Mesodermal fate decisions of a stem cell: the Wnt switch

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis

The calcium signal in human neutrophils and its relation to exocytosis investigated by patch-clamp capacitance and Fura-2 measurements.

Intracellular calcium ([Ca2+]i) and exocytosis of human neutrophils were investigated with patch-clamp capacitance and Fura-2 fluorescence measurements. Intracellular application of GTP gamma S induces a calcium transient and exocytosis. The onset of degranulation occurs at the time where the maximal [Ca2+]i is reached. Despite the close correlation in time, buffering [Ca2+]i at the resting level or at approximately 2 microM leaves the extent and the time course of degranulation unchanged. The decay of the calcium transient is due to diffusional equilibration between the cytosol and the pipette volume. GTP gamma S activates no cellular mechanisms for Ca2+ reuptake or extrusion. The endogenous calcium buffer capacity can be estimated to be as low as that of approximately 90 microM Fura-2. Stimulation with fMLP also induces degranulation and a calcium transient. The decay of fMLP-induced calcium transients is much faster than that of GTP gamma S-induced transients and is independent of diffusion indicating that fMLP also induces rapid reuptake or extrusion of Ca2+. Degranulation but not the calcium transient requires the presence of intracellular GTP. Different signalling pathways appear to be involved in GTP gamma S- and fMLP-stimulated calcium signals. The intracellular calcium release is not an essential signal to initiate exocytosis in neutrophils

GTPγS-induced calcium transients and exocytosis in human neutrophils.

Exocytosis and intracellular free calcium ([Ca2+]in) were simultaneously recorded in single human neutrophils using patch-clamp capacitance measurements and the fura-2 fluorescence ratio method. Intracellular application of guanosine-5′-O(3-thiotriphosphate) (GTPγS) stimulates both exocytosis and a calcium transient. The calcium transient starts to develop after a lag phase of ∼40s and normally appears to trigger the onset of exocytosis indicated by the beginning of the capacitance increase. After this delay [Ca2+]in increases from ∼150 nM to ∼600 nM with a sigmoidal time course. The peak concentration is reached within ∼30 s but the main increase occurs during ∼ 3s. [Ca2+]in subsequently decays within 1–2 min to a level which is close to the resting value. This calcium transient is due to calcium release from inositoltrisphosphate-sensitive intracellular stores. Exocytosis also occurs if the calcium transient is abolished by intracellular EGTA but the lag phase is markedly prolonged. The GTPγS-induced calcium transient is very similar to that observed after stimulation with N-formyl-methionyl-leucyl-phenylalanine. The interplay between guanine nucleotides, [Ca2+]in and exocytosis in neutrophils closely resembles previous results obtained in mast cells suggesting a similar regulation of exocytosis in both cell types

Herstellung und Charakterisierung von photonischen Kristall Kavitäten im Sichtbaren Fabrication and Characterization of Photonic Crystal Cavities in the Visible

In diesem Beitrag berichten wir über die Herstellung und optische Charakterisierung von photonischen Kristall Kavitäten für sichtbare Wellenlängen auf der Basis von Siliziumnitrid. Es finden signifikante Verbesserungen im Hers tellungsprozess im Vergleich zu unseren bisherigen Studien Erwähnung. Die intrinsische Lumineszenz der Nitridmembranen wurde als interne Lichtquelle verwendet, um den Qualitätsfaktor der Kavitätsmoden zu untersuchen. Wir beobach teten Werte um 3400, die bis vor kurzem unübertroffen waren. Numerische Simulationen deuten an, dass eine Verbesserung des Q Faktors um den Faktor zwei durch weitere Optimierung des Herstellungsprozesses möglich ist. Wir be schreiben eine Methode zur deterministischen Ankopplung von Emittern oder anderen nanoskaligen Objekten auf der Basis von Rasterkraftmikroskopi

Fabrication and characterization of photonic crystal cavities in the visible

We report on the fabrication and optical characterization of photonic crystal cavities for visible wavelengths made from silicon nitride SiN . We note significant improvements in fabrication process with respect to our previous studies. The intrinsic luminescence of the SiN membranes was used as an internal light source to study the quality factor of the cavity modes. We experimentally found values as high as 3400, which are up to the present unsurpassed for photonic crystal resonators in the visible spectra range. Finite difference time domain FDTD simulations suggest another boost by a factor of two is possible by further optimizing the fabrication process. We describe a method by which arbitrary emitters or other nanoscopic objects can be coupled in a deterministic way by using the manipulation capabilities of an atomic force microscope

Intracellular application of guanosine-5'-O-(3-thiotriphosphate) induces exocytotic granule fusion in guinea pig eosinophils.

The mechanism of eosinophil secretion was studied in guinea pig eosinophils by measuring release of hexosaminidase from cell suspensions (greater than 98% pure) permeabilized with streptolysin-O and by whole-cell patch-clamp capacitance measurements. It is shown that release of eosinophil granule components occurs by an exocytotic mechanism in which individual granules fuse with the plasma membrane. Exocytosis can be induced by intracellular application of the nonhydrolyzable GTP analog guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S), suggesting the involvement of a GTP-binding protein. The activation is modulated by the intracellular calcium concentration, with activation by GTP-gamma-S inducing transient elevations in the concentration of Ca2+. Thus, the nature and regulation of the release mechanism appear to be very similar to that of the mast cell and neutrophil

Controlled coupling of nanoparticles to photonic crystal cavities

We demonstrate a hybrid approach for the realization of novel nanophotonic devices by combining lithographic fabrication techniques with a nano manipulation method. In particular, we report on the fabrication of photonic crystal cavities as a platform to which arbitrary emitters or other nanoscopic objects can be coupled in a deterministic way by exploiting the manipulation capabilities of an atomic force microscope. In addition, the optical properties of such particle cavity systems are analyzed with regard to changes of the quality factor and resonance wavelength of the cavity mode. Our approach is well suited to create improved single photon sources and also complex photonic devices with several emitters coupled coherently via shared cavity mode

Ca2+-induced exocytosis in individual human neutrophils: high- and low-affinity granule populations and submaximal responses.

We have investigated Ca2+-induced exocytosis from human neutrophils using the whole cell patch-clamp capacitance technique. Microperfusion of Ca2+ buffer solutions (<30 nM to 5 mM free Ca2+) through the patch-clamp pipette revealed a biphasic activation of exocytosis by Ca2+. The first phase was characterized by high affinity (1.5-5 microM) and low apparent cooperativity (<=2) for Ca2+, and the second phase by low affinity (approximately 100 microM) and high cooperativity (>6). Only the second phase was accompanied by loss of myeloperoxidase, suggesting that the low-affinity exocytosis reflected release of peroxidase-positive (primary) granules, while the high-affinity exocytosis reflected release of peroxidase-negative (secondary and tertiary) granules. At submaximal Ca2+ concentrations, only a fraction of a given granule population was released. This submaximal release cannot simply be explained by Ca2+ modulation of the rate of exocytosis, and it suggests that the secretory response of individual cells is adjusted to the strength of the stimulus. The Ca2+ dependence of the high- and low-affinity phases of neutrophil exocytosis bears a resemblance to endocrine and neuronal exocytosis, respectively. The occurrence of such high- and low-affinity exocytosis in the same cell is novel, and suggests that the Ca2+ sensitivity of secretion is granule-, rather than cell-specific