Journal: Annals of Neurology

Article Title: COVID ‐19‐Associated Critical Illness Myopathy with Direct Viral Effects

doi: 10.1002/ana.26318

Figure Lengend Snippet: Case 1. A vastus lateralis muscle biopsy showing severe myopathic changes with atrophic fibers, degenerative/necrotic fibers with loss of normal staining (A, hematoxylin and eosin; arrows ), increased adipose tissue (A,*), fibers with mitochondrial proliferation (B, Gomori trichrome staining; arrows ), vacuoles containing debris (C, Gomori trichrome staining; arrow ), and fibers with increased reactivity for NADH (D,*). Immunohistochemistry revealing focally scattered CD68+ macrophages (E), CD8+ T‐cells (F), MHC‐1 sarcolemmal and sarcoplasmic staining in necrotic and non‐necrotic fibers (G), occasional p62+ puncta/vacuoles (H, arrows ), and SARS‐CoV+ fibers containing strongly positive granules (I, arrows ). Electron microscopy demonstrating extensive loss of A bands with retained Z lines and I bands (J,*), virus‐like particles in necrotic fibers (K, arrows , with characteristic surface projections/spikes; an inset of the left arrowed area ), and degenerative fibers (L‐O, a series of magnifications focusing on a virus‐like particle, arrows ; L, with identifiable myofibrils,*; M, the rectangle in L; N, higher magnification of the arrowed area in M; O, higher magnification of the arrowed area in N), numerous abnormal mitochondria with electron‐dense granules (P–R), focal to extensive loss of cristae, crystalline inclusions (Q, arrow ), and virus‐like particles (R, arrow , with an inset of the arrowed area). Scale bars: 20 μm (A), 10 μm (B–I), 800 nm (J), 100 nm (K, N), 600 nm (L, P), 200 nm (M, Q), 50 nm (O), and 500 nm (R). NADH = nicotinamide adenine dinucleotide; SARS‐CoV = severe acute respiratory syndrome‐coronavirus.

Article Snippet: The following antibodies were used: p62/SQSTM1 (Progen Biotechnik; 1:100 dilution following antigen retrieval), LC3 (clone 5F10, Enzo; 1:200 dilution following antigen retrieval), and SARS‐CoV nucleoprotein (SARS‐CoV, Sino Biological; NB100‐56576, raised by a synthetic peptide corresponding to amino acids 399 to 411 [ADMDDFSRQLQNS‐C] of the human SARS coronavirus nucleocapsid protein; 1:5000 dilution following antigen retrieval).

Techniques: Staining, Immunohistochemistry, Electron Microscopy

Journal: Annals of Neurology

Article Title: COVID ‐19‐Associated Critical Illness Myopathy with Direct Viral Effects

doi: 10.1002/ana.26318

Figure Lengend Snippet: Case 2. A vastus lateralis muscle biopsy showing marked myopathic changes with vacuoles (A, loss of hematoxylin and eosin staining; B, lost or increased Gomori trichrome staining; arrows ), rubbed‐out centers on NADH staining with granular/vacuolar positivity preferentially at the periphery (C, arrows ), punctate immunostaining for p62 (D, arrows ) and LC3 (E, arrows ) in the fiber centers, focal MHC‐1 immunostaining (F), SARS‐CoV+ fibers containing strongly positive granules (G, arrows ), and electron microscopy findings of fibers with loss of A bands and retained Z lines (H–J, *), vacuoles containing granules/particles (H), virus‐like particles in the subsarcolemmal areas (I–K, a series of magnifications focusing on a virus‐like particle; J, the rectangle in I, with an inset of the arrowed virus‐like particle; K, the rectangle in J, containing the arrowed virus‐like particle), and disrupted myofibrils (L; M, the rectangle in L, containing the arrowed virus‐like particle), and abnormal mitochondria in an atrophic/degenerative fiber (N, with focally identifiable myofibrils and Z lines, *; O, the rectangle in L, containing abnormal mitochondria, *). Scale bars: 10 μm (A–G), 1 μm (H, I), 400 nm (J), 100 nm (K, M), 600 nm (L), 2 μm (N), and 200 nm (O). NADH = nicotinamide adenine dinucleotide; SARS‐CoV = severe acute respiratory syndrome‐coronavirus.

Article Snippet: The following antibodies were used: p62/SQSTM1 (Progen Biotechnik; 1:100 dilution following antigen retrieval), LC3 (clone 5F10, Enzo; 1:200 dilution following antigen retrieval), and SARS‐CoV nucleoprotein (SARS‐CoV, Sino Biological; NB100‐56576, raised by a synthetic peptide corresponding to amino acids 399 to 411 [ADMDDFSRQLQNS‐C] of the human SARS coronavirus nucleocapsid protein; 1:5000 dilution following antigen retrieval).

Techniques: Staining, Immunostaining, Electron Microscopy

Journal: Annals of Neurology

Article Title: COVID ‐19‐Associated Critical Illness Myopathy with Direct Viral Effects

doi: 10.1002/ana.26318

Figure Lengend Snippet: Case 3. A biopsy of the vastus lateralis muscle showing fibro‐adipose tissue with rare residual atrophic fibers (A, hematoxylin and eosin; arrows ; inset in A, ATPase 4.3 histochemical staining: negative atrophic fibers compared to the arrowheaded positive granules), occasional vacuoles containing muscle debris positive for myoglobin immunostaining (B, arrows ), focally scattered CD68+ macrophages (C) and CD8+ T‐cells (D), but negative SARS‐CoV immunostaining (E). Electron microscopy demonstrating abnormal mitochondria with focal degenerative changes (F; arrows , compared to a relatively preserved mitochondrion, arrowhead ). Scale bars: 20 μm (A), 10 μm (B–E), and 200 nm (F). SARS‐CoV = severe acute respiratory syndrome‐coronavirus.

Article Snippet: The following antibodies were used: p62/SQSTM1 (Progen Biotechnik; 1:100 dilution following antigen retrieval), LC3 (clone 5F10, Enzo; 1:200 dilution following antigen retrieval), and SARS‐CoV nucleoprotein (SARS‐CoV, Sino Biological; NB100‐56576, raised by a synthetic peptide corresponding to amino acids 399 to 411 [ADMDDFSRQLQNS‐C] of the human SARS coronavirus nucleocapsid protein; 1:5000 dilution following antigen retrieval).

Techniques: Staining, Immunostaining, Electron Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Intensity and duration of TCR signaling is limited by p38 phosphorylation of ZAP-70 T293 and destabilization of the signalosome

doi: 10.1073/pnas.1713301115

Figure Lengend Snippet: Phosphorylation of ZAP-70T293 enhances TCR signaling. (A) Jurkat cells stably expressing YFP-tagged ZAP-70 or (B) purified primary human CD4+ T cells were plated on coverslips coated with anti-CD45 (nonactivating) or anti-CD3 (activating) for 3 min, fixed, and immunostained. ZAP-YFP is shown in green and anti-pY323 p38 is shown in red for Jurkat–ZAP-70–YFP cells; ZAP-70 is shown in green and anti-pY323 p38 is shown in red for human T cells. (C and D) ZAP-70–deficient P116 cells expressing WT ZAP-70 or ZAP-70T293A were stimulated with cross-linked anti-CD3 for the indicated times and cell lysates immunoblotted for the indicated proteins. The data are representative of three (A, C, and D) and two (B) independent experiments.

Article Snippet: Human CD4 + T cells were isolated from buffy coats of healthy volunteers (NIH Blood Bank) using the human CD4 cell recovery column kit (Cedarlane), according to the manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Purification

Journal: Journal of Controlled Release

Article Title: Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection

doi: 10.1016/j.jconrel.2021.05.049

Figure Lengend Snippet: Construction of SARS-CoV-2 S protein RBD-tagged EVs. (A) Detection of SARS-CoV-2 Spike protein in cell lysates or EV pellets by western blot. 293T cells were transfected with plasmid expressing the FLAG-tagged Spike protein. EVs pelleted from the media were analyzed by western blot for the presence of the Spike protein using the FLAG M2 antibody. Purified EVs were characterized for EV markers CD9 and Alix, and non-EV markers calnexin (endoplasmic reticulum marker) and GM130 (Golgi matrix marker). (B) Schematic illustration of RBD-VSVG fusion-loaded EVs. The VSVG ectodomain was replaced by the RBD domain of SARS-CoV-2 (right, red), followed by a transmembrane helix and a cytoplasmic tail of VSVG (right, green). (C) Western blot analysis of EVs purified from 293T cells transfected with or without RBD-VSVG-vector for 48 h. CD9 or Alix proteins served as markers for the presence of EVs.(D, E) Schematic presentation of affinity pull-down of EVs by anti-RBD coupled magnetic beads. (D)EVs isolated from supernatants of transfected or non-transfected 293T cells were subjected to anti-RBD pull-down assay, then total protein of EVs was analyzed by western blot (E). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells, purified EVs or tissues were lysed with RIPA buffer (Santa Cruz,USA) and cleared lysate was collected by centrifugation for protein separation on SDS-polyacrylamide gel(10%), followed by transfer onto PVDF membranes(Millipore), blocked with 5% nonfat dry milk in TBST and reacted with primary antibodies recognizing FLAG M2 tag(Sigma,1:2000), SARS-CoV-2 RBD(Sinobiological,1:500), CD9(Abcam,1:2000), Alix(Cell Signaling Technology,1:1000), GM130(Abcam,1:1000), Calnexin(Abcam,1:1000), hACE2(Abcam,# ab108209,only react with human species,1:2000) and GAPDH(Santa Cruz,1:2000).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Purification, Marker, Magnetic Beads, Isolation, Pull Down Assay

Journal: Journal of Controlled Release

Article Title: Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection

doi: 10.1016/j.jconrel.2021.05.049

Figure Lengend Snippet: Characterization of physical properties of SARS-CoV-2 S protein RBD-tagged EVs. (A) The ratio of particles to protein for each sample is shown. (B)Histogram showing the size distribution of the purified EVs as analyzed by NTA. (C) TEM or SEM images of RBD-tagged or control EVs purified from 293T cells. (D and E) EV protein and RNA concentration measurement. EV protein or RNA was extracted from 1 × 10 8 EVs and protein concentration was measured by BCA protein quantification (D) and RNA concentration was determined by NanoDrop (E). Data are shown as mean ± SEM of four independent experiments, each experiment performs triplicate.

Article Snippet: Cells, purified EVs or tissues were lysed with RIPA buffer (Santa Cruz,USA) and cleared lysate was collected by centrifugation for protein separation on SDS-polyacrylamide gel(10%), followed by transfer onto PVDF membranes(Millipore), blocked with 5% nonfat dry milk in TBST and reacted with primary antibodies recognizing FLAG M2 tag(Sigma,1:2000), SARS-CoV-2 RBD(Sinobiological,1:500), CD9(Abcam,1:2000), Alix(Cell Signaling Technology,1:1000), GM130(Abcam,1:1000), Calnexin(Abcam,1:1000), hACE2(Abcam,# ab108209,only react with human species,1:2000) and GAPDH(Santa Cruz,1:2000).

Techniques: Purification, Concentration Assay, Protein Concentration

Journal: Journal of Controlled Release

Article Title: Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection

doi: 10.1016/j.jconrel.2021.05.049

Figure Lengend Snippet: ACE2 is required for the cellular uptake and targeting of SARS-CoV-2 S protein RBD-tagged EVs. (A)Western blot analysis of human ACE2 was performed with cell lysates from various cell lines or organoid as where indicated using antibody against ACE2. (B) Schematic illustration of RBD-GFP-loaded EVs. Cultured cells were transfected with RBD-VSVG vector or empty vector (NC-vector) in combination with CD9-GFP vector. 48 h post transfection, the EV fraction that was labeled with GFP was collected(EV-RBD-GFP + /EV-NC-GFP + ). (C) Flow cytometric analysis of GFP ratio in different cell lines after incubating with 1 × 10 9 of EV-RBD-GFP+ or EV-NC-GFP+ for 6 h. Different symbols correspond to independent experiments. Data are shown as mean ± SEM of three independent experiments, each individual experiment performs triplicate. (D, E and F) Representative images of Caco-2 cells (D), iPS cells-derived cardiomyocytes (E) and intestinal organoids (F) after application of 1 × 10 9 EV-RBD-GFP + or EV-NC-GFP + for 6 h. The GFP ratio was determined by flow cytometric analysis. (G) The binding affinity for RBD-tagged EVs with ACE2 protein was quantified by ELISA. Different concentration of EVs were incubated and followed by the anti-CD9 as the detector antibody. Each concentration was repeated with six wells. Data are shown as mean ± SEM of three independent experiments. (H and I) Representative images of ACE2-A549 cells showed an earlier plasma membrane distribution of EV-RBD-GFP + or EV-NC-GFP + incubation for 1 h (H) and 6 h (I) with or without pre-treated with ACE2 antibody. Fluorescence intensity of GFP was analyzed by ImageJ software were averaged from six different fields for each. Data are shown as mean ± SEM of three independent experiments. ** p < 0.01 *** p < 0.001.ns: no significant.

Article Snippet: Cells, purified EVs or tissues were lysed with RIPA buffer (Santa Cruz,USA) and cleared lysate was collected by centrifugation for protein separation on SDS-polyacrylamide gel(10%), followed by transfer onto PVDF membranes(Millipore), blocked with 5% nonfat dry milk in TBST and reacted with primary antibodies recognizing FLAG M2 tag(Sigma,1:2000), SARS-CoV-2 RBD(Sinobiological,1:500), CD9(Abcam,1:2000), Alix(Cell Signaling Technology,1:1000), GM130(Abcam,1:1000), Calnexin(Abcam,1:1000), hACE2(Abcam,# ab108209,only react with human species,1:2000) and GAPDH(Santa Cruz,1:2000).

Techniques: Western Blot, Cell Culture, Transfection, Plasmid Preparation, Labeling, Derivative Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Fluorescence, Software

Journal: Journal of Controlled Release

Article Title: Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection

doi: 10.1016/j.jconrel.2021.05.049

Figure Lengend Snippet: SARS-CoV-2 S protein RBD-tagged EVs specifically target tissues in vivo . (A) Western blot analysis of tissue distribution of hACE2 in humanized mice using antibody against human ACE2. (B,C,D) After intravenous injection of DiD-labeled EV-RBD or EV-NC in hACE2 mice( n = 4), the red-fluorescence of whole animal was detected at the indicated time points by IVIS spectrum (B) Representative IVIS images of various organs were acquired at the indicated time points through intravenous injection of EVs(C). Radiant efficiency was measured using Living Image software. Four mice were sacrificed in each time-point for tissue collection(D). (E, F, G) Serum cytokines concentrations of IL-6 (E), TNF-α (F) and IFN-β (G) were measured by ELISA. Serum samples were from hACE2 mice after intravenous injection of EV-RBD or EV-NC at 24 h. Each mouse serum was repeated with four wells. All data expressed as mean ± SEM of four independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells, purified EVs or tissues were lysed with RIPA buffer (Santa Cruz,USA) and cleared lysate was collected by centrifugation for protein separation on SDS-polyacrylamide gel(10%), followed by transfer onto PVDF membranes(Millipore), blocked with 5% nonfat dry milk in TBST and reacted with primary antibodies recognizing FLAG M2 tag(Sigma,1:2000), SARS-CoV-2 RBD(Sinobiological,1:500), CD9(Abcam,1:2000), Alix(Cell Signaling Technology,1:1000), GM130(Abcam,1:1000), Calnexin(Abcam,1:1000), hACE2(Abcam,# ab108209,only react with human species,1:2000) and GAPDH(Santa Cruz,1:2000).

Techniques: In Vivo, Western Blot, Injection, Labeling, Fluorescence, Software, Enzyme-linked Immunosorbent Assay

Journal: Journal of Controlled Release

Article Title: Tagged extracellular vesicles with the RBD of the viral spike protein for delivery of antiviral agents against SARS-COV-2 infection

doi: 10.1016/j.jconrel.2021.05.049

Figure Lengend Snippet: Delivery of siRNAs as a model system to identify inhibition of SARS-CoV-2 pseudovirus infection in vivo . (A) The efficiency of siRNA loading in EVs after electroporation as measured by real-time PCR. Each sample contained mixture of 150 μg siRNA-GFP and 150 μg EVs or 150 μg naked siRNA. After electroporation, purified EVs by ultracentrifugation to remove unencapsulated siRNA and each EV pellet treated with or without Benzonase. All electroporation samples were prepared in triplicate and each RNA isolate was analyzed in duplicate. (B) Scheme of the possibility of loading unmodified or modified EVs with specific siRNA into target cells. (C) Representative images of pseudotyped SARS-CoV-2-GFP-infected ACE2-A549 cells after incubation with different origin of EVs. The ACE2-A549 cells was infected with SARS-CoV-2-GFP for 24 h(MOI = 2), and then incubated for 24 h with naked si-GFP, EV-NC or EV-RBD electroporated with the siRNA, respectively. The GFP ratio was determined by flow cytometric analysis. Data are shown as mean ± SEM of three independent experiments in triplicates. (D) hACE2 mice(n = 4) were inoculated intranasally with pseudotyped SARS-CoV-2-GFP at a multiplicity of infection (MOI) of 50 per 50 μl inoculum volume per mouse. At 24 h post-infection,150 μg RBD-tagged EVs(EV-RBD-si-GFP) or control EVs (EV-NC-si-GFP) loaded with 150 μg GFP siRNA were injected into tail veins, and all mice were sacrificed at 48 h post injection for lung tissues collection.(E) Immunofluorescence staining of mouse lung paraffin sections for pseudotyped SARS-CoV-2-GFP (green, white arrows) and DAPI(blue). (F) Each hACE2 mouse(n = 4) injected with 150 μg RBD-tagged EVs encapsulated GFP siRNA or control EVs in 24 h and later inoculated intranasally with pseudotyped SARS-CoV-2-GFP at MOI of 50. All mice were sacrificed at 48 h post infection for lung tissues collection (G) Immunofluorescence staining analysis for pseudotyped SARS-CoV-2-GFP infected cells in lung paraffin sections (green, white arrows). Fluorescence intensity of GFP in the regions were analyzed through ImageJ software. Data are shown as mean ± SEM of four independent experiments in triplicates.**p < 0.01, ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells, purified EVs or tissues were lysed with RIPA buffer (Santa Cruz,USA) and cleared lysate was collected by centrifugation for protein separation on SDS-polyacrylamide gel(10%), followed by transfer onto PVDF membranes(Millipore), blocked with 5% nonfat dry milk in TBST and reacted with primary antibodies recognizing FLAG M2 tag(Sigma,1:2000), SARS-CoV-2 RBD(Sinobiological,1:500), CD9(Abcam,1:2000), Alix(Cell Signaling Technology,1:1000), GM130(Abcam,1:1000), Calnexin(Abcam,1:1000), hACE2(Abcam,# ab108209,only react with human species,1:2000) and GAPDH(Santa Cruz,1:2000).

Techniques: Inhibition, Infection, In Vivo, Electroporation, Real-time Polymerase Chain Reaction, Purification, Modification, Incubation, Injection, Immunofluorescence, Staining, Fluorescence, Software