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161.
Pollyanna A. Goh Sara Caxaria Catharina Casper Cecilia Rosales Thomas T. Warner Pete J. Coffey Amit C. Nathwani 《PloS one》2013,8(11)
A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells. 相似文献
162.
Raymond J. Holt Alexis S. Lefevre Ian J. Flatters Pete Culmer Richard M. Wilkie Brian W. Henson Geoff P. Bingham Mark Mon-Williams 《PloS one》2013,8(7)
Old age is associated with reduced mobility of the hand. To investigate age related decline when reaching-to-lift an object we used sophisticated kinematic apparatus to record reaches carried out by healthy older and younger participants. Three objects of different widths were placed at three different distances, with objects having either a high or low friction surface (i.e. rough or slippery). Older participants showed quantitative differences to their younger counterparts – movements were slower and peak speed did not scale with object distance. There were also qualitative differences with older adults showing a greater propensity to stop the hand and adjust finger position before lifting objects. The older participants particularly struggled to lift wide slippery objects, apparently due to an inability to manipulate their grasp to provide the level of precision necessary to functionally enclose the object. These data shed light on the nature of age related changes in reaching-to-grasp movements and establish a powerful technique for exploring how different product designs will impact on prehensile behavior. 相似文献
163.
Marion Pfeifer Bertrand Schatz F. Xavier Pic Nicodemo G. Passalacqua Michael F. Fay Pete D. Carey Florian Jeltsch 《Journal of Biogeography》2009,36(12):2353-2365
Aim This study aims to link demographic traits and post‐glacial recolonization processes with genetic traits in Himantoglossum hircinum (L.) Spreng (Orchidaceae), and to test the implications of the central–marginal concept (CMC) in Europe. Location Twenty sites covering the entire European distribution range of this species. Methods We employed amplified fragment length polymorphism (AFLP) markers and performed a plastid microsatellite survey to assess genetic variation in 20 populations of H. hircinum located along central–marginal gradients. We measured demographic traits to assess population fitness along geographical gradients and to test for correlations between demographic traits and genetic diversity. We used genetic diversity indices and analyses of molecular variance (AMOVA) to test hypotheses of reduced genetic diversity and increased genetic differentiation and isolation from central to peripheral sites. We used Bayesian simulations to analyse genetic relationships among populations. Results Genetic diversity decreased significantly with increasing latitudinal and longitudinal distance from the distribution centre when excluding outlying populations. The AMOVA revealed significant genetic differentiation among populations (FST = 0.146) and an increase in genetic differentiation from the centre of the geographical range to the margins (except for the Atlantic group). Population fitness, expressed as the ratio NR/N, decreased significantly with increasing latitudinal distance from the distribution centre. Flower production was lower in most eastern peripheral sites. The geographical distribution of microsatellite haplotypes suggests post‐glacial range expansion along three major migratory pathways, as also supported by individual membership fractions in six ancestral genetic clusters (C1–C6). No correlations between genetic diversity (e.g. diversity indices, haplotype frequency) and population demographic traits were detected. Main conclusions Reduced genetic diversity and haplotype frequency in H. hircinum at marginal sites reflect historical range expansions. Spatial variation in demographic traits could not explain genetic diversity patterns. For those sites that did not fit into the CMC, the genetic pattern is probably masked by other factors directly affecting either demography or population genetic structure. These include post‐glacial recolonization patterns and changes in habitat suitability due to climate change at the northern periphery. Our findings emphasize the importance of distinguishing historical effects from those caused by geographical variation in population demography of species when studying evolutionary and ecological processes at the range margins under global change. 相似文献
164.
Tae-Jin Lee Pete E. Pascuzzi Sharon B. Settlage Randall W. Shultz Milos Tanurdzic Pablo D. Rabinowicz Margit Menges Ping Zheng Dorrie Main James A. H. Murray Bryon Sosinski George C. Allen Robert A. Martienssen Linda Hanley-Bowdoin Matthew W. Vaughn William F. Thompson 《PLoS genetics》2010,6(6)
DNA replication programs have been studied extensively in yeast and animal systems, where they have been shown to correlate with gene expression and certain epigenetic modifications. Despite the conservation of core DNA replication proteins, little is known about replication programs in plants. We used flow cytometry and tiling microarrays to profile DNA replication of Arabidopsis thaliana chromosome 4 (chr4) during early, mid, and late S phase. Replication profiles for early and mid S phase were similar and encompassed the majority of the euchromatin. Late S phase exhibited a distinctly different profile that includes the remaining euchromatin and essentially all of the heterochromatin. Termination zones were consistent between experiments, allowing us to define 163 putative replicons on chr4 that clustered into larger domains of predominately early or late replication. Early-replicating sequences, especially the initiation zones of early replicons, displayed a pattern of epigenetic modifications specifying an open chromatin conformation. Late replicons, and the termination zones of early replicons, showed an opposite pattern. Histone H3 acetylated on lysine 56 (H3K56ac) was enriched in early replicons, as well as the initiation zones of both early and late replicons. H3K56ac was also associated with expressed genes, but this effect was local whereas replication time correlated with H3K56ac over broad regions. The similarity of the replication profiles for early and mid S phase cells indicates that replication origin activation in euchromatin is stochastic. Replicon organization in Arabidopsis is strongly influenced by epigenetic modifications to histones and DNA. The domain organization of Arabidopsis is more similar to that in Drosophila than that in mammals, which may reflect genome size and complexity. The distinct patterns of association of H3K56ac with gene expression and early replication provide evidence that H3K56ac may be associated with initiation zones and replication origins. 相似文献
165.
Raymond Campbell Simon D. A. Pont Jenny A. Morris Gaynor McKenzie Sanjeev Kumar Sharma Pete E. Hedley Gavin Ramsay Glenn J. Bryan Mark A. Taylor 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2014,127(9):1917-1933
Key message
Genome-wide QTL analysis of potato tuber carotenoid content was investigated in populations of Solanum tuberosum Group Phureja that segregate for flesh colour, revealing a novel major QTL on chromosome 9.Abstract
The carotenoid content of edible plant storage organs is a key nutritional and quality trait. Although the structural genes that encode the biosynthetic enzymes are well characterised, much less is known about the factors that determine overall storage organ content. In this study, genome-wide QTL mapping, in concert with an efficient ‘genetical genomics’ analysis using bulked samples, has been employed to investigate the genetic architecture of potato tuber carotenoid content. Two diploid populations of Solanum tuberosum Group Phureja were genotyped (AFLP, SSR and DArT markers) and analysed for their tuber carotenoid content over two growing seasons. Common to both populations were QTL that explained relatively small proportions of the variation in constituent carotenoids and a major QTL on chromosome 3 explaining up to 71 % of the variation in carotenoid content. In one of the populations (01H15), a second major carotenoid QTL was identified on chromosome 9, explaining up to 20 % of the phenotypic variation. Whereas the major chromosome 3 QTL was likely to be due to an allele of a gene encoding β-carotene hydroxylase, no known carotenoid biosynthetic genes are located in the vicinity of the chromosome 9 QTL. A unique expression profiling strategy using phenotypically distinct bulks comprised individuals with similar carotenoid content provided further support for the QTL mapping to chromosome 9. This study shows the potential of using the potato genome sequence to link genetic maps to data arising from eQTL approaches to enhance the discovery of candidate genes underlying QTLs. 相似文献166.
167.
Summary . Statistical methods have been developed and applied to estimating populations that are difficult or too costly to enumerate. Known as multilist methods in epidemiological settings, individuals are matched across lists and estimation of population size proceeds by modeling counts in incomplete multidimensional contingency tables (based on patterns of presence/absence on lists). As multilist methods typically assume that lists are compiled instantaneously, there are few options available for estimating the unknown size of a closed population based on continuously (longitudinally) compiled lists. However, in epidemiological settings, continuous time lists are a routine byproduct of administrative functions. Existing methods are based on time-to-event analyses with a second step of estimating population size. We propose an alternative approach to address the twofold epidemiological problem of estimating population size and of identifying patient factors related to duration (in days) between visits to a health care facility. A Bayesian framework is proposed to model interval lengths because, for many patients, the data are sparse; many patients were observed only once or twice. The proposed method is applied to the motivating data to illustrate the methods' applicability. Then, a small simulation study explores the performance of the estimator under a variety of conditions. Finally, a small discussion section suggests opportunities for continued methodological development for continuous time population estimation. 相似文献
168.
Adventitious shoot regeneration from leaf explants of southern highbush blueberry cultivars 总被引:1,自引:0,他引:1
Cheng Liu Pete Callow Lisa J. Rowland James F. Hancock Guo-qing Song 《Plant Cell, Tissue and Organ Culture》2010,103(1):137-144
Protocols were developed to optimize adventitious shoot regeneration from four southern highbush blueberry cultivars. Leaf
explants from 6 week-old shoots of the four cultivars were excised and cultured on woody plant medium each containing thidiazuron
(4.54 or 9.08 μM), zeatin (18.2 μM), or zeatin riboside (5.7 or 11.4 μM) either separately or in combination with α-naphthaleneacetic
acid at 2.69 μM. Optimum medium for shoot regeneration was genotype-dependent. Efficient regeneration was obtained at frequencies
of 88.9% for ‘Jewel’, 87.8% for ‘Emerald’, 53.3% for ‘Jubilee’ and 87.8% for ‘Biloxi’. Leaf explants of newly developed shoots
from the cultures having undergone five subcultures had higher regeneration frequencies than those having undergone two subcultures.
Regenerated shoots, 80–100% for each cultivar, rooted in 8 weeks after transplantation to soil. The regeneration systems described
have potential use in genetic transformation of southern highbush blueberry cultivars. 相似文献
169.
A need exists for broadly applicable biomarkers of pregnancy outcome in population-based studies that assess environmental hazards to human reproduction. Previous studies have demonstrated that during the periimplantation period, measures of the circulating levels of immunoreactive hCG (IhCG) are not predictive of pregnancy outcome, whereas measurements of the circulating levels of bioactive hCG (BhCG) provide information relating to pregnancy outcome and might provide the basis for an early biomarker of pregnancy outcome. However, for this biomarker to have broad application in population-based studies, it must be adapted to urinary hCG metabolites. The principle objective of the present study was to characterize the periimplantation excretion patterns of urinary hCG metabolites of pregnancies that resulted in live birth (LB), early pregnancy loss (EPL), and recognized clinical abortion (CAB) with an immunoenzymometric assay specific to intact hCG and an LH/chorionic gonadotropin cellular bioassay as the basis for a preliminary comparison between successful (LB) and failing (EPL and CAB) outcome groups. Automated immunoassays for FSH and hCG were used to define each conceptive cycle's implantation window. The timing of first hCG detection was significantly later for the EPL group. Pregnancies that resulted in LB had consistently rising average daily IhCG and BhCG levels, with no significant differences when average daily IhCG and BhCG measurements were compared (Student t-test, P>0.05), whereas pregnancies that resulted in CAB and EFL had lower average daily IhCG and BhCG levels that increased inconsistently. These findings demonstrate that critical information related to pregnancy outcome may be present when multiple urinary hCG isoforms are measured. Further data suggest that the rate of change for the ratio of daily BhCG over IhCG levels might be useful as the basis of a broadly applicable early biomarker for pregnancy outcome. 相似文献
170.
Phenol hydroxylase (PH) belongs to a family of bacterial multicomponent monooxygenases (BMMs) with carboxylate-bridged diiron active sites. Included are toluene/o-xylene (ToMO) and soluble methane (sMMO) monooxygenase. PH hydroxylates aromatic compounds, but unlike sMMO, it cannot oxidize alkanes despite having a similar dinuclear iron active site. Important for activity is formation of a complex between the hydroxylase and a regulatory protein component. To address how structural features of BMM hydroxylases and their component complexes may facilitate the catalytic mechanism and choice of substrate, we determined X-ray structures of native and SeMet forms of the PH hydroxylase (PHH) in complex with its regulatory protein (PHM) to 2.3 A resolution. PHM binds in a canyon on one side of the (alphabetagamma)2 PHH dimer, contacting alpha-subunit helices A, E, and F approximately 12 A above the diiron core. The structure of the dinuclear iron center in PHH resembles that of mixed-valent MMOH, suggesting an Fe(II)Fe(III) oxidation state. Helix E, which comprises part of the iron-coordinating four-helix bundle, has more pi-helical character than analogous E helices in MMOH and ToMOH lacking a bound regulatory protein. Consequently, conserved active site Thr and Asn residues translocate to the protein surface, and an approximately 6 A pore opens through the four-helix bundle. Of likely functional significance is a specific hydrogen bond formed between this Asn residue and a conserved Ser side chain on PHM. The PHM protein covers a putative docking site on PHH for the PH reductase, which transfers electrons to the PHH diiron center prior to O2 activation, suggesting that the regulatory component may function to block undesired reduction of oxygenated intermediates during the catalytic cycle. A series of hydrophobic cavities through the PHH alpha-subunit, analogous to those in MMOH, may facilitate movement of the substrate to and/or product from the active site pocket. Comparisons between the ToMOH and PHH structures provide insights into their substrate regiospecificities. 相似文献