3D-cultured neural stem cell microarrays on a micropillar chip for high-throughput developmental neurotoxicology.

Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system.

Suramin binds and inhibits infection of SARS-CoV-2 through both spike protein-heparan sulfate and ACE2 receptor interactions.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19.

Designer DNA nanostructures for viral inhibition.

Emerging viral diseases can substantially threaten national and global public health. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Since these antigens are arranged in a defined geometric pattern that is unique to each virus, the structure of the DDN is designed to mirror the spatial arrangement of antigens on the viral particle, providing very high viral binding avidity. We describe how available structural data can be used to identify unique spatial patterns of antigens on the surface of a viral particle. We then present a procedure for synthesizing DDNs using a combination of in silico design principles, self-assembly, and characterization using gel electrophoresis, atomic force microscopy and surface plasmon resonance spectroscopy. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. We expect this protocol to take 2-3 d to complete virus antigen pattern identification from existing cryogenic electron microscopy data, ~2 weeks for DDN design, synthesis, and virus binding characterization, and ~2 weeks for DDN cytotoxicity and antiviral efficacy assays.

Designer DNA architecture offers precise and multivalent spatial pattern-recognition for viral sensing and inhibition.

DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometre precision. Here we demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. We show that this structure is a potent viral inhibitor and that it can act as a sensor by including a fluorescent output to report binding. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.

Cell-Based Assay Design for High-Content Screening of Drug Candidates.

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.