ABSTRACT
Receptor tyrosine kinase (RTK)-RAS signalling through the downstream mitogen-activated protein kinase (MAPK) cascade regulates cell proliferation and survival. The SHOC2-MRAS-PP1C holophosphatase complex functions as a key regulator of RTK-RAS signalling by removing an inhibitory phosphorylation event on the RAF family of proteins to potentiate MAPK signalling1. SHOC2 forms a ternary complex with MRAS and PP1C, and human germline gain-of-function mutations in this complex result in congenital RASopathy syndromes2-5. However, the structure and assembly of this complex are poorly understood. Here we use cryo-electron microscopy to resolve the structure of the SHOC2-MRAS-PP1C complex. We define the biophysical principles of holoenzyme interactions, elucidate the assembly order of the complex, and systematically interrogate the functional consequence of nearly all of the possible missense variants of SHOC2 through deep mutational scanning. We show that SHOC2 binds PP1C and MRAS through the concave surface of the leucine-rich repeat region and further engages PP1C through the N-terminal disordered region that contains a cryptic RVXF motif. Complex formation is initially mediated by interactions between SHOC2 and PP1C and is stabilized by the binding of GTP-loaded MRAS. These observations explain how mutant versions of SHOC2 in RASopathies and cancer stabilize the interactions of complex members to enhance holophosphatase activity. Together, this integrative structure-function model comprehensively defines key binding interactions within the SHOC2-MRAS-PP1C holophosphatase complex and will inform therapeutic development .
Subject(s)
Cryoelectron Microscopy , Intracellular Signaling Peptides and Proteins , Multiprotein Complexes , Protein Phosphatase 1 , ras Proteins , Amino Acid Motifs , Binding Sites , Guanosine Triphosphate/metabolism , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , MAP Kinase Signaling System , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Multiprotein Complexes/ultrastructure , Mutation, Missense , Phosphorylation , Protein Binding , Protein Phosphatase 1/chemistry , Protein Phosphatase 1/metabolism , Protein Phosphatase 1/ultrastructure , Protein Stability , raf Kinases , ras Proteins/chemistry , ras Proteins/metabolism , ras Proteins/ultrastructureABSTRACT
Abscission is the final stage of cytokinesis whereby the midbody, a thin intercellular bridge, is resolved to separate the daughter cells. Cytokinetic abscission is mediated by the endosomal sorting complex required for transport (ESCRT), a conserved membrane remodelling machinery. The midbody organiser CEP55 recruits early acting ESCRT factors such as ESCRT-I and ALIX (also known as PDCD6IP), which subsequently initiate the formation of ESCRT-III polymers that sever the midbody. We now identify UMAD1 as an ESCRT-I subunit that facilitates abscission. UMAD1 selectively associates with VPS37C and VPS37B, supporting the formation of cytokinesis-specific ESCRT-I assemblies. TSG101 recruits UMAD1 to the site of midbody abscission, to stabilise the CEP55-ESCRT-I interaction. We further demonstrate that the UMAD1-ESCRT-I interaction facilitates the final step of cytokinesis. Paradoxically, UMAD1 and ALIX co-depletion has synergistic effects on abscission, whereas ESCRT-III recruitment to the midbody is not inhibited. Importantly, we find that both UMAD1 and ALIX are required for the dynamic exchange of ESCRT-III subunits at the midbody. Therefore, UMAD1 reveals a key functional connection between ESCRT-I and ESCRT-III that is required for cytokinesis.
Subject(s)
Cytokinesis , Endosomal Sorting Complexes Required for Transport , Endosomal Sorting Complexes Required for Transport/genetics , Cell Cycle ProteinsABSTRACT
Regulatory T (Treg) cells are important in maintaining self-tolerance and immune homeostasis. The Treg cell transcription factor Foxp3 works in concert with other co-regulatory molecules, including Eos, to determine the transcriptional signature and characteristic suppressive phenotype of Treg cells. Here, we report that the inflammatory cytokine interleukin-6 (IL-6) actively repressed Eos expression through microRNA-17 (miR-17). miR-17 expression increased in Treg cells in the presence of IL-6, and its expression negatively correlated with that of Eos. Treg cell suppressive activity was diminished upon overexpression of miR-17 in vitro and in vivo, which was mitigated upon co-expression of an Eos mutant lacking miR-17 target sites. Also, RNAi of miR-17 resulted in enhanced suppressive activity. Ectopic expression of miR-17 imparted effector-T-cell-like characteristics to Treg cells via the de-repression of genes encoding effector cytokines. Thus, miR-17 provides a potent layer of Treg cell control through targeting Eos and additional Foxp3 co-regulators.
Subject(s)
Carrier Proteins/metabolism , Colitis/immunology , Interleukin-6/metabolism , MicroRNAs/metabolism , Nerve Tissue Proteins/metabolism , T-Lymphocytes, Regulatory/immunology , Animals , Carrier Proteins/genetics , Cells, Cultured , DNA-Binding Proteins , Disease Models, Animal , Forkhead Transcription Factors/metabolism , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-6/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , MicroRNAs/genetics , Nerve Tissue Proteins/genetics , Self ToleranceABSTRACT
Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United -States. Despite the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies, we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment of db/db mice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion, and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing the progression of diabetic kidney disease.
Subject(s)
Diabetes Mellitus, Experimental , Diabetes Mellitus, Type 2 , Diabetic Nephropathies , Mice , Animals , Diabetic Nephropathies/metabolism , NAD/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 2/metabolism , Mitochondria/metabolism , DNA, Mitochondrial/metabolism , Nucleotidyltransferases/metabolism , Inflammation/metabolism , Interferons/metabolismABSTRACT
Chronic kidney disease progresses through the replacement of functional tissue compartments with fibrosis, a maladaptive repair process. Shifting kidney repair toward a physiologically intact architecture, rather than fibrosis, is key to blocking chronic kidney disease progression. Much research into the mechanisms of fibrosis is performed in rodent models with less attention to the human genetic context. Recently, human induced pluripotent stem cell (iPSC)-derived organoids have shown promise in overcoming the limitation. In this study, we developed a fibrosis model that uses human iPSC-based 3-dimensional renal organoids, in which exogenous transforming growth factor-ß1 (TGF-ß1) induced the production of extracellular matrix. TGF-ß1-treated organoids showed tubulocentric collagen 1α1 production by regulating downstream transcriptional regulators, Farnesoid X receptor, phosphorylated mothers against decapentaplegic homolog 3 (p-SMAD3), and transcriptional coactivator with PDZ-binding motif (TAZ). Increased nuclear TAZ expression was confirmed in the tubular epithelium in human kidney biopsies with tubular injury and early fibrosis. A dual bile acid receptor agonist (INT-767) increased Farnesoid X receptor and reduced p-SMAD3 and TAZ, attenuating TGF-ß1-induced fibrosis in kidney organoids. Finally, we show that TAZ interacted with TEA-domain transcription factors and p-SMAD3 with TAZ and TEA-domain transcription factor 4 coregulating collagen 1α1 gene transcription. In summary, we establish a novel, readily manipulable fibrogenesis model and posit a role for bile acid receptor agonism early in renal parenchymal fibrosis.
Subject(s)
Fibrosis , Induced Pluripotent Stem Cells , Kidney , Organoids , Transforming Growth Factor beta1 , Humans , Organoids/metabolism , Organoids/drug effects , Induced Pluripotent Stem Cells/metabolism , Transforming Growth Factor beta1/metabolism , Kidney/metabolism , Kidney/pathologyABSTRACT
BACKGROUND: Clinical trials of the KRAS inhibitors adagrasib and sotorasib have shown promising activity in cancers harboring KRAS glycine-to-cysteine amino acid substitutions at codon 12 (KRASG12C). The mechanisms of acquired resistance to these therapies are currently unknown. METHODS: Among patients with KRASG12C -mutant cancers treated with adagrasib monotherapy, we performed genomic and histologic analyses that compared pretreatment samples with those obtained after the development of resistance. Cell-based experiments were conducted to study mutations that confer resistance to KRASG12C inhibitors. RESULTS: A total of 38 patients were included in this study: 27 with non-small-cell lung cancer, 10 with colorectal cancer, and 1 with appendiceal cancer. Putative mechanisms of resistance to adagrasib were detected in 17 patients (45% of the cohort), of whom 7 (18% of the cohort) had multiple coincident mechanisms. Acquired KRAS alterations included G12D/R/V/W, G13D, Q61H, R68S, H95D/Q/R, Y96C, and high-level amplification of the KRASG12C allele. Acquired bypass mechanisms of resistance included MET amplification; activating mutations in NRAS, BRAF, MAP2K1, and RET; oncogenic fusions involving ALK, RET, BRAF, RAF1, and FGFR3; and loss-of-function mutations in NF1 and PTEN. In two of nine patients with lung adenocarcinoma for whom paired tissue-biopsy samples were available, histologic transformation to squamous-cell carcinoma was observed without identification of any other resistance mechanisms. Using an in vitro deep mutational scanning screen, we systematically defined the landscape of KRAS mutations that confer resistance to KRASG12C inhibitors. CONCLUSIONS: Diverse genomic and histologic mechanisms impart resistance to covalent KRASG12C inhibitors, and new therapeutic strategies are required to delay and overcome this drug resistance in patients with cancer. (Funded by Mirati Therapeutics and others; ClinicalTrials.gov number, NCT03785249.).
Subject(s)
Acetonitriles/therapeutic use , Carcinoma, Non-Small-Cell Lung/drug therapy , Colorectal Neoplasms/drug therapy , Drug Resistance, Neoplasm/genetics , Lung Neoplasms/drug therapy , Mutation , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/genetics , Pyrimidines/therapeutic use , Appendiceal Neoplasms/drug therapy , Appendiceal Neoplasms/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Colorectal Neoplasms/genetics , Humans , Lung Neoplasms/genetics , Protein Conformation , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/ultrastructure , Pyridines/therapeutic useABSTRACT
Microwave absorbers with high efficiency and mechanical robustness are urgently desired to cope with more complex and harsh application scenarios. However, manipulating the trade-off between microwave absorption performance and mechanical properties is seldom realized in microwave absorbers. Here, a chemistry-tailored charge dynamic engineering strategy is proposed for sparking hetero-interfacial polarization and thus coordinating microwave attenuation ability with the interfacial bonding, endowing polymer-based composites with microwave absorption efficiency and mechanical toughness. The absorber designed by this new conceptual approach exhibits remarkable Ku-band microwave absorption efficiency (-55.3 dB at a thickness of 1.5 mm) and satisfactory effective absorption bandwidth (5.0 GHz) as well as desirable interfacial shear strength (97.5 MPa). The calculated differential charge density depicts the uneven distribution of space charge and the intense hetero-interfacial polarization, clarifying the structure-performance relationship from a theoretical perspective. This work breaks through traditional single performance-oriented design methods and ushers a new direction for next-generation microwave absorbers.
ABSTRACT
Signaling desensitization is key to limiting signal transduction duration and intensity. Signal transducer and activator of transcription 1 (STAT1) can mediate type II interferon (IFNγ)-induced immune responses, which are enhanced and inhibited by STAT1 phosphorylation and sumoylation, respectively. Here, we identified an N-MYC interacting protein, NMI, which can enhance STAT1 phosphorylation and STAT1-mediated IFNγ immune responses by binding and sequestering the E2 SUMO conjugation enzyme, UBC9, and blocking STAT1 sumoylation. NMI facilitates UBC9 nucleus-to-cytoplasm translocation in response to IFNγ, thereby inhibiting STAT1 sumoylation. STAT1 phosphorylation at Y701 and sumoylation at K703 are mutually exclusive modifications that regulate IFNγ-dependent transcriptional responses. NMI could not alter the phosphorylation level of sumoylation-deficient STAT1 after IFNγ treatment. Thus, IFNγ signaling is modulated by NMI through sequestration of UBC9 in the cytoplasm, leading to inhibition of STAT1 sumoylation. Hence, NMI functions as a switch for STAT1 activation/inactivation cycles by modulating an IFNγ-induced desensitization mechanism.
Subject(s)
Interferon-gamma , Sumoylation , Interferon-gamma/metabolism , Signal Transduction , Phosphorylation , STAT1 Transcription Factor/geneticsABSTRACT
A cube-like Zn(II)-Eu(III) nanocluster 1 (molecular sizes: 1.8 × 2.0 × 2.0 nm) was constructed by the use of a new long-chain Schiff base ligand. It shows a ratiometric fluorescence response to levofloxacin (LFX) with high sensitivity and selectivity, which can be expressed as I615 nm/I550 nm = A*[LFX]2 + B*[LFX] + C. It is used to quantitatively detect the LFX concentrations in fetal calf serum (FCS) and tablets sold in pharmacy. Filter paper strips bearing 1 can be used to qualitatively detect LFX by a color change to red under a UV lamp. 1 and its hybrid with sodium alginate (SA), 1@SA, display potential applications in the qualitative detection of LFX in FCS and the medicine. The limit of detection of 1 to LFX is as low as 2.1 × 10-2 nM.
Subject(s)
Alginates , Europium , Levofloxacin , Zinc , Alginates/chemistry , Zinc/chemistry , Zinc/blood , Levofloxacin/blood , Levofloxacin/analysis , Europium/chemistry , Spectrometry, Fluorescence , Animals , Humans , Cattle , Tablets , Fluorescent Dyes/chemistryABSTRACT
Meloxicam (MLX) is a novel nonsteroidal anti-inflammatory drug, but on the other hand, it has become one of the common microcontaminants in surface waters and sewage. Herein, we report the preparation of a ternary-metal Zn(II)-Cd(II)-Eu(III) nanocluster 1 for the response of MLX through the enhancement of lanthanide luminescence. The luminescence sensing behavior of 1 is expressed by the equation I615nm = 3060 × [MLX] + 46,604, which can be used in the quantitative analysis of MLX concentrations in meloxicam dispersible tablets. Filter paper strips bearing 1 can be used to qualitatively detect MLX by a color change to red under a UV lamp. The luminescence response time is no more than five s, and the detection limit is as low as 2.31 × 10-2 nM.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Europium , Meloxicam , Zinc , Meloxicam/analysis , Zinc/chemistry , Zinc/analysis , Europium/chemistry , Anti-Inflammatory Agents, Non-Steroidal/analysis , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Luminescent Measurements , Luminescence , Nanostructures/chemistry , Limit of DetectionABSTRACT
A nine-metal Zn(II)-Eu(III) nanoring 1 with a diameter of about 2.3 nm was constructed by the use of a long-chain Schiff base ligand. It shows a luminescence response to neopterin (Neo) through the enhancement of lanthanide emission with high selectivity and sensitivity, which can be used to quantitatively analyze the concentrations of Neo in fetal calf serum and urine. The luminescence sensing of 1 to Neo is temperature-dependent, and it displays more obvious response behavior at lower temperatures. Filter paper strips bearing 1 can be used to qualitatively detect Neo by the color change from chartreuse to red under a UV lamp. The limit of detection is as low as 3.77 × 10-2 nM.
Subject(s)
Europium , Nanostructures , Neopterin , Temperature , Zinc , Zinc/chemistry , Zinc/analysis , Neopterin/analysis , Neopterin/urine , Neopterin/blood , Europium/chemistry , Nanostructures/chemistry , Humans , Luminescence , Luminescent Measurements , Biomarkers/analysis , Biomarkers/blood , Limit of Detection , AnimalsABSTRACT
BACKGROUND: Citrus flower-green tea (CT) is a scented tea processed from green tea (GT) and fresh citrus flower, which is favored by consumers due to its potential health benefits and unique citrus flavor. This study evaluated the quality of CT and revealed the mechanism of its quality formation. RESULTS: The CT had a significant citrus flavor and a good antioxidant activity, and its sensory quality was superior to that of GT. Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the scenting process resulted in a significant increase of alkenes such as ß-pinene, trans-ß-ocimene, α-farnesene, isoterpinolene, and γ-terpinene, as well as a significant decrease of alcohols such as α-terpineol, l-menthol, and linalool in CT in comparison with GT. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the levels of flavonoids (such as neohesperidin, hesperidin, tangeritin, hesperetin 5-O-glucoside, and nobiletin) and alkaloids (such as trigonelline and theobromine) in CT increased significantly after scenting process, while the levels of amino acids (such as valine and l-phenylalanine) and organic acids (such as ascorbic acid) decreased significantly. CONCLUSION: These observations showed that the scenting process promoted the absorption of aroma from citrus flowers by GT and the changes in its non-volatile metabolites, leading to the formation of citrus flavor quality in CT. © 2024 Society of Chemical Industry.
Subject(s)
Citrus , Flowers , Gas Chromatography-Mass Spectrometry , Metabolomics , Solid Phase Microextraction , Taste , Flowers/chemistry , Flowers/metabolism , Citrus/chemistry , Citrus/metabolism , Flavoring Agents/chemistry , Flavoring Agents/metabolism , Humans , Tea/chemistry , Tandem Mass Spectrometry , Odorants/analysis , Antioxidants/analysis , Antioxidants/metabolism , Antioxidants/chemistry , Flavonoids/analysis , Flavonoids/metabolism , Flavonoids/chemistry , Volatile Organic Compounds/chemistry , Volatile Organic Compounds/metabolism , Volatile Organic Compounds/analysisABSTRACT
Oral diseases concern almost every individual and are a serious health risk to the population. The restorative treatment of tooth and jaw defects is an important means to achieve oral function and support the appearance of the contour. Based on the principle of "learning from the nature", Deng Xuliang's group of Peking University School and Hospital of Stomatology has proposed a new concept of "microstructural biomimetic design and tissue adaptation of tooth/jaw materials" to address the worldwide problems of difficulty in treating dentine hypersensitivity, poor prognosis of restoration of tooth defects, and vertical bone augmentation of alveolar bone after tooth loss. The group has broken through the bottleneck of multi-stage biomimetic technology from the design of microscopic features to the enhancement of macroscopic effects, and invented key technologies such as crystalline/amorphous multi-level assembly, ion-transportation blocking, and multi-physical properties of the micro-environment reconstruction, etc. The group also pioneered the cationic-hydrogel desensitizer, digital stump and core integrated restorations, and developed new crown and bridge restorative materials, gradient functionalisation guided tissue regeneration membrane, and electrically responsive alveolar bone augmentation restorative membranes, etc. These products have established new clinical strategies for tooth/jaw defect repair and achieved innovative results. In conclusion, the research results of our group have strongly supported the theoretical improvement of stomatology, developed the technical system of oral hard tissue restoration, innovated the clinical treatment strategy, and led the progress of the stomatology industry.
Subject(s)
Bionics , Dental Restoration, Permanent , Mouth Diseases , HumansABSTRACT
Bladder cancer remains the 10th most common cancer worldwide. In recent years, metformin has been found to have potential anti-bladder cancer activity while high concentration of IC50 at millimolar level is needed, which could not be reached by regular oral administration route. Thus, higher efficient agent is urgently demanded for clinically treating bladder cancer. Here, by conjugating artesunate to metformin, a novel artesunate-metformin dimer triazine derivative AM2 was designed and synthesized. The inhibitory effect of AM2 on bladder cancer cell line T24 and the mechanism underlying was determined. Anti-tumor activity of AM2 was assessed by MTT, cloning formation and wound healing assays. Decreasing effect of AM2 on lipogenesis was determined by oil red O staining. The protein expressions of Clusterin, SREBP1 and FASN in T24 cells were evaluated by Western blotting. The results show that AM2 significantly inhibited cell proliferation and migration at micromolar level, much higher than parental metformin. AM2 reduced lipogenesis and down-regulated the expressions of Clusterin, SREBP1 and FASN. These results suggest that AM2 inhibits the growth of bladder cancer cells T24 by inhibiting cellular lipogenesis associated with the Clusterin/SREBP1/FASN signaling pathway.
ABSTRACT
There is an unmet need for easy-to-visualize drug carriers that can deliver therapeutic cargoes deep into solid tumors. Herein, we report the preparation of ultrasmall luminescent imine-based lanthanide nanocages, Eu60 and Tb60 (collectively Ln60 ), designed to encapsulate anticancer chemotherapeutics for tumor therapy. The as-prepared nanocages possess large cavities suitable for the encapsulation of doxorubicin (DOX), yielding DOX@Ln60 nanocages with diameters around 5â nm. DOX@Ln60 are efficiently internalized by breast cancer cells, allowing the cells to be visualized via the intrinsic luminescent property of Ln(III). Once internalized, the acidic intracellular microenvironment promotes imine bond cleavage and the release of the loaded DOX. DOX@Ln60 inhibits DNA replication and triggers tumor cell apoptosis. In a murine triple negative breast cancer (TNBC) model, DOX@Ln60 was found to inhibit tumor growth with negligible side effects on normal tissues. It proved more effective than various controls, including DOX and Ln60 . The present nanocages thus point the way to the development of precise nanomedicines for tumor imaging and therapy.
Subject(s)
Lanthanoid Series Elements , Nanoparticles , Animals , Mice , Cell Line, Tumor , Drug Delivery Systems/methods , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Carriers/chemistry , Imines , Nanoparticles/chemistryABSTRACT
Regulatory T cells (Tregs) are crucial mediators of immune control. The characteristic gene expression and suppressive functions of Tregs depend considerably on the stable expression and activity of the transcription factor FOXP3. Transcriptional regulation of the Foxp3 gene has been studied in depth, but both the expression and function of this factor are also modulated at the protein level. However, the molecular players involved in posttranslational FOXP3 regulation are just beginning to be elucidated. Here, we found that TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to implanted tumors and displayed enhanced anti-tumor immunity. We further determined that FOXP3 undergoes K63-linked ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. In the absence of TRAF6 activity or upon mutation of the ubiquitination site, FOXP3 displayed aberrant, perinuclear accumulation and disrupted regulatory function. Thus, K63-linked ubiquitination by TRAF6 ensures proper localization of FOXP3 and facilitates the transcription factor's gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing regulator that may be targeted in novel tolerance-breaking therapies.
Subject(s)
Colitis/immunology , Forkhead Transcription Factors/physiology , Lysine/metabolism , Melanoma, Experimental/immunology , T-Lymphocytes, Regulatory/immunology , TNF Receptor-Associated Factor 6/physiology , Ubiquitination , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/pathology , Colitis/chemically induced , Colitis/metabolism , Colitis/pathology , Disease Models, Animal , Gene Expression Regulation , Melanoma, Experimental/metabolism , Melanoma, Experimental/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/pathologyABSTRACT
Single-cell DNA methylation sequencing is highly effective for identifying cell subpopulations and constructing epigenetic regulatory networks. Existing methylome analyses require extensive starting materials and are costly, complex, and susceptible to contamination, thereby impeding the development of single-cell methylome technology. In this work, we report digital microfluidics-based single-cell reduced representation bisulfite sequencing (digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, effective, reproducible, and reagent-efficient technique to dissect the single-cell methylome. Using our digital microfluidic chip, we isolated single cells in 15 s and successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. Digital-scRRBS demonstrates a high capacity for distinguishing cell identity and tracking DNA methylation during drug administration. Digital-scRRBS expands the applicability of single-cell methylation methods as a versatile tool for epigenetic analysis of rare cells and populations with high levels of heterogeneity.
Subject(s)
Epigenome , Microfluidics , Cost-Benefit Analysis , DNA Methylation , Cloning, MolecularABSTRACT
BACKGROUND: Flavor contributes to the sensory quality of fruits, including taste and aroma aspects. The quality of foods is related to their flavor-associated compounds. Pear fruits have a fruity sense of smell, and esters are the main contributor of the aroma. Korla pear are well known due to its unique aroma, but the mechanism and genes related to volatile synthesis have not been fully investigated. RESULTS: Flavor-associated compounds, including 18 primary metabolites and 144 volatiles, were characterized in maturity fruits of ten pear cultivars from five species, respectively. Based on the varied metabolites profiles, the cultivars could be grouped into species, respectively, by using orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 14 volatiles were selected as biomarkers to discriminate Korla pear (Pyrus sinkiangensis) from others. Correlation network analysis further revealed the biosynthetic pathways of the compounds in pear cultivars. Furthermore, the volatile profile in Korla pear throughout fruit development was investigated. Aldehydes were the most abundant volatiles, while numerous esters consistently accumulated especially at the maturity stages. Combined with transcriptomic and metabolic analysis, Ps5LOXL, PsADHL, and PsAATL were screened out as the key genes in ester synthesis. CONCLUSION: Pear species can be distinguished by their metabolic profiles. The most diversified volatiles as well as esters was found in Korla pear, in which the enhancement of lipoxygenase pathway may lead to the high level of volatile esters at maturity stages. The study will benefit the fully usage of pear germplasm resources to serve fruit flavor breeding goals.
Subject(s)
Pyrus , Volatile Organic Compounds , Esters/metabolism , Plant Breeding , Fruit , Metabolome , Gene Expression Profiling , Volatile Organic Compounds/metabolismABSTRACT
The study aimed to determine whether ULBP2 was associated with prognosis and immune infiltration in colon cancer (CC) and provided important molecular basis in order to early non-invasive diagnosis and immunotherapy of CC. Using The Cancer Genome Atlas database (TCGA) and ImmPort database, we extracted messenger RNA (mRNA) data of CC and immune-related genes, then we used "limma" package, "survival" package, and Venn overlap analysis to obtain the differentially expressed mRNA (DEmRNA) associated with prognosis and immunity of CC patients. "pROC" package was used to analyze receiver operating characteristics (ROC) of target gene. We used chi-square test and two-class logistics model to identify clinicopathological parameters that correlated with target gene expression. In order to determine the effects of target gene expression and clinicopathological parameters on survival, univariate and multivariate cox regression analyses were performed. We analyzed the related functions and signaling pathways of target gene by enrichment analysis. Finally, the correlation between target gene and tumor immune infiltrating was explored by ssGSEA and spearman correlation analysis. Results showed that ULBP2 was a target gene associated with immunity and prognosis in CC patients. CC patients with higher ULBP2 expression had poor outcomes. In terms of ROC, ULBP2 had an area under the curve (AUC) of 0.984. ULBP2 was associated with T stage, N stage, and pathologic stage of CC patients, and served as an independent predictor of overall survival in CC patients. Functional enrichment analysis revealed ULBP2 was obviously enriched in pathways connected with carcinogenesis and immunosuppression. The expression of ULBP2 was significantly associated with tumor immune cells and immune checkpoints according to ssGSEA and spearman correlation analysis. To conclude, our study suggested that ULBP2 was associated with tumor immunity, and might be a biomarker associated with the diagnosis and prognosis of CC patients, and a potential target of CC immunotherapy.
Subject(s)
Colonic Neoplasms , Humans , Colonic Neoplasms/genetics , Biomarkers , Immunotherapy , Carcinogenesis , Logistic Models , Intercellular Signaling Peptides and Proteins , GPI-Linked ProteinsABSTRACT
The purpose of this study was to identify the role of FEZF1-AS1 in colon cancer and predicted the underlying mechanism. We extracted sequencing data of colon cancer patients from The Cancer Genome Atlas database, identified the differential expression of long noncoding RNA, microRNA, and messenger RNA, constructed a competitive endogenous RNA network, and then analyzed prognosis. Then, we used the enrichment analysis databases for functional analysis. Finally, we studied the FEZF1-AS1/miR-92b-3p/ZIC5 axis. We detected the expression of FEZF1-AS1, miR-92b-3p, and ZIC5 via quantitative reverse transcription-PCR, transfected colon cancer cell RKO with lentivirus and conducted FEZF1-AS1 knockdown, and performed cancer-related functional assays. It indicated that many RNA in the competitive endogenous RNA network, such as ZIC5, were predicted to be related to overall survival of colon cancer patients, and enrichment analysis showed cancer-related signaling pathways, such as PI3K/AKT signaling pathway. The expression of FEZF1-AS1 and ZIC5 was significantly higher and that of miR-92b-3p was lower in the colon cancer than in the normal colon tissues. FEZF1-AS1 promoted the migration, proliferation, as well as invasion of RKO. According to the prediction, FEZF1-AS1 and ZIC5 might competitively bind to miR-92b-3p, leading to the weakening of the inhibitory impact of miR-92b-3p on ZIC5 and increasing expression of ZIC5, thus further activating the PI3K/AKT signaling pathway, which led to the occurrence and development of colon cancer. The study suggested that FEZF1-AS1 might be an effective diagnosis biomarker for colon cancer.