Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Nef expression and HIV-1 infection induce vesicle formation. TEM image of conditioned media from HEK293 cells transfected with (A) Nef expression plasmid or (B) mock-transfected. Conditioned media collected 3 days posttransfection and subjected to ultracentrifugation (400,000 × g). Pellets were fixed, embedded, and stained with 2% uranyl acetate. Samples were visualized using a JEOL 1200 EX transmission microscope. (C) Culture supernatant from HEK293 transfected with a Nef expression plasmid was subjected high-speed differential centrifugation. Supernatants were centrifuged for 10 min at 300 × g, followed by 30 min at 10,000 × g, and then centrifuged for 1 h at 300,000 × g. Nef was detected in vesicles pelleted at 300,000 by immunoblot using anti-Nef monoclonal. S, supernatants; P, pellet.

Article Snippet: Human plasma samples Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Expressing, Infection, Transfection, Plasmid Preparation, Staining, Transmission Assay, Microscopy, Centrifugation, Western Blot

Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Nef vesicles captured using anti-CD45 magnetic bead separation. Supernatants from HIV-1-infected cells were harvested 15 days postinfection and vesicles/virions were concentrated by centrifugation at 200,000 × g for 1 h on a 20% sucrose cushion. The pellet was resuspended in 1 × TNE buffer and subjected to CD45 magnetic bead separation. Each fraction was collected, ultracentrifuged at 400,000 × g for 1 h, and then separated by SDS-PAGE. Fractions (SM = starting material, FT = flow through, W = wash, and E = eluate) were analyzed by Western blot for HIV proteins using anti-HIV sera (upper panel). The singular band present in the eluate was confirmed to be Nef via an immunoblot using an anti-Nef monoclonal antibody (lower panel). Nef+, recombinant Nef protein.

Article Snippet: Human plasma samples Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Infection, Centrifugation, SDS Page, Western Blot, Recombinant

Journal: AIDS Research and Human Retroviruses

Article Title: HIV Type 1 Nef Is Released from Infected Cells in CD45 + Microvesicles and Is Present in the Plasma of HIV-Infected Individuals

doi: 10.1089/aid.2009.0170

Figure Lengend Snippet: Production of CD45+ Nef microvesicles is cell-type dependent. Concentrations of p24 (A) microvesicles and Nef (B) were measured by ELISA in culture supernatants and CD45 captured microvesicles of HIV-1-infected Jurkat T-lymphocytes, THP-1 monocytes, and PMA-treated THP-1 (macrophage-like). T-lymphocytes release the most HIV-1 virus as measured by p24 ELISA whereas macrophage-like THP-1 releases the most Nef microvesicles.

Article Snippet: Human plasma samples Plasma from HIV-1 + /HCV − /HBV − individuals ( n = 10) and HIV − / HCV − /HBV − controls ( n = 10) was obtained from Bioreclamation (Long Island, NY).

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal: bioRxiv

Article Title: Robust and High-Throughput Analytical Flow Proteomics Analysis of Cynomolgus Monkey and Human Matrices with Zeno SWATH Data Independent Acquisition

doi: 10.1101/2022.11.30.518440

Figure Lengend Snippet: Volcano plots representing differential proteins in individuals diagnosed with non-alcoholic steatohepatitis (NASH, n = 20) ( A ) and type 2 diabetes (T2D, n = 10) ( B ) in comparison to healthy individuals (n = 20). A two-sided Welch’s t-test was used for differential analysis and proteins with FDR < 0.1 were considered significant. Proteins underlined and in bold are uniquely significant in Zeno SWATH mode. ( C ) Pie charts representing the proportion of significant proteins (FDR < 0.1) in Zeno SWATH DIA mode that were i) not detected, ii) detected and significant, and iii) detected but not significant in SWATH DIA mode. ( D ) Comparison of pathway significance using significant proteins identified in SWATH and Zeno SWATH DIA modes in NASH and T2D studies. Ingenuity Pathway Analysis was used for enrichment analysis and pathways with P-values < 0.05 were considered significant.

Article Snippet: Commercial human EDTA-plasma samples from 20 individuals self-reported as healthy were purchased from BioIVT and EDTA-plasma samples from 20 individuals diagnosed with NASH, and 10 individuals diagnosed with T2D were purchased from ProteoGenex (lot numbers and subject demographics information are available in Table S4 ).

Techniques: