Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 1. Rapid testing microfluidic platform for early thrombosis recapitulates SARS-CoV-2 and Spike-mediated technologies thrombotic effect. a) Schematic of SARS-CoV-2 infection-mediated thrombus formation in the microcirculation. In response to viral infection, inflammatory cells are recruited to activate the extrinsic and intrinsic coagulation pathways, leading to thrombin production. Thrombin cleaves fibrinogen to fibrin, which in turn, pro- motes platelet aggregation and fibrin deposition to form blood clots in the microcirculation. b) Similarly, SARS-CoV-2 vaccination therapies Spike-based technologies have the potential role to increment blood coagulation. c) SARS-CoV-2, Spike protein, and Spike variant for mimicking microcirculation environment were assessed for their thrombotic phenotypes in multiple endothelialized microfluidic channels (2 cm x 400 mm x 100 mm). Antibody anti-IL6 and decoy nanoliposome-hACE2 were also tested together with the aforementioned conditions. SARS-CoV-2, Spike protein, and Spike variant expressed using viral vectors were incubated in the PDMS-based microfluidic channels for 12 h at 37 °C, followed by a thrombosis assay in the presence of human subject-specific whole blood at wall shear stress of 25 dyne cm−2. Thrombus formation was quantified in terms of fibrin and platelet deposition.

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Infection, Coagulation, Variant Assay, Incubation, Shear

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 3. SARS-CoV-2 and Spike-mediated inflammatory cytokines regulates coagulation cascade. a) Tissue factor (TF) binds to coagulation factor VII (FVII) to initiate the thrombosis. SARS-CoV-2 infection also induces endothelial release of cytokines (such as IL-1, IL-6, and TNF-𝛼) that mediate platelet activation and coagulation cascades. SARS-COV-2 treatment for 48 h significantly upregulated HAEC mRNA expression of TNF-𝛼, IL-1, IL-6, and IL-15 as demonstrated by the heatmap (*p < 0.05 CTRL vs SARS-COV-2, n = 4 by qRT-PCR). The heat map was constructed by using Euclidean distance with average linkage. The Z-score centered log2-transformed gene in each sample is presented by a color scale, and gene upregulation is denoted in blue, and downregulation in red. b) Spike mutation D614G inflammatory effect was tested HAECs. A microarray heatmap represents 22 genes and selected control genes in HAECs in response to Spike D614G. Hierarchical clustering heatmap reveal differentially expressed genes in response to Lenti-S mutation (in the presence or absence of Lipo-hACE2), normalized to the lenti-CTRL respectively. The heatmap was constructed as previously described, and the Z-score centered log2-transformed gene in each sample was presented as a color scale. Each condition was performed in triplicate (n = 3). In addition to increased cytokines and chemokines mRNA expression level, higher mRNA expression level of endothelial marker of thrombosis, such as vWF and PAI-1, was also observed. Furthermore, the activation of the Toll-like receptor signaling pathway suggests its crucial role in enhancing downstream inflammation and thrombosis (see Figure S5, Supporting Information) (*p < 0.05: n = 3). c) Immunocytochemical analysis showed Lenti-S D614G increasing protein level of IL-6 (in red). Nuclei were stained with DAPI (scale bar: 50 μm).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Coagulation, Infection, Activation Assay, Expressing, Quantitative RT-PCR, Construct, Transformation Assay, Mutagenesis, Microarray, Control, Marker, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 4. Lipo-hACE2 and anti-IL-6 attenuate SARS-CoV-2-mediated inflammation and thrombosis. a) In the endothelialized microfluidic platform, HAECs were exposed to SARS-CoV-2, Lenti-S D614G, or Lipo-S in the presence or absence of Lipo-hACE2 or anti-IL-6 (scale bar = 100 μm). Lipo-hACE2 and anti-IL-6 attenuated SARS-CoV-2, Lenti-S, or Lipo-S-mediated fibrin deposition. b) Both Lipo-hACE2 and anti-IL-6 reduced SARS-CoV-2, Lipo-S, and Lenti-S mediated increases in surface coverage of fibrin and platelet deposition (*p < 0.05 vs Lipo-S or Lenti-S mutation; ***p < 0.001 vs SARS-CoV-2, n = 4).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Mutagenesis