Affinity measurement of strong ligands with NMR spectroscopy: Limitations and ways to overcome them.

NMR spectroscopy is currently extensively used in binding assays for hit identification, but its use in dissociation constant determination is more limited when compared to other biophysical techniques, in particular for tight binders. Although NMR is quite suitable for measuring the binding strength of weak to medium affinity ligands with dissociation constant KD > 1 µM, it has some limitations in the determination of the binding strength of tight binders (KD < 1 µM). A theoretical analysis of the binding affinity determination of strong ligands using different types of NMR experiments is provided and practical guidelines are given for overcoming the limitations and for the proper set-up of the experiments. Some approaches require reagents with unique properties or highly specialized equipment, while others can be applied quite generally. We describe all approaches in detail, but give higher emphasis to the more general methods, like competition experiments, where we include actual experimental data and discuss the practical aspects.

Surgical Treatment of Patients with Sacroiliac Joint Syndrome: Comparative Study of Two Implants. / Operative Behandlung von Patienten mit Iliosakralgelenksyndrom: Vergleichsstudie zweier Implantate.

The treatment of patients with ISJ dysfunction is difficult due to the multifactorial causes of pain and various problems in clarification. Treatment includes physical therapy, corticosteroids, prolotherapy, radiofrequency denervation and sacroiliac joint fusion. A new option for the surgical treatment of ISG dysfunction is the Torpedo implant system. For a safe fusion, only 2 implants are needed, which are available in lengths of 30-50 mm. The new implant system has been tested in pilot studies for efficacy and biocompatibility with good results. For further documentation for the Torpedo implant system, a comparative study against the iFuse system was carried out.Two different implants were used: Group 1: Deltacor Torpedo, Group 2: iFuse implants (Si-Bone). The data generated during admission and subsequent check-ups (VAS, ODI, opioid use) were entered into an evaluation file set up for this purpose. Follow-up appointments were set at 1 month, 3, 6 and 12 months postoperatively.The data of 65 patients were evaluated comparatively. In all comparisons, only very small effect sizes were found with regard to the differences in the decrease in pain intensities, so that equivalent effectiveness of the two methods could initially be postulated from a clinical point of view. Most patients in both groups reported taking opioids to treat pain before surgery. According to the decrease in pain intensity, opioid treatment could be discontinued in some patients after the operation. After 12 months, the number of patients treated with opioids decreases to 23% in group 1 and to 17% in group 2. The success of the fusions with the two methods can also be proven by image documentation, from which the position of the implants can also be clearly recognised. In no case was there any loosening.Overall, the evaluation of this study allows the conclusion that both implant systems can be successfully used for the treatment of patients with ISJ syndrome. The present results should be confirmed in further comparative studies with the proposed evaluation methods.

Infiltration therapy in the context of cartilage surgery.

Guideline-based surgical cartilage therapy for focal cartilage damage offers highly effective possibilities to sustainably reduce patients' complaints and to prevent or at least delay the development of early osteoarthritis. In the knee joint, it has the potential to reduce almost a quarter of the arthroses requiring joint replacement caused by cartilage damage. Biologically effective injection therapies could further improve these results. Based on the currently available literature and preclinical studies, intra- and postoperative injectables may have a positive effect of platelet-rich plasma/fibrin (PRP/PRF) and hyaluronic acid (HA) on cartilage regeneration and, in the case of HA injections, also on the clinical outcome can be assumed. The role of a combination therapy with use of intra-articular corticosteroids is lacking in the absence of adequate study data and cannot be defined yet. With regard to adipose tissue-based cell therapy, the current scientific data do not yet justify any recommendation for its use. Further studies also regarding application intervals, timing and differences in different joints are required.

Empfehlungen der AG Klinische Geweberegeneration zur Behandlung von Knorpelschäden am Kniegelenk. / Empfehlungen der AG Klinische Geweberegeneration zur Behandlung von Knorpelschäden am Kniegelenk.

The Working Group of the German Orthopedic and Trauma Society (DGOU) on Tissue Regeneration has published recommendations on the indication of different surgical approaches for treatment of full-thickness cartilage defects in the knee joint in 2004, 2013 and 2016. Based upon new scientific knowledge and new developments, this recommendation is an update based upon the best clinical evidence available. In addition to prospective randomised controlled clinical trials, this also includes studies with a lower level of evidence. In the absence of evidence, the decision is based on a consensus process within the members of the working group.The principle of making decision dependent on defect size has not been changed in the new recommendation either. The indication for arthroscopic microfracturing has been reduced up to a defect size of 2 cm2 maximum, while autologous chondrocyte implantation is the method of choice for larger cartilage defects. Additionally, matrix-augmented bone marrow stimulation (mBMS) has been included in the recommendation for defects ranging from 1 to 4.5 cm2. For the treatment of smaller osteochondral defects, in addition to osteochondral transplantation (OCT), mBMS is also recommended. For larger defects, matrix-augmented autologous chondrocyte implantation (mACI/mACT) in combination with augmentation of the subchondral bone is recommended.

Nanobody GPS by PCS: An Efficient New NMR Analysis Method for G Protein Coupled Receptors and Other Large Proteins.

NMR chemical shift changes can report on the functional dynamics of biomacromolecules in solution with sizes >1 MDa. However, their interpretation requires chemical shift assignments to individual nuclei, which for large molecules often can only be obtained by tedious point mutations that may interfere with function. We present here an efficient pseudocontact shift NMR method to assign biomacromolecules using bound antibodies tagged with lanthanoid DOTA chelators. The stability of the antibody allows positioning the DOTA tag at many surface sites, providing triangulation of the macromolecule nuclei at distances >60 Å. The method provides complete assignments of valine and tyrosine 1H-15N resonances of the ß1-adrenergic receptor in various functional forms. The detected chemical shift changes reveal strong forces exerted onto the backbone of transmembrane helix 3 during signal transmission, which are absorbed by its electronic structure. The assignment method is applicable to any soluble biomacromolecule for which suitable complementary binders exist.

GPCR Activation States Induced by Nanobodies and Mini-G Proteins Compared by NMR Spectroscopy.

In this work, we examine methyl nuclear magnetic resonance (NMR) spectra of the methionine ε-[13CH3] labelled thermostabilized ß1 adrenergic receptor from turkey in association with a variety of different effectors, including mini-Gs and nanobody 60 (Nb60), which have not been previously studied in complex with ß1 adrenergic receptor (ß1AR) by NMR. Complexes with pindolol and Nb60 induce highly similar inactive states of the receptor, closely resembling the resting state conformational ensemble. We show that, upon binding of mini-Gs or nanobody 80 (Nb80), large allosteric changes throughout the receptor take place. The conformation of tß1AR stabilized by the native-like mini-Gs protein is highly similar to the conformation induced by the currently used surrogate Nb80. Interestingly, in both cases residual dynamics are present, which were not observed in the resting states. Finally, we reproduce a pharmaceutically relevant situation, where an antagonist abolishes the interaction of the receptor with the mini-G protein in a competitive manner, validating the functional integrity of our preparation. The presented system is therefore well suited for reproducing the individual steps of the activation cycle of a G protein-coupled receptor (GPCR) in vitro and serves as a basis for functional and pharmacological characterizations of more native-like systems in the future.

Enabling NMR Studies of High Molecular Weight Systems Without the Need for Deuteration: The XL-ALSOFAST Experiment with Delayed Decoupling.

Current biological research increasingly focusses on large human proteins and their complexes. Such proteins are difficult to study by NMR spectroscopy because they often can only be produced in higher eukaryotic expression systems, where deuteration is hardly feasible. Here, we present the XL-ALSOFAST-[13 C,1 H]-HMQC experiment with much improved sensitivity for fully protonated high molecular weight proteins. For the tested systems ranging from 100 to 240 kDa in size, 3-fold higher sensitivity was obtained on average for fast relaxing signals compared to current state-of-the-art experiments. In the XL-ALSOFAST approach, non-observed magnetisation is optimally exploited and transverse relaxation is minimized by the newly introduced concept of delayed decoupling. The combination of high sensitivity and superior artefact suppression makes it ideal for studying inherently unstable membrane proteins or for analysing therapeutic antibodies at natural 13 C abundance. The XL-ALSOFAST and delayed decoupling will therefore expand the range of biomolecular systems accessible to NMR spectroscopy.

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family.

Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses.IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.

Acetabular defects in revision hip arthroplasty: a therapy-oriented classification.

INTRODUCTION: The treatment of severe acetabular bone loss remains a difficult challenge. No classification system is available that combines intuitive use, structured design and offers a therapeutic recommendation according to the current literature and modern state of the art treatment options. The goal of this study is to introduce an intuitive, reproducible and reliable guideline for the evaluation and treatment of acetabular defects. METHODS: The proposed Acetabular Defect Classification (ADC) is based on the integrity of the acetabular rim and supporting structures. It consists of 4 main types of defects ascending in severity and subdivisions narrowing down-defect location. Type 1 presents an intact acetabular rim, type 2 includes a noncontained defect of the acetabular rim ≤ 10 mm, in type 3 the rim defect exceeds 10 mm and type 4 includes different kinds of pelvic discontinuity. A collective of 207 preoperative radiographs were graded according to ADC and correlated with intraoperative findings. Additionally, a randomized sample of 80 patients was graded according to ADC by 5 observers to account for inter- and intra-rater reliability. RESULTS: We evaluated the agreement of preoperative, radiographic grading and intraoperative findings presenting with a k value of 0.74. Interobserver agreement presented with a k value of 0.62 and intraobserver at a k value of 0.78. CONCLUSION: The ADC offers an intuitive, reliable and reproducible classification system. It guides the surgeon pre- and intraoperatively through a complex field of practice.

3-D Optics for Thoracoscopic Vertebral Body Replacement - Essential Technical Progress or Just Nice to Have? / Die dreidimensionale Optik für den thorakoskopischen ventralen Wirbelkörperersatz ­ notwendiger Fortschritt oder nur "nice to have"?

BACKGROUND: Three-dimensional (3-D) endoscopic optics use 2 cameras to simulate the different perspectives of the right and left eye, creating the illusion of spatial depth. Optimised orientation as well as improved hand-eye coordination compared to 2-D-optics could be proven in standardised test setups (black box) and in laparoscopic use. This retrospective study examines whether these results can also be applied to thoracoscopic vertebral body replacement at the thoracolumbar junction. HYPOTHESES: 1. Ventral vertebral body replacement using 3-D-thoracoscopy results in a shorter operation time than with 2-D-thoracoscopy. 2. Perioperative blood loss is less, due to better spatial orientation (faster haemostasis) and reduced tissue laceration. MATERIAL AND METHODS: 29 patients met the inclusion criteria of this retrospective study. Between 08 - 2012 and 08 - 2017, all of these received ventral thoracoscopic vertebral replacement at the thoracolumbar junction (Th11 to L2). Patients with additional anterior procedures (e.g. anterolateral plate) were excluded. Perioperative data such as blood loss, duration of surgery and length of hospital stay were analysed. Conventional 2-D-optics were used in n = 14 patients and 3-D-optics in 15 patients. Aesculap EinsteinVision® 2.0 was used as the 3-D-optics. Statistical significance was calculated using Student's t-test. RESULTS: The most common diagnosis was a L1 fracture (n = 18, 62%). Mean OR time was 24 minutes shorter in the 3-D group (149 ± 29, 107 - 198 min) than in the 2-D group (173 ± 39, 125 - 260 min), but this difference was not significant. Total perioperative blood loss in the 3-D group was significantly lower than in the 2-D group (**p = 0.043). Proportional intraoperative blood loss in the 3-D group was also lower (mean around 115 ml), but not significantly so. Significantly lower values were found for the delivery rate of the thoracic drainage in the 3-D group (248 vs. 560 ml, *p = 0.195). Inpatient stay with the 3-D group was on average 1.5 days (d) shorter (8.7 d for the 3-D group, 10.2 d for the 2-D group) but this difference was not significant (p = 0.27). CONCLUSION: Thoracoscopic-assisted vertebral body replacement at the thoracolumbar junction is a safe and reliable surgical procedure using conventional 2-D-optics or the new 3-D-optics. Both methods allow thoracoscopic vertebral body replacement in comparable operation times but in our study the 3-D group presented with significantly lower postoperative blood loss. Due to the small number of cases and because of the retrospective design, the present study is considered to be a pilot study only.

Spondylitis - Spondylodiscitis - an Update. / Spondylitis ­ Spondylodiszitis ­ ein aktuelles Update.

Spondylodiscitis is an infection of the intervertebral disc with subsequent infection of the adjacent vertebral bodies. The main causes are 3 pathogen groups: bacteria, particularly tuberculosis pathogens, fungi and parasites. In pyogenic spondylodiscitis, infections with Staphylococcus aureus are the most common, with an incidence of up to 80%. Mortality is around 2 - 3%. Infections with tuberculosis are often associated with psoas and paravertebral abscesses. Neurological deficits are registered in up to 50% of patients. For microbiological diagnostic testing, blood cultures are used for aerobic and anaerobic bacteria. However, histological examination leads significantly more frequently to positive pathogen detection. In tissue samples, results with 16S rRNA PCR results are clearly superior to results from microbiological examination. The MRI exhibits high sensitivity and specificity and is therefore superior to other radiological methods. Elimination of the infection, pain reduction and stabilisation of the spine are the main objectives of any treatment. A standardised antibiotic therapy for spondylodiscitis has not been clearly defined. Pathogen detection is important, with focused antibiotic therapy. Antibiotic therapy should initially be administered intravenously for 2 - 4 weeks. This should be followed by oral administration for 6 - 12 weeks. In the case of antibiotic-resistant infections with neurological deficiencies, it is recommended to perform a surgical procedure, with careful debridement and instrumentation as well as i. v. administered antibiotics for 3 weeks followed by three month oral antibiotic treatment. Surgical procedures are indicated with neurological deficits, progressive increase in spinal deformities, failure of conservative therapy with insufficient pain relief and unreliable pathogen identification. The selection of the surgical procedure should mainly be based on the extent and localisation of bone destruction and the individual circumstances of the patients. The prognosis is good if there is a clear reduction in CRP and ESR in the first few weeks.

Author Correction: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 3. The HTML version of the Article has been updated to include a corrected version of the Supplementary Information.

The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10's unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.

[Basic Clinical Examination of the Shoulder]. / Klinische Basisuntersuchung der Schulter.

OBJECTIVE: Successful treatment of shoulder disease requires an accurate diagnosis. In addition to differentiated history taking, clinical examination is the most important component in the diagnosis of shoulder diseases. METHOD: The present video explains the common provocation tests and functional tests that are used in the basic clinical examination of the shoulder. In addition to general inspection and palpation, the focus is on different diagnostic tests and clinical signs that improve diagnostic accuracy. CONCLUSION: The present basic clinical examination methods allow a structured approach to clinical issues and can be a good basis, if supplemented by further specific and individual tests.

[Influence of trauma mechanisms on thoracic and lumbar spinal fractures]. / Einfluss des Unfallmechanismus auf die Wirbelkörperfraktur.

BACKGROUND: Vertebral body fractures (VBF) can be caused by various trauma mechanisms. The AOSpine classification system differentiates three main types of fractures according to the grade of instability. How the increasing energy of various accident mechanisms changes the complexity of the individual fracture, its localization and the occurrence of further fractures has not yet been finally investigated. OBJECTIVE: What influence do traumatic events with different kinematics have on the localization, complexity and number of VBF in the thoracic and lumbar spine? MATERIAL AND METHODS: In this retrospective study data from patients with a freshly traumatized VBF were analyzed. The patients were divided into six trauma groups (UFG) depending on the trauma mechanism. The VBF were classified on the basis of computed tomography (CT) imaging according to the AOSpine classification system. Testing was performed bilaterally and a significance level of 5% was used. The statistical calculations were carried out using IBM SPSS Statistics. RESULTS: A significant increase in the severity of fractures (AO classification) was found in the high energy trauma groups (UFG III and V). In addition, the incidence of thoracic (TH) VBF was significantly increased for TH7 (p = 0.011) and TH10 (p = 0.001). In comparison to the other low energy trauma groups, the risk of a TH7 fracture was 7­times higher after a high energy trauma (odds ratio OR = 7.0; 95% confidence interval CI = 1.4; 35.2). The UFG III (falls > 3 m) showed the highest number of fractures with a median of 2.5 (SD 1.84) VBF. CONCLUSION: An exact reproduction of the traumatic event enables a distinction between high and low energy trauma groups to be made. In previous studies traffic accidents were recorded as one group, so an influence of the increasing kinematic energy could not be assessed. The accident kinematics can be taken into account by differentiating between high and low-energy trauma groups. In high-energy accidents the TH7 and TH10 vertebrae were found to be at risk vertebrae. In addition to the force direction, the force strength also has a decisive influence on the distribution pattern of VBF.

[A Novel Technique for the Preparation of a 3- or 5-Stranded Hamstring Graft for Individualised Single-Bundle ACL or PCL Reconstruction]. / Neuartige Technik zur Präparation eines 3- oder 5-strängigen Hamstring-Transplantats für die individualisierte Single-Bundle-VKB- oder -HKB-Rekonstruktion.

Objective Development of a preparation technique for hamstring tendons to ensure a maximum of intraoperative flexibility during individualised cruciate ligament surgery. Indications Primary ACL- and PCL-reconstruction as well as cases of later revision. Method Three- or five-stranded grafts can be obtained with the use of semitendinosus and gracilis tendons with fiber tape enforcement and use of a conventional femoral cortical suspensory fixation device. Fibre tape and knotting style ensure free motion of the continous loop. Through its simplicity, the technique offers the highest grade of intraoperative scalability.