T cells from the brain of <i>Il21</i>^−/− mice produce less IFN-γ during chronic toxoplasmic encephalitis.

<p>A) Flow cytometry of BMNCs from WT and <i>Il21</i>^−/− mice infected for 35 days; cells were stimulated <i>ex vivo</i> with PMA and ionomycin in the presence of brefeldin A for 4 h and then stained intracellularly for IFN-γ. Numbers in boxed areas represent percent IFN-γ⁺ CD4⁺ (left) or CD8⁺ (right) T cells, while bold numbers indicate MFI. B) Total IFN-γ⁺CD4⁺ or CD8⁺ T cells isolated from BMNC preparations of WT (black bars) and <i>Il21</i>^−/− (white bars) mice infected for 35 days, calculated from the percentages determined by flow cytometry. Data are representative of four independent experiments with similar results. Error bars represent SEM. Significance was determined by a two-tailed unpaired Student’s <i>t</i> test. **<i>P</i><0.01.</p

Relationship Between Molecular Contact Thermodynamics and Surface Contact Mechanics

Measurements have been made of the adhesion and friction forces between organic monolayers in heptane/acetone mixtures using an atomic force microscope (AFM). It has been found that the contact mechanics are best modeled by treating the friction force as the sum of a load-dependent term (attributed to “molecular plowing”) and an area-dependent term attributed to shearing (adhesion). The relative contributions of plowing and shearing are determined by the coefficient of friction, μ, and the surface shear strength τ. The transition from adhesion- to load-determined friction is controlled by the solvation state of the surface: solvated surfaces represent a limiting case in which the shear term approaches zero, and the friction-load relationship is linear, while in other circumstances, the friction-load relationship is nonlinear and consistent with Derjaguin–Muller–Toporov mechanics. A striking correlation has been observed between the concentration-dependence of the association constant (<i>K</i>_a) for the formation of 1:1 hydrogen-bonded complexes and the pull-off force <i>F</i>_a and surface shear strength τ for the same molecules when one partner is immobilized by attachment to an AFM probe and the other is adsorbed to a surface. Analysis of the concentration-dependence of <i>F</i>_a and τ enables the prediction of <i>K</i>_S with remarkably high precision, indicating that for these hydrogen bonding systems, the tip–sample adhesion is dominated by the H-bond thermodynamics. For mixed monolayers, H-bond thermodynamics dominate the interaction even at very low concentrations of the H-bond acceptor. Even for weakly adhering systems, a nonlinear friction-load relationship results. The variation in τ with the film composition is correlated very closely with the variation in <i>F</i>_a. However, the coefficient of friction varies little with the film composition and is invariant with the strength of tip–sample adhesion, being dominated by molecular plowing and, for sufficiently large concentrations of hydroxyl terminated adsorbates, the disruption of intramonolayer hydrogen bonding interactions

<i>Il21</i>^−/− mice have reduced inflammatory cell numbers in the brain during chronic <i>T. gondii</i> infection.

<p>A) The percentage of CD4⁺ (left) and CD8⁺ (right) T cells expressing the cytokine IL-10 in the brain of individual WT and <i>Il21</i>^−/− mice infected for 35 days. Data are representative of two or three independent experiments with three to four mice per group. B) Enumeration of total cell numbers recovered from the brain of WT and <i>Il21</i>^−/− mice infected for 35 days. Data are representative of three experiments with similar results. C) Total CD4⁺ T cells, CD8⁺ T cells, macrophages and microglia in each BMNC preparation from (B), calculated by the percentages determined by flow cytometry. D) Total IL-10⁺ CD4⁺ and CD8⁺ T cells in each BMNC preparation from (A), calculated by the percentages determined by flow cytometry. E) Flow cytometry of CD4⁺ T cells isolated from WT and <i>Il21</i>^−/− mice, and activated with anti-CD3 and anti-CD28 in cRPMI (left) or IMDM (right) media in the presence of IL-27 for 4 days. Cells were then stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A before staining for intracellular IL-10. Numbers in boxed areas indicate percent IL-10⁺CD4⁺ T cells. Results are representative of three independent experiments with similar results. Significance was determined by a two-tailed unpaired Student’s <i>t</i> test. Error bars represent SEM. *<i>P</i><0.05, **<i>P</i><0.01.</p

<i>Il21</i>^−/− mice have an impaired antibody-mediated immune response during chronic toxoplasmic encephalitis.

<p>A) ELISA of IgM, IgG and IgG2c STAg-specific Ab titers in the serum of WT and <i>Il21</i>^−/− mice infected for 56 days. B, C) Flow cytometry of splenocytes isolated from day 35 infected WT or <i>Il21</i>^−/− mice and stained for (B) germinal center (GC) B cells (CD19⁺B220⁺PNA⁺) or (C) T_FH cells (CD4⁺ICOS⁺CXCR5⁺). Numbers in the box represent the percent of (B) PNA⁺CD19⁺ B cells or (C) CD4⁺ICOS⁺CXCR5⁺ T_FH cells. Quantitation of (B) GC B cells, or (C) T_FH cells on day 35 and 56 p.i. from the spleen of WT and <i>Il21</i>^−/− mice. Error bars represent SEM. Data are representative of two (A) or three (B, C) independent experiments with similar results.</p

Relationship between Chemical Structure and Supramolecular Effective Molarity for Formation of Intramolecular H‑Bonds

Effective molarity (EM) is a key parameter that determines the efficiency of a range of supramolecular phenomena from the folding of macromolecules to multivalent ligand binding. Coordination complexes formed between zinc porphyrins equipped H-bond donor sites and pyridine ligands equipped with H-bond acceptor sites have allowed systematic quantification of EM values for the formation of intramolecular H-bonds in 240 different systems. The results provide insights into the relationship of EM to supramolecular architecture, H-bond strength, and solvent. Previous studies on ligands equipped with phosphonate diester and ether H-bond acceptors were inconclusive, but the experiments described here on ligands equipped with phosphine oxide, amide, and ester H-bond acceptors resolve these ambiguities. Chemical double-mutant cycles were used to dissect the thermodynamic contributions of individual H-bond interactions to the overall stabilities of the complexes and hence determine the values of EM, which fall in the range 1–1000 mM. Solvent has little effect on EM, and the values measured in toluene and 1,1,2,2-tetrachloroethane are similar. For H-bond acceptors that have similar geometries but different H-bond strengths (amide and ester), the values of EM are very similar. For H-bond acceptors that have different geometries but similar H-bond strengths (amide and phosphonate diester), there is little correlation between the values of EM. These results imply that supramolecular EMs are independent of solvent and intrinsic H-bond strength but depend on supramolecular architecture and geometric complementarity

Metal Hydrides Form Halogen Bonds: Measurement of Energetics of Binding

The formation of halogen bonds from iodopentafluorobenzene and 1-iodoperfluorohexane to a series of bis­(η⁵-cyclopentadienyl)­metal hydrides (Cp₂TaH₃, <b>1</b>; Cp₂MH₂, M = Mo, <b>2</b>, M = W, <b>3</b>; Cp₂ReH, <b>4</b>; Cp₂Ta­(H)­CO, <b>5</b>; Cp = η⁵-cyclopentadienyl) is demonstrated by ¹H NMR spectroscopy. Interaction enthalpies and entropies for complex <b>1</b> with C₆F₅I and C₆F₁₃I are reported (Δ<i>H</i>° = −10.9 ± 0.4 and −11.8 ± 0.3 kJ/mol; Δ<i>S</i>° = −38 ± 2 and −34 ± 2 J/(mol·K), respectively) and found to be stronger than those for <b>1</b> with the hydrogen-bond donor indole (Δ<i>H</i>° = −7.3 ± 0.1 kJ/mol, Δ<i>S</i>° = −24 ± 1 J/(mol·K)). For the more reactive complexes <b>2</b>–<b>5</b>, measurements are limited to determination of their low-temperature (212 K) association constants with C₆F₅I as 2.9 ± 0.2, 2.5 ± 0.1, <1.5, and 12.5 ± 0.3 M^–1, respectively

Validation of a Computational Cocrystal Prediction Tool: Comparison of Virtual and Experimental Cocrystal Screening Results

A virtual cocrystal screening method based on calculated gas phase molecular electrostatic potential surfaces (MEPS) of the individual components has been validated using experimental cocrystal screens reported in the literature. The noncovalent interactions of a molecule with its environment are described by a discrete set of independent surface site interaction points (SSIPs), whose properties can be calculated from the <i>ab initio</i> MEPS. The stability of a crystal is estimated based on pairing SSIPs such that the sum of the pairwise interaction energies is optimized. This provides a means of calculating the relative stability of a cocrystal compared with the pure components without knowing anything about the three-dimensional structures of the crystalline states. For a set of potential crystal coformers (CCF), the difference between interaction site pairing energies of different solid forms (Δ<i>E</i>) provides a method for ranking CCFs based on the calculated probability of cocrystal formation. The method was applied to cocrystal screens of 18 compounds that reported both hits and misses, and in most cases, the virtual cocrystal screen reproduces experimental results well. In lists of CCFs ranked by Δ<i>E</i>, the experimentally observed hits were significantly enriched at the top, and this indicates that virtual screening is a promising tool for focusing experimental efforts on the most promising CCF candidates

Disruption of <i>TgPHIL1</i> Alters Specific Parameters of <i>Toxoplasma gondii</i> Motility Measured in a Quantitative, Three-Dimensional Live Motility Assay

<div><p><i>T. gondii</i> uses substrate-dependent gliding motility to invade cells of its hosts, egress from these cells at the end of its lytic cycle and disseminate through the host organism during infection. The ability of the parasite to move is therefore critical for its virulence. <i>T. gondii</i> engages in three distinct types of gliding motility on coated two-dimensional surfaces: twirling, circular gliding and helical gliding. We show here that motility in a three-dimensional Matrigel-based environment is strikingly different, in that all parasites move in irregular corkscrew-like trajectories. Methods developed for quantitative analysis of motility parameters along the smoothed trajectories demonstrate a complex but periodic pattern of motility with mean and maximum velocities of 0.58±0.07 µm/s and 2.01±0.17 µm/s, respectively. To test how a change in the parasite's crescent shape might affect trajectory parameters, we compared the motility of Δ<i>phil1</i> parasites, which are shorter and wider than wild type, to the corresponding parental and complemented lines. Although comparable percentages of parasites were moving for all three lines, the Δ<i>phil1</i> mutant exhibited significantly decreased trajectory lengths and mean and maximum velocities compared to the parental parasite line. These effects were either partially or fully restored upon complementation of the Δ<i>phil1</i> mutant. These results show that alterations in morphology may have a significant impact on <i>T. gondii</i> motility in an extracellular matrix-like environment, provide a possible explanation for the decreased fitness of Δ<i>phil1</i> parasites <i>in vivo</i>, and demonstrate the utility of the quantitative three-dimensional assay for studying parasite motility.</p></div

3D motility of the Δ<i>phil1</i> parasites.

<p>(<b>A</b>) MIPs for wild-type (RH), <i>TgPHIL1</i> knockout (Δ<i>phil1</i>) and complemented (Comp) parasites. Scale bar = 50 µm. The colour scheme for all MIPs was inverted for better visualization of parasite trajectories. The percentage of total parasites moving (<b>B</b>) was comparable for the three parasite lines, but the cumulative frequency distribution (<b>C</b>) and histogram (<b>D</b>) of the smoothed trajectory lengths for RH (black), Δ<i>phil1</i> (red) and Comp parasites (grey) reveal that the Δ<i>phil1</i> parasites do not move as far as the RH or Comp parasites within the same timeframe (Kolmogorov-Smirnov test, D = 0.199, p<0.0001 and D = 0.114, p<0.0001, respectively). The Δ<i>phil1</i> parasites also exhibited significantly decreased mean velocity compared to the RH parasites (<b>E</b>) and significantly reduced maximum velocity compared to both RH and Comp parasites (<b>F</b>) (paired t-test, significance indicated by asterisks). Closed data points are the results from five independent experiments comparing RH and Δ<i>phil1</i> parasites; open data points are the results from four independent experiments comparing Δ<i>phil1</i> and Comp parasites. Each of the independent experiments (assigned a different colour in the scatter plot) was performed in either triplicate or quadruplicate. The total number of parasites analyzed was 6,467 for RH, 9,305 for Δ<i>phil1</i> and 3,743 for Comp. * p<0.05, ** p<0.001, ns = not significant.</p