Journal: ACS Nano

Article Title: Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses

doi: 10.1021/acsnano.4c03679

Figure Lengend Snippet: Production and characterization of HIV. (A) Schematic diagram depicting the differential ultracentrifugation workflow used to produce concentrated HIV for downstream analysis. (B) Bar graph showing the results of a p24 ELISA assay performed with unconcentrated HIV CM and the concentrated HIV preparation. The differential ultracentrifugation workflow concentrated the HIV ∼ 350×. (C) Immunoblot analysis of the concentrated HIV preparation. HIV gp120 was visible as a single band ∼120 kDa. HIV Gag p24 was detected in two bands; a lower band (∼24 kDa) corresponding to the mature protein, and an upper band corresponding to unprocessed Gag polyprotein p55. Bovine serum albumin, a contaminant derived from FBS used in the cell culture media, was also detected. (D) Left panel: nanoflow cytometry plots showing the size distribution of particles collected at various steps of the differential ultracentrifugation process. Different samples were run at different dilutions to avoid cytometer clogging, therefore particle counts represented on the Y -axis are not representative of true particle counts. Right panel: nanoflow cytometry plots representing the concentrated HIV sample and the control PM1 EVs. Samples were corrected for dilution factors before plotting. (E) Representative transmission electron micrographs of media control, PM1 EVs, and concentrated HIV. Individual scale bars are provided under each image. (F) Line chart representing the results of a TZM-Bl reporter luciferase assay measuring HIV infectivity at varying doses in the presence (orange line) or absence (purple line) of virus-inactivating UV-light. Results are represented as fold change compared to an uninfected cell control.

Article Snippet: Chips were then incubated with fluorescent antibodies against CD63 (CD63-CF647, from ExoView kit), CD81 (CD81-CF555, from ExoView kit), and HIV-1 gp120 (gp120-AF488, from Novus Biologicals, #NBP3-06630AF488).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Cell Culture, Cytometry, Control, Transmission Assay, Luciferase, Infection, Virus

Journal: ACS Nano

Article Title: Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses

doi: 10.1021/acsnano.4c03679

Figure Lengend Snippet: SP-IRIS analysis of HIV virions. (A) Left panel: bar graph comparing the total particle capture of media control, PM1 EVs, and HIV virions on three different antitetraspanin antibody capture spots. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. Right panels: Bar graphs indicating the number of particles on each antitetraspanin antibody capture spot measured as positive for a target protein via fluorescent antibody binding. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. (B) Grouped bar graph comparing the number of CD81+ particles (determined by capture spot) that are detected by interferometry, or detected by interferometry and additionally are determined to be gp120+. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. Statistical testing was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test, within each category (for representation purposes, the data are combined into a single graph). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (C) Combined representative size histogram/fluorescent scatter plots of particles captured on the CD81 antitetraspanin antibody capture spots, for each condition. The green line represents the total size distribution of all the particles detected interferometrically on the capture spot, with size plotted on the X -axis and particle count frequency on the left Y -axis. The blue dots represent gp120+ particles that were codetected interferometrically, plotted with size on the X -axis and gp120 fluorescence intensity on the right Y -axis. (D) Representative blue channel fluorescent images of the CD81 capture spots on the SP-IRIS microchips, across the three conditions. In this experiment, the blue channel detects gp120+ particles.

Article Snippet: Chips were then incubated with fluorescent antibodies against CD63 (CD63-CF647, from ExoView kit), CD81 (CD81-CF555, from ExoView kit), and HIV-1 gp120 (gp120-AF488, from Novus Biologicals, #NBP3-06630AF488).

Techniques: Control, MicroChIP Assay, Standard Deviation, Binding Assay, Fluorescence

Journal: ACS Nano

Article Title: Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses

doi: 10.1021/acsnano.4c03679

Figure Lengend Snippet: SPIRFISH analysis of HIV virions. (A) Left panel: bar graph comparing the total particle capture of media control, PM1 EVs, and HIV virions across three different antitetraspanin antibody capture spots on SP-IRIS microchips. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. Right panels: bar graphs indicating the number of particles on each antitetraspanin antibody capture spot measured as positive for a target protein or target RNA via fluorescent antibody or fluorescent anti-HIV-1 BaL gRNA smFISH-probe binding, respectively. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. (B) Grouped bar graph comparing the number of CD81+ particles (determined by capture spot) that are detected by interferometry and additionally are determined to be gp120+/smFISH+. Bars show the mean particle counts across three independent SP-IRIS microchip experiments; error bars represent standard deviation. Statistical testing was performed by ordinary one-way ANOVA with Tukey’s multiple comparisons test, within each category (for representation purposes, the data are combined into a single graph). ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. (C) Combined representative size histogram/fluorescent scatter plots of particles captured on the CD81 antitetraspanin antibody capture spots, for each condition. The green line represents the total size distribution of all the particles detected interferometrically on the capture spot, with size plotted on the X -axis and particle count frequency on the left Y -axis. The blue dots represent gp120+ particles that were codetected interferometrically, plotted with size on the X -axis. The magenta dots represent smFISH+ particles that were codetected interferometrically, also plotted with size on the X -axis. Fluorescence intensity for both gp120 and smFISH signals is represented on the right Y -axis. (D) Representative red channel fluorescent images of the CD81 capture spots on the SP-IRIS microchips, across the three conditions. In this experiment, the red channel detects anti-HIV-1 BaL gRNA smFISH+ particles.

Article Snippet: Chips were then incubated with fluorescent antibodies against CD63 (CD63-CF647, from ExoView kit), CD81 (CD81-CF555, from ExoView kit), and HIV-1 gp120 (gp120-AF488, from Novus Biologicals, #NBP3-06630AF488).

Techniques: Control, MicroChIP Assay, Standard Deviation, Binding Assay, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Increased neuroinflammatory and arachidonic acid cascade markers, and reduced synaptic proteins, in brain of HIV-1 transgenic rats

doi: 10.1186/1742-2094-8-101

Figure Lengend Snippet: (A) Representative immunoblot of gp120 and tat protein in HIV-1 Tg rat brain (A), detected as described in Methods . mRNA levels of brain GFAP (B) and CD11b (D) in control and HIV-1 Tg rat brain, measured using real time RT-PCR, normalized to β-globulin and relative to control level (calibrator) using the ΔΔC T method. Representative immunoblots of (C) GFAP and (E) CD11b protein in control and HIV-1 Tg rat brain. Bar graphs are ratios of optical densities of individual protein bands to β-actin, expressed as percent of control. Data represent mean ± SEM, statistical significance: **p < 0.01, ***p < 0.001 as determined by unpaired t-test.

Article Snippet: Cytosolic protein blots were incubated overnight in Tris-buffered-saline containing 5% nonfat dried milk and 0.1% Tween-20, with specific primary antibodies for proinflammatory cytokines: IL-1β (1:500), TNFα (1:500); astrocytes: glial fibrillary acidic protein (GFAP) (1:1000); CD11b (1:1000); AA cascade proteins: cPLA 2 -IVA, sPLA 2 -IIA, iPLA 2 -VIA, COX-1 (1:1000), COX-2 (1:1000), cytochrome p450 epoxygenase, TXS, 5-, 12-, 15-LOX, mPGES, cPGES (1:1000); gp120 (1:100); tat (1:100); drebrin (1:1000), BDNF (1:1000) (Santa Cruz, Santa Cruz, CA);); neurofilament-L (1:500) (Cell Signaling Technology, Danvers, MA) and β-actin (1:10,000) (Sigma Aldrich, St. Louis, MO).

Techniques: Western Blot, Control, Quantitative RT-PCR

Journal: PLoS Pathogens

Article Title: Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

doi: 10.1371/journal.ppat.1003278

Figure Lengend Snippet: (A) FIP1C depletion was achieved using shRNA in HeLa cells (left) and the H9 T cell line (right). Populations of FIP1C-depleted HeLa and H9 cells were then divided and infected with VSV-pseudotyped NL4-3 or NL4-3delCT144. Released viruses were harvested 2 days after infection (HeLa) or 4 days after infection (H9) and evaluated for protein content by Western blotting. Note that results using specific antiserum used for detection of gp120 and gp160 in cells and particles are shown; gp41 particle blots were probed with a gp41-specific monoclonal antibody. (B) Infectivity of released viral particles from the experiment shown in (A) was measured in the TZM-bl reporter cell line, expressed as infectious units/ng p24. (C) Plasma membrane (left) and total cellular (permeabilized) levels of HIV-1 Env (right) were detected using flow cytometry 4 days after infection with VSV-G- pseudotyped NL4-3 WT or NL4-3delCT144 viruses. The relative level of Env on plasma membrane compared to total Env was calculated and plotted in panel (D) for control and FIP1C-depleted H9 cells. (E) FIP1C-depleted (open circles) and control shRNA-treated H9 cells (filled squares) were infected with VSV-pseudotyped viruses indicated at MOI 0.5. Viral growth was monitored over time by detection of p24 in the culture supernatant. CT144 growth (triangles) shown in control H9 cells.

Article Snippet: Goat polyclonal antibody against HIV-1 gp120 and gp160 (used in Western blotting) was AHP2204 from AbD Serotec (Oxford, UK).

Techniques: shRNA, Infection, Western Blot, Clinical Proteomics, Membrane, Flow Cytometry, Control

Journal: PLoS Pathogens

Article Title: Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

doi: 10.1371/journal.ppat.1003278

Figure Lengend Snippet: (A) HeLa cells were transfected with proviral plasmid pNL4-3 or NL4-3 CT144 and either a dominant negative form of Rab14 (Rab14S25N) or a constitutively active form of Rab14 (Rab14Q70L). 48 hours after transfection, cellular and particle content of HIV-1 Env was examined by Western blot. Numbers above Env blots represent densitometry values with leftmost lanes set to 100. (B) shRNA-mediated depletion of Rab11 or Rab14 was performed in HeLa cells. Following selection in puromycin, cells were transfected with either pNL4-3 or pNL4-3 CT144 plasmid. Cellular and particle-associated Env was detected by immunoblotting 48 hours following transfection. (C) shRNA-resistant GFP-FIP1C* WT and GFP-FIP1C* (S580N/S582L) were transfected together with pNL4-3 in control HeLa cells or FIP1C-depleted HeLa cells as in the repletion experiment described in . Input FIP1C* plasmid levels were 2 µg in lanes 3, 5, 7, and 8 and increased to 4 µg in lanes 4 and 6. In a separate experiment the ability of Rab11 binding mutant GFP-FIP1C* I62E and GFP-FIP1C* D622N to rescue Env particle incorporation in FIP1C-depleted HeLa cells (lanes 7 and 8). Note that gp160 and gp120 were detected with polyclonal antiserum; gp41 particle blots were probed with a monoclonal specific for gp41.

Article Snippet: Goat polyclonal antibody against HIV-1 gp120 and gp160 (used in Western blotting) was AHP2204 from AbD Serotec (Oxford, UK).

Techniques: Transfection, Plasmid Preparation, Dominant Negative Mutation, Western Blot, shRNA, Selection, Control, Binding Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: Rab11-FIP1C and Rab14 Direct Plasma Membrane Sorting and Particle Incorporation of the HIV-1 Envelope Glycoprotein Complex

doi: 10.1371/journal.ppat.1003278

Figure Lengend Snippet: (A) MDMs were prepared from peripheral blood of healthy donors and depleted of FIP1C using two administrations of siRNA. MDMs were then infected with VSV-G-pseudotyped NL4-3 or NL4-3delCT144. Cell lysates and purified particles were examined 8 days following infection. Shown are Western blot results from two donors. (B) Particle infectivity from the experiments described above was measured in TZM-bl reporter cells as before. (C) Parental HeLa T-REx cells (Invitrogen) and HeLa-T-REx cells stably transfected with plasmids for inducible expression of FIP1C and Rab14Q70L were transfected with a constant amount of Gag expression plasmid and increasing amounts of NL4-3 Env plasmid (numbers above gp160 blot represent µg of input plasmid). Cell lysates and purified particles were examined by Western blotting. In these experiments, virus lanes were loaded with equal amounts of p24 as measured by ELISA. The bottom Env immunoblot represents cell surface Env as determined by biotinylation and precipitation with streptavidin beads, using the same input Gag and Env DNA as above. (D) Env and Gag content of released viral particles was quantified by densitometry using the LiCor instrument and plotted as background-subtracted Env pixel intensity to Gag pixel intensity (left) and by p24 and gp120 antigen-capture ELISA (right). The number of Env molecules per particle was calculated as an approximation, assuming that 5000 CA molecules are present in one HIV-1 virion.

Article Snippet: Goat polyclonal antibody against HIV-1 gp120 and gp160 (used in Western blotting) was AHP2204 from AbD Serotec (Oxford, UK).

Techniques: Infection, Purification, Western Blot, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Virus, Enzyme-linked Immunosorbent Assay

Journal: Advanced materials (Deerfield Beach, Fla.)

Article Title: Tunable Fano-resonant Metasurfaces on a Disposable Plastic-template for Multi-modal and Multiplex Biosensing

doi: 10.1002/adma.201907160

Figure Lengend Snippet: Surface sensitivity of plastic-templated metasurface for layer-by-layer modifications. a Summary of surface chemistry steps. b Resonance peak wavelength shifts demonstrated on surfaces etched for 30, 60 and 90 sec. c Layer-by-layer binding response of bio-molecules on the 60 sec etched surface is monitored in real-time. d-f Resonance wavelength shifts as a function various concentrations of protein G, anti-gp120 antibody and rec-gp120 protein are represented via Box-Whisker plots. I-shaped box indicates 25th and 75th, red-line median, and whiskers the 95th and 5th percentiles. Dots represent data points for each concentration. Non-parametric Kruskal-Wallis analysis followed by Dunn’s multiple comparison test was performed to evaluate the collected data for each concentration at Protein G, anti-gp120 antibody and rec. gp120 protein experiments. The minimum, first quartile, median, third quartile, and maximum of each concentration were presented in Supplementary Table 1. In Protein G experiment, statistical difference was observed between 75 and 1000 μg/mL concentrations (n=3, p<0.05). In anti-gp120 antibody experiments, statistical difference was observed between 30 and 75 μg/mL concentrations (n=3, p<0.05). In rec-gp120 protein experiments, statistical difference was observed between 25 and 200 μg/mL concentrations (n=3, p<0.05).

Article Snippet: This step was followed by a 100 μL PBS wash. (2) Anti-gp120 antibody layer: 100 μL of anti-gp120 (immunoaffinity purified rabbit polyclonal IgG, 1 mg/mL, #11233-RP02, Sino Biological, Pennsylvania, US) was captured on the protein G layer and the surface was washed using 100 μL PBS. (3) HIV capture: The functionalized metasurface was used to capture recombinant gp120 proteins and HIV-1 particles.

Techniques: Binding Assay, Whisker Assay, Concentration Assay