Treatment of scaphoid nonunion by arthroscopic cancellous bone grafting.

INTRODUCTION: In order to avoid Scaphoid Nonunion Advanced Collapse (SNAC) type osteoarthritis, which progressively affects the radial and midcarpal joints, several vascularized and non-vascularized grafting techniques have been described. Over the past decade, there has been growing interest in arthroscopic cancellous bone grafts for scaphoid nonunion. The aim of this novel prospective study was to assess the healing rate of scaphoid grafts under arthroscopy, and the prognostic factors for healing. MATERIAL AND METHODS: This prospective study was carried out across 10 centers between September 2019 and April 2021, in patients aged 16 to 65. Scaphoid nonunion grafting was performed arthroscopically. Union was assessed on CT scans and displacement correction angles were measured preoperatively and then at 3 and 6months. We assessed mobility, Jamar wrist strength, functional results as per the Patient Related Wrist Score (PRWE) and the Quick Disabilities of the Arm, Shoulder and Hand (Quick DASH) score. Risk factors for nonunion were assessed. RESULTS: We included 77 patients with a mean age of 24years (18 to 55years) with a mean time between trauma and treatment of nonunion of 34.8months (6 to 180months). The population was represented by 46 manual workers and 20 were smokers. In 42 cases, the nonunion was proximal, in Schernberg zone I or II. At the last follow-up of 12.9months on average (Standard Deviation: 8.7months), union was achieved in 72 patients (93.5%). The average duration of union was 3.4months (Standard Deviation 1.6). Among the 5 patients who did not heal, grafting was performed in addition to the fixation. We did not identify any contributory factors for nonunion. CONCLUSION: This study demonstrated the effectiveness of arthroscopic treatment of scaphoid nonunion with a union rate at least equivalent to pedicled vascularized grafts. Smoking and delayed treatment were no longer considered unfavorable prognostic factors in the context of arthroscopic treatment. LEVEL OF EVIDENCE: III.

Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter.

Tripartite ATP-independent periplasmic (TRAP) transporters are found widely in bacteria and archaea and consist of three structural domains, a soluble substrate-binding protein (P-domain), and two transmembrane domains (Q- and M-domains). HiSiaPQM and its homologs are TRAP transporters for sialic acid and are essential for host colonization by pathogenic bacteria. Here, we reconstitute HiSiaQM into lipid nanodiscs and use cryo-EM to reveal the structure of a TRAP transporter. It is composed of 16 transmembrane helices that are unexpectedly structurally related to multimeric elevator-type transporters. The idiosyncratic Q-domain of TRAP transporters enables the formation of a monomeric elevator architecture. A model of the tripartite PQM complex is experimentally validated and reveals the coupling of the substrate-binding protein to the transporter domains. We use single-molecule total internal reflection fluorescence (TIRF) microscopy in solid-supported lipid bilayers and surface plasmon resonance to study the formation of the tripartite complex and to investigate the impact of interface mutants. Furthermore, we characterize high-affinity single variable domains on heavy chain (VHH) antibodies that bind to the periplasmic side of HiSiaQM and inhibit sialic acid uptake, providing insight into how TRAP transporter function might be inhibited in vivo.

A new versatile MR-guided high-intensity focused ultrasound (HIFU) device for the treatment of musculoskeletal tumors.

Magnetic Resonance (MR) Imaging-guided High Intensity focused Ultrasound (MRgHIFU) is a non-invasive, non-ionizing thermal ablation therapy that is particularly interesting for the palliative or curative treatment of musculoskeletal tumors. We introduce a new modular MRgHIFU device that allows the ultrasound transducer to be positioned precisely and interactively over the body part to be treated. A flexible, MR-compatible supporting structure allows free positioning of the transducer under MRI/optical fusion imaging guidance. The same structure can be rigidified using pneumatic depression, holding the transducer rigidly in place. Targeting accuracy was first evaluated in vitro. The average targeting error of the complete process was found to be equal to 5.4 ± 2.2 mm in terms of focus position, and 4.7° ± 2° in terms of transducer orientation. First-in-man feasibility is demonstrated on a patient suffering from important, uncontrolled pain from a bone metastasis located in the forearm. The 81 × 47 × 34 mm3 lesion was successfully treated using five successive positions of the transducer, under real-time monitoring by MR Thermometry. Significant pain palliation was observed 3 days after the intervention. The system described and characterized in this study is a particularly interesting modular, low-cost MRgHIFU device for musculoskeletal tumor therapy.

Structural basis for safe and efficient energy conversion in a respiratory supercomplex.

Proton-translocating respiratory complexes assemble into supercomplexes that are proposed to increase the efficiency of energy conversion and limit the production of harmful reactive oxygen species during aerobic cellular respiration. Cytochrome bc complexes and cytochrome aa3 oxidases are major drivers of the proton motive force that fuels ATP generation via respiration, but how wasteful electron- and proton transfer is controlled to enhance safety and efficiency in the context of supercomplexes is not known. Here, we address this question with the 2.8 Å resolution cryo-EM structure of the cytochrome bcc-aa3 (III2-IV2) supercomplex from the actinobacterium Corynebacterium glutamicum. Menaquinone, substrate mimics, lycopene, an unexpected Qc site, dioxygen, proton transfer routes, and conformational states of key protonable residues are resolved. Our results show how safe and efficient energy conversion is achieved in a respiratory supercomplex through controlled electron and proton transfer. The structure may guide the rational design of drugs against actinobacteria that cause diphtheria and tuberculosis.

Why do we use arthroscopy for distal radius fractures?

The management of distal radius fractures needs to be adapted to the increasingly complex traumas in patients with greater functional requirements. The goal remains to restore normal anatomy in order to preserve function. A pre-operative assessment using x-rays and thin-slice CT scans with three-dimensional reconstructions enables the best possible understanding of the fracture prior to surgery and planning of the pre-operative strategy. Arthroscopy is a technique that allows visualisation of the bone fragments and their displacement, as well as their reduction. It is the only tool that allows diagnostic and prognostic assessment of the associated injuries. Arthroscopy is the gold standard for identifying and grading scapholunate injuries. It allows treatment of the lesions where necessary and a dynamic appreciation of the stability of the osteosynthesis. Recognition of articular fragmentation patterns and instability features can therefore aid treatment choice to prevent poor outcomes due to malunion and degenerative arthritis. We recommend arthroscopic-assisted internal fixation for articular fractures for any active patient, not only for young adults, displaced or a gap by more than 2 mm, potential scapholunate ligament injuries, and fractures of the ulnar styloid. A strong initial learning and a minimal experience are recommended to avoid the complications of this invaluable procedure.

Structure of the transcription activator target Tra1 within the chromatin modifying complex SAGA.

The transcription co-activator complex SAGA is recruited to gene promoters by sequence-specific transcriptional activators and by chromatin modifications to promote pre-initiation complex formation. The yeast Tra1 subunit is the major target of acidic activators such as Gal4, VP16, or Gcn4 but little is known about its structural organization. The 430 kDa Tra1 subunit and its human homolog the transformation/transcription domain-associated protein TRRAP are members of the phosphatidyl 3-kinase-related kinase (PIKK) family. Here, we present the cryo-EM structure of the entire SAGA complex where the major target of activator binding, the 430 kDa Tra1 protein, is resolved with an average resolution of 5.7 Å. The high content of alpha-helices in Tra1 enabled tracing of the majority of its main chain. Our results highlight the integration of Tra1 within the major epigenetic regulator SAGA.

Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization.

Multi-subunit SMC complexes control chromosome superstructure and promote chromosome disjunction, conceivably by actively translocating along DNA double helices. SMC subunits comprise an ABC ATPase "head" and a "hinge" dimerization domain connected by a 49 nm coiled-coil "arm." The heads undergo ATP-dependent engagement and disengagement to drive SMC action on the chromosome. Here, we elucidate the architecture of prokaryotic Smc dimers by high-throughput cysteine cross-linking and crystallography. Co-alignment of the Smc arms tightly closes the interarm space and misaligns the Smc head domains at the end of the rod by close apposition of their ABC signature motifs. Sandwiching of ATP molecules between Smc heads requires them to substantially tilt and translate relative to each other, thereby opening up the Smc arms. We show that this mechanochemical gating reaction regulates chromosome targeting and propose a mechanism for DNA translocation based on the merging of DNA loops upon closure of Smc arms.

Structural insight into the mechanism of stabilization of the 7SK small nuclear RNA by LARP7.

The non-coding RNA 7SK is the scaffold for a small nuclear ribonucleoprotein (7SKsnRNP) which regulates the function of the positive transcription elongation factor P-TEFb in the control of RNA polymerase II elongation in metazoans. The La-related protein LARP7 is a component of the 7SKsnRNP required for stability and function of the RNA. To address the function of LARP7 we determined the crystal structure of its La module, which binds a stretch of uridines at the 3'-end of 7SK. The structure shows that the penultimate uridine is tethered by the two domains, the La-motif and the RNA-recognition motif (RRM1), and reveals that the RRM1 is significantly smaller and more exposed than in the La protein. Sequence analysis suggests that this impacts interaction with 7SK. Binding assays, footprinting and small-angle scattering experiments show that a second RRM domain located at the C-terminus binds the apical loop of the 3' hairpin of 7SK, while the N-terminal domains bind at its foot. Our results suggest that LARP7 uses both its N- and C-terminal domains to stabilize 7SK in a closed structure, which forms by joining conserved sequences at the 5'-end with the foot of the 3' hairpin and has thus functional implications.

Mapping the deubiquitination module within the SAGA complex.

The molecular organization of the yeast transcriptional coactivator Spt-Ada-Gcn5 acetyltransferase (SAGA) was analyzed by single-particle electron microscopy. Complete or partial deletion of the Sgf73 subunit disconnects the deubiquitination (DUB) module from SAGA and favors in our conditions the cleavage of the C-terminal ends of the Spt7 subunit and the loss of the Spt8 subunit. The structural comparison of the wild-type SAGA with two deletion mutants positioned the DUB module and enabled the fitting of the available atomic models. The localization of the DUB module close to Gcn5 defines a chromatin-binding interface within SAGA, which could be demonstrated by the binding of nucleosome core particles. The TATA-box binding protein (TBP)-interacting subunit Spt8 was found to be located close to the DUB but in a different domain than Spt3, also known to contact TBP. A flexible protein arm brings both subunits close enough to interact simultaneously with TBP.

Structure, assembly and dynamics of macromolecular complexes by single particle cryo-electron microscopy.

BACKGROUND: Proteins in their majority act rarely as single entities. Multisubunit macromolecular complexes are the actors in most of the cellular processes. These nanomachines are hold together by weak protein-protein interactions and undergo functionally important conformational changes. TFIID is such a multiprotein complex acting in eukaryotic transcription initiation. This complex is first to be recruited to the promoter of the genes and triggers the formation of the transcription preinitiation complex involving RNA polymerase II which leads to gene transcription. The exact role of TFIID in this process is not yet understood. METHODS: Last generation electron microscopes, improved data collection and new image analysis tools made it possible to obtain structural information of biological molecules at atomic resolution. Cryo-electron microscopy of vitrified samples visualizes proteins in a fully hydrated, close to native state. Molecular images are recorded at liquid nitrogen temperature in low electron dose conditions to reduce radiation damage. Digital image analysis of these noisy images aims at improving the signal-to-noise ratio, at separating distinct molecular views and at reconstructing a three-dimensional model of the biological particle. RESULTS: Using these methods we showed the early events of an activated transcription initiation process. We explored the interaction of the TFIID coactivator with the yeast Rap1 activator, the transcription factor TFIIA and the promoter DNA. We demonstrated that TFIID serves as an assembly platform for transient protein-protein interactions, which are essential for transcription initiation. CONCLUSIONS: Recent developments in electron microscopy have provided new insights into the structural organization and the dynamic reorganization of large macromolecular complexes. Examples of near-atomic resolutions exist but the molecular flexibility of macromolecular complexes remains the limiting factor in most case. Electron microscopy has the potential to provide both structural and dynamic information of biological assemblies in order to understand the molecular mechanisms of their functions.