Adenosine, ‘pertussis-sensitive’ G-proteins, and K+ conductance in central mammalian neurones under energy deprivation

There is a striking similarity between the effects of adenosine and of hypoxia or glucose depletion on membrane potential and conductance of hippocampal neurones in tissue slices of rat brain. Both induce a membrane hyperpolarization by an increase in potassium conductance. It seemed likely, therefore, that a rise in extracellular adenosine concentration during energy deprivation may link neuronal metabolism with membrane K+ conductance. To test this hypothesis, we have now investigated the effects of hypoxia/glucose deprivation on hippocampal neurones from pertussis toxin-treated rats. In such slices adenosine had no effect on postsynaptic membrane potential and input resistance. Nevertheless, hypoxia or glucose depletion were as effective as in controls. These data provide evidence against adenosine as the main mediator between cell metabolism and potassium conductance

Cromakalim (BRL 34915) restores in vitro the membrane potential of depolarized human skeletal muscle fibres

The purpose of the present study was to analyze the effects of cromakalim (BRL 34915), a potent drug from a new class of drugs characterized as K+ channel openers, on the electrical activity of human skeletal muscle. Therefore, intracellular recordings were used to measure the effects of cromakalim on the membrane potential and input conductance of fibres from human skeletal muscle biopsies. Cromakalim in a concentration above 1 mol/l induced an increase in membrane K+ conductance. This effect resulted in a membrane hyperpolarization. The magnitude of this polarization depended on the difference between resting and K+ equilibrium potential. The effect had a rapid onset and was quickly reversible after washing. Fibres from two patients with hyperkalaemic periodic paralysis showed an excessive membrane depolarization during and also after exposure to an slightly elevated extracellular K+ concentration. In the latter situation, cromakalim repolarized the fibres to the normal resting potential. Tolbutamide (1 mmol/l) and Ba2+ (3 mmol/l) strongly antagonized the effect of cromakalim. The data show that cromakalim hyperpolarizes depolarized human skeletal muscle fibres maintained in vitro. The underlying mechanism is probably an activation of otherwise silent, ATP-regulated K+ channels. Such an effect may be of therapeutic benefit in a situation in which a membrane depolarization causes muscle paralysis

Altered expression of cyclin A 1 in muscle of patients with facioscapulohumeral muscle dystrophy (FSHD-1)

Objectives Cyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD). Methods Muscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy. Results cDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients. Conclusions In most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease

Full-length dysferlin transfer by the hyperactive Sleeping Beauty transposase restores dysferlin-deficient muscle

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy

Global Estimation of Range Resolved Thermodynamic Profiles from MicroPulse Differential Absorption Lidar

We demonstrate thermodynamic profile estimation with data obtained using the MicroPulse DIAL such that the retrieval is entirely self contained. The only external input is surface meteorological variables obtained from a weather station installed on the instrument. The estimator provides products of temperature, absolute humidity and backscatter ratio such that cross dependencies between the lidar data products and raw observations are accounted for and the final products are self consistent. The method described here is applied to a combined oxygen DIAL, potassium HSRL, water vapor DIAL system operating at two pairs of wavelengths (nominally centered at 770 and 828 nm). We perform regularized maximum likelihood estimation through the Poisson Total Variation technique to suppress noise and improve the range of the observations. A comparison to 119 radiosondes indicates that this new processing method produces improved temperature retrievals, reducing total errors to less than 2 K below 3 km altitude and extending the maximum altitude of temperature retrievals to 5 km with less than 3 K error. The results of this work definitively demonstrates the potential for measuring temperature through the oxygen DIAL technique and furthermore that this can be accomplished with low-power semiconductor-based lidar sensors

Human satellite cells have regenerative capacity and are genetically manipulable

Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon-mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies

2D Signal Estimation for Sparse Distributed Target Photon Counting Data

In this study, we explore the utilization of maximum likelihood estimation for the analysis of sparse photon counting data obtained from distributed target lidar systems. Specifically, we adapt the Poisson Total Variation processing technique to cater to this application. By assuming a Poisson noise model for the photon count observations, our approach yields denoised estimates of backscatter photon flux and related parameters. This facilitates the processing of raw photon counting signals with exceptionally high temporal and range resolutions (demonstrated here to 50 Hz and 75 cm resolutions), including data acquired through time-correlated single photon counting, without significant sacrifice of resolution. Through examination involving both simulated and real-world 2D atmospheric data, our method consistently demonstrates superior accuracy in signal recovery compared to the conventional histogram-based approach commonly employed in distributed target lidar applications

Human muscle-derived CLEC14A-positive cells regenerate muscle independent of PAX7

Skeletal muscle stem cells, called satellite cells and defined by the transcription factor PAX7, are responsible for postnatal muscle growth, homeostasis and regeneration. Attempts to utilize the regenerative potential of muscle stem cells for therapeutic purposes so far failed. We previously established the existence of human PAX7-positive cell colonies with high regenerative potential. We now identified PAX7-negative human muscle-derived cell colonies also positive for the myogenic markers desmin and MYF5. These include cells from a patient with a homozygous PAX7 c.86-1G > A mutation (PAX7null). Single cell and bulk transcriptome analysis show high intra- and inter-donor heterogeneity and reveal the endothelial cell marker CLEC14A to be highly expressed in PAX7null cells. All PAX7-negative cell populations, including PAX7null, form myofibers after transplantation into mice, and regenerate muscle after reinjury. Transplanted PAX7neg cells repopulate the satellite cell niche where they re-express PAX7, or, strikingly, CLEC14A. In conclusion, transplanted human cells do not depend on PAX7 for muscle regeneration

Free fatty acids link metabolism and regulation of the insulin-sensitizing fibroblast growth factor-21

OBJECTIVE—Fibroblast growth factor (FGF)-21 improves insulin sensitivity and lipid metabolism in obese or diabetic animal models, while human studies revealed increased FGF-21 levels in obesity and type 2 diabetes. Given that FGF-21 has been suggested to be a peroxisome proliferator–activator receptor (PPAR) –dependent regulator of fasting metabolism, we hypothesized that free fatty acids (FFAs), natural agonists of PPAR, might modify FGF-21 levels. RESEARCH DESIGN AND METHODS—The effect of fatty acids on FGF-21 was investigated in vitro in HepG2 cells. Within a randomized controlled trial, the effects of elevated FFAs were studied in 21 healthy subjects (13 women and 8 men). Within a clinical trial including 17 individuals, the effect of insulin was analyzed using an hyperinsulinemic-euglycemic clamp and the effect of PPAR activation was studied subsequently in a rosiglitazone treatment trial over 8 weeks. RESULTS—Oleate and linoleate increased FGF-21 expression and secretion in a PPAR-dependent fashion, as demonstrated by small-interfering RNA–induced PPAR knockdown, while palmitate had no effect. In vivo, lipid infusion induced an increase of circulating FGF-21 in humans, and a strong correlation between the change in FGF-21 levels and the change in FFAs was observed. An artificial hyperinsulinemia, which was induced to delineate the potential interaction between elevated FFAs and hyperinsulinemia, revealed that hyperinsulinemia also increased FGF-21 levels in vivo, while rosiglitazone treatment had no effect. CONCLUSIONS—The results presented here offer a mechanism explaining the induction of the metabolic regulator FGF-21 in the fasting situation but also in type 2 diabetes and obesity