Effects of Simulated Microgravity on Embryonic Stem Cells

There have been many studies on the biological effects of simulated microgravity (SMG) on differentiated cells or adult stem cells. However, there has been no systematic study on the effects of SMG on embryonic stem (ES) cells. In this study, we investigated various effects (including cell proliferation, cell cycle distribution, cell differentiation, cell adhesion, apoptosis, genomic integrity and DNA damage repair) of SMG on mouse embryonic stem (mES) cells. Mouse ES cells cultured under SMG condition had a significantly reduced total cell number compared with cells cultured under 1 g gravity (1G) condition. However, there was no significant difference in cell cycle distribution between SMG and 1G culture conditions, indicating that cell proliferation was not impaired significantly by SMG and was not a major factor contributing to the total cell number reduction. In contrast, a lower adhesion rate cultured under SMG condition contributed to the lower cell number in SMG. Our results also revealed that SMG alone could not induce DNA damage in mES cells while it could affect the repair of radiation-induced DNA lesions of mES cells. Taken together, mES cells were sensitive to SMG and the major alterations in cellular events were cell number expansion, adhesion rate decrease, increased apoptosis and delayed DNA repair progression, which are distinct from the responses of other types of cells to SMG

Non-Overlapping Functions for Pyk2 and FAK in Osteoblasts during Fluid Shear Stress-Induced Mechanotransduction

Mechanotransduction, the process by which cells convert external mechanical stimuli such as fluid shear stress (FSS) into biochemical changes, plays a critical role in maintenance of the skeleton. We have proposed that mechanical stimulation by FSS across the surfaces of bone cells results in formation of unique signaling complexes called mechanosomes that are launched from sites of adhesion with the extracellular matrix and with other bone cells [1]. Deformation of adhesion complexes at the cell membrane ultimately results in alteration of target gene expression. Recently, we reported that focal adhesion kinase (FAK) functions as a part of a mechanosome complex that is required for FSS-induced mechanotransduction in bone cells. This study extends this work to examine the role of a second member of the FAK family of non-receptor protein tyrosine kinases, proline-rich tyrosine kinase 2 (Pyk2), and determine its role during osteoblast mechanotransduction. We use osteoblasts harvested from mice as our model system in this study and compared the contributions of Pyk2 and FAK during FSS induced mechanotransduction in osteoblasts. We exposed Pyk2+/+ and Pyk2−/− primary calvarial osteoblasts to short period of oscillatory fluid flow and analyzed downstream activation of ERK1/2, and expression of c-fos, cyclooxygenase-2 and osteopontin. Unlike FAK, Pyk2 was not required for fluid flow-induced mechanotransduction as there was no significant difference in the response of Pyk2+/+ and Pyk2−/− osteoblasts to short periods of fluid flow (FF). In contrast, and as predicted, FAK−/− osteoblasts were unable to respond to FF. These data indicate that FAK and Pyk2 have distinct, non-redundant functions in launching mechanical signals during osteoblast mechanotransduction. Additionally, we compared two methods of generating FF in both cell types, oscillatory pump method and another orbital platform method. We determined that both methods of generating FF induced similar responses in both primary calvarial osteoblasts and immortalized calvarial osteoblasts

Paxillin and Hic-5 Interaction with Vinculin Is Differentially Regulated by Rac1 and RhoA

Cell migration is of paramount importance to organism development and maintenance as well as multiple pathological processes, including cancer metastasis. The RhoGTPases Rac1 and RhoA are indispensable for cell migration as they regulate cell protrusion, cell-extracellular matrix (ECM) interactions and force transduction. However, the consequences of their activity at a molecular level within the cell remain undetermined. Using a combination of FRET, FRAP and biochemical analyses we show that the interactions between the focal adhesion proteins vinculin and paxillin, as well as the closely related family member Hic-5 are spatially and reciprocally regulated by the activity of Rac1 and RhoA. Vinculin in its active conformation interacts with either paxillin or Hic-5 in adhesions in response to Rac1 and RhoA activation respectively, while inactive vinculin interacts with paxillin in the membrane following Rac1 inhibition. Additionally, Rac1 specifically regulates the dynamics of paxillin as well as its binding partner and F-actin interacting protein actopaxin (α-parvin) in adhesions. Furthermore, FRET analysis of protein:protein interactions within cell adhesions formed in 3D matrices revealed that, in contrast to 2D systems vinculin interacts preferentially with Hic-5. This study provides new insight into the complexity of cell-ECM adhesions in both 2D and 3D matrices by providing the first description of RhoGTPase-coordinated protein:protein interactions in a cellular microenvironment. These data identify discrete roles for paxillin and Hic-5 in Rac1 and RhoA-dependent cell adhesion formation and maturation; processes essential for productive cell migration

Facteurs prédictifs d'amélioration clinique après traitement rTMS chez des patients atteints de dépression pharmaco-résistante

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Deletion of osteopontin or bone sialoprotein induces opposite bone responses to mechanical stimulation in mice

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Comment les lasers ultra-courts aident à créer des reliefs hiérarchiques guidant efficacement les cellules sur des motifs nanostructurés

International audienceUltra-short laser texturing allows us to produce various surface reliefs on both nano- and micro-scales as complex hierarchical patterns. This kind of surface treatment has also advantaged in the reduction of thermal effects, of debris, and, particularly, in the minimization of surface contamination. Particularly, surface micro-patterns with additional nano-reliefs, or so-called “hierarchical reliefs” can be quite easily produced considerably alternating not only surface roughness and wettability but allowing additional opportunities to better guide the behavior of different objects ranging from nanoparticles and viruses to larger droplets and/or living microorganisms. Previously, a clear connection between cell behavior and wetting properties on laser-structured patterns has been revealed. However, the optimum laser treatment is still under discussion. In fact, laser interactions affect not only surface relief and morphology but also composition, phase state, etc. One of the known challenges is also the fact that surface properties can be unstable and evolve with time. They can be also strongly affected by additional heating, sterilization, ultra-sound or cold plasma treatment. The reasons for these changes are not yet well understood. For this, femtosecond laser irradiation of titanium-based surfaces is used. As a result, multi-scale textures are produced with high precision. Additionally, computer simulations were also performed to examine surface chemistry and particle and small droplet behavior on such surfaces. Then, wetting properties are analyzed. Finally, the capacities to capture and guide human stem cell cultures (HSC) were evaluated. Several patterns with different sizes and motifs have been examined. In general, the results confirm that wettability maps can help predict cellular behavior. The obtained results have numerous applications in bioengineering, cellular tests, the treatment of dental implants, and various prosthesis.La texturation laser ultra-courte nous permet de produire divers reliefs de surface à la fois à l'échelle nano et micro sous forme de motifs hiérarchiques complexes. Ce type de traitement de surface présente également des avantages dans la réduction des effets thermiques, des débris et, en particulier, dans la minimisation de la contamination de surface. En particulier, des micro-motifs de surface avec des nano-reliefs supplémentaires, ou soi-disant «reliefs hiérarchiques», peuvent être assez facilement produits en alternant considérablement non seulement la rugosité de surface et la mouillabilité, mais permettant des opportunités supplémentaires pour mieux guider le comportement de différents objets allant des nanoparticules aux virus. à des gouttelettes plus grosses et/ou à des micro-organismes vivants. Auparavant, un lien clair entre le comportement des cellules et les propriétés de mouillage sur les motifs structurés au laser a été révélé. Cependant, le traitement au laser optimal est encore en discussion. En fait, les interactions laser affectent non seulement le relief et la morphologie de la surface, mais aussi sa composition, son état de phase, etc. L'un des défis connus est également le fait que les propriétés de surface peuvent être instables et évoluer avec le temps. Ils peuvent également être fortement affectés par un chauffage supplémentaire, une stérilisation, un traitement aux ultrasons ou au plasma froid. Les raisons de ces changements ne sont pas encore bien comprises. Pour cela, une irradiation laser femtoseconde de surfaces à base de titane est utilisée. En conséquence, des textures multi-échelles sont produites avec une grande précision. De plus, des simulations informatiques ont également été effectuées pour examiner la chimie de surface et le comportement des particules et des petites gouttelettes sur ces surfaces. Ensuite, les propriétés de mouillage sont analysées. Enfin, les capacités de capture et de guidage des cultures de cellules souches humaines (CSH) ont été évaluées. Plusieurs modèles avec des tailles et des motifs différents ont été examinés. En général, les résultats confirment que les cartes de mouillabilité peuvent aider à prédire le comportement cellulaire. Les résultats obtenus ont de nombreuses applications dans la bio-ingénierie, les tests cellulaires, le traitement des implants dentaires et diverses prothèses