Structural biology of flavivirus NS1 protein and its antibody complexes.

The genus of flavivirus includes many mosquito-borne human pathogens, such as Zika (ZIKV) and the four serotypes of dengue (DENV1-4) viruses, that affect billions of people as evidenced by epidemics and endemicity in many countries and regions in the world. Among the 10 viral proteins encoded by the viral genome, the nonstructural protein 1 (NS1) is the only secreted protein and has been used as a diagnostic biomarker. NS1 has also been an attractive target for its biotherapeutic potential as a vaccine antigen. This review focuses on the recent advances in the structural landscape of the secreted NS1 (sNS1) and its complex with monoclonal antibodies (mAbs). NS1 forms an obligatory dimer, and upon secretion, it has been reported to be hexametric (trimeric dimers) that could dissociate and bind to the epithelial cell membrane. However, high-resolution structural information has been missing about the high-order oligomeric states of sNS1. Several cryoEM studies have since shown that DENV and ZIKV recombinant sNS1 (rsNS1) are in dynamic equilibrium of dimer-tetramer-hexamer states, with tetramer being the predominant form. It was recently revealed that infection-derived sNS1 (isNS1) forms a complex of the NS1 dimer partially embedded in a High-Density Lipoprotein (HDL) particle. Structures of NS1 in complexes with mAbs have also been reported which shed light on their protective roles during infection. The biological significance of the diversity of NS1 oligomeric states remains to be further studied, to inform future research on flaviviral pathogenesis and the development of therapeutics and vaccines. Given the polymorphism of flavivirus NS1 across sample types with variations in antigenicity, we propose a nomenclature to accurately define NS1 based on the localization and origin.

Molecular basis for differential Igk versus Igh V(D)J joining mechanisms.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1-3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.

The step-by-step assembly mechanism of secreted flavivirus NS1 tetramer and hexamer captured at atomic resolution.

Flaviviruses encode a conserved, membrane-associated nonstructural protein 1 (NS1) with replication and immune evasion functions. The current knowledge of secreted NS1 (sNS1) oligomers is based on several low-resolution structures, thus hindering the development of drugs and vaccines against flaviviruses. Here, we revealed that recombinant sNS1 from flaviviruses exists in a dynamic equilibrium of dimer-tetramer-hexamer states. Two DENV4 hexameric NS1 structures and several tetrameric NS1 structures from multiple flaviviruses were solved at atomic resolution by cryo-EM. The stacking of the tetrameric NS1 and hexameric NS1 is facilitated by the hydrophobic ß-roll and connector domains. Additionally, a triacylglycerol molecule located within the central cavity may play a role in stabilizing the hexamer. Based on differentiated interactions between the dimeric NS1, two distinct hexamer models (head-to-head and side-to-side hexamer) and the step-by-step assembly mechanisms of NS1 dimer into hexamer were proposed. We believe that our study sheds light on the understanding of the NS1 oligomerization and contributes to NS1-based therapies.

Structural insights into the activation and inhibition of CXC chemokine receptor 3.

The chemotaxis of CD4+ type 1 helper cells and CD8+ cytotoxic lymphocytes, guided by interferon-inducible CXC chemokine 9-11 (CXCL9-11) and CXC chemokine receptor 3 (CXCR3), plays a critical role in type 1 immunity. Here we determined the structures of human CXCR3-DNGi complexes activated by chemokine CXCL11, peptidomimetic agonist PS372424 and biaryl-type agonist VUF11222, and the structure of inactive CXCR3 bound to noncompetitive antagonist SCH546738. Structural analysis revealed that PS372424 shares a similar orthosteric binding pocket to the N terminus of CXCL11, while VUF11222 buries deeper and activates the receptor in a distinct manner. We showed an allosteric binding site between TM5 and TM6, accommodating SCH546738 in the inactive CXCR3. SCH546738 may restrain the receptor at an inactive state by preventing the repacking of TM5 and TM6. By revealing the binding patterns and the pharmacological properties of the four modulators, we present the activation mechanisms of CXCR3 and provide insights for future drug development.

A method for structure determination of GPCRs in various states.

G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the ß2-adrenergic receptor (ß2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For ß2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structure‒function relationships and drug development.

Accumulation, potential risk and source identification of toxic metal elements in soil: a case study of a coal-fired power plant in Western China.

Coal-fired power plants (CPP) usually release massive numerous amounts of potentially toxic metal(loid)s (PTMs) into nearby ecosystems. There have been relatively few studies targeted on the ecological influences of PTMs related to the CPP in arid area. In this work, the distribution pattern, source apportionment and environmental risks of As, Cd, Cr, Hg, Pb and a couple of seldom monitored PTMs (Se, Zn, Co, Cu, Fe, Mn and Ni) in the soils near a coal electricity integration base were investigated in Hami, a city in northwestern China. Nemerow synthesis pollution index, geo-accumulation index and ecological risk index were used to assess pollution state of these PTMs in soils, and ordinary Kriging interpolation was used to analyze the spatial distribution for these elements. Methods of CA, PCA, CA and PAM were carried out for quantitative source analysis. The research outcome includes: (1) the contents of individual PTMs in most samples were greater than the background values, the pollution degrees of Se, Pb, Hg, Cd and As were significant, and some areas exceeded the warning threshold value; (2) the main sources of these PTMs were natural sources (35%), coal mine sewage (11%), atmospheric release during coal combustion (21%), dust generated from coal and combustion products (33%); (3) attention should be paid to the open-pit coal mines, shaft coal mines and ash dumps where the contents of metal elements were significantly polluted; and (4) wind is the main driving forces of PTMs migration in arid areas.

Mechanism of agonist-induced activation of the human itch receptor MRGPRX1.

Mas-related G-protein-coupled receptors X1-X4 (MRGPRX1-X4) are 4 primate-specific receptors that are recently reported to be responsible for many biological processes, including itch sensation, pain transmission, and inflammatory reactions. MRGPRX1 is the first identified human MRGPR, and its expression is restricted to primary sensory neurons. Due to its dual roles in itch and pain signaling pathways, MRGPRX1 has been regarded as a promising target for itch remission and pain inhibition. Here, we reported a cryo-electron microscopy (cryo-EM) structure of Gq-coupled MRGPRX1 in complex with a synthetic agonist compound 16 in an active conformation at an overall resolution of 3.0 Å via a NanoBiT tethering strategy. Compound 16 is a new pain-relieving compound with high potency and selectivity to MRGPRX1 over other MRGPRXs and opioid receptor. MRGPRX1 was revealed to share common structural features of the Gq-mediated receptor activation mechanism of MRGPRX family members, but the variable residues in orthosteric pocket of MRGPRX1 exhibit the unique agonist recognition pattern, potentially facilitating to design MRGPRX1-specific modulators. Together with receptor activation and itch behavior evaluation assays, our study provides a structural snapshot to modify therapeutic molecules for itch relieving and analgesia targeting MRGPRX1.

High-Quality Genome Assembly and Annotation Resource of Elsinoë annonae, Causing Fruit Scab on Camellia oleifera.

Elsinoë annonae is a fungal pathogen that causes fruit scab disease in the edible-oil (tea oil) plant (Camellia oleifera Abel). The absence of genome resources for this fungus hampers functional genetic studies of the pathogenesis mechanism of E. annonae. This study reports the genome assembly of E. annonae strain SM-YC-2 collected from tea oil tree fruit with scab disease in Fujian Province, China. Combining 16.44 Gb of PacBio Sequel II long reads and 5.13 Gb of Illumina NovaSeq reads, we generated a 25.93-Mb (99.19% of expected genome size) high-quality genome assembly with 52.66% GC content, 5.05% repeats, and over 98% Benchmarking Universal Single-Copy Orthologs completeness for E. annonae strain SM-YC-2. These high-quality genome assembly resources will facilitate functional genomic characterization studies, enhance insights into the pathogenicity mechanism of E. annonae, and support the development of molecular-based control strategies.

Risk factors for postoperative pulmonary complications in elderly patients receiving elective colorectal surgery: A retrospective study.

Study objective: Postoperative pulmonary complications (PPCs) are common and associated with adverse outcomes impairing long-term survival and quality of recovery. This single-centered retrospective study aimed to examine factors associated with PPCs in patients receiving elective colorectal surgery aged ≥60 years. Methods: Between January 2019 and December 2019, 638 patients at the Shanghai Changhai Hospital who had received elective surgery for colorectal cancer were enrolled in this study. Patients were divided into the PPC group (n=38) and non-PPC group (n=600). Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), red blood cell distribution width (RDW), and systemic inflammatory index (SII) were selected and caculated to indicate preoperative and postoperative inflammatory status. Receiver operating characteristic curve and bivariate correlation analyses were performed to evaluate the identified risk factors. Main results: The overall incidence of PPCs was approximately 5.96%. Multivariate regression analysis identified age (OR = 1.094, 95%CI 1.038-1.153, P = 0.001), preoperative RDW (OR = 1.159, 95%CI 1.025-1.309, P = 0.018), and preoperative SII (OR = 1.001, 95%CI 1.000-1.003, P = 0.035) as independent risk factors for PPCs. The cut-off values of age, preoperative RDW, and preoperative SII for predicting PPCs were 69.5 (sensitivity 0.658, specificity 0.653), 13.2 (sensitivity 0.789, specificity 0.552) and 556.1 (sensitivity 0.579, specificity 0.672), respectively. Conclusions: Age, preoperative RDW, and preoperative SII were identified as independent risk factors for PPC occurrence in elderly patients receiving elective colorectal surgery. Further studies are warranted to evaluate whether normalization of preoperative RDW and SII, as modifiable risk factors, are associated with improved surgical outcomes.

The First Telomere-to-Telomere Chromosome-Level Genome Assembly of Stagonospora tainanensis Causing Sugarcane Leaf Blight.

The sexual morph Leptosphaeria taiwanensis Yen and Chi and its asexual morph Stagonospora tainanensis W. H. Hsieh is an important necrotrophic fungal phytopathogen, which causes sugarcane leaf blight, resulting in loss of cane tonnage and sucrose in susceptible sugarcane varieties. Decoding the genome and understanding of the basis of virulence is vitally important for devising effective disease control strategies. Here, we present a 38.25-Mb high-quality genome assembly of S. tainanensis strain StFZ01, denovo assembled with 10.19 Gb Nanopore sequencing long reads (~267×) and 3.82 Gb Illumina short reads (~100×). The genome assembly consists of 12 contigs with N50 of 2.86 Mb of which 5 belong to the telomere to telomere (T2T) chromosome. It contains 13.20% repeat sequences, 12,543 proteins, and 12,206 protein-coding genes with the BUSCO completeness 99.18% at fungi (n = 758) and 99.87% at ascomycota (n = 1706), indicating the high accuracy and completeness of our gene annotations. The virulence analysis in silico revealed the presence of 2379 PHIs, 599 CAZys, 248 membrane transport proteins, 191 cytochrome P450 enzymes, 609 putative secreted proteins, and 333 effectors in the StFZ01 genome. The genomic resources presented here will not only be helpful for development of specific molecular marker and diagnosis technique, population genetics, molecular taxonomy, and disease managements, it can also provide a significant precise genomic reference for investigating the ascomycetous genome, the necrotrophic lifestyle, and pathogenicity in the future.

Structural basis for recognition of N-formyl peptides as pathogen-associated molecular patterns.

The formyl peptide receptor 1 (FPR1) is primarily responsible for detection of short peptides bearing N-formylated methionine (fMet) that are characteristic of protein synthesis in bacteria and mitochondria. As a result, FPR1 is critical to phagocyte migration and activation in bacterial infection, tissue injury and inflammation. How FPR1 distinguishes between formyl peptides and non-formyl peptides remains elusive. Here we report cryo-EM structures of human FPR1-Gi protein complex bound to S. aureus-derived peptide fMet-Ile-Phe-Leu (fMIFL) and E. coli-derived peptide fMet-Leu-Phe (fMLF). Both structures of FPR1 adopt an active conformation and exhibit a binding pocket containing the R2015.38XXXR2055.42 (RGIIR) motif for formyl group interaction and receptor activation. This motif works together with D1063.33 for hydrogen bond formation with the N-formyl group and with fMet, a model supported by MD simulation and functional assays of mutant receptors with key residues for recognition substituted by alanine. The cryo-EM model of agonist-bound FPR1 provides a structural basis for recognition of bacteria-derived chemotactic peptides with potential applications in developing FPR1-targeting agents.

Effect of Perioperative Intravenous Lidocaine on Postoperative Recovery in Patients Undergoing Ileostomy Closure: Study Protocol for a Randomized Controlled Trial.

Introduction: Opioids have been widely used clinically as the first choice for pain management. Ileostomy closure usually leads to temporary intestinal paralysis, which manifests as abdominal distension and pain, delayed defecation, nausea, and vomiting. Intraoperative and postoperative use of opioids inhibit gastrointestinal function and aggravate intestinal paralysis, and are notoriously addictive. Thus, reducing perioperative opioid use is important for patients undergoing ileostomy closure to restore the continuity and integrity of the intestine. Intravenous lidocaine has been shown to have anti-inflammatory properties and analgesic effects. We consider minimizing the use of opioids for such patients, and perioperative intravenous injection of lidocaine may be beneficial to the recovery of intestinal function in patients with ileostomy closure. Methods and Analysis: This is a randomized double-blind placebo-controlled trial to investigate the effectiveness and safety of intravenous lidocaine in patients undergoing ileostomy closure. The time of first postoperative anal venting, postoperative opioids use, postoperative recovery, intraoperative adverse effects and postoperative complications will be collected and analyzed.

Structural insights into sphingosine-1-phosphate receptor activation.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step "shallow to deep" transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

The role of chromatin loop extrusion in antibody diversification.

Cohesin mediates chromatin loop formation across the genome by extruding chromatin between convergently oriented CTCF-binding elements. Recent studies indicate that cohesin-mediated loop extrusion in developing B cells presents immunoglobulin heavy chain (Igh) variable (V), diversity (D) and joining (J) gene segments to RAG endonuclease through a process referred to as RAG chromatin scanning. RAG initiates V(D)J recombinational joining of these gene segments to generate the large number of different Igh variable region exons that are required for immune responses to diverse pathogens. Antigen-activated mature B cells also use chromatin loop extrusion to mediate the synapsis, breakage and end joining of switch regions flanking Igh constant region exons during class-switch recombination, which allows for the expression of different antibody constant region isotypes that optimize the functions of antigen-specific antibodies to eliminate pathogens. Here, we review recent advances in our understanding of chromatin loop extrusion during V(D)J recombination and class-switch recombination at the Igh locus.

High-Quality Genome Assembly of Pseudopestalotiopsis theae, the Pathogenic Fungus Causing Tea Gray Blight.

Tea gray blight is one of the most serious foliar diseases of tea tree, caused by the plant-pathogenic fungus Pseudopestalotiopsis theae, which can affect production and quality of tea worldwide. We generated a highly contiguous, 50.41-Mbp genome assembly (N50 = 1.30 Mbp) of P. theae strain CYF27 by combining PacBio long-read and Illumina short-read sequencing technologies. We identified a total of 15,626 gene models, of which 1,038 genes encode putative secreted proteins. The high-quality genome assembly and annotation resource reported here will be useful for the study of fungal infection mechanisms and pathogen-host interaction.

Loop extrusion mediates physiological Igh locus contraction for RAG scanning.

RAG endonuclease initiates Igh V(D)J recombination in progenitor B cells by binding a JH-recombination signal sequence (RSS) within a recombination centre (RC) and then linearly scanning upstream chromatin, presented by loop extrusion mediated by cohesin, for convergent D-RSSs1,2. The utilization of convergently oriented RSSs and cryptic RSSs is intrinsic to long-range RAG scanning3. Scanning of RAG from the DJH-RC-RSS to upstream convergent VH-RSSs is impeded by D-proximal CTCF-binding elements (CBEs)2-5. Primary progenitor B cells undergo a mechanistically undefined contraction of the VH locus that is proposed to provide distal VHs access to the DJH-RC6-9. Here we report that an inversion of the entire 2.4-Mb VH locus in mouse primary progenitor B cells abrogates rearrangement of both VH-RSSs and normally convergent cryptic RSSs, even though locus contraction still occurs. In addition, this inversion activated both the utilization of cryptic VH-RSSs that are normally in opposite orientation and RAG scanning beyond the VH locus through several convergent CBE domains to the telomere. Together, these findings imply that broad deregulation of CBE impediments in primary progenitor B cells promotes RAG scanning of the VH locus mediated by loop extrusion. We further found that the expression of wings apart-like protein homologue (WAPL)10, a cohesin-unloading factor, was low in primary progenitor B cells compared with v-Abl-transformed progenitor B cell lines that lacked contraction and RAG scanning of the VH locus. Correspondingly, depletion of WAPL in v-Abl-transformed lines activated both processes, further implicating loop extrusion in the locus contraction mechanism.