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Nucleic acid–sensing Toll-like receptors (TLRs) play a pivotal role in innate immunity by recognizing foreign DNA and RNA. Compartmentalization of these TLRs in the endosome limits their activation by self-derived nucleic acids and reduces the possibility of autoimmune reactions. Although chaperone Unc-93 homolog B1, TLR signaling regulator (UNC93B1) is indispensable for the trafficking of TLRs from the endoplasmic reticulum to the endosome, mechanisms of UNC93B1-mediated TLR regulation remain largely unknown. Here, we report two cryo-EM structures of human and mouse TLR3–UNC93B1 complexes and a human TLR7–UNC93B1 complex. UNC93B1 exhibits structural similarity to the major facilitator superfamily transporters. Both TLRs interact with the UNC93B1 amino-terminal six-helix bundle through their transmembrane and luminal juxtamembrane regions, but the complexes of TLR3 and TLR7 with UNC93B1 differ in their oligomerization state. The structural information provided here should aid in designing compounds to combat autoimmune diseases. Cryo-EM structures of nucleic acid–sensing Toll-like receptors in complex with their trafficking chaperone UNC93B1, a protein that mediates TLR compartmentalization important for self versus non-self discrimination, provide insights into their interaction.

Artificial lipid-bilayer membranes are valuable tools for the study of membrane structure and dynamics. For applications such as the study of vesicular transport and drug delivery, there is a pressing need for artificial vesicles with controlled size. However, controlling vesicle size and shape with nanometre precision is challenging, and approaches to achieve this can be heavily affected by lipid composition. Here, we present a bio-inspired templating method to generate highly monodispersed sub-100-nm unilamellar vesicles, where liposome self-assembly was nucleated and confined inside rigid DNA nanotemplates. Using this method, we produce homogeneous liposomes with four distinct predefined sizes. We also show that the method can be used with a variety of lipid compositions and probe the mechanism of templated liposome formation by capturing key intermediates during membrane self-assembly. The DNA nanotemplating strategy represents a conceptually novel way to guide lipid bilayer formation and could be generalized to engineer complex membrane/protein structures with nanoscale precision. Precise control of vesicle size is highly desirable both for basic biochemical research and biomedical applications. Now, monodispersed sub-100-nm vesicles with predefined sizes have been produced using a method based on membrane self-assembly within a DNA-nanostructure guide.

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40–50 base pairs of dsRNA is necessary for TLR3 binding and dimerization. However, efficient TLR3 activation requires longer dsRNA and the molecular mechanism underlying its dsRNA length-dependent activation remains unknown. Here, we report cryo-electron microscopy analyses of TLR3 complexed with longer dsRNA. TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction. TLR3 activates a potent immune response by binding to dsRNA. Here the authors report cryo-EM analyses to show that TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction.

Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually 1 , 2 . For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes 3 . However, the molecular basis for the virus–transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs. Cryo-electron microscopy structures of the bile acid transporter NTCP in the apo state and in complex with the preS1 domain of hepatitis B virus (HBV) provide insight into NTCP substrate transport and HBV recognition mechanisms.

Toll-like receptor 7 (TLR7) recognizes both microbial and endogenous RNAs and nucleosides. Aberrant activation of TLR7 has been implicated in several autoimmune diseases including systemic lupus erythematosus (SLE). Here, by modifying potent TLR7 agonists, we develop a series of TLR7-specific antagonists as promising therapeutic agents for SLE. These compounds protect mice against lethal autoimmunity. Combining crystallography and cryo-electron microscopy, we identify the open conformation of the receptor and reveal the structural equilibrium between open and closed conformations that underlies TLR7 antagonism, as well as the detailed mechanism by which TLR7-specific antagonists bind to their binding pocket in TLR7. Our work provides small-molecule TLR7-specific antagonists and suggests the TLR7-targeting strategy for treating autoimmune diseases. A series of Toll-like receptor 7 (TLR7)-specific antagonists and extensive structural analysis reveal the open conformation of the receptor and the structural basis of TLR7 antagonism. One of the compounds shows efficacy in treating mouse model of systemic lupus erythematosus.

Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αβ-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules. Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.

Background Diffuse idiopathic skeletal hyperostosis (DISH) is a systemic non-inflammatory disorder characterized by enthesopathy and osteophyte formation. DISH can also cause several other symptoms. Limited range of motion (ROM) is the most common symptom; however, dysphagia and respiratory distress are clinically important symptoms. Dysphagia caused by cervical DISH is initially treated conservatively, but surgical treatment is performed when conservative treatment is ineffective. Although there are many reports on the surgical excision of osteophytes for refractory dysphagia, only a few reports on surgery for dysphagia caused by DISH associated with ossification of the posterior longitudinal ligament (OPLL) exist. Here, we report a rare case of cervical spinal cord injury following osteophyte excision for a respiratory distress and dysphagia caused by DISH associated with OPLL. Case presentation A 76-year-old male with hypertension and diabetes presented with dysphagia, respiratory insufficiency, and palpitations. Four months later, he experienced severe dyspnea and was hospitalized. His vital signs indicated respiratory distress, which led to intubation and tracheotomy due to his worsening condition. Imaging revealed massive anterior cervical osteophytes and multisegmental OPLL that caused spinal canal stenosis and tracheal compression. Surgical excision of the osteophytes was performed, but the patient later developed tetraplegia attributed to C5/C6 instability. Posterior fusion and laminoplasty were performed, resulting in neurological improvement but persistent dysphagia and motor deficits. He was transferred to another hospital for rehabilitation but died of aspiration pneumonia. Conclusions Patients with cervical OPLL and spinal cord compression may experience spinal cord injury when intervertebral mobility is slightly increased due to osteophyte excision. If dysphagia or respiratory distress occur in patients with DISH and OPLL, decompression and fusion surgery at the mobile segment is required, in addition to osteophyte excision surgery. Posterior decompression and fusion surgery should be performed before anterior osteophyte excision surgery to avoid implant infection, particularly in patients with respiratory distress who have undergone tracheostomy. Patients receiving long-term mechanical ventilation are less likely to recover their swallowing function and should undergo a total laryngectomy.

Background Transverse sacral fractures with spinopelvic dissociation are highly unstable and can cause severe neurological compromise. In cases combining an H-shaped sacral fracture, a floating Roy-Camille type 3 component, and a free canal fragment, posterior plating, standard spinopelvic fixation, or transiliac-transsacral screw fixation may be unsafe, particularly after decompression, due to a dorsal cortical defect. Method A 49-year-old woman sustained polytrauma after a fall from the fifth floor, including an AO/OTA 61-C3.2 unstable pelvic ring injury. Computed tomography (CT) demonstrated an H-shaped sacral fracture with an S1–2 transverse Roy-Camille type 3 component and a large canal fragment with bilateral fractures and a floating distal sacral fragment. After angioembolization for active pelvic hemorrhage and damage control orthopedics, definitive posterior surgery was performed on post-injury day 5. Decompression was achieved via laminectomy (L5–S2) and removal of the canal fragment. The floating sacral fragment was reduced using a Kapandji-like maneuver with bilateral neurodissectors. Minimally invasive crab-shaped spinopelvic fixation was performed using bilateral iliac screws and percutaneous L5 pedicle screws connected using transverse rods and longitudinal offset connectors. Offset connectors were positioned along the dorsal sacral cortex as buttresses to prevent redisplacement of the distal fragment and secured around the S2 spinous process. Results The operative time was 270 min, with 100 mL of blood loss. Postoperative imaging confirmed a satisfactory reduction. Mild anterior shortening occurred within three months without progression, union at six months, or complete union without implant failure at 1 year. The patient regained independent ambulation with mild residual sensory disturbances and urinary dysfunction. Conclusion Modified crab-shaped fixation using offset connectors as dorsal buttresses can provide stable reduction and maintain alignment until union in complex spinopelvic dissociation patterns where standard constructs or transiliac transsacral screw (TITSS) are not feasible.

PurposeTo determine the factors associated with 6-week postoperative global coronal balance and delayed global coronal balance at 2-year follow-up after anterior spinal fusion for Lenke 5C curves.MethodsA total of 124 consecutive Lenke 5C curves with minimum 2-year follow-up was studied. Radiographic parameters were studied preoperatively, 6 weeks postoperatively, and 2 years postoperatively. Coronal balance was measured by C7-CSVL and trunk shift < 20 mm. The study outcomes were patients with early coronal balance and those who had immediate imbalance but developed delayed balance. Multivariate regression analyses of associated factors were performed with cutoffs determined by receiver operating characteristic curve. Results31.5% patients attained global coronal balance immediate postoperatively and 89.4% of the early imbalance cases showed spontaneous coronal balance at 2-year follow-up. Increased preoperative UIV tilt (OR 1.093; p = 0.026; 95% CI: 1.011–1.182) and reduced immediate postoperative RSH difference (OR 0.963; p = 0.015; 95% CI: 0.935–0.993) were associated with immediate postoperative balance. For those with immediate imbalance, larger preoperative major Cobb angle (OR 1.226; p = 0.047; 95% CI: 1.003–1.499), less preoperative C7-CSVL (OR 0.829; p = 0.016; 95% CI: 0.712–0.966), and less immediate postoperative LIV tilt (OR 0.728; p = 0.013; 95% CI: 0.567–0.934) were associated with 2-year coronal balance. There was significant improvement in function (p = 0.006), self-image (p = 0.039) and total score domains (p = 0.014) in immediate imbalance to 2-year balance and imbalance groups.ConclusionSuccessful balance is achieved with a parallel fusion mass when performing anterior spinal fusion for Lenke 5C curves. Patients should be reassured that most attain eventual coronal balance despite the early imbalance.Level of evidence Therapeutic III

Epilepsy is a common brain disorder throughout history. Epilepsy-related ligand–receptor complex, LGI1–ADAM22, regulates synaptic transmission and has emerged as a determinant of brain excitability, as their mutations and acquired LGI1 autoantibodies cause epileptic disorders in human. Here, we report the crystal structure of human LGI1–ADAM22 complex, revealing a 2:2 heterotetrameric assembly. The hydrophobic pocket of the C-terminal epitempin-repeat (EPTP) domain of LGI1 binds to the metalloprotease-like domain of ADAM22. The N-terminal leucine-rich repeat and EPTP domains of LGI1 mediate the intermolecular LGI1–LGI1 interaction. A pathogenic R474Q mutation of LGI1, which does not exceptionally affect either the secretion or the ADAM22 binding, is located in the LGI1–LGI1 interface and disrupts the higher-order assembly of the LGI1–ADAM22 complex in vitro and in a mouse model for familial epilepsy. These studies support the notion that the LGI1–ADAM22 complex functions as the trans-synaptic machinery for precise synaptic transmission. LGI1 is an epilepsy-related gene that encodes a secreted neuronal protein. Here the authors present the crystal structure of LGI1 bound to its receptor ADAM22, which provides structural insights into epilepsy-causing LGI1 mutations and might facilitate the development of novel anti-epilepsy drugs.