COVID-19 detection from red blood cells using highly comparative time-series analysis (HCTSA) in digital holographic microscopy.

We present an automated method for COVID-19 screening based on reconstructed phase profiles of red blood cells (RBCs) and a highly comparative time-series analysis (HCTSA). Video digital holographic data -was obtained using a compact, field-portable shearing microscope to capture the temporal fluctuations and spatio-temporal dynamics of live RBCs. After numerical reconstruction of the digital holographic data, the optical volume is calculated at each timeframe of the reconstructed data to produce a time-series signal for each cell in our dataset. Over 6000 features are extracted on the time-varying optical volume sequences using the HCTSA to quantify the spatio-temporal behavior of the RBCs, then a linear support vector machine is used for classification of individual RBCs. Human subjects are then classified for COVID-19 based on the consensus of their cells' classifications. The proposed method is tested on a dataset of 1472 RBCs from 24 human subjects (10 COVID-19 positive, 14 healthy) collected at UConn Health Center. Following a cross-validation procedure, our system achieves 82.13% accuracy, with 92.72% sensitivity, and 73.21% specificity (area under the receiver operating characteristic curve: 0.8357). Furthermore, the proposed system resulted in 21 out of 24 human subjects correctly labeled. To the best of our knowledge this is the first report of a highly comparative time-series analysis using digital holographic microscopy data.

Highly efficient intercellular spreading of protein misfolding mediated by viral ligand-receptor interactions.

Protein aggregates associated with neurodegenerative diseases have the ability to transmit to unaffected cells, thereby templating their own aberrant conformation onto soluble homotypic proteins. Proteopathic seeds can be released into the extracellular space, secreted in association with extracellular vesicles (EV) or exchanged by direct cell-to-cell contact. The extent to which each of these pathways contribute to the prion-like spreading of protein misfolding is unclear. Exchange of cellular cargo by both direct cell contact or via EV depends on receptor-ligand interactions. We hypothesized that enabling these interactions through viral ligands enhances intercellular proteopathic seed transmission. Using different cellular models propagating prions or pathogenic Tau aggregates, we demonstrate that vesicular stomatitis virus glycoprotein and SARS-CoV-2 spike S increase aggregate induction by cell contact or ligand-decorated EV. Thus, receptor-ligand interactions are important determinants of intercellular aggregate dissemination. Our data raise the possibility that viral infections contribute to proteopathic seed spreading by facilitating intercellular cargo transfer.

Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.

The etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

Microvascular dysfunction in COVID-19: the MYSTIC study.

RATIONALE: Pre-clinical and autopsy studies have fueled the hypothesis that a dysregulated vascular endothelium might play a central role in the pathogenesis of ARDS and multi-organ failure in COVID-19. OBJECTIVES: To comprehensively characterize and quantify microvascular alterations in patients with COVID-19. METHODS: Hospitalized adult patients with moderate-to-severe or critical COVID-19 (n = 23) were enrolled non-consecutively in this prospective, observational, cross-sectional, multi-center study. Fifteen healthy volunteers served as controls. All participants underwent intravital microscopy by sidestream dark field imaging to quantify vascular density, red blood cell velocity (VRBC), and glycocalyx dimensions (perfused boundary region, PBR) in sublingual microvessels. Circulating levels of endothelial and glycocalyx-associated markers were measured by multiplex proximity extension assay and enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: COVID-19 patients showed an up to 90% reduction in vascular density, almost exclusively limited to small capillaries (diameter 4-6 µm), and also significant reductions of VRBC. Especially, patients on mechanical ventilation showed severe glycocalyx damage as indicated by higher PBR values (i.e., thinner glycocalyx) and increased blood levels of shed glycocalyx constituents. Several markers of endothelial dysfunction were increased and correlated with disease severity in COVID-19. PBR (AUC 0.75, p = 0.01), ADAMTS13 (von Willebrand factor-cleaving protease; AUC 0.74, p = 0.02), and vascular endothelial growth factor A (VEGF-A; AUC 0.73, p = 0.04) showed the best discriminatory ability to predict 60-day in-hospital mortality. CONCLUSIONS: Our data clearly show severe alterations of the microcirculation and the endothelial glycocalyx in patients with COVID-19. Future therapeutic approaches should consider the importance of systemic vascular involvement in COVID-19.