design-to-implementation-study-for-development-patient-validation

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Access to low-burden molecular diagnostics that can be deployed into the community for testing is increasingly important and has meaningful wider ...

University of Toronto

The global spread of SARS-CoV-2 has shaken our health care and economic systems, prompting re-evaluation of long-held views on how best to deliver care. This is especially the case for our global diagnostic strategy. While current laboratory-based centralized RT-qPCR will continue to serve as a gold standard diagnostic into the foreseeable future, the shortcomings of our dependence on this method have been laid bare. It is now clear that a robust diagnostics pandemic response strategy, like any disaster planning, must include adaptive, diverse and de-centralized solutions. Here we look at how the COVID-19 pandemic, and previous outbreaks, have set the stage for a new innovative phase in diagnostics and a re-thinking of pandemic preparedness.

Adaptive, diverse and de-centralized diagnostics are key to the future of outbreak response.

Recent advances in cell-free synthetic biology have given rise to gene circuit-based sensors with the potential to provide decentralized and low-cost molecular diagnostics. However, it remains a challenge to deliver this sensing capacity into the hands of users in a practical manner. Here, we leverage the glucose meter, one of the most widely available point-of-care sensing devices, to serve as a universal reader for these decentralized diagnostics. We describe a molecular translator that can convert the activation of conventional gene circuit-based sensors into a glucose output that can be read by off-the-shelf glucose meters. We show the development of new glucogenic reporter systems, multiplexed reporter outputs and detection of nucleic acid targets down to the low attomolar range. Using this glucose-meter interface, we demonstrate the detection of a small-molecule analyte; sample-to-result diagnostics for typhoid, paratyphoid A/B; and show the potential for pandemic response with nucleic acid sensors for SARS-CoV-2.

A glucose meter interface for point-of-care gene circuit-based diagnostics.

The field of cell-free synthetic biology is an emerging branch of engineered biology that allows for rapid prototyping of biological designs and, in its own right, is becoming a venue for the  operation of gene circuit-based sensors and biomanufacturing. To date, the related DNA encoded tools that operate in cell-free reactions have primarily relied on plasmid DNA inputs, as linear templates are highly susceptible to degradation by exonucleases present in cell-free extracts. This incompatibility has precluded significant throughput, time and cost benefits that could be gained with the use of linear DNA in the cell-free expression workflow. Here to tackle this limitation, we report that terminal incorporation of Ter binding sites for the DNA-binding protein Tus enables highly efficient protection of linear expression templates encoding mCherry and deGFP. In  extracts, our method compares favorably with the previously reported GamS-mediated protection scheme. Importantly, we extend the Tus-Ter system to  extracts, and demonstrate that this simple and easily implemented method can enable an unprecedented plasmid-level expression from linear templates in this emerging chassis organism.

High-Efficiency Protection of Linear DNA in Cell-Free Extracts from  and .

Vimentin expression contributes to cellular mechanoprotection and is a widely recognized marker of fibroblasts and of epithelial-mesenchymal transition. But it is not understood how vimentin affects signaling that controls cell migration and extracellular matrix (ECM) remodeling. Recent data indicate that vimentin controls collagen deposition and ECM structure by regulating contractile force application to the ECM and through post-transcriptional regulation of ECM related genes. Binding of cells to the ECM promotes the association of vimentin with cytoplasmic domains of adhesion receptors such as integrins. After initial adhesion, cell-generated, myosin-dependent forces and signals that impact vimentin structure can affect cell migration. Post-translational modifications of vimentin determine its adaptor functions, including binding to cell adhesion proteins like paxillin and talin. Accordingly, vimentin regulates the growth, maturation and adhesive strength of integrin-dependent adhesions, which enables cells to tune their attachment to collagen, regulate the formation of cell extensions and control cell migration through connective tissues. Thus, vimentin tunes signaling cascades that regulate cell migration and ECM remodeling. Here we consider how specific properties of vimentin serve to control cell attachment to the underlying ECM and to regulate mesenchymal cell migration and remodeling of the ECM by resident fibroblasts.

Masoud Norouzi

Leslie Dan Faculty of Pharmacy

University of Toronto

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Masoud Norouzi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Leslie Dan Faculty of Pharmacy

University of Toronto

Design to Implementation Study for Development and Patient Validation of Paper-Based Toehold Switch Diagnostics

Leslie Dan Faculty of Pharmacy