The caveolae-mediated sv40 entry pathway bypasses the golgi complex en route to the endoplasmic reticulum

BACKGROUND: Simian virus 40 (SV40) enters cells via an atypical caveolae-mediated endocytic pathway, which delivers the virus to a new intermediary compartment, the caveosome. The virus then is believed to go directly from the caveosome to the endoplasmic reticulum. Cholera toxin likewise enters via caveolae and traffics to caveosomes. But, in contrast to SV40, cholera toxin is transported from caveosomes to the endoplasmic reticulum via the Golgi. For that reason, and because the caveosome and Golgi may have some common markers, we revisited the issue of whether SV40 might access the endoplasmic reticulum via the Golgi. RESULTS: We confirmed our earlier finding that SV40 co localizes with the Golgi marker β-COP. However, we show that the virus does not co localize with the more discriminating Golgi markers, golgin 97 and BODIPY-ceramide. CONCLUSION: The caveolae-mediated SV40 entry pathway does not intersect the Golgi. SV40 is seen to co localize with β-COP because that protein is a marker for caveosomes as well as the Golgi. Moreover, these results are consistent with the likelihood that the caveosome is a sorting organelle. In addition, there are at least two distinct but related routes by which a ligand might traffic from the caveosome to the ER; one route involving transport through the Golgi, and another pathway that does not involve the Golgi

Speeding up VP9 Intra Encoder with Hierarchical Deep Learning Based Partition Prediction

In VP9 video codec, the sizes of blocks are decided during encoding by recursively partitioning 64$\times$64 superblocks using rate-distortion optimization (RDO). This process is computationally intensive because of the combinatorial search space of possible partitions of a superblock. Here, we propose a deep learning based alternative framework to predict the intra-mode superblock partitions in the form of a four-level partition tree, using a hierarchical fully convolutional network (H-FCN). We created a large database of VP9 superblocks and the corresponding partitions to train an H-FCN model, which was subsequently integrated with the VP9 encoder to reduce the intra-mode encoding time. The experimental results establish that our approach speeds up intra-mode encoding by 69.7% on average, at the expense of a 1.71% increase in the Bjontegaard-Delta bitrate (BD-rate). While VP9 provides several built-in speed levels which are designed to provide faster encoding at the expense of decreased rate-distortion performance, we find that our model is able to outperform the fastest recommended speed level of the reference VP9 encoder for the good quality intra encoding configuration, in terms of both speedup and BD-rate

A Collaborative Clearinghouse for Data Management Training and Education Resources

Objective: The main objectives of this breakout session are for the Data Management Training (DMT) Clearinghouse team to: 1) introduce the Clearinghouse and its current design and implementation, 2) solicit submissions to its learning resource inventory, and 3) collect feedback upon its web interface and future development. Features of the Clearinghouse that will be demonstrated include how to search and browse its inventory as well as submit a learning resource to the Clearinghouse using the LRMI (Learning Resource Metadata Initiative) metadata format. The team will also share the roadmap for the Clearinghouse’s upcoming features. In order to provide feedback regarding the Clearinghouse’s usability, the team will invite the session attendees to test the Clearinghouse’s services and will encourage comments to guide its future development. Setting/Participants/Resources: Since the DMT Clearinghouse is entirely accessible via the web, in order to demonstrate the Clearinghouse successfully, a reliable (and preferably free of charge) internet connection, and an overhead projecting capability will need to be available to the presenter. It would also be very useful for the attendees of the session to have access to the same internet connection, so that if they desire, the attendees can follow along with the steps of the demonstration, and contribute to the Clearinghouse inventory. The main presenter will plan to bring her own laptop with built-in standard HDMI and USB ports. As a result, it will be helpful if a HDMI or USB cable could also be provided for the presenter to connect her laptop to the projecting equipment. Method: Many research organizations, government agencies, and academic institutions have been developing excellent learning resources in order to support and meet the needs for data management training. However, these learning resources are often hosted on various websites and spread across various scientific domains. Consequently, these resources can be difficult to locate, especially by those who are not already familiar with the creators/authors. This is a barrier to the use and reuse of these resources, and can have significant impact on the promotion and propagation of best practices for data management. To address this need within the Earth sciences, the U.S. Geological Survey’s (USGS) Community for Data Integration (CDI), the Federation of Earth Science Information Partners (ESIP), and the Data Observation Network for Earth (DataONE) have collaborated to create a web-based Clearinghouse1 for collecting data management learning resources that are focused on the Earth sciences. The initial seed funding for the effort was provided by a grant received from the USGS CDI earlier in 2016, and ESIP’s Drupal site provided the hosting infrastructure for the Clearinghouse. Members from the USGS, DataONE, ESIP’s Data Stewardship Committee and its Data Management Training Working Group, Knowledge Motifs LLC, as well as Blue Dot Lab met regularly between April and October, 2016 in order to discuss, create, and implement the content structure and infrastructure components necessary to build the current revision of the Clearinghouse. 1. http://dmtclearinghouse.esipfed.org Results: As a registry of information about the educational resources on topics related to research data management (initially focused on Earth sciences), the Clearinghouse serves as a centralized location for searching or browsing an inventory of these learning resources. Currently, the Clearinghouse offers search and browse functionality that is open to all, and submission of information about educational resources by login with a free ESIP account. To assist with discoverability, the learning resources are described using Learning Resource Metadata Initiative (LRMI) schema. Additionally, the resources may be associated with the steps of data and research life cycles, such as the USGS CDI’s Science Support Framework2 and DataONE’s Data Life Cycle3. Leveraging the team’s collective experience in creating, presenting and distributing data management learning resources, the Clearinghouse included the learning resources from USGS, ESIP, and DataONE as its initial inventory, but is expanding to resources from NASA and others. Crowdsourcing is currently the main mechanism for sustaining the Clearinghouse. Going forward, in addition to the built-in workflow to allow anyone from the public to submit descriptive information about the data management learning resources that s/he wishes to share, future capabilities will be added to enable contributions to review, edit, and rank the submissions, as desired. 2. https://my.usgs.gov/confluence/display/cdi/CDI+Science+Support+Framework3. https://www.dataone.org/data-life-cycle Discussion/Conclusion: The DMT Clearinghouse team was successful in completing the initial development phase as scheduled for the first six months of its funding, including some informal usability testing of the interface. The team aims to continue to develop and enhance the Clearinghouse’s capabilities, including the evaluation of its usability, through collaboration with additional communities, and if feasible, adding the capability for bulk-loading of learning resources. Being able to present the Clearinghouse at the eScience Symposium would not only allow those who are involved with or would like to learn about data management to leverage the Clearinghouse’s resources, but also connect those who would like to contribute to the project with the Clearinghouse team. Ultimately, the Clearinghouse is designed so that the resources from its inventory could be used in a variety of data management training and education environments. By exposing the Clearinghouse to diverse users and communities, the Clearinghouse team can better assess how the Clearinghouse can be updated and what technological enhancements to pursue in the future in order to improve our support of research data management training needs

Inter- and intra-rater reliabilities of the Beighton Score compared to the Contompasis Score to assess Generalised Joint Hypermobility

Objectives: Generalized Joint Hypermobility [GJH] is a common connective tissue disorder associated with a range of musculoskeletal complaints. An effective screening tool to assess GJH may influence our understanding and choice of management. Diagnosis is clinical, using tools such as the Beighton Hypermobility Score and the Contompasis Scoring System. The comparable reliability of these tools has not been previously reported. The aim of the present study was to compare the intra- and the inter-rater reliability of the Beighton Score to the Contompasis Score to assess GJH. Methods: This was an observational study assessing 36 pain-free participants; 27 females and nine males; aged 18–32 years. Participants were assessed in random order, by two researchers over two sessions to determine intra- and inter-rater analyses. Intraclass Correlation Coefficient [ICC] and weighted Kappa statistics were used to calculate the level of agreement. Results: The intra- [ICC: 0.71–0.82] and the inter- [ICC: 0.72–0.80] rater reliability of the Beighton Score was substantial to almost perfect. The Contompasis Score displayed substantial to almost perfect intra-rater [ICC: 0.73–0.82] reliability and moderate to substantial inter-rater [ICC: 0.58–0.62] reliability. Conclusions: The present study provides an indication of the measurement capabilities of the Beighton and Contompasis Scores. The Beighton score appears to be superior compared with the Contompasis score particularly based on inter-rater reliability

Caveolin-1 and -2 in airway epithelium: expression and in situ association as detected by FRET-CLSM

BACKGROUND: Caveolae are involved in diverse cellular functions such as signal transduction, cholesterol homeostasis, endo- and transcytosis, and also may serve as entry sites for microorganisms. Hence, their occurrence in epithelium of the airways might be expected but, nonetheless, has not yet been examined. METHODS: Western blotting, real-time quantitative PCR analysis of abraded tracheal epithelium and laser-assisted microdissection combined with subsequent mRNA analysis were used to examine the expression of cav-1 and cav-2, two major caveolar coat proteins, in rat tracheal epithelium. Fluorescence immunohistochemistry was performed to locate caveolae and cav-1 and -2 in the airway epithelium of rats, mice and humans. Electron-microscopic analysis was used for the identification of caveolae. CLSM-FRET analysis determined the interaction of cav-1α and cav-2 in situ. RESULTS: Western blotting and laser-assisted microdissection identified protein and transcripts, respectively, of cav-1 and cav-2 in airway epithelium. Real-time quantitative RT-PCR analysis of abraded tracheal epithelium revealed a higher expression of cav-2 than of cav-1. Immunoreactivities for cav-1 and for cav-2 were co-localized in the cell membrane of the basal cells and basolaterally in the ciliated epithelial cells of large airways of rat and human. However, no labeling for cav-1 or cav-2 was observed in the epithelial cells of small bronchi. Using conventional double-labeling indirect immunofluorescence combined with CLSM-FRET analysis, we detected an association of cav-1α and -2 in epithelial cells. The presence of caveolae was confirmed by electron microscopy. In contrast to human and rat, cav-1-immunoreactivity and caveolae were confined to basal cells in mice. Epithelial caveolae were absent in cav-1-deficient mice, implicating a requirement of this caveolar protein in epithelial caveolae formation. CONCLUSION: These results show that caveolae and caveolins are integral membrane components in basal and ciliated epithelial cells, indicating a crucial role in these cell types. In addition to their physiological role, they may be involved in airway infection

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BACKGROUND: Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. METHODS: We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. RESULTS: We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. CONCLUSION: Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen

Reliability of two goniometric methods of measuring active inversion and eversion range of motion at the ankle

BACKGROUND: Active inversion and eversion ankle range of motion (ROM) is widely used to evaluate treatment effect, however the error associated with the available measurement protocols is unknown. This study aimed to establish the reliability of goniometry as used in clinical practice. METHODS: 30 subjects (60 ankles) with a wide variety of ankle conditions participated in this study. Three observers, with different skill levels, measured active inversion and eversion ankle ROM three times on each of two days. Measurements were performed with subjects positioned (a) sitting and (b) prone. Intra-class correlation coefficients (ICC([2,1])) were calculated to determine intra- and inter-observer reliability. RESULTS: Within session intra-observer reliability ranged from ICC([2,1] )0.82 to 0.96 and between session intra-observer reliability ranged from ICC([2,1] )0.42 to 0.80. Reliability was similar for the sitting and the prone positions, however, between sessions, inversion measurements were more reliable than eversion measurements. Within session inter-observer measurements in sitting were more reliable than in prone and inversion measurements were more reliable than eversion measurements. CONCLUSION: Our findings show that ankle inversion and eversion ROM can be measured with high to very high reliability by the same observer within sessions and with low to moderate reliability by different observers within a session. The reliability of measures made by the same observer between sessions varies depending on the direction, being low to moderate for eversion measurements and moderate to high for inversion measurements in both positions