Structure-based design of non-hypertrophic apelin receptor modulator.

Apelin is a key hormone in cardiovascular homeostasis that activates the apelin receptor (APLNR), which is regarded as a promising therapeutic target for cardiovascular disease. However, adverse effects through the ß-arrestin pathway limit its pharmacological use. Here, we report cryoelectron microscopy (cryo-EM) structures of APLNR-Gi1 complexes bound to three agonists with divergent signaling profiles. Combined with functional assays, we have identified "twin hotspots" in APLNR as key determinants for signaling bias, guiding the rational design of two exclusive G-protein-biased agonists WN353 and WN561. Cryo-EM structures of WN353- and WN561-stimulated APLNR-G protein complexes further confirm that the designed ligands adopt the desired poses. Pathophysiological experiments have provided evidence that WN561 demonstrates superior therapeutic effects against cardiac hypertrophy and reduced adverse effects compared with the established APLNR agonists. In summary, our designed APLNR modulator may facilitate the development of next-generation cardiovascular medications.

Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer.

Tumor-resident intracellular microbiota is an emerging tumor component that has been documented for a variety of cancer types with unclear biological functions. Here, we explored the functional significance of these intratumor bacteria, primarily using a murine spontaneous breast-tumor model MMTV-PyMT. We found that depletion of intratumor bacteria significantly reduced lung metastasis without affecting primary tumor growth. During metastatic colonization, intratumor bacteria carried by circulating tumor cells promoted host-cell survival by enhancing resistance to fluid shear stress by reorganizing actin cytoskeleton. We further showed that intratumor administration of selected bacteria strains isolated from tumor-resident microbiota promoted metastasis in two murine tumor models with significantly different levels of metastasis potential. Our findings suggest that tumor-resident microbiota, albeit at low biomass, play an important role in promoting cancer metastasis, intervention of which might therefore be worth exploring for advancing oncology care.

The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

Identifying tumor-induced leukocyte subsets and their derived circulating factors has been instrumental in understanding cancer as a systemic disease. Nevertheless, how primary tumor-induced non-leukocyte populations in distal organs contribute to systemic spread remains poorly defined. Here, we report one population of tumor-inducible, erythroblast-like cells (Ter-cells) deriving from megakaryocyte-erythroid progenitor cells with a unique Ter-119+CD45-CD71+ phenotype. Ter-cells are enriched in the enlarged spleen of hosts bearing advanced tumors and facilitate tumor progression by secreting neurotrophic factor artemin into the blood. Transforming growth factor ß (TGF-ß) and Smad3 activation are important in Ter-cell generation. In vivo blockade of Ter-cell-derived artemin inhibits hepatocellular carcinoma (HCC) growth, and artemin deficiency abolishes Ter-cells' tumor-promoting ability. We confirm the presence of splenic artemin-positive Ter-cells in human HCC patients and show that significantly elevated serum artemin correlates with poor prognosis. We propose that Ter-cells and the secreted artemin play important roles in cancer progression with prognostic and therapeutic implications.

Conformational transitions and activation of the adhesion receptor CD97.

Adhesion G protein-coupled receptors (aGPCRs) are evolutionarily ancient receptors involved in a variety of physiological and pathophysiological processes. Modulators of aGPCR, particularly antagonists, hold therapeutic promise for diseases like cancer and immune and neurological disorders. Hindered by the inactive state structural information, our understanding of antagonist development and aGPCR activation faces challenges. Here, we report the cryo-electron microscopy structures of human CD97, a prototypical aGPCR that plays crucial roles in immune system, in its inactive apo and G13-bound fully active states. Compared with other family GPCRs, CD97 adopts a compact inactive conformation with a constrained ligand pocket. Activation induces significant conformational changes for both extracellular and intracellular sides, creating larger cavities for Stachel sequence binding and G13 engagement. Integrated with functional and metadynamics analyses, our study provides significant mechanistic insights into the activation and signaling of aGPCRs, paving the way for future drug discovery efforts.

Methyltransferase Dnmt3a upregulates HDAC9 to deacetylate the kinase TBK1 for activation of antiviral innate immunity.

The DNA methyltransferase Dnmt3a has high expression in terminally differentiated macrophages; however, its role in innate immunity remains unknown. Here we report that deficiency in Dnmt3a selectively impaired the production of type I interferons triggered by pattern-recognition receptors (PRRs), but not that of the proinflammatory cytokines TNF and IL-6. Dnmt3a-deficient mice exhibited enhanced susceptibility to viral challenge. Dnmt3a did not directly regulate the transcription of genes encoding type I interferons; instead, it increased the production of type I interferons through an epigenetic mechanism by maintaining high expression of the histone deacetylase HDAC9. In turn, HDAC9 directly maintained the deacetylation status of the key PRR signaling molecule TBK1 and enhanced its kinase activity. Our data add mechanistic insight into the crosstalk between epigenetic modifications and post-translational modifications in the regulation of PRR signaling and activation of antiviral innate immune responses.

Utilizing the oxygen-atom trapping effect of Co3O4 with oxygen vacancies to promote chlorite activation for water decontamination.

Heterogeneous high-valent cobalt-oxo [≡Co(IV)=O] is a widely focused reactive species in oxidant activation; however, the relationship between the catalyst interfacial defects and ≡Co(IV)=O formation remains poorly understood. Herein, photoexcited oxygen vacancies (OVs) were introduced into Co3O4 (OV-Co3O4) by a UV-induced modification method to facilitate chlorite (ClO2-) activation. Density functional theory calculations indicate that OVs result in low-coordinated Co atom, which can directionally anchor chlorite under the oxygen-atom trapping effect. Chlorite first undergoes homolytic O-Cl cleavage and transfers the dissociated O atom to the low-coordinated Co atom to form reactive ≡Co(IV)=O with a higher spin state. The reactive ≡Co(IV)=O rapidly extracts one electron from ClO2- to form chlorine dioxide (ClO2), accompanied by the Co atom returning a lower spin state. As a result of the oxygen-atom trapping effect, the OV-Co3O4/chlorite system achieved a 3.5 times higher efficiency of sulfamethoxazole degradation (~0.1331 min-1) than the pristine Co3O4/chlorite system. Besides, the refiled OVs can be easily restored by re-exposure to UV light, indicating the sustainability of the oxygen atom trap. The OV-Co3O4 was further fabricated on a polyacrylonitrile membrane for back-end water purification, achieving continuous flow degradation of pollutants with low cobalt leakage. This work presents an enhancement strategy for constructing OV as an oxygen-atom trapping site in heterogeneous advanced oxidation processes and provides insight into modulating the formation of ≡Co(IV)=O via defect engineering.

Isolation, characterization, and circulation sphere of a filovirus in fruit bats.

Bats are associated with the circulation of most mammalian filoviruses (FiVs), with pathogenic ones frequently causing deadly hemorrhagic fevers in Africa. Divergent FiVs have been uncovered in Chinese bats, raising concerns about their threat to public health. Here, we describe a long-term surveillance to track bat FiVs at orchards, eventually resulting in the identification and isolation of a FiV, Dehong virus (DEHV), from Rousettus leschenaultii bats. DEHV has a typical filovirus-like morphology with a wide spectrum of cell tropism. Its entry into cells depends on the engagement of Niemann-Pick C1, and its replication is inhibited by remdesivir. DEHV has the largest genome size of filoviruses, with phylogenetic analysis placing it between the genera Dianlovirus and Orthomarburgvirus, suggesting its classification as the prototype of a new genus within the family Filoviridae. The continuous detection of viral RNA in the serological survey, together with the wide host distribution, has revealed that the region covering southern Yunnan, China, and bordering areas is a natural circulation sphere for bat FiVs. These emphasize the need for a better understanding of the pathogenicity and potential risk of FiVs in the region.

Accelerating the design of pili-enabled living materials using an integrative technological workflow.

Bacteria can be programmed to create engineered living materials (ELMs) with self-healing and evolvable functionalities. However, further development of ELMs is greatly hampered by the lack of engineerable nonpathogenic chassis and corresponding programmable endogenous biopolymers. Here, we describe a technological workflow for facilitating ELMs design by rationally integrating bioinformatics, structural biology and synthetic biology technologies. We first develop bioinformatics software, termed Bacteria Biopolymer Sniffer (BBSniffer), that allows fast mining of biopolymers and biopolymer-producing bacteria of interest. As a proof-of-principle study, using existing pathogenic pilus as input, we identify the covalently linked pili (CLP) biosynthetic gene cluster in the industrial workhorse Corynebacterium glutamicum. Genetic manipulation and structural characterization reveal the molecular mechanism of the CLP assembly, ultimately enabling a type of programmable pili for ELM design. Finally, engineering of the CLP-enabled living materials transforms cellulosic biomass into lycopene by coupling the extracellular and intracellular bioconversion ability.

Local and global consequences of reward-evoked striatal dopamine release.

The neurotransmitter dopamine is required for the reinforcement of actions by rewarding stimuli1. Neuroscientists have tried to define the functions of dopamine in concise conceptual terms2, but the practical implications of dopamine release depend on its diverse brain-wide consequences. Although molecular and cellular effects of dopaminergic signalling have been extensively studied3, the effects of dopamine on larger-scale neural activity profiles are less well-understood. Here we combine dynamic dopamine-sensitive molecular imaging4 and functional magnetic resonance imaging to determine how striatal dopamine release shapes local and global responses to rewarding stimulation in rat brains. We find that dopamine consistently alters the duration, but not the magnitude, of stimulus responses across much of the striatum, via quantifiable postsynaptic effects that vary across subregions. Striatal dopamine release also potentiates a network of distal responses, which we delineate using neurochemically dependent functional connectivity analyses. Hot spots of dopaminergic drive notably include cortical regions that are associated with both limbic and motor function. Our results reveal distinct neuromodulatory actions of striatal dopamine that extend well beyond its sites of peak release, and that result in enhanced activation of remote neural populations necessary for the performance of motivated actions. Our findings also suggest brain-wide biomarkers of dopaminergic function and could provide a basis for the improved interpretation of neuroimaging results that are relevant to learning and addiction.

Key roles for phosphorylation and the Coiled-coil domain in TRIM56-mediated positive regulation of TLR3-TRIF-dependent innate immunity.

Tripartite-motif protein-56 (TRIM56) positively regulates the induction of type I interferon response via the TLR3 pathway by enhancing IRF3 activation and depends on its C-terminal residues 621-750 for interacting with the adaptor TRIF. However, the precise underlying mechanism and detailed TRIM56 determinants remain unclear. Herein, we show ectopic expression of murine TRIM56 also enhances TLR3-dependent interferon-ß promoter activation, suggesting functional conservation. We found that endogenous TRIM56 and TRIF formed a complex early (0.5-2 h) after poly-I:C stimulation and that TRIM56 overexpression also promoted activation of NF-κB by poly-I:C but not that by TNF-α or IL-1ß, consistent with a specific effect on TRIF prior to the bifurcation of NF-κB and IRF3. Using transient transfection and Tet-regulated cell lines expressing various TRIM56 mutants, we demonstrated the Coiled-coil domain and a segment spanning residues ∼434-610, but not the B-box or residues 355-433, were required for TRIM56 augmentation of TLR3 signaling. Moreover, alanine substitution at each putative phosphorylation site, Ser471, Ser475, and Ser710, abrogated TRIM56 function. Concordantly, mutants bearing Ser471Ala, Ser475Ala, or Ser710Ala, or lacking the Coiled-coil domain, all lost the capacity to enhance poly-I:C-induced establishment of an antiviral state. Furthermore, the Ser710Ala mutation disrupted the TRIM56-TRIF association. Using phospho-specific antibodies, we detected biphasic phosphorylation of TRIM56 at Ser471 and Ser475 following TLR3 stimulation, with the early phase occurring at ∼0.5 to 1 h, prior to IRF3 phosphorylation. Together, these data reveal novel molecular details critical for the TRIM56 augmentation of TLR3-dependent antiviral response and highlight important roles for TRIM56 scaffolding and phosphorylation.

Developmental programming and lineage branching of early human telencephalon.

The dorsal and ventral human telencephalons contain different neuronal subtypes, including glutamatergic, GABAergic, and cholinergic neurons, and how these neurons are generated during early development is not well understood. Using scRNA-seq and stringent validations, we reveal here a developmental roadmap for human telencephalic neurons. Both dorsal and ventral telencephalic radial glial cells (RGs) differentiate into neurons via dividing intermediate progenitor cells (IPCs_div) and early postmitotic neuroblasts (eNBs). The transcription factor ASCL1 plays a key role in promoting fate transition from RGs to IPCs_div in both regions. RGs from the regionalized neuroectoderm show heterogeneity, with restricted glutamatergic, GABAergic, and cholinergic differentiation potencies. During neurogenesis, IPCs_div gradually exit the cell cycle and branch into sister eNBs to generate distinct neuronal subtypes. Our findings highlight a general RGs-IPCs_div-eNBs developmental scheme for human telencephalic progenitors and support that the major neuronal fates of human telencephalon are predetermined during dorsoventral regionalization with neuronal diversity being further shaped during neurogenesis and neural circuit integration.

Identify gestational diabetes mellitus by deep learning model from cell-free DNA at the early gestation stage.

Gestational diabetes mellitus (GDM) is a common complication of pregnancy, which has significant adverse effects on both the mother and fetus. The incidence of GDM is increasing globally, and early diagnosis is critical for timely treatment and reducing the risk of poor pregnancy outcomes. GDM is usually diagnosed and detected after 24 weeks of gestation, while complications due to GDM can occur much earlier. Copy number variations (CNVs) can be a possible biomarker for GDM diagnosis and screening in the early gestation stage. In this study, we proposed a machine-learning method to screen GDM in the early stage of gestation using cell-free DNA (cfDNA) sequencing data from maternal plasma. Five thousand and eighty-five patients from north regions of Mainland China, including 1942 GDM, were recruited. A non-overlapping sliding window method was applied for CNV coverage screening on low-coverage (~0.2×) sequencing data. The CNV coverage was fed to a convolutional neural network with attention architecture for the binary classification. The model achieved a classification accuracy of 88.14%, precision of 84.07%, recall of 93.04%, F1-score of 88.33% and AUC of 96.49%. The model identified 2190 genes associated with GDM, including DEFA1, DEFA3 and DEFB1. The enriched gene ontology (GO) terms and KEGG pathways showed that many identified genes are associated with diabetes-related pathways. Our study demonstrates the feasibility of using cfDNA sequencing data and machine-learning methods for early diagnosis of GDM, which may aid in early intervention and prevention of adverse pregnancy outcomes.

Histone tyrosine sulfation by SULT1B1 regulates H4R3me2a and gene transcription.

Tyrosine sulfation is a common posttranslational modification in mammals. To date, it has been thought to be limited to secreted and transmembrane proteins, but little is known about tyrosine sulfation on nuclear proteins. Here we report that SULT1B1 is a histone sulfotransferase that can sulfate the tyrosine 99 residue of nascent histone H3 in cytosol. The sulfated histone H3 can be transported into the nucleus and majorly deposited in the promoter regions of genes in chromatin. While the H3Y99 residue is buried inside octameric nucleosome, dynamically regulated subnucleosomal structures provide chromatin-H3Y99sulf the opportunity of being recognized and bound by PRMT1, which deposits H4R3me2a in chromatin. Disruption of H3Y99sulf reduces PRMT1 binding to chromatin, H4R3me2a level and gene transcription. These findings reveal the mechanisms underlying H3Y99 sulfation and its cross-talk with H4R3me2a to regulate gene transcription. This study extends the spectrum of tyrosine sulfation on nuclear proteins and the repertoire of histone modifications regulating chromatin functions.

Does oncolytic viruses-mediated metabolic reprogramming benefit or harm the immune microenvironment?

Oncolytic virus immunotherapy as a new tumor therapy has made remarkable achievements in clinical practice. And metabolic reprogramming mediated by oncolytic virus has a significant impact on the immune microenvironment. This review summarized the reprogramming of host cell glucose metabolism, lipid metabolism, oxidative phosphorylation, and glutamine metabolism by oncolytic virus and illustrated the effects of metabolic reprogramming on the immune microenvironment. It was found that oncolytic virus-induced reprogramming of glucose metabolism in tumor cells has both beneficial and detrimental effects on the immune microenvironment. In addition, oncolytic virus can promote fatty acid synthesis in tumor cells, inhibit oxidative phosphorylation, and promote glutamine catabolism, which facilitates the anti-tumor immune function of immune cells. Therefore, targeted metabolic reprogramming is a new direction to improve the efficacy of oncolytic virus immunotherapy.

Interaction of Nmi and IFP35 Promotes Mutual Protein Stabilization and IRF3 and IRF7 Degradation to Suppress Type I IFN Production in Teleost Fish.

IFN-stimulated genes (ISGs) can act as effector molecules against viral infection and can also regulate pathogenic infection and host immune response. N-Myc and STAT interactor (Nmi) is reported as an ISG in mammals and in fish. In this study, the expression of Nmi was found to be induced significantly by the infection of Siniperca chuatsi rhabdovirus (SCRV), and the induced expression of type I IFNs after SCRV infection was reduced following Nmi overexpression. It is observed that Nmi can interact with IRF3 and IRF7 and promote the autophagy-mediated degradation of these two transcription factors. Furthermore, Nmi was found to be interactive with IFP35 through the CC region to inhibit IFP35 protein degradation, thereby enhancing the negative role in type I IFN expression after viral infection. In turn, IFP35 is also capable of protecting Nmi protein from degradation through its N-terminal domain. It is considered that Nmi and IFP35 in fish can also interact with each other in regulating negatively the expression of type I IFNs, but thus in enhancing the replication of SCRV.

Transcriptional Regulation and Signaling of Type IV IFN with Identification of the ISG Repertoire in an Amphibian Model, Xenopus laevis.

The type IV IFN (IFN-υ) is reported in vertebrates from fish to primary mammals with IFN-υR1 and IL-10R2 as receptor subunits. In this study, the proximal promoter of IFN-υ was identified in the amphibian model, Xenopus laevis, with functional IFN-sensitive responsive element and NF-κB sites, which can be transcriptionally activated by transcription factors, such as IFN regulatory factor (IRF)1, IRF3, IRF7, and p65. It was further found that IFN-υ signals through the classical IFN-stimulated gene (ISG) factor 3 (ISGF3) to induce the expression of ISGs. It seems likely that the promoter elements of the IFN-υ gene in amphibians is similar to type III IFN genes, and that the mechanism involved in IFN-υ induction is very much similar to type I and III IFNs. Using recombinant IFN-υ protein and the X. laevis A6 cell line, >400 ISGs were identified in the transcriptome, including ISGs homologous to humans. However, as many as 268 genes were unrelated to human or zebrafish ISGs, and some of these ISGs were expanded families such as the amphibian novel TRIM protein (AMNTR) family. AMNTR50, a member in the family, was found to be induced by type I, III, and IV IFNs through IFN-sensitive responsive element sites of the proximal promoter, and this molecule has a negative role in regulating the expression of type I, III, and IV IFNs. It is considered that the current study contributes to the understanding of transcription, signaling, and functional aspects of type IV IFN at least in amphibians.

Precise genome editing of the Kozak sequence enables bidirectional and quantitative modulation of protein translation to anticipated levels without affecting transcription.

None of the existing approaches for regulating gene expression can bidirectionally and quantitatively fine-tune gene expression to desired levels. Here, on the basis of precise manipulations of the Kozak sequence, which has a remarkable influence on translation initiation, we proposed and validated a novel strategy to directly modify the upstream nucleotides of the translation initiation codon of a given gene to flexibly alter the gene translation level by using base editors and prime editors. When the three nucleotides upstream of the translation initiation codon (named KZ3, part of the Kozak sequence), which exhibits the most significant base preference of the Kozak sequence, were selected as the editing region to alter the translation levels of proteins, we confirmed that each of the 64 KZ3 variants had a different translation efficiency, but all had similar transcription levels. Using the ranked KZ3 variants with different translation efficiencies as predictors, base editor- and prime editor-mediated mutations of KZ3 in the local genome could bidirectionally and quantitatively fine-tune gene translation to the anticipated levels without affecting transcription in vitro and in vivo. Notably, this strategy can be extended to the whole Kozak sequence and applied to all protein-coding genes in all eukaryotes.

Structural basis of peptidomimetic agonism revealed by small- molecule GLP-1R agonists Boc5 and WB4-24.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are effective in treating type 2 diabetes and obesity with proven cardiovascular benefits. However, most of these agonists are peptides and require subcutaneous injection except for orally available semaglutide. Boc5 was identified as the first orthosteric nonpeptidic agonist of GLP-1R that mimics a broad spectrum of bioactivities of GLP-1 in vitro and in vivo. Here, we report the cryoelectron microscopy structures of Boc5 and its analog WB4-24 in complex with the human GLP-1R and Gs protein. Bound to the extracellular domain, extracellular loop 2, and transmembrane (TM) helices 1, 2, 3, and 7, one arm of both compounds was inserted deeply into the bottom of the orthosteric binding pocket that is usually accessible by peptidic agonists, thereby partially overlapping with the residues A8 to D15 in GLP-1. The other three arms, meanwhile, extended to the TM1-TM7, TM1-TM2, and TM2-TM3 clefts, showing an interaction feature substantially similar to the previously known small-molecule agonist LY3502970. Such a unique binding mode creates a distinct conformation that confers both peptidomimetic agonism and biased signaling induced by nonpeptidic modulators at GLP-1R. Further, the conformational difference between Boc5 and WB4-24, two closed related compounds, provides a structural framework for fine-tuning of pharmacological efficacy in the development of future small-molecule therapeutics targeting GLP-1R.