Structural basis of hepatitis B virus receptor binding.

Hepatitis B virus (HBV), a leading cause of developing hepatocellular carcinoma affecting more than 290 million people worldwide, is an enveloped DNA virus specifically infecting hepatocytes. Myristoylated preS1 domain of the HBV large surface protein binds to the host receptor sodium-taurocholate cotransporting polypeptide (NTCP), a hepatocellular bile acid transporter, to initiate viral entry. Here, we report the cryogenic-electron microscopy structure of the myristoylated preS1 (residues 2-48) peptide bound to human NTCP. The unexpectedly folded N-terminal half of the peptide embeds deeply into the outward-facing tunnel of NTCP, whereas the C-terminal half formed extensive contacts on the extracellular surface. Our findings reveal an unprecedented induced-fit mechanism for establishing high-affinity virus-host attachment and provide a blueprint for the rational design of anti-HBV drugs targeting virus entry.

How coronal malalignment affects the surgical outcome in corrective spine surgery for adult symptomatic lumbar deformity.

PURPOSE: The importance of coronal alignment is unclear, while the importance of sagittal alignment in the treatment of adult patients with spinal deformities is well described. This study sought to elucidate the impact of global coronal malalignment (GCMA) in surgically treated adult symptomatic lumbar deformity (ASLD) patients. METHODS: A multicentre retrospective analysis of a prospective ASD database. GCMA was defined as GCA (C7PL-CSVL) ≥ 3 cm. GCMA is categorized based on the Obeid-Coronal Malalignment Classification (O-CM). Demographic, surgical, radiographic, HRQOL, and complication data were analysed. The risk for postoperative GCMA was analysed by univariate and multivariate analyses. RESULTS: Of 230 surgically treated ASLD patients, 96 patients showed GCMA preoperatively and baseline GCA was correlated with the baseline SRS-22 pain domain score (r = - 30). Postoperatively, 62 patients (27%, O-CM type 1: 41[18%], type 2: 21[9%]) developed GCMA. The multivariate risk analysis indicated dementia (OR 20.1[1.2-304.4]), diabetes (OR 5.9[1.3-27.3]), and baseline O-CM type 2 (OR 2.1[1.3-3.4]) as independent risk factors for postoperative GCMA. The 2-year SRS-22 score was not different between the 2 groups, while 4 GCMA patients required revision surgery within 1 year after surgery due to coronal decompensation (GCMA+ vs. GCMA- function: 3.6 ± 0.6 vs. 3.7 ± 0.7, pain: 3.7 ± 0.8 vs. 3.8 ± 0.8, self-image: 3.6 ± 0.8 vs. 3.6 ± 0.8, mental health: 3.7 ± 0.8 vs. 3.8 ± 0.9, satisfaction: 3.9 ± 0.9 vs. 3.9 ± 0.8, total: 3.7 ± 0.7 vs. 3.7 ± 0.7). Additionally, the comparisons of 2-yr SRS-22 between GCMA ± showed no difference in any UIV and LIV level or O-CM type. CONCLUSIONS: In ASLD patients with corrective spine surgery, GCMA at 2 years did not affect HRQOL or major complications at any spinal fusion extent or O-CM type of malalignment, whereas GCA correlated with pain intensity before surgery. These findings may warrant further study of the impact of GCMA on HRQOL in the surgical treatment of ASLD patients.

Optimal dose for the efficacy of asenapine in patients with schizophrenia: Real-world data.

AIMS: A meta-analysis of short-term studies revealed no significant differences between the doses of asenapine, 10 and 20 mg/day, in the acute treatment of schizophrenia. However, it should be noted that many patients from clinical practice were excluded, and the dose-response to asenapine in a real-world setting is still unclear. Additionally, the dose-response in the maintenance phase is not clear. This study aimed to evaluate the differences in the efficacy of different asenapine doses in patients with maintenance phase of schizophrenia in a real-world setting. METHODS: This study conducted post-marketing surveillance of asenapine in clinical settings in Japan. It followed patients diagnosed with schizophrenia who received asenapine for the first time for a maximum of 52 weeks. These patients were divided into two categories based on their average daily asenapine dosage: ≤10 mg/day and >10 mg/day. Asenapine efficacy was assessed by adjusting for patient demographics using multivariate logistic regression analysis, employing the Clinical Global Impression-Global Improvement (CGI-I) scale, which has seven categories. RESULTS: A total of 2774 patients were included in the analysis. Of these, 1689 and 1085 patients were treated with asenapine ≤10 mg/day and >10 mg/day, respectively. The CGI-I improvement rate was significantly higher in the asenapine >10 group (p = 0.012) after adjusting for patient background factors. CONCLUSION: These results suggest that asenapine doses >10 mg/day may be more effective than 10 mg/day in the treatment of schizophrenia; however, further studies are needed to confirm these findings.

Positional relationships between a tracheal diverticulum and the tracheal tube under general anesthesia: a single-center observational and simulation study.

BACKGROUND: Incomplete sealing of tracheal diverticula by a tracheal tube cuff during positive-pressure ventilation causes barotrauma but the concrete possibility of incomplete sealing has not been indicated. We aimed to assess the possibility of incomplete sealing in a simulated situation of tracheal intubation for patients with tracheal diverticula with tube fixation where the tracheal tube's vocal cord guide overlaps with the patient's vocal cord. METHODS: We retrospectively assessed the characteristics of tracheal diverticula based on thoracic computed tomography data in our institution from January 2018 to July 2020. Then, we assessed the structural parameters of three single-lumen tracheal tubes (Parker Flex-Tip [Parker Medical, Bridgewater, CT, USA], Portex Soft Seal [ICU Medical, San Clemente, CA, USA], and Shiley TaperGuard [Medtronic, Dublin, Ireland]; 6.0-8.0 mm inner diameter size) and simulated the positional relationships between tracheal diverticula and the tracheal tube during tracheal intubation where the vocal cord guide overlaps with the patient's vocal cord. We assessed each tube product's possibility of incompletely sealing tracheal diverticula and the possibility of unintended bronchial intubation. RESULTS: In 5,854 patients, the prevalence of tracheal diverticula was 5.7%. The mean (SD) length from the vocal cord to the distal end of the tracheal diverticula was 52.2 (12.8) mm. Tracheal tubes with length from the distal end of the tracheal cuff to the vocal cord guide of ≥ 70 mm had a low risk of incompletely sealing tracheal diverticula (< 5%) and length from the distal end of the tube to the vocal cord guide of ≤ 95 mm had a low risk of unintended bronchial intubation (< 5%). No products in this study satisfied both outcomes. CONCLUSIONS: Tube fixation, where the vocal cord guide overlaps with the patient's vocal cord, is associated with risk of incompletely sealing of tracheal diverticula depending on the tube's manufacturer and tube's inner diameter size, although it was not a high risk. The use of small inner diameter sized tube relative to patient's body size is high risk of incomplete sealing of tracheal diverticula. TRIAL REGISTRATION: This trial was prospectively registered at University Hospital Medical Information Network (UMIN). CLINICAL TRIAL NUMBER AND REGISTRY URL: UMIN000043317 (URL: https://center6.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000048055 ).

Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5'-phosphate (PLP) via Schiff base formation.

D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ-lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5'-phosphate)-dependent enzymes such as methionine γ-lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC50 = 13 ± 1 µM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine ß-synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.

Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

A novel Toll-like receptor 7/8-specific antagonist E6742 ameliorates clinically relevant disease parameters in murine models of lupus.

The sensing of self RNA by the endosomal Toll-like receptors (TLRs) 7 and 8 initiates pathogenic mechanisms underlying the autoimmune disease lupus. A blockade of the TLR7/8 signals may, therefore, be a novel therapeutic intervention for lupus. To test the hypothesis, a novel compound E6742 that blocks TLR7/8 activation was identified. The mode of action of E6742 was investigated by analysis of the tertiary structure of TLR7 and 8 in complex with E6742. The in vitro activities of the compound were examined in cellular systems and its therapeutic potential was evaluated in murine lupus models. Tertiary structures of the extracellular domain of TLR7 and 8 in complex with E6742 showed that E6742 binds specifically and non-covalently to the hydrophobic pocket located at the interface of TLR7 or TLR8 homodimers. E6742 potently and selectively inhibited several TLR7/8-mediated cytokine responses in human PBMC. In two mouse models of lupus, oral dosing of E6742 after the onset of disease suppressed increase in autoantibodies and blocked the advance of organ damage. Collectively, the data show that TLR7/8 activation contributes to disease progression and its blocking by E6742 has potential as a therapeutic intervention for lupus.

A screening method for visual attention disabilities in cerebral palsy with periventricular leukomalacia.

PURPOSE: Patients with periventricular leukomalacia (PVL) have been reported to have a variety of complications; however, whether these involve impaired visual attention disabilities remains unclear. Therefore, this study aimed to investigate the presence or absence and degree of visual attention disabilities in patients with PVL and propose a screening test that would allow anyone to check for visual attention disabilities easily. METHODS: The study participants were 14 patients with PVL and seven controls with dyskinetic cerebral palsy. All participants performed three types of visual attention tasks: spatial attention tasks, feature-based attention tasks, and object-based attention tasks. The participants also performed counting tasks to determine how many squares of the same size and color could be counted (up to nine). Receiver operating characteristic analysis was used to calculate cutoff values, with disability as the objective variable and the value of the counting task as the explanatory variable. RESULTS: The results revealed that patients with PVL often had visual attention disabilities, as indicated by a significant reduction in tasks requiring divided attention. Visual attention disabilities could be detected by a score of ≤8 in the square counting task. CONCLUSIONS: These findings suggest that family members and teachers of patients with PVL can easily screen for visual attention disabilities at home and school to improve mobility precautions in patients with this disability.

A comprehensive Drosophila resource to identify key functional interactions between SARS-CoV-2 factors and host proteins.

Development of effective therapies against SARS-CoV-2 infections relies on mechanistic knowledge of virus-host interface. Abundant physical interactions between viral and host proteins have been identified, but few have been functionally characterized. Harnessing the power of fly genetics, we develop a comprehensive Drosophila COVID-19 resource (DCR) consisting of publicly available strains for conditional tissue-specific expression of all SARS-CoV-2 encoded proteins, UAS-human cDNA transgenic lines encoding established host-viral interacting factors, and GAL4 insertion lines disrupting fly homologs of SARS-CoV-2 human interacting proteins. We demonstrate the utility of the DCR to functionally assess SARS-CoV-2 genes and candidate human binding partners. We show that NSP8 engages in strong genetic interactions with several human candidates, most prominently with the ATE1 arginyltransferase to induce actin arginylation and cytoskeletal disorganization, and that two ATE1 inhibitors can reverse NSP8 phenotypes. The DCR enables parallel global-scale functional analysis of SARS-CoV-2 components in a prime genetic model system.

TLR7/8 stress response drives histiocytosis in SLC29A3 disorders.

Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3-/- mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3-/- mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic monocytes failed to activate inflammatory responses. Enhanced production of proinflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8-antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.

Long-Term Clinical, Radiographic, and Cost Analysis of Corrective Spine Surgery for Adult Symptomatic Lumbar Deformity With a Mean of 7.5 years Follow-Up.

STUDY DESIGN: Multicenter retrospective study. OBJECTIVE: This study reports long-term clinical and radiographic outcomes in surgically treated patients with adult symptomatic lumbar deformity (ASLD). SUMMARY OF BACKGROUND DATA: The short-term results of corrective spinal surgery for ASLD are often favorable despite a relatively high complication profile. However, long-term outcomes have not been completely characterized. METHODS: A total of 169 surgically treated consecutive ASLD patients (≥50 yr) who achieved minimum 5 year follow-up were included (average 7.5 yr observation window, average age 67±8 yr, 96% female). The subjects were stratified by current age (50s, 60s, and 70s) and compared. Kaplan-Meier analysis was used to estimate the cumulative incidence of unplanned reoperation stratified by age group. Initial and overall direct costs of surgery were also analyzed. RESULTS: The SRS-22 at final follow-up was similar among the three groups (50s, 60s, and 70s; 4.0±0.5 vs. 3.8±0.7 vs. 3.8±0.7, respectively). The overall major complication rate was 56%, and 12% experienced late complications. The cumulative reoperation rate was 23%, and 4% required late reoperation. Patients in their 70s had a significantly higher reoperation rate (33%) and overall complication rate (65%). However, the late complication rate was not significantly different between the three groups (9% vs. 12% vs. 13%). Sagittal alignment was improved at two years and maintained to the final follow-up, whereas reciprocal thoracic kyphosis developed in all age groups. The direct cost of initial surgery was $45K±9K and increased by 13% ($53K±13K) at final follow-up. CONCLUSIONS: Long-term surgical outcomes for ASLDs were favorable, with a relatively low rate of late-stage complications and reoperations, as well as reasonable direct costs. Despite the higher reoperation and complication rate, ASLD patients of more advanced age achieved similar improvement to those in the younger age groups.

TLR3 forms a laterally aligned multimeric complex along double-stranded RNA for efficient signal transduction.

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.

Structural insight into the activation of an Arabidopsis organellar C-to-U RNA editing enzyme by active site complementation.

RNA-binding pentatricopeptide repeat (PPR) proteins catalyze hundreds of cytidine to uridine RNA editing events in plant organelles; these editing events are essential for proper gene expression. More than half of the PPR-type RNA editing factors, however, lack the DYW cytidine deaminase domain. Genetic analyses have suggested that their cytidine deaminase activity arises by association with a family of DYW1-like proteins that contain an N-terminally truncated DYW domain, but their molecular mechanism has been unclear. Here, we report the crystal structure of the Arabidopsis thaliana DYW1 deaminase domain at 1.8 Å resolution. DYW1 has a cytidine deaminase fold lacking the PG box. The internal insertion within the deaminase fold shows an α-helical fold instead of the ß-finger reported for the gating domain of the A. thaliana ORGANELLE TRANSCRIPT PROCESSING 86. The substrate-binding pocket is incompletely formed and appears to be complemented in the complex by the E2 domain and the PG box of the interacting PPR protein. In vivo RNA editing assays corroborate the activation model for DYW1 deaminase. Our study demonstrates the common activation mechanism of the DYW1-like proteins by molecular complementation of the DYW domain and reconstitution of the substrate-binding pocket.

Normal pressure hydrocephalus due to an iatrogenic giant lumbar pseudomeningocele after posterior lumbar interbody fusion: illustrative case.

BACKGROUND: Iatrogenic pseudomeningocele incidence after lumbar surgery is 0.068%-2%, and most lumbar pseudomeningoceles are smaller than 5 cm; however, in rare cases, "giant" pseudomeningoceles greater than 8 cm in size may develop. Normal pressure hydrocephalus (NPH) is another rare condition in which the ventricles expand despite the presence of normal intracranial pressure. To date, pseudomeningocele associated with NPH has not been reported. OBSERVATIONS: An 80-year-old woman underwent L3-5 laminectomy and posterior lumbar interbody fusion, and her symptoms improved after surgery. However, dementia appeared 1 month after surgery. Repeated brain computed tomography showed ventricular enlargement, and lumbar magnetic resonance imaging showed a long pseudomeningocele in the subcutaneous tissues at the L4 level. Here, the authors report a rare case of an iatrogenic giant pseudomeningocele accompanied by NPH after lumbar surgery. The symptoms of NPH in the present case occurred after spinal surgery and recovered after dural repair surgery, indicating that the changes in cerebrospinal fluid circulation and/or pressure due to pseudomeningoceles may cause NPH. LESSONS: The prevention of dural tears through precise surgical technique and primary repair of dural tears are the best approaches to prevent pseudomeningocele incidence and subsequent events.

Structure of SARS-CoV-2 membrane protein essential for virus assembly.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A).

N-Glycosylation is a common post-translational modification, and the number of GlcNAc branches in N-glycans impacts glycoprotein functions. N-Acetylglucosaminyltransferase-IVa (GnT-IVa, also designated as MGAT4A) forms a ß1-4 GlcNAc branch on the α1-3 mannose arm in N-glycans. Downregulation or loss of GnT-IVa causes diabetic phenotypes by dysregulating glucose transporter-2 in pancreatic ß-cells. Despite the physiological importance of GnT-IVa, its structure and catalytic mechanism are poorly understood. Here, we identify the lectin domain in mouse GnT-IVa's C-terminal region. The crystal structure of the lectin domain shows structural similarity to a bacterial GlcNAc-binding lectin. Comprehensive glycan binding assay using 157 glycans and solution NMR reveal that the GnT-IVa lectin domain selectively interacts with the product N-glycans having a ß1-4 GlcNAc branch. Point mutation of the residue critical to sugar recognition impairs the enzymatic activity, suggesting that the lectin domain is a regulatory subunit for efficient catalytic reaction. Our findings provide insights into how branching structures of N-glycans are biosynthesized.

Structure of the bile acid transporter and HBV receptor NTCP.

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 annually1,2. 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 hepatocytes3. 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.

Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation.

SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.

The structure of NLRP9 reveals a unique C-terminal region with putative regulatory function.

Nucleotide-binding and oligomerisation domain-like receptors (NLRs) can form inflammasomes that activate caspase-1 and pro-interleukin-1ß and induce pyroptosis. NLR family pyrin domain-containing 9 (NLRP9) forms an inflammasome and activates innate immune responses during virus infection, but little is known about this process. Here, we report the crystal and cryo-electron microscopy structures of NLRP9 in an ADP-bound state, revealing inactive and closed conformations of NLRP9 and its similarities to other structurally characterised NLRs. Moreover, we found a C-terminal region interacting with the concave surface of the leucine-rich repeat domain of NLRP9. This region is unique among NLRs and might be involved in the specific function of NLRP9. These data provide the structural basis for understanding the mechanism of NLRP9 regulation and activation.