Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

There is an urgent and unprecedented need for sensitive and high-throughput molecular diagnostic tests to combat the SARS-CoV-2 pandemic. Here we present a generalized version of the RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) assay, called "capture RASL-seq" (cRASL-seq), which enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid purification or reverse transcription, steps that have introduced supply bottlenecks into standard assay workflows. Our simplified protocol additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

UW Supplementation with AP39 Improves Liver Viability Following Static Cold Storage.

Static cold storage of donor livers at 4°C incompletely arrests metabolism, ultimately leading to decreases in ATP levels, oxidative stress, cell death, and organ failure. Hydrogen Sulfide (H2S) is an endogenously produced gas, previously demonstrated to reduce oxidative stress, reduce ATP depletion, and protect from ischemia and reperfusion injury. H2S is difficult to administer due to its rapid release curve, resulting in cellular death at high concentrations. AP39, a mitochondrially targeted, slow-release H2S donor, has been shown to reduce ischemia-reperfusion injury in hearts and kidneys. Thus, we investigated whether the addition of AP39 during 3-day static cold storage can improve liver graft viability. At the end of storage, livers underwent six hours of acellular normothermic machine perfusion, a model of transplantation. During simulated transplantation, livers stored with AP39 showed reduced resistance, reduced cellular damage (ALT and AST), and reduced apoptosis. Additionally, bile production and glucose, as well as energy charge were improved by the addition of AP39. These results indicate that AP39 supplementation improves liver viability during static cold storage.

Valve Endothelial Cell Exposure to High Levels of Flow Oscillations Exacerbates Valve Interstitial Cell Calcification.

The aortic valve facilitates unidirectional blood flow to the systemic circulation between the left cardiac ventricle and the aorta. The valve's biomechanical function relies on thin leaflets to adequately open and close over the cardiac cycle. A monolayer of valve endothelial cells (VECs) resides on the outer surface of the aortic valve leaflet. Deeper within the leaflet are sublayers of valve interstitial cells (VICs). Valve tissue remodeling involves paracrine signaling between VECs and VICs. Aortic valve calcification can result from abnormal paracrine communication between these two cell types. VECs are known to respond to hemodynamic stimuli, and, specifically, flow abnormalities can induce VEC dysfunction. This dysfunction can subsequently change the phenotype of VICs, leading to aortic valve calcification. However, the relation between VEC-exposed flow oscillations under pulsatile flow to the progression of aortic valve calcification by VICs remains unknown. In this study, we quantified the level of flow oscillations that VECs were exposed to under dynamic culture and then immersed VICs in VEC-conditioned media. We found that VIC-induced calcification was augmented under maximum flow oscillations, wherein the flow was fully forward for half the cardiac cycle period and fully reversed for the other half. We were able to computationally correlate this finding to specific regions of the aortic valve that experience relatively high flow oscillations and that have been shown to be associated with severe calcified deposits. These findings establish a basis for future investigations on engineering calcified human valve tissues and its potential for therapeutic discovery of aortic valve calcification.

Highly multiplexed oligonucleotide probe-ligation testing enables efficient extraction-free SARS-CoV-2 detection and viral genotyping.

The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.