Selective Perturbation of Apical Membrane Traffic by Expression of Influenza M2, an Acid-activated Ion Channel, in Polarized Madin–Darby Canine Kidney Cells

The function of acidification along the endocytic pathway is not well understood, in part because the perturbants used to modify compartmental pH have global effects and in some cases alter cytoplasmic pH. We have used a new approach to study the effect of pH perturbation on postendocytic traffic in polarized Madin–Darby canine kidney (MDCK) cells. Influenza M2 is a small membrane protein that functions as an acid-activated ion channel and can elevate the pH of the trans-Golgi network and endosomes. We used recombinant adenoviruses to express the M2 protein of influenza virus in polarized MDCK cells stably transfected with the polymeric immunoglobulin (Ig) receptor. Using indirect immunofluorescence and immunoelectron microscopy, M2 was found to be concentrated at the apical plasma membrane and in subapical vesicles; intracellular M2 colocalized partly with internalized IgA in apical recycling endosomes as well as with the trans-Golgi network marker TGN-38. Expression of M2 slowed the rate of IgA transcytosis across polarized MDCK monolayers. The delay in transport occurred after IgA reached the apical recycling endosome, consistent with the localization of intracellular M2. Apical recycling of IgA was also slowed in the presence of M2, whereas basolateral recycling of transferrin and degradation of IgA were unaffected. By contrast, ammonium chloride affected both apical IgA and basolateral transferrin release. Together, our data suggest that M2 expression selectively perturbs acidification in compartments involved in apical delivery without disrupting other postendocytic transport steps.

MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane–mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH4Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that ∼30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.

Feature selection by multi-objective optimisation: Application to network anomaly detection by hierarchical self-organising maps

Feature selection is an important and active issue in clustering and classification problems. By choosing an adequate feature subset, a dataset dimensionality reduction is allowed, thus contributing to decreasing the classification computational complexity, and to improving the classifier performance by avoiding redundant or irrelevant features. Although feature selection can be formally defined as an optimisation problem with only one objective, that is, the classification accuracy obtained by using the selected feature subset, in recent years, some multi-objective approaches to this problem have been proposed. These either select features that not only improve the classification accuracy, but also the generalisation capability in case of supervised classifiers, or counterbalance the bias toward lower or higher numbers of features that present some methods used to validate the clustering/classification in case of unsupervised classifiers. The main contribution of this paper is a multi-objective approach for feature selection and its application to an unsupervised clustering procedure based on Growing Hierarchical Self-Organising Maps (GHSOMs) that includes a new method for unit labelling and efficient determination of the winning unit. In the network anomaly detection problem here considered, this multi-objective approach makes it possible not only to differentiate between normal and anomalous traffic but also among different anomalies. The efficiency of our proposals has been evaluated by using the well-known DARPA/NSL-KDD datasets that contain extracted features and labelled attacks from around 2 million connections. The selected feature sets computed in our experiments provide detection rates up to 99.8% with normal traffic and up to 99.6% with anomalous traffic, as well as accuracy values up to 99.12%.

Net Neutrality and Mandatory Network-Sharing: How to disconnect the continent. CEPS Policy Brief No. 309, 18 December 2013

A lively debate emerged on the proposed “Connected Continent” legislative package presented by the European Commission in September 2013. The package contains a proposed rule on the ‘open Internet’, which was heavily discussed in European Parliament hearings in early December. This commentary argues that while the proposed rule is in principle balanced and appealing, it is utterly impractical due to the enormous uncertainty that its application would entail. At the same time, the rule is very far from what neutrality proponents have argued for almost a decade: rather than the place for internet freedom, it would transform the Web into a place requiring constant micro-management and tutoring of user behaviour. Both arguments lead to the conclusion that the current proposal should be at once reformed and analysed under a more holistic lens. On the one hand, Europe should launch an ambitious project for the future, converged infrastructure by mobilising resources and reforming rules to encourage investment into ubiquitous, converged, ‘always on’ connectivity. On the other hand, enhanced legal certainty for broadband investment could justify a more neutrality-oriented approach to traffic management practices on the Internet. The author proposes a new approach to Internet regulation which, altogether, will lead to a more balanced and sustainable model for the future, without jeopardising user freedom.

Construction of a comprehensive causal network. Volume 2: literature review and bibliographies. Phase II report.

National Highway Traffic Safety Administration, Washington, D.C.

A methodology for the assessment of truck lane needs in the Texas highway network. Final report.

Texas State Department of Highways and Public Transportation, Transportation Planning Division, Austin

Network assignment methods for the analysis of truck-related highway improvements. Interim report.

Texas State Department of Highways and Public Transportation, Transportation Planning Division, Austin

Truck lane needs methodology: a heuristic approach to solve a five option discrete network design problem. Interim report.

Texas State Department of Highways and Public Transportation, Transportation Planning Division, Austin

Construction of a comprehensive causal network, phase III: final report (volume I).

National Highway Traffic Safety Administration, Washington, D.C.

Traffic safety and health care : state and national estimates of employer costs /

"DOT HS 808 234"--P. [4] of cover.

Privacy in the Smart City: Implications of Sensor Network Design, Law, and Policy for Locational Privacy

Thesis (Master's)--University of Washington, 2016-06

A Novel Framework for Real-time Traffic Flow Parameter Estimation from Aerial Videos

Thesis (Master's)--University of Washington, 2016-06

Layer 2.5 Network Coding to Improve 802.11 Network Efficiency

Thesis (Master's)--University of Washington, 2016-06