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981.
Michael Y. Sha Mark Yamanaka Ian D. Walton Scott M. Norton Rebecca L. Stoermer Christine D. Keating Michael J. Natan Sharron G. Penn 《NanoBioTechnology》2005,1(4):327-335
In this paper we describe a molecular beacon format assay in which encoded nanowire particles are used to achieve multiplexing.
We demonstrate this principle with the detection of five viral pathogens; Hepatitis A virus, Hepatitis C virus, West Nile
Virus, Human Immune Deficiency virus and Severe Acute Respiratory Syndrome virus. Oligonucleotides are designed complementary
to a target sequence of interest containing a 3′ universal fluorescence dye. A 5′ thiol causes the oligonucleotides to self-assemble
onto the metal nanowire. The single-stranded oligonucleotide contains a self-complementary hairpin stem sequence of 10 bases
that forces the 3′ fluorophore to come into contact with the metallic nanowire surface, thereby quenching the fluorescence.
Upon addition of target DNA, there is hybridization with the complementary oligonucleotides. The resulting DNA hybrid is rigid,
unfolds the hairpin structure, and causes the fluorophore to be moved away from the surface such that it is no longer quenched.
By using differently encoded nanowires, each conjugated with a different oligonucleotide sequence, multiplexed DNA assays
are possible using a single fluorophore, from a multiplexed RT-PCR reaction. 相似文献
982.
983.
984.
Changwang Zhang Shi Zhou Elisabetta Groppelli Pierre Pellegrino Ian Williams Persephone Borrow Benjamin M. Chain Clare Jolly 《PLoS computational biology》2015,11(4)
HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments’ influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies. 相似文献
985.
986.
987.
Several vector systems are available for tissue-specific transactivation or chemical induction of transgene expression in plants. The choice facing researchers is which promoter system to commit to as this determines the range and characteristics of the expression resources available. The decision will not be the same for all species or applications. We present some general discussion on the use of these technologies and review in detail the properties in various (mainly angiosperm) species of the most promising: mGal4:VP16/UAS and pOp/LhG4 for transactivation, and the alc-switch, GVE/VGE, GVG, pOp6/LhGR, and XVE systems for chemical induction. 相似文献
988.
Gateway technology is a powerful system for converting a single entry vector into a wide variety of expression vectors. We expressed recombinant influenza matrix protein M1 (FMP), a potent antigen for cytotoxic T cells, using the Gateway vector pET-DEST42 containing the FMP cDNA, and purified the expressed FMP as a single 32 kDa recombinant protein. N-terminal and internal protein sequencing, however, showed that the recombinant FMP contained an extra 10 amino acids fused to the N-terminal of native FMP. Further investigation of the DNA sequence adjacent to the 5'-FMP cDNA indicated that the "TTG" in the attB1 site (30 bp upstream of the "ATG" in the 5'-FMP cDNA) behaved as a dominant translation start site, resulting in a 10 amino acid extension of the recombinant FMP. Thus, it is possible that recombinant proteins produced by this Gateway vector contain unexpected vector-derived peptides, which may affect experimental outcomes. 相似文献
989.
Starch synthesis and CO2 evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with 14C-labelled hexose-phosphates. Amyloplasts converted [U-14C]glucose 1-phosphate (Glc1P) but not [U-14C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was
stimulated, both [U-14C]Glc1P and [U-14C]Glc6P were metabolized to CO2, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand
the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured
in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts
incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When
amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux
through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites
involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate
oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase
is significantly displaced from equilibrium.
Received: 29 March 1997 / Accepted: 5 June 1997 相似文献
990.
Juli O'Connor Dean F. Revell Karen E. Masters Ian F. Connerton Ian G. Sumner 《Protein expression and purification》1996,7(4):377-383
The gene encoding the 67-kDa cocoa storage protein precursor has been cloned fromTheobroma cacaoand expressed inEscherichia coliusing the pET expression system. The recombinant storage protein has been renatured from inclusion bodies at 30°C using 20 m
glycine–NaOH buffer, pH 10.0, containing 1 m
oxidized glutathione and 0.1% Brij. The renatured protein was purified and demonstrated to adopt a stable native conformation by optical spectroscopy. Secondary structure analysis from circular dichroism indicated the protein to be 23% α-helix and 38% β-sheet, in close agreement with values obtained using a secondary structure prediction program. 相似文献