Neutrophils from p40phox−/− mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing

The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91phox, p22phox, p47phox, p67phox, p40phox, and rac-2. Mutations in each of the phox components, except p40phox, have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40phox by investigating the NADPH oxidase responses of neutrophils isolated from p40phox−/− mice. In the absence of p40phox, the expression of p67phox is reduced by ∼55% and oxidase responses to tumor necrosis factor α/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by ∼97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40phox−/− neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40phox as an essential component in bacterial killing

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

SBOL Visual: A Graphical Language for Genetic Designs

Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual

The PX domain: a new phosphoinositide-binding module

The PX domain, which until recently was an orphan domain, has emerged as the latest member of the phosphoinositide-binding module superfamily. Structural studies have revealed that it has a novel fold and identified key residues that interact with the bound phosphoinositide, enabling some prediction of phosphoinositide-binding specificity. Specificity for PtdIns(3)P appears to be the most common, and several proteins containing PX domains localise to PtdIns(3)P-rich endosomal and vacuolar structures through their PX domains: these include the yeast t-SNARE Vam7p, mammalian sorting nexins (involved in membrane trafficking events) and the Ser/Thr kinase CISK, which is implicated in cell survival. Additionally,phosphoinositide binding to the PX domains of p40phox and p47phox appears to play a critical role in the active assembly of the neutrophil oxidase complex.</jats:p

Determinate Assembly of Tooling Allows Concurrent Design of Airbus Wings and Major Assembly Fixtures

Most new aircraft programs encounter the challenge of balancing the time required for design optimization with product delivery constraints. The high cost and long lead times of traditional tooling makes it difficult for aircraft manufactures to efficiently meet ever-changing market demands. The large size, low relative stiffness and high positional tolerances required for aircraft components drive the requirement for rigid fixed tooling to maintain the precision part relationships over time. Use of today’s advance 3-Dimensional CAD systems coupled with the high accuracy of CNC machines enables the success of the determinate assembly approach for aircraft tooling. This approach provides the aircraft manufacturer significant lead-time reductions while at the same time it supports enhanced system flexibility. Determinate assembly for aircraft tooling has proven to be highly successful for tooling manufacture on large-scale system such as the A380 and A340-600 wing assembly projects

Kinetics and Role of Plasma Matrix Metalloproteinase-9 Expression in Acute Lung Injury and the Acute Respiratory Distress Syndrome

Primed neutrophils that are capable of releasing matrix metalloproteinases (MMPs) into the circulation are thought to play a significant role in the pathophysiology of acute respiratory distress syndrome (ARDS). We hypothesized that direct measurement of plasma MMP-9 activity may be a predictor of incipient tissue damage and subsequent lung injury, which was investigated in both an animal model of ARDS and a small cohort of 38 critically ill human patients. In a mouse model of ARDS involving instillation of intratracheal lipopolysaccharide (LPS) to induce lung inflammation, we measured neutrophil-mediated inflammation, along with MMP-9 activity in the airways and lung tissue and MMP-9 expression in the plasma. Neutrophil recruitment, inflammation, and MMP-9 activity in the airways and lung tissue increased throughout the 72 h after LPS instillation, whereas plasma MMP-9 expression was greatest at 12 to 24 h after LPS instillation. The results suggest that the peak in plasma MMP-9 activity may precede the peak of neutrophil inflammation in the airways and lung tissue in the setting of ARDS. Based on this animal study, a retrospective observational cohort study involving 38 patients admitted to a surgical intensive care unit at a tertiary care university hospital with acute respiratory failure requiring intubation and mechanical ventilation was conducted. Plasma samples were collected daily, and MMP-9 activity was compared with lung function as determined by the PaO[subscript 2]/FiO[subscript 2] ratio. In patients who developed ARDS, a notable increase in plasma MMP-9 activity on a particular day correlated with a decrease in the PaO[subscript 2]/FiO[subscript 2] ratio on the following day (r = -0.503, P < 0.006). Taken together, these results suggest that plasma MMP-9 activity changes, as a surrogate for primed neutrophils may have predictive value for the development of ARDS in a selected subset of critically ill patients.National Institutes of Health (U.S.) (Grants R01-GM59281, P50-GM68762 and UM1-HL120877)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award

Use of the GRP1 PH domain as a tool to measure the relative levels of PtdIns(3,4,5)P3 through a protein-lipid overlay approach

We describe a novel approach to the relative quantification of phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] and its application to measure, in neutrophils, the activation of phosphoinositide 3-kinase (PI3K). This protein-lipid overlay-based assay allowed us to confirm and extend the observations, first, that N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation of primed human neutrophils leads to a transient and biphasic increase in PtdIns(3,4,5)P(3) levels and, second, that the ability of fMLP to stimulate PtdIns(3,4,5)P(3) accumulation in neutrophils isolated from mice carrying a Ras-insensitive ('DASAA') knock-in of PI3Kgamma (p110gamma(DASAA/DASAA)) is substantially dependent on the Ras binding domain of PI3Kgamma

Abrogation of Antibody-Induced Arthritis in Mice by a Self-Activating Viridin Prodrug and Association with Impaired Neutrophil and Endothelial Cell Function

Objective. To test a novel self-activating viridin (SAV) prodrug that slowly releases wortmannin, a potent phosphoinositide 3-kinase inhibitor, in a model of antibody-mediated inflammatory arthritis.Methods. The SAV prodrug was administered to K/BxN mice or to C57BL/6 (B6) mice that had been injected with K/BxN serum. Ankle thickness was measured, and histologic changes were scored after a 10-day disease course (serum-transfer arthritis). Protease activity was measured by a near-infrared imaging approach using a cleavable cathepsin-selective probe. Further near-infrared imaging techniques were used to analyze early changes in vascular permeability after serum injection, as well as neutrophil-endothelial cell interactions. Neutrophil functions were assessed using an oxidative burst assay as well as a degranulation assay. Results. SAV prevented ankle swelling in mice with serum-transfer arthritis in a dose-dependent manner. It also markedly reduced the extent of other features of arthritis, such as protease activity and histology scores for inflammation and joint erosion. Moreover, SAV was an effective therapeutic agent. The underlying mechanisms for the antiinflammatory activity were manifold. Endothelial permeability after serum injection was reduced, as was firm neutrophil attachment to endothelial cells. Endothelial cell activation by tumor necrosis factor alpha was impeded by SAV, as measured by the expression of vascular cell adhesion molecule. Crucial neutrophil functions, such as generation of reactive oxygen species and degranulation of protease-laden vesicles, were decreased by SAV administration. Conclusion. A novel SAV prodrug proved strongly antiinflammatory in a murine model of antibody-induced inflammatory arthritis. Its activity could be attributed, at least in part, to the inhibition of neutrophil and endothelial cell functions.Accepted Manuscrip