Identifying a second component from simulated mixture data <i>via</i> incorrect single component analysis.

<p>These curves shown represent the chi-squared contours for the different fitting methods when fitting two-component data with a single component model. When the χ² value exceeds ∼1.3 we conclude that the single component analysis fails, indicating the presence of a second component in the sample. Four χ² surfaces demonstrate the boundary in parameter space for 1) FCS (green lines); 2) Fluorescence Lifetime (yellow lines); 3) Fluorescence Lifetime with average intensity constraint (blue lines); and 4) τFCS with average intensity constraint (red lines).</p

Molecular parameter values recovered using Global-τFCS compared to known parameter values.

<p>The accuracy of the recovered information goes well beyond previous experimental capabilities, which is more remarkable given that no fitting constraints or <i>a priori</i> assumptions were required for these fitting results.</p

τFCS analysis of binary dye mixtures with known R6G and RhB concentrations.

<p>(a) Recovery of molecular concentrations across nine known concentration ratios using τFCS. Measured concentrations are shown as red and blue squares, and the solid lines show the known concentration of each dye mixture. (b) Sample parameters detailing concentrations, diffusion coefficients, molecular brightnesses and fluorescence lifetimes of the prepared R6G and RhB mixtures. Recovery of RhB diffusion coefficient (c), molecular brightness (d), and fluorescence lifetime (e) respectively. Data points and error bars represent the average and standard deviation of three repeated experiments.</p

Phase Networks of Cross-β Peptide Assemblies

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimer’s disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid–liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid–solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies

Design of Asymmetric Peptide Bilayer Membranes

Energetic insights emerging from the structural characterization of peptide cross-β assemblies have enabled the design and construction of robust asymmetric bilayer peptide membranes. Two peptides differing only in their N-terminal residue, phosphotyrosine vs lysine, coassemble as stacks of antiparallel β-sheets with precisely patterned charged lattices stabilizing the bilayer leaflet interface. Either homogeneous or mixed leaflet composition is possible, and both create nanotubes with dense negative external and positive internal solvent exposed surfaces. Cross-seeding peptide solutions with a preassembled peptide nanotube seed leads to domains of different leaflet architecture within single nanotubes. Architectural control over these cross-β assemblies, both across the bilayer membrane and along the nanotube length, provides access to highly ordered asymmetric membranes for the further construction of functional mesoscale assemblies

Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity

<div><p>Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for <i>Plasmodium falciparum</i>. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary <i>Plasmodium</i> infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal <i>P</i>. <i>yoelii</i> XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of <i>P</i>. <i>yoelii</i> XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for <i>Plasmodium</i> transmission.</p></div

MHV68 and <i>Plasmodium</i> co-infection results in defective splenic T follicular helper (Tfh) response.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Representative flow plots for gating strategies used to define the global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+). (B) Absolute values for all three Tfh subsets are plotted for the <i>P</i>. <i>yoelii</i> XNL (Day 23, all Tfh subsets, <i>P</i>. <i>yoelii</i> vs. co-infected, p<0.05 Mann Whitney U-test) or (C) <i>P</i>. <i>chabaudi</i> co-infection models at multiple time points (Day 23, all Tfh subsets, <i>P</i>. <i>chabaudi</i> vs. co-infected, p<0.05 Mann Whitney U-test).</p

Acute, but not latent, MHV68 infection results in suppressed humoral response.

<p>(A) Timeline of infection. C57BL/6 mice were infected with 1000 PFU of MHV68 IN at day -60, -30, -15 or -7 and challenged with 10⁵ pRBCs on day 0. Absolute number of (B) splenic GC B cell (B220+ GL7+ CD95+) and plasma cell (CD3- B220int CD138+) populations at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (For GC and PC: Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) MHV68 and <i>P</i>. <i>yoelii</i> XNL specific IgG responses at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (D) Global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+) in the spleen at day 16 post <i>P</i>. <i>yoelii</i> XNL infection.</p

MHV68 co-infection with the non-lethal <i>P</i>. <i>yoelii</i> XNL in C57BL/6 results in lethal malarial disease and suppressed <i>Plasmodium</i> specific IgG response.

<p>(A) Timeline of infection. 6–8 week old C57BL/6 mice were infected with 1000 PFU of MHV68 on day -7 followed by infection with 10⁵pRBCs of non-lethal <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Infections consisted of 5 experimental groups: MHV68 + <i>Plasmodium</i>, <i>Plasmodium</i>, MHV68 or mock infected. Each experimental group consisted of n = 5 and was repeated twice. Animals were sacrificed at days 8, 12, 16 and 23 post <i>P</i>. <i>yoelii</i> XNL infection or day 7, 11, 15 and 23 post <i>P</i>. <i>chabaudi</i> AS infection for collection of spleen, lung and blood. (B) Survival analysis of animals co-infected with MHV68 and <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Total IgG and IgM levels in serum in (C) <i>P</i>. <i>yoelii</i> XNL (Day 23 IgG—<i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (D) <i>P</i>. <i>chabaudi</i> AS co-infection model (Day 11 IgG—<i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test). Parasite specific IgG levels in serum during (E) <i>P</i>. <i>yoelii</i> XNL (day 23 post infection, <i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (F) <i>P</i>. <i>chabaudi</i> AS (day 11 post infection, <i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test) co-infection.</p

MHV68 suppresses splenic B cell responses during co-infection with <i>Plasmodium</i>.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Absolute numbers of splenic GC B cell populations (B220+ GL7+ CD95+) during <i>P</i>. <i>yoelii</i> XNL and <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 15 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (B) Absolute numbers of splenic plasma cell populations (CD3- B220int CD138+) during <i>P</i>. <i>yoelii</i> XNL AND <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 11 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (C) Spleen section for mock infected, MHV68 infected, <i>P</i>. <i>yoelii</i> XNL infected and MHV68 and <i>P</i>. <i>yoelii</i> XNL co-infected animals at day 8 post infection with <i>P</i>. <i>yoelii</i> XNL (or day 15 post-infection with MHV68). Green: B220-FITC (B cells), Blue: GL7-AF660 (Germinal center B cells) and Red: CD3-AF568 (T cells).</p