Capturing the blue-light activated state of the Phot-LOV1 domain from Chlamydomonas reinhardtii using time-resolved serial synchrotron crystallography.

Light-oxygen-voltage (LOV) domains are small photosensory flavoprotein modules that allow the conversion of external stimuli (sunlight) into intracellular signals responsible for various cell behaviors (e.g. phototropism and chloroplast relocation). This ability relies on the light-induced formation of a covalent thioether adduct between a flavin chromophore and a reactive cysteine from the protein environment, which triggers a cascade of structural changes that result in the activation of a serine/threonine (Ser/Thr) kinase. Recent developments in time-resolved crystallography may allow the activation cascade of the LOV domain to be observed in real time, which has been elusive. In this study, we report a robust protocol for the production and stable delivery of microcrystals of the LOV domain of phototropin Phot-1 from Chlamydomonas reinhardtii (CrPhotLOV1) with a high-viscosity injector for time-resolved serial synchrotron crystallography (TR-SSX). The detailed process covers all aspects, from sample optimization to data collection, which may serve as a guide for soluble protein preparation for TR-SSX. In addition, we show that the crystals obtained preserve the photoreactivity using infrared spectroscopy. Furthermore, the results of the TR-SSX experiment provide high-resolution insights into structural alterations of CrPhotLOV1 from Δt = 2.5 ms up to Δt = 95 ms post-photoactivation, including resolving the geometry of the thioether adduct and the C-terminal region implicated in the signal transduction process.

Spine surgery in a state-of-the-art hybrid operating room: an experience of 1745 implanted pedicle screws in the thoracolumbar spine.

Hybrid-operating rooms (hybrid-OR) combine high-resolution 2D images and 3D-scans with the possibility of 3D-navigation and allow minimal invasive pedicle screw placement even in the upper thoracic spine. The disadvantage of high cost and increased radiation needs to be compensated with high accuracy and safety. The hybrid operating room consists of a floor-based flat-panel robotic C-arm with 3D-scan capability (Artis Zeego, Siemens; Germany) combined with navigation (BrainLAB Curve, BrainLAB; Germany). Through a minimally invasive incision, a Jamshidi needle was advanced through the pedicle and a K-wire was placed. If 2D image quality did not allow safe placement 3D-navigation was used to place the K-wire. Position was controlled through a 3D-Scan and corrected if necessary before screw placement. Postoperative CTs evaluated screw perforation grade with grade I when completely within the pedicle, II < 2 mm, III 2-4 mm, and IV > 4 mm outside the pedicle. Overall, 354 screws were placed in T1-T6, 746 in the lower thoracic spine T7-T12 and 645 in the L1-L5. Navigation was mainly used in upper thoracic spine cases (31 of 57). In 63 out of 326 cases K-wire was corrected after the 3D-Scan. Overall, 99.1% of the screws showed perforation less than 2 mm. Mean radiation was 13.3 ± 11.7 mSv and significantly higher in the upper thoracic spine and in navigated procedures. Despite higher costs and radiation, the hybrid-OR allows highest accuracy and therefore patient safety in minimal invasive pedicle screw placement in the thoracic and lumbar spine.

Exploring the signaling space of a GPCR using bivalent ligands with a rigid oligoproline backbone.

G protein-coupled receptors (GPCRs) are one of the most important drug-target classes in pharmaceutical industry. Their diversity in signaling, which can be modulated with drugs, permits the design of more effective and better-tolerated therapeutics. In this work, we have used rigid oligoproline backbones to generate bivalent ligands for the gastrin-releasing peptide receptor (GRPR) with a fixed distance between their recognition motifs. This allows the stabilization of GPCR dimers irrespective of their physiological occurrence and relevance, thus expanding the space for medicinal chemistry. Specifically, we observed that compounds presenting agonists or antagonists at 20- and 30-Å distance induce GRPR dimerization. Furthermore, we found that 1) compounds with two agonists at 20- and 30-Å distance that induce dimer formation show bias toward Gq efficacy, 2) dimers with 20- and 30-Å distance have different potencies toward ß-arrestin-1 and ß-arrestin-2, and 3) the divalent agonistic ligand with 10-Å distance specifically reduces Gq potency without affecting ß-arrestin recruitment, pointing toward an allosteric effect. In summary, we show that rigid oligoproline backbones represent a tool to develop ligands with biased GPCR signaling.

C-Graphs Tool with Graphical User Interface to Dissect Conserved Hydrogen-Bond Networks: Applications to Visual Rhodopsins.

Dynamic hydrogen-bond networks provide proteins with structural plasticity required to translate signals such as ligand binding into a cellular response or to transport ions and larger solutes across membranes and, thus, are of central interest to understand protein reaction mechanisms. Here, we present C-Graphs, an efficient tool with graphical user interface that analyzes data sets of static protein structures or of independent numerical simulations to identify conserved, vs unique, hydrogen bonds and hydrogen-bond networks. For static structures, which may belong to the same protein or to proteins with different sequences, C-Graphs uses a clustering algorithm to identify sites of the hydrogen-bond network where waters are conserved among the structures. Using C-Graphs, we identify an internal protein-water hydrogen-bond network common to static structures of visual rhodopsins and adenosine A2A G protein-coupled receptors (GPCRs). Molecular dynamics simulations of a visual rhodopsin indicate that the conserved hydrogen-bond network from static structure can recruit dynamic hydrogen bonds and extend throughout most of the receptor. We release with this work the code for C-Graphs and its graphical user interface.

Accuracy of screw stabilization of the dorsal pelvic ring using a hybrid operating room: 5 Year experience in a level 1 trauma center.

INTRODUCTION: Accuracy for screw placement in the dorsal pelvic ring can be enhanced using intraoperative 3D navigation. Advances in intraoperative imaging lead to benefits for pelvic surgery. New c-arms are equipped with flat panel detectors, which have a larger detector and assure higher image quality with accompanying dose reduction. A hybrid OR is defined by a fixed imaging system in an operating room providing the benefit of the surgical environment in combination with advanced intraoperative imaging. Aim of our investigation was to analyze the accuracy of navigated sacroiliac (SI) and transsacral transiliac (TSTI) screws in the dorsal pelvic ring, which were implanted with a hybrid OR in the first five years of use. MATERIAL AND METHODS: All percutaneous SI or TSTI screws implanted in the hybrid OR using intraoperative navigation in the first 5 years of utilization (between June 2012 to June 2017) were included. Intraoperative 3D-scans and postoperative computed tomography were examined for screw perforation. RESULTS: 210 SI and TSTI screws were implanted in 187 patients using intraoperative navigation in the hybrid-OR. 90.6 % of SI screws showed no cortical perforation. 6,3 % had a grade 1, 2.4 % a grade 2 and 0.8 % a grade 3 perforation. In 80.7 % of TSTI screws no perforation and in 13.3 % a grade 1 perforation was detected. 3.6 % showed a grade 2 and 2.4 % a grade 3 perforation. No significant difference between both screw types regarding the grade of cortical perforation could be seen. No significant relation between perforation rate and year of operation could be detected. CONCLUSION: Intraoperative navigation in a hybrid OR ensures a high accuracy for SI screws. Due to the large field of view and high image quality TSTI screws can be safely implanted in S1 and S2. Utilization of a hybrid-OR is accompanied with a steep learning curve.

Structural basis of the activation of the CC chemokine receptor 5 by a chemokine agonist.

The human CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) that plays a major role in inflammation and is involved in cancer, HIV, and COVID-19. Despite its importance as a drug target, the molecular activation mechanism of CCR5, i.e., how chemokine agonists transduce the activation signal through the receptor, is yet unknown. Here, we report the cryo-EM structure of wild-type CCR5 in an active conformation bound to the chemokine super-agonist [6P4]CCL5 and the heterotrimeric Gi protein. The structure provides the rationale for the sequence-activity relation of agonist and antagonist chemokines. The N terminus of agonist chemokines pushes onto specific structural motifs at the bottom of the orthosteric pocket that activate the canonical GPCR microswitch network. This activation mechanism differs substantially from other CC chemokine receptors that bind chemokines with shorter N termini in a shallow binding mode involving unique sequence signatures and a specialized activation mechanism.

Cement augmentation of the proximal femur nail antirotation: is it safe?

BACKGROUND: Cement augmentation of the proximal femur nail antirotation (PFNA; Fa. DePuy Synthes) showed good biomechanical and clinical results regarding increased stability and functional outcome [Linden et al. in J Orthop Res 24:2230-2237, 2006;Kammerlander et al. in Injury 49:1436-1444, 2018;]. Cement-associated complications are well known in orthopedic procedures like hip arthroplasty, vertebra- and kyphoplasty. This study investigates outcome and safety of augmentation of the proximal femur nail blade. MATERIALS AND METHODS: The retrospective review of the 299 patients (mean age 80 ± 13 years; 205 women and 94 men) focused on perioperative complications after augmentation which was performed with Traumacem V+ Cement (Fa. DePuy Synthes) in 152 cases. The decision for augmentation of the blade was made by the attending surgeon and based on the factors age, bone quality, and fracture pattern. Primary outcome measures were changes in blood pressure, heart rate or oxygen saturation, and the number of needed vasoactive drugs during augmentation. Secondary outcome measures where the rate of cement leakage into the joint, mechanical failure, and perioperative complications like pulmonary embolism, stroke, or heart attack. RESULTS: In 152 augmented cases, no leakage of cement into the joint could be detected. No signs of mechanical failure like cut-out of the blade were seen after 6 weeks and 3 months. Also, augmentation did not show a higher rate of mortality or postoperative complications like stroke, heart attack, embolism, or infection. 57 of 152 augmented cases received an intraoperative intervention with vasoactive medication at the time of augmentation either prophylactically or because of a blood pressure fall. Out of the non-augmented cases, 21 of 147 needed vasoactive medication in the second half of the operation. The difference between these groups was significant (p < 0.05). In the cases without an intervention, there was a significant blood pressure fall of about 8 ± 7.4 mmHg during the augmentation (p < 0.001). Still, none of the augmented cases showed a change in heart rate or oxygen saturation. CONCLUSION: The augmentation of the PFNA blade proved to be a safe procedure. Cement augmentation will not increase postoperative complications or mortality. The risk for leakage of cement into the joint is low and mechanical cut-out might be prevented. The decision for augmentation should be made carefully and always be declared loud and in advance to allow the anesthetist to prepare, because blood pressure changes can occur.

Femtosecond-to-millisecond structural changes in a light-driven sodium pump.

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Impact of oral anticoagulation on proximal femur fractures treated within 24 h - A retrospective chart review.

BACKGROUND: About one third of all patients with proximal femur fractures take oral anticoagulation like aspirin (ASS), direct platelet aggregation inhibitors like Clopidogrel and Ticagrelor (PAI), vitamin-K-antagonists like Warfarin (VKA) and direct oral anticoagulants like Rivaroxaban, Dabigatran and Apixaban (DOAC). The management and timing of fracture stabilization of these patients is a rising challenge in orthopedic trauma. Our objective was to determine the effect of oral anticoagulation on patients with proximal femur fractures, which received a proximal femur nail antirotation (PFNA) within 24 h after trauma. MATERIAL AND METHODS: A retrospective chart review of 327 patients (mean age 80 ± 13 years; 223 women and 104 men) with sub- or intertrochanteric fractures between January 2013 and December 2017 was performed. All patients underwent surgery in the first 24 h after admission. Solely patients without or with only one type of oral anticoagulation were included. There were 74 patients with ASS, 30 with PAI, 52 with DOAC and 25 with VKA medication. All patients taking VKA received high dose Vitamin K or coagulation factors to normalize INR prior to surgery. Primary outcome measures were transfusion rate and pre- and postoperative hemoglobin (Hb) difference. Secondary outcome measures were mortality and complications like infection, hematoma and acute cardiovascular events. RESULTS: Patients undergoing treatment with DOAC had a 3.4-fold increased risk for intraoperative blood transfusion. The risk for blood transfusion for patients taking ASS, PAI or VKA did no differ from the control group. Patients without an intraoperative blood transfusion on oral anticoagulation showed no increase in pre- and postoperative Hb-difference compared with controls. Anticoagulation showed no significant effect on complication rates and mortality in patients operated within the first 24 h. CONCLUSION: Early surgical care of proximal femur fractures is safe even in patients with anticoagulant medication. All patients should be preoperatively prepared for possibly intraoperative transfusion, especially patients on DOAC.

Radiation exposure for the surgical team in a hybrid-operating room.

Hybrid-operating rooms enable the surgeon to acquire intraoperative high-resolution 2- and 3D images and use them for navigation. The radiation dose of the operating personal and the patient remains the major concern. In 9 months, 109 pelvic and spine cases were performed using a hybrid operating room. Radiation dose of the surgeon and the assisting nurse was recorded using real-time dosimeters. Lower radiation doses for the main surgeon in navigated dorsal instrumentations of the thoracic spine were recorded. Standing between the C-arm during screw placement increased the radiation dose sixfold. Lumbar dorsal instrumentation showed a similar radiation dose compared to the previous studies in traditional operating room settings. The use of a hybrid-operating room for dorsal spine instrumentation showed no increase in radiation dose compared to traditional settings. Intraoperative navigation can help to reduce the radiation dosage for the operating personnel.

Correction to: Radiation exposure for intraoperative 3D scans in a hybrid operating room: how to reduce radiation exposure for the surgical team.

The original version of this article unfortunately contained a mistake. In abstract, results section should read.

Radiation exposure for intraoperative 3D scans in a hybrid operating room: how to reduce radiation exposure for the surgical team.

BACKGROUND: Hybrid operating rooms are used in different fields of surgery. In orthopedic surgery, the possibility of a 3D scan of difficult anatomical regions (spine, pelvis) showed promising results not only in navigated screw placement. The associated exposure to radiation raises questions regarding potential risks for the operating room personal and the patient. The present study focuses on scatter radiation during 3D scans in a hybrid operating room, the adjacent rooms, and methods to reduce radiation exposure. MATERIAL AND METHODS: [Formula: see text] i2 dosimeters were used to measure scatter radiation during 3D scans of different anatomical regions in different distances and heights in a hybrid operating room. The 3D scans were performed with a floor-based flat-panel robotic C-arm with 3D scan capability (Artis Zeego, Siemens; Germany). The 3D scans were performed using a human cadaver. The 3D scans were performed using a standard and a dose reduction protocol (DRP). RESULTS: The highest scatter radiation was measured during 3D scans of the pelvis on the side of the surgical assistant (39.5 µSv in height of 1 m) compared to the side of the main surgeon (23 µSv in height of 1 m). Scatter radiation was less on the position of the scrub nurse (6.8 µSv in height of 1 m) and during 3D scans of the other anatomical regions. The radiation dosage was about 66% less with the DRP. Low values of scatter radiation were measured behind a radiation protection wall and with open doors in the adjacent rooms. CONCLUSION: While performing a scan scatter radiation was measured everywhere in the operating room especially during 3D scans of the pelvic girdle. Therefore, settings with lower dosage should be used whenever possible. Personnel should stand behind a radiation safety wall or leave the operating room and close the doors. For this study, six behavioral rules to avoid radiation in a hybrid operating room were established.

Mesenchymal stem cells in peripheral blood of severely injured patients.

PURPOSE: Mesenchymal stem cells (MSCs) are primarily stromal cells present in bone marrow and other tissues that are crucial for tissue regeneration and can be mobilized into peripheral blood after different types of organ damage. However, little is known about MSC appearance in blood in the setting of polytrauma. METHODS: We conducted a monocentered and longitudinal observational clinical study in 11 polytraumatized patients with an injury severity score (ISS) ≥ 24 to determine the numbers of MSCs in peripheral blood. Blood was collected from healthy volunteers and patients after polytrauma in the emergency room and 4, 12, 24, 48 h, 5 and 10 day later, and cells carrying MSC-surface markers (negative for CD45, positive for CD29, CD73, CD90, CD105, and CD166 in different combinations also employing the more stringent markers STRO1 and MSCA1) were detected and characterized using flow cytometry. Relative numbers of MSC-like cells were correlated with clinical parameters to evaluate if specific injury patterns had an influence on their presence in the blood cell pool. RESULTS: We were able to detect MSC marker-positive cells in both cohorts; however, the percentage of those cells present in the blood of patients during the first 10 day after injury was mostly similar to healthy volunteers, and significantly lowers starting at 4 h post trauma for one marker combination when compared to controls. Furthermore, the presence of a pelvis fracture was partly correlated with reduced relative numbers of MSC-like cells detectable in blood. CONCLUSIONS: Polytrauma in humans was associated with partly reduced relative numbers of MSC-like cells detected in peripheral blood in the time course after injury. Further studies need to define if this reduction was due to lower mobilization from the bone marrow or to active migration to the sites of injury.

Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

Feasibility of laser-guided percutaneous pedicle screw placement in the lumbar spine using a hybrid-OR.

PURPOSE: Innovations in intraoperative imaging lead to major changes in orthopaedic surgery. In our setting, a 3D flat-panel c-arm (Artis zeego) is mounted on a robotic arm offering an integrated aiming tool (Syngo iGuide). Our aim was to investigate the feasibility of Syngo iGuide for pedicle screw placement in comparison with fluoroscopic screw implantation. METHODS: In 10 lumbar models, 100 screws were implanted. In 5 models, a standard fluoroscopic technique was used. Syngo iGuide was used in all other models. Afterwards, CT-scans were performed and screw accuracy was investigated. RESULTS: The procedure time for the new technique was significantly longer in comparison with the standard technique. The post-operative CT showed the same accuracy in both groups. CONCLUSIONS: Syngo iGuide proofed feasible for percutaneous implantation of pedicle screws in anatomic models. Syngo iGuide can be a help for screw implantation in difficult anatomic regions without the need of an additional navigation system.

[The Ulm trauma track : Trauma care and research as focal points for medical students]. / Der Ulmer Trauma-Track : Traumaversorgung und -forschung als Schwerpunktsetzung im Medizinstudium.

BACKGROUNDS AND OBJECTIVES: As part of the expansion of the site-specific education profile of the medical curriculum MED@ULM of the University of Ulm, a new track "trauma care and trauma research" was established in the winter semester 2012/2013. The acceptance of the track was evaluated during the winter semester 2013/2014. MATERIAL AND METHODS: The 6-semester track extends the existing curriculum by offering subjects in trauma management and trauma research to students of human medicine. A central aim of the track is to promote medical professional competence, expertise in emergency care and competence in trauma-related scientific work and research. Central learning contents could be intensified in newly established emergency simulation training. Additionally, participating students have to perform a doctoral thesis on an obligatory trauma-related experimental subject. A first analysis study focusing on the learning style of the participating students (n = 17) and a control group consisting of members of the same semester (n = 20) was performed using the Kolb learning style inventory. In a validated evaluation in the winter semesters 2013/2014 and 2014/2015, the students were asked about their expectations and experience with the track, criticisms, suggestions and satisfaction with the study conditions. The data were analyzed using descriptive statistics. RESULTS: The analysis of the students' preferred learning styles revealed no differences between track students and the control group. Most of the students considered the track as a form of personal further education. The students had high expectations of practical skills with relevance to the clinical daily routine, learning scientific methods and preparing their thesis. The track students were more critical with regard to the study conditions than the control group students, although the track students of the third semester still judged their studies to be more interesting than the track students of the first semester and the control group. CONCLUSION: With the introduction of the new trauma track into the curriculum of the medical curriculum MED@ULM of the University of Ulm, a further possibility for medical students to focus on their own individual options was established. At least half of the track students wanted to be later active in the triad of patient care, teaching and research. Further investigations are necessary to determine whether the establishment of the trauma track has a positive influence on the number of new recruits in trauma surgery and anesthesiology.