Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration

Distinct cell populations with regenerative capacity have been reported to contribute to myofibres after skeletal muscle injury, including non-satellite cells as well as myogenic satellite cells. However, the relative contribution of these distinct cell types to skeletal muscle repair and homeostasis and the identity of adult muscle stem cells remain unknown. We generated a model for the conditional depletion of satellite cells by expressing a human diphtheria toxin receptor under control of the murine Pax7 locus. Intramuscular injection of diphtheria toxin during muscle homeostasis, or combined with muscle injury caused by myotoxins or exercise, led to a marked loss of muscle tissue and failure to regenerate skeletal muscle. Moreover, the muscle tissue became infiltrated by inflammatory cells and adipocytes. This localised loss of satellite cells was not compensated for endogenously by other cell types, but muscle regeneration was rescued after transplantation of adult Pax7(+) satellite cells alone. These findings indicate that other cell types with regenerative potential depend on the presence of the satellite cell population, and these observations have important implications for myopathic conditions and stem cell-based therapeutic approaches.

Determination of the persistence of dimetridazole, metronidazole and ronidazole residues in black tiger shrimp (Penaeus monodon) tissue and stability during cooking

The depletion of three banned nitroimidazole drugs (dimetridazole (DMZ), metronidazole (MNZ) and ronidazole (RNZ)) was investigated in black tiger shrimp (Penaeus monodon) following in-water medication. The highest concentrations of residues were measured immediately after the 24 h immersion (day 0). At this time, MNZ and MNZ-OH residues were measured in shrimp tissue samples at concentrations ranging from 361–4189 and 0.28–6.6 μg kg−1, respectively. DMZ and its metabolites HMMNI ranged in concentration between 31509–37780 and 15.0–31.9 μg kg−1, respectively. RNZ and HMMNI concentrations ranged 14530–24206 and 25.0–55 μg kg−1, respectively. MNZ, DMZ and RNZ were the more persistent marker residues and can be detected for at least eight days post-treatment. MNZ-OH was only detectable on day 0 following treatment with MNZ. HMMNI residues were only detectable up to day 1 (0.97–3.2 μg kg−1) or 2 (1.2–4.5 μg kg−1) following DMZ and RNZ treatment, respectively. The parent drugs, MNZ, DMZ and RNZ were still measureable on day 8 at 0.12–1.00, 40.5–55 and 8.8–18.7 μg kg−1, respectively. The study also investigated the stability of nitroimidazole residues under various cooking procedures (frying, grilling, boiling and boiling followed by microwaving). The experiments were carried out in shrimp muscle tissue containing both high and low concentrations of these residues. Different cooking procedures showed the impact on nitroimidazole residue concentration in shrimp tissuetheir concentration depleted significantly, but partially, by boiling and/or microwaving but the compounds were largely resistant to conventional grilling or frying. Cooking cannot therefore be considered as a safeguard against harmful nitroimidazole residues in shrimp.

Network signatures based on gene pair expression ratios improve classification and the analysis of muscle-invasive urothelial cancer

Urothelial cancer (UC) is highly recurrent and can progress from non-invasive (NMIUC) to a more aggressive muscle-invasive (MIUC) subtype that invades the muscle tissue layer of the bladder. We present a proof of principle study that network-based features of gene pairs can be used to improve classifier performance and the functional analysis of urothelial cancer gene expression data. In the first step of our procedure each individual sample of a UC gene expression dataset is inflated by gene pair expression ratios that are defined based on a given network structure. In the second step an elastic net feature selection procedure for network-based signatures is applied to discriminate between NMIUC and MIUC samples. We performed a repeated random subsampling cross validation in three independent datasets. The network signatures were characterized by a functional enrichment analysis and studied for the enrichment of known cancer genes. We observed that the network-based gene signatures from meta collections of proteinprotein interaction (PPI) databases such as CPDB and the PPI databases HPRD and BioGrid improved the classification performance compared to single gene based signatures. The network based signatures that were derived from PPI databases showed a prominent enrichment of cancer genes (e.g., TP53, TRIM27 and HNRNPA2Bl). We provide a novel integrative approach for large-scale gene expression analysis for the identification and development of novel diagnostical targets in bladder cancer. Further, our method allowed to link cancer gene associations to network-based expression signatures that are not observed in gene-based expression signatures.

Lipid extraction has little effect on the delta N-15 of aquatic consumers

Proper application of stable isotopes (e. g., delta N-15 and delta C-13) to food web analysis requires an understanding of all nondietary factors that contribute to isotopic variability. Lipid extraction is often used during stable isotope analysis (SIA), because synthesized lipids have a low delta C-13 and can mask the delta C-13 of a consumer's diet. Recent studies indicate that lipid extraction intended to adjust delta C-13 may also cause shifts in delta N-15, but the magnitude of and reasons for the shift are highly uncertain. We examined a large data set (n = 854) for effects of lipid extraction (using Bligh and dyer's [ 1959] chloroform-methanol solvent mixtures) on the delta N-15 of aquatic consumers. We found no effect of chemically extracting lipids on the delta N-15 of whole zooplankton, unionid mussels, and fish liver samples, and found a small increase in fish muscle delta N-15 of similar to 0.4%. We also detected a negative relationship between the shift in delta N-15 following extraction and the C:N ratio in muscle tissue, suggesting that effects of extraction were greater for tissue with lower lipid content. As long as appropriate techniques such as those from Bligh and dyer (1959) are used, effects of lipid extraction on delta N-15 of aquatic consumers need not be a major consideration in the SIA of food webs.

Antennas for over-body-surface communication at 2.45 GHz

In this paper, the on-body performance of a range of wearable antennas was investigated by measuring vertical bar S-21 vertical bar path gain between two devices mounted on tissue-equivalent numerical and experimental phantoms, representative of human muscle tissue at 2.45 GHz. In particular, the study focused on the performance of a compact higher mode microstrip patch antenna (HMMPA) with a profile as low as lambda/20. The 5- and 10-mm-high HMMPA prototypes had an impedance bandwidth of 6.7% and 8.6%, respectively, sufficient for the operating requirements of the 2.45-GHz industrial, scientific, and medical (ISM) band and both antennas offered 11-dB higher path gain compared to a fundamental-mode microstrip patch antenna. It was also dernonstrated that a 7-dB improvement in path gain can be obtained for a fundamental-mode patch through the addition of a shortening wall. Notably, on-body HMMPA performance was comparable to a quarter wave monopole antenna on the same size of ground-plane, mounted normal to the tissue surface, indicating that the low-profile and physically more robust antenna is a promising solution for bodyworn antenna applications.

An Antennas and Propagation Approach to Improving Physical Layer Performance in Wireless Body Area Networks

A combined antennas and propagation study has been undertaken with a view to directly improving link conditions for wireless body area networks. Using tissue-equivalent numerical and experimental phantoms representative of muscle tissue at 2.45 GHz, we show that the node to node [S-21] path gain performance of a new wearable integrated antenna (WIA) is up to 9 dB better than a conventional compact Printed-F antenna, both of which are suitable for integration with wireless node circuitry. Overall, the WIA performed extremely well with a measured radiation efficiency of 38% and an impedance bandwidth of 24%. Further benefits were also obtained using spatial diversity, with the WIA providing up to 7.7 dB of diversity gain for maximal ratio combining. The results also show that correlation was lower for a multipath environment leading to higher diversity gain. Furthermore, a diversity implementation with the new antenna gave up to 18 dB better performance in terms of mean power level and there was a significant improvement in level crossing rates and average fade durations when moving from a single-branch to a two-branch diversity system.

Continuous variation in the pattern of marine v. freshwater foraging in brown trout Salmo trutta L. from Loch Lomond, Scotland

Carbon stable-isotope analysis showed that individual brown trout Salmo trutta in Loch Lomond adopted strategies intermediate to that of freshwater residency or anadromy, suggesting either repeated movement between freshwater and marine environments, or estuarine residency. Carbon stable-isotope (delta C-13) values from Loch Lomond brown trout muscle tissue ranged from those indicative of assimilation of purely freshwater-derived carbon to those reflecting significant utilization of marine-derived carbon. A single isotope, two-source mixing model indicated that, on average, marine C made a 33% contribution to the muscle tissue C of Loch Lomond brown trout. Nitrogen stable isotope, delta N-15, but not delta C-13 was correlated with fork length suggesting that larger fish were feeding at a higher trophic level but that marine feeding was not indicated by larger body size. These results are discussed with reference to migration patterns in other species. (c) 2008 The Authors Journal compilation (c) 2008 The Fisheries Society of the British Isles.

Has habitat heterogeneity promoted phenotypic and ecological sub-structuring among a Coregonus lavaretus population in a large Scottish lake?

Differences in stable-isotope values, morphology and ecology in whitefish Coregonus lavaretus were investigated between the three basins of Loch Lomond. The results are discussed with reference to a genetic investigation to elucidate any substructuring or spawning site fidelity. Foraging fidelity between basins of Loch Lomond was indicated by delta 13C and delta 15N values of C. lavaretus muscle tissue. There was, however, no evidence of the existence of sympatric morphs in the C. lavaretus population. A previous report of two C. lavaretus 'species' in Loch Lomond probably reflects natural variation between individuals within a single mixed population.

Systemic oxidative-nitrosative-inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity

New Findings

What is the central question of this study?Exercise performance is limited during hypoxia by a critical reduction in cerebral and skeletal tissue oxygenation. To what extent an elevation in systemic free radical accumulation contributes to microvascular deoxygenation and the corresponding reduction in maximal aerobic capacity remains unknown.What is the main finding and its importance?We show that altered free radical metabolism is not a limiting factor for exercise performance in hypoxia, providing important insight into the fundamental mechanisms involved in the control of vascular oxygen transport.

Exercise performance in hypoxia may be limited by a critical reduction in cerebral and skeletal tissue oxygenation, although the underlying mechanisms remain unclear. We examined whether increased systemic free radical accumulation during hypoxia would be associated with elevated microvascular deoxygenation and reduced maximal aerobic capacity (). Eleven healthy men were randomly assigned single-blind to an incremental semi-recumbent cycling test to determine  in both normoxia (21% O_{2) and hypoxia (12% O2) separated by a week. Continuous-wave near-infrared spectroscopy was employed to monitor concentration changes in oxy- and deoxyhaemoglobin in the left vastus lateralis muscle and frontal cerebral cortex. Antecubital venous blood samples were obtained at rest and at  to determine oxidative (ascorbate radical by electron paramagnetic resonance spectroscopy), nitrosative (nitric oxide metabolites by ozone-based chemiluminescence and 3-nitrotyrosine by enzyme-linked immunosorbent assay) and inflammatory stress biomarkers (soluble intercellular/vascular cell adhesion 1 molecules by enzyme-linked immunosorbent assay). Hypoxia was associated with increased cerebral and muscle tissue deoxygenation and lower  (P < 0.05 versus normoxia). Despite an exercise-induced increase in oxidative–nitrosative–inflammatory stress, hypoxia per se did not have an additive effect (P > 0.05 versus normoxia). Consequently, we failed to observe correlations between any metabolic, haemodynamic and cardiorespiratory parameters (P > 0.05). Collectively, these findings suggest that altered free radical metabolism cannot explain the elevated microvascular deoxygenation and corresponding lower  in hypoxia. Further research is required to determine whether free radicals when present in excess do indeed contribute to the premature termination of exercise in hypoxia.}

Smooth Muscle Cells Differentiated from Reprogrammed Embryonic Lung Fibroblasts Through DKK3 Signalling are Potent for Tissue Engineering of Vascular Grafts

Rationale: Smooth muscle cells (SMCs) are a key component of tissue-engineered vessels. However, the sources by which they can be isolated are limited.

Objective: We hypothesized that a large number of SMCs could be obtained by direct reprogramming of fibroblasts, that is, direct differentiation of specific cell lineages before the cells reaching the pluripotent state.

Methods and Results: We designed a combined protocol of reprogramming and differentiation of human neonatal lung fibroblasts. Four reprogramming factors (OCT4, SOX2, KLF4, and cMYC) were overexpressed in fibroblasts under reprogramming conditions for 4 days with cells defined as partially-induced pluripotent stem (PiPS) cells. PiPS cells did not form tumors in vivo after subcutaneous transplantation in severe combined immunodeficiency mice and differentiated into SMCs when seeded on collagen IV and maintained in differentiation media. PiPS-SMCs expressed a panel of SMC markers at mRNA and protein levels. Furthermore, the gene dickkopf 3 was found to be involved in the mechanism of PiPS-SMC differentiation. It was revealed that dickkopf 3 transcriptionally regulated SM22 by potentiation of Wnt signaling and interaction with Kremen1. Finally, PiPS-SMCs repopulated decellularized vessel grafts and ultimately gave rise to functional tissue-engineered vessels when combined with previously established PiPS-endothelial cells, leading to increased survival of severe combined immunodeficiency mice after transplantation of the vessel as a vascular graft.

Conclusions: We developed a protocol to generate SMCs from PiPS cells through a dickkopf 3 signaling pathway, useful for generating tissue-engineered vessels. These findings provide a new insight into the mechanisms of SMC differentiation with vast therapeutic potential.

Morphology and localization of interstitial cells in the guinea pig bladder: structural relationships with smooth muscle and neurons.

PURPOSE: In the current study we examined the location of interstitial cell of Cajal (ICC)-like cells in the guinea pig bladder wall and studied their structural interactions with nerves and smooth muscle cells. MATERIALS AND METHODS: Whole mount samples and cryosections of bladder tissue were labeled with primary and fluorescent secondary antibodies, and imaged using confocal and multiphoton microscopy. RESULTS: Kit positive ICC-like cells were located below the urothelium, in the lamina propria region and throughout the detrusor. In the suburothelium they had a stellate morphology and appeared to network. They made connections with nerves, as shown by double labeling experiments with anti-kit and anti-protein gene product 9.5. A network of vimentin positive cells was also found, of which many but not all were kit positive. In the detrusor kit positive cells were most often seen at the edge of smooth muscle bundles. They were elongated with lateral branches, running in parallel with the bundles and closely associated with intramural nerves. Another population of kit positive cells was seen in the detrusor between muscle bundles. These cells had a more stellate-like morphology and made connections with each other. Kit positive cells were seen tracking nerve bundles and close to intramural ganglia. Vimentin positive cells were present in the detrusor, of which some were also kit positive. CONCLUSIONS: There are several populations of ICC-like cells throughout the guinea pig bladder wall. They differ in morphology and orientation but all make connections with intramural nerves and in the detrusor they are closely associated with smooth muscle cells.

Ca(2+)-activated Cl(-) current in sheep lymphatic smooth muscle.

Freshly dispersed sheep mesenteric lymphatic smooth muscle cells were studied at 37 degrees C using the perforated patch-clamp technique with Cs(+)- and K(+)-filled pipettes. Depolarizing steps evoked currents that consisted of L-type Ca(2+) [I(Ca(L))] current and a slowly developing current. The slow current reversed at 1 +/- 1.5 mV with symmetrical Cl(-) concentrations compared with 23.2 +/- 1.2 mV (n = 5) and -34.3 +/- 3.5 mV (n = 4) when external Cl(-) was substituted with either glutamate (86 mM) or I(-) (125 mM). Nifedipine (1 microM) blocked and BAY K 8644 enhanced I(Ca(L)), the slow-developing sustained current, and the tail current. The Cl(-) channel blocker anthracene-9-carboxylic acid (9-AC) reduced only the slowly developing inward and tail currents. Application of caffeine (10 mM) to voltage-clamped cells evoked currents that reversed close to the Cl(-) equilibrium potential and were sensitive to 9-AC. Small spontaneous transient depolarizations and larger action potentials were observed in current clamp, and these were blocked by 9-AC. Evoked action potentials were triphasic and had a prominent plateau phase that was selectively blocked by 9-AC. Similarly, fluid output was reduced by 9-AC in doubly cannulated segments of spontaneously pumping sheep lymphatics, suggesting that the Ca(2+)-activated Cl(-) current plays an important role in the electrical activity underlying spontaneous activity in this tissue. PMID: 11029279 [PubMed - indexed for MEDLINE]

The smooth muscle pharmacology of maximakinin, a receptor-selective, bradykinin-related nonadecapeptide from the venom of the Chinese toad, Bombina maxima.

Structural homologues of vertebrate regulatory peptides found in defensive skin secretions of anuran amphibians often display enhanced bioactivity and receptor binding when compared with endogenous mammalian peptide ligands. Maximakinin, a novel N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the skin venom of a Chinese toad (Bombina maxima), displays such activity enhancement when compared with bradykinin but is additionally highly selective for mammalian arterial smooth muscle bradykinin receptors displaying a 50-fold increase in molar potency in this smooth muscle type. In contrast, a 100-fold decrease in molar potency was observed at bradykinin receptors in intestinal and uterine smooth muscle preparations. Maximakinin has thus evolved as a “smart” defensive weapon in the toad with receptor/tissue selective targeting. Natural selection of amphibian skin venom peptides for antipredator defence, through inter-species delivery by an exogenous secretory mode, produces subtle structural stabilisation modifications that can potentially provide new insights for the design of selectively targeted peptide therapeutics.

Kit-positive interstitial cells in the rabbit urethra: structural relationships with nerves and smooth muscle.

OBJECTIVE: To identify interstitial cells (ICs) in the wall of the rabbit urethra using antibodies to the Kit receptor, and to examine their location, morphology and relationship with nerves and smooth muscle cells (SMCs), as studies of enzymatically isolated cells from the rabbit urethra have established that there are specialized cells that show spontaneous electrical activity and have morphological properties of ICs. MATERIALS AND METHODS: Urethral tissues from rabbits were fixed, labelled with antibodies and examined with confocal microscopy. Some specimens were embedded in paraffin wax and processed for histology. Histological sections from the most proximal third and mid-third region of rabbit urethra were stained with Masson's Trichrome to show their cellular arrangement. RESULTS: Sections from both regions had outer longitudinal and inner circular layers of SM, and a lamina propria containing connective tissue and blood vessels; the lumen was lined with urothelial cells. The mid-third region had a more developed circular SM layer than the most-proximal samples, and had extensive inner longitudinal SM bundles in the lamina propria. Labelling with anti-Kit revealed immunopositive cells within the wall of the rabbit urethra, in the circular and longitudinal layers of the muscularis. Double-labelling with an antibody to SM myosin showed Kit-positive cells on the boundary of the SM bundles, orientated parallel to the axis of the bundles. Others were in spaces between the bundles and often made contact with each other. Kit-positive cells were either elongated, with several lateral branches, or stellate with branches coming from a central soma. Similar cells could be labelled with vimentin antibodies. Their relationship with intramural nerves was examined by double immunostaining with an anti-neurofilament antibody. There were frequent points of contact between Kit-positive cells and nerves, with similar findings in specimens double-immunostained with anti-neuronal nitric oxide synthase (nNOS). CONCLUSION: Kit-positive ICs were found within the SM layers of the rabbit urethra, in association with nerves, on the edge of SM bundles and in the interbundle spaces. The contact with nNOS-containing neurones might imply participation in the nitrergic inhibitory neurotransmission of the urethra. PMID: 17212607 [PubMed - indexed for MEDLINE]

Muscle-specific mutations accumulate with aging in critical human mtDNA control sites for replication.

The recently discovered aging-dependent large accumulation of point mutations in the human fibroblast mtDNA control region raised the question of their occurrence in postmitotic tissues. In the present work, analysis of biopsied or autopsied human skeletal muscle revealed the absence or only minimal presence of those mutations. By contrast, surprisingly, most of 26 individuals 53 to 92 years old, without a known history of neuromuscular disease, exhibited at mtDNA replication control sites in muscle an accumulation of two new point mutations, i.e., A189G and T408A, which were absent or marginally present in 19 individuals younger than 34 years. These two mutations were not found in fibroblasts from 22 subjects 64 to 101 years of age (T408A), or were present only in three subjects in very low amounts (A189G). Furthermore, in several older individuals exhibiting an accumulation in muscle of one or both of these mutations, they were nearly absent in other tissues, whereas the most frequent fibroblast-specific mutation (T414G) was present in skin, but not in muscle. Among eight additional individuals exhibiting partial denervation of their biopsied muscle, four subjects >80 years old had accumulated the two muscle-specific point mutations, which were, conversely, present at only very low levels in four subjects <or =40 years old. The striking tissue specificity of the muscle mtDNA mutations detected here and their mapping at critical sites for mtDNA replication strongly point to the involvement of a specific mutagenic machinery and to the functional relevance of these mutations.