Dynamin- and Rab5-Dependent Endocytosis of a Ca²⁺-Activated K⁺ Channel, KCa2.3

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca2+-activated K+ channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane. © 2012 Gao et al

Easy as Pi:A Network Coding Raspberry Pi Testbed

In the near future, upcoming communications and storage networks are expected to tolerate major difficulties produced by huge amounts of data being generated from the Internet of Things (IoT). For these types of networks, strategies and mechanisms based on network coding have appeared as an alternative to overcome these difficulties in a holistic manner, e.g., without sacrificing the benefit of a given network metric when improving another. There has been recurrent issues on: (i) making large-scale deployments akin to the Internet of Things; (ii) assessing and (iii) replicating the obtained results in preliminary studies. Therefore, finding testbeds that can deal with large-scale deployments and not lose historic data in order to evaluate these mechanisms are greatly needed and desirable from a research perspective. However, this can be hard to manage, not only due to the inherent costs of the hardware, but also due to maintenance challenges. In this paper, we present the required key steps to design, setup and maintain an inexpensive testbed using Raspberry Pi devices for communications and storage networks with network coding capabilities. This testbed can be utilized for any applications requiring results replicability

DEVELOPMENT OF AN ALL-PURPOSE FREE PHOTOGRAMMETRIC TOOL

Photogrammetry is currently facing some challenges and changes mainly related to automation, ubiquitous processing and variety of applications. Within an ISPRS Scientific Initiative a team of researchers from USAL, UCLM, FBK and UNIBO have developed an open photogrammetric tool, called GRAPHOS (inteGRAted PHOtogrammetric Suite). GRAPHOS allows to obtain dense and metric 3D point clouds from terrestrial and UAV images. It encloses robust photogrammetric and computer vision algorithms with the following aims: (i) increase automation, allowing to get dense 3D point clouds through a friendly and easy-to-use interface; (ii) increase flexibility, working with any type of images, scenarios and cameras; (iii) improve quality, guaranteeing high accuracy and resolution; (iv) preserve photogrammetric reliability and repeatability. Last but not least, GRAPHOS has also an educational component reinforced with some didactical explanations about algorithms and their performance. The developments were carried out at different levels: GUI realization, image pre-processing, photogrammetric processing with weight parameters, dataset creation and system evaluation. The paper will present in detail the developments of GRAPHOS with all its photogrammetric components and the evaluation analyses based on various image datasets. GRAPHOS is distributed for free for research and educational needs

Pelvic floor dysfunction in Mexican women

Background: Pelvic floor dysfunction (PFD) is a term used to describe a variety of disorders that involve moderate to severe impairment of the pelvic floor muscles. Throughout their lives, up to 46% of women will present at least one form of PFD and may even have a combination of this pathology. These afflictions have a profound influence on women’s general well-being and quality of life, as well as being an immense economic burden for global health systems. Methods: This was a populational study which used surveys made in Google Forms of different questionnaires validated in Spanish [Urinary Incontinence Questionnaire (ICIQ), Female Sexual Function Index (IFSF), CPPQ Questionnaire for chronic pelvic pain, Wexner anal incontinence scale and quality of life] which were taken by women with access to social networks and who agreed to carry out an anonymous survey. Results: Seven hundred and twenty-one participants were evaluated, a total of 61.4% (443) responded positively to involuntary loss of urine, 35% had presented discomfort during sexual activity or lack of sexual interest, 14% had genital pain and 16% reported involuntary loss of feces or gas. Of all the patients that answered any of these questioners positively, only 33% had sought medical attention in the past. Conclusions: We concluded that in our population PFD is underdiagnosed. Our populations prevalence of urinary incontinence and pelvic pain is within the internationally described margins; anal incontinence is above international reports, and our population presented a lower prevalence of sexual dysfunction. With this new information we must impulse awareness to guide various preventive behaviors

LHC sensitivity to the resonance spectrum of a minimal strongly interacting electroweak symmetry breaking sector

We present a unified analysis of the two main production processes of vector boson pairs at the LHC, VV-fusion and qqbar annihilation, in a minimal strongly interacting electroweak symmetry breaking sector. Using a unitarized electroweak chiral Lagrangian formalism and modeling the final V_L V_L strong rescattering effects by a form factor, we describe qqbar annihilation processes in terms of the two chiral parameters that govern elastic V_L V_L scattering. Depending on the values of these two chiral parameters, the unitarized amplitudes may present resonant enhancements in different angular momentum-isospin channels. Scanning this two parameter space, we generate the general resonance spectrum of a minimal strongly interacting electroweak symmetry breaking sector and determine the regions that can be probed at the LHC.Comment: Final version to appear in Phys. Rev. D, including a more detailed exposition and a few more references. Conclusions and results unchanged. 14 pages, 5 figure

Lentiviral gene therapy reverts GPIX expression and phenotype in Bernard-Soulier syndrome type C

Bernard-Soulier syndrome (BSS) is a rare congenital disease characterized by macrothrombocytopenia and frequent bleeding. It is caused by pathogenic variants in three genes (GP1BA, GP1BB, or GP9) that encode for the GPIbα, GPIbβ, and GPIX subunits of the GPIb-V-IX complex, the main platelet surface receptor for von Willebrand factor, being essential for platelet adhesion and aggregation. According to the affected gene, we distinguish BSS type A1 (GP1BA), type B (GP1BB), or type C (GP9). Pathogenic variants in these genes cause absent, incomplete, or dysfunctional GPIb-V-IX receptor and, consequently, a hemorrhagic phenotype. Using gene-editing tools, we generated knockout (KO) human cellular models that helped us to better understand GPIb-V-IX complex assembly. Furthermore, we developed novel lentiviral vectors capable of correcting GPIX expression, localization, and functionality in human GP9-KO megakaryoblastic cell lines. Generated GP9-KO induced pluripotent stem cells produced platelets that recapitulated the BSS phenotype: absence of GPIX on the membrane surface and large size. Importantly, gene therapy tools reverted both characteristics. Finally, hematopoietic stem cells from two unrelated BSS type C patients were transduced with the gene therapy vectors and differentiated to produce GPIX-expressing megakaryocytes and platelets with a reduced size. These results demonstrate the potential of lentiviral-based gene therapy to rescue BSS type C

Advances in greater amberjack (Seriola dumerili) research: the DIVERSIFY project

The greater amberjack (Seriola dumerili) is a species with high potential for the EU aquaculture due to its fast growth (6 kg in 2.5 years), excellent flesh quality and global market. Its farming in the Mediterranean region started in the 1990s with wild-caught juveniles, but the production is still negligible, as several bottlenecks exist for its industrial production. These include the absence of reliable reproduction, limited availability of juveniles, lack of knowledge on the nutrient requirements and pathology of the species. The EU FP7-funded DIVERSIFY project (www.diversifyfish.eu) examines the major aspects of greater amberjack aquaculture in order to overcome these bottlenecks and develop appropriate rearing methods for commercial production. This article provides some highlights from the first 2 years of the project.Postprin

Hyperspectral imaging benchmark based on machine learning for intraoperative brain tumour detection

publishedVersio

Participation of Actin on Giardia lamblia Growth and Encystation

BACKGROUND:Microfilaments play a determinant role in different cell processes such as: motility, cell division, phagocytosis and intracellular transport; however, these structures are poorly understood in the parasite Giardia lamblia. METHODOLOGY AND PRINCIPAL FINDINGS:By confocal microscopy using TRITC-phalloidin, we found structured actin distributed in the entire trophozoite, the label stand out at the ventral disc, median body, flagella and around the nuclei. During Giardia encystation, a sequence of morphological changes concurrent to modifications on the distribution of structured actin and in the expression of actin mRNA were observed. To elucidate whether actin participates actively on growth and encystation, cells were treated with Cytochalasin D, Latrunculin A and Jasplakinolide and analyzed by confocal and scanning electron microscopy. All drugs caused a growth reduction (27 to 45%) and changes on the distribution of actin. Besides, 60 to 80% of trophozoites treated with the drugs, exhibited damage at the caudal region, alterations in the flagella and wrinkles-like on the plasma membrane. The drugs also altered the cyst-yield and the morphology, scanning electron microscopy revealed diminished cytokinesis, cysts with damages in the wall and alterations in the size and on the intermembranal space. Furthermore, the drugs caused a significant reduction of the intensity of fluorescence-labeled CWP1 on ESV and on cyst wall, this was coincident with a reduction of CWP1 gene expression (34%). CONCLUSIONS AND SIGNIFICANCE:All our results, indicated an important role of actin in the morphology, growth and encystation and indirectly suggested an actin role in gene expression