Structured analysis and modeling of complex systems

The Aircrew Evaluation Sustained Operations Performance (AESOP) facility at Brooks AFB, Texas, combines the realism of an operational environment with the control of a research laboratory. In recent studies we collected extensive data from the Airborne Warning and Control Systems (AWACS) Weapons Directors subjected to high and low workload Defensive Counter Air Scenarios. A critical and complex task in this environment involves committing a friendly fighter against a hostile fighter. Structured Analysis and Design techniques and computer modeling systems were applied to this task as tools for analyzing subject performance and workload. This technology is being transferred to the Man-Systems Division of NASA Johnson Space Center for application to complex mission related tasks, such as manipulating the Shuttle grappler arm

Identification of hepatitis C virus core protein residues critical for the interaction with the cellular DEAD-Box Helicase DDX3 and their functional relevance

Hepatitis C virus (HCV) is a single-stranded RNA virus belonging to the Flaviviridae and infects approximately 170 million people worldwide. Unlike other known RNA viruses, HCV causes a persistent infection in the majority of infected people and can lead to cirrhosis of the liver and hepatocellular carcinoma. For these reasons, HCV is rightly classified as a major human pathogen. HCV core protein is believed to form, by analogy with other members of the Flaviviridae family, the nucleocapsid of the virus. As well as this, core has been shown to modulate many cellular processes via interactions with numerous host-cell proteins. One such protein shown to interact with HCV core is the DEAD-box RNA helicase DDX3. In cells expressing either HCV core alone, or as part of the full length HCV polyprotein, DDX3 is redistributed from its normal diffuse cytoplasmic localisation to lipid droplets where it colocalises with core. The cellular function of DDX3 is still unknown although it has been suggested to be involved in processes such as splicing, translation and RNA transport. The aim of this study was to investigate the role of DDX3 in the life cycle of HCV. This was aided by the recent discovery of a fully infectious HCV genotype 2a clone (strain JFH-1), allowing previously inaccessible aspects of the virus life cycle to be studied such as particle assembly and release. A library of HCV core mutants (residues 1-59 only) was produced by error-prone PCR and subsequently expressed in bacteria and analysed for their ability to bind bacterially expressed DDX3 using a rapid, high throughput ELISA screen. Six HCV core residues, conserved throughout all genotypes, were identified as being critical for interaction with DDX3. These residues were confirmed as being critical for the interaction by transfection of mutant core (together with E1 and E2 to ensure correct processing of core) into Huh7 cells. None of the 6 mutant core proteins were able to redistribute cellular DDX3. In order to study the effects of abolishing the core-DDX3 interaction in terms of a fully infectious HCV life cycle, the 6 critical residues were individually mutated to alanine in the cell culture infectious strain JFH-1 genome. All 6 mutant JFH-1 RNAs were capable of replication and being translated. Further investigation however, suggested that replication rate of mutant JFH-1 RNA was >50-fold lower than that of wild type JFH-1 RNA replication. Mutant core proteins colocalised with the lipid droplet marker ADRP, indicating correct subcellular localisation of the viral protein. Western-immunoblot analysis of mutant cores also confirmed that core proteins of same molecular weight to that of wild type core were produced, suggesting mutant cores were correctly processed. Of the 6 mutant JFH-1 clones analysed, 5 of them were capable of secreting infectious HCV particles that could subsequently infect naïve Huh7 cells, as detected by immunofluorescence and RT-PCR. However, one mutant, in which residue 33 of core had been changed from glycine to alanine, was initially unable to produce infectious particles. Upon passaging of cells electroporated with this mutant, infectious particles were eventually produced. The production of infectious particles consistently coincided with the presence of a second mutation in the surrounding area of the originally mutated residue 33. However, JFH-1 RNA containing both the mutation at residue 33 and the second identified mutation nearby, was unable to produce infectious particles upon electroporation, suggesting another lesion elsewhere in the HCV genome may also be required in order to overcome the effect of mutating residue 33. A recent report has indicated that DDX3 may be a nucleo-cytoplasmic shuttling protein, utilising the CRM1 export pathway. To confirm this, DDX3 localisation was analysed in the presence of the CRM1 inhibitor leptomycin B (LMB). In the absence of LMB, DDX3 was seen to have a diffuse cytoplasmic localisation while a small proportion was also seen in the nucleus. In the presence of LMB however, a build-up of DDX3 was seen in the nucleus, confirming that DDX3 uses the CRM1 pathway to shuttle from the nucleus to the cytoplasm. The results of this study indicate that the interaction of the cellular DEAD-box helicase DDX3 with core protein is not essential for the life cycle of HCV. It has been shown here however, that the replication rates of mutant HCV RNA are lower than that of wild type, suggesting that DDX3 may enhance either replication itself, or translation (which in turn provides the machinery required for viral RNA replication). Investigating this possibility is the subject of our future work. The identification of glycine 33 of core protein as being essential for production of infectious virus particles (without abolishing replication) will provide the basis for further studies on the production of infectious particles and the role that core protein plays in this process. The panel of JFH-1 core mutants will also be useful in studying the core-DDX3 interaction in a much wider context involving the role of DDX3 in normal cells. This study has uncovered important details regarding the interaction between core and DDX3 and, together with the reagents produced throughout this investigation, should enable further successful study into the role of DDX3 in the life cycle of HCV

Quality control of the sheep bacterial artificial chromosome library, CHORI-243

<p>Abstract</p> <p>Background</p> <p>The sheep CHORI-243 bacterial artificial chromosome (BAC) library is being used in the construction of the virtual sheep genome, the sequencing and construction of the actual sheep genome assembly and as a source of DNA for regions of the genome of biological interest. The objective of our study is to assess the integrity of the clones and plates which make up the CHORI-243 library using the virtual sheep genome.</p> <p>Findings</p> <p>A series of analyses were undertaken based on the mapping the sheep BAC-end sequences (BESs) to the virtual sheep genome. Overall, very few plate specific biases were identified, with only three of the 528 plates in the library significantly affected. The analysis of the number of tail-to-tail (concordant) BACs on the plates identified a number of plates with lower than average numbers of such BACs. For plates 198 and 213 a partial swap of the BESs determined with one of the two primers appear to have occurred. A third plate, 341, also with a significant deficit in tail-to-tail BACs, appeared to contain a substantial number of sequences determined from contaminating eubacterial 16 S rRNA DNA. Additionally a small number of eubacterial 16 S rRNA DNA sequences were present on two other plates, 111 and 338, in the library.</p> <p>Conclusions</p> <p>The comparative genomic approach can be used to assess BAC library integrity in the absence of fingerprinting. The sequences of the sheep CHORI-243 library BACs have high integrity, especially with the corrections detailed above. The library represents a high quality resource for use by the sheep genomics community.</p

Sea Level Rise für the U.S. West Coast

Source: ICHE Conference Archive - https://mdi-de.baw.de/icheArchiv

The importance of donor population identity and habitat type when creating new populations of small Melampyrum sylvaticum from seed in Perthshire, Scotland

Small cow-wheat Melampyrum sylvaticum, a nationally scarce annual identified as a priority species in the UK Biodiversity Action Plan, was the focus of a translocation attempt aiming to establish new populations within the extent of its former Scottish range. Seeds were collected (from wild Scottish populations) in three phases (in the years 2005, 2006 and 2008) and sown at six receptor sites where the species was absent but habitat seemed suitable. Each phase used increasing numbers of seed after the results of the first phase (89 to 103 seeds sown per site) suggested that many more than 100 seeds are needed to establish the species (at least in the short-term) at a site. Comparisons of the suitability of seed from three different wild populations showed that one resulted in higher germination rates. This donor population was associated with environmental conditions more similar to those at the receptor sites than those of the other donors. Receptor sites also differed in their suitability; those that were climatically and edaphically more similar to sites supporting wild populations appear to be more favourable to M.sylvaticum longer-term survival. Together, these can be seen to suggest that future seed translocation should be to sites that are ecologically similar to the donor population and within sites that fall into the cooler and wetter range of environmental conditions currently supporting Scottish populations of M. sylvaticum

Post-hoc derivation of SOHO Michelson doppler imager flat fields

&lt;p&gt;&lt;b&gt;Context:&lt;/b&gt; The SOHO satellite now offers a unique perspective on the Sun as it is the only space-based instrument that can provide large, high-resolution data sets over an entire 11-year solar cycle. This unique property enables detailed studies of long-term variations in the Sun. One significant problem when looking for such changes is determining what component of any variation is due to deterioration of the instrument and what is due to the Sun itself. One of the key parameters that changes over time is the apparent sensitivity of individual pixels in the CCD array. This can change considerably as a result of optics damage, radiation damage, and aging of the sensor itself. In addition to reducing the sensitivity of the telescope over time, this damage significantly changes the uniformity of the flat field of the instrument, a property that is very hard to recalibrate in space. For procedures such as feature tracking and intensity analysis, this can cause significant errors.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Aims:&lt;/b&gt; We present a method for deriving high-precision flat fields for high-resolution MDI continuum data, using analysis of existing continuum and magnetogram data sets.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Methods:&lt;/b&gt; A flat field is constructed using a large set (1000-4000 frames) of cospatial magnetogram and continuum data. The magnetogram data is used to identify and mask out magnetically active regions on the continuum data, allowing systematic biases to be avoided. This flat field can then be used to correct individual continuum images from a similar time.&lt;/p&gt; &lt;p&gt;&lt;b&gt;Results:&lt;/b&gt; This method allows us to reduce the residual flat field error by around a factor 6-30, depending on the area considered, enough to significantly change the results from correlation-tracking analysis. One significant advantage of this method is that it can be done retrospectively using archived data, without requiring any special satellite operations.&lt;/p&gt

Behavioural Consequences of Frontal Cortex Grafts and Enriched Environments after Sensorimotor Cortex Lesions

Past studies have experienced difficulty in achieving graft survival and behavioural recovery after sensorimotor cortex lesions. In the present work, adult female rats trained preoperatively to cross a narrow beam for food reward were maintained in standard group cages or an enriched environment, commencing one week after a unilateral lesion. One month post-lesion, half of these rats received multiple suspension grafts of (E20) fetal frontal cortex, placed adjacent to the lesion cavity, and 8 days later recovery of beam-walking skills was examined for a six-week period. The grafts survived in all cases with an appropriate lesion, a notable result given the one month lesion-graft delay, but graft volume was not influenced by postoperative environment. The substantial lesion-induced deficits evident just prior to differential housing showed a marked reduction by the start of post-graft testing, but relative to intact controls a persistent deficit in foot slip errors occurred in all lesion groups. Irrespective of graft status, postoperative enrichment prevented the occurrence of severe foot slips, especially early in retraining. The frontal grafts, however, enhanced beam-walking recovery by reducing the overall frequency of foot slips on early post-grafting sessions, an effect we suggest is related to graft-derived trophic influences, but this measure was not significantly improved by postoperative enrichment