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141.
Sukhija K Pyne M Ali S Orr V Abedi D Moo-Young M Chou CP 《Molecular biotechnology》2012,51(2):109-118
Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method
was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets
(FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P
trc
and P
araB
), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol)
under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation
using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive
genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic
engineering. 相似文献
142.
143.
Linda Larcombe Neeloffer Mookherjee Joyce Slater Caroline Slivinski Matthew Singer Chris Whaley Lizette Denechezhe Sara Matyas Emily Turner-Brannen Peter Nickerson Pamela Orr 《PloS one》2012,7(11)
The wide spectrum of vitamin D activity has focused attention on its potential role in the elevated burden of disease in a northern Canadian First Nations (Dené) cohort. Vitamin D insufficiency, and gene polymorphisms in the vitamin D receptor (VDR) and vitamin D binding protein (VDBP) have been implicated in susceptibility to infectious and chronic diseases. The objectives of this study were to determine the contribution of vitamin D from food, and measure the serum concentrations of 25-hydroxyvitamin D3 (25-OHD3) and VDBP in Dené participants. Single nucleotide polymorphisms (SNPs) associated with the dysregulation of the innate immune response were typed and counted. Potential correlations between the SNPs and serum concentrations of 25-OHD3 and VDBP were evaluated. Venous blood was collected in summer and winter over a one-year period and analyzed for 25-OHD3 and VDBP concentrations (N = 46). A questionnaire was administered to determine the amount of dietary vitamin D consumed. Sixty-one percent and 30% of the participants had 25-OHD3 serum concentrations <75 nmol/L in the winter and summer respectively. Mean vitamin D binding protein concentrations were within the normal range in the winter but below normal in the summer. VDBP and VDR gene polymorphisms affect the bioavailability and regulation of 25-OHD3. The Dené had a high frequency of the VDBP D432E-G allele (71%) and the Gc1 genotype (90%), associated with high concentrations of VDBP and a high binding affinity to 25-OHD3. The Dené had a high frequency of VDR Fok1-f allele (82%), which has been associated with a down-regulated Th1 immune response. VDBP and VDR polymorphisms, and low winter 25-OHD3 serum concentrations may be risk factors for infectious diseases and chronic conditions related to the dysregulation of the vitamin D pathway. 相似文献
144.
Russell J. S. Orr Shauna A. Murray Anke Stüken Lesley Rhodes Kjetill S. Jakobsen 《PloS one》2012,7(11)
The dinoflagellates are a diverse lineage of microbial eukaryotes. Dinoflagellate monophyly and their position within the group Alveolata are well established. However, phylogenetic relationships between dinoflagellate orders remain unresolved. To date, only a limited number of dinoflagellate studies have used a broad taxon sample with more than two concatenated markers. This lack of resolution makes it difficult to determine the evolution of major phenotypic characters such as morphological features or toxin production e.g. saxitoxin. Here we present an improved dinoflagellate phylogeny, based on eight genes, with the broadest taxon sampling to date. Fifty-five sequences for eight phylogenetic markers from nuclear and mitochondrial regions were amplified from 13 species, four orders, and concatenated phylogenetic inferences were conducted with orthologous sequences. Phylogenetic resolution is increased with addition of support for the deepest branches, though can be improved yet further. We show for the first time that the characteristic dinoflagellate thecal plates, cellulosic material that is present within the sub-cuticular alveoli, appears to have had a single origin. In addition, the monophyly of most dinoflagellate orders is confirmed: the Dinophysiales, the Gonyaulacales, the Prorocentrales, the Suessiales, and the Syndiniales. Our improved phylogeny, along with results of PCR to detect the sxtA gene in various lineages, allows us to suggest that this gene was probably acquired separately in Gymnodinium and the common ancestor of Alexandrium and Pyrodinium and subsequently lost in some descendent species of Alexandrium. 相似文献
145.
146.
Mullen DG Desai AM Waddell JN Cheng XM Kelly CV McNerny DQ Majoros IJ Baker JR Sander LM Orr BG Banaszak Holl MM 《Bioconjugate chemistry》2008,19(9):1748-1752
Stochastic synthesis of a ligand coupled to a nanoparticle results in a distribution of populations with different numbers of ligands per nanoparticle. This distribution was resolved and quantified using HPLC and is in excellent agreement with the ligand/nanoparticle average measured by 1H NMR, gel permeation chromatography (GPC), and potentiometric titration, and yet significantly more disperse than commonly held perceptions of monodispersity. Two statistical models were employed to confirm that the observed heterogeneity is consistent with theoretical expectations. 相似文献
147.
Shalchian-Tabrizi K Minge MA Espelund M Orr R Ruden T Jakobsen KS Cavalier-Smith T 《PloS one》2008,3(5):e2098
Animals are evolutionarily related to fungi and to the predominantly unicellular protozoan phylum Choanozoa, together known as opisthokonts. To establish the sequence of events when animals evolved from unicellular ancestors, and understand those key evolutionary transitions, we need to establish which choanozoans are most closely related to animals and also the evolutionary position of each choanozoan group within the opisthokont phylogenetic tree. Here we focus on Ministeria vibrans, a minute bacteria-eating cell with slender radiating tentacles. Single-gene trees suggested that it is either the closest unicellular relative of animals or else sister to choanoflagellates, traditionally considered likely animal ancestors. Sequencing thousands of Ministeria protein genes now reveals about 14 with domains of key significance for animal cell biology, including several previously unknown from deeply diverging Choanozoa, e.g. domains involved in hedgehog, Notch and tyrosine kinase signaling or cell adhesion (cadherin). Phylogenetic trees using 78 proteins show that Ministeria is not sister to animals or choanoflagellates (themselves sisters to animals), but to Capsaspora, another protozoan with thread-like (filose) tentacles. The Ministeria/Capsaspora clade (new class Filasterea) is sister to animals and choanoflagellates, these three groups forming a novel clade (filozoa) whose ancestor presumably evolved filose tentacles well before they aggregated as a periciliary collar in the choanoflagellate/sponge common ancestor. Our trees show ichthyosporean choanozoans as sisters to filozoa; a fusion between ubiquitin and ribosomal small subunit S30 protein genes unifies all holozoa (filozoa plus Ichthyosporea), being absent in earlier branching eukaryotes. Thus, several successive evolutionary innovations occurred among their unicellular closest relatives prior to the origin of the multicellular body-plan of animals. 相似文献
148.
Mannaioni G Orr AG Hamill CE Yuan H Pedone KH McCoy KL Berlinguer Palmini R Junge CE Lee CJ Yepes M Hepler JR Traynelis SF 《The Journal of biological chemistry》2008,283(29):20600-20611
Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons. 相似文献
149.
Wear paths produced by individual hip-replacement patients--a large-scale, long-term follow-up study
Bennett D Humphreys L O'Brien S Kelly C Orr JF Beverland DE 《Journal of biomechanics》2008,41(11):2474-2482
Wear particle accumulation is one of the main contributors to osteolysis and implant failure in hip replacements. Altered kinematics produce significant differences in wear rates of hip replacements in simulator studies due to varying degrees of multidirectional motion. Gait analysis data from 153 hip-replacement patients 10-years post-operation were used to model two- and three-dimensional wear paths for each patient. Wear paths were quantified in two dimensions using aspect ratios and in three dimensions using the surface areas of the wear paths, with wear-path surface area correlating poorly with aspect ratio. The average aspect ratio of the patients wear paths was 3.97 (standard deviation=1.38), ranging from 2.13 to 10.86. Sixty percent of patients displayed aspect ratios between 2.50 and 3.99. However, 13% of patients displayed wear paths with aspect ratios >5.5, which indicates reduced multidirectional motion. The majority of total hip replacement (THR) patients display gait kinematics which produce multidirectional wear paths, but a significant minority display more linear paths. 相似文献
150.