ABSTRACT
Lymphoid cells that produce interleukin (IL)-17 cytokines protect barrier tissues from pathogenic microbes but are also prominent effectors of inflammation and autoimmune disease. T helper 17 (Th17) cells, defined by RORγt-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ T cells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, Th17 cell behaviors vary markedly according to their environment. Here, we show that SAAs additionally direct a pathogenic pro-inflammatory Th17 cell differentiation program, acting directly on T cells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting Th17-mediated inflammatory diseases. These studies suggest that T cell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.
Subject(s)
Irritable Bowel Syndrome/metabolism , Serum Amyloid A Protein/metabolism , Th17 Cells/metabolism , Adult , Animals , Autoimmune Diseases/metabolism , Cell Differentiation/immunology , Cytokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Humans , Inflammation/metabolism , Interleukin-17/metabolism , Irritable Bowel Syndrome/blood , Male , Mice , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Th1 Cells , Th17 Cells/immunologyABSTRACT
Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.
Subject(s)
Cell Differentiation/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Glucose-6-Phosphate Isomerase/metabolism , Glycolysis/genetics , Oxidative Phosphorylation , Pentose Phosphate Pathway/physiology , Th17 Cells/metabolism , Animals , Cell Hypoxia/genetics , Cell Hypoxia/immunology , Chimera/genetics , Chromatography, Gas , Chromatography, Liquid , Clostridium Infections/immunology , Cytokines/deficiency , Cytokines/genetics , Cytokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/metabolism , Glucose-6-Phosphate Isomerase/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/genetics , Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism , Glycolysis/immunology , Homeostasis/genetics , Homeostasis/immunology , Inflammation/genetics , Inflammation/immunology , Mass Spectrometry , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Mucous Membrane/immunology , Mucous Membrane/metabolism , Mucous Membrane/microbiology , Pentose Phosphate Pathway/genetics , Pentose Phosphate Pathway/immunology , RNA-Seq , Single-Cell Analysis , Th17 Cells/immunology , Th17 Cells/pathologyABSTRACT
T helper 17 (Th17) cells regulate mucosal barrier defenses but also promote multiple autoinflammatory diseases. Although many molecular determinants of Th17 cell differentiation have been elucidated, the transcriptional programs that sustain Th17 cells in vivo remain obscure. The transcription factor RORγt is critical for Th17 cell differentiation; however, it is not clear whether the closely related RORα, which is co-expressed in Th17 cells, has a distinct role. Here, we demonstrated that although dispensable for Th17 cell differentiation, RORα was necessary for optimal Th17 responses in peripheral tissues. The absence of RORα in T cells led to reductions in both RORγt expression and effector function among Th17 cells. Cooperative binding of RORα and RORγt to a previously unidentified Rorc cis-regulatory element was essential for Th17 lineage maintenance in vivo. These data point to a non-redundant role of RORα in Th17 lineage maintenance via reinforcement of the RORγt transcriptional program.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Nuclear Receptor Subfamily 1, Group F, Member 3 , Cell Differentiation , Encephalomyelitis, Autoimmune, Experimental/metabolism , Gene Expression Regulation , Nuclear Receptor Subfamily 1, Group F, Member 3/genetics , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Th17 Cells/metabolism , Transcription Factors/metabolismABSTRACT
Over half of hepatocellular carcinoma (HCC) cases diagnosed worldwide are in China1-3. However, whole-genome analysis of hepatitis B virus (HBV)-associated HCC in Chinese individuals is limited4-8, with current analyses of HCC mainly from non-HBV-enriched populations9,10. Here we initiated the Chinese Liver Cancer Atlas (CLCA) project and performed deep whole-genome sequencing (average depth, 120×) of 494 HCC tumours. We identified 6 coding and 28 non-coding previously undescribed driver candidates. Five previously undescribed mutational signatures were found, including aristolochic-acid-associated indel and doublet base signatures, and a single-base-substitution signature that we termed SBS_H8. Pentanucleotide context analysis and experimental validation confirmed that SBS_H8 was distinct to the aristolochic-acid-associated SBS22. Notably, HBV integrations could take the form of extrachromosomal circular DNA, resulting in elevated copy numbers and gene expression. Our high-depth data also enabled us to characterize subclonal clustered alterations, including chromothripsis, chromoplexy and kataegis, suggesting that these catastrophic events could also occur in late stages of hepatocarcinogenesis. Pathway analysis of all classes of alterations further linked non-coding mutations to dysregulation of liver metabolism. Finally, we performed in vitro and in vivo assays to show that fibrinogen alpha chain (FGA), determined as both a candidate coding and non-coding driver, regulates HCC progression and metastasis. Our CLCA study depicts a detailed genomic landscape and evolutionary history of HCC in Chinese individuals, providing important clinical implications.
Subject(s)
Carcinoma, Hepatocellular , Genome, Human , High-Throughput Nucleotide Sequencing , Liver Neoplasms , Mutation , Whole Genome Sequencing , Humans , Aristolochic Acids/metabolism , Carcinogenesis , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/virology , China , Chromothripsis , Disease Progression , DNA, Circular/genetics , East Asian People/genetics , Evolution, Molecular , Genome, Human/genetics , Hepatitis B virus/genetics , INDEL Mutation/genetics , Liver/metabolism , Liver Neoplasms/genetics , Liver Neoplasms/virology , Mutation/genetics , Neoplasm Metastasis/genetics , Open Reading Frames/genetics , Reproducibility of ResultsABSTRACT
Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.
Subject(s)
Disease/genetics , Drosophila melanogaster/genetics , Genetic Testing , Inheritance Patterns , RNA Interference , Animals , Disease Models, Animal , Humans , X ChromosomeABSTRACT
BACKGROUND: Standard treatment with neoadjuvant nivolumab plus chemotherapy significantly improves outcomes in patients with resectable non-small-cell lung cancer (NSCLC). Perioperative treatment (i.e., neoadjuvant therapy followed by surgery and adjuvant therapy) with nivolumab may further improve clinical outcomes. METHODS: In this phase 3, randomized, double-blind trial, we assigned adults with resectable stage IIA to IIIB NSCLC to receive neoadjuvant nivolumab plus chemotherapy or neoadjuvant chemotherapy plus placebo every 3 weeks for 4 cycles, followed by surgery and adjuvant nivolumab or placebo every 4 weeks for 1 year. The primary outcome was event-free survival according to blinded independent review. Secondary outcomes were pathological complete response and major pathological response according to blinded independent review, overall survival, and safety. RESULTS: At this prespecified interim analysis (median follow-up, 25.4 months), the percentage of patients with 18-month event-free survival was 70.2% in the nivolumab group and 50.0% in the chemotherapy group (hazard ratio for disease progression or recurrence, abandoned surgery, or death, 0.58; 97.36% confidence interval [CI], 0.42 to 0.81; P<0.001). A pathological complete response occurred in 25.3% of the patients in the nivolumab group and in 4.7% of those in the chemotherapy group (odds ratio, 6.64; 95% CI, 3.40 to 12.97); a major pathological response occurred in 35.4% and 12.1%, respectively (odds ratio, 4.01; 95% CI, 2.48 to 6.49). Grade 3 or 4 treatment-related adverse events occurred in 32.5% of the patients in the nivolumab group and in 25.2% of those in the chemotherapy group. CONCLUSIONS: Perioperative treatment with nivolumab resulted in significantly longer event-free survival than chemotherapy in patients with resectable NSCLC. No new safety signals were observed. (Funded by Bristol Myers Squibb; CheckMate 77T ClinicalTrials.gov number, NCT04025879.).
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Neoadjuvant Therapy , Nivolumab , Humans , Nivolumab/therapeutic use , Nivolumab/adverse effects , Nivolumab/administration & dosage , Lung Neoplasms/drug therapy , Lung Neoplasms/mortality , Lung Neoplasms/surgery , Lung Neoplasms/pathology , Male , Middle Aged , Female , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/surgery , Carcinoma, Non-Small-Cell Lung/pathology , Aged , Double-Blind Method , Chemotherapy, Adjuvant , Progression-Free Survival , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Adult , Antineoplastic Agents, Immunological/therapeutic use , Antineoplastic Agents, Immunological/adverse effects , Antineoplastic Agents, Immunological/administration & dosage , Neoplasm Staging , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/adverse effects , PneumonectomyABSTRACT
The transcriptional repression of alternative lineage genes is critical for cell fate commitment. Mechanisms by which locus-specific gene silencing is initiated and heritably maintained during cell division are not clearly understood. To study the maintenance of silent gene states, we investigated how the Cd4 gene is stably repressed in CD8+ T cells. Through CRISPR and shRNA screening, we identified the histone chaperone CAF-1 as a critical component for Cd4 repression. We found that the large subunit of CAF-1, Chaf1a, requires the N-terminal KER domain to associate with the histone deacetylases HDAC1/2 and the histone demethylase LSD1, enzymes that also participate in Cd4 silencing. When CAF-1 was lacking, Cd4 derepression was markedly enhanced in the absence of the de novo DNA methyltransferase Dnmt3a but not the maintenance DNA methyltransferase Dnmt1. In contrast to Dnmt1, Dnmt3a deficiency did not significantly alter levels of DNA methylation at the Cd4 locus. Instead, Dnmt3a deficiency sensitized CD8+ T cells to Cd4 derepression mediated by compromised functions of histone-modifying factors, including the enzymes associated with CAF-1. Thus, we propose that the heritable silencing of the Cd4 gene in CD8+ T cells exploits cooperative functions among the DNA methyltransferases, CAF-1, and histone-modifying enzymes.
Subject(s)
CD4 Antigens/genetics , Chromatin Assembly Factor-1/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , Retinoblastoma-Binding Protein 4/metabolism , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Animals , CD4 Antigens/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA Methyltransferase 3A , Female , Gene Expression Regulation , Gene Silencing , Histone Chaperones/metabolism , Histone Deacetylases/metabolism , Histones/metabolism , Male , Mice , Protein DomainsABSTRACT
ABSTRACT: Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is a lethal syndrome because of persistent EBV infection. When diagnosed as CAEBV, EBV infection was observed in multiple hematopoietic lineages, but the etiology of CAEBV is still elusive. Bone marrow and peripheral cells derived from 5 patients with CAEBV, 1 patient with EBV-associated hemophagocytic lymphohistiocytosis, and 2 healthy controls were analyzed. Multiple assays were applied to identify and characterize EBV-infected cells, including quantitative polymerase chain reaction, PrimeFlow, and single-cell RNA-sequencing (scRNA-seq). Based on scRNA-seq data, alterations in gene expression of particular cell types were analyzed between patients with CAEBV and controls, and between infected and uninfected cells. One patient with CAEBV was treated with allogeneic hematopoietic stem cell transplantation (HSCT), and the samples derived from this patient were analyzed again 6 months after HSCT. EBV infected the full spectrum of the hematopoietic system including both lymphoid and myeloid lineages, as well as the hematopoietic stem cells (HSCs) of the patients with CAEBV. EBV-infected HSCs exhibited a higher differentiation rate toward downstream lineages, and the EBV infection had an impact on both the innate and adaptive immunity, resulting in inflammatory symptoms. EBV-infected cells were thoroughly removed from the hematopoietic system after HSCT. Taken together, multiple lines of evidence presented in this study suggest that CAEBV disease originates from the infected HSCs, which might potentially lead to innovative therapy strategies for CAEBV.
Subject(s)
Epstein-Barr Virus Infections , Lymphohistiocytosis, Hemophagocytic , Humans , Herpesvirus 4, Human/genetics , Chronic Disease , Lymphohistiocytosis, Hemophagocytic/complications , Hematopoietic Stem CellsABSTRACT
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
NCOA4 is a selective cargo receptor for ferritinophagy, the autophagic turnover of ferritin (FTH), a process critical for regulating intracellular iron bioavailability. However, how ferritinophagy flux is controlled through NCOA4 in iron-dependent processes needs to be better understood. Here, we show that the C-terminal FTH-binding domain of NCOA4 harbors a [3Fe-4S]-binding site with a stoichiometry of approximately one labile [3Fe-4S] cluster per NCOA4 monomer. By analyzing the interaction between NCOA4 and HERC2 ubiquitin ligase or NCOA4 and FTH, we demonstrate that NCOA4 regulates ferritinophagy by sensing the intracellular iron-sulfur cluster levels. Under iron-repletion conditions, HERC2 recognizes and recruits holo-NCOA4 as a substrate for polyubiquitination and degradation, favoring ferritin iron storage. Under iron-depletion conditions, NCOA4 exists in the form of apo-protein and binds ferritin to promote the occurrence of ferritinophagy and release iron. Thus, we identify an iron-sulfur cluster [3Fe-4S] as a critical cofactor in determining the fate of NCOA4 in favoring iron storage in ferritin or iron release via ferritinophagy and provide a dual mechanism for selective interaction between HERC2 and [3Fe-4S]-NCOA4 for proteasomal degradation or between ferritin and apo-NCOA4 for ferritinophagy in the control of iron homeostasis.
Subject(s)
Homeostasis , Iron , Nuclear Receptor Coactivators , Autophagy , Ferritins/metabolism , Iron/chemistry , Iron/metabolism , Nuclear Receptor Coactivators/genetics , Nuclear Receptor Coactivators/metabolism , Sulfur/chemistry , Sulfur/metabolism , Humans , Animals , Mice , Protein Domains , Cell Line , Cells, Cultured , Ubiquitin-Protein Ligases/metabolism , Protein Stability , Proteasome Endopeptidase Complex/metabolismABSTRACT
B-cell receptor (BCR) knock-in (KI) mouse models play an important role in vaccine development and fundamental immunological studies. However, the time required to generate them poses a bottleneck. Here we report a one-step CRISPR/Cas9 KI methodology to combine the insertion of human germline immunoglobulin heavy and light chains at their endogenous loci in mice. We validate this technology with the rapid generation of three BCR KI lines expressing native human precursors, instead of computationally inferred germline sequences, to HIV broadly neutralizing antibodies. We demonstrate that B cells from these mice are fully functional: upon transfer to congenic, wild type mice at controlled frequencies, such B cells can be primed by eOD-GT8 60mer, a germline-targeting immunogen currently in clinical trials, recruited to germinal centers, secrete class-switched antibodies, undergo somatic hypermutation, and differentiate into memory B cells. KI mice expressing functional human BCRs promise to accelerate the development of vaccines for HIV and other infectious diseases.
Subject(s)
B-Lymphocytes/metabolism , CRISPR-Cas Systems/genetics , Receptors, Antigen, B-Cell/metabolism , Animals , B-Lymphocytes/immunology , Broadly Neutralizing Antibodies/immunology , CRISPR-Cas Systems/immunology , Cell Line , Gene Knock-In Techniques/methods , Germinal Center/immunology , Germinal Center/metabolism , HEK293 Cells , HIV-1/immunology , Humans , Male , Mice , Mice, Inbred C57BL , Models, Animal , Receptors, Antigen, B-Cell/immunologyABSTRACT
Bile acids are abundant in the mammalian gut, where they undergo bacteria-mediated transformation to generate a large pool of bioactive molecules. Although bile acids are known to affect host metabolism, cancer progression and innate immunity, it is unknown whether they affect adaptive immune cells such as T helper cells that express IL-17a (TH17 cells) or regulatory T cells (Treg cells). Here we screen a library of bile acid metabolites and identify two distinct derivatives of lithocholic acid (LCA), 3-oxoLCA and isoalloLCA, as T cell regulators in mice. 3-OxoLCA inhibited the differentiation of TH17 cells by directly binding to the key transcription factor retinoid-related orphan receptor-γt (RORγt) and isoalloLCA increased the differentiation of Treg cells through the production of mitochondrial reactive oxygen species (mitoROS), which led to increased expression of FOXP3. The isoalloLCA-mediated enhancement of Treg cell differentiation required an intronic Foxp3 enhancer, the conserved noncoding sequence (CNS) 3; this represents a mode of action distinct from that of previously identified metabolites that increase Treg cell differentiation, which require CNS1. The administration of 3-oxoLCA and isoalloLCA to mice reduced TH17 cell differentiation and increased Treg cell differentiation, respectively, in the intestinal lamina propria. Our data suggest mechanisms through which bile acid metabolites control host immune responses, by directly modulating the balance of TH17 and Treg cells.
Subject(s)
Cell Differentiation/drug effects , Lithocholic Acid/pharmacology , T-Lymphocytes, Regulatory/drug effects , Th17 Cells/drug effects , Animals , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/immunology , Lithocholic Acid/chemistry , Mice , Mice, Inbred C57BL , Reactive Oxygen Species/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Th17 Cells/cytology , Th17 Cells/immunology , Th17 Cells/metabolismABSTRACT
BACKGROUND: Clubfoot, presenting as a rigid inward and downward turning of the foot, is one of the most common congenital musculoskeletal anomalies. The aetiology of clubfoot is poorly understood and variants in known clubfoot disease genes account for only a small portion of the heritability. METHODS: Exome sequence data were generated from 1190 non-syndromic clubfoot cases and their family members from multiple ethnicities. Ultra-rare variant burden analysis was performed comparing 857 unrelated clubfoot cases with European ancestry with two independent ethnicity-matched control groups (1043 in-house and 56 885 gnomAD controls). Additional variants in prioritised genes were identified in a larger cohort, including probands with non-European ancestry. Segregation analysis was performed in multiplex families when available. RESULTS: Rare variants in 29 genes were enriched in clubfoot cases, including PITX1 (a known clubfoot disease gene), HOXD12, COL12A1, COL9A3 and LMX1B. In addition, rare variants in posterior HOX genes (HOX9-13) were enriched overall in clubfoot cases. In total, variants in these genes were present in 8.4% (100/1190) of clubfoot cases with both European and non-European ancestry. Among these, 3 are de novo and 22 show variable penetrance, including 4 HOXD12 variants that segregate with clubfoot. CONCLUSION: We report HOXD12 as a novel clubfoot disease gene and demonstrate a phenotypic expansion of known disease genes (myopathy gene COL12A1, Ehlers-Danlos syndrome gene COL9A3 and nail-patella syndrome gene LMX1B) to include isolated clubfoot.
Subject(s)
Clubfoot , Exome Sequencing , Homeodomain Proteins , Female , Humans , Male , Clubfoot/genetics , Clubfoot/pathology , Exome/genetics , Genetic Predisposition to Disease , Homeodomain Proteins/genetics , Pedigree , Transcription Factors/geneticsABSTRACT
Localized surface plasmon resonances (LSPRs) can enhance the electromagnetic fields on metallic nanostructures upon light illumination, providing an approach for manipulating light-matter interactions at the sub-wavelength scale. However, currently, there is no thorough investigation of the physical mechanism in the dynamic formation of the strongly coupled LSPRs on sub-5 nm plasmonic cavities at the sub-picosecond scale. In this work, through femtosecond broadband transient absorption spectroscopy, we reveal the dynamic ultrastrong coupling processes in a nanoparticle-in-trench (NPiT) structure containing 2 nm gap cavities, and demonstrate a coherent motional coupling between vibrating AuNPs and the nanogaps. We achieve a maximum Rabi splitting energy of â¼660 meV in the sub-picosecond hot-electron relaxation time scale under the resonant excitation of the nanogap cavity's LSPR, reaching the ultrastrong coupling regime. This leads to a change of global vibration modes for the 2 nm gap cavity, potentially related to the dynamical Casimir effect with nanogap resonators.
ABSTRACT
Senescence of vascular smooth muscle cells (VSMCs) is a key contributor to plaque vulnerability in atherosclerosis (AS), which is affected by endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production. However, the crosstalk between ER stress and ROS production in the pathogenesis of VSMC senescence remains to be elucidated. ER-associated degradation (ERAD) is a complex process that clears unfolded or misfolded proteins to maintain ER homeostasis. HRD1 is the major E3 ligase in mammalian ERAD machineries that catalyzes ubiquitin conjugation to the unfolded or misfolded proteins for degradation. Our results showed that HRD1 protein levels were reduced in human AS plaques and aortic roots from ApoE-/- mice fed with high-fat diet (HFD), along with the increased ER stress response. Exposure to cholesterol in VSMCs activated inflammatory signaling and induced senescence, while reduced HRD1 protein expression. CRISPR Cas9-mediated HRD1 knockout (KO) exacerbated cholesterol- and thapsigargin-induced cell senescence. Inhibiting ER stress with 4-PBA (4-Phenylbutyric acid) partially reversed the ROS production and cell senescence induced by HRD1 deficiency in VSMCs, suggesting that ER stress alone could be sufficient to induce ROS production and senescence in VSMCs. Besides, HRD1 deficiency led to mitochondrial dysfunction, and reducing ROS production from impaired mitochondria partly reversed HRD1 deficiency-induced cell senescence. Finally, we showed that the overexpression of HDR1 reversed cholesterol-induced ER stress, ROS production, and cellular senescence in VSMCs. Our findings indicate that HRD1 protects against senescence by maintaining ER homeostasis and mitochondrial functionality. Thus, targeting HRD1 function may help to mitigate VSMC senescence and prevent vascular aging related diseases. TRIAL REGISTRATION: A real-world study based on the discussion of primary and secondary prevention strategies for coronary heart disease, URL:https://www.clinicaltrials.gov, the trial registration number is [2022]-02-121-01.
Subject(s)
Atherosclerosis , Muscle, Smooth, Vascular , Animals , Humans , Mice , Atherosclerosis/metabolism , Cellular Senescence , Endoplasmic Reticulum Stress/physiology , Endoplasmic Reticulum-Associated Degradation , Mammals/metabolism , Muscle, Smooth, Vascular/metabolism , Proteins/metabolism , Reactive Oxygen Species/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Nature uses compact but functionalized biosynthetic fragments as building blocks to generate complex natural products. To leverage this strategy for the discovery of natural products with new scaffolds, we performed genome mining to identify biosynthetic gene clusters (BGCs) in fungi that embed genes that can synthesize targeted fragments. The three-enzyme pathway that biosynthesizes the strained dityrosine cyclophane in the herquline A pathway was used to identify a large number of potential BGCs that may use the cyclophane as a fragment. Characterization of a conserved BGC from fungal strains led to the isolation of octacyclin A, an octacyclic natural product with an unprecedented structure, including two hetero-[3.3.1]bicycles and a combination of fused, bridged, and macrocyclic rings. Biosynthetic steps leading to octacyclin A were fully elucidated using pathway reconstitution and enzymatic assays, unveiling intriguing chemical logic and new enzymatic reactions in building the octacyclic core. Our work demonstrates the potential utility of fragment-guided genome mining in expanding natural product chemical space.
ABSTRACT
Conjugation between three-dimensional (3D) carboranes and the attached substituents is commonly believed to be very weak. In this paper, we report that reducing 1,12-bis(BMes2)-p-carborane (B2pCab) with one electron gives a radical anion with a centrosymmetric semiquinoidal structure. This radical anion shows extensive electron delocalization between the two boron centers over the p-carborane bridge due to the overlap of carborane lowest unoccupied molecular orbital (LUMO) and the BMes2 LUMO. Unlike dianions of other C2B10H12 carboranes, which rearrange to a nido-form, two-electron reduction of B2pCab leads to a rearrangement into a basket-shaped intermediate.
ABSTRACT
BACKGROUND: Cadonilimab is a bispecific antibody that simultaneously targets programmed cell death receptor-1 and cytotoxic T lymphocyte-associated antigen-4. This study aimed to assess the safety and efficacy of cadonilimab plus anlotinib for the first-line treatment of advanced non-small cell lung cancer (NSCLC) without sensitizing EGFR/ALK/ROS1 mutations. METHODS: Patients received cadonilimab 15 mg/kg and 10 mg/kg every three weeks (Q3W) plus anlotinib at doses of 10 or 12 mg once daily for two weeks on a one-week-off schedule. The primary endpoints included safety and objective response rate (ORR). RESULTS: Sixty-nine treatment-naïve patients received cadonilimab 15 mg/kg Q3W combination (n = 49) and 10 mg/kg Q3W combination (n = 20). Treatment-related adverse events (TRAEs) were reported in 48 (98.0%) and 19 (95.0%) patients, with grade ≥3 TRAEs occurring in 29 (59.2%) and five (25.0%) patients, respectively. TRAEs leading to cadonilimab discontinuation occurred in eight (16.3%) and one (5.0%) patients in the cadonilimab 15 mg/kg Q3W and 10 mg/kg Q3W dosing groups. The confirmed ORRs were 51.0% (25/49) and 60.0% (12/20) accordingly. CONCLUSIONS: Cadonilimab 10 mg/kg Q3W plus anlotinib showed manageable safety and promising efficacy as a first-line chemo-free treatment for advanced NSCLC. GOV IDENTIFIER: NCT04646330.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Indoles , Lung Neoplasms , Quinolines , Humans , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , CTLA-4 Antigen , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Programmed Cell Death 1 Receptor/therapeutic use , Protein-Tyrosine Kinases , Proto-Oncogene ProteinsABSTRACT
Transportin-2 (TNPO2) mediates multiple pathways including non-classical nucleocytoplasmic shuttling of >60 cargoes, such as developmental and neuronal proteins. We identified 15 individuals carrying de novo coding variants in TNPO2 who presented with global developmental delay (GDD), dysmorphic features, ophthalmologic abnormalities, and neurological features. To assess the nature of these variants, functional studies were performed in Drosophila. We found that fly dTnpo (orthologous to TNPO2) is expressed in a subset of neurons. dTnpo is critical for neuronal maintenance and function as downregulating dTnpo in mature neurons using RNAi disrupts neuronal activity and survival. Altering the activity and expression of dTnpo using mutant alleles or RNAi causes developmental defects, including eye and wing deformities and lethality. These effects are dosage dependent as more severe phenotypes are associated with stronger dTnpo loss. Interestingly, similar phenotypes are observed with dTnpo upregulation and ectopic expression of TNPO2, showing that loss and gain of Transportin activity causes developmental defects. Further, proband-associated variants can cause more or less severe developmental abnormalities compared to wild-type TNPO2 when ectopically expressed. The impact of the variants tested seems to correlate with their position within the protein. Specifically, those that fall within the RAN binding domain cause more severe toxicity and those in the acidic loop are less toxic. Variants within the cargo binding domain show tissue-dependent effects. In summary, dTnpo is an essential gene in flies during development and in neurons. Further, proband-associated de novo variants within TNPO2 disrupt the function of the encoded protein. Hence, TNPO2 variants are causative for neurodevelopmental abnormalities.