A predictive self-organizing multicellular computational model of infant skin permeability to topically applied substances

Computational models of skin permeability are typically based on assumptions of fixed geometry and homogeneity of the whole epidermis or of epidermal strata and are often limited to adult skin. Infant skin differs quantitatively from adult in its structure and its functional properties, including its barrier function to permeation. To address this problem, we developed a self-organizing multicellular epidermis model of barrier formation with realistic cell morphology. By modulating parameters relating to cell turnover reflecting those in adult or infant epidermis, we were able to generate accordingly two distinct models. Emerging properties of these models reflect the corresponding experimentally measured values of epidermal and Stratum Corneum thickness. Diffusion of an externally applied substance (e.g. caffeine) was simulated by molecular exchange between the model “agents”, defined by the individual cells and their surrounding extracellular space. By adjusting the surface concentration and the intercellular exchange rate, the model can recapitulate experimental permeability data, following topical exposure. By applying these parameters to an infant model, we were able to predict the caffeine concentration profile in infant skin, closely matching experimental results. This work paves the way for better understanding of skin physiology and function during the first years of life

ORAI1 calcium channel orchestrates skin homeostasis

To achieve and maintain skin architecture and homeostasis, keratinocytes must intricately balance growth, differentiation, and polarized motility known to be governed by calcium. Orai1 is a pore subunit of a store-operated Ca(2+) channel that is a major molecular counterpart for Ca(2+) influx in nonexcitable cells. To elucidate the physiological significance of Orai1 in skin, we studied its functions in epidermis of mice, with targeted disruption of the orai1 gene, human skin sections, and primary keratinocytes. We demonstrate that Orai1 protein is mainly confined to the basal layer of epidermis where it plays a critical role to control keratinocyte proliferation and polarized motility. Orai1 loss of function alters keratinocyte differentiation both in vitro and in vivo. Exploring underlying mechanisms, we show that the activation of Orai1-mediated calcium entry leads to enhancing focal adhesion turnover via a PKCβ-Calpain-focal adhesion kinase pathway. Our findings provide insight into the functions of the Orai1 channel in the maintenance of skin homeostasis

Remodelling of Cortical Actin Where Lytic Granules Dock at Natural Killer Cell Immune Synapses Revealed by Super-Resolution Microscopy

Super-resolution 3D imaging reveals remodeling of the cortical actin meshwork at the natural killer cell immune synapse, which is likely to be important for secretion of lytic granules

A minimally invasive optical trapping system to understand cellular interactions at onset of an immune response

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T- cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated

Studies of contacts between T7 RNA polymerase and its promoter reveal features in common with multisubunit RNA polymerases.

International audienceWe have used UV-laser mediated cross-linking, DNase I footprinting and KMnO4 reactivity to probe the interaction between T7 RNA polymerase (RNAP) and a consensus promoter during the early stages of transcription. In a binary complex formed in the absence of substrate on a supercoiled plasmid, direct contacts were observed on the template (T) strand at positions -17, -5, and +3 and on the nontemplate (NT) strand at position -8. These contacts lie within the DNase I cleavage footprint from positions -21 to +11 on the T strand and from positions -17 to +16 on the NT strand and straddle sites of enhanced reactivity of thymines to KMnO4 at position -3 on the T strand and position -2 on the NT strand. Use of supercoiled plasmid templates has allowed the mapping of contacts in the initiation region of the promoter in the binary complex for the first time. Upon addition of GTP, T7 RNAP enters a reiterative mode of synthesis, producing a ladder of poly(G) products. Under these conditions the downstream contact on the T strand switched from position +3 to +4 and +5 while the contact at position -17 was maintained. Under conditions in which the synthesis of transcription products is limited to 6-7 nucleotides, only the contact at position -17 on the T strand was preserved. A comparison of these results with the interaction of Escherichia coli RNA polymerase at the lac promoter reveals strong similarities in the manner in which these polymerases recognize their promoters

Human mRNA polyadenylate binding protein: evolutionary conservation of a nucleic acid binding motif.

We have isolated a full length cDNA (cDNA) coding for the human poly(A) binding protein. The cDNA derived 73 kd basic translation product has the same Mr, isoelectric point and peptidic map as the poly(A) binding protein. DNA sequence analysis reveals a 70,244 dalton protein. The N terminal part, highly homologous to the yeast poly(A) binding protein, is sufficient for poly(A) binding activity. This domain consists of a four-fold repeated unit of approximately 80 amino acids present in other nucleic acid binding proteins. In the C terminal part there is, as in the yeast protein, a sequence of approximately 150 amino acids, rich in proline, alanine and glutamine which together account for 48% of the residues. A 2,9 kb mRNA corresponding to this cDNA has been detected in several vertebrate cell types and in Drosophila melanogaster at every developmental stage including oogenesis