Targeted sensors for glutamatergic neurotransmission.

Optical report of neurotransmitter release allows visualisation of excitatory synaptic transmission. Sensitive genetically-encoded fluorescent glutamate reporters operating with a range of affinities and emission wavelengths are available. However, without targeting to synapses, the specificity of the fluorescent signal is uncertain, compared to sensors directed at vesicles or other synaptic markers. We fused the state-of-the-art reporter iGluSnFR to glutamate receptor auxiliary proteins in order to target it to postsynaptic sites. Chimeras of Stargazin and gamma-8 that we named SnFR-γ2 and SnFR-γ8, were enriched at synapses, retained function and reported spontaneous glutamate release in rat hippocampal cells, with apparently diffraction-limited spatial precision. In autaptic mouse neurons cultured on astrocytic microislands, evoked neurotransmitter release could be quantitatively detected at tens of synapses in a field of view whilst evoked currents were recorded simultaneously. These experiments revealed a specific postsynaptic deficit from Stargazin overexpression, resulting in synapses with normal neurotransmitter release but without postsynaptic responses. This defect was reverted by delaying overexpression. By working at different calcium concentrations, we determined that SnFR-γ2 is a linear reporter of the global quantal parameters and short-term synaptic plasticity, whereas iGluSnFR is not. On average, half of iGluSnFR regions of interest (ROIs) showing evoked fluorescence changes had intense rundown, whereas less than 5% of SnFR-γ2 ROIs did. We provide an open-source analysis suite for extracting quantal parameters including release probability from fluorescence time series of individual and grouped synaptic responses. Taken together, postsynaptic targeting improves several properties of iGluSnFR and further demonstrates the importance of subcellular targeting for optogenetic actuators and reporters.

Essential steps in the development, implementation, evaluation and quality assurance of the written part of the Swiss federal licensing examination for human medicine.

Purpose: This report describes the essential steps in the development, implementation, evaluation and quality assurance of the written part of the Swiss Federal Licensing Examination for Human Medicine (FLE) and the insights gained since its introduction in 2011. Methods: Based on existing scientific evidence, international expertise, and experience gained from previous examinations, the FLE is developed by experts from all five medical faculties in Switzerland with the support of the Institute for Medical Education and is held simultaneously at five locations. The exam organisers document and review every examination held and continuously optimise the processes; they have summarised the results in this report. Results: The essential steps comprise the development, revision and translation of questions; construction of the exam and production of materials; candidate preparation; implementation and analysis. The quality assurance measures consist of guideline coherence in the development of the questions and implementation of the exam, revision processes, construction of the exam based on the national blueprint, multiphase review of the translations and exam material, and statistical analysis of the exam and the comments from candidates. The intensive collaboration, especially on the part of representatives from all the participating faculties and a central coordination unit, which provides methodological support throughout and oversees the analysis of the exam, has proven successful. Successfully completed examinations and reliable results in the eleven examinations so far implemented represent the outcomes of the quality assurance measures. Significant insights in recent years are the importance of appreciating the work of those involved and the central organisation of exam development, thus ensuring the long-term success of the process. Conclusion: Common guidelines and workshops, quality assurance measures accompanied by the continuous improvement of all processes, and appreciation of everyone involved, are essential to carrying out such an examination at a high-quality level in the long term.

LRRC8 channel activation and reduction in cytosolic chloride concentration during early differentiation of C2C12 myoblasts.

Leucine-rich repeat containing family 8 (LRRC8) proteins form the volume-regulated anion channel (VRAC). Recently, they were shown to be required for normal differentiation and fusion of C2C12 myoblasts, by promoting membrane hyperpolarization and intracellular Ca2+ signals. However, the mechanism by which they are involved remained obscure. Here, using a FRET-based sensor for VRAC activity, we show temporary activation of VRAC within the first 2 h of myogenic differentiation. During this period, we also observed a significant decrease in the intracellular Cl- concentration that was abolished by the VRAC inhibitor carbenoxolone. However, lowering the intracellular Cl- concentration by extracellular Cl- depletion did not promote differentiation as judged by the percentage of myogenin-positive nuclei or total myogenin levels in C2C12 cells. Instead, it inhibited myosin expression and myotube formation. Together, these data suggest that VRAC is activated and mediates Cl- efflux early on during myogenic differentiation, and a moderate intracellular Cl- concentration is necessary for myoblast fusion.

A FRET sensor of C-terminal movement reveals VRAC activation by plasma membrane DAG signaling rather than ionic strength.

Volume-regulated anion channels (VRACs) are central to cell volume regulation. Recently identified as hetero-hexamers formed by LRRC8 proteins, their activation mechanism remains elusive. Here, we measured Förster resonance energy transfer (FRET) between fluorescent proteins fused to the C-termini of LRRC8 subunits. Inter-subunit FRET from LRRC8 complexes tracked VRAC activation. With patch-clamp fluorometry, we confirmed that the cytoplasmic domains rearrange during VRAC opening. With these FRET reporters, we determined VRAC activation, non-invasively, in live cells and their subcompartments. Reduced intracellular ionic strength did not directly activate VRACs, and VRACs were not activated on endomembranes. Instead, pharmacological manipulation of diacylglycerol (DAG), and protein kinase D (PKD) activity, activated or inhibited plasma membrane-localized VRACs. Finally, we resolved previous contradictory reports concerning VRAC activation, using FRET to detect robust activation by PMA that was absent during whole-cell patch clamp. Overall, non-invasive VRAC measurement by FRET is an essential tool for unraveling its activation mechanism.

More than just a pressure relief valve: physiological roles of volume-regulated LRRC8 anion channels.

The volume-regulated anion channel (VRAC) is a key player in the volume regulation of vertebrate cells. This ubiquitously expressed channel opens upon osmotic cell swelling and potentially other cues and releases chloride and organic osmolytes, which contributes to regulatory volume decrease (RVD). A plethora of studies have proposed a wide range of physiological roles for VRAC beyond volume regulation including cell proliferation, differentiation and migration, apoptosis, intercellular communication by direct release of signaling molecules and by supporting the exocytosis of insulin. VRAC was additionally implicated in pathological states such as cancer therapy resistance and excitotoxicity under ischemic conditions. Following extensive investigations, 5 years ago leucine-rich repeat-containing family 8 (LRRC8) heteromers containing LRRC8A were identified as the pore-forming components of VRAC. Since then, molecular biological approaches have allowed further insight into the biophysical properties and structure of VRAC. Heterologous expression, siRNA-mediated downregulation and genome editing in cells, as well as the use of animal models have enabled the assessment of the proposed physiological roles, together with the identification of new functions including spermatogenesis and the uptake of antibiotics and platinum-based cancer drugs. This review discusses the recent molecular biological insights into the physiology of VRAC in relation to its previously proposed roles.

Biophysics and Structure-Function Relationships of LRRC8-Formed Volume-Regulated Anion Channels.

Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B-E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.

Computer assisted evaluation of plate osteosynthesis of diaphyseal femur fracture considering interfragmentary movement: a finite element study.

A semi-automated workflow for evaluation of diaphyseal fracture treatment of the femur has been developed and implemented. The aim was to investigate the influence of locking compression plating with diverse fracture-specific screw configurations on interfragmentary movements (IFMs) with the use of finite element (FE) analysis. Computed tomography (CT) data of a 22-year-old non-osteoporotic female were used for patient specific modeling of the inhomogeneous material properties of bone. Hounsfield units (HU) were exported and assigned to elements of a FE mesh and converted to mechanical properties such as the Young's modulus followed by a linear FE analysis performed in a semi-automated fashion. IFM on the near and far cortex was evaluated. A positive correlation between bridging length and IFM was observed. Optimal healing conditions with IFMs between 0.5 mm and 1 mm were found in a constellation with a medium bridging length of 80 mm with three unoccupied screw holes around the fracture gap. Usage of monocortical screws instead of bicortical ones had negligible influence on the evaluated parameters when modeling non-osteoporotic bone. Minimal user input, automation of the procedure and an efficient computation time ensured quick delivery of results which will be essential in a future clinical application.

Single-source dual-energy computed tomography: use of monoenergetic extrapolation for a reduction of metal artifacts.

OBJECTIVES: The objective of this study was to retrospectively determine the potential of virtual monoenergetic (ME) reconstructions for a reduction of metal artifacts using a new-generation single-source computed tomographic (CT) scanner. MATERIALS AND METHODS: The ethics committee of our institution approved this retrospective study with a waiver of the need for informed consent. A total of 50 consecutive patients (29 men and 21 women; mean [SD] age, 51.3 [16.7] years) with metal implants after osteosynthetic fracture treatment who had been examined using a single-source CT scanner (SOMATOM Definition Edge; Siemens Healthcare, Forchheim, Germany; consecutive dual-energy mode with 140 kV/80 kV) were selected. Using commercially available postprocessing software (syngo Dual Energy; Siemens AG), virtual ME data sets with extrapolated energy of 130 keV were generated (medium smooth convolution kernel D30) and compared with standard polyenergetic images reconstructed with a B30 (medium smooth) and a B70 (sharp) kernel. For quantification of the beam hardening artifacts, CT values were measured on circular lines surrounding bone and the osteosynthetic device, and frequency analyses of these values were performed using discrete Fourier transform. A high proportion of low frequencies to the spectrum indicates a high level of metal artifacts. The measurements in all data sets were compared using the Wilcoxon signed rank test. RESULTS: The virtual ME images with extrapolated energy of 130 keV showed significantly lower contribution of low frequencies after the Fourier transform compared with any polyenergetic data set reconstructed with D30, B70, and B30 kernels (P < 0.001). CONCLUSIONS: Sequential single-source dual-energy CT allows an efficient reduction of metal artifacts using high-energy ME extrapolation after osteosynthetic fracture treatment.

Intra- and postoperative complications of navigated and conventional techniques in percutaneous iliosacral screw fixation after pelvic fractures: Results from the German Pelvic Trauma Registry.

BACKGROUND: Percutaneous iliosacral screw placement following pelvic trauma is a very demanding technique involving a high rate of screw malpositions possibly associated with the risk of neurological damage or inadequate stability. In the conventional technique, the screw's correct entry point and the small target corridor for the iliosacral screw may be difficult to visualise using an image intensifier. 2D and 3D navigation techniques may therefore be helpful tools. The aim of this multicentre study was to evaluate the intra- and postoperative complications after percutaneous screw implantation by classifying the fractures using data from a prospective pelvic trauma registry. The a priori hypothesis was that the navigation techniques have lower rates of intraoperative and postoperative complications. METHODS: This study is based on data from the prospective pelvic trauma registry introduced by the German Society of Traumatology and the German Section of the AO/ASIF International in 1991. The registry provides data on all patients with pelvic fractures treated between July 2008 and June 2011 at any one of the 23 Level I trauma centres contributing to the registry. RESULTS: A total of 2615 patients were identified. Out of these a further analysis was performed in 597 patients suffering injuries of the SI joint (187×with surgical interventions) and 597 patients with sacral fractures (334×with surgical interventions). The rate of intraoperative complications was not significantly different, with 10/114 patients undergoing navigated techniques (8.8%) and 14/239 patients in the conventional group (5.9%) for percutaneous screw implantation (p=0.4242). Postoperative complications were analysed in 30/114 patients in the navigated group (26.3%) and in 70/239 patients (29.3%) in the conventional group (p=0.6542). Patients who underwent no surgery had with 66/197 cases (33.5%) a relatively high rate of complications during their hospital stay. The rate of surgically-treated fractures was higher in the group with more unstable Type-C fractures, but the fracture classification had no significant influence on the rate of complications. DISCUSSION: In this prospective multicentre study, the 2D/3D navigation techniques revealed similar results for the rate of intraoperative and postoperative complications compared to the conventional technique. The rate of neurological complications was significantly higher in the navigated group.

Safe surgical technique: cement-augmented pedicle screw instrumentation and balloon-guided kyphoplasty for a lumbar burst fracture in a 97-year-old patient.

BACKGROUND: During the last few years, an increasing number of unstable thoracolumbar fractures, especially in elderly patients, has been treated by dorsal instrumentation combined with a balloon kyphoplasty. This combination provides additional stabilization to the anterior spinal column without any need for a second ventral approach. CASE PRESENTATION: We report the case of a 97-year-old male patient with a lumbar burst fracture (type A3-1.1 according to the AO Classification) who presented prolonged neurological deficits of the lower limbs - grade C according to the modified Frankel/ASIA score. After a posterior realignment of the fractured vertebra with an internal screw fixation and after an augmentation with non-absorbable cement in combination with a balloon kyphoplasty, the patient regained his mobility without any neurological restrictions. CONCLUSION: Especially in older patients, the presented technique of PMMA-augmented pedicle screw instrumentation combined with balloon-assisted kyphoplasty could be an option to address unstable vertebral fractures in "a minor-invasive way". The standard procedure of a two-step dorsoventral approach could be reduced to a one-step procedure.

Balloon osteoplasty--a new technique for reduction and stabilisation of impression fractures in the tibial plateau: a cadaver study and first clinical application.

PURPOSE: Fractures of the tibial plateau are among the most severe injuries of the knee joint and lead to advanced gonarthrosis if the reduction does not restore perfect joint congruency. Many different reduction techniques focusing on open surgical procedures have been described in the past. In this context we would like to introduce a novel technique which was first tested in a cadaver setup and has undergone its successful first clinical application. METHODS: Since kyphoplasty demonstrated effective ways of anatomical correction in spine fractures, we adapted the inflatable instruments and used the balloon technique to reduce depressed fragments of the tibial plateau. RESULTS: The technique enabled us to restore a congruent cartilage surface and bone reduction. CONCLUSIONS: In this technique we see a useful new method to reduce depressed fractures of the tibial plateau with the advantages of low collateral damage as it is known from minimally invasive procedures.

Image guidance in pelvic and acetabular surgery--expectations, success and limitations.

During the last decade navigation techniques in pelvic and acetabular surgery have been described. Nowadays, available techniques include CT-based navigation, 2D C-arm navigation and 3D C-arm navigation. The main indication is the navigated percutaneous SI screw fixation, but acetabular screw fixations are also reported. In this article, based upon a literature review and our own clinical experiences, the indications for and limitations of navigated techniques in pelvic and acetabular surgery are described.