Engineering a human P2X2 receptor with altered ligand selectivity in yeast.

P2X receptors are a family of ligand gated ion channels found in a range of eukaryotic species including humans but are not naturally present in the yeast Saccharomyces cerevisiae. We demonstrate the first recombinant expression and functional gating of the P2X2 receptor in baker's yeast. We leverage the yeast host for facile genetic screens of mutant P2X2 by performing site saturation mutagenesis at residues of interest, including SNPs implicated in deafness and at residues involved in native binding. Deep mutational analysis and rounds of genetic engineering yield mutant P2X2 F303Y A304W, which has altered ligand selectivity toward the ATP analog AMP-PNP. The F303Y A304W variant shows over 100-fold increased intracellular calcium amplitudes with AMP-PNP compared to the WT receptor and has a much lower desensitization rate. Since AMP-PNP does not naturally activate P2X receptors, the F303Y A304W P2X2 may be a starting point for downstream applications in chemogenetic cellular control. Interestingly, the A304W mutation selectively destabilizes the desensitized state, which may provide a mechanistic basis for receptor opening with suboptimal agonists. The yeast system represents an inexpensive, scalable platform for ion channel characterization and engineering by circumventing the more expensive and time-consuming methodologies involving mammalian hosts.

The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells.

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Assessing the effect of cleansing products on artificially polluted human hairs and skin through in vivo and in vitro models.

OBJECTIVE: Based on in vivo data, in vitro models and new methods are created to mimic the impact of aerial pollution onto the hair surface and assess the efficacy of different formulae prototypes. MATERIAL AND METHODS: Two protocols are developed to mimic the pollution effect, in vitro, on purchased swatches, and in vivo, on scalps and forearms. First, with an artificial sebum mixed with Carbon Black particles, named "sebollution," we evaluated, through an instrumental color measurement, the cleansing efficacy of some shampoo on scalp and hair. The second protocol allowed to assess the interaction between hair care product deposit (shampoo, conditioner, mask, and leave-on) on hair and carbon black particles spread on fiber. The quantification of particle coverage allowed to evaluate the efficiency of a formula to limit the aerial pollution deposit on hair fiber. RESULTS: To simplify and accelerate the evaluation of 42 shampoo formulae, an extrapolation of the scalp cleaning process was validated on forearm. The respective cleanabilities were calculated and covered a large range of efficacy, from 5%, for a basic bland shampoo generally used to reset swatches, to a strong deep cleansing efficacy of 100%. On hair swatches, cleanability efficiencies of five shampoo were also evaluated to eliminate the deposited of sebollution, in a range of 40%-80%. To quantify the efficacy of preventing the deposition of carbon particle on hair surface, the percentage of coverage of 45 different products was measured, from 2% to 16%. The performance depended of the product category (shampoo, conditioner, mask, and leave-on), driven by the performance of the product deposit, and the capacity of this deposit to interact with aerial pollution. CONCLUSION: Three new protocols and evaluation methods are proposed to evaluate and quantify the performance of hair care product, to remove/clean, limit, and protect the hair fibers against the aerial pollution that could interact with hair, scalp and sebum. The validation of these approaches was done through the testing of a large panel of hair care product leading to a complete and sincere evaluation of cleansing and anti-deposit efficacy. Combining the knowledge acquired on pollution impact on hair and the development of specific way of evaluation, this work reinforced the rationale of using and developing new cosmetic products that reduced the impact of pollution upon some hair properties.