Proton-Detected Solid-State NMR of the Cell-Free Synthesized α-Helical Transmembrane Protein NS4B from Hepatitis C Virus

Proton-detected 100 kHz magic-angle-spinning (MAS) solid-state NMR is an emerging analysis method for proteins with only hundreds of microgram quantities, and thus allows structural investigation of eukaryotic membrane proteins. This is the case for the cell-free synthesized hepatitis C virus (HCV) nonstructural membrane protein 4B (NS4B). We demonstrate NS4B sample optimization using fast reconstitution schemes that enable lipid-environment screening directly by NMR. 2D spectra and relaxation properties guide the choice of the best sample preparation to record 2D ¹H-detected ¹H,¹⁵N and 3D ¹H,¹³C,¹⁵N correlation experiments with linewidths and sensitivity suitable to initiate sequential assignments. Amino-acid-selectively labeled NS4B can be readily obtained using cell-free synthesis, opening the door to combinatorial labeling approaches which should enable structural studies.     

On keys' meanings and modes: the impact of different key solutions on children's efficiency using a mobile phone

The present study investigates the impact of different key solutions of mobile phones on users' effectiveness and efficiency using the devices. In the first experiment, 36 children (9 - 14 years) and in the second experiment 45 young adults (19 - 33 years) completed four common phone tasks twice consecutively on three simulated phones that had identical menus, but different key solutions. An approach was undertaken to quantify the complexity of keys in three models, incorporating different factors contributing to the keys' complexity (number of key options, number of modes and number of modes with a semantically dissimilar meaning), in order to predict users' performance decrements. As a further main factor, the degree of the users' locus of control (LOC) was measured and interactions with performance outcomes were studied. As dependent measures, the number of inefficient keystrokes, the number of tasks solved and the processing time were determined. Results showed a significant effect of control key solutions on users' efficiency and effectiveness for both children and young adults. Moreover, children's LOC values significantly interacted with performance: children with low LOC values showed the lowest performance and no learnability, especially when using keys with a high complexity. From the three factors contributing to the complexity of keys, keys exerting different functions with semantically inconsistent meanings had the worst effect on performance. It is concluded that in mobile user interface design keys with semantically inconsistent meanings should be generally avoided.     

Expansion in bioreactors of human progenitor populations from cord blood and mobilized peripheral blood

Umbilical cord blood (UCB) and mobilized peripheral blood (MPB) provide an alternate source to bone marrow for transplantation. Expansion in vitro of stem/progenitor cell populations from these sources may provide adult-sized grafts otherwise not attainable because of the limited cell numbers available in the case of UCB or because of numerous rounds of apheresis required for sufficient MPB cells. We asked whether continuous perfusion culture could be employed in ex vivo expansion to produce clinically relevant numbers of stem/progenitor cells from these sources. To evaluate MPB, 1-10 million leukocytes, from patients who had received either granulocyte colony-stimulating factor (G-CSF) or cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF), were inoculated into bioreactors, with or without irradiated, allogeneic stroma. The growth factor combination in the perfusion medium consisted of interleukin-3 (IL-3), stem cell factor (SCF), GM-CSF and erythropoietin (Epo). Under the best conditions tested, total cell numbers, granulocyte-macrophage colony-forming units (CFU-GM), and long-term culture-initiating cell (LTC-IC) populations were expanded by about 50-, 80-, and 20-fold, respectively, over 14 days. At low cell inocula (1 million), the presence of stroma enhanced the expansion of total cells and CFU-GM but not of LTC-IC. When SCF was not included in the medium, both total cells and CFU-GM expanded to a much lesser extent, but again the expansion of LTC-IC was not affected. At the higher cell inoculum (10 million), expansions of total cells and CFU-GM were equivalent with or without stroma. To evaluate UCB, cells were placed into bioreactors with or without irradiated, allogeneic stroma, and the bioreactors were perfused with medium containing the four standard growth factors. After 6-14 days, in several independent experiments, 20-24 million cells were harvested from bioreactors perfused with SCF-containing medium, irrespective of the presence or absence of preformed stroma. Similarly, in reactors perfused with SCF-containing medium (with or without stroma), an average 40- to 60-fold expansion of CFU-GM was obtained, yielding an average of 1.5-1.8 x 10(5) CFU-GM per reactor. Harvested cells were thus up to 40-fold enriched in CFU-GM in comparison to the inoculum. In the absence of SCF, cell expansions averaged 1.5- to 2-fold, and CFU-GM were expanded only 10- to 14-fold by day 14. As before, the presence of preformed stroma did not affect either cell or CFU-GM yields, provided the cell inoculum was at least 4.5 million cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

A Study of Integrated Document and Connection Caching in the WWW

Document caching and connection caching are extensively studied problems. In document caching, one has to maintain caches containing documents accessible in a network. In connection caching, one has to maintain a set of open network connections that handle data transfer. Previous work investigated these two problems separately while in practice the problems occur together: In order to load a document, one has to establish a connection between network nodes if the required connection is not already open. In this paper we present the first study that integrates document and connection caching. We first consider a very basic model in which all documents have the same size and the cost of loading a document or establishing a connection is equal to 1. We present deterministic and randomized online algorithms that achieve nearly optimal competitive ratios unless the size of the connection cache is extremely small. We then consider general settings where documents have varying sizes. We investigate a FAULT model in which the loading cost of a document is 1 as well as a BIT model in which the loading cost is equal to the size of the document.