Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment

The protocols described here are comprehensive instructions for deriving human embryonic stem (hES) cell lines in xeno-free conditions from cryopreserved embryos. Details are included for propagation, cryopreservation and characterization. Initial derivation is on feeder cells and is followed by adaptation to a feeder-free environment; competent technicians can perform these simplified methods easily. From derivation to cryopreservation of fully characterized initial stocks takes 3-4 months. These protocols served as the basis for standard operating procedures (SOPs), with both operational and technical components, that we set to meet good manufacturing practice (GMP) and UK regulatory body requirements for derivation of clinical-grade cells. As such, these SOPs are currently used in our current GMP compliant facility to derive hES cell lines ab initio, in an animal product-free environment; these lines are suitable for research and potentially for clinical use in cell therapy. So far, we have derived eight clinical-grade lines, which will be freely available to the scientific community after submission/accession to the UK Stem Cell Bank.     

Molecular population genetics of ref(2)P, a locus which confers viral resistance in Drosophila

The ref(2)P locus (2-54.2) is polymorphic for two allelic forms in natural populations of Drosophila melanogaster, ref(2)Po and ref(2)Pp. The latter allele confers resistance to the rhabdovirus sigma infecting wild populations. Previous work, based on a small sample of prescreened restrictive (resistant) and permissive (susceptible) alleles, identified a large number of amino acid replacement changes (7) relative to synonymous changes (1). Such protein variability could be the result of variation-enhancing selection. To further test the selection hypothesis, we have examined the DNA sequences of ten randomly chosen lines of D. melanogaster and one line of D. simulans. Nine of the ten lines are permissive; D. simulans does not harbor the virus. The melanogaster alleles contain 4 synonymous changes, 19 noncoding changes, and 13 amino acid replacement changes, indicating a relatively high level of polymorphism. Three sequenced restrictive alleles have nearly identical sequences, indicating that they are relatively young. Compared to the permissive alleles, they share only a complex deletion at codon 34, CAG-AAT to GGA, which our analysis indicates to be the site conferring the restrictive phenotype. Patterns of polymorphism and divergence differ from neutral predictions by several criteria for the amino terminal region, which contains the complex deletion (codons 1-91), but not the remainder of the protein (codons 92-599). We find a higher rate of evolution on the D. melanogaster lineage than on the D. simulans lineage. The relatively large amount of both replacement and silent polymorphism in the permissive alleles and the lack of divergence between permissive and restrictive alleles suggests that the sigma virus and ref(2)P may be engaged in an evolutionary race in which new restrictive alleles are continually arising but are relatively short-lived.      

Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers

Considerable interest has been focused on the role of myosin light chain LC(2) in the contraction of vertebrate striated muscle. A study was undertaken to further our investigations (Moss, R.L., G.G. Giulian, and M.L. Greaser, 1981, J. Biol. Chem., 257:8588-8591) of the effects of LC(2) removal upon contraction in skinned fibers from rabbit psoas muscles. Isometric tension and maximum velocity of shortening, V(max), were measured in fiber segments prior to LC(2) removal. The segments were then bathed at 30 degrees C for up to 240 min in a buffer solution containing 20 mM EDTA in order to extract up to 60 percent of the LC(2). Troponin C (TnC) was also partially removed by this procedure. Mechanical measurements were done following the EDTA extraction and the readditions of first TnC and then LC(2) to the segments. The protein subunit compositions of the same fiber segments were determined following each of these procedures by SDS PAGE of small pieces of the fiber. V(max) was found to decrease as the LC(2) content of the fiber segments was reduced by increasing the duration of extraction. EDTA treatment also resulted in substantial reductions in tension due mainly to the loss of TnC, though smaller reductions due to the extraction of LC(2) were also observed. Reversal of the order of recombination of LC(2) and TnC indicated that the reduction in V(max) following EDTA treatment was a specific effect of LC(2) removal. These results strongly suggest that LC(2) may have roles in determining the kinetics and extent of interaction between myosin and actin.     

Role of vitamin E in glutathione-induced oxidant stress: methemoglobin, lipid peroxidation, and hemolysis.

Red blood cells (RBC) from normal and vitamin E-deficient rats were incubated in a hypertonic solution of reduced glutathione adjusted to pH 8. Methemoglobin formation occurred in intact RBC from both normal and vitamin E-deficient rats. Hemolysis was significantly greater in RBC from vitamin E-deficient rats. Experiments with catalase, superoxide dismutase, and methional showed that H(2)O(2) was the primary extracellular source of oxidant stress. Extracellular superoxide and hydroxyl radical were not involved in oxidant stress. Experiments with dimethyl sulfoxide showed that intracellular hydroxyl radical, generated from H(2)O(2), was the hemolytic agent. Neither methemoglobin formation nor lipid peroxidation involved hydroxyl radical. Indeed, lipid peroxidation and hemolysis in RBC from vitamin E-deficient rats were concurrent rather than consecutive events. Phase contrast microscopy showed that rigid, crenated RBC with a precipitate around the interior periphery formed during glutathione-induced oxidant stress. The precipitate dissolved slowly as the crenated RBC were converted to smooth ghosts. It appeared that protein precipitates involving mixed disulfide bonds were reduced and solubilized when extracellular glutathione penetrated the ruptured cell. Comparisons between normal RBC and vitamin E-deficient RBC suggest that vitamin E has little effect on the inward diffusion of extra-cellular H(2)O(2). Vitamin E apparently interacts with different oxidant species derived from intracellular H(2)O(2) in preventing lipid peroxidation and the sulfhydryl group oxidation leading to hemolysis.     

Expression of the bilateral deficit during reflexively evoked contractions.

During maximal contractions, the sum of forces exerted by homonymous muscles unilaterally is typically larger than the sum of forces exerted by the same muscles bilaterally. This phenomenon is known as the bilateral deficit (BLD), and it is suggested that this deficit is due to neural inhibition. It remains unclear, however, whether such inhibition is mediated by supraspinal mechanisms or by reflex pathways at the level of spinal cord. To further study the origin of likely neural influences, we tested for the presence of BLD under the condition of reflexive force generation. Force output and integrated electromyogram (iEMG) (quadriceps femoris) were measured in 17 male participants after initiation of the myotatic patellar reflex under unilateral and bilateral conditions. A significant BLD of 9.26 +/- 1.19 (P = 0.004) and 16.76 +/- 4.69% (P = 0.001) was found for force and iEMG, respectively. However, because similar findings were not evident during maximal isometric knee extensions, it is difficult to predict the contribution of a spinal mechanism to the BLD under the condition of maximal voluntary activation.     

A Geographic Mosaic of Climate Change Impacts on Terrestrial Vegetation: Which Areas Are Most at Risk?

Changes in climate projected for the 21^st century are expected to trigger widespread and pervasive biotic impacts. Forecasting these changes and their implications for ecosystem services is a major research goal. Much of the research on biotic responses to climate change has focused on either projected shifts in individual species distributions or broad-scale changes in biome distributions. Here, we introduce a novel application of multinomial logistic regression as a powerful approach to model vegetation distributions and potential responses to 21^st century climate change. We modeled the distribution of 22 major vegetation types, most defined by a single dominant woody species, across the San Francisco Bay Area. Predictor variables included climate and topographic variables. The novel aspect of our model is the output: a vector of relative probabilities for each vegetation type in each location within the study domain. The model was then projected for 54 future climate scenarios, spanning a representative range of temperature and precipitation projections from the CMIP3 and CMIP5 ensembles. We found that sensitivity of vegetation to climate change is highly heterogeneous across the region. Surprisingly, sensitivity to climate change is higher closer to the coast, on lower insolation, north-facing slopes and in areas of higher precipitation. While such sites may provide refugia for mesic and cool-adapted vegetation in the face of a warming climate, the model suggests they will still be highly dynamic and relatively sensitive to climate-driven vegetation transitions. The greater sensitivity of moist and low insolation sites is an unexpected outcome that challenges views on the location and stability of climate refugia. Projections provide a foundation for conservation planning and land management, and highlight the need for a greater understanding of the mechanisms and time scales of potential climate-driven vegetation transitions.     

Brittle ductile transition in carbon nanotube bundles

The superior strength and stiffness of carbon nanotubes (CNTs) make them attractive for many structural applications. Although the strength and stiffness of CNTs are extremely high, fibres of aligned CNTs have been found to date to be far weaker than the constituent CNTs. The intermolecular interactions between the CNTs in the fibres are governed by weak van der Waals forces, resulting in slippage between CNTs which occurs at tensions well below the breaking strength of the CNTs. Both theoretical and experimental studies show that by introducing chemical bonds between the CNTs increases load transfer and prevents the CNTs from slipping.     

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.