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IE1 Sequence Alignment in Several Primary GBM Tissues (S1); HCMV IE is enriched in the SSEA1+ fraction of primary GBM cells and co-localizes with stemness markers in situ (S2); IE siRNA Optimization (S3); HCMV transcripts are enriched in the GSC fraction (S4); HCMV IE modulates Sox2 expression in GBM via miR145 (S5); IE1 overexpression induces Sox2 via miRNA145 inhibition in GSCs (S6); IE attenuation induces a mesenchymal and pro-inflammatory phenotype in HCMV-positive GSC (S7); Immunohistochemical analyses of IE1+/- mouse gliomas (S8); GSC lines used in this study are tumorigenic in vivo (S9); CR208, an IE1 deficient HCMV does not promote GBM stemness to the same extent as Towne parental strain (S10).

Detection of HCMV and cellular markers in 40 patient samples included in this study (S1); HCMV serostatus in a subset of GBM cases (S2); Histopathological and immunohistochemical characterization of spontaneous mouse gliomas (S3).

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Description of additional methods and procedures used in the study. Also includes Supplementary References.

Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression. Cancer Res; 72(10); 2578–88. ©2012 AACR.

Legend for Supplementary Figures S1-S10.

Research has made clear that neighbourhood conditions affect racial inequality. We examine how living in minority neighbourhoods affects ease of access to conventional banks versus alternative financial institutions (AFIs) such as check cashers and payday lenders, which some have called predatory. Based on more than 6 million queries, we compute the difference in the time required to walk, drive or take public transport to the nearest bank versus AFI from the middle of every block in each of 19 of the largest cities in the United States. The results suggest that race is strikingly more important than class: even after numerous conditions are accounted for, the AFI is more often closer than the bank in low-poverty racial/ethnic minority neighbourhoods than in high-poverty white ones. Results are driven not by the absence of banks but by the prevalence of AFIs in minority areas. Gaps appear too large to reflect simple differences in preferences.

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP_HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies.

Diabetes mellitus is a medical condition examined by data miners for reasons such as significant health complications in affected people, the economic impact on healthcare networks, and so on. In order to find the main causes of this disease, researchers look into the patient's lifestyle, hereditary information, etc. The goal of data mining in this context is to find patterns that make early detection of the disease and proper treatment easier. Due to the high volume of data involved in therapeutic contexts and disease diagnosis, provision of the intended treatment method become almost impossible over a short period of time. This justifies the use of pre-processing techniques and data reduction methods in such contexts. In this regard, clustering and meta-heuristic algorithms maintain important roles. In this paper, a method based on the k-means clustering algorithm is first utilized to detect and delete outliers. Then, in order to select significant and effective features, four bi-objective meta-heuristic algorithms are employed to choose the least number of significant features with the highest classification accuracy using support vector machines (SVM). In addition, the 10-fold cross validation (CV) method is used to validate the constructed model. Using real case data, it is concluded that the multi-objective firefly (MOFA) and multi-objective imperialist competitive algorithm (MOICA) with a 100% classification accuracy outperform the non-dominated sorting genetic algorithm (NSGA-II) and multi-objective particle swarm optimization (MOPSO) with the accuracies of 98.2% and 94.6%, respectively.

Recent research has reported that residents from different types of neighborhoods exhibit divergent patterns of urban mobility. However, these analyses have not considered the accessibility enabled by transportation networks. Furthermore, the data are typically not collected at a fine-grained enough temporal resolution, which impedes upon the accurate identification of more frequently visited locations. Here, we develop a data fusion framework to integrate individuals' mobility, transportation routing, and census data to quantify urban dwellers' accessibility to their frequently visited locations. The framework has been applied to a random sample of 1000 Houstonians. The analytical results show that Houston residents visit a median of nine locations five times or more in the period of one week. Residents from primarily poor and black neighborhoods experience longer travel times compared to individuals from nonpoor and white neighborhoods when using public transit, even though their travel durations by driving are similar. The differences are problematic given that residents from poor and black neighborhoods often have lower rates of car ownership. The results indicate that urban dwellers' accessibility is strongly impacted by their neighborhood characteristics, which has implications for equitable access in urban environments.

In this research, a maximal covering location problem (MCLP) with real-world constraints such as multiple types of facilities and vehicles with different setup costs is taken into account. An original mixed integer linear programming (MILP) model is constructed in order to find the optimal solution. Since the problem at hand is shown to be NP-hard, a constructive heuristic method and a meta-heuristic approach based on genetic algorithm (GA) are developed to solve the problem. To find the most effective solution technique, a set of problems of different sizes is randomly generated and solved by the proposed solution methods. Computational results demonstrate that the heuristic method is capable of producing optimal or near-optimal solutions in a rational execution time.

Research has made clear that neighbourhood conditions affect racial inequality. We examine how living in minority neighbourhoods affects ease of access to conventional banks versus alternative financial institutions (AFIs) such as check cashers and payday lenders, which some have called predatory. Based on more than 6 million queries, we compute the difference in the time required to walk, drive or take public transport to the nearest bank versus AFI from the middle of every block in each of 19 of the largest cities in the United States. The results suggest that race is strikingly more important than class: even after numerous conditions are accounted for, the AFI is more often closer than the bank in low-poverty racial/ethnic minority neighbourhoods than in high-poverty white ones. Results are driven not by the absence of banks but by the prevalence of AFIs in minority areas. Gaps appear too large to reflect simple differences in preferences.

Purpose The purpose of this study is to develop a novel and practical series-parallel inventory-redundancy allocation system in a green supply chain including a single manufacturer and multiple retailers operating in several positions without any conflict of interests. The manufacturer first produces multi-product and then dispatches them to the retailers at different wholesale prices based on a common replenishment cycle policy. In contrast, the retailers sell the purchased products to customers at different retail prices. In this way, the manufacturer encounters a redundancy allocation problem (RAP), in which the solution subsequently enhances system production reliability. Furthermore, to emphasize on global warming and human health concerns, this paper pays attention both the tax cost of industrial greenhouse gas (GHG) emissions of all produced products and the limitation for total GHG emissions. Design/methodology/approach The manufacturer intends not only to maximize the total net profit but also to minimize the mean time to failure of his production system using a RAP. To achieve these objectives, the max-min approach associated with the solution method known as the interior point method is utilized to maximize the minimum (the worst) value of the objective functions. Finally, numerical experiments are presented to further demonstrate the applicability of the proposed methodology. Sensitivity analysis on the green supply chain approach is also performed to obtain more insight. Findings The computational results showed that increasing the number of products and retailers might lead into a substantial increase in the total net profit. This indicated that the manufacturer would feel adding a new retailer to the green supply chain strongly. Moreover, an increase in the number of machines provides significant improvement in the reliability of the production system. Furthermore, the results of the performed sensitivity analysis on the green approach indicated that increasing the number of machines has a substantial impact on both the total net profit and the total tax cost. In addition, not only the proposed green supply chain was more efficient than the supply chain without green but also the proposed green supply chain was very sensitive to the tax cost of GHG emission rather than the number of machines. Originality/value In summary, the motivations are as follows: the development of a bi-objective series-parallel inventory-RAP in a green supply chain; proposing a hybrid inventory-RAP; and considering the interior point solution method. The novel method comes from both theoretical and experimental techniques. The paper also has industrial applications. The advantage of using the proposed approach is to generate additional opportunities and cost effectiveness for businesses and companies that operate utilizing the green supply chain under an inventory model.

Emerging micromobility services provide not only new transportation options, but also valuable sources of data for researchers and planners seeking to understand traveler behavior and preferences and to improve transportation networks. Dockless systems for shared bicycles and electric scooters have recently begun operating in many American cities, providing data not only on trip start and end, but also on the route followed. In this study, we analyze data from more than 110,000 dockless bikeshare trips in Boston’s suburbs. About 15% of trip ends were within 100 m of a transit station, indicating that bikeshare serves many more functions than access to transit. More than 40% ended in a town or village center, suggesting that bikeshare may support the local economy. From examining GPS traces, it was clear that – contrary to the assumptions of common bike routing algorithms – bike riders often use sidewalks, other footways and soft-surfaced paths, parking lots, and driveways, and 36% ride the wrong way on one-way streets. An examination of the streets with high contraflow volumes indicates two common profiles. One is streets whose travel channel is so narrow that either the bike or vehicle has to pull into a parking lane to let the other pass; those streets typically have low traffic speeds and very low traffic volumes. The other is streets whose travel channel is wide enough for a bike and car to pass without either yielding to the other; they typically have greater vehicular speed and volume, but still only one lane. After classifying streets by level of traffic stress, we find that only 7 percent of trips use exclusively low-stress links, and that about 40 percent of bike-miles are ridden on low-stress links. These low percentages are consistent with an underdeveloped bike network that forces riders to use through streets for all except ultra-local trips; it may also reflect a prevalence of sidewalk riding on major arterials.

This paper introduces a new hub-and-center transportation network problem for a new company competing against an operating company. The new company intends to locate p hubs and assign the center nodes to the located hubs in order to form origin–destination pairs. It desires not only to maximize the total captured flow in the market but also aims to minimize the total transportation cost. Three competition rules are established between the companies which must be abided. According to the competition rules, the new company can capture the full percentage of the traffic in each origin-destination pair if its transportation cost for each route is significantly less than of the competitor. If its transportation cost for each route is not significantly less than one of the competitors, only a certain percentage of the traffic can be captured. A bi-objective optimization model is proposed for the hub location problem on hand under a competitive environment. As the problem is shown to be NP-hard, a novel meta-heuristic algorithm called multi-objective biogeography-based optimization is developed. As there is no benchmark in the literature, a popular non-dominated sorting algorithm is utilized to validate the results obtained. Moreover, to enhance the performance of the proposed Pareto-based algorithms, this paper intends to develop a binary opposition-based learning as a diversity mechanism for both algorithms. The algorithms are tuned to solve the problem, based on which their performances are compared, ranked, and analyzed statistically. Finally, the applicability of the proposed approach and the solution methodologies are demonstrated in three steps.

The rise of location positioning technologies has generated enormous volumes of digital footprints. Translating this big data into understandable trip patterns plays a crucial role in estimating infrastructure demands. Previous studies were unable to correctly represent commuting patterns on smaller urban scales due to insufficient spatial accuracy. In this study, we investigated if, and to what extent, estimated commuting patterns identified from GPS data can replicate the results from transportation surveys and to what degree these estimates improve the estimates of trips distribution pattern on census tract level using higher resolution data. We inferred average daily home-to-work trips by analyzing phone GPS data and compared these patterns with U.S. Census summary tables. We found that trips detected by GPS data highly correlate with census trips. Furthermore, GPS data is a better proxy for Census tract-pairs with larger numbers of trips.

The redundancy allocation problem (RAP) is a useful method to enhance system reliability. In most works involving RAP, failure rates of the system components are assumed to follow either exponential or k-Erlang distributions. In real world problems however, many systems have components with increasing failure rates. This indicates that as time passes by, the failure rates of the system components increase in comparison to their initial failure rates. In this paper, the redundancy allocation problem of a series–parallel system with components having an increasing failure rate based on Weibull distribution is investigated. An optimization method via simulation is proposed for modeling and a genetic algorithm is developed to solve the problem.

Many cities in the United States are working to become more “bike-friendly” through the provision of new bike infrastructure that is safe and attractive for all types of cyclists, from the timid to assured. These efforts are supported by evidence associating low level of traffic stress facilities with increased cycling activity rates and co-benefits related to the economy, environment, and public health. However, not every mile of bike infrastructure provides the same utility, prompting planning agencies with finite financial resources to search for empirical methods to help evaluate what projects will provide the greatest network connectivity benefit and how disparate projects can complement one another to produce a complete bike network. In this study, we introduce the Cyclist Routing Algorithm for Network Connectivity (CRANC), an accessibility-oriented decision-support tool designed to quantify the benefits of new bike facilities for various populations and neighborhoods. Unlike prior tools, this method simulates the route preferences of different cyclist types and trade-offs in travel time and level of traffic stress to model potential changes in destination accessibility that may result from multiple scenarios of citywide and regional bike network expansion. Here, CRANC is applied to the Boston region’s bike network to determine how a proposed shared-use path in Cambridge, Massachusetts will improve accessibility to regional job opportunities and to labor force for employment sites in Cambridge. Our introduced decision-support tool produces unique, meaningful results relevant to a variety of stakeholders, and holds promise as a new resource for transportation researchers and practitioners.

The primary objective of this research is to obtain an accurate forecasting model for the US presidential election. To identify a reliable model, artificial neural networks (ANN) and support vector regression (SVR) models are compared based on some specified performance measures. Moreover, six independent variables such as GDP, unemployment rate, the president’s approval rate, and others are considered in a stepwise regression to identify significant variables. The president’s approval rate is identified as the most significant variable, based on which eight other variables are identified and considered in the model development. Preprocessing methods are applied to prepare the data for the learning algorithms. The proposed procedure significantly increases the accuracy of the model by 50%. The learning algorithms (ANN and SVR) proved to be superior to linear regression based on each method’s calculated performance measures. The SVR model is identified as the most accurate model among the other models as this model successfully predicted the outcome of the election in the last three elections (2004, 2008, and 2012). The proposed approach significantly increases the accuracy of the forecast.

Glioblastoma (GBM) is the most common and aggressive human brain tumor. Human cytomegalovirus (HCMV) immediate-early (IE) proteins that are endogenously expressed in GBM cells are strong viral transactivators with oncogenic properties. Here, we show how HCMV IEs are preferentially expressed in glioma stem–like cells (GSC), where they colocalize with the other GBM stemness markers, CD133, Nestin, and Sox2. In patient-derived GSCs that are endogenously infected with HCMV, attenuating IE expression by an RNAi-based strategy was sufficient to inhibit tumorsphere formation, Sox2 expression, cell-cycle progression, and cell survival. Conversely, HCMV infection of HMCV-negative GSCs elicited robust self-renewal and proliferation of cells that could be partially reversed by IE attenuation. In HCMV-positive GSCs, IE attenuation induced a molecular program characterized by enhanced expression of mesenchymal markers and proinflammatory cytokines, resembling the therapeutically resistant GBM phenotype. Mechanistically, HCMV/IE regulation of Sox2 occurred via inhibition of miR-145, a negative regulator of Sox2 protein expression. In a spontaneous mouse model of glioma, ectopic expression of the IE1 gene (UL123) specifically increased Sox2 and Nestin levels in the IE1-positive tumors, upregulating stemness and proliferation markers in vivo. Similarly, human GSCs infected with the HCMV strain Towne but not the IE1-deficient strain CR208 showed enhanced growth as tumorspheres and intracranial tumor xenografts, compared with mock-infected human GSCs. Overall, our findings offer new mechanistic insights into how HCMV/IE control stemness properties in GBM cells. Cancer Res; 75(15); 3065–76. ©2015 AACR.

Perturbations in the composition and assembly of extracellular matrices (ECM) contribute to progression of numerous diseases, including cancers. Anchoring of laminins at the cell surface enables assembly and signaling of many ECMs, but the possible contributions of altered laminin anchoring to cancer progression remain undetermined. In this study, we investigated the prominence and origins of defective laminin anchoring in cancer cells and its association with cancer subtypes and clinical outcomes. We found loss of laminin anchoring to be widespread in cancer cells. Perturbation of laminin anchoring originated from several distinct defects, which all led to dysfunctional glycosylation of the ECM receptor dystroglycan. In aggressive breast and brain cancers, defective laminin anchoring was often due to suppressed expression of the glycosyltransferase LARGE. Reduced expression of LARGE characterized a broad array of human tumors in which it was associated with aggressive cancer subtypes and poor clinical outcomes. Notably, this defect robustly predicted poor survival in patients with brain cancers. Restoring LARGE expression repaired anchoring of exogenous and endogenous laminin and modulated cell proliferation and tumor growth. Together, our findings suggest that defects in laminin anchoring occur commonly in cancer cells, are characteristic of aggressive cancer subtypes, and are important drivers of disease progression. Cancer Res; 72(10); 2578–88. ©2012 AACR.

We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I). Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.

Dystroglycan (DG) is an extracellular matrix receptor implicated in muscular dystrophies and cancers. DG belongs to the membrane-tethered mucin family and is composed of extracellular (alpha-DG) and transmembrane (beta-DG) subunits stably coupled at the cell surface. These two subunits are generated by autoproteolysis of a monomeric precursor within a distinctive protein motif called sea urchin-enterokinase-agrin (SEA) domain, yet the purpose of this cleavage and heterodimer creation is uncertain. In this study, we identify a functional nuclear localization signal within beta-DG and show that, in addition to associating with alpha-DG at the cell surface, the full-length and glycosylated beta-DG autonomously traffics to the cytoplasm and nucleoplasm in a process that occurs independent of alpha-DG ligand binding. The trafficking pattern of beta-DG mirrors that of MUC1-C, the transmembrane subunit of the related MUC1 oncoprotein, also a heterodimeric membrane-tethered mucin created by SEA autoproteolysis. We show that the transmembrane subunits of both MUC1 and DG transit the secretory pathway prior to nuclear targeting and that their monomeric precursors maintain the capacity for nuclear trafficking. A screen of breast carcinoma cell lines of distinct pathophysiological origins revealed considerable variability in the nuclear partitioning of beta-DG, indicating that nuclear localization of beta-DG is regulated, albeit independent of extracellular ligand binding. These findings point to novel intracellular functions for beta-DG, with possible disease implications. They also reveal an evolutionarily conserved role for SEA autoproteolysis, serving to enable independent functions of mucin transmembrane subunits, enacted by a shared and poorly understood pathway of segregated subunit trafficking.

Human platelet derived growth factor-alpha (PDGFR-α) receptor has been found to be a requirement for the infection of human cells by human cytomegalovirus (HCMV), the herpesvirus that's the most common cause of congenital human infections, and causes life threatening disease in HIV-infected and transplant patients. If the receptor is inactivated, HCMV cannot enter the cell and viral genes are not expressed. Importantly, two currently available pharmaceutical agents — the anticancer drug imatinib (Gleevec) and PDGFR-α blocking antibody IMC-3G3 — are active against the virus in human cells suggesting that they might prove useful in the treatment of HCMV infection. Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host1. Upon attachment to the cell, the virus induces robust inflammatory, interferon- and growth-factor-like signalling2,3,4,5,6,7,8,9. The mechanisms facilitating viral entry and gene expression are not clearly understood4. Here we show that platelet-derived growth factor-α receptor (PDGFR-α) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-3-kinase (PI(3)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-α associated with the p85 regulatory subunit of PI(3)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-α was genetically deleted10 or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-α blocking antibody (IMC-3G3)11 or targeted inhibition of its kinase activity with a small molecule (Gleevec)12 completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-α was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-α, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies13 inhibit HCMV-induced PDGFR-α phosphorylation. Taken together, these data indicate that PDGFR-α is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.

Post-translational modifications of the extracellular matrix receptor dystroglycan (DG) determine its functional state, and defects in these modifications are linked to muscular dystrophies and cancers. A prominent feature of DG biosynthesis is a precursor cleavage that segregates the ligand-binding and transmembrane domains into the noncovalently attached alpha- and beta-subunits. We investigate here the structural determinants and functional significance of this cleavage. We show that cleavage of DG elicits a conspicuous change in its ligand-binding activity. Mutations that obstruct this cleavage result in increased capacity to bind laminin, in part, due to enhanced glycosylation of alpha-DG. Reconstitution of DG cleavage in a cell-free expression system demonstrates that cleavage takes place in the endoplasmic reticulum, providing a suitable regulatory point for later processing events. Sequence and mutational analyses reveal that the cleavage occurs within a full SEA (sea urchin, enterokinase, agrin) module with traits matching those ascribed to autoproteolysis. Thus, cleavage of DG constitutes a control point for the modulation of its ligand-binding properties, with therapeutic implications for muscular dystrophies. We provide a structural model for the cleavage domain that is validated by experimental analysis and discuss this cleavage in the context of mucin protein and SEA domain evolution.

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.

The dynamic interactions between cells and basement membranes serve as essential regulators of tissue architecture and function in metazoans, and perturbation of these interactions contributes to the progression of a wide range of human diseases, including cancers. Here, we reveal the pathway and mechanism for the endocytic trafficking of a prominent basement membrane protein, laminin-111 (referred to here as laminin), and their disruption in disease. Live-cell imaging of epithelial cells revealed pronounced internalization of laminin into endocytic vesicles. Laminin internalization was receptor mediated and dynamin dependent, and laminin proceeded to the lysosome through the late endosome. Manipulation of laminin receptor expression revealed that the dominant regulator of laminin internalization is dystroglycan, a laminin receptor that is functionally perturbed in muscular dystrophies and in many cancers. Correspondingly, laminin internalization was found to be deficient in aggressive cancer cells displaying non-functional dystroglycan, and restoration of dystroglycan function strongly enhanced the endocytosis of laminin in both breast cancer and glioblastoma cells. These results establish previously unrecognized mechanisms for the modulation of cell–basement-membrane communication in normal cells and identify a profound disruption of endocytic laminin trafficking in aggressive cancer subtypes.

1. Long QT syndrome (LQT) is an electrophysiological disorder that can lead to sudden death from cardiac arrhythmias. One form of LQT has been attributed to mutations in the human ether-a-go-go-related gene (HERG) that encodes a voltage-gated cardiac K+ channel. While a recent report indicates that LQT in some patients is associated with a mutation of HERG at a consensus extracellular N-linked glycosylation site (N629), earlier studies failed to identify a role for N-linked glycosylation in the functional expression of voltage-gated K+ channels. In this study we used pharmacological agents and site-directed mutagenesis to assess the contribution of N-linked glycosylation to the surface localization of HERG channels. 2. Tunicamycin, an inhibitor of N-linked glycosylation, blocked normal surface membrane expression of a HERG-green fluorescent protein (GFP) fusion protein (HERGGFP) transiently expressed in human embryonic kidney (HEK 293) cells imaged with confocal microscopy. 3. Immunoblot analysis revealed that N-glycosidase F shifted the molecular mass of HERGGFP, stably expressed in HEK 293 cells, indicating the presence of N-linked carbohydrate moieties. Mutations at each of the two putative extracellular N-linked glycosylation sites (N598Q and N629Q) led to a perinuclear subcellular localization of HERGGFP stably expressed in HEK 293 cells, with no surface membrane expression. Furthermore, patch clamp analysis revealed that there was a virtual absence of HERG current in the N-glycosylation mutants. 4. Taken together, these results strongly suggest that N-linked glycosylation is required for surface membrane expression of HERG. These findings may provide insight into a mechanism responsible for LQT2 due to N-linked glycosylation-related mutations of HERG.

As primary conductors of ionic charge across virtually every cell in our body, ion channels play a fundamental role in various aspects of mammalian physiology. The role of ion channels in physiology is particularly evident in the function of the heart and the brain which operate primarily by conducting electrical impulses. Not surprisingly, investigations of mechanisms associated with ion channel function have dominated basic science research in areas of cardiology and neurology. However, we know very little about the mechanisms involved in intracellular delivery and the localization of ion channels. A mislocalized ion channel is not only physiologically worthless, its altered localization may be detrimental to the cell and the overall survival of the organism. In a recent issue of The Journal of Physiology, Zadeh et al. (2009) investigated the molecular mechanisms implicated in the delivery of a cardiac voltage-gated potassium channel (Kv1.5) to its final destination, the plasma membrane. They found that a particular isoform (Kif5b) of the kinesin superfamily is involved in the transport of the Kv1.5 channels to the cell surface. Prior to this study, it was well known that kinesin motor proteins are critical for the transport of ion channels across lengthy distances in axons of nerve cells (Rivera et al. 2007). However, the study of Zadeh et al. (2009) suggests that kinesins are equally involved in the transport of ion channels in cardiac cells. Kinesins and dyneins are motor proteins which deliver a large cargo to their final cellular destination by moving along the microtubule tracks. As depicted in Fig. 1, in general, kinesins move from the centre of the cell towards the periphery (or the plus end of microtubules) and dyneins move in the reverse direction. Kinesin-1 (also known as conventional kinesin) is perhaps the most prominent member of the kinesin superfamily. Kinesin-1 is a tetrameric complex composed of two heavy chains (KHC) and two light chains (KLC). The amino terminal end of KHC interacts with the microtubule and hydrolyses ATP to generate the force for movement. Binding to cargo is mediated by either KHC or KLC (reviewed in Hirokawa & Noda, 2008). Figure 1 Synthesis and transport of Kv1.5 channels Zadeh et al. (2009) focused their investigation on Kif5b, the only KHC subunit of kinesin-1 which is ubiquitously expressed (Hirokawa & Noda, 2008). They found that overexpression of Kif5b results in a significant increase in functional expression of Kv1.5, presumably because more channels are delivered to the plasma membrane. The delivery of Kv1.5 channels by Kif5b is expected to take place between the Golgi and the plasma membrane (as shown in Fig. 1). Therefore, manipulations that block transit to Golgi should lessen or eliminate the increase in functional expression of Kv1.5 by Kif5b. Zadeh et al. (2009) show that blocking the exit of Kv1.5 channels from the endoplasmic reticulum by brefeldin A (BFA) attenuates the increase in functional expression of Kv1.5 by Kif5b. In order to more directly test the specific role of Kif5b, in several of their experiments the authors compared the effect of overexpressing Kif5bDN, a dominant negative construct of Kif5b. As expected, the presence of BFA had marginal effect on the influence of Kif5bDN on the current density of Kv1.5. In addition, in a tetracycline-inducible expression system, the authors demonstrate that induced expression of Kv1.5 subsequent to expression of Kif5bDN significantly diminishes the magnitude of currents generated by Kv1.5 channels, presumably due to a block of channel delivery to the plasma membrane. Peculiarly, however, when investigated at steady-state level, coexpression of Kif5bDN and Kv1.5 resulted in a significant increase in the function of Kv1.5 channels. Zadeh et al. (2009) argue that the increase in surface expression of Kv1.5 upon coexpression of Kif5bDN is mediated through a distinct mechanism unrelated to that observed with coexpression of Kif5b. A previous study by the same group has demonstrated that inhibition of dynein increases the surface expression of Kv1.5 (Choi et al. 2005), prompting the authors to suggest that the increase in the current density of Kv1.5 at steady-state level by Kif5bDN is mediated by the inhibition of dynein function. Indeed, Zadeh et al. (2009) show that several manipulations that reduce channel internalization, potentially by interfering with dynein function, diminish the effect of Kif5bDN on the functional expression of Kv1.5. The effect of Kif5b, however, persisted even after inhibiting channel internalization. Zadeh et al. (2009) have concluded that Kif5b is essential for the delivery of Kv1.5 channels to the plasma membrane in cardiac cells, which is perhaps the most likely and the simplest conclusion that can be drawn from their data. Several issues, however, remain to be addressed and some important experiments are needed to strengthen the conclusion of this study. In particular, the authors clearly demonstrate a dramatic increase in current density in two different cell lines upon coexpression of Kv1.5 and Kif5b. However, as evident from their data, the cell lines that they use express endogenous channels, the traffic of which may also be influenced by Kif5b. Therefore the contribution of Kv1.5 channels to the increase in current density observed upon expression of Kif5b is unclear. Since the endogenous channels appear to have similar kinetics to Kv1.5, it is particularly important to subtract their contribution from electrophysiological measurements in order to accurately and quantitatively estimate changes in Kv1.5 function. Furthermore, the authors note that they have been unable to demonstrate a direct interaction between Kv1.5 and Kif5b. It is therefore possible that the increase in current density associated with overexpression of Kif5b is due to increased surface expression of a ubiquitously expressed putative auxiliary subunit required for the function of Kv1.5. Thus, it is critical to quantitatively and directly measure the surface expression of exogenously expressed Kv1.5 channels before and after overexpression of Kif5b. Similarly, it is equally important to define the specificity of the effect of Kif5b on cardiac ion channel trafficking. Are Kv1.5 channels unique in their dependence on Kif5b or do other cardiac channels also require Kif5b for their trafficking to the plasma membrane? Is KLC a player in the delivery of Kv1.5 channels to the plasma membrane? What is the amino acid sequence in Kv1.5 that is required for enhanced functional expression by Kif5b and is that sequence conserved in any other voltage-gated potassium channel? Answers to these questions, amongst others, are central to solidifying the implication of kinesin-1 in the delivery of ion channels to the plasma membrane of cardiac cells. In addition, these answers may help in identifying novel therapeutic targets for cardiac disorders arising from deficient delivery of ion channels to the plasma membrane.

The C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) bind to the ebolavirus glycoprotein (EBOV-GP) and augment viral infectivity. DC-SIGN/R strongly enhance infection driven by the GP of EBOV subspecies. Zaire (ZEBOV) but have a much less pronounced effect on infection mediated by the GP of EBOV subspecies. Sudan (SEBOV). For this study, we analyzed the determinants of the differential DC-SIGN/R interactions with ZEBOV- and SEBOV-GP. The efficiency of DC-SIGN engagement by ZEBOV-GP was dependent on the rate of GP incorporation into lentiviral particles, while appreciable virion incorporation of SEBOV-GP did not allow robust DC-SIGN/R usage. Forced incorporation of high-mannose carbohydrates into SEBOV-GP augmented the engagement of DC-SIGN/R to the levels observed with ZEBOV-GP, indicating that appropriate glycosylation of SEBOV-GP is sufficient for efficient DC-SIGN/R usage. However, neither signals for N-linked glycosylation unique to SEBOV- or ZEBOV-GP nor the highly variable and heavily glycosylated mucin-like domain modulated the interaction with DC-SIGN/R. In contrast, analysis of chimeric GPs identified the signal peptide as a determinant of DC-SIGN/R engagement. Thus, ZEBOV- but not SEBOV-GP was shown to harbor high-mannose carbohydrates, and GP modification with these glycans was controlled by the signal peptide. These results suggest that the signal peptide governs EBOV-GP interactions with DC-SIGN/R by modulating the incorporation of high-mannose carbohydrates into EBOV-GP. In summary, we identified the level of GP incorporation into virions and signal peptide-controlled glycosylation of GP as determinants of attachment factor engagement.

Mutations of a putative cyclic-nucleotide-binding domain (CNBD) can disrupt the function of the hyperpolarization-activated cyclic-nucleotide-gated channel (HCN2) and the human ether-a-go-go-related gene potassium channel (HERG). Loss of function caused by C-terminal truncation, which includes all or part of the CNBD in HCN and HERG, has been related to abnormal channel trafficking. Similar defects have been reported for several of the missense mutations of HERG associated with long QT syndrome type 2 (LQT2). Thus, we postulate that normal processing of these channels depends upon the presence of the CNBD. Here, we show that removal of the entire CNBD prevents Golgi transit, surface localization and function of HERG channel tetramers. This is also true when any of the structural motifs of the CNBD is deleted, suggesting that deletion of any highly conserved region along the entire length of the CNBD can disrupt channel trafficking. Furthermore, we demonstrate that defective trafficking is a consequence of all LQT2 mutations in the CNBD, including two mutations not previously assessed and two others for which there are conflicting results in the literature. The trafficking sensitivity of the CNBD might be of general significance for other ion channels because complete deletion of the CNBD or mutations at highly conserved residues within the CNBD of the related ERG3 channel and HCN2 also prevent Golgi transit. These results broadly implicate the CNBD in ion-channel trafficking that accounts for the commonly observed loss of function associated with CNBD mutants and provides a rationale for distinct genetic disorders.

Mutations in the potassium channel encoded by the human ether-a-go-go-related gene (HERG) have been linked to the congenital long QT syndrome (LQTS), a cardiac disease associated with an increased preponderance of ventricular arrhythmias and sudden death. The COOH terminus of HERG harbors a large number of LQTS mutations and its removal prevents functional expression for reasons that remain unknown. In this study, we show that the COOH terminus of HERG is required for normal trafficking of the ion channel. We have identified a region critical for trafficking between residues 860 and 899 that includes a novel missense mutation at amino acid 861 (HERGN861I). Truncations or deletion of residues 860–899, characterized in six different expression systems including a cardiac cell line, resulted in decreased expression levels and an absence of the mature glycosylated form of the HERG protein. Deletion of this region did not interfere with the formation of tetramers but caused retention of the assembled ion channels within the endoplasmic reticulum. Consequently, removal of residues 860–899 resulted in the absence of the ion channels from the cell surface and a more rapid turnover rate than the wild type channels, which was evident very early in biogenesis. This study reveals a novel role of the COOH terminus in the normal biogenesis of HERG channels and suggests defective trafficking as a common mechanism for abnormal channel function resulting from mutations of critical COOH-terminal residues, including the LQTS mutant HERGN861I.

The biogenesis and processing of membrane and secretory proteins takes place at the endoplasmic reticulum (ER) where they are integrated into or translocated across the ER membrane. A remarkable element implicated in biogenesis of this class of protein is that they are almost always processed by various cotranslational and posttranslational modifications which ultimately determine protein stability, localization and function. Defaults in biogenesis, processing or trafficking of membrane proteins are often the direct cause of various human diseases. In particular, some forms of cardiac arrhythmias, such as the long QT syndrome (LQTS), are caused by aberrant processing and defective trafficking of ion channels to the plasma membrane (Anderson et al. 2006). The LQTS is a congenital heart disease characterized by an abnormally long QT interval measured with a body surface electrocardiogram and increase risk of torsade de pointes, a fatal cardiac arrhythmia. Several forms of LQTS arise from mutations in genes encoding main or auxiliary subunits of potassium channels implicated in the repolarization phase of cardiac action potential. Some of these mutations generate dysfunctional ion channels, thus delaying the repolarization phase of the cardiac action potential and prolonging the QT interval. In contrast, other mutations have no or little effect on the function of potassium channels but instead result in misprocessing and defective trafficking of the channel protein to the plasma membrane where they normally function (Anderson et al. 2006). In human heart, KCNE1 and KCNQ1 co-assemble to form the slow outward current (IKs) of the repolarization phase of the action potential. Homozygous mutations in KCNE1 or KCNQ1 genes gives rise to the Jervell and Lange–Nielsen syndrome (JLNS), a form of LQTS associated with congenital deafness (Splawski et al. 1997). In a recent issue of The Journal of Physiology, Chandrasekhar et al. (2011) investigated the molecular mechanisms by which one mutation in KCNE1 diminish the contribution of IKs to the repolarization phase of the cardiac action potential, giving rise to JLNS. They found a novel posttranslational modification in the KCNE1 subunit and they suggest that abolishing this modification by a natural mutation is associated with defective trafficking of the channel protein to the plasma membrane. One common type of modification of ion channels involves addition of oligosaccharide chains to asparagine (N) residues, a process referred to as N-linked glycosylation. Several cardiac ion channels undergo N-linked glycosylation and mutations in consensus N-linked glycosylation sites have been identified in potassium channels implicated in LQTS (Anderson et al. 2006). In contrast, much less if known about processing of cardiac ion channels by O-linked glycosylation. In eukaryotic cells, several types of O-linked glycosylation exist but they all involve addition of carbohydrate moieties to serine (S) or threonine (T) amino acids. In their study, Chandrasekhar et al. (2011) pointed to multiple consensus sites for O-linked glycosylation in KCNE1 and experimentally identified the actual T residue that undergoes glycosylation. They showed KCNE1 undergoes both N- and O-linked glycosylation when heterologously expressed with KCNQ1 in cells. By exploring a transgenic mouse model stably expressing concatenated KCNE1–KCNQ1 fusion protein, they found that the cardiac IKs complex acquires O-linked glycosylation in vivo. Mutational analysis identified a T at position 7 (T7) as the sole site for O-linked glycosylation. Interestingly, this same site is critical for N-linked glycosylation at N5, allowing abrogation of both modifications with a single mutation. Indeed, one such mutation exists in patients with LQTS and it severely compromises the glycosylation and trafficking of KCNE1–KCNQ1 channel complexes (Bas et al. 2001). In contrast, mutations that eliminate either N- or O-linked glycans separately have minimal effect on the functional activity, biogenesis and trafficking of the channel proteins. Notably, the latter conclusions were based on investigations of glycosylation sites that were created by mutagenesis. The authors argue that the consensus N- and O-linked glycosylation sites overlap in native KCNE1 sequence, thus making it difficult to investigate these glycosylation types separately by mutational analysis. However, in the absence of additional information, it is unclear as to why it is not possible to create mutants that would allow separate analysis of native N- and O-linked glycosylation sites. Theoretically, mutation of N5 should abrogate N-linked glycosylation without affecting O-linked glycosylation, thereby enabling investigations aimed at identifying the consequence of N-linked glycosylation on biogenesis and function of the KCNE1–KCNQ1 complex. Another alternative to separately investigate the consequence of N- and O-linked glycosylation in biogenesis and trafficking of KCNE1 involves pharmacological treatments which inhibit distinct glycosylation types. For example, treatment of live cells with tunicamycine specifically abolishes N-linked glycosylation, thereby allowing investigation of this defect without interference from defects associated with O-linked glycans. One important novelty of the studies reported by Chandrasekhar et al. (2011) is the discovery that the cardiac IKs complex is modified by O-linked glycans. However, the precise consequence of this modification for the function and biogenesis of the ion channel remains unknown. The authors suggest that this modification is potentially important for subcellular localization of the IKs complex in cardiac cells. This hypothesis can be tested by investigating the localization of mutant channels in cardiac cell lines (such as the HL-1 cell line) or in a transgenic mouse model similar to the one described by Chandrasekhar et al. (2011). An alternative function for this modification is to allow regulation of IKs based on physiological demand. It is well known that the IKs channel is modulated by phosphorylation as a result of adrenergic stimuli. Thus, specific protein modifications can allow interaction with molecular machinery involved in regulation of IKs or the specific targeting of the ion channel to the proximity of adrenergic receptors (Marx et al. 2002). Undoubtedly, a better understanding of the implication of O-linked glycosylation for IKs function requires investigations in native cardiac cells and under physiological conditions.