H-Ras Nanocluster Stability Regulates the Magnitude of MAPK Signal Output

H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability.     

Perception of Graphical Virtual Environments by Blind Users via Sensory Substitution

Graphical virtual environments are currently far from accessible to blind users as their content is mostly visual. This is especially unfortunate as these environments hold great potential for this population for purposes such as safe orientation, education, and entertainment. Previous tools have increased accessibility but there is still a long way to go. Visual-to-audio Sensory-Substitution-Devices (SSDs) can increase accessibility generically by sonifying on-screen content regardless of the specific environment and offer increased accessibility without the use of expensive dedicated peripherals like electrode/vibrator arrays. Using SSDs virtually utilizes similar skills as when using them in the real world, enabling both training on the device and training on environments virtually before real-world visits. This could enable more complex, standardized and autonomous SSD training and new insights into multisensory interaction and the visually-deprived brain. However, whether congenitally blind users, who have never experienced virtual environments, will be able to use this information for successful perception and interaction within them is currently unclear.We tested this using the EyeMusic SSD, which conveys whole-scene visual information, to perform virtual tasks otherwise impossible without vision. Congenitally blind users had to navigate virtual environments and find doors, differentiate between them based on their features (Experiment1:task1) and surroundings (Experiment1:task2) and walk through them; these tasks were accomplished with a 95% and 97% success rate, respectively. We further explored the reactions of congenitally blind users during their first interaction with a more complex virtual environment than in the previous tasks–walking down a virtual street, recognizing different features of houses and trees, navigating to cross-walks, etc. Users reacted enthusiastically and reported feeling immersed within the environment. They highlighted the potential usefulness of such environments for understanding what visual scenes are supposed to look like and their potential for complex training and suggested many future environments they wished to experience.     

A Robust Method of Measuring Other-Race and Other-Ethnicity Effects: The Cambridge Face Memory Test Format

Other-race and other-ethnicity effects on face memory have remained a topic of consistent research interest over several decades, across fields including face perception, social psychology, and forensic psychology (eyewitness testimony). Here we demonstrate that the Cambridge Face Memory Test format provides a robust method for measuring these effects. Testing the Cambridge Face Memory Test original version (CFMT-original; European-ancestry faces from Boston USA) and a new Cambridge Face Memory Test Chinese (CFMT-Chinese), with European and Asian observers, we report a race-of-face by race-of-observer interaction that was highly significant despite modest sample size and despite observers who had quite high exposure to the other race. We attribute this to high statistical power arising from the very high internal reliability of the tasks. This power also allows us to demonstrate a much smaller within-race other ethnicity effect, based on differences in European physiognomy between Boston faces/observers and Australian faces/observers (using the CFMT-Australian).     

Expression and Regulation of the Arabidopsis thaliana Cel1 Endo 1,4 �� Glucanase Gene During Compatible Plant-Nematode Interactions

The root-knot nematode Meloidogyne incognita is an obligate endoparasite of plant roots and stimulates elaborate modifications of selected root vascular cells to form giant cells for feeding. An Arabidopsis thaliana endoglucanase (Atcel1) promoter is activated in giant cells that were formed in Atcel1::UidA transgenic tobacco and Arabidopsis plants. Activity of the full-length Atcel1 promoter was detected in root and shoot elongation zones and in the lateral root primordia. Different 5’ and internal deletions of regions of the 1,673 bp Atcel1 promoter were each fused to the UidA reporter gene and transformed in tobacco, and roots of the transformants were inoculated with M. incognita to assay for GUS expression in giant cells and noninfected plant tissues. Comparison of the Atcel1 promoter deletion constructs showed that the region between −1,673 and −1,171 (fragment 1) was essential for Atcel1 promoter activity in giant cells and roots. Fragment 1 alone, however, was not sufficient for Atcel1 expression in giant cells or roots, suggesting that cis-acting elements in fragment 1 may function in consort with other elements within the Atcel1 promoter. Root-knot nematodes and giant cells developed normally within roots of Arabidopsis that expressed a functional antisense construct to Atcel1, suggesting that a functional redundancy in endoglucanase activity may represent another level of regulatory control of cell wall-modifying activity within nematode feeding cells.     

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.