Invariance of Structure in an Aging Colloidal Glass

We study concentrated colloidal suspensions, a model system which has a glass transition. The non-equilibrium nature of the glassy state is most clearly highlighted by aging -- the dependence of the system's properties on the time elapsed since vitrification. Fast laser scanning confocal microscopy allows us to image a colloidal glass and track the particles in three dimensions. We analyze the static structure in terms of tetrahedral packing. We find that while the aging of the suspension clearly affects its dynamics, none of the geometrical quantities associated with tetrahedra change with age.Comment: Submitted to the proceedings of "The 3rd International Workshop on Complex Systems" in Sendai, Japa

Dynamics and structure of an aging binary colloidal glass

We study aging in a colloidal suspension consisting of micron-sized particles in a liquid. This system is made glassy by increasing the particle concentration. We observe samples composed of particles of two sizes, with a size ratio of 1:2.1 and a volume fraction ratio 1:6, using fast laser scanning confocal microscopy. This technique yields real-time, three-dimensional movies deep inside the colloidal glass. Specifically, we look at how the size, motion and structural organization of the particles relate to the overall aging of the glass. Particles move in spatially heterogeneous cooperative groups. These mobile regions tend to be richer in small particles, and these small particles facilitate the motion of nearby particles of both sizes.Comment: 7 pages; submitted to Phys. Rev. E. Revised with 1 new figure, improved tex

Correlations of Structure and Dynamics in an Aging Colloidal Glass

We study concentrated colloidal suspensions, a model system which has a glass transition. Samples in the glassy state show aging, in that the motion of the colloidal particles slows as the sample ages from an initial state. We study the relationship between the static structure and the slowing dynamics, using confocal microscopy to follow the three-dimensional motion of the particles. The structure is quantified by considering tetrahedra formed by quadruplets of neighboring particles. We find that while the sample clearly slows down during aging, the static properties as measured by tetrahedral quantities do not vary. However, a weak correlation between tetrahedron shape and mobility is observed, suggesting that the structure facilitates the motion responsible for the sample aging.Comment: Submitted to Solid State Communication

Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission

To gain insight into female-to-male HIV sexual transmission and how male circumcision protects against this mode of transmission, we visualized HIV-1 interactions with foreskin and penile tissues in ex vivo tissue culture and in vivo rhesus macaque models utilizing epifluorescent microscopy. 12 foreskin and 14 cadaveric penile specimens were cultured with R5-tropic photoactivatable (PA)-GFP HIV-1 for 4 or 24 hours. Tissue cryosections were immunofluorescently imaged for epithelial and immune cell markers. Images were analyzed for total virions, proportion of penetrators, depth of virion penetration, as well as immune cell counts and depths in the tissue. We visualized individual PA virions breaching penile epithelial surfaces in the explant and macaque model. Using kernel density estimated probabilities of localizing a virion or immune cell at certain tissue depths revealed that interactions between virions and cells were more likely to occur in the inner foreskin or glans penis (from local or cadaveric donors, respectively). Using statistical models to account for repeated measures and zero-inflated datasets, we found no difference in total virions visualized at 4 hours between inner and outer foreskins from local donors. At 24 hours, there were more virions in inner as compared to outer foreskin (0.0495 +/- 0.0154 and 0.0171 +/- 0.0038 virions/image, p = 0.001). In the cadaveric specimens, we observed more virions in inner foreskin (0.0507 +/- 0.0079 virions/image) than glans tissue (0.0167 +/- 0.0033 virions/image, p<0.001), but a greater proportion was seen penetrating uncircumcised glans tissue (0.0458 +/- 0.0188 vs. 0.0151 +/- 0.0100 virions/image, p = 0.099) and to significantly greater mean depths (29.162 +/- 3.908 vs. 12.466 +/- 2.985 μm). Our in vivo macaque model confirmed that virions can breach penile squamous epithelia in a living model. In summary, these results suggest that the inner foreskin and glans epithelia may be important sites for HIV transmission in uncircumcised men

CCR5 Conformations Are Dynamic and Modulated by Localization, Trafficking and G Protein Association

<div><p>CCR5 acts as the principal coreceptor during HIV-1 transmission and early stages of infection. Efficient HIV-1 entry requires a series of processes, many dependent on the conformational state of both viral envelope protein and cellular receptor. Monoclonal antibodies (MAbs) are able to identify different CCR5 conformations, allowing for their use as probes to distinguish CCR5 populations. Not all CCR5 MAbs are able to reduce HIV-1 infection, suggesting the use of select CCR5 populations for entry. In the U87.CD4.CCR5-GFP cell line, we used such HIV-1-restricting MAbs to probe the relation between localization, trafficking and G protein association for individual CCR5 conformations. We find that CCR5 conformations not only exhibit different localization and abundance patterns throughout the cell, but that they also display distinct sensitivities to endocytosis inhibition. Using chemokine analogs that vary in their HIV-1 inhibitory mechanisms, we also illustrate that responses to ligand engagement are conformation-specific. Additionally, we provide supporting evidence for the select sensitivity of conformations to G protein association. Characterizing the link between the function and dynamics of CCR5 populations has implications for understanding their selective targeting by HIV-1 and for the development of inhibitors that will block CCR5 utilization by the virus.</p></div

CCR5 conformation changes upon engagement.

<p>U87.CD4.CCR5-GFP cells were treated with 100 nM RANTES analogs, 0.45 M sucrose or 5µg/mL chlorpromazine, or pretreated with an endocytosis inhibitor followed by addition of analog in inhibitor-containing medium. Cells were then stained for surface (grey bars) or total (black bars) conformation expression using MAb PA11 (A,C) or MC-5 (B,D). Graphs display averages of 2 (B,D) or 4 (A,C) experiments (N = 35 per experimental condition). Statistical analysis was performed using analysis of variance (ANOVA). ^*p<.05 between untreated and analog-treated, ^†p<.05 between analog-treated only and in combination with endocytosis inhibitor.</p

Surface expression of CCR5 conformations upon blocking endocytosis.

<p>U87.CD4.CCR5-GFP cells were treated with 0.45 M sucrose, 5µM Dynasore, 5µg/mL chlorpromazine, or 50µg/mL nystatin, inhibitors of both clathrin- and caveolae-mediated endocytosis. Cells were then surface stained with (A) 2D7, (B) 45531 or (C) PA11. Graphs display averages of 3 experiments (N = 30 per experimental condition). Statistical analysis was performed using analysis of variance (ANOVA). ^***p<.001, ^**p<.01, ^*p<.05, where significantly different than ‘Untreated.’</p

Surface and Total CCR5 conformation expression within U87.CD4.CCR5-GFP cells.

<p>U87.CD4.CCR5-GFP cells were incubated with 2D7, 45531, PA11 or MC-5 for surface (grey bars) or total labeling (black bars). CCR5 MAb labeling was measured using an IDL program and represented as a percentage of total CCR5. Graphs display averages of 4 independent experiments (N = 30 cells per experimental condition). Statistical analysis was performed using an unpaired <i>t</i> test. Error bars represent Standard Error of the Mean (SEM), ^****p<.0001, ^**p<.01, ^*p<.05</p

Surface CCR5 conformation is sensitive to pertussis toxin treatment.

<p>Cells were treated with 1µg/mL pertussis toxin and then surface stained with MAbs 2D7, 45531 or PA11. Changes in MAb 45531 labeling represent sensitivity to PTX treatment. Statistical analysis was performed using a paired student's t-test. ^**p<.01</p