Identification of myopia-associated WNT7B polymorphisms provides insights into the mechanism underlying the development of myopia

10.1038/ncomms7689Nature Communications

Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius.

In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections

Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius.

In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogen Staphylococcus pseudintermedius for their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsL mutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants. Lactococcus lactis transformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1 markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520-846 and SpsL538-823 regions harboring the major fibronectin-binding sites inhibited S. pseudintermedius internalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections

Semi-loose seal Neurobiotin electroporation for combined structural and functional analysis of neurons

Intracellular sharp-electrode, whole-cell patch clamp and juxtacellular labeling methods have previously been developed for combined analysis of neuronal structure and function. We describe a novel electroporation technique for labeling neurons with Neurobiotin, using patch electrodes in a semi-loose seal configuration (R = 100-300 MOmega) with very small amplitude pulses (50 mV). The addition of 2% Neurobiotin to the intracellular solution in the patch electrode reduces the dielectric membrane breakdown voltage threshold by about threefold. The resulting pore formation allows for (1) the stable recording of spontaneous and light-evoked postsynaptic potentials without significant cytoplasmic washout and (2) the passage of dye without spillover. The efficiency and reliability of the method makes it particularly suitable for the serial recording and labeling of multiple neurons in a small area of tissue