illustrate this simultaneity need., (© 2019 médecine/sciences – Inserm.)

Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay., Study Design and Methods: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500., Results: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03., Conclusion: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions., (© 2019 AABB.)

In this work, we propose an innovative high throughput (HT) apparatus using the bar adsorptive microextraction (BAμE) technique, which enables the simultaneous enrichment of up to 100 samples. This novel configuration was combined with microliquid desorption and high-performance liquid chromatography-diode array detection to monitor trace levels of eight benzodiazepines (diazepam, prazepam, bromazepam, oxazepam, lorazepam, alprazolam, temazepam and loflazepate) in biological samples. The proposed methodology was fully developed, optimized and validated, resulting in suitable intraday and interday precision (RSD ≤ 15%), with recovery yields ranging from 33.0% to 104.5%. The lower limits of quantification were between 20.0 and 100.0 µg L -1 , using 1.0 mL of urine and 0.5 mL of plasma or serum samples. The application of the proposed methodology to real matrices resulted in average sample preparation time of around 2 min per sample, demonstrating that it is user-friendly, cost-effective and a rapid decision-making tool, whenever large number of samples are involved., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Flavones are known as an inhibitor of tankyrase, a potential drug target of cancer. We here expedited the use of different computational approaches and presented a fast, easy, cost-effective and high throughput screening method to identify flavones analogs as potential tankyrase inhibitors. For this, we developed a field point based (3D-QSAR) quantitative structure-activity relationship model. The developed model showed acceptable predictive and descriptive capability as represented by standard statistical parameters r 2 (0.89) and q 2 (0.67). This model may help to explain SAR data and illustrated the key descriptors which were firmly related with the anticancer activity. Using the QSAR model a dataset of 8000 flavonoids were evaluated to classify the bioactivity, which resulted in the identification of 1480 compounds with the IC 50 value of less than 5 µM. Further, these compounds were scrutinized through molecular docking and ADMET risk assessment. Total of 25 compounds identified which further analyzed for drug-likeness, oral bioavailability, synthetic accessibility, lead-likeness, and alerts for PAINS & Brenk. Besides, metabolites of screened compounds were also analyzed for pharmacokinetics compliance. Finally, compounds F2, F3, F8, F11, F13, F20, F21 and F25 with predicted activity (IC 50 ) of 1.59, 1, 0.62, 0.79, 3.98, 0.79, 0.63 and 0.64, respectively were find as top hit leads. This study is offering the first example of a computationally-driven tool for prioritization and discovery of novel flavone scaffold for tankyrase receptor affinity with high therapeutic windows.

Protein kinases are attractive targets for both biological research and drug development. Several assay kits, especially for the detection of adenosine diphosphate (ADP), which is universally produced by kinases, are commercially available for high-throughput screening (HTS) of kinase inhibitors, but their cost is quite high for large-scale screening. Here, we report a new enzyme-coupled fluorescence assay for ADP detection, which uses just 10 inexpensive, commercially available components. The assay protocol is very simple, requiring only the mixing of test solutions with ADP detection solution and reading the fluorescence intensity of resorufin produced by coupling reaction. To validate the assay, we focused on CDC2-like kinase 1 (CLK1), a dual-specificity kinase that plays an important role in alternative splicing, and we used the optimized assay to screen an in-house chemical library of about 215,000 compounds for CLK1 inhibitors. We identified and validated 12 potent inhibitors of CLK1, including a novel inhibitory scaffold. The results demonstrate that this assay platform is not only simple and cost-effective, but also sufficiently robust, showing good reproducibility and giving similar results to those obtained with the widely used ADP-Glo bioluminescent assay.

Reliable species identification methods are important for industrial environmental monitoring programs. Probe based real-time quantitative polymerase chain reaction (qPCR) provides an accurate, cost-effective and high-throughput method for species identification. Here we present the development and validation of species-specific primers and probes for the cytochrome c oxidase (COI) gene for the identification of eight ecologically and economically important freshwater fish species: lake whitefish (Coregonus clupeaformis), yellow perch (Perca flavescens), rainbow smelt (Osmerus mordax), brook trout (Salvelinus fontinalis), smallmouth bass (Micropterus dolomieu), round whitefish (Prosopium cylindraceum), spottail shiner (Notropis hudsonius) and deepwater sculpin (Myoxocephalus thompsonii). In order to identify novel primer-probe sets with maximum species-specificity, two separate primer-probe design criteria were employed. Highest ranked primer-probe sets from both methods were assayed to identify sequences that demonstrated highest specificity. Specificity was determined using control species from same genus and non-target species from different genus. Selected primer-probe sets were optimized for annealing temperature and primer-probe concentrations to identify minimum reagent parameters. The selected primer-probe sets were highly sensitive, with DNA concentrations as low as 1 ng adequate for positive species identification. A decoder algorithm was developed based on the cumulative qPCR results that allowed for full automation of species identification. Blinded experiments revealed that the combination of the species-specific primer/probes sets with the automated species decoder resulted in target species identification with 100% accuracy. We also conducted a cost/time comparison analysis between the qPCR assays established in this study with other species identification methods. The qPCR technique was the most cost-effective and least time consuming method of species identification. In summary, probe-based multiplex qPCR assays provide a rapid and accurate method for freshwater fish species identification, and the methodology established in this study can be utilized for various other species identification initiatives., Competing Interests: Dr. Boreham reports other financial contribution from Bruce Power during the conduct of this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Response to anticancer agents is often restricted to subsets of patients. The recognition of factors underlying this heterogeneity and the identification of biomarkers associated with response to drugs would greatly improve the efficacy of drug treatment. Platforms that can comprehensively map drug response in high-throughput ex vivo provide a unique tool to identify associated biomarkers and provide hypotheses for mechanisms underlying variable response. Such screens can be performed on cell lines and short-term cultures of primary cells to take advantage of the respective models' strength, which include, e.g., the ability to silence genes in cell lines and the "indefinite" supply of primary cells where clonal selection can be avoided. Cohorts of such samples represent the natural diversity of cancers, including rarer mutations and combinatorial patterns of mutations.We here summarize a simple and scalable method for the measurement of viability after drug exposure based on ATP measurements as a surrogate for viability, which we use to measure and understand drug response in cell lines and primary cells.

The world is heading toward a dangerous post-antibiotic era where antibiotics fail to treat infections. Staphylococcus aureus is the leading cause of healthcare-associated infections worldwide, and an ever-increasing percentage of them are methicillin-resistant (MRSA). New strategies are urgently needed to combat this pathogen. Wall teichoic acids (WTA) in S. aureus are polyribitol phosphate polymers that play important roles in virulence and resistance to β-lactam antibiotics. Here, we describe a high-throughput whole-cell screening platform for inhibitors targeting WTA biosynthesis. This platform takes advantage of the unique dispensability patterns of genes encoding WTA biosynthesis. We further describe follow-up dose-response assays to identify WTA inhibitors among the primary bioactives. WTA inhibitors offer an exciting opportunity for the development of novel antibacterial leads of unique mechanism in the fight against drug-resistant staphylococcal infections.

Human herpesviruses (HHV) cause a variety of clinically relevant conditions upon primary infection of typically young and immunocompetent hosts. Both primary infection and reactivation after latency can lead to more severe disease, such as encephalitis, congenital defects and cancer. Infections with HHV are also associated with cardiovascular and neurodegenerative disease. However, most of the associations are based on retrospective case-control analyses and well-powered prospective cohort studies are needed for assessing temporality and causality. To enable comprehensive investigations of HHV-related disease etiology in large prospective population-based cohort studies, we developed HHV Multiplex Serology. This methodology represents a low-cost, high-throughput technology that allows simultaneous measurement of specific antibodies against five HHV species: Herpes simplex viruses 1 and 2, Varicella zoster virus, Epstein-Barr virus, and Cytomegalovirus. The newly developed HHV species-specific ('Monoplex') assays were validated against established gold-standard reference assays. The specificity and sensitivity of the HHV species-specific Monoplex Serology assays ranged from 92.3% to 100.0% (median 97.4%) and 91.8% to 98.7% (median 96.6%), respectively. Concordance with reference assays was very high with kappa values ranging from 0.86 to 0.96 (median kappa 0.93). Multiplexing the Monoplex Serology assays resulted in no loss of performance and allows simultaneous detection of antibodies against the 5 HHV species in a high-throughput manner., Competing Interests: The authors have declared that no competing interests exist.

Protein purification is essential in the study of protein structure and function, and the development of novel therapeutics. Many studies require purifying multiple proteins at once, increasing the demand for improved purification methods. We hypothesized that multiple chromatography columns could be interfaced with a multi-well collection plate for rapid and convenient protein purification without the need of expensive instrumentation. As such, we developed a multi-column plate adapter (MCPA), which provides an economical yet versatile and time efficient, high-throughput protein purification system. The MCPA system simultaneously purified milligrams of different proteins under gravity or under vacuum for faster purification. The MCPA handles up to twenty-four 12 mL columns and multiple MCPA's in sequence allow milligram-scale purification of 96 different samples with relative ease. We also used the MCPA system for large scale affinity purification of four proteins, providing sufficient yields and purity for protein crystallization and biophysical characterization. The MCPA system is ideal for optimizing resin type and volume or any other purification parameter by customizing individual columns during the same purification. The high-throughput and versatile nature of this system should prove to be useful in obtaining adequate amounts of protein for subsequent analyses in any laboratory setting., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pathogenic contamination and resistant bacterial infections remain critical concerns in both developed and developing countries. Rapid and sensitive detection of pathogens is still a key requirement for both environmental and clinical settings. This article introduces a simple, colorimetric, cost-effective, and high-throughput system based on a positively charged iron oxide/enzyme complex for the detection of both gram-positive and gram-negative bacteria in water between 10 3 and 10 8 cfu/mL. This study provides an effective strategy for the identification and purification of pathogen contamination in drinking water.

High-throughput top-down proteomic experiments directly identify proteoforms in complex mixtures, making high quality tandem mass spectra necessary to deeply characterize proteins with many sources of variation. Collision-based dissociation methods offer expedient data acquisition but often fail to extensively fragment proteoforms for thorough analysis. Electron-driven dissociation methods are a popular alternative approach, especially for precursor ions with high charge density. Combining infrared photoactivation concurrent with electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), can significantly improve ETD characterization of intact proteins, but benefits of AI-ETD have yet to be quantified in high-throughput top-down proteomics. Here, we report the first application of AI-ETD to LC-MS/MS characterization of intact proteins (<20 kDa), highlighting improved proteoform identification the method offers over higher energy-collisional dissociation (HCD), standard ETD, and ETD followed by supplemental HCD activation (EThcD). We identified 935 proteoforms from 295 proteins from human colorectal cancer cell line HCT116 using AI-ETD compared to 1014 proteoforms, 915 proteoforms, and 871 proteoforms with HCD, ETD, and EThcD, respectively. Importantly, AI-ETD outperformed each of the three other methods in MS/MS success rates and spectral quality metrics (e.g., sequence coverage achieved and proteoform characterization scores). In all, this four-method analysis offers the most extensive comparisons to date and demonstrates that AI-ETD both increases identifications over other ETD methods and improves proteoform characterization via higher sequence coverage, positioning it as a premier method for high-throughput top-down proteomics.

Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aim: The EGFR inhibitors represent the first-line treatment of non-small-cell lung cancer. However, the emergence of resistance urgently requires the development of new inhibitors targeting drug-resistant mutants., Methodology: A recently released structure of an EGFR kinase domain in complex with an allosteric inhibitor and a mutant protein model derived from it were used to set up a low-cost high-throughput docking protocol for the fast identification of EGFR allosteric inhibitors., Conclusion: The virtual screening of commercially available compounds led to the identification of interesting new hit compounds. The most promising hit was confirmed to be a new allosteric inhibitor of wild-type and T790M/L858R double mutant EGFR which was able to inhibit the growth of  non-small-cell lung cancer cell lines.

Objectives: Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness., Methods: We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision., Results: The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r = 0.79, P = 1.44 × 10 -15 ). In an independent set of samples (n = 550), the new assay showed a negative correlation of RTL with age (r = -0.41), a result providing external validation for the method., Conclusion: We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies., (© 2018 Wiley Periodicals, Inc.)

Screening drugs on patient biopsies from solid tumours has immense potential, but is challenging due to the small amount of available material. To address this, we present here a plug-based microfluidics platform for functional screening of drug combinations. Integrated Braille valves allow changing the plug composition on demand and enable collecting >1200 data points (56 different conditions with at least 20 replicates each) per biopsy. After deriving and validating efficient and specific drug combinations for two genetically different pancreatic cancer cell lines and xenograft mouse models, we additionally screen live cells from human solid tumours with no need for ex vivo culturing steps, and obtain highly specific sensitivity profiles. The entire workflow can be completed within 48 h at assay costs of less than US$ 150 per patient. We believe this can pave the way for rapid determination of optimal personalized cancer therapies.

When excessively activated, C1 is insufficiently regulated, which results in tissue damage. Such tissue damage causes the complement system to become further activated to remove the resulting tissue damage, and a vicious cycle of activation/tissue damage occurs. Current Food and Drug Administration approved treatments include supplemental recombinant C1 inhibitor, but these are extremely costly and a more economical solution is desired. In our work, we have utilized an existing data set of 136 compounds that have been previously tested for activity against C1. Using these compounds and the activity data, we have created models using principal component analysis, genetic algorithm, and support vector machine approaches to characterize activity. The models were then utilized to virtually screen the 72 million compound PubChem repository. This first round of virtual high-throughput screening identified many economical and promising inhibitor candidates, a subset of which was tested to validate their biological activity. These results were used to retrain the models and rescreen PubChem in a second round vHTS. Hit rates for the first round vHTS were 57%, while hit rates for the second round vHTS were 50%. Additional structure⁻property analysis was performed on the active and inactive compounds to identify interesting scaffolds for further investigation.

The use of AlphaScreen ® detection has allowed researchers to examine a wide variety of molecular interactions for use in high-throughput screening. However, the cost of Alpha reagents can often be prohibitory for extended screening campaigns or for young investigators with limited funding. To reduce assay costs, many labs have focused on miniaturization, while there have been limited efforts to scale down Alpha reagents. Thus, we describe the optimization of an AlphaScreen detection platform by systematically reducing the Alpha reagents down to 2.5 μg/ml beads, without compromising assay integrity. We demonstrate that reducing bead concentration reduces detection costs substantially while yielding robust statistics. We believe this simple new protocol will enhance the future utilization of AlphaScreen technology in high-throughput screening.

Aim: To simplify the recognition of Actinobacteria, at different stages of the growth phase, from a mixed culture to facilitate the isolation of novel strains of these bacteria for drug discovery purposes., Materials & Methods: A method was developed based on Gabor transform, and machine learning using k-Nearest Neighbors and Naive Bayes classifier, Logitboost, Bagging and Random Forest to automatically categorize the colonies., Results: A signature pattern was inferred by the model, making the differentiation of identical strains possible. Additionally, higher performance, compared with other classification methods was achieved., Conclusion: This automated approach can contribute to the acceleration of the drug discovery process while it simultaneously can diminish the loss of budget due to the redundancy occurred by the inexperienced researchers.

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-μLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by μLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the μLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

Phenotype-based small molecule screens in zebrafish embryos and larvae have been successful in accelerating pathway and therapeutic discovery for diverse biological processes. Yet, the application of chemical screens to adult physiologies has been relatively limited due to additional demands on cost, space, and labor associated with screens in adult animals. In this study, we present a 3D printed system and methods for intermittent drug dosing that enable rapid and cost-effective chemical administration in adult zebrafish. Using prefilled screening plates, the system enables dosing of 96 fish in ∼3 min, with a 10-fold reduction in drug quantity compared to that used in previous chemical screens in adult zebrafish. We characterize water quality kinetics during immersion in the system and use these kinetics to rationally design intermittent dosing regimens that result in 100% fish survival. As a demonstration of system fidelity, we show the potential to identify two known chemical inhibitors of adult tail fin regeneration, cyclopamine and dorsomorphin. By developing methods for rapid and cost-effective chemical administration in adult zebrafish, this study expands the potential for small molecule discovery in postembryonic models of development, disease, and regeneration.

Up to now, knowledge of enzymes capable of degrading various contaminants of emerging concern (CEC) is limited, which is especially due to the lack of rapid screening methods. Thus, a miniaturized high-throughput setup using a chip-based robotic nanoelectrospray ionization system coupled to mass spectrometry has been developed to rapidly screen enzymatic reactions with environmentally relevant CECs. Three laccases, two tyrosinases, and two peroxidases were studied for their ability to transform ten pharmaceuticals and benzotriazole. Acetaminophen was most susceptible to enzymatic conversion by horseradish peroxidase (HRP), laccase from Trametes versicolor (LccTV), and a tyrosinase from Agaricus bisporus (TyrAB). Diclofenac and mefenamic acid were converted by HRP and LccTV, whereas sotalol was solely amenable to HRP conversion. Benzotriazole, carbamazepine, gabapentin, metoprolol, primidone, sulfamethoxazole, and venlafaxine remained persistent in this study. The results obtained here emphasize that enzymes are highly selective catalysts and more effort is required in the use of fast monitoring technologies to find suitable enzyme systems. Despite the methodological limitations discussed in detail, the automated tool provides a routine on-line screening of various enzymatic reactions to identify potential enzymes that degrade CECs. Graphical abstract A chip-based robotic nano-ESI-MS tool to rapidly monitor enzymatic degradation of environmentally relevant emerging contaminants.

General anesthetics are considered among the most significant advances in modern medicine; however, they are also some of the most dangerous commonly administered drugs. Despite this, the discovery of novel anesthetics has been slow, with few clinically used agents regardless of their nearly 200-year history. Xenopus laevis frogs have a long history as a model organism and provide a vital bridge between in vitro and preclinical mammalian assays. The provided protocols are efficient and cost-effective and therefore readily amendable for high-throughput evaluation of novel anesthetic ligands. By using the X. laevis bioassay, a researcher is capable of determining relative general anesthetic tolerance and/or cross-tolerance for candidate nonvolatile and/or volatile ligands., (© 2018 Elsevier Inc. All rights reserved.)

The new paradigm of the big data raises many expectations, particularly in the field of health. Curiously, even though medical biology laboratories generate a great amount of data, the opportunities offered by this new field are poorly documented. For better understanding the clinical context of chronical disease follow-up, for leveraging preventive and/or personalized medicine, the contribution of big data analytics seems very promising. It is within this framework that we have explored to use data of a Breton group of laboratories of medical biology to analyze the possible contributions of their exploitation in the improvement of the clinical practices and to anticipate the evolution of pathologies for the benefit of patients. We report here three practical applications derived from routine laboratory data from a period of 5 years (February 2010-August 2015): follow-up of patients treated with AVK according to the recommendations of the High authority of health (HAS), use of the new troponin markers HS and NT-proBNP in cardiology. While the risks and difficulties of using algorithms in the health domain should not be underestimated - quality, accessibility, and protection of personal data in particular - these first results show that use of tools and technologies of the big data repository could provide decisive support for the concept of "evidence based medicine".

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research., (Copyright © 2017 Elsevier B.V. All rights reserved.)

HTMD is a programmable scientific platform intended to facilitate simulation-based research in molecular systems. This paper presents the functionalities of HTMD for the preparation of a molecular dynamics simulation starting from PDB structures, building the system using well-known force fields, and applying standardized protocols for running the simulations. We demonstrate the framework's flexibility for high-throughput molecular simulations by applying a preparation, building, and simulation protocol with multiple force-fields on all of the seven hundred eukaryotic membrane proteins resolved to-date from the orientation of proteins in membranes (OPM) database. All of the systems are available on www.playmolecule.org .

Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory., (© 2017 The Protein Society.)

Objectives: To develop an efficient cost-effective screening process to improve production of glucoamylase in Aspergillus niger., Results: The cultivation of A. niger was achieved with well-dispersed morphology in 48-deep-well microtiter plates, which increased the throughput of the samples compared to traditional flask cultivation. There was a close negative correlation between glucoamylase and its pH of the fermentation broth. A novel high-throughput analysis method using Methyl Orange was developed. When compared to the conventional analysis method using 4-nitrophenyl α-D-glucopyranoside as substrate, a correlation coefficient of 0.96 by statistical analysis was obtained., Conclusion: Using this novel screening method, we acquired a strain with an activity of 2.2 × 10 3  U ml -1 , a 70% higher yield of glucoamylase than its parent strain.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost., (Published by Elsevier Inc.)

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.