Exercise running and tetracycline as means to enhance skeletal muscle stem cell performance after external fixation

Prolonged limb immobilization, which is often the outcome of injury and illness, results in the atrophy of skeletal muscles. The basis of muscle atrophy needs to be better understood in order to allow development of effective countermeasures. The present study focused on determining whether skeletal muscle stem cells, satellite cells, are directly affected by long-term immobilization as well as on investigating the potential of pharmacological and physiological avenues to counterbalance atrophy-induced muscle deterioration. We used external fixation (EF), as a clinically relevant model, to gain insights into the relationships between muscle degenerative and regenerative conditions to the myogenic properties and abundance of bona fide satellite cells. Rats were treated with tetracycline (Tet) through the EF period, or exercise trained on a treadmill for 2 weeks after the cessation of the atrophic stimulus. EF induced muscle mass loss; declined expression of the muscle specific regulatory factors (MRFs) Myf5, MyoD, myogenin, and also of satellite cell numbers and myogenic differentiation aptitude. Tet enhanced the expression of MRFs, but did not prevent the decline of the satellite cell pool. After exercise running, however, muscle mass, satellite cell numbers (enumerated through the entire length of myofibers), and myogenic differentiation aptitude (determined by the lineal identity of clonal cultures of satellite cells) were re-gained to levels prior to EF. Together, our results point to Tet and exercise running as promising and relevant approaches for enhancing muscle recovery after atrophy.     

A new genus of soft coral of the family Alcyoniidae (Cnidaria, Octocorallia) with re-description of a new combination and description of a new species

A new genus, Aldersladum (family Alcyoniidae), is established to accommodate a previously described species, Efflatounaria sodwanae Benayahu, 1993 (family Xeniidae) from Sodwana Bay, South Africa that was wrongly assigned to the latter genus. This species is redescribed and a second new species, Aldersladum jengi from Penghu Is., Taiwan, is described. The diagnostic features of the new genus include the presence of only figure-eight shaped platelets in all parts of the colony, thus differentiating it from all known genera of the Alcyoniidae. Based on examination of additional material from other localities, the zoogeographical distribution of the genus is confirmed to include the coral reefs of South Africa, Kenya, Gulf of Oman, Taiwan and Japan. Phylogenetic analyses of two mitochondrial genes strongly support its placement in the family Alcyoniidae.     

Adipocyte Stiffness Increases with Accumulation of Lipid Droplets

Adipogenesis and increase in fat tissue mass are mechanosensitive processes and hence should be influenced by the mechanical properties of adipocytes. We evaluated subcellular effective stiffnesses of adipocytes using atomic force microscopy (AFM) and interferometric phase microscopy (IPM), and we verified the empirical results using finite element (FE) simulations. In the AFM studies, we found that the mean ratio of stiffnesses of the lipid droplets (LDs) over the nucleus was 0.83 ± 0.14, from which we further evaluated the ratios of LDs over cytoplasm stiffness, as being in the range of 2.5 to 8.3. These stiffness ratios, indicating that LDs are stiffer than cytoplasm, were verified by means of FE modeling, which simulated the AFM experiments, and provided good agreement between empirical and model-predicted structural behavior. In the IPM studies, we found that LDs mechanically distort their intracellular environment, which again indicated that LDs are mechanically stiffer than the surrounding cytoplasm. Combining these empirical and simulation data together, we provide in this study evidence that adipocytes stiffen with differentiation as a result of accumulation of LDs. Our results are relevant to research of adipose-related diseases, particularly overweight and obesity, from a mechanobiology and cellular mechanics perspectives.     

Estimating Cell Depth from Somatic Mutations

The depth of a cell of a multicellular organism is the number of cell divisions it underwent since the zygote, and knowing this basic cell property would help address fundamental problems in several areas of biology. At present, the depths of the vast majority of human and mouse cell types are unknown. Here, we show a method for estimating the depth of a cell by analyzing somatic mutations in its microsatellites, and provide to our knowledge for the first time reliable depth estimates for several cells types in mice. According to our estimates, the average depth of oocytes is 29, consistent with previous estimates. The average depth of B cells ranges from 34 to 79, linearly related to the mouse age, suggesting a rate of one cell division per day. In contrast, various types of adult stem cells underwent on average fewer cell divisions, supporting the notion that adult stem cells are relatively quiescent. Our method for depth estimation opens a window for revealing tissue turnover rates in animals, including humans, which has important implications for our knowledge of the body under physiological and pathological conditions.     

Effect of raloxifene-analog (LY 117018-Hcl) on the bone marrow of ovariectomized mice

The effects of LY117018-Hcl (Ralox-A) on body metabolism and differentiation of bone marrow cells were studied in ovariectomized (OVX) mice. We used a mouse model in which estrogen depletion was established for a period of three months before treatment. After that period the animals were divided into three experimental groups consisting of sham-operated, OVX, and OVX-Ralox-A-treated mice. The OVX animals received daily treatment of Ralox-A during two time periods (35 and 65 days). After the treatment we measured the serum levels of protein, ion(s), lipid content, liver, and kidney functions. Our findings indicated that a change in hormonal state did not affect basic body metabolism except for causing an increase in triglycerides (TG) in the OVX mice, which was lowered by the Ralox-A. A higher alkaline phosphatase (ALK-P) level was observed in serum of the OVX-Ralox-A-treated mice than in serum of the OVX mice. We investigated the effects of estrogen depletion on the differentiation of hematopoietic and stromal cells that directly affect bone resorption and formation. OVX and OVX-treated mice were compared with the sham group and assessed for the alteration of these cells' differentiation. The proliferation of stromal stem cells was measured by CFU-F assay in vitro. A decrease in CFU-F colonies derived from OVX mice was observed and after the Ralox-A treatment the number of CFU-F reached sham levels. On the contrary, an upregulation of myeloid cells was observed when analyzed by FACS and by granulocyte/macrophage-colony forming unit (G/M-CFU) assay in selective culture conditions. The G/M-CFUs were increased in the OVX mice and were reduced to sham levels after Ralox-A treatment. In this study, we demonstrated cellular changes of stromal and hemopoietic cells in OVX mice and a beneficial Ralox-A effect that protected such cellular changes.     

Cell-based screening for membranal and cytoplasmatic markers using dielectric spectroscopy

Dielectric spectroscopy (DS) of living biological cells is based on the analysis of the complex dielectric permittivity of cells suspended in a physiological medium. It provides knowledge on the polarization–relaxation response of cells to external electric field as function of the excitation frequency. This response is strongly affected by both structural and molecular properties of cells and therefore, can reveal rare insights on cell physiology and behaviour. This study demonstrates the mapping potential of DS after cytoplasmatic and membranal markers for cell-based screening analysis. The effect of membrane permittivity and cytoplasm conductivity was examined using tagged MBA and MDCK cell lines respectively. Comparing the permittivity spectra of tagged and native cell lines reveals clear differences between the analyzed suspensions. In addition, differences on the matching dielectric properties of cells were obtained. Those findings support the high distinction resolution and sensitivity of DS after fine molecular and cellular changes, and hence, highlight the high potential of DS as non invasive screening tool in cell biology research.     

Comparative Proteomics of Symbiotic and Aposymbiotic Juvenile Soft Corals

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral–algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.     

Planulae brooding and acquisition of zooxanthellae inXenia macrospiculata (Cnidaria: Octocorallia)

Brooding in the octocoralXenia macrospiculata is described. Young planulae ofX. macrospiculata were found in brooding pouches located below the anthocodia among the polyps’ cavities. These cavities are connected by and lined with ectoderm. Detached zooxanthellae were present within the brooding pouches, and are most probably acquired later by the planulae. The zooxanthellae enter into ectodermal ameboid cells by phagocytosis, and are then transferred to the endoderm.     

Reconstruction of cell lineage trees in mice

The cell lineage tree of a multicellular organism represents its history of cell divisions from the very first cell, the zygote. A new method for high-resolution reconstruction of parts of such cell lineage trees was recently developed based on phylogenetic analysis of somatic mutations accumulated during normal development of an organism. In this study we apply this method in mice to reconstruct the lineage trees of distinct cell types. We address for the first time basic questions in developmental biology of higher organisms, namely what is the correlation between the lineage relation among cells and their (1) function, (2) physical proximity and (3) anatomical proximity. We analyzed B-cells, kidney-, mesenchymal- and hematopoietic-stem cells, as well as satellite cells, which are adult skeletal muscle stem cells isolated from their niche on the muscle fibers (myofibers) from various skeletal muscles. Our results demonstrate that all analyzed cell types are intermingled in the lineage tree, indicating that none of these cell types are single exclusive clones. We also show a significant correlation between the physical proximity of satellite cells within muscles and their lineage. Furthermore, we show that satellite cells obtained from a single myofiber are significantly clustered in the lineage tree, reflecting their common developmental origin. Lineage analysis based on somatic mutations enables performing high resolution reconstruction of lineage trees in mice and humans, which can provide fundamental insights to many aspects of their development and tissue maintenance.