RESUMO
Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Metástase Neoplásica , Neoplasias da Próstata/patologia , Microambiente Tumoral , Animais , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Proliferação de Células , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.
Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Fusão de Membrana/fisiologia , Actinas/metabolismo , Animais , Cálcio/metabolismo , Bovinos , Membrana Celular/química , Células Cromafins/citologia , Células Cromafins/metabolismo , Dinaminas/metabolismo , Estimulação Elétrica , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Masculino , Microscopia Confocal , Modelos Biológicos , Técnicas de Patch-Clamp , Vesículas Secretórias/fisiologiaRESUMO
The gastrointestinal tract is known as the largest endocrine organ that encounters and integrates various immune stimulations and neuronal responses due to constant environmental challenges. Enterochromaffin (EC) cells, which function as chemosensors on the gut epithelium, are known to translate environmental cues into serotonin (5-HT) production, contributing to intestinal physiology. However, how immune signals participate in gut sensation and neuroendocrine response remains unclear. Interleukin-33 (IL-33) acts as an alarmin cytokine by alerting the system of potential environmental stresses. We here demonstrate that IL-33 induced instantaneous peristaltic movement and facilitated Trichuris muris expulsion. We found that IL-33 could be sensed by EC cells, inducing release of 5-HT. IL-33-mediated 5-HT release activated enteric neurons, subsequently promoting gut motility. Mechanistically, IL-33 triggered calcium influx via a non-canonical signaling pathway specifically in EC cells to induce 5-HT secretion. Our data establish an immune-neuroendocrine axis in calibrating rapid 5-HT release for intestinal homeostasis.
Assuntos
Células Enterocromafins/fisiologia , Interleucina-33/metabolismo , Intestinos/fisiologia , Neurônios/fisiologia , Serotonina/metabolismo , Tricuríase/imunologia , Trichuris/fisiologia , Animais , Sinalização do Cálcio , Homeostase , Interleucina-33/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroimunomodulação , PeristaltismoRESUMO
ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.
Assuntos
Bacillus subtilis/citologia , Citidina Trifosfato/metabolismo , Pirofosfatases/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Divisão Celular/genética , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Cromossomos Bacterianos/genética , Citidina Trifosfato/fisiologia , Proteínas do Citoesqueleto/genética , Pirofosfatases/fisiologiaRESUMO
Single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) data provided new insights into the understanding of epigenetic heterogeneity and transcriptional regulation. With the increasing abundance of dataset resources, there is an urgent need to extract more useful information through high-quality data analysis methods specifically designed for scATAC-seq. However, analyzing scATAC-seq data poses challenges due to its near binarization, high sparsity and ultra-high dimensionality properties. Here, we proposed a novel network diffusion-based computational method to comprehensively analyze scATAC-seq data, named Single-Cell ATAC-seq Analysis via Network Refinement with Peaks Location Information (SCARP). SCARP formulates the Network Refinement diffusion method under the graph theory framework to aggregate information from different network orders, effectively compensating for missing signals in the scATAC-seq data. By incorporating distance information between adjacent peaks on the genome, SCARP also contributes to depicting the co-accessibility of peaks. These two innovations empower SCARP to obtain lower-dimensional representations for both cells and peaks more effectively. We have demonstrated through sufficient experiments that SCARP facilitated superior analyses of scATAC-seq data. Specifically, SCARP exhibited outstanding cell clustering performance, enabling better elucidation of cell heterogeneity and the discovery of new biologically significant cell subpopulations. Additionally, SCARP was also instrumental in portraying co-accessibility relationships of accessible regions and providing new insight into transcriptional regulation. Consequently, SCARP identified genes that were involved in key Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to diseases and predicted reliable cis-regulatory interactions. To sum up, our studies suggested that SCARP is a promising tool to comprehensively analyze the scATAC-seq data.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Cromatina , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Cromatina/genética , Genoma , Epigenômica , Análise de DadosRESUMO
As sessile organisms, plants experience variable environments and encounter diverse stresses during their growth and development. Adventitious rooting, orchestrated by multiple coordinated signaling pathways, represents an adaptive strategy evolved by plants to adapt to cope with changing environmental conditions. This study uncovered the role of the miR159a-PeMYB33 module in the formation of adventitious roots (ARs) synergistically with abscisic acid (ABA) signaling in poplar. Overexpression of miR159a increased the number of ARs and plant height while reducing sensitivity to ABA in transgenic plants. In contrast, inhibition of miR159a (using Short Tandem Target Mimic) or overexpression of PeMYB33 decreased the number of ARs in transgenic plants. Additionally, miR159a targets and cleaves transcripts of PeMYB33 using degradome analysis, which was further confirmed by a transient expression experiment of poplar protoplast. We show the miR159a-PeMYB33 module controls ARs development in poplar through ABA signaling. In particular, we demonstrated that miR159a promotes the expression of genes in the ABA signaling pathway. The findings from this study shed light on the intricate regulatory mechanisms governing the development of ARs in poplar plants. The miR159a-PeMYB33 module, in conjunction with ABA signaling, plays a crucial role in modulating AR formation and subsequent plant growth.
Assuntos
Ácido Abscísico , Regulação da Expressão Gênica de Plantas , MicroRNAs , Proteínas de Plantas , Raízes de Plantas , Plantas Geneticamente Modificadas , Populus , Transdução de Sinais , Ácido Abscísico/metabolismo , Populus/genética , Populus/crescimento & desenvolvimento , Populus/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Chromatin in eukaryotes folds into a complex three-dimensional (3D) structure that is essential for controlling gene expression and cellular function and is dynamically regulated in biological processes. Studies on plant phosphorus signaling have concentrated on single genes and gene interactions. It is critical to expand the existing signaling pathway in terms of its 3D structure. In this study, low-Pi treatment led to greater chromatin volume. Furthermore, low-Pi stress increased the insulation score and the number of TAD-like domains, but the effects on the A/B compartment were not obvious. The methylation levels of target sites (hereafter as RdDM levels) peaked at specific TAD-like boundaries, whereas RdDM peak levels at conserved TAD-like boundaries shifted and decreased sharply. The distribution pattern of RdDM sites originating from the Helitron transposons matched that of genome-wide RdDM sites near TAD-like boundaries. RdDM pathway genes were upregulated in the middle or early stages and downregulated in the later stages under low-Pi conditions. The RdDM pathway mutant ddm1a showed increased tolerance to low-Pi stress, with shortened and thickened roots contributing to higher Pi uptake from the shallow soil layer. ChIP-seq results revealed that ZmDDM1A could bind to Pi- and root development-related genes. Strong associations were found between interacting genes in significantly different chromatin-interaction regions and root traits. These findings not only expand the mechanisms by which plants respond to low-Pi stress through the RdDM pathway but also offer a crucial framework for the analysis of biological issues using 3D genomics.
Assuntos
Cromatina , Zea mays , Cromatina/genética , Zea mays/genética , Metilação de DNA , Montagem e Desmontagem da Cromatina/genética , Inativação Gênica , Regulação da Expressão Gênica de PlantasRESUMO
Fertilization is a fundamental process of development, and the blocking mechanisms act at the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, permeating and fusing after fertilization. In clinical practice, some couples undergoing recurrent IVF failures that mature oocytes had abnormal fertilization for unknown reason. Ovastacin encoded by ASTL cleave the ZP protein ZP2 and play a key role in preventing polyspermy. Here, we identified bi-allelic variants in ASTL that are mainly characterized by fertilization problems in humans. All four independent affected individuals had bi-allelic frameshift variants or predicted damaging missense variants, which follow a Mendelian recessive inheritance pattern. The frameshift variants significantly decreased the quantity of ASTL protein in vitro. And all missense variants affected the enzymatic activity that cleaves ZP2 in mouse egg in vitro. Three knock-in female mice (corresponding to three missense variants in patients) all show subfertility due to low embryo developmental potential. This work presents strong evidence that pathogenic variants in ASTL cause female infertility and provides a new genetic marker for the diagnosis of fertilization problems.
Assuntos
Infertilidade Feminina , Sêmen , Humanos , Masculino , Feminino , Camundongos , Animais , Glicoproteínas da Zona Pelúcida/genética , Glicoproteínas da Zona Pelúcida/metabolismo , Sêmen/metabolismo , Oócitos/metabolismo , Infertilidade Feminina/genética , Fertilização/genética , Metaloproteases/genéticaRESUMO
MOTIVATION: Gene regulatory networks (GRNs) are vital tools for delineating regulatory relationships between transcription factors and their target genes. The boom in computational biology and various biotechnologies has made inferring GRNs from multi-omics data a hot topic. However, when networks are constructed from gene expression data, they often suffer from false-positive problem due to the transitive effects of correlation. The presence of spurious noise edges obscures the real gene interactions, which makes downstream analyses, such as detecting gene function modules and predicting disease-related genes, difficult and inefficient. Therefore, there is an urgent and compelling need to develop network denoising methods to improve the accuracy of GRN inference. RESULTS: In this study, we proposed a novel network denoising method named REverse Network Diffusion On Random walks (RENDOR). RENDOR is designed to enhance the accuracy of GRNs afflicted by indirect effects. RENDOR takes noisy networks as input, models higher-order indirect interactions between genes by transitive closure, eliminates false-positive effects using the inverse network diffusion method, and produces refined networks as output. We conducted a comparative assessment of GRN inference accuracy before and after denoising on simulated networks and real GRNs. Our results emphasized that the network derived from RENDOR more accurately and effectively captures gene interactions. This study demonstrates the significance of removing network indirect noise and highlights the effectiveness of the proposed method in enhancing the signal-to-noise ratio of noisy networks. AVAILABILITY AND IMPLEMENTATION: The R package RENDOR is provided at https://github.com/Wu-Lab/RENDOR and other source code and data are available at https://github.com/Wu-Lab/RENDOR-reproduce.
Assuntos
Algoritmos , Biologia Computacional , Redes Reguladoras de Genes , Biologia Computacional/métodos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Radiation-induced enteritis, a significant concern in abdominal radiation therapy, is associated closely with gut microbiota dysbiosis. The mucus layer plays a pivotal role in preventing the translocation of commensal and pathogenic microbes. Although significant expression of REGγ in intestinal epithelial cells is well established, its role in modulating the mucus layer and gut microbiota remains unknown. The current study revealed notable changes in gut microorganisms and metabolites in irradiated mice lacking REGγ, as compared to wild-type mice. Concomitant with gut microbiota dysbiosis, REGγ deficiency facilitated the infiltration of neutrophils and macrophages, thereby exacerbating intestinal inflammation after irradiation. Furthermore, fluorescence in situ hybridization assays unveiled an augmented proximity of bacteria to intestinal epithelial cells in REGγ knockout mice after irradiation. Mechanistically, deficiency of REGγ led to diminished goblet cell populations and reduced expression of key goblet cell markers, Muc2 and Tff3, observed in both murine models, minigut organoid systems and human intestinal goblet cells, indicating the intrinsic role of REGγ within goblet cells. Interestingly, although administration of broad-spectrum antibiotics did not alter the goblet cell numbers or mucin 2 (MUC2) secretion, it effectively attenuated inflammation levels in the ileum of irradiated REGγ absent mice, bringing them down to the wild-type levels. Collectively, these findings highlight the contribution of REGγ in counteracting radiation-triggered microbial imbalances and cell-autonomous regulation of mucin secretion.
Assuntos
Enterite , Microbioma Gastrointestinal , Células Caliciformes , Homeostase , Camundongos Knockout , Mucina-2 , Complexo de Endopeptidases do Proteassoma , Animais , Humanos , Camundongos , Disbiose/microbiologia , Disbiose/metabolismo , Enterite/microbiologia , Enterite/metabolismo , Enterite/patologia , Células Caliciformes/patologia , Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos Endogâmicos C57BL , Mucina-2/metabolismo , Proteínas Associadas a Pancreatite/metabolismo , Lesões por Radiação/metabolismo , Lesões por Radiação/microbiologia , Lesões por Radiação/patologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Lesões Experimentais por Radiação/microbiologia , Fator Trefoil-3/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/efeitos da radiação , Autoantígenos/genética , Autoantígenos/metabolismo , Autoantígenos/efeitos da radiaçãoRESUMO
Act1 is an essential adaptor in interleukin 17 (IL-17)-mediated signaling and is recruited to the receptor for IL-17 after stimulation with IL-17. Here we found that Act1 was a 'client' protein of the molecular chaperone hsp90. The D10N variant of Act1 (Act1(D10N)) that is linked to susceptibility to psoriasis was defective in its interaction with hsp90, which resulted in a global loss of Act1 function. Act1-deficient mice modeled the mechanistic link between loss of Act1 function and susceptibility to psoriasis. Although Act1 was necessary for IL-17-mediated inflammation, Act1-deficient mice had a hyperactive response of the T(H)17 subset of helper T cells and developed spontaneous IL-22-dependent skin inflammation. In the absence of IL-17 signaling, IL-22 was the main contributor to skin inflammation, which provides a molecular mechanism for the association of Act1(D10N) with psoriasis susceptibility.
Assuntos
Conexina 43/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Fragmentos de Peptídeos/metabolismo , Psoríase/imunologia , Células Th17/imunologia , Animais , Linhagem Celular , Conexina 43/genética , Conexina 43/imunologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Mutação/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Polimorfismo Genético , Ligação Proteica/genética , Ligação Proteica/imunologia , Psoríase/genética , Transdução de SinaisRESUMO
The bone marrow microenvironment (BMM) can regulate leukemia stem cells (LSCs) via secreted factors. Increasing evidence suggests that dissecting the mechanisms by which the BMM maintains LSCs may lead to the development of effective therapies for the eradication of leukemia. Inhibitor of DNA binding 1 (ID1), a key transcriptional regulator in LSCs, previously identified by us, controls cytokine production in the BMM, but the role of ID1 in acute myeloid leukemia (AML) BMM remains obscure. Here, we report that ID1 is highly expressed in the BMM of patients with AML, especially in BM mesenchymal stem cells, and that the high expression of ID1 in the AML BMM is induced by BMP6, secreted from AML cells. Knocking out ID1 in mesenchymal cells significantly suppresses the proliferation of cocultured AML cells. Loss of Id1 in the BMM results in impaired AML progression in AML mouse models. Mechanistically, we found that Id1 deficiency significantly reduces SP1 protein levels in mesenchymal cells cocultured with AML cells. Using ID1-interactome analysis, we found that ID1 interacts with RNF4, an E3 ubiquitin ligase, and causes a decrease in SP1 ubiquitination. Disrupting the ID1-RNF4 interaction via truncation in mesenchymal cells significantly reduces SP1 protein levels and delays AML cell proliferation. We identify that the target of Sp1, Angptl7, is the primary differentially expression protein factor in Id1-deficient BM supernatant fluid to regulate AML progression in mice. Our study highlights the critical role of ID1 in the AML BMM and aids the development of therapeutic strategies for AML.
Assuntos
Proteína 7 Semelhante a Angiopoietina , Proteína 1 Inibidora de Diferenciação , Leucemia Mieloide Aguda , Animais , Camundongos , Proteína 7 Semelhante a Angiopoietina/genética , Proteína 7 Semelhante a Angiopoietina/metabolismo , Medula Óssea/metabolismo , Modelos Animais de Doenças , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Microambiente Tumoral , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismoRESUMO
Many prokaryotic operons encode a processive antitermination (P-AT) system. Transcription complexes associated with an antitermination factor can bypass multiple transcription termination signals regardless of their sequences. However, to avoid compromising transcriptional regulation of downstream regions, the terminator at the end of the operon needs to be resistant to antitermination. So far, no studies on the mechanism of resistance to antitermination have been reported. The recently discovered conAn P-AT system is composed of two components that are encoded at the start of many conjugation operons on plasmids of Gram-positive bacteria. Here we report the identification of a conAn-resistant terminator, named TerR, in the conjugation operon of the Bacillus subtilis plasmid pLS20, re-defining the end of the conjugation operon. We investigated the various characteristics of TerR and show that its extraordinary long stem is the determining feature for resistance to antitermination. This is the first P-AT resistance mechanism to be reported.
Assuntos
Células Procarióticas , Regiões Terminadoras Genéticas , Óperon/genética , Plasmídeos/genética , Fatores de Transcrição , Transcrição Gênica , Células Procarióticas/metabolismoRESUMO
SMC complexes, loaded at ParB-parS sites, are key mediators of chromosome organization in bacteria. ParA/Soj proteins interact with ParB/Spo0J in a pathway involving adenosine triphosphate (ATP)-dependent dimerization and DNA binding, facilitating chromosome segregation in bacteria. In Bacillus subtilis, ParA/Soj also regulates DNA replication initiation and along with ParB/Spo0J is involved in cell cycle changes during endospore formation. The first morphological stage in sporulation is the formation of an elongated chromosome structure called an axial filament. Here, we show that a major redistribution of SMC complexes drives axial filament formation in a process regulated by ParA/Soj. Furthermore, and unexpectedly, this regulation is dependent on monomeric forms of ParA/Soj that cannot bind DNA or hydrolyze ATP. These results reveal additional roles for ParA/Soj proteins in the regulation of SMC dynamics in bacteria and yet further complexity in the web of interactions involving chromosome replication, segregation and organization, controlled by ParAB and SMC.
Assuntos
Bacillus subtilis , Cromossomos Bacterianos , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Segregação de Cromossomos , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos MultiproteicosRESUMO
Protection of the stalled replication fork is crucial for responding to replication stress and minimizing its impact on chromosome instability, thus preventing diseases, including cancer. We found a new component, Abro1, in the protection of stalled replication fork integrity. Abro1 deficiency results in increased chromosome instability, and Abro1-null mice are tumor-prone. We show that Abro1 protects stalled replication fork stability by inhibiting DNA2 nuclease/WRN helicase-mediated degradation of stalled forks. Depletion of RAD51 prevents the DNA2/WRN-dependent degradation of stalled forks in Abro1-deficient cells. This mechanism is distinct from the BRCA2-dependent fork protection pathway, in which stable RAD51 filament formation prevents MRE11-dependent degradation of the newly synthesized DNA at stalled forks. Thus, our data reveal a new aspect of regulated protection of stalled replication forks that involves Abro1.
Assuntos
Replicação do DNA , Instabilidade Genômica , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteases Específicas de Ubiquitina/fisiologia , Animais , Proteína BRCA2/genética , Linhagem Celular , Células Cultivadas , DNA/biossíntese , DNA Helicases/fisiologia , Endodesoxirribonucleases/fisiologia , Proteína Homóloga a MRE11/fisiologia , Camundongos Knockout , Enzimas Multifuncionais/fisiologia , Neoplasias Experimentais/genética , Proteínas Associadas à Matriz Nuclear/genética , Rad51 Recombinase/genética , Estresse Fisiológico , Proteases Específicas de Ubiquitina/genéticaRESUMO
Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.
Assuntos
Fibrose Cística , Lipídeo A , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/imunologia , Humanos , Lipídeo A/metabolismo , Lipídeo A/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Líquido da Lavagem Broncoalveolar/imunologia , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/metabolismoRESUMO
Preimplantation embryonic arrest is an important pathogenesis of female infertility, but little is known about the genetic factors behind this phenotype. MEI4 is an essential protein for DNA double-strand break formation during meiosis, and Mei4 knock-out female mice are viable but sterile, indicating that MEI4 plays a crucial role in reproduction. To date, MEI4 has not been found to be associated with any human reproductive diseases. Here, we identified six compound heterozygous and homozygous MEI4 variants-namely, c.293C > T, p.(Ser98Leu), c.401C > G, p.(Pro134Arg), c.391C > G, p.(Pro131Ala), c.914A > T, p.(Tyr305Phe), c.908C > G, p.(Ala303Gly), and c.899A > T, p.(Gln300Leu)-in four independent families that were responsible for female infertility mainly characterized by preimplantation embryonic arrest. In vitro, we found that these variants reduced the interaction between MEI4 and DNA. In vivo, we generated a knock-in mouse model and demonstrated that female mice were infertile and were characterized by developmental defects during oogenesis. Our findings reveal the important roles of MEI4 in human reproduction and provide a new diagnostic marker for genetic counseling of clinical infertility patients.
RESUMO
Both controllable regulation of the conformational structure of a polypeptide and specific recognition of an amino acid are still arduous challenges. Here, a novel dual-mode (electrochemical and colorimetric) biosensor was built for arginine (Arg) recognition based on a conformation switch, utilizing controllable and synergistic self-assembly of a ferrocene-grafted hexadecapeptide (P16Fc) with gold nanoparticles (AuNPs). Benefiting from the flexibility and unique topological structure of P16Fc formed nanospheres, the assembly and disassembly can undergo a conformation transition induced by Arg through controlling the distance and number of Fc detached from the gold surface, producing on-off electrical signals. Also, they can induce aggregation and dispersion of AuNPs in solution, causing a color change. The mechanism of Arg recognition with polypeptide conformation regulation was well explored by combining microstructure characterizations with molecular mechanics calculations. The electrochemical and colorimetric assays for Arg were successfully established in sensitive and selective manner, not only obtaining a very low detection limit, but also effectively eliminating the interference from other amino acids and overcoming the limitation of AuNP aggregation. Notably, the conformational change-based assay with the peptide regulated by the target will make a powerful tool for the amino acid biosensing and health diagnosis.
Assuntos
Arginina , Técnicas Eletroquímicas , Compostos Ferrosos , Ouro , Nanopartículas Metálicas , Metalocenos , Peptídeos , Arginina/química , Compostos Ferrosos/química , Metalocenos/química , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Técnicas Biossensoriais/métodos , Colorimetria/métodos , Conformação Proteica , Limite de DetecçãoRESUMO
BACKGROUND: Immune cell homeostasis plays a crucial role in cancer research and therapeutic response. While chemotherapy and immunotherapy hold promise in treating osteosarcoma (OS), identifying patients who are likely to respond would significantly improve clinical practices. Necroptosis, a fundamental mechanism mediating chemotherapy and immunotherapy efficacy, offers valuable insights. In this context, subtypes based on necroptosis-related genes have been established to predict the response of OS patients to immunotherapy and chemotherapy. METHODS: We conducted a high-throughput screening test to identify necroptosis-associated genes that regulate the development of osteosarcoma. Subsequently, the ConsensusClusterPlus package was employed to classify OS patients into subtypes, enabling comparisons of prognosis and clinical information between these subtypes. Patients from the TARGET-OS and GSE21257 datasets were stratified into high-risk and low-risk groups, and their prognoses were compared. Additionally, we assessed the accuracy of the Risk Scoring Model in predicting prognosis, identified independent prognostic factors and explored potential chemotherapeutic agents and immunotherapy drugs. RESULTS: Through the intersection of expression profiles from the TARGET-OS and GSE21257 datasets, we have identified a total of 92 genes associated with necroptosis. Based on differences in the expression of these genes, patients were divided into three subtypes, and we investigated the differences in tumor-infiltrating immune cells, immune-related pathways, and prognosis among these subtypes. Our nomogram effectively differentiated subtypes with distinct responses to chemotherapy and immunotherapy. The established signature demonstrated superior prediction ability compared with single clinical indicators. CONCLUSIONS: This pioneering study unveils the prognostic role of necroptosis-related genes in OS patients, providing a promising alternative for prognostic prediction in clinical disease management. Moreover, our findings highlight the significance of immune cell homeostasis in cancer research and therapeutic response, underscoring its relevance in advancing current treatment strategies.
Assuntos
Neoplasias Ósseas , Osteossarcoma , Criança , Humanos , Apoptose/genética , Osteossarcoma/genética , Osteossarcoma/terapia , Imunoterapia , Diferenciação Celular , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/genética , Neoplasias Ósseas/terapiaRESUMO
MAIN CONCLUSION: This study reveals that TRM21 acts as a positive regulator of flavonoid biosynthesis at the translational level in Arabidopsis, impacting both secondary metabolites and genes associated with root hair growth. TRM (TONNEAU1-recruiting motif) superfamily proteins are reported to be involved in microtubule assembly. However, the functions of this protein family are just beginning to be uncovered. Here, we provide metabolomic and genetic evidence that 1 of the 34 TRM members, TRM21, positively regulates the biosynthesis of flavonoids at the translational level in Arabidopsis thaliana. A loss-of-function mutation in TRM21 led to root hair growth defects and stunted plant growth, accompanied by significant alterations in secondary metabolites, particularly a marked reduction in flavonoid content. Interestingly, our study revealed that the transcription levels of genes involved in the flavonoid biosynthesis pathway remained unchanged in the trm21 mutants, but there was a significant downregulation in the translation levels of certain genes [flavanone 3-hydroxylase (F3H), dihydroflavonol-4-reductase (DFR), anthocyanidin reductase (ANR), flavanone 3'-hydroxylase (F3'H), flavonol synthase (FLS), chalcone synthase (CHS)]. Additionally, the translation levels of some genes related to root hair growth [RHO-related GTPases of plant 2 (ROP2), root hair defective 6 (RHD6), root hair defective 2 (RHD2)] were also reduced in the trm21 mutants. Taken together, these results indicate that TRM21 functions as a positive regulator of flavonoid biosynthesis at the translational level in Arabidopsis.