Tonnesenfranco3549

Although Escherichia coli has been a popular tool for plasmid construction, this bacterium was believed to be "unsuitable" for constructing a large plasmid whose size exceeds 500 kilobases. We assumed that traditional plasmid vectors may lack some regulatory DNA elements required for the stable replication and segregation of such a large plasmid. In addition, the use of a few site-specific recombination systems may facilitate cloning of large DNA segments. selleck chemicals llc Here we show two strategies for constructing 1-megabase (1-Mb) secondary chromosomes by using new bacterial artificial chromosome (BAC) vectors. First, the 3-Mb genome of a genome-reduced E. coli strain was split into two chromosomes (2-Mb and 1-Mb), of which smaller one has the origin of replication and the partitioning locus of the Vibrio tubiashii secondary chromosome. This chromosome fission method (Flp-POP cloning) works via flippase-mediated excision which coincides with the reassembly of a split chloramphenicol resistance gene, allowing chloramphenicol selection. Next, we developed a new cloning method (oriT-POP cloning) and a fully-equipped BAC vector (pMegaBAC1H) for developing a 1-Mb plasmid. Two 0.5-Mb genomic regions were sequentially transferred from two donor strains to a recipient strain via conjugation and captured by pMegaBAC1H in the recipient strain to produce a 1-Mb plasmid. This 1-Mb plasmid was transmissible to another E. coli strain via conjugation. Furthermore, these 1-Mb secondary chromosomes were amplifiable in vitro by using the reconstituted E. coli chromosome replication cycle reaction (RCR). These strategies and technologies would make popular E. coli cells a productive factory for designer chromosome engineering.Aims Interleukin-13 (IL-13) is a Th2-associated cytokine that typically induces gut contractility and mucus secretion to eliminate helminth parasites from the digestive tract. Little evidence exists of IL-13's direct effect on Haemonchus contortus larvae (L3), and thus were the objective of this study. Methods To test effects of IL-13 on H. contortus, L3 were treated with ovine recombinant (r)IL-13 (1 μg/ml); motility and morbidity were assessed. Monocytes isolated from H. contortus-resistant St. Croix (STC) and -susceptible Suffolk (SUF) sheep were treated with anti-IL-13 blocking antibody to elucidate differences in host immune response. Results rIL-13 treatment reduced L3 speed (27μm/s) and distance (7.5 μm) compared to untreated L3 (speed 94 μm/s; distance 27 μm) (P less then 0.001). Comparison of larval speed to known paralytic levamisole (LEV) revealed no difference between treatments (rIL13 23 μm/s; LEV 27 μm/s). Additionally, rIL-13 had no effect on larval morbidity. Blocking IL-13 reduced monocyte-driven larval morbidity (0.13 μM ATP) and increased larval motility (88 μm/s; 27 μm) compared to larvae treated with STC-monocytes alone (0.07 μM ATP; 34 μm/s; 8 μm) (P less then 0.05). Conclusions These data indicate IL-13 has a dual capability paralyzing L3 and contributing to monocyte-driven larval morbidity, and also indicate breed differences.The adult visual system was traditionally thought to be relatively hard-wired, but recent studies have challenged this view by demonstrating plasticity following short-term monocular deprivation. Depriving one eye of spatial information for 2-3 h increased subsequent sensory dominance of that eye. However, the mechanism underlying this phenomenon is unclear. The present study sought to address this issue and determine the consequences of short-term monocular deprivation on inter-ocular suppression of each eye. Sensory eye dominance was examined before and after depriving an eye of all input using an opaque patch for 2.5 h, in six adult participants with normal binocular vision. We used a percept tracking task during binocular rivalry (BR) to assess the relative eye dominance, and an objective probe detection task under continuous flash suppression (CFS) to quantify each eye's susceptibility to inter-ocular suppression. The monocular contrast increment threshold of each eye was also measured using the probe task to ascertain if the altered eye dominance is accompanied by changes in monocular perception. Our BR results replicated previous findings of a shift of relative dominance towards the eye that has been deprived of form information. More crucially, using CFS we demonstrated reduced inter-ocular suppression of the deprived eye with no complementary changes in the other eye, and no monocular changes in increment threshold. These findings imply that short-term monocular deprivation alters binocular interactions. The differential effect on inter-ocular suppression between eyes may have important implications for the use of patching as a therapy to recover visual function in amblyopia.Grating-based X-ray phase-contrast imaging provides three simultaneous image channels originating from a single image acquisition. While the phase signal provides direct access to the electron density in tomography, there is additional information on sub-resolutional structural information which is called dark-field signal in analogy to optical microscopy. The additional availability of the conventional attenuation image qualifies the method for implementation into existing diagnostic routines. The simultaneous access to the attenuation coefficient and the electron density allows for quantitative two-material discrimination as demonstrated lately for measurements at a quasi-monochromatic compact synchrotron source. Here, we investigate the transfer of the method to conventional polychromatic X-ray sources and the additional inclusion of the dark-field signal for three-material decomposition. We evaluate the future potential of grating-based X-ray phase-contrast CT for quantitative three-material discrimination for the specific case of early stroke diagnosis at conventional polychromatic X-ray sources. Compared to conventional CT, the method has the potential to discriminate coagulated blood directly from contrast agent extravasation within a single CT acquisition. Additionally, the dark-field information allows for the clear identification of hydroxyapatite clusters due to their micro-structure despite a similar attenuation as the applied contrast agent. This information on materials with sub-resolutional microstructures is considered to comprise advantages relevant for various pathologies.