Sustained performance and some effects on the design and operation of complex systems

Man, increasingly the limiting element in the military man-machine system, must often operate for several days in a high-risk environment with little or no sleep. It is necessary, therefore, to have some knowledge of the likely effects of sleep deprivation to predict his behaviour and minimize the adverse effects of sleep loss. The early work of the Army Personnel Research Establishment (APRE) concentrated on studying the infantryman in field trials, characterized by more realism and of greater length than previously attempted. Although measures of cognitive functioning were included in these trials, continuous cognitive performance was not assessed, nor was performance on complex tasks. An opportunity to remedy this situation arose because of a newer study concerned with controlling a removely-piloted air vehicle from a ground control station (GCS). A 65-hour experiment was designed during which subjects performed continuously either on the GCS simulator or on a battery of cognitive tests, mood scales, and physiological assessments. Results showed that whereas performance showed the usual deterioration in the test battery, it held up remarkably well on the simulator. Several reasons for this difference are suggested.     

Echocardiographic assessment and percutaneous closure of multiple atrial septal defects

Percutaneous coronary intervention can be associated with distal embolization of thrombotic material causing myocardial necrosis and infarction. We discuss the role of intravascular imaging to guide the use of a distal protection device by describing the outcome of a young woman presenting with non-ST elevation myocardial infarction. Coronary angiography demonstrated an isolated minor stenosis in the proximal left anterior descending coronary artery with slight haziness beyond the lesion. Intravascular ultrasound confirmed an extensive thrombus overlying a bulky atherosclerotic plaque. A distal filter wire was therefore successfully used to reduce the risk of distal embolization. The use of intravascular ultrasound in patients presenting with acute coronary syndrome may reveal large thrombi that are difficult to image using conventional angiographic techniques. Intravascular ultrasound can therefore be used as a tool to select lesions requiring distal protection.     

Glycomics analysis of Schistosoma mansoni egg and cercarial secretions

The parasitic helminth Schistosoma mansoni is a major public health concern in many developing countries. Glycoconjugates, and in particular the carbohydrate component of these products, represent the main immunogenic challenge to the host and could therefore represent one of the crucial determinants for successful parasite establishment. Here we report a comparative glycomics analysis of the N- and O-glycans derived from glycoproteins present in S. mansoni egg (egg-secreted protein) and cercarial (0-3-h released protein) secretions by a combination of mass spectrometric techniques. Our results show that S. mansoni secrete glycoproteins with glycosylation patterns that are complex and stage-specific. Cercarial stage secretions were dominated by N-glycans that were core-xylosylated, whereas N-glycans from egg secretions were predominantly core-difucosylated. O-Glycan core structures from cercarial secretions primarily consisted of the core sequence Galbeta1-->3(Galbeta1-->6)GalNAc, whereas egg-secreted O-glycans carried the mucin-type core 1 (Galbeta1-->3GalNAc) and 2 (Galbeta1-->3(GlcNAcbeta1-->6)GalNAc) structures. Additionally we identified a novel O-glycan core in both secretions in which a Gal residue is linked to the protein. Terminal structures of N- and O-glycans contained high levels of fucose and include stage-specific structures. These glycan structures identified in S. mansoni secretions are potentially antigenic motifs and ligands for carbohydrate-binding proteins of the host immune system.     

Metabolic engineering of the omega-3 long chain polyunsaturated fatty acid biosynthetic pathway into transgenic plants

Omega-3 (ω-3) very long chain polyunsaturated fatty acids (VLC-PUFAs) such as eicosapentaenoic acid (EPA; 20:5 Δ5,8,11,14,17) and docosahexaenoic acid (DHA; 22:6 Δ4,7,10,13,16,19) have been shown to have significant roles in human health. Currently the primary dietary source of these fatty acids are marine fish; however, the increasing demand for fish and fish oil (in particular the expansion of the aquaculture industry) is placing enormous pressure on diminishing marine stocks. Such overfishing and concerns related to pollution in the marine environment have directed research towards the development of a viable alternative sustainable source of VLC-PUFAs. As a result, the last decade has seen many genes encoding the primary VLC-PUFA biosynthetic activities identified and characterized. This has allowed the reconstitution of the VLC-PUFA biosynthetic pathway in oilseed crops, producing transgenic plants engineered to accumulate ω-3 VLC-PUFAs at levels approaching those found in native marine organisms. Moreover, as a result of these engineering activities, knowledge of the fundamental processes surrounding acyl exchange and lipid remodelling has progressed. The application of new technologies, for example lipidomics and next-generation sequencing, is providing a better understanding of seed oil biosynthesis and opportunities for increasing the production of unusual fatty acids. Certainly, it is now possible to modify the composition of plant oils successfully, and, in this review, the most recent developments in this field and the challenges of producing VLC-PUFAs in the seed oil of higher plants will be described.     

Enhancing the accumulation of omega-3 long chain polyunsaturated fatty acids in transgenic Arabidopsis thaliana via iterative metabolic engineering and genetic crossing

The synthesis and accumulation of long chain polyunsaturated fatty acids such as eicosapentaenoic acid has previously been demonstrated in the seeds of transgenic plants. However, the obtained levels are relatively low, indicating the need for further studies and the better definition of the interplay between endogenous lipid synthesis and the non-native transgene-encoded activities. In this study we have systematically compared three different transgenic configurations of the biosynthetic pathway for eicosapentaenoic acid, using lipidomic profiling to identify metabolic bottlenecks. We have also used genetic crossing to stack up to ten transgenes in Arabidopsis. These studies indicate several potential approaches to optimize the accumulation of target fatty acids in transgenic plants. Our data show the unexpected channeling of heterologous C20 polyunsaturated fatty acids into minor phospholipid species, and also the apparent negative metabolic regulation of phospholipid-dependent ??6-desaturases. Collectively, this study confirms the benefits of iterative approaches to metabolic engineering of plant lipid synthesis.     

Glycomic studies of Drosophila melanogaster embryos

With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galβ1-3GalNAc sequence.     

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes.Protein O glycosylation is a posttranslational modification implicated in a wide range of physiological processes, including cell adhesion and trafficking, T-cell apoptosis, cell signaling, endocytosis and pathogen-host interaction (1, 6, 27, 30, 54, 61, 71). Core-type protein O glycosylation is initiated in the secretory pathway by the covalent addition of a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues by one of multiple polypeptide GalNAc transferases (ppGalNAcTs) (20, 44, 57, 58). After linkage of the GalNAc monosaccharide to serine or threonine, other glycosyltransferases sequentially and sometimes competitively elaborate the repertoire of O-glycan structures to include different core subtypes (31, 42, 48, 49).The core 2 β1,6-N-acetylglucosaminyltransferases (C2GnTs) and the Core 2 O-glycans they generate are widely expressed among cells of mammalian species. The C2GnTs act after the core 1 β-1,3-galactosyltransferase adds a galactose in a β1,3-linkage to the GalNAc-Ser/Thr generating the initial core 1 O-glycan disaccharide structure (26). Then, one of the three C2GnTs (C2GnT1, C2GnT2, and C2GnT3) can add an N-acetylglucosamine (GlcNAc) in a β1,6-linkage to the GalNAc to initiate what is known as the core 2 O-glycan branch (Fig. ​(Fig.1a)1a) (7, 50, 51, 69). In a distinct pathway, core 3 β-1,3-N-acetylglucosaminyltransferase (C3GnT) can add a GlcNAc to the unmodified GalNAc to generate a core 3 O-glycan (24). In this case, C2GnT2 can add a GlcNAc in β1,6-linkage to the GalNAc of the core 3 O-glycan disaccharide to initiate the formation of a core 4 O-glycan (Fig. ​(Fig.1b)1b) (50, 69). In addition, both C2GnT2 and the I β-1,6-N-acetylglucosaminyltransferase (IGnT) are independently capable of forming branched polylactosamine structures (I-branches) from otherwise linear polylactosamine glycan chains (Fig. ​(Fig.1c)1c) (69).Open in a separate windowFIG. 1.Activity and expression of C2GnTs. (a to c) Monosaccharides are depicted as geometric shapes, with GalNAc as a yellow square, galactose as a yellow circle, and GlcNAc as a blue square. In addition, the vertical arrows indicate that each branch can be further elaborated by additional saccharide linkages. (a) Biantennary core 2 O-glycans are generated when any of the three C2GnTs acts on the core 1 O-glycan disaccharide. (b) C2GnT2 can generate core 4 O-glycans from core 3 O-glycans by adding a GlcNAc to the initiating GalNAc. (c) C2GnT2, in addition to IGnT, also has the ability to generate branched polylactosamine repeats from linear polylactosamine repeats. The figure depicts distal I-branching as the GlcNAc is transferred to the predistal galactose, the preferential I-branching activity of C2GnT2. However, IGnT preferentially has central I-branching activity that adds GlcNAc on the internal galactose in Galβ1→4GlcNAcβ1→3Gal-R (69). (d) RNA expression of murine Gcnt3 (left panel) and Gcnt4 (right panel), which code for C2GnT2 and C2GnT3, respectively, as determined by qPCR. The data on single animals are graphed relative to testes expression. All values are means ± the standard errors of the mean (SEM).C2GnT1-deficient mice have been shown to have an unexpected phenotype first observed as leukocytosis reflecting neutrophilia (14). This appears to be due to a severe but selective defect in selectin ligand biosynthesis among myeloid cells, leading to decreased recruitment of neutrophils that attenuates inflammation and vascular disease pathogenesis (14, 64). C2GnT1-deficient mice also exhibit a partial reduction in L-selectin ligand biosynthesis on high endothelial venules, resulting in reduced B-cell homing and colonization of peripheral lymph nodes (18, 21). Furthermore, thymic progenitors from C2GnT1-deficient mice have a reduced ability to home to the thymus due to the loss of P-selectin ligands on these cells (46). However, as of yet, C2GnT2 and C2GnT3 have not been similarly investigated, and their biological functions remain to be elucidated. To further investigate why multiple glycosyltransferases capable of core 2 O-glycan formation have been conserved, we have generated mice singly and multiply deficient in the three known C2GnTs and characterized the resulting physiology and alterations to the glycome.     

The MAP kinase‐activated protein kinase 2 (MK2) contributes to the Shiga toxin‐induced inflammatory response

Infection with Shiga toxin (STx)‐producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen‐activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx‐induced inflammatory response. From a siRNA screen of the human kinome adapted to a high‐throughput format, we found that knock‐down of the MAPK‐activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activates the p38‐MK2 stress response pathway in both p38‐ and MK2‐dependent manners in two distinct cell lines. MK2 activation was specific to damage to the ribosome by an enzymatically active toxin and did not result from translational inhibition per se. Genetic and chemical inhibition of MK2 significantly decreased the inflammatory response to STx. These findings suggest that MK2 inhibition might play a valuable role in decreasing the immuopathological component of STx‐mediated disease.