Adipose tissue‐derived progenitors for engineering osteogenic and vasculogenic grafts

The current need for bone grafts in orthopedic and reconstructive surgery cannot be satisfied by autologous tissue transplant due to its limited availability and significant associated morbidity. Tissue engineering approaches could supply sufficient amounts of bone substitutes by exploiting the ability to harvest autologous osteogenic progenitors associated with suitable porous materials. However, the generation of clinically relevant‐sized constructs is critically hampered by limited vascularization, with consequent engraftment and survival only of a thin outer shell, upon in vivo implantation. To overcome this limitation, different non‐mutually exclusive approaches have recently been developed to promote or accelerate graft vascularization, from angiogenic growth factor gene delivery to surgical pre‐vascularization of the construct before implantation. A simple, promising strategy involves the co‐culture of vasculogenic cells to form an intrinsic vascular network inside the graft in vitro, which can rapidly anastomose with the host blood vessels in vivo. Recent data have shown that adipose tissue‐derived stromal vascular fraction (SVF) may provide an efficient, convenient, and autologous source for both osteogenic and endothelial cells. When SVF progenitors were cultured in appropriate bioreactor systems and ectopically implanted, a functional vascular network connected to the host was formed concomitantly to bone formation. Future studies should aim at demonstrating that this approach effectively supports survival of scaled up cell‐based bone grafts at an orthotopic site. The procedure should also be adapted to become compatible with an intra‐operative timeline and complemented with the definition of suitable potency markers, to facilitate its development into a simplified, reproducible, and cost‐effective clinical treatment. J. Cell. Physiol. 225: 348–353, 2010. © 2010 Wiley‐Liss, Inc.     

Diet fat alters expression of genes for enzymes of lipogenesis in lean and obese mice

The objective of this experiment was to determine the effect of polyunsaturated fatty acids on gene expression for fatty acid synthase, acetyl CoA-carboxylase, malic enzyme, pyruvate kinase, and phosphoenolpyruvate carboxykinase in obese mice. Eight-week-old female lean and obese mice were fed semi-purified diets containing 20% (w/w) fat of either high or low polyunsaturated to saturated (P/S) fatty acid ratio for four weeks. Total RNA was isolated from liver and was hybridized to cDNA probes for the above enzymes. Consumption of a high P/S diet decreased mRNA levels for all the lipogenic enzymes studied in both lean and obese mice. Compared to lean mice, obese mice exhibited a higher mRNA level for fatty acid synthase, acetyl CoA-carboxylase, malic enzyme, and pyruvate kinase in animals fed either a high or low P/S diet. Enzyme-specific activities followed the same profile as the mRNA levels in both lean and obese mice fed a high or low P/S diet. The decrease in liver fatty acid synthase mRNA level was more pronounced in lean mice compared to obese mice, suggesting that the obese mice may be more resistant to polyunsaturated fatty acid feedback control of gene expression.     

Temporal and tissue‐specific disruption of LINC complexes in vivo

Migration and anchorage of nuclei within developing and adult tissues rely on Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes). These macromolecular assemblies span the nuclear envelope and physically couple chromatin and nuclear lamina to cytoplasmic cytoskeletal networks. LINC complexes assemble within the perinuclear space through direct interactions between the respective evolutionary‐conserved SUN and KASH domains of Sun proteins, which reside within the inner nuclear membrane, and Nesprins, which reside within the outer nuclear membrane. Here, we describe and validate a dominant‐negative transgenic strategy allowing for the disruption of endogenous SUN/KASH interactions through the inducible expression of a recombinant KASH domain. Our approach, which is based on the Cre/Lox system, allows for the targeted disruption of LINC complexes in a wide array of mouse tissues or specific cell types thereof and bypasses the perinatal lethality and potential cell nonautonomous effects of current mouse models based on germline inactivation of genes encoding Sun proteins and Nesprins. For these reasons, this mouse model provides a useful tool to evaluate the physiological relevance of LINC complexes integrity during development and homeostasis in a wide array of mammalian tissues. genesis 52:359–365, 2014. © 2014 Wiley Periodicals, Inc.     

Sex‐ and tissue‐specific changes in mTOR signaling with age in C57BL/6J mice

Inhibition of the mTOR (mechanistic Target Of Rapamycin) signaling pathway robustly extends the lifespan of model organisms including mice. The precise molecular mechanisms and physiological effects that underlie the beneficial effects of rapamycin are an exciting area of research. Surprisingly, while some data suggest that mTOR signaling normally increases with age in mice, the effect of age on mTOR signaling has never been comprehensively assessed. Here, we determine the age‐associated changes in mTORC1 (mTOR complex 1) and mTORC2 (mTOR complex 2) signaling in the liver, muscle, adipose, and heart of C57BL/6J.Nia mice, the lifespan of which can be extended by rapamycin treatment. We find that the effect of age on several different readouts of mTORC1 and mTORC2 activity varies by tissue and sex in C57BL/6J.Nia mice. Intriguingly, we observed increased mTORC1 activity in the liver and heart tissue of young female mice compared to male mice of the same age. Tissue and substrate‐specific results were observed in the livers of HET3 and DBA/2 mouse strains, and in liver, muscle and adipose tissue of F344 rats. Our results demonstrate that aging does not result in increased mTOR signaling in most tissues and suggest that rapamycin does not promote lifespan by reversing or blunting such an effect.     

Sex‐ And tissue‐specific differences in telomere length in a reptile

The usage of telomere length (TL) in blood as a proxy for the TL of other tissues relies on the assumption that telomere dynamics across all tissues are similar. However, telomere attrition can be caused by reactive oxygen species (ROS) which may vary with metabolic rate, which itself varies across organs depending upon the life history strategy of an organism. Thus, we chose to measure the telomeres of various cell types in juvenile painted dragon lizards, Ctenophorus pictus, given their unusual life history strategy. Individuals typically only experience a single mating season. We measured the TL of male and female dragons using qPCR and observed that TL varied with tissue type and sex. Telomeres of blood cells were longer than those of liver, heart, brain, and spleen, and females had longer telomeres than males. Brain telomeres in males were approximately half the length of those in females. Telomeric attrition in the male brain may be due to the need for rapid learning of reproductive tactics (territory patrol and defense, mate‐finding). Significant correlations between the TL of tissue types suggest that blood TL may be a useful proxy for the TL of other tissues. Our comparison of organ‐specific telomere dynamics, the first in a reptile, suggests that the usage of blood TL as a proxy requires careful consideration of the life history strategy of the organism.     

Shade‐induced stem elongation in rice seedlings: Implication of tissue‐specific phytohormone regulation

A better understanding of shade avoidance syndrome(SAS) is an urgent need because of its effect on energy reallocation. Leverage-related mechanism in crops is of potential economic interest for agricultural applications. Here we report the SAS phenotype at tissue level rice seedlings. Tissue-specific RNA-sequencing indicates auxin plays different roles between coleoptile and the first leaf.Phenotypes of wild type treated by gibberellin and brassinosteroid biosynthesis inhibitors and of related mutants suggest these two hormones positively regulate SAS. Our work reveals the diversity of hormone responses in different organs and different species in shade conditions.     

Reversible and tissue‐specific activation of MAP kinase signaling by tamoxifen in brafV637ERT2 mice

Deregulated MAP kinase (MAPK) signaling plays key roles in developmental and adult disease processes, but the experimental activation of MAPK is a currently unresolved task. For the reversible induction of MAPK signaling, we generated transgenic mice harboring a tamoxifen inducible BRAF^V637EER^T2 fusion protein. The expression of the inducible BRAF kinase can be directed by Cre/loxP‐mediated recombination to selected cell types and enables the highly specific activation of MAPK signalling in vivo. We show that MAPK signaling can be transiently activated in the brain, liver, or kidney of Braf^V637EER^T2 mice by a single injection of tamoxifen. Braf^V637EER^T2 mice provide a new versatile tool to study disease mechanisms elicited by MAPK activation, complementing gene knockout technology that is restricted to the analysis of loss‐of‐function phenotypes. genesis 51:448–455. © 2013 Wiley Periodicals, Inc.     

A new mouse model for temporal‐ and tissue‐specific control of extracellular superoxide dismutase

The extracellular isoform of superoxide dismutase (EC‐SOD, Sod3) plays a protective role against various diseases and injuries mediated by oxidative stress. To investigate the pathophysiological roles of EC‐SOD, we generated tetracycline‐inducible Sod3 transgenic mice and directed the tissue‐specific expression of transgenes by crossing Sod3 transgenic mice with tissue‐specific transactivator transgenics. Double transgenic mice with liver‐specific expression of Sod3 showed increased EC‐SOD levels predominantly in the plasma as the circulating form, whereas double transgenic mice with neuronal‐specific expression expressed higher levels of EC‐SOD in hippocampus and cortex with intact EC‐SOD as the dominant form. EC‐SOD protein levels also correlated well with increased SOD activities in double transgenic mice. In addition to enabling tissue‐specific expression, the transgene expression can be quickly turned on and off by doxycycline supplementation in the mouse chow. This mouse model, thus, provides the flexibility for on–off control of transgene expression in multiple target tissues. genesis 47:142–154, 2009. © 2009 Wiley‐Liss, Inc.     

A tissue‐specific screen of ceramide expression in aged mice identifies ceramide synthase‐1 and ceramide synthase‐5 as potential regulators of fiber size and strength in skeletal muscle

Loss of skeletal muscle mass is one of the most widespread and deleterious processes in aging humans. However, the mechanistic metabolic principles remain poorly understood. In the framework of a multi‐organ investigation of age‐associated changes of ceramide species, a unique and distinctive change pattern of C_16:0 and C_18:0 ceramide species was detected in aged skeletal muscle. Consistently, the expression of CerS1 and CerS5 mRNA, encoding the ceramide synthases (CerS) with substrate preference for C_16:0 and C_18:0 acyl chains, respectively, was down‐regulated in skeletal muscle of aged mice. Similarly, an age‐dependent decline of both CerS1 and CerS5 mRNA expression was observed in skeletal muscle biopsies of humans. Moreover, CerS1 and CerS5 mRNA expression was also reduced in muscle biopsies from patients in advanced stage of chronic heart failure (CHF) suffering from muscle wasting and frailty. The possible impact of CerS1 and CerS5 on muscle function was addressed by reversed genetic analysis using CerS1^Δ/Δ and CerS5^Δ/Δ knockout mice. Skeletal muscle from mice deficient of either CerS1 or CerS5 showed reduced caliber sizes of both slow (type 1) and fast (type 2) muscle fibers, fiber grouping, and fiber switch to type 1 fibers. Moreover, CerS1‐ and CerS5‐deficient mice exhibited reduced twitch and tetanus forces of musculus extensor digitorum longus. The findings of this study link CerS1 and CerS5 to histopathological changes and functional impairment of skeletal muscle in mice that might also play a functional role for the aging skeletal muscle and for age‐related muscle wasting disorders in humans.     

The phosphorylation status of T522 modulates tissue‐specific functions of SIRT1 in energy metabolism in mice

SIRT1, the most conserved mammalian NAD⁺‐dependent protein deacetylase, is an important metabolic regulator. However, the mechanisms by which SIRT1 is regulated in vivo remain unclear. Here, we report that phosphorylation modification of T522 on SIRT1 is crucial for tissue‐specific regulation of SIRT1 activity in mice. Dephosphorylation of T522 is critical for repression of its activity during adipogenesis. The phospho‐T522 level is reduced during adipogenesis. Knocking‐in a constitutive T522 phosphorylation mimic activates the β‐catenin/GATA3 pathway, repressing PPARγ signaling, impairing differentiation of white adipocytes, and ameliorating high‐fat diet‐induced dyslipidemia in mice. In contrast, phosphorylation of T522 is crucial for activation of hepatic SIRT1 in response to over‐nutrition. Hepatic SIRT1 is hyperphosphorylated at T522 upon high‐fat diet feeding. Knocking‐in a SIRT1 mutant defective in T522 phosphorylation disrupts hepatic fatty acid oxidation, resulting in hepatic steatosis after high‐fat diet feeding. In addition, the T522 dephosphorylation mimic impairs systemic energy metabolism. Our findings unveil an important link between environmental cues, SIRT1 phosphorylation, and energy homeostasis and demonstrate that the phosphorylation of T522 is a critical element in tissue‐specific regulation of SIRT1 activity in vivo.     

Comprehensive construction strategy of bidirectional green tissue‐specific synthetic promoters

Bidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue‐specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue‐specific synthetic promoters. It is the first report on bidirectional green tissue‐specific promoters that could be efficiently applied in genetic engineering.     

Time-dependent hepatic proteome analysis in lean and diet-induced obese mice

C57BL/6J mice have been widely used as a diet-induced obesity model because they trigger common features of the human metabolic syndrome. In the present study, C57BL/6J male mice were fed either a high-fat diet (HFD) or normal diet (ND) during a 24-week period, and then the age-dependent liver proteome of mice in two groups was analyzed using 2-DE combined with MALDI-TOF-MS. Among identified proteins, up-regulated proteins were subdivided to early (during the first 4 weeks) and late (20~24 weeks) markers that played a role in diet-induced obesity development. Important early markers included ketohexokinase and prohibitin, and late markers included the 75 kDa glucose-regulated protein, citrate synthase, and selenium-binding liver protein. Of these, the 75 kDa glucoseregulated protein has already been linked to obesity; however, prohibitin protein involved in obesity was identified for the first time in this study. In order to validate the proteomic results and gain insight into metabolic changes between the two groups, we further confirmed the expression pattern of some proteins of interest by Western blot analysis. Combined results of proteomic analysis with Western blot analysis revealed that antioxidant enzymes were progressively decreased, whereas cytoskeletal proteins were time-dependently increased in HFD mice.     

Ral GTPase and the exocyst regulate autophagy in a tissue‐specific manner

Autophagy traffics cellular components to the lysosome for degradation. Ral GTPase and the exocyst have been implicated in the regulation of stress‐induced autophagy, but it is unclear whether they are global regulators of this process. Here, we investigate Ral function in different cellular contexts in Drosophila and find that it is required for autophagy during developmentally regulated cell death in salivary glands, but does not affect starvation‐induced autophagy in the fat body. Furthermore, knockdown of exocyst subunits has a similar effect, preventing autophagy in dying cells but not in cells of starved animals. Notch activity is elevated in dying salivary glands, this change in Notch signaling is influenced by Ral, and decreased Notch function influences autophagy. These data indicate that Ral and the exocyst regulate autophagy in a context‐dependent manner, and that in dying salivary glands, Ral mediates autophagy, at least in part, by regulation of Notch.