Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Experimental layout of the ARDS model: After 4 hours of intratracheal (IT) LPS injection (3 mg/kg, 100 μl), 30 μg of hep-EVs, hep-SPLEVs, or MSC-EVs, or vehicle [phosphate-buffered saline (PBS)] was injected into the tail vein. Samples were collected and analyzed at 20 hours. h, hours; iv, intravenous. ( B ) Counts of total inflammatory cells and neutrophils in BALF [control ( n = 6), LPS only ( n = 7), LPS + hep-EVs ( n = 4), and LPS + MSC-EVs ( n = 3)]. ( C ) Lung Injury scores were calculated in 10 fields in each sample [control ( n = 4), LPS only ( n = 5), LPS + hep-EVs ( n = 6), and LPS + MSC-EVs ( n = 3)]. ( D ) Hematoxylin and eosin (H&E)–stained images of the control or LPS-challenged lung (left) and lung injury score [LPS only ( n = 5), LPS + hep-EVs ( n = 6), LPS + hep-SPLEVs ( n = 8), and LPS + MSC-EVs ( n = 3)] (right) following each treatment. Scale bars, 100 μm. ( E ) Counts of neutrophils in BALF of LPS-challenged mice following indicated treatments [LPS only ( n = 7), LPS + hep-EVs ( n = 4), LPS + hep-SPLEVs ( n = 5), and LPS + MSC-EVs ( n = 3)]. ( F ) Enzyme-linked immunosorbent assay (ELISA) of cytokines (IL-6 and TNFα) in sera of ARDS-induced mice treated with hep-EVs or hep-SPLEVs ( n = 4 for each). ( G ) Localization of hep-EVs and hep-SPLEVs in the lung. Red: F4/80-positive macrophages; green: hep-EVs or hep-SPLEVs prestained with PKH67; blue: DAPI. Scale bar, 20 μm. A tag-free recombinant human sPLA 2 -X was used in (D) to (G) under the condition shown in table S3. * P < 0.033, ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Injection, Saline, Control, Staining, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis of phospholipids extracted from LPS-challenged mouse lungs with or without 30 μg of sPLA 2 -X, hep-EVs, hep-SPLEVs, or vehicle (PBS) treatment for 20 hours ( n = 3 for each). Colors indicate fold changes in the levels of individual phospholipids [PC (phosphatidylcholine), pPC (plasmalogen-type PC), PE (phosphatidylethanolamine), pPE (plasmalogen-type PE), PS (phosphatidylserine), PA (phosphatidic acid), PG (phosphatidylglycerol), and PI (phosphatidylinositol)] following each treatment relative to those in control. ( B ) LC-MS/MS analysis of free fatty acids in the lung with same treatment as (A). DPA, docosapentaenoic acid. ( C ) LC-MS/MS analysis of PUFA metabolites in the lung. Colors indicate fold changes in the levels of individual metabolites following each treatment relative to those in control. ( D ) Representative LipiORDER images for phase transition of lipids in the lung of LPS-challenged mice with sPLA 2 -X, hep-EVs, or hep-SPLEVs treatment for 20 hours and normal control (NC). Scale bar, 50 μm. ( E and F ) Representative LipiORDER images for phase transition of lipids and immunostaining of surfactant protein C (SFTPC) in primary mouse alveolar cells (E) and A549 cells (F) with or without LPS stimulation for 6 hours in the presence or absence of hep-EVs or hep-SPLEVs (5 μg/ml). The cells were cultured on collagen-coated ϕ18-mm cover glasses and photographed under a confocal microscope. Scale bar, 50 μm. A tag-free recombinant human sPLA 2 -X was used under the condition shown in tables S3 and S4.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Control, Sublimation, Immunostaining, Cell Culture, Microscopy, Recombinant

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Experimental layout of the BLM-induced acute lung injury model: CL or PBS liposomes (PL) were preinjected into mice 24 hours before IT BLM administration, and after 24 hours, PBS or hep-SPLEVs were injected into the tail vein. After additional 24 hours, samples were collected and analyzed. ( B ) Counts of total inflammatory cells in BALF of mouse lungs challenged with BLM for 24 hours in the presence or absence of hep-SPLEVs [PBS ( n = 5), PL + hep-SPLEVs ( n = 4), CL + hep-SPLEVs ( n = 4), and CL ( n = 4)]. ( C ) H&E-stained images of the lungs (left) and lung injury scores (right) following BLM-induced acute lung injury [PBS ( n = 4), PL + hep-SPLEVs ( n = 4), CL + hep-SPLEVs ( n = 3), and CL ( n = 3)]. N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in table S3. ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Liposomes, Injection, Staining

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Gene Ontology singular enrichment analysis based on RNA-seq analysis at 24 hours in the lungs of ARDS-challenged mice treated with 30 μg of hep-EVs, hep-SPLEVs, or with PBS. MAPK, mitogen-activated protein kinase. ( B and C ) Quantitative polymerase chain reaction (qPCR) of Srebf1 in mouse lungs at 20 hours (B) and SREBF1 in A549 cells at 6 hours (C) following each treatment ( n = 3 for each). ( D ) Western blotting of SREBP1, AKT, and phosphorylated AKT (pAKT), as well as α-tubulin as a loading control, in A549 cells following each treatment. Arrow indicates cleaved SREBP1. Right panels show the quantified ratio of cleaved SREBP1 to α-tubulin (top) or pAKT to AKT (bottom) ( n = 3 for each). ( E and F ) The quantified ratio of cleaved SREBP1 to β-actin as a loading control ( n = 3 for each) (E) and Western blotting of SREBP1 and β-actin (F) in mouse lungs after treatment with hep-EVs (5 μg/ml), hep-SPLEVs, or with PBS in the presence or absence of LPS. ( G ) LipiORDER analysis of A549 cells following each treatment. Hep-EVs or hep-SPLEVs were used at 5 μg/ml. Representative images for phase transition of lipids (top) and fluorescence intensity (bottom) are shown. ( H ) LipiORDER images for phase transition of lipids (left) and fluorescence intensity (right) in the lungs of WT and Srebf1 –/– mice following each treatment ( n = 3 for each). Hep-EVs or hep-SPLEVs were used at 30 μg per mouse. ( I ) Analysis of representative phospholipid species in lungs of WT and Srebf1 –/– mice following each treatment. Y axis shows relative peak areas of individual lipids in LPS-challenged mice compared to control mice. Nontagged sPLA 2 -X was used in (A), and N-terminally His-tagged sPLA 2 -X bv was used in (B) to (I) under the condition shown in tables S3 and S4. * P < 0.033, ** P < 0.002, and *** P < 0.001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: RNA Sequencing, Real-time Polymerase Chain Reaction, Western Blot, Control, Sublimation, Fluorescence

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Lung injury scores following treatment with LPS in the presence of hep-EVs, Akata-EVs, hep-SPLEVs, Akata-SPLEVs, or vehicle (PBS). ( B ) Comparison of the ratio of phospholipid classes in hep-EVs versus Akata-EVs. ( C ) qNano analysis of PG PL. Histogram of size distribution (left) and average particle diameter (right) of PG PL before and after treatment with sPLA 2 -X (right) is shown. ( D and E ) Counts of total inflammatory cells and neutrophils in BALF [PBS ( n = 4), hep-EVs ( n = 4), hep-SPLEVs ( n = 5), PG PL ( n = 6), and PG-SPLEVs ( n = 6)] (D) and qPCR of Il6 in the lung ( n = 3 for each) (E) following each treatment in the LPS-induced ARDS model. ( F ) LipiORDER images for transition images of lipids (top) and fluorescence intensity (bottom) in A549 cells following each treatment. Hep-EVs or hep-SPLEVs were used at 5 μg/ml. ( G ) Effects of LPG18:1 on SREBP-1 cleavage and AKT phosphorylation in A549 cells, as evaluated by Western blotting of SREBP1, AKT and pAKT, as well as α-tubulin as a loading control. Quantified ratios of cleaved SREBP1 to α-tubulin ( n = 4) or pAKT to AKT ( n = 3) are shown. Values are mean ± SEM in [(A), (C), (D), (E), and (G)]. The results were analyzed by analysis of variance (ANOVA) test and Tukey’s multiple-comparison test [(A), (C), (D), (E), and (G)] or by Student’s t test (C). N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in tables S3 and S4. * P < 0.033, ** P < 0.002, *** P < 0.001, and **** P < 0.0001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Comparison, Fluorescence, Phospho-proteomics, Western Blot, Control

Journal: Science Advances

Article Title: sPLA 2 -reacted extracellular vesicles (SPLEVs) as a therapeutic modality for cytokine storm syndromes

doi: 10.1126/sciadv.adr9135

Figure Lengend Snippet: ( A ) Survival curves of mice treated with PBS ( n = 9), hep-EVs ( n = 8), or hep-SPLEVs ( n = 8) in the moderate to severe sepsis model. ( B ) qPCR evaluation of splenic Il6 and Tnf expression in mice treated for 10 days with hep-EVs or hep-SPLEVs after cecum puncture. ( C ) Survival curves after treatment with PBS, hep-EVs, or hep-SPLEVs ( n = 9 for each) in the LPS-induced coagulopathy model. ( D ) Serum ALT and AST levels with or without administration of hep-SPLEVs at 48 hours in the CCl 4 -induced hepatitis model ( n = 3 to 4). ( E to G ) Compared effects of hep-SPLEVs ( n = 9) and hep-EVs ( n = 6) as DSS alone ( n = 10) in DSS-challenged mice. Each mice treated 7 days. (E) Representative photos of colon of mice (left). Decrease in colon length (%) after treatments (right). (F) H&E staining of the colon in mice. Black arrowheads show inflammatory cells, white arrowhead shows loss of intestinal structure, double-headed arrow shows thinning, and black arrow shows edema. Scale bar, 100 μm. (G) Inflammatory and DAI scores in mouse colon. ( H ) H&E staining of mouse lungs with or without H3 influenza infection for 7 days in the presence or absence of hep-SPLEVs (left). Lung injury scores in these mice [naïve ( n = 3), influenza ( n = 4), and influenza + hep-SPLEVs ( n = 5)] are shown (right). ( I ) Viral titers as monitored by qPCR of the H3 influenza gene in the lungs in (H) [naïve ( n = 9), influenza ( n = 9), and influenza + hep-SPLEVs ( n = 13)]. N-terminally His-tagged sPLA 2 -X bv was used under the condition shown in table S3. * P < 0.033, ** P < 0.002, *** P < 0.001, and **** P < 0.0001.

Article Snippet: As required for experiments, 1 μM varespladib (MedChemExpress) was added during the treatment of EVs with sPLA 2 -X.

Techniques: Expressing, Staining, Infection

Journal: Viruses

Article Title: Procyanidins A1 and B1 Suppress PEDV CV777 by Modulating Mitophagy

doi: 10.3390/v18070758

Figure Lengend Snippet: The chemical structures of procyanidins A1, A2, B1, and B2. ( A ) Procyanidin A1. ( B ) Procyanidin A2. ( C ) Procyanidin B1. ( D ) Procyanidin B2.

Article Snippet: Procyanidin A1 (CAS No. 103883-03-0, purity ≥ 99.96%, HY-N2344, 1 μg/mL = 1.735 μM), procyanidin A2 (CAS No. 41743-41-3, purity ≥ 99.85%, HY-N2343, 1 μg/mL = 1.735 μM), procyanidin B1 (CAS No. 20315-25-7, purity ≥ 99.28%, HY-N0795, 1 μg/mL = 1.729 μM), procyanidin B2 (CAS No. 29106-49-8, purity ≥ 99.79%, HY-N0796, 1 μg/mL = 1.729 μM), and ribavirin (CAS No. 36791-04-5, purity ≥ 99.96%, HY-B0434) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques:

Journal: Viruses

Article Title: Procyanidins A1 and B1 Suppress PEDV CV777 by Modulating Mitophagy

doi: 10.3390/v18070758

Figure Lengend Snippet: Inhibitory effects of different procyanidin configurations on PEDV infection ( n = 3). ( A , B ) Vero cells were infected with PEDV (MOI = 0.5) for 2 h and treated with the indicated concentrations of procyanidins for 20 h. The cells were fixed, permeabilized, and immunostained for indirect immunofluorescence imaging. PEDV-N: green; DAPI: blue. ( A ) Procyanidin A1 and A2 at 5, 25, and 50 μg/mL. ( B ) Procyanidin B1 and B2 at 5, 25, and 50 μg/mL. ( C , D ) Vero cells were infected with PEDV (MOI = 0.5) for 2 h and subsequently treated with the indicated concentrations of procyanidins for 20 h. Total protein was extracted for Western blot analysis. ( C ) Procyanidin A1 at 10, 20 and 30 μg/mL. ( D ) Procyanidin B1 at 25, 37.5, and 50 μg/mL. ns: p > 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Procyanidin A1 (CAS No. 103883-03-0, purity ≥ 99.96%, HY-N2344, 1 μg/mL = 1.735 μM), procyanidin A2 (CAS No. 41743-41-3, purity ≥ 99.85%, HY-N2343, 1 μg/mL = 1.735 μM), procyanidin B1 (CAS No. 20315-25-7, purity ≥ 99.28%, HY-N0795, 1 μg/mL = 1.729 μM), procyanidin B2 (CAS No. 29106-49-8, purity ≥ 99.79%, HY-N0796, 1 μg/mL = 1.729 μM), and ribavirin (CAS No. 36791-04-5, purity ≥ 99.96%, HY-B0434) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques: Infection, Immunofluorescence, Imaging, Western Blot

Journal: Viruses

Article Title: Procyanidins A1 and B1 Suppress PEDV CV777 by Modulating Mitophagy

doi: 10.3390/v18070758

Figure Lengend Snippet: Drug selectivity index of procyanidins A1 and B1 ( n = 3). ( A ) CC 50 of procyanidin A1 in Vero cells. ( B ) EC 50 of procyanidin A1 in Vero cells. ( C ) CC 50 of procyanidin B1 in Vero cells. ( D ) EC 50 of procyanidin B1 in Vero cells.

Article Snippet: Procyanidin A1 (CAS No. 103883-03-0, purity ≥ 99.96%, HY-N2344, 1 μg/mL = 1.735 μM), procyanidin A2 (CAS No. 41743-41-3, purity ≥ 99.85%, HY-N2343, 1 μg/mL = 1.735 μM), procyanidin B1 (CAS No. 20315-25-7, purity ≥ 99.28%, HY-N0795, 1 μg/mL = 1.729 μM), procyanidin B2 (CAS No. 29106-49-8, purity ≥ 99.79%, HY-N0796, 1 μg/mL = 1.729 μM), and ribavirin (CAS No. 36791-04-5, purity ≥ 99.96%, HY-B0434) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques:

Journal: Viruses

Article Title: Procyanidins A1 and B1 Suppress PEDV CV777 by Modulating Mitophagy

doi: 10.3390/v18070758

Figure Lengend Snippet: Procyanidins A1 and B1 antagonize the effects of PEDV on autophagy, mitochondrial dynamics, and mitophagy-related proteins ( n = 3). ( A , B ) Vero cells were infected with PEDV (MOI = 0.5) for 2 h, followed by treatment with procyanidins A1 or B1. ( A ) Cells were treated with procyanidin A1 (10, 20, or 30 μg/mL) for 20 h, and total protein was extracted for Western blot. ( B ) Cells were treated with procyanidin B1 (25, 37.5, or 50 μg/mL) for 20 h, and total protein was extracted for Western blot. ( C – H ) Densitometric analysis of the p62 ( C ), LC3-II ( D ), PINK1 ( E ), Parkin ( F ), DRP1 ( G ), and MFN1 ( H ) protein bands was performed via ImageJ. ( I , J ) IPEC-J2 cells were transfected with the mCherry-GFP-LC3 plasmid, and the effects of procyanidins on autophagic flux during PEDV infection were evaluated via fluorescence microscopy (scale bar 10 μm). Arrows indicate representative yellow or red puncta of mCherry-GFP-LC3, reflecting autophagosomes or autolysosomes. ( I ) Procyanidin A1 treatment group. ( J ) Procyanidin B1 treatment group. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Article Snippet: Procyanidin A1 (CAS No. 103883-03-0, purity ≥ 99.96%, HY-N2344, 1 μg/mL = 1.735 μM), procyanidin A2 (CAS No. 41743-41-3, purity ≥ 99.85%, HY-N2343, 1 μg/mL = 1.735 μM), procyanidin B1 (CAS No. 20315-25-7, purity ≥ 99.28%, HY-N0795, 1 μg/mL = 1.729 μM), procyanidin B2 (CAS No. 29106-49-8, purity ≥ 99.79%, HY-N0796, 1 μg/mL = 1.729 μM), and ribavirin (CAS No. 36791-04-5, purity ≥ 99.96%, HY-B0434) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques: Infection, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Microscopy

Journal: Viruses

Article Title: Procyanidins A1 and B1 Suppress PEDV CV777 by Modulating Mitophagy

doi: 10.3390/v18070758

Figure Lengend Snippet: Molecular docking simulations of procyanidins A1 and B1 with proteins related to mitophagy and mitochondrial membrane dynamics. Panels ( A – H ) Display the molecular docking results (binding poses) and 2D diagrams illustrating hydrogen bonds and detailed intermolecular interactions between the procyanidins and target proteins. ( A ) Procyanidin A1 with DRP1. ( B ) Procyanidin B1 with DRP1. ( C ) Procyanidin A1 with MFN1. ( D ) Procyanidin B1 with MFN1. ( E ) Procyanidin A1 with Parkin. ( F ) Procyanidin B1 with Parkin. ( G ) Procyanidin A1 with PINK1. ( H ) Procyanidin B1 with PINK1.

Article Snippet: Procyanidin A1 (CAS No. 103883-03-0, purity ≥ 99.96%, HY-N2344, 1 μg/mL = 1.735 μM), procyanidin A2 (CAS No. 41743-41-3, purity ≥ 99.85%, HY-N2343, 1 μg/mL = 1.735 μM), procyanidin B1 (CAS No. 20315-25-7, purity ≥ 99.28%, HY-N0795, 1 μg/mL = 1.729 μM), procyanidin B2 (CAS No. 29106-49-8, purity ≥ 99.79%, HY-N0796, 1 μg/mL = 1.729 μM), and ribavirin (CAS No. 36791-04-5, purity ≥ 99.96%, HY-B0434) were purchased from MedChemExpress (South Brunswick, NJ, USA).

Techniques: Membrane, Binding Assay