Rac1 activation can generate untemplated, lamellar membrane ruffles.

BACKGROUND: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. RESULTS: Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles. CONCLUSIONS: Combining LLSM and optogenetics enables superior spatial and temporal control for studying such dynamic mechanisms. Demonstrated here, the techniques implemented provide insight into the complex nature of the molecular interplay involved in dynamic actin machinery, revealing that Rac1 activation can generate untemplated, lamellar protrusions.

Rapid whole cell imaging reveals a calcium-APPL1-dynein nexus that regulates cohort trafficking of stimulated EGF receptors.

The endosomal system provides rich signal processing capabilities for responses elicited by growth factor receptors and their ligands. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors. Activated EGFRs are trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated and degraded. The details of the mechanisms that govern the movements of stimulated EGFRs towards the PNR, are not completely known. Here, exploiting the advantages of lattice light-sheet microscopy, we show that EGFR activation by EGF triggers a transient calcium increase causing a whole-cell level redistribution of Adaptor Protein, Phosphotyrosine Interacting with PH Domain And Leucine Zipper 1 (APPL1) from pre-existing endosomes within one minute, the rebinding of liberated APPL1 directly to EGFR, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within ten minutes. The cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGF receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking and have evolved processes that provide the temporal predictability that typify canonical signaling.

A comparative study about motivations, expectations and future plans for professional development in four European radiography programs.

INTRODUCTION: This study aims to compare motivations, expectations and work plans of students and teaching-staff from four different European radiography programs, it aims also to explore areas that could be included to advance post graduate studies. METHODS: Two different questionnaires (open- and closed-end questions) were applied to key-informants, students who had just completed their bachelor thesis and teaching-staff, to collect data regarding motivations, expectations, challenges and potentials for radiography education and, plans for further work. Descriptive statistics and thematic analysis were performed according to the nature of the questions. RESULTS: The response rates were 45% (students) and 68% (teaching-staff). The motivations to study radiography were similar between students: to work in a healthcare-service, helping people, manipulating high-end technologies, providing service while combining different knowledge (physics, patient-care, physiology, anatomy). 75% of the students did not reach all their expectations due to the lack of focused and updated content for some areas. The teaching-staff were expecting an extension of the radiographers' role. The development of advance studies in computed tomography and magnetic resonance was highlighted as important by students. Future work plans included: self-improvement, continuation of studies, specialization, research and collaborations. CONCLUSIONS: This study increased the understanding of radiography education and provides insights into future perspectives. Participants have similar motivations, expectations and future plans. Improvements in education should focus on technological developments and meeting job market demands. Further studies should be performed to identify approaches that acknowledge the specific needs of each country, while also providing strategies to harmonize radiography education in Europe.

Curricula, attributes and clinical experiences of radiography programs in four European educational institutions.

INTRODUCTION: The aim of this study was to compare radiography curricula, teaching/learning strategies, skill development, clinical practice outcomes and research development delivered by four European educational institutions. METHODS: This study was carried out in two phases: the first focused on curricula analysis; the second involved online questionnaires to ascertain data from two key-informants: students who had recently completed their bachelor thesis and teaching-staff. Questionnaires were designed to capture teaching and learning strategies, skill acquisition and outcomes of clinical practice and research. Descriptive statistics and thematic analysis were performed according to the nature of the questions. RESULTS: The European Credits Transfer System dedicated per core subject area (natural sciences, clinical practice, research, imaging technology, humanities) differed between institutions. Students classified technical, practical and communication skills as the most important, teaching-staff highlighted also critical thinking. The students defined as "very good" their experience in radiography (58.5%) and computed-tomography (45%). Magnetic resonance imaging practice was considered "Average" by 53% of the UK-students and "Good" by the other European students (40%). According to 71% (55/78) of the students, research work contributed to the development of critical/reflective thinking. CONCLUSIONS: The four radiography programs presented variations in curricula, contact-hours, clinical experience and outcomes. Research units allowed the participant-students to develop their critical thinking capabilities. The outcomes from clinical practice differ across the institutions, mainly due to differences in background and access to specialities. Further work is necessary to assess the real impact of different radiography programs on professional and academic mobility across Europe.

The 2018 correlative microscopy techniques roadmap.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy.

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50 ± 15 nm and 120 ± 40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Self-organizing actin patterns shape membrane architecture but not cell mechanics.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties.

Reliability and relatedness of peak VO2 assessments during body weight supported treadmill training and arm cycle ergometry in individuals with chronic motor incomplete spinal cord injury.

STUDY DESIGN: Prospective assessment as part of initial evaluations for randomized-controlled trial participation. OBJECTIVES: To determine the test-retest reliability of peak VO2 testing during both robotically assisted body weight supported treadmill training (RABWSTT) and arm cycle ergometry and to assess whether a relationship exists between these two measurements in individuals with chronic motor incomplete spinal cord injury (CMISCI). METHODS: Twenty-one participants with CMISCI enrolled in a 3- month, RABWSTT randomized-controlled trial. As part of their baseline assessments, individuals underwent VO2 peak assessments twice on separate days during both RABWSTT and arm cycle ergometry using a metabolic cart. RESULTS: Peak oxygen consumption measured at baseline correlated significantly between repeated tests in the RABWSTT (r=0.96, P<0.01) and the arm ergometer (r=0.95, P<0.01). A Pearson correlation (r=0.87, P<0.01) existed between the peak VO2 measurements obtained using RABWSTT and arm ergometry, although Bland-Altman analysis suggested a more limited relationship with a bias of 1.1 favoring arm ergometry. This relationship was stronger for individuals with tetraplegia than for people with paraplegia. CONCLUSION/CLINICAL RELEVANCE: Determination of VO2 peak during both RABWSTT and arm ergometry in individuals with CMISCI is highly reproducible. Furthermore, a moderate correlation exists between peak VO2 measured during RABWSTT and arm cycle ergometry in this population for individuals with tetraplegia. This correlation offers implications for future cardiovascular testing of individuals with CMISCI, as two reliable peak VO2 measurement techniques are possible.

Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus.

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.

Does renal impairment protect from gout?

OBJECTIVE: To test whether renal impairment protects from the development of gout. METHODS: We conducted a retrospective cohort study in which 40 hyperuricemic patients (serum uric acid > 0.54 mM/l) with renal impairment (serum creatinine > 200 microM/l) and 40 equally hyperuricemic patients with normal renal function (serum creatinine < 120 microM/l) were given a telephone questionnaire eliciting a history of gout, its pattern and severity, and other features of medical and family history. RESULTS: There was no significant difference among the prevalence of gout (relative risk 1.1, confidence interval 0.73-1.67), the pattern and severity of gout, and the presence of tophi between the 2 groups. A positive family history of gout was significantly increased in the patients with gouty arthritis (p < 0.05). CONCLUSION: Renal impairment does not protect from gout. There may be a familial factor in the development of gout that is independent from the familial tendency for hyperuricemia.

Tick-borne encephalitis virus envelope protein E-specific monoclonal antibodies for the study of low pH-induced conformational changes and immature virions.

A set of ten monoconal antibodies (mabs) specific for the tick-borne encephalitis (TBE) virus envelope protein E were prepared and characterized with respect to their functional activities, the location of their binding sites on protein E and the involvement of their epitopes in acid pH-induced conformational changes and interactions with the precursor to the membrane protein (prM) in immature virions. The majority of these mabs mapped to the previously defined antigenic domain A. All of the mabs recognize parts of the E protein which undergo low pH-induced structural rearrangements believed to be necessary for the fusion activity of the virus, and six of the mabs define epitopes which are affected by the prM-E interaction in immature virions. They are therefore of potential value as specific reagents for studying the structure and function of protein E, as well as the function of the prM-E association. Five of the mabs exhibited neutralizing activity, and can therefore be used for the selection of escape mutants.

Reproducibility of synovial fluid examination for crystals.

A quality assurance survey of synovial fluid examination for crystals was performed at six teaching hospitals. Aliquots of 12 different fluids (three with no crystals, one with betamethasone crystals, four with calcium pyrophosphate dihydrate (CPPD) crystals and four with monosodium urate monohydrate [urate] crystals) were examined by all six laboratories. Four laboratories performed well (10 or more correct out of 12) but two did poorly (50% or less correct). False positive crystal identification occurred in eight of 72 samples, but all false positive reports were from two laboratories. CPPD and urate crystals were missed in seven of 24 (29%) and five of 24 (21%) samples respectively. The standard of synovial fluid examination for crystals in Sydney teaching hospitals is not uniform and in some cases appears unsatisfactory.

Preservative activity in diluted corticosteroid creams.

The preservative efficacy of both 'Betnovate' and 'Synalar' creams diluted 1:1 with 'Unguentum Merck' was investigated. Each formulation was challenged with Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at an initial inoculum level of approximately 1 X 10(6) viable organisms per gram of cream. All formulations tested were found to be effectively preserved against the organisms used and no viable bacteria were detected 7 days after inoculation.