Colonic Mucosal Proteome Signature Reveals Reduced Energy Metabolism and Protein Synthesis but Activated Autophagy during Anorexia‐Induced Malnutrition in Mice

Anorexia nervosa is an eating disorder often associated with intestinal disorders. To explore the underlying mechanisms of these disorders, the colonic proteome was evaluated during activity‐based anorexia. Female C57Bl/6 mice were randomized into three groups: Control, Limited Food Access (LFA) and Activity‐Based Anorexia (ABA). LFA and ABA mice had a progressive limited access to food but only ABA mice had access to an activity wheel. On colonic mucosal protein extracts, a 2D PAGE‐based comparative proteomic analysis was then performed and differentially expressed proteins were identified by LC‐ESI‐MS/MS. Twenty‐seven nonredundant proteins that were differentially expressed between Control, LFA, and ABA groups were identified. ABA mice exhibited alteration of several mitochondrial proteins involved in energy metabolism such as dihydrolipoyl dehydrogenase and 3‐mercaptopyruvate sulfurtransferase. In addition, a downregulation of mammalian target of rapamycin (mTOR) pathway was observed leading, on the one hand, to the inhibition of protein synthesis, evaluated by puromycin incorporation and mediated by the increased phosphorylation of eukaryotic elongation factor 2, and on the other hand, to the activation of autophagy, assessed by the increase of the marker of autophagy, form LC3‐phosphatidylethanolamine conjugate/Cytosolic form of Microtubule‐associated protein 1A/1B light chain 3 (LC3II/LC3I) ratio. Colonic mucosal proteome is altered during ABA suggesting a downregulation of energy metabolism. A decrease of protein synthesis and an activation of autophagy were also observed mediated by mTOR pathway.     

Pluripotency of male germline stem cells

The ethical issues and public concerns regarding the use of embryonic stem (ES) cells in human therapy have motivated considerable research into the generation of pluripotent stem cell lines from non-embryonic sources. Numerous reports have shown that pluripotent cells can be generated and derived from germline stem cells (GSCs) in mouse and human testes during in vitro cultivation. The gene expression patterns of these cells are similar to those of ES cells and show the typical self-renewal and differentiation patterns of pluripotent cells in vivo and in vitro. However, the mechanisms underlying the spontaneous dedifferentiation of GSCs remain to be elucidated. Studies to identify master regulators in this reprogramming process are of critical importance for understanding the gene regulatory networks that sustain the cellular status of these cells. The results of such studies would provide a theoretical background for the practical use of these cells in regenerative medicine. Such studies would also help elucidate the molecular mechanisms underlying certain diseases, such as testicular germ cell tumors.     

Establishment of hepatic and neural differentiation platforms of Wilson’s disease specific induced pluripotent stem cells

The combination of disease-specific human induced pluripotent stem cells (iPSC) and directed cell differentiation offers an ideal platform for modeling and studying many inherited human diseases. Wilson’s disease (WD) is a monogenic disorder of toxic copper accumulation caused by pathologic mutations of the ATP7B gene. WD affects multiple organs with primary manifestations in the liver and central nervous system (CNS). In order to better investigate the cellular pathogenesis of WD and to develop novel therapies against various WD syndromes, we sought to establish a comprehensive platform to differentiate WD patient iPSC into both hepatic and neural lineages. Here we report the generation of patient iPSC bearing a Caucasian population hotspot mutation of ATP7B. Combining with directed cell differentiation strategies, we successfully differentiated WD iPSC into hepatocyte-like cells, neural stem cells and neurons. Gene expression analysis and cDNA sequencing confirmed the expression of the mutant ATP7B gene in all differentiated cells. Hence we established a platform for studying both hepatic and neural abnormalities of WD, which may provide a new tool for tissue-specific disease modeling and drug screening in the future.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

The continuity of living matter and the discontinuities of its constituents: do plankton and benthos really exist?

Plankton and benthos are popular concepts identifying two ways of life of aquatic organisms. Their spatial separation led to the development of different sampling techniques and to separate conceptualizations of the principles governing these subsets of the aquatic environment. Reciprocal connections between plankton and benthos, however, are very strong both from a functional (energy fluxes) and a structural (life cycle dynamics) point of view. A full appreciation of such links is forcing marine ecology towards a more integrated approach.     

Mesenchyme with fgf-10 expression is responsible for regenerative capacity in Xenopus limb buds

A young tadpole of an anuran amphibian can completely regenerate an amputated limb, and it exhibits an ontogenetic decline in the ability to regenerate its limbs. However, whether mesenchymal or epidermal tissue is responsible for this decrease of the capacity remains unclear. Moreover, little is known about the molecular interactions between these two tissues during regeneration. The results of this study showed that fgf-10 expression in the limb mesenchymal cells clearly corresponds to the regenerative capacity and that fgf-10 and fgf-8 are synergistically reexpressed in regenerating blastemas. However, neither fgf-10 nor fgf-8 is reexpressed after amputation of a nonregenerative limb. Nevertheless, nonregenerative epidermal tissue can reexpress fgf-8 under the influence of regenerative mesenchyme, as was demonstrated by experiments using a recombinant limb composed of regenerative limb mesenchyme and nonregenerative limb epidermis. Taken together, our data demonstrate that the regenerative capacity depends on mesenchymal tissue and suggest that fgf-10 is likely to be involved in this capacity.     

Unveiling the establishment of left-right asymmetry in the chick embryo

Vertebrates display striking left-right asymmetries in the placement of internal organs, which are concealed by a seemingly bilaterally symmetric body plan. The establishment of asymmetries about the left-right axis occurs early during embryo development and requires the concerted and sequential action of several epigenetic, genetic and cellular mechanisms. Experiments in the chick embryo model have contributed crucially to our current understanding of such mechanisms and are reviewed here. Particular emphasis is given to the elucidation of a genetic network that conveys left-right information from Hensen's node to the organ primordia, characterized to a significant degree of detail in the chick embryo. We also point out a number of early and late events in the determination of left-right asymmetries that are currently poorly understood and for whose study the chick embryo model presents several advantages. We anticipate that the availability of the chick genome sequence will be combined with multidisciplinary approaches from experimental embryology, biophysics, live-cell imaging, and mathematical modeling to boost up our knowledge of left-right organ asymmetry in the near future.