The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

Background: Duchenne muscular dystrophy (DMD) is an X-linked disease characterized by skeletal muscle degeneration and a significant cardiomyopathy secondary to cardiomyocyte damage and myocardial loss. The molecular basis of DMD lies in the absence of the protein dystrophin, which plays critical roles in mechanical membrane integrity and protein localization at the sarcolemma. A popular mouse model of DMD is the mdx mouse, which lacks dystrophin and displays mild cardiac and skeletal pathology that can be exacerbated to advance the disease state. In clinical and pre-clinical studies of DMD, angiotensin signaling pathways have emerged as therapeutic targets due to their adverse influence on muscle remodeling and oxidative stress. Here we aim to establish a physiologically relevant cardiac injury model in the mdx mouse, and determine whether acute blockade of the angiotensin II type 1 receptor (AT 1 R) may be utilized for prevention of dystrophic injury. Methods and Results: A single IP injection of isoproterenol (Iso, 10 mg/kg) was used to induce cardiac stress and injury in mdx and wild type (C57Bl/10) mice. Mice were euthanized 8 hours, 30 hours, 1 week, or 1 month following the injection, and hearts were harvested for injury evaluation. At 8 and 30 hours post-injury, mdx hearts showed 2.2-fold greater serum cTnI content and 3-fold more extensive injury than wild type hearts. Analysis of hearts 1 week and 1 month after injury revealed significantly higher fibrosis in mdx hearts, with a more robust and longerlasting immune response compared to wild type hearts. In the 30-hour group, losartan treatment initiated 1 hour before Iso injection protected dystrophic hearts from cardiac damage, reducing mdx acute injury area by 2.8-fold, without any significant effect on injury in wild type hearts. However, both wild type and dystrophic hearts showed a 2-fold reduction in the magnitude of the macrophage response to injury 30 hours after Iso with losartan.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3′,5′-difluoro-4′-hydroxybenzylidene)-2-methyl-1-(2″,2″,2″-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research.

Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. Here we present a modified T7 RNA polymerase promoter that acts to significantly increase the yields of both transcription and translation within in vitro systems. The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system. Unlike other methods of limiting linear template degradation, the T7Max promoter increases transcript concentration in a T7 transcription reaction, providing more mRNA for translation.

Background Efficient cell-free protein expression from linear DNA templates has remained a challenge primarily due to template degradation. In addition, the yields of transcription in cell-free systems lag behind transcriptional efficiency of live cells. Most commonly used in vitro translation systems utilize T7 RNA polymerase, which is also the enzyme included in many commercial kits. Results Here we present characterization of a variant of T7 RNA polymerase promoter that acts to significantly increase the yields of gene expression within in vitro systems. We have demonstrated that T7Max increases the yield of translation in many types of commonly used in vitro protein expression systems. We also demonstrated increased protein expression yields from linear templates, allowing the use of T7Max driven expression from linear templates. Conclusions The modified promoter, termed T7Max, recruits standard T7 RNA polymerase, so no protein engineering is needed to take advantage of this method. This technique could be used with any T7 RNA polymerase- based in vitro protein expression system.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

Structural biology education commonly employs molecular visualization software, such as PyMol, RasMol, and VMD, to allow students to appreciate structure-function relationships in biomolecules. In on-ground, classroombased education, these programs are commonly used on University-owned devices with software preinstalled. Remote education typically involves the use of student-owned devices, which complicates the use of such software, owing to the fact that (a) student devices have differing configurations (e.g., Windows vs MacOS) and processing power, and (b) not all student devices are suitable for use with such software. Smartphones are near-ubiquitous devices, with smartphone ownership exceeding personal computer ownership, according to a recent survey. Here, we show the use of a smartphone-based augmented reality app, Augment, in a structural biology classroom exercise, which students installed independently without IT support. Post-lab attitudinal survey results indicate positive student experiences with this app. Based on our experiences, we suggest that smartphone-based molecular visualization software, such as that used in this exercise, is a powerful educational tool that is particularly well-suited for use in remote education.