Medium-throughput Screening Research Articles

Discovery of a small-molecule inhibitor of KSHV lytic replication from the MMV pandemic response box

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for primary effusion lymphoma (PEL), multicentric Castleman's disease (MCD) and Kaposi's sarcoma (KS). KSHV is one of the oncoviruses that contribute to 1.5 million new infection-related cancer cases annually. Currently, there are no targeted therapies for KSHV-associated diseases. Through the development of a medium-throughput phenotype-based ELISA screening platform based on KSHV ORF57 protein detection, we screened the Medicines for Malaria Venture (MMV) Pandemic Response Box for non-cytotoxic inhibitors of KSHV lytic replication. MMV1645152 was identified as a promising inhibitor of KSHV lytic replication, suppressing KSHV immediate-early and late lytic gene expression and blocking the production of infectious KSHV virion particles at non-cytotoxic concentrations in cell line models of KSHV infection with or without EBV coinfection. MMV1645152 is a promising hit compound for the development of future therapeutic agents against KSHV-associated malignancies.

Cardiac glycosides restore autophagy flux in an iPSC-derived neuronal model of WDR45 deficiency.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Development of a live cell assay for the zinc transporter ZnT8

Zinc is an essential trace element that is involved in many biological processes and in cellular homeostasis. In pancreatic β-cells, zinc is crucial for the synthesis, processing, and secretion of insulin, which plays a key role in glucose homeostasis and which deficiency is the cause of diabetes. The accumulation of zinc in pancreatic cells is regulated by the solute carrier transporter SLC30A8 (or Zinc Transporter 8, ZnT8), which transports zinc from cytoplasm in intracellular vesicles. Allelic variants of SLC30A8 gene have been linked to diabetes. Given the physiological intracellular localization of SLC30A8 in pancreatic β-cells and the ubiquitous endogenous expression of other Zinc transporters in different cell lines that could be used as cellular model for SLC30A8 recombinant over-expression, it is challenging to develop a functional assay to measure SLC30A8 activity. To achieve this goal, we have firstly generated a HEK293 cell line stably overexpressing SLC30A8, where the over-expression favors the partial localization of SLC30A8 on the plasma membrane. Then, we used the combination of this cell model, commercial FluoZin-3 cell permeant zinc dye and live cell imaging approach to follow zinc flux across SLC30A8 over-expressed on plasma membrane, thus developing a novel functional imaging- based assay specific for SLC30A8. Our novel approach can be further explored and optimized, paving the way for future small molecule medium-throughput screening.

Optical redox imaging to screen synthetic hydrogels for stem cell-derived cardiomyocyte differentiation and maturation.

Heart disease is the leading cause of death in the United States, yet research is limited by the inability to culture primary cardiac cells. Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSCs) are a promising solution for drug screening and disease modeling. Induced pluripotent stem cell-derived CM (iPSC-CM) differentiation and maturation studies typically use heterogeneous substrates for growth and destructive verification methods. Reproducible, tunable substrates and touch-free monitoring are needed to identify ideal conditions to produce homogenous, functional CMs. We generated synthetic polyethylene glycol-based hydrogels for iPSC-CM differentiation and maturation. Peptide concentrations, combinations, and gel stiffness were tuned independently. Label-free optical redox imaging (ORI) was performed on a widefield microscope in a 96-well screen of gel formulations. We performed live-cell imaging throughout differentiation and early to late maturation to identify key metabolic shifts. Label-free ORI confirmed the expected metabolic shifts toward oxidative phosphorylation throughout the differentiation and maturation processes of iPSC-CMs on synthetic hydrogels. Furthermore, ORI distinguished high and low differentiation efficiency cell batches in the cardiac progenitor stage. We established a workflow for medium throughput screening of synthetic hydrogel conditions with the ability to perform repeated live-cell measurements and confirm expected metabolic shifts. These methods have implications for reproducible iPSC-CM generation in biomanufacturing.

ZebraReg-a novel platform for discovering regulators of cardiac regeneration using zebrafish.

Cardiovascular disease is the leading cause of death worldwide with myocardial infarction being the most prevalent. Currently, no cure is available to either prevent or revert the massive death of cardiomyocytes that occurs after a myocardial infarction. Adult mammalian hearts display a limited regeneration capacity, but it is insufficient to allow complete myocardial recovery. In contrast, the injured zebrafish heart muscle regenerates efficiently through robust proliferation of pre-existing myocardial cells. Thus, zebrafish allows its exploitation for studying the genetic programs behind cardiac regeneration, which may be present, albeit dormant, in the adult human heart. To this end, we have established ZebraReg, a novel and versatile automated platform for studying heart regeneration kinetics after the specific ablation of cardiomyocytes in zebrafish larvae. In combination with automated heart imaging, the platform can be integrated with genetic or pharmacological approaches and used for medium-throughput screening of presumed modulators of heart regeneration. We demonstrate the versatility of the platform by identifying both anti- and pro-regenerative effects of genes and drugs. In conclusion, we present a tool which may be utilised to streamline the process of target validation of novel gene regulators of regeneration, and the discovery of new drug therapies to regenerate the heart after myocardial infarction.

Image-based quantification of mitochondrial iron uptake via Mitoferrin-2

Iron is a trace element that is critical for most living organisms and plays a key role in a wide variety of metabolic processes. In the mitochondrion, iron is involved in producing iron-sulfur clusters and synthesis of heme and kept within physiological ranges by concerted activity of multiple molecules. Mitochondrial iron uptake is mediated by the solute carrier transporters Mitoferrin-1 (SLC25A37) and Mitoferrin-2 (SLC25A28). While Mitoferrin-1 is mainly involved in erythropoiesis, the cellular function of the ubiquitously expressed Mitoferrin-2 remains less well defined. Furthermore, Mitoferrin-2 is associated with several human diseases, including cancer, cardiovascular and metabolic diseases, hence representing a potential therapeutic target. Here, we developed a robust approach to quantify mitochondrial iron uptake mediated by Mitoferrin-2 in living cells. We utilize HEK293 cells with inducible expression of Mitoferrin-2 and measure iron-induced quenching of rhodamine B[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA) fluorescence and validate this assay for medium-throughput screening. This assay may allow identification and characterization of Mitoferrin-2 modulators and could enable drug discovery for this target.

Screening of factors inducing alveolar type 1 epithelial cells using human pluripotent stem cells

Alveolar type 2 (AT2) epithelial cells are tissue stem cells capable of differentiating into alveolar type 1 (AT1) cells for injury repair and maintenance of lung homeostasis. However, the factors involved in human AT2-to-AT1 cell differentiation are not fully understood. Here, we established SFTPCGFP and AGERmCherry-HiBiT dual-reporter induced pluripotent stem cells (iPSCs), which detected AT2-to-AT1 cell differentiation with high sensitivity and identified factors inducing AT1 cell differentiation from AT2 and their progenitor cells. We also established an "on-gel" alveolar epithelial spheroid culture suitable for medium-throughput screening. Among the 274 chemical compounds, several single compounds, including LATS-IN-1, converted AT1 cells from AT2 and their progenitor cells. Moreover, YAP/TAZ signaling activation and AKT signaling suppression synergistically recapitulated the induction of transcriptomic, morphological, and functionally mature AT1 cells. Our findings provide novel insights into human lung development and lung regenerative medicine.

Abstract 4235: PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction

Abstract Physiologically relevant in vitro tumor models are crucial in any research setting from preclinical drug development to functional precision oncology. Patient-derived 3D cell culture models (PD3D®) are validated cancer models which recapitulate the biology of the original tumor tissue. PD3D® can be used to model intratumoral heterogeneity in medium and high throughput screens. Using a reverse clinical engineering approach, PD3D® models are already applied in personalized oncology to identify treatment for an individual patient. We successfully established a living biobank of more than 500 PD3D® models, spanning from common cancers like colorectal, breast (incl. TNBC), non-small cell lung and pancreatic carcinoma, to orphan indications including various subtypes of sarcomas. Not surprisingly, PD3D® model morphology and culture requirements differ between tumor entities. Treating PD3D® models with standard of care drugs in a semi-automated high-throughput assay, we observed heterogeneity in drug sensitivity between models of the same cancer type, recapitulating clinical response of patients. Combining drug sensitivity profiles with genomic and proteomic data of the same PD3D® models, we successfully identified a biomarker for predicting chemosensitivity towards MEK-targeting drugs. For application of PD3D® in truly personalized oncology, we developed a protocol that allows us to generate a PD3D® culture and perform a drug sensitivity assay for an individual patient within a therapy-relevant timeframe. Within 38 days we identified a systemic therapy for a young patient with a relapsed metastasized synovial sarcoma. After surgery and initiation of recommended chemotherapy, the patient is now under remission. To further expand the scope of PD3D® for therapy response prediction, we established a method for irradiation of PD3D® cultures with different dosages of photon and proton radiation. We observed significant, model specific differences in radiosensitivity between PD3D® sarcoma models which also reflect clinically heterogenous responses. Additionally, PD3D® models can be employed for a tumor organoid-on-chip platform (TumOC), delivering real-time information on physiological parameters like drug response-mediated oxygen consumption in a microfluidic system. In conclusion, PD3D® models act as jacks-of-all-trades in current and future cancer research, delivering robust and reliable data that not only is of academic value, but speeds up the drug development process, and are ready for prime time in functional precision oncology. Citation Format: Lena Wedeken, Samantha Forbrig, Katja Herrera-Glomm, Juergen Loskutov, Ulrike Pfohl, Irina Piven, Michael Poehle, Manuela J. Regenbrecht, Barbara Seller, Cynthia Yapto, Sabine Finkler, Larissa Ruhe, Quirin Graf Adelmann, Christoph Reinhard, Marie Flechner, Katja Uhlig, David Kaul, Siyer Roohani, Maya Niethard, Rica Sauer, Christian R. Regenbrecht. PD3D®models as jacks-of-all-trades for cancer research and therapy response prediction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4235.

HcHCR: High-Throughput Single-Cell Imaging of RNA in Human Primary Immune Cells.

Functional genomics and chemical screens can identify and characterize novel cellular factors regulating signaling networks and chemical tools to modulate their function for the treatment of disease. Screening methods have relied primarily on immortalized and/or transformed cancer cell lines, which can limit the generalization of results to more physiologically relevant systems. Most have also relied on immunofluorescence, or on stably expressed recombinant fluorescent proteins, to detect specific protein markers using high-content imaging readouts. In comparison, high-throughput methods to visualize and measure RNA species have been less explored. To address this, we have adapted an isothermal signal amplification chemistry for RNA FISH known as hybridization chain reaction (HCR) to an automated, high-content imaging assay format. We present a detailed protocol for this technique, which we have named high-content HCR (hcHCR). The protocol focuses on the measurement of changes in mRNA abundance at the single-cell level in human primary cells, but it can be applied to a variety of primary cell types and perturbing agents. We anticipate that hcHCR will be most suitable for low- to medium-throughput screening experiments in which changes in transcript abundance are the desired output measure.

Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches.

Testing drugs in vivo and in vitro have been essential elements for the discovery of new therapeutics. Due to the recent advances in in vitro cell culture models, such as human-induced pluripotent stem cell-derived cardiomyocytes and 3D multicell type organoid culture methods, the detection of adverse cardiac events prior to human clinical trials has improved. However, there are still numerous therapeutics whose adverse cardiac effects are not detected until human trials due to the inability of these cell cultures to fully model the complex multicellular organization of an intact human myocardium. Cardiac tissue slices are a possible alternative solution. Myocardial slices are a 300-micron thin snapshot of the myocardium, capturing a section of the adult heart in a 1×1cm section. Using a culture method that incorporates essential nutrients and electrical stimulation, tissue slices can be maintained in culture for 6days with full viability and functionality. With the addition of mechanical stimulation and humoral cues, tissue slices can be cultured for 12days. Here we provide detailed methods for how to culture cardiac tissue slices under continuous mechanical stimulation in the cardiac tissue culture model (CTCM) device. The CTCM incorporates four essential factors for maintaining tissue slices in culture for 12days: mechanical stimulation, electrical stimulation, nutrients, and humoral cues. The CTCM can also be used to model disease conditions, such as overstretch-induced cardiac hypertrophy. The versatility of the CTCM illustrates its potential to be a medium-throughput screening platform for personalized drug testing.

Phenotypical Screening of an MMV Open Box Library and Identification of Compounds with Antiviral Activity against St. Louis Encephalitis Virus.

St. Louis encephalitis virus (SLEV) is a neglected mosquito-borne Flavivirus that may cause severe neurological disease in humans and other animals. There are no specific treatments against SLEV infection or disease approved for human use, and drug repurposing may represent an opportunity to accelerate the development of treatments against SLEV. Here we present a scalable, medium-throughput phenotypic cell culture-based screening assay on Vero CCL81 cells to identify bioactive compounds that could be repurposed against SLEV infection. We screened eighty compounds from the Medicines for Malaria Venture (MMV) COVID Box library to identify nine (11%) compounds that protected cell cultures from SLEV-induced cytopathic effects, with low- to mid-micromolar potencies. We validated six hit compounds using viral plaque-forming assays to find that the compounds ABT-239, Amiodarone, Fluphenazine, Posaconazole, Triparanol, and Vidofludimus presented varied levels of antiviral activity and selectivity depending on the mammalian cell type used for testing. Importantly, we identified and validated the antiviral activity of the anti-flavivirus nucleoside analog 7DMA against SLEV. Triparanol and Fluphenazine reduced infectious viral loads in both Vero CCL81 and HBEC-5i cell cultures and, similar to the other validated compounds, are likely to exert antiviral activity through a molecular target in the host.

Abstract A071: Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma

Abstract Background Metastatic pancreas adenocarcinoma (PDAC) is highly lethal and minimally responsive to immunotherapy. Gamma delta T cells (gDTs) are a unique T cell subset found within the PDAC microenvironment. Animal models have shown that gDTs promote oncogenic progression and ablating gDT delays cancer progression and improves survival. Understanding the activity of immune effector cells against cancer organoids provides a scalable platform to understand the impact of activation and small molecule modulation to redirect gDT killing in PDAC. Methods Patient-derived organoids (PDOs) were generated from fresh tissue sampling and exapanded using a defined, serum-free media. Upon passaging, media was collected from PDAC PDOs. Allogeneic gDTs isolated from healthy donors were cultured in PDAC PDO conditioned media from 3 separate PDOs, in 3 separate biological replicates, and then subjected to bulk RNAseq. Allogeneic gDTs were isolated and cultured with PDAC PDOs at ratio of 5:1 (gDTs:PDAC PDOs) for 48 hours. After 48 hours, staining was performed for induction of apoptosis (cleaved caspase-3/7FITC). Images were acquired by high content imaging (Cytation 5, BioTek) and quantified for response across organoid populations. Based on RNAseq nominated candidates, low density lipoprotein (LDL) was added (10 ug/mL) as well as a small molecule inverse agonist of the Liver X Receptor (LXR) beta transcription factor (SR9243) to assess the ability of these factors to modulate gDT mediated PDAC killing. Results Exposure of gDTs to PDAC PDO secreted factors results in differential regulation of cholesterol handling, including upregulation of cholesterol efflux, and downregulation of cholesterol uptake and biosynthesis related genes in conditioned T cell populations. gDTs cause marked apoptosis in PDAC PDOs, including 40% staining of % organoid area versus negligible staining in control populations of 1.9% organoid area (effect size = 9.5, p<0.001). This response was directly compared to standard chemotherapy at the time of clinical resistance to FOLFIRINOX which conferred only 2% staining of mean organoid area (effect size = 0.11, NS). Given RNAseq data demonstrating cholesterol handling changes after exposure to PDAC secreted factors, we investigated LDL supplementation in our model. Supplementation of LDL significantly improves gDT mediated killing, with induction of apoptosis seen in 52% of the PDAC PDO area (p=0.003). This effect is mimicked by the small molecule SR9243 (an LXR inverse agonist), with 56% of PDAC PDO demonstrating apoptotic induction at 48 hours. Conclusions A PDAC PDO model system can be used to investigate gDT mediated PDAC killing when applied in low-volume medium throughput screening applications. Modulation of LXR activity and microenvironment LDL results in substantially increased immune-mediated killing of PDAC. This screening platform identifies potentially clinically actionable modulators of immune response to PDAC, and suggests potential future immunologically relevant targets that may improve the efficacy of immunotherapy. Citation Format: Johnathan D Ebben, David Turicek, Md Shahadat Hossan, Austin Stram, Ethan Lin, Nicholas Hess, Zachary Mayhew, Melissa A Kinney, Devin Burpee, Anikait Patel, Christian M Capitini, Jeremy D Kratz. Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A071.

Drug Repurposing in a Zebrafish AML Model

Acute myeloid leukemia (AML) is a genetically heterogeneous hematological malignancy. AML arises from the stepwise accumulation of mutations in hematopoietic stem cells (HSCs). The t(9;11)(p22;q23) translocation is a translocation detected in about 5 to 7% of AML patients, which gives rise to the KMT2A/MLLT3 or MLL/AF9 ( MA9) fusion gene. Zebrafish (ZF) are well-established model organisms for investigating human hematopoiesis. Approximately 70% of human genes have corresponding orthologues in zebrafish, and over 80% of disease-related genes are conserved between the two species. Previously, we established a transgenic MA9 zebrafish AML model (Runx1+23:MA9), in which the AML was serially transplantable. Our investigations revealed that MA9 expression alters hematopoiesis in transgenic embryos before the onset of AML, leading to myeloid expansion. Whole-mount in situ hybridization (WISH) of embryos showed increased expression of granulocyte markers such as lyz 24 and 48 hours after fertilization (hpf). Based on these findings, we developed a Runx1+23:MA9/lyz-EGFP zebrafish leukemia line, in which lyz-expressing myeloid cells are EGFP positive. This model provides a phenotype-based platform to evaluate the therapeutic potential of any drug that can affect the myeloid cell population. We used this ZF model to investigate the therapeutic potential of nine drugs in MA9-driven AML. MA9 transgenic fish (Runx1+23:MA9) were crossed with lyz-EGFP fish to generate a MA9/lyz-EFGP line. In this line, myeloid cells can be visualized with a fluorescent microscope. The expression of lyz in the MA9/lyz-EFGP transgenic embryos was compared to that in the lyz-EGFP line. In our previous study on MA9 transgenic leukemic fish, RNA-seq analysis had revealed higher expression of genes acting in the EGFR, MEK, RAF, KRAS pathways and downregulation of genes in TP53. Based on these results, we selected nine drugs that are known to affect some of these pathways as well as drugs that are used to treat leukemia for screening in our model. Control and MA9/lyz-EFGP ZF 48 hpf embryos were treated with 10 µM of each drug. 24 hrs after drug treatment, the number of lyz expressing cells was analyzed by visualizing the embryos under a fluorescent microscope, and lyz transcript levels were measured by qPCR. Treatment with erlotinib, lapatinib, cytarabine, and gefitinib led to a visible reduction in the number of lyz-expressing cells in the MA9 transgenic embryos but not in the controls (Figure 1). In comparison, treatment with decitabine resulted in a decrease in the number of lyz expressing cells in both the MA9/lyz-EGFP and the control embryos. Treatment with doxorubicin, zoledronic and arsenic trioxide did not have an effect on the number of lyz-expressing population in the transgenic nor in the control embryos. These results were confirmed by qPCR, which showed that expression of lyz was reduced by 33% to 51% in the erlotinib, lapatinib, cytarabine, and gefitinib treated MA9 transgenic embryos (Figure 2). Treatment with these drugs did not significantly change the lyz expression in lyz-EFGP control embryos. Our results provide strong evidence for the successful establishment of a zebrafish model of KMT2A-rearranged AML that can be used for low to medium-throughput drug screening by employing the expression level of an endogenous gene (lyz) as a read-out. This model is a valuable tool to assess the drug's specificity in targeting oncogene-expressing cells and normal hematopoietic cells and, therefore, to identify novel therapeutic agents that can target the cancer cells with fewer side effects on normal hematopoietic cells.

Contractility measurements for cardiotoxicity screening with ventricular myocardial slices of pigs.

Cardiotoxicity is one major reason why drugs do not enter or are withdrawn from the market. Thus, approaches are required to predict cardiotoxicity with high specificity and sensitivity. Ideally, such methods should be performed within intact cardiac tissue with high relevance for humans and detect acute and chronic side effects on electrophysiological behaviour, contractility, and tissue structure in an unbiased manner. Herein, we evaluate healthy pig myocardial slices and biomimetic cultivation setups (BMCS) as a new cardiotoxicity screening approach. Pig left ventricular samples were cut into slices and spanned into BMCS with continuous electrical pacing and online force recording. Automated stimulation protocols were established to determine the force-frequency relationship (FFR), frequency dependence of contraction duration, effective refractory period (ERP), and pacing threshold. Slices generated 1.3 ± 0.14 mN/mm2 force at 0.5 Hz electrical pacing and showed a positive FFR and a shortening of contraction duration with increasing pacing rates. Approximately 62% of slices were able to contract for at least 6 days while showing stable ERP, contraction duration-frequency relationship, and preserved cardiac structure confirmed by confocal imaging and X-ray diffraction analysis. We used specific blockers of the most important cardiac ion channels to determine which analysis parameters are influenced. To validate our approach, we tested five drug candidates selected from the Comprehensive in vitro Proarrhythmia Assay list as well as acetylsalicylic acid and DMSO as controls in a blinded manner in three independent laboratories. We were able to detect all arrhythmic drugs and their respective mode of action on cardiac tissue including inhibition of Na+, Ca2+, and hERG channels as well as Na+/Ca2+ exchanger. We systematically evaluate this approach for cardiotoxicity screening, which is of high relevance for humans and can be upscaled to medium-throughput screening. Thus, our approach will improve the predictive value and efficiency of preclinical cardiotoxicity screening.

Screening Mosquito Larvae Under a Fluorescence Binocular Microscope.

Transgenic mosquitoes are used in many aspects of mosquito research, and convenient selection markers are crucial to identifying transgenic individuals even among large numbers of wild types. Visual markers, in the form of fluorescent proteins expressed in larval and adult mosquito tissues, are the most commonly used. This requires observing large numbers of mosquitoes under the fluorescence microscope and recognizing positive individuals expressing the fluorescent genetic marker. As research models, mosquito larvae possess the following two advantages over many other insects, greatly facilitating fluorescence screening: (1) Being aquatic, they can be isolated in a drop of clear water, an ideal medium for live observations under the binocular microscope; and (2) synchronous hatching from many eggs is easily obtained, so that large populations of larvae can be screened in batches of several tens of thousands at a time. Screening at the neonate stage, when larvae are ∼1-mm-long, allows the concentration of hundreds of larvae in a drop of water that fits in the observation field of the microscope at medium magnification. Thus, many individuals can be screened rapidly. We strongly recommend screening larvae at the neonate stage and before any feeding for two reasons, as follows: (1) Food particles can be strongly autofluorescent, thereby dramatically increasing the fluorescence background noise around larvae, and (2) tissue autofluorescence increases during development, notably in the digestive tract, significantly decreasing the specific signal-to-noise ratio. In this protocol, we guide the experimenter step-by-step for a fast and efficient medium-throughput manual screening for fluorescent larvae.

Screening for chemicals with thyroid hormone-disrupting effects using zebrafish embryo

Thyroid disruption is an increasingly recognized issue in the use and development of chemicals and new drugs, especially to help toxicologist to complement the reproductive and developmental toxicology information of chemicals. Still, adequate assessment methods are scarce and often suffer a trade-off between physiological relevance and labor- and cost-intensive assays. Here, we present a tiered approach for a medium-throughput screening of chemicals to identify their thyroid disrupting potential in zebrafish embryos as a New Approach Methodology (NAM). After identifying the maximum tolerated concentrations, we exposed zebrafish larvae to sub-adverse effect levels of the reference compounds benzophenone-2, bisphenol A, phenylthiourea, potassium perchlorate, propylthiouracil, and phloroglucinol to exclude any systemic toxicity. Applying the transgenic zebrafish line that carries a gene for the red fluorescence protein (Tg(tg:mCherry)) under the thyroglobulin promoter, we could identify the thyroid disrupting effects of the chemicals by a time and cost-effective image analysis measuring the fluorescence levels in the thyroid glands. Our observations could be confirmed by altered expression patterns of genes involved in the hypothalamus-pituitary-thyroid (HPT) axis. Finally, to anchor the observed thyroid disruption, we determined some changes in the Thyroid hormone levels of triiodothyronine (T3) and Thyroxine (T4) using a newly developed liquid chromatography mass spectrometric (LCMS) method. The presented approach carries the potential to extend the toolbox for legislative authorities and chemical producers for the assessment of thyroid-specific endocrine disruption and to overcome current challenges in the evaluation of endocrine disruptors.

High-resolution bioenergetics correlates the length of continuous protonatable diaminoethane motif of four-armed oligo(ethanamino)amide transfectants to cytotoxicity

Recent clinical success with Onpattro and cationic ionizable lipid nanoparticle-based mRNA vaccines has rejuvenated research in the design and engineering of broader synthetic cationic vectors for nucleic acid compaction and transfection. However, perturbation of metabolic processes and cytotoxicity are still of concern with synthetic cationic vectors. Here, through an integrated bioenergetic and biomembrane integrity probing in three different human cell lines we reveal the dynamic effect of a library of sequence-defined four-arm oligo(ethanamino)amide transfectant on cell homeostasis, and identify metabolically safe building units over wide concentration ranges. The results show differential effects of the oligo(ethanamino)amide structure of comparable molecular weight on cell energetics. The severity of polycation effect on bioenergetic crisis follows with the length of continuous protonatable diaminoethane motif in the ascending order of glutaryl-triethylene tetramine, succinyl-tetraethylene pentamine and succinyl-pentaethylene hexamine. We further identify oligomeric structures that do not induce bioenergetic crisis even at high concentrations. Finally, transfection studies with a library of polyplexes carrying a reporter gene show no correlation between transfection efficiency and cytotoxicity. These observations demonstrate the usefulness of integrated high-resolution respirometry and plasma membrane integrity probing as a highly sensitive medium-throughput screening strategy for identification and selection of safe building units for transfectant engineering.

Genome-wide association study for phosphate responsive root hair length and density in bread wheat

The increase in root hair length and density is considered an effective way to deal with external phosphorous (P) limitation. The identification of useful loci for root hair traits is crucial for wheat breeding. Here, We employed a simple, cost-effective, and medium-throughput screening method using cigar rolls to assess P-responsive root hair length (RHL) and density (RHD) in bread wheat at the seedling stage. A set of 113 historical bread wheat cultivars was screened for root hair length and density (RHL/D) under low (0.005 mM KH2PO4) and high P-levels (0.25 mM KH2PO4). Significant variations (p < 0.001) were found among genotypes for length and density under both P treatments. Genotyping was performed using 50 K SNP array data to conduct the genome-wide association study (GWAS) using a multi-locus model (mrMLM) and 67 SNPs linked to RHL/D under phosphorous treatment were identified. Of these, 33 SNPs were associated with RHL and 34 were linked with RHD. The maximum number of SNPs (43) linked to target traits were observed under high-P levels following 24 were identified under low-P conditions. We also identified 6 SNPs associated with target traits causing missense mutations and predicted their candidate genes of which 5 were protein coding. RNA-seq based expression analysis indicated that all genes were expressed in roots of 24 wheat varieties ranging from 0.38 to 101.7 tpm. The least expression (0.41–10.28 tpm) was observed in TraesCS4D02G066200 which consisted of non-translating CDS and was highly expressed in leaf tissues with 14.55–53.67 tpm. Of these genes, TraesCS1A02G313600 solely expressed in roots with no expression (0 tpm) in leaf tissues. This first GWAS study on root hairs in bread wheat illustrates the genetic basis of RHL and RHD and identifies loci that could be an invaluable resource for further functional investigation.

In Utero Electroporated Neurons for Medium-Throughput Screening of Compounds Regulating Neuron Morphology.

Several neurodevelopmental disorders are associated with increased mTOR activity that results in pathogenic neuronal dysmorphogenesis (i.e., soma and dendrite overgrowth), leading to circuit alterations associated with epilepsy and neurologic disabilities. Although an mTOR analog is approved for the treatment of epilepsy in one of these disorders, it has limited efficacy and is associated with a wide range of side effects. There is a need to develop novel agents for the treatment of mTOR-pathway related disorders. Here, we developed a medium-throughput phenotypic assay to test drug efficacy on neurite morphogenesis of mouse neurons in a hyperactive mTOR condition. Our assay involved in utero electroporation (IUE) of a selective population of cortical pyramidal neurons with a plasmid encoding the constitutively active mTOR activator, Rheb, and tdTomato. Labeled neurons from the somatosensory cortex (SSC) were cultured onto 96-well plates and fixed at various days in vitro or following Torin 1 treatment. Automated systems were used for image acquisition and neuron morphologic measurements. We validated our automated approach using traditional manual methods of neuron morphologic assessment. Both automated and manual analyses showed increased neurite length and complexity over time, and decreased neurite overgrowth and soma size with Torin 1. These data validate the accuracy of our automated approach that takes hours compared with weeks when using traditional manual methods. Taken together, this assay can be scaled to screen 32 compounds simultaneously in two weeks, highlighting its robustness and efficiency for medium-throughput screening of candidate therapeutics on a defined population of wild-type or diseased neurons.

Suspended hydrogel culture as a method to scale up intestinal organoids

Primary tissue-derived epithelial organoids are a physiologically relevant in vitro intestinal model that have been implemented for both basic research and drug development applications. The existing method of culturing intestinal organoids in surface-attached native extracellular matrix (ECM) hydrogel domes is not readily amenable to large-scale culture and contributes to culture heterogeneity. We have developed a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries, which streamlines the protocol, increases the scalability, enables kinetic sampling, and improves culture uniformity without specialized equipment or additional expertise. We demonstrate the compatibility of this method with multiple culture formats, and provide examples of suspended BME hydrogel organoids in downstream applications: implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation. The suspended BME hydrogel culture method will allow intestinal organoids, and potentially other organoid types, to be used more widely and at higher throughputs than previously possible.