96-well Format Research Articles

High-Efficient Transient Gene Transfection for Transcriptional Activity Assay Using Rice Protoplasts on Large-Scale Platform.

Transient gene expression in protoplasts offers a straightforward and efficient approach for investigating gene function. Here, I introduce a method that utilizes a 96-well plate format to assess transcriptional activation capacity of transcription factors through transient expression system in rice protoplasts. This method includes plant cultivation, protoplast preparation, transfection, and transactivation assay and allows for the simultaneous measurement and validation of 23 samples with 4 replicates per sample at a time.

A Novel High-Throughput Immunoaffinity LC-MS/MS Assay for P-III-NP and Other Fragments of Type III Procollagen in Human Serum.

The amino-terminal propeptide of type III procollagen (P-III-NP) is used with IGF-I to detect the illicit use of growth hormone and to monitor growth hormone therapy. However, the only currently available assays for P-III-NP are immunoassays, which are not well harmonized. In addition, other fragments of type III procollagen may better evaluate collagen turnover. We aimed to develop a high-throughput assay using immunoaffinity enrichment coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify peptides belonging to three different regions of type III procollagen in human serum simultaneously. To facilitate higher throughput, we transferred the assay from microcentrifuge tubes to a 96-well plate format with partially automated pipetting. The method was linear (Pearson's R ≥ 0.994) over an estimated concentration range of 1.35-13.3 nM, 0.04-2.28 nM, and 0.26-5.1 nM for each surrogate peptide of P-III-NP, collagen degradation products, and the carboxyl-terminal propeptide, respectively. Intra-day and inter-day imprecision were both < 13.6%, and the results of robustness testing were also encouraging. The method was successfully applied to capillary blood samples obtained using Tasso+ microsampling devices. Modest correlation of P-III-NP concentration was observed between our new method and a WADA-approved immunoassay (N = 40, Pearson's R = 0.789) with a significant bias of -87.8%. Our method simultaneously quantifies four peptides belonging to three regions of type III procollagen in human serum. High bias between assays highlights the need for common higher-order calibrators or reference materials to help improve the comparability of results across laboratories.

Quantitative High-Throughput and High-Sensitivity Glycoprofile Analysis of Therapeutic Glycoproteins Using HILIC-FLD and ESI-MS.

Protein glycosylation is recognized as a Critical Quality Attribute for the biological and therapeutic activity of many recombinant proteins. Therefore, glycosylation should be monitored rigorously to ensure the desired quality, safety, and potency of monoclonal antibodies and other therapeutic glycoproteins. However, glycans are highly heterogeneous structures in proteins, and this poses a challenge for glycoprofile analysis. Hydrophilic interaction liquid chromatography with fluorescence detection has been recognized as a standard method for separation and quantification of released N-glycans after conventional 2-aminobenzamide labeling, but the sample preparation procedure is time-consuming with low sensitivity and poor reproducibility. Here we present a streamlined 96-well plate format platform for high-throughput and high-sensitivity glycan profiling. By taking advantage of rapid glycoprotein denaturation, enzymatic deglycosylation, highly sensitive fluorescent derivation, and on-matrix glycan cleanup, the reaction time has been shortened significantly to approximately 10min. A detailed workflow including two-dimensional chromatography fractionation, exoglycosidase sequential digestion, mass spectroscopy glycan mass confirmation, and data interpretation is described. Potential pitfalls and precautions to be taken to ensure data accuracy are also discussed in this chapter.

High-resolution acoustic ejection mass spectrometry for high-throughput library screening

An approach is described for high-throughput quality assessment of drug candidate libraries using high-resolution acoustic ejection mass spectrometry (AEMS). Sample introduction from 1536-well plates is demonstrated for this application using 2.5 nL acoustically dispensed sample droplets into an Open Port Interface (OPI) with pneumatically assisted electrospray ionization at a rate of one second per sample. Both positive and negative ionization are shown to be essential to extend the compound coverage of this protease inhibitor-focused library. Specialized software for efficiently interpreting this data in 1536-well format is presented. A new high-throughput method for quantifying the concentration of the components (HTQuant) is proposed that neither requires adding an internal standard to each well nor further encumbers the high-throughput workflow. This approach for quantitation requires highly reproducible peak areas, which is shown to be consistent within 4.4 % CV for a 1536-well plate analysis. An approach for troubleshooting the workflow based on the background ion current signal is also presented. The AEMS data is compared to the industry standard LC/PDA/ELSD/MS approach and shows similar coverage but at 180-fold greater throughput. Despite the same ionization process, both methods confirmed the presence of a small percentage of compounds in wells that the other did not. The data for this relatively small, focused library is compared to a larger, more chemically diverse library to indicate that this approach can be more generally applied beyond this single case study. This capability is particularly timely considering the growing implementation of artificial intelligence strategies that require the input of large amounts of high-quality data to formulate predictions relevant to the drug discovery process. The molecular structures of the 872-compound library analyzed here are included to begin the process of correlating molecular structures with ionization efficiency and other parameters as an initial step in this direction.

Anticancer Efficacy of Biosynthesized Zinc Oxide and Gold Nanoparticles

Abstract Introduction/Objective Introduction: Advancements in nanoscale materials have led to the exploration of metal oxides for cancer diagnosis and therapy. Zinc oxide nanoparticles (ZnO NPs) and gold nanoparticles (Au NPs) are particularly promising due to their biocompatibility and anticancer properties demonstrated across various cancer cell lines. Objectives This study aimed to isolate fungal endophytes from Eucalyptus sideroxylon leaves and utilize them for the biosynthesis of nanoparticles (ZnO NPs and Au NPs) to assess their anticancer activity. Methods/Case Report Methods: Healthy Eucalyptus sideroxylon samples were collected from a desert research center, and endophytes were isolated and cultivated. The fungal biomass was processed to synthesize ZnO NPs and Au NPs. Cell lines were propagated for cytotoxicity experiments using the MTT assay in a 96-well plate format. Results (if a Case Study enter NA) Results: Biosynthesized ZnO and Au NPs exhibited anticancer activity against HCT-116 and CACO2 cell lines. The IC50 values for ZnO NPs were 5.06 μg/mL for HCT-116 and 6.07 μg/mL for CACO2, while Au NPs showed IC50 values of 5.9 μg/mL for HCT-116 and 1.1 μg/mL for CACO2. ZnO NPs demonstrated dose-dependent toxicity on HCT-116 cells, with increasing concentrations leading to a reduction in cell viability. Similarly, Au NPs exhibited dose-dependent cytotoxicity on both cell lines. Conclusion Conclusion: This study underscores the potential of endophytic fungi for biosynthesizing metal nanoparticles with significant anticancer effects. ZnO NPs displayed superior efficacy against colon cancer cells compared to Au NPs, indicating their potential as therapeutic agents. The findings highlight the importance of exploring natural sources for nanoparticle synthesis and their application in cancer therapy.

Development of an orthotopic medulloblastoma zebrafish model for rapid drug testing.

Medulloblastoma (MB) is one of the most common malignant brain tumors in children. Current preclinical in vivo model systems for MB have increased our understanding of molecular mechanisms regulating MB development. However, they may not be suitable for large-scale studies. The aim of this study was to investigate if a zebrafish-based xenograft model can recapitulate MB growth and enable rapid drug testing. Nine different MB cell lines or patient-derived cells were transplanted into blastula-stage zebrafish embryos. Tumor development and migration were then monitored using live imaging. RNA sequencing was performed to investigate transcriptome changes after conditioning cells in neural stem cell-like medium. Furthermore, drug treatments were tested in a 96-well format. We demonstrate here that transplantation of MB cells into the blastula stage of zebrafish embryos leads to orthotopic tumor growth that can be observed within 24 hours after transplantation. Importantly, the homing of transplanted cells to the hindbrain region and the aggressiveness of tumor growth are enhanced by pre-culturing cells in a neural stem cell-like medium. The change in culture conditions rewires the transcriptome towards a more migratory and neuronal phenotype, including the expression of guidance molecules SEMA3A and EFNB1, both of which correlate with lower overall survival in MB patients. Furthermore, we highlight that the orthotopic zebrafish MB model has the potential to be used for rapid drug testing. Blastula-stage zebrafish MB xenografts present an alternative to current MB mouse xenograft models, enabling quick evaluation of tumor cell growth, neurotropism, and drug efficacy.

Unbiased High-Throughput Screening of Drug-Repurposing Libraries Identifies Small-Molecule Inhibitors of Clot Retraction

Platelet clot retraction, the ultimate phase of platelet thrombus formation, is critical for clot stabilization. It requires functional αIIbβ3 receptors, fibrin, and the integrated actions of the actin-myosin contractile and cytoskeletal systems. Disturbances in clot retraction have been associated with both bleeding and thrombosis. We recently demonstrated that platelets treated with the αIIbβ3 antagonist peptide RGDW, which eliminates fibrinogen-mediated platelet aggregation, are still able to retract clots. We have exploited this observation to developed an unbiased, functional high-throughput assay to identify small molecule inhibitors of fibrin-mediated clot retraction adapted for a 384-well plate format. We tested 9,710 compounds from drug-repurposing libraries (DRL’s). These libraries contain compounds that are either FDA-approved or have undergone preclinical/clinical development. We identified 27 compounds from the LOPAC library as inhibitors of clot retraction of which 14 are known inhibitors of platelet function. From the DRL we identified 135 compounds (1.6% hit rate). After extensive curation, these compounds were categorized based on the activity of their reported target. Multiple kinase and phosphodiesterase inhibitors with known antiplatelet effects were identified, along with multiple deubiquitination and receptor inhibitors, as well as compounds that have not previously been reported to have antiplatelet activity. Studies of one of the deubiquitination inhibitors (degrasyn) suggests that its effects are downstream of thrombin-induced platelet-fibrinogen interactions and thus may permit the separation of platelet thrombin-induced aggregation-mediated events from clot retraction. Additional studies of the identified compounds may lead to novel mechanisms of inhibiting thrombosis.

12422 Linsitinib Inhibits Proliferation And Induces Apoptosis Of IGF-1R And TSH-R Expressing Cells

Abstract Disclosure: M. Luffy: None. A. Ganz: None. J. Ropertz: None. R. Douglas: Employee; Self; Sling Therapeutics. R. Zeidan: Employee; Self; Sling Therapeutics. J. Kent: Employee; Self; Sling Therapeutics. J. Wolf: None. G.J. Kahaly: Grant Recipient; Self; Sling Therapeutics. Background: The insulin-like growth factor 1 receptor (IGF-1R) and the thyrotropin receptor (TSH-R) are expressed on orbital target cells and thyrocytes. Targeting these pathways effectively improves thyroid eye disease (TED). Methods: The inhibitory effect of Linsitinib, a small molecule targeting the IGF-1R (Sling Therapeutics, Ann Arbor, MI, USA), on cell proliferation was investigated in an IGF-1R expressing cell line, NCI-H295R (ATCC, Manassas, VA, USA) and a Chinese Hamster Ovary (CHO) cell line overexpressing the TSH-R (QuidelOrtho, San Diego, CA, USA). An IGF-1R antagonist, Teprotumumab (BioVision, Milpitas, CA, USA) served as control. Both cell lines were plated in a 96-well format and treated with four concentrations of both compounds for 24 hours. After addition of tetrazolium, absorbance was measured (GloMax luminometer, Promega, Madison, WI, USA). Furthermore, the half-maximal inhibitory concentration (IC50) of TSH-R-Ab induced stimulation (stimulatory monoclonal antibody, mAb, M22, RSR, Cardiff, UK) of the TSH-R cell line was evaluated with a cell-based bioassay for blocking TSH-R-Ab (Thyretain TBI, QuidelOrtho). Cells were treated with ten rising concentrations of either Linsitinib, Linsitinib + Metformin, Teprotumumab, or a blocking TSH-R mAb (K1-70, RSR). The Kruskal-Wallis and/or the two-tailed Mann-Whitney tests were used for statistical comparisons. Results: Linsitinib strongly inhibited the proliferation of both cell lines at the following concentrations: 31,612.5 ng/mL (-78%, P=0.0031, IGF-1R cell line and -75%, P=0.0059, TSH-R cell line), as well as at 63,225 ng/mL (-73%, P=0.0073, IGF-1R cell line and -73%, P=0.0108, TSH-R cell line). Linsitinib induced morphologically confirmed apoptosis of both cell lines across all tested concentrations (Luna-FL cell counter, Logos Biosystems, Annandale, VA, USA). Compared to Teprotumumab, Linsitinib inhibited markedly more the proliferation of the IGF-1R cell line at all concentrations (Linsitinib versus Teprotumumab, P=0.0286). Teprotumumab inhibition ranged from -21 to -31% of the IGF-1R cell line across all concentrations, while it was significant only at 15,806.25 ng/mL with the TSH-R cell line (-15%, P=0.0396). In addition, in the TSH-R-Ab blocking bioassay, all tested compounds demonstrated strong inhibition across all ten dilutions (100%). The IC50 of Linsitinib was lower than Teprotumumab (147.1 ng/mL versus 160.6 ng/mL). Combining Metformin to Linsitinib did not further lower the IC50. Not surprisingly, the TSH-R blocking mAb K1-70 had the lowest IC50 of 20.57 ng/mL. Conclusions: Linsitinib effectively induces apoptosis and inhibits proliferation of both IGF-1R and TSH-R expressing target cells, also displaying a lower IC50 in the blocking TSH-R-Ab bioassay compared to Teprotumumab, hence demonstrating its therapeutic potential for TED. Presentation: 6/2/2024

Rapid Lipid Mediator Profiling by Convergence Chromatography-MS/MS.

Bioactive lipid mediators derived from arachidonic acid constitute an attractive pool of metabolites that reflect cellular function and signaling, as well as potential biomarkers that may respond quantitatively to disease progression or pharmacological treatment. Their quantitative measurement in biological samples is complicated by the number of isomers that share common structural features, which are not easily distinguished by immunoassays or reverse phase chromatography-tandem mass spectrometry. Here, we present a method that enables the rapid analysis of a panel of over 25 biologically important eicosanoids in a 96-well format for cell culture supernatants, plasma, and organ tissues using convergence chromatography-tandem mass spectrometry to resolve these analytes of interest.

B-040 Direct Aspiration of Cryogenic Matrix Tubes on the Siemens Atellica System With Epic-Beaker Integration of External Barcode IDs for Analysis Small Volume Biobanked Specimens

Abstract Background Most core chemistry and immunochemistry clinical analyzers are designed for direct sampling of primary blood tubes with volumes ranging from 3.5-10 mL. This limits analysis of low volume pediatric specimens and biobanked aliquoted samples from clinical trials without manual technologist intervention. This work outlines a microsampling and data transmission scheme for analysis of 500 uL aliquots of biobanked serum collected for the Nutrition for Precision Health study powered by the NIH All of Us Research Program without relabeling secondary aliquot tubes after receipt. Methods Commercial plastic clip bearings sourced from IGUS (LCM-08-02) were used to stabilize the matrix tube in a standard 5 mL Sarstedt aliquot tube. 96-well format Matrix Tubes (ThermoFisher 3719-11) with pre-printed 10-digit side barcodes were programmed in the Atellica software as a custom false bottom tube with specifications height 75 mm, diameter 12 mm, bottom offset 41 mm and were loaded using a dedicated false bottom tray. Manifest files (.csv) containing the sample ID, clinical trials participant ID, collection date/time, age and sex were ingested into Epic via an Incoming Orders HL7 interface. Non-human patient records were generated using the participant ID as an external patient ID and patient name and calculating the Date of Birth based on the age in years given in the manifest file. Test orders were created based on assigned testing for the sample ID in the manifest. The sample ID corresponding the preprinted Matrix Tube barcode ID was inserted into the Epic container identifier field. Results Test orders were automatically transmitted to the Atellica Data Manger (ADM) allowing specimens to be run on the Atellica System (CH and IM modules) and results transmitted to Epic utilizing the established Data Innovations (DI) interface and processes used for patient samples without the need for Epic generated labels. Successful scanning and recognition of Matrix Tube barcodes was 100%. Complete aspiration of a Comprehensive Metabolic Panel, Lipid Panel, Iron, TIBC, hsCRP, insulin, and H, I, L indices occurred for 50% of samples tested without intervention. Approximately 250 uL of the 500 uL aliquot remained after aspiration of all tests (247.7 uL total volume) from the Matrix Tube. No probe crashes were observed despite the narrower 8 mm diameter of the Matrix Tubes versus the standard 12- or 13-mm diameter of primary blood collection tubes. Conclusions This Epic-DI-ADM data workflow and microsampling adaptation for Matrix Tube aspiration allows greater container type flexibility for the receipt and analysis of external samples delivered in non-primary blood tubes for clinical laboratories. Future work involving adjustment of the false bottom tube offset depth may reduce unusable sample volume allowing for a higher percentage of complete aspiration without intervention. This work is significant because it suggests existing clinical analyzers are easily adaptable to microsampling smaller volume tubes without significant effort.

Innovative hierarchical porous hydrophilic molecularly imprinted resin for high-throughput detection of perfluorocarboxylic acids in milk using 96-well plate SPE-LC-MS/MS

The accumulation of perfluorocarboxylic acids (PFCAs) in environment and foods represents a significant threat to public health due to the long-term ingestion of contaminated food. This study introduces a novel adsorbent, the hierarchical porous hydrophilic molecularly imprinted resin (HPHMIR), which was synthesized by integrating molecular imprinting techniques with hydrophilic resins. The HPHMIR, characterized by its extensive mesoporous structure (average pore width ∼9.71 nm) and favorable imprinting factors (2.6–5.0), facilitates the effective adsorption of PFCAs from complex matrices through multiple interaction mechanisms, including hydrogen bonding and electrostatic interactions. This innovative material was employed in a 96-well plate format for solid-phase extraction (SPE), and combined with LC-MS/MS, a high-throughput method for the determination of PFCAs in milk was developed. The proposed method demonstrated exceptional performance, including excellent linearity (0.48–240 ng mL−1; r ≥ 0.9986), low detection limits (0.04–0.11 ng mL−1), high precision (relative standard deviation ≤ 9.9 %), and satisfactory recovery (75.7–118.1 %). These results highlight the efficacy of the method in extracting trace levels of PFCAs from complicated sample matrices, presenting a promising alternative for monitoring PFCA contamination and advancing public health standards.

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.

TIRTL-seq: Deep, quantitative, and affordable paired TCR repertoire sequencing.

α/β T cells are key players in adaptive immunity. The specificity of T cells is determined by the sequences of the hypervariable T cell receptor (TCR) α and β chains. Although bulk TCR sequencing offers a cost-effective approach for in-depth TCR repertoire profiling, it does not provide chain pairings, which are essential for determining T cell specificity. In contrast, single-cell TCR sequencing technologies produce paired chain data, but are limited in throughput to thousands of cells and are cost-prohibitive for cohort-scale studies. Here, we present TIRTL-seq (Throughput-Intensive Rapid TCR Library sequencing), a novel approach that generates ready-to-sequence TCR libraries from live cells in less than 7 hours. The protocol is optimized for use with non-contact liquid handlers in an automation-friendly 384-well plate format. Reaction volume miniaturization reduces library preparation costs to <$0.50 per well. The core principle of TIRTL-seq is the parallel generation of hundreds of libraries providing multiple biological replicates from a single sample that allows precise inference of both frequencies of individual clones and TCR chain pairings from well-occurrence patterns. We demonstrate scalability of our approach up to 1 million unique paired αβTCR clonotypes corresponding to over 30 million T cells per sample at a cost of less than $2000. For a sample of 10 million cells the cost is ~$200. We benchmarked TIRTL-seq against state-of-the-art 5'RACE bulk TCR-seq and 10x Genomics Chromium technologies on longitudinal samples. We show that TIRTL-seq is able to quantitatively identify expanding and contracting clonotypes between timepoints while providing accurate TCR chain pairings, including distinct temporal dynamics of SARS-CoV-2-specific and EBV-specific CD8+ T cell responses after infection. While clonal expansion was followed by sharp contraction for SARS-CoV-2 specific TCRs, EBV-specific TCRs remained stable once established. The sequences of both α and β TCR chains are essential for determining T cell specificity. As the field moves towards greater applications in diagnostics and immunotherapy that rely on TCR specificity, we anticipate that our scalable paired TCR sequencing methodology will be instrumental for collecting large paired-chain datasets and ultimately extracting therapeutically relevant information from the TCR repertoire.

A 96-position platform for magnetic sorbent-based dispersive microextraction: Application to the determination of tetrahydrocannabinol and cannabidiol in saliva of cannabis smokers

BackgroundIn order to obtain sustainable analytical methods, it is essential to develop kinetically efficient sample preparation strategies in which equilibria are reached faster, as dispersive extraction techniques. In addition, the higher the reduction in size, the higher number of extraction vessels can be located and thus the higher number of samples can be simultaneously treated. All this increases sample throughput, and contributes to the reduction of chemical waste, and energy and sample consumption. In this sense, multiposition extraction platforms are smart strategies to achieve these goals, but they are scarcely developed for dispersive extraction techniques. ResultsTaking miniaturized stir bar sorptive dispersive microextraction (mSBSDME) as a starting point, a 96-position extraction platform has been developed using a 96-position stirring plate and a tailor-designed 3D-printed support for locating the miniaturized extraction vessels, achieving a high-throughput miniaturized sample preparation strategy. In order to show the applicability of this novel platform, the determination of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in human saliva has been carried out and applied to samples collected after the consumption of marijuana and legal CBD-rich cannabis. Only 100 μL of saliva were needed for the analysis and good analytical features in terms of linearity (at least up to 500 ng mL−1), limits of detection (0.7 and 2.8 ng mL−1 for THC and CBD, respectively), and precision (RSD ≤ 14 %) were achieved. SignificanceThe miniaturization of the vessel allows the use of small volumes of sample (i.e., a few microliters) and the treatment of 96 samples in parallel, being the first proposal for carrying out dispersive sorbent-based microextraction under the concept of 96-well format. Additionally, this new workflow contributes to the development of analytical methods that meet the three pillars of sustainability, i.e., greenness and easily affordable in terms of economics and applicability.

High Content Screening Assay of Inhibitors of the Legionella Pneumophila Histone Methyltransferase RomA in Infected Cells.

Resistance to anti-microbial agents is a world-wide health threat. Thus, there is an urgent need for new treatments. An alternative approach to disarm pathogens consists in developing drugs targeting epigenetic modifiers. Bacterial pathogens can manipulate epigenetic regulatory systems of the host to bypass defences to proliferate and survive. One example is Legionella pneumophila, a Gram-negative intracellular pathogen that targets host chromatin with a specific, secreted bacterial SET-domain methyltransferase named RomA. This histone methyltransferase specifically methylates H3 K14 during infection and is responsible for changing the host epigenetic landscape upon L. pneumophila infection. To inhibit RomA activity during infection, we developed a reliable high-content imaging screening assay, which we used to screen an in-house chemical library developed to inhibit DNA and histone methyltransferases. This assay was optimised using monocytic leukemic THP-1 cells differentiated into macrophages infected with L. pneumophila in a 96- or 384-well plate format using the Opera Phenix (Perkin Elmer) confocal microscope, combined with Columbus software for automated image acquisition and analysis. H3 K14 methylation was followed in infected, single cells and cytotoxicity was assessed in parallel. A first pilot screening of 477 compounds identified a potential starting point for inhibitors of H3 K14 methylation.

High-throughput analysis of topographical cues for the expansion of murine pluripotent stem cells

The expansion of pluripotent stem cells (PSCs)in vitroremains a critical barrier to their use in tissue engineering and regenerative medicine. Biochemical methods for PSC expansion are known to produce heterogeneous cell populations with varying states of pluripotency and are cost-intensive, hindering their clinical translation. Engineering biomaterials to physically control PSC fate offers an alternative approach. Surface or substrate topography is a promising design parameter for engineering biomaterials. Topographical cues have been shown to elicit profound effects on stem cell differentiation and proliferation. Previous reports have shown isotropic substrate topographies to be promising in expanding PSCs. However, the optimal feature to promote PSC proliferation and the pluripotent state has not yet been determined. In this work, the MultiARChitecture (MARC) plate is developed to conduct a high-throughput analysis of topographical cues in a 96-well plate format. The MARC plate is a reproducible and customizable platform for the analysis of multiple topographical patterns and features and is compatible with both microscopic assays and molecular biology techniques. The MARC plate is used to evaluate the expression of pluripotency markersOct4, Nanog, andSox2and the differentiation markerLmnAas well as the proliferation of murine embryonic stem (mES) cells. Our systematic analyses identified three topographical patterns that maintain pluripotency in mES cells after multiple passages: 1µm pillars (1µm spacing, square arrangement), 2µm wells (c-c (x, y) = 4, 4µm), and 5µm pillars (c-c (x, y) = 7.5, 7.5µm). This study represents a step towards developing a biomaterial platform for controlled murine PSC expansion.

Development of a time-resolved fluorescence resonance energy transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z’ factor > 0.8 and signal-to-background (S/B) ratio > 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein–protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.

Identification of small molecule antagonists of sonic hedgehog/heparin binding with activity in hedgehog functional assays

Sonic hedgehog (Shh) is a morphogen with important roles in embryonic development and in the development of a number of cancers. Its activity is modulated by interactions with binding partners and co-receptors including heparin and heparin sulfate proteoglycans (HSPG). To identify antagonists of Shh/heparin binding, a diverse collection of 34,560 chemicals was screened in single point 384-well format. We identified and confirmed twenty six novel small molecule antagonists with diverse structures including four scaffolds that gave rise to multiple hits. Nineteen of the confirmed hits blocked binding of the N-terminal fragment of Shh (ShhN) to heparin with IC50 values < 50 μM. In the Shh-responsive C3H10T1/2 cell model, four of the compounds demonstrated the ability to block ShhN-induced alkaline phosphatase activity. To demonstrate a direct and selective effect on ShhN ligand mediated activity, two of the compounds were able to block induction of Gli1 mRNA, a primary downstream marker for Shh signaling activity, in Shh-mediated but not Smoothened agonist (SAG)-mediated C3H10T1/2 cells. Direct binding of the two compounds to ShhN was confirmed by thermal shift assay and molecular docking simulations, with both compounds docking with the N-terminal heparin binding domain of Shh. Overall, our findings indicate that small molecule compounds that block ShhN binding to heparin and act to inhibit Shh mediated activity in vitro can be identified. We propose that the interaction between Shh and HSPGs provides a novel target for identifying small molecules that bind Shh, potentially leading to novel tool compounds to probe Shh ligand function.

Screening persistent organic pollutants for effects on testosterone and estrogen synthesis at human-relevant concentrations using H295R cells in 96-well plates.

Many persistent organic pollutants (POPs) are suspected endocrine disruptors and it is important to investigate their effects at low concentrations relevant to human exposure. Here, the OECD test guideline #456 steroidogenesis assay was downscaled to a 96-well microplate format to screen 24 POPs for their effects on viability, and testosterone and estradiol synthesis using the human adrenocortical cell line H295R. The compounds (six polyfluoroalkyl substances, five organochlorine pesticides, ten polychlorinated biphenyls and three polybrominated diphenyl ethers) were tested at human-relevant levels (1 nM to 10 µM). Increased estradiol synthesis, above the OECD guideline threshold of 1.5-fold solvent control, was shown after exposure to 10 µM PCB-156 (153%) and PCB-180 (196%). Interestingly, the base hormone synthesis varied depending on the cell batch. An alternative data analysis using a linear mixed-effects model that include multiple independent experiments and considers batch-dependent variation was therefore applied. This approach revealed small but statistically significant effects on estradiol or testosterone synthesis for 17 compounds. Increased testosterone levels were demonstrated even at 1 nM for PCB-74 (18%), PCB-99 (29%), PCB-118 (16%), PCB-138 (19%), PCB-180 (22%), and PBDE-153 (21%). The MTT assay revealed significant effects on cell viability after exposure to 1 nM of perfluoroundecanoic acid (12%), 3 nM PBDE-153 (9%), and 10 µM of PCB-156 (6%). This shows that some POPs can interfere with endocrine signaling at concentrations found in human blood, highlighting the need for further investigation into the toxicological mechanisms of POPs and their mixtures at low concentrations relevant to human exposure.

A comparison of phage susceptibility testing with two liquid high-throughput methods.

Phage therapy is a promising antibacterial strategy, especially given that drug-resistant bacterial infections are escalating worldwide. Because phages are not active against all strains of a given species, phages being considered for therapeutic use would ideally be tested against bacterial isolates from individual patients prior to administration. Standardized, clinically validated phage susceptibility testing (PST) methods are needed for assessing in vitro phage activity. This study compared two high-throughput liquid-based PST assays. The first, using the Biolog Omnilog™, assessed changes in microbial respiration leading to color changes based on a tetrazolium dye. The second, Agilent BioTek Cytation 7, assessed changes in optical density. Both used 96-well microtiter plate formats. A total of 55 diverse phages with activity against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, or Enterococcus faecalis were studied against their respective susceptible bacterial hosts and non-susceptible controls, with susceptibility defined based on plaque assay. PST was performed by both assays in replicates, with results compared in terms of hold times (time through which bacterial growth is inhibited by phage compared to controls). Coefficients of variance and interclass correlation coefficients were used to assess inter- and intra-assay reproducibility. Based on a ≤50% coefficient of variance cutpoint, 87% of Biolog and 84% of Agilent assays were considered valid for susceptible bacteria, with 100% considered valid for non-susceptible bacteria by both systems. Using a 8 h hold time cutpoint, 100% of the results matched between the two assays. The interclass correlation coefficient showed 26% excellent agreement, 35% good agreement, and 17% moderate agreement between the two assays for susceptible isolates and 100% excellent agreement for non-susceptible isolates. Overall, the assays compared provided good/fair statistical reproducibility for the assessment of phage susceptibility.