RESUMO
Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors for a wide range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed "DrugMap," an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF-κB1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF-κB1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription-factor activity.
Assuntos
Cisteína , Neoplasias , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Cisteína/metabolismo , Cisteína/química , Ligantes , Melanoma/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , NF-kappa B/química , NF-kappa B/metabolismo , Oxirredução , Transdução de Sinais , Fatores de Transcrição SOXE/química , Fatores de Transcrição SOXE/metabolismoRESUMO
Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.
Assuntos
RNA Helicases DEAD-box/metabolismo , RNA Polimerase I/metabolismo , Precursores de RNA/genética , RNA Longo não Codificante/metabolismo , Sítio Alostérico , Animais , Carcinogênese , Linhagem Celular , Linhagem Celular Tumoral , RNA Helicases DEAD-box/química , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos , Camundongos Nus , Precursores de RNA/metabolismo , Transcrição GênicaRESUMO
VAP (VAPA and VAPB) is an evolutionarily conserved endoplasmic reticulum (ER)-anchored protein that helps generate tethers between the ER and other membranes through which lipids are exchanged across adjacent bilayers. Here, we report that by regulating PI4P levels on endosomes, VAP affects WASH-dependent actin nucleation on these organelles and the function of the retromer, a protein coat responsible for endosome-to-Golgi traffic. VAP is recruited to retromer budding sites on endosomes via an interaction with the retromer SNX2 subunit. Cells lacking VAP accumulate high levels of PI4P, actin comets, and trans-Golgi proteins on endosomes. Such defects are mimicked by downregulation of OSBP, a VAP interactor and PI4P transporter that participates in VAP-dependent ER-endosomes tethers. These results reveal a role of PI4P in retromer-/WASH-dependent budding from endosomes. Collectively, our data show how the ER can control budding dynamics and association with the cytoskeleton of another membrane by direct contacts leading to bilayer lipid modifications.
Assuntos
Endossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Actinas/metabolismo , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Receptores de Esteroides/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Proteínas de Transporte Vesicular/genéticaRESUMO
Protein interactions form a network whose structure drives cellular function and whose organization informs biological inquiry. Using high-throughput affinity-purification mass spectrometry, we identify interacting partners for 2,594 human proteins in HEK293T cells. The resulting network (BioPlex) contains 23,744 interactions among 7,668 proteins with 86% previously undocumented. BioPlex accurately depicts known complexes, attaining 80%-100% coverage for most CORUM complexes. The network readily subdivides into communities that correspond to complexes or clusters of functionally related proteins. More generally, network architecture reflects cellular localization, biological process, and molecular function, enabling functional characterization of thousands of proteins. Network structure also reveals associations among thousands of protein domains, suggesting a basis for examining structurally related proteins. Finally, BioPlex, in combination with other approaches, can be used to reveal interactions of biological or clinical significance. For example, mutations in the membrane protein VAPB implicated in familial amyotrophic lateral sclerosis perturb a defined community of interactors.
Assuntos
Mapas de Interação de Proteínas , Proteômica/métodos , Esclerose Lateral Amiotrófica/genética , Humanos , Espectrometria de Massas , Mapeamento de Interação de Proteínas , Proteínas/química , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
The proper function of the genome relies on spatial organization of DNA, RNA, and proteins, but how transcription contributes to the organization is unclear. Here, we show that condensates induced by transcription inhibition (CITIs) drastically alter genome spatial organization. CITIs are formed by SFPQ, NONO, FUS, and TAF15 in nucleoli upon inhibition of RNA polymerase II (RNAPII). Mechanistically, RNAPII inhibition perturbs ribosomal RNA (rRNA) processing, releases rRNA-processing factors from nucleoli, and enables SFPQ to bind rRNA. While accumulating in CITIs, SFPQ/TAF15 remain associated with active genes and tether active chromatin to nucleoli. In the presence of DNA double-strand breaks (DSBs), the altered chromatin compartmentalization induced by RNAPII inhibition increases gene fusions in CITIs and stimulates the formation of fusion oncogenes. Thus, proper RNAPII transcription and rRNA processing prevent the altered compartmentalization of active chromatin in CITIs, suppressing the generation of gene fusions from DSBs.
Assuntos
Cromatina , Transcrição Gênica , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is the sole enzyme that catalyzes all O-GlcNAcylation reactions intracellularly. Previous investigations have found that OGT levels oscillate during the cell division process. Specifically, OGT abundance is downregulated during mitosis, but the underlying mechanism is lacking. Here we demonstrate that OGT is ubiquitinated by the ubiquitin E3 ligase, anaphase promoting complex/cyclosome (APC/C)-cell division cycle 20 (Cdc20). We show that APC/CCdc20 interacts with OGT through a conserved destruction box (D-box): Arg-351/Leu-354, the abrogation of which stabilizes OGT. As APC/CCdc20-substrate binding is often preceded by a priming ubiquitination event, we also used mass spectrometry and mapped OGT Lys-352 to be a ubiquitination site, which is a prerequisite for OGT association with APC/C subunits. Interestingly, in The Cancer Genome Atlas, R351C is a uterine carcinoma mutant, suggesting that mutations of the D-box are linked with tumorigenesis. Paradoxically, we found that both R351C and the D-box mutants (R351A/L354A) inhibit uterine carcinoma in mouse xenograft models, probably due to impaired cell division and proliferation. In sum, we propose a model where OGT Lys-352 ubiquitination primes its binding with APC/C, and then APC/CCdc20 partners with OGT through the D-box for its mitotic destruction. Our work not only highlights the key mechanism that regulates OGT during the cell cycle, but also reveals the mutual coordination between glycosylation and the cell division machinery.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase , Mitose , N-Acetilglucosaminiltransferases , Ubiquitinação , Humanos , N-Acetilglucosaminiltransferases/metabolismo , N-Acetilglucosaminiltransferases/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Animais , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Camundongos , Proteólise , Células HeLa , Células HEK293 , FemininoRESUMO
Communication between neurons relies on neurotransmission that takes place at synapses. Excitatory synapses are located primarily on dendritic spines that possess diverse morphologies, ranging from elongated filopodia to mushroom-shaped spines. Failure in the proper development of dendritic spines has detrimental consequences on neuronal connectivity, but the molecular mechanism that controls the balance of filopodia and mushroom spines is not well understood. G3BP1 is the key RNA-binding protein that assembles the stress granules in non-neuronal cells to adjust protein synthesis upon exogenous stress. Emerging evidence suggests that the biological significance of G3BP1 extends beyond its role in stress response, especially in the nervous system. However, the mechanism underlying the regulation and function of G3BP1 in neurons remains elusive. Here we found that G3BP1 suppresses protein synthesis and binds to the translation initiation factor eIF4E via its NTF2-like domain. Notably, the over-production of filopodia caused by G3BP1 depletion can be alleviated by blocking the formation of the translation initiation complex. We further found that the interaction of G3BP1 with eIF4E is regulated by arginine methylation. Knockdown of the protein arginine methyltransferase PRMT8 leads to elevated protein synthesis and filopodia production, which is reversed by the expression of methylation-mimetic G3BP1. Our study, therefore, reveals arginine methylation as a key regulatory mechanism of G3BP1 during dendritic spine morphogenesis and identifies eIF4E as a novel downstream target of G3BP1 in neuronal development independent of stress response.
Assuntos
DNA Helicases , Espinhas Dendríticas , Fator de Iniciação 4E em Eucariotos , Neurônios , Arginina/metabolismo , Proteínas de Transporte/metabolismo , Espinhas Dendríticas/metabolismo , DNA Helicases/metabolismo , Hipocampo/metabolismo , Metilação , Neurônios/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Animais , Ratos , Fator de Iniciação 4E em Eucariotos/metabolismoRESUMO
The chimeric antigen receptor (CAR) directs T cells to target and kill specific cancer cells. Despite the success of CAR T therapy in clinics, the intracellular signaling pathways that lead to CAR T cell activation remain unclear. Using CD19 CAR as a model, we report that, similar to the endogenous T cell receptor (TCR), antigen engagement triggers the formation of CAR microclusters that transduce downstream signaling. However, CAR microclusters do not coalesce into a stable central supramolecular activation cluster (cSMAC). Moreover, LAT, an essential scaffold protein for TCR signaling, is not required for microcluster formation, immunological synapse formation, nor actin remodeling following CAR activation. However, CAR T cells still require LAT for an optimal production of the cytokine IL-2. Together, these data show that CAR T cells can bypass LAT for a subset of downstream signaling outputs, thus revealing a rewired signaling pathway as compared to native T cells.
Assuntos
Sinapses Imunológicas/imunologia , Interleucina-2/imunologia , Receptores de Antígenos Quiméricos/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Células HEK293 , Humanos , Sinapses Imunológicas/genética , Interleucina-2/genética , Células Jurkat , Receptores de Antígenos Quiméricos/genética , Transdução de Sinais/genéticaRESUMO
Autoimmune demyelinating diseases can be induced by an immune response against myelin peptides; however, the exact mechanism underlying the development of such diseases remains unclear. In experimental autoimmune encephalomyelitis, we found that the clearance of exogenous myelin antigen at the peak of the primary immune response is key to the pathogenesis of the disease. The generation of effector T cells requires continuous antigen stimulation, whereas redundant antigen traps and exhausts effector T cells in the periphery, which induces resistance to the disease. Moreover, insufficient antigenic stimulation fails to induce disease efficiently owing to insufficient numbers of effector T cells. When myelin antigen is entirely cleared, the number of effector T cells reaches a peak, which facilitates infiltration of more effector T cells into the central nervous system. The peripheral antigen clearance initiates the first wave of effector T cell entry into the central nervous system and induces chronic inflammation. The inflamed central nervous system recruits the second wave of effector T cells that worsen inflammation, resulting in loss of self-tolerance. These findings provide new insights into the mechanism underlying the development of autoimmune demyelinating diseases, which may potentially impact future treatments.
Assuntos
Encefalomielite Autoimune Experimental , Animais , Linfócitos T , Sistema Nervoso Central/patologia , Inflamação , ImunidadeRESUMO
BACKGROUND: Observational studies have suggested a suspected association between gastroesophageal reflux disease (GERD) and respiratory diseases, but the causality remains equivocal. The goal of this study was to evaluate the causal role of GERD in respiratory diseases by employing Mendelian randomization (MR) studies. METHODS: We conducted Mendelian randomization analysis based on summary data of genome-wide association studies (GWASs) and three MR statistical techniques (inverse variance weighted, weighted median and MR-Egger) were employed to assess the probable causal relationship between GERD and the risk of respiratory diseases. Sensitivity analysis was also carried out to ensure more trustworthy results, which involves examining the heterogeneity, pleiotropy and leave-one-SNP-out method. We also identified 33 relevant genes and explored their distribution in 26 normal tissues. RESULTS: In the analysis, for every unit increase in developing GERD, the odds ratio for developing COPD, bronchitis, pneumonia, lung cancer and pulmonary embolism rose by 72% (ORIVW = 1.72, 95% CI 1.50; 1.99), 19% (ORIVW = 1.19, 95% CI 1.11; 1.28), 16% (ORIVW = 1.16, 95% CI 1.07; 1.26), 0. 3% (ORIVW = 1.003, 95% CI 1.0012; 1.0043) and 33% (ORIVW = 1.33, 95% CI 1.12; 1.58), respectively, in comparison with non-GERD cases. In addition, neither heterogeneity nor pleiotropy was found in the study. This study also found that gene expression was higher in the central nervous system and brain tissue than in other normal tissues. CONCLUSIONS: This study provided evidence that people who developed GERD had a higher risk of developing COPD, bronchitis, pneumonia, lung cancer and pulmonary embolism. Our research suggests physicians to give effective treatments for GERD on respiratory diseases. By exploring the gene expression, our study may also help to reveal the role played by the central nervous system and brain tissue in developing respiratory diseases caused by GERD.
Assuntos
Bronquite , Refluxo Gastroesofágico , Neoplasias Pulmonares , Pneumonia , Doença Pulmonar Obstrutiva Crônica , Embolia Pulmonar , Doenças Respiratórias , Humanos , Estudo de Associação Genômica Ampla , Análise da Randomização Mendeliana , Refluxo Gastroesofágico/complicações , Refluxo Gastroesofágico/genética , Doença Pulmonar Obstrutiva Crônica/genéticaRESUMO
BACKGROUND: Prompt and precise differential diagnosis of biliary atresia (BA) among cholestatic patients is of great importance. Matrix metalloproteinase-7 (MMP-7) holds great promise as a diagnostic marker for BA. This study aimed to investigate the accuracy of age-specific serum MMP-7 for discriminating BA from other cholestatic pediatric patients. METHODS: This was a single center diagnostic accuracy and validation study including both retrospective and prospective cohorts. Serum MMP-7 concentrations were measured using an ELISA kit, the trajectory of which with age was investigated in a healthy infants cohort aged 0 to 365 days without hepatobiliary diseases (n = 284). Clinical BA diagnosis was based on intraoperative cholangiography and subsequent histological examinations. The diagnostic accuracy of age-specific cutoffs of serum MMP-7 were assessed in a retrospective cohort of cholestatic patients (n = 318, with 172 BA) and validated in a prospective cohort (n = 687, including 395 BA). RESULTS: The MMP-7 concentration declines non-linearly with age, showing higher levels in healthy neonates as well as higher cutoff value in neonatal cholestasis. The area under the ROC curve (AUROC) was 0.967 (95% confidence interval [CI]: 0.946-0.988) for the retrospective cohort, and the cutoff of 18 ng/mL yielded 93.0% (95%CI: 88.1-96.3%), 93.8% (95%CI: 88.6-97.1%), 94.7% (95%CI: 90.1-97.5%), and 91.9% (95%CI: 86.4-95.8%) for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), respectively. The performance of MMP-7 was successfully validated in the larger prospective cohort, resulting in a diagnostic sensitivity of 95.9% (379/395; 95% CI: 93.5-97.7%), a specificity of 87.3% (255/292; 95% CI: 83.0-90.9%), a PPV of 91.1% (379/416; 95% CI: 87.9-93.7%), and a NPV of 94.1% (255/271; 95% CI: 90.6-96.6%), respectively. Besides, higher cutoff value of 28.1 ng/mL achieved the best sensitivity, specificity, PPV, and NPV for infants aged 0-30 days, which was 86.4% (95% CI: 75.0-94.0%), 95.5% (95% CI: 77.2-99.9%), 98.1% (95% CI: 89.7-100%), and 72.4% (95% CI: 52.8-87.3%), respectively. CONCLUSIONS: The serum MMP-7 is accurate and reliable in differentiating BA from non-BA cholestasis, showing its potential application in the diagnostic algorithm for BA and significant role in the future research regarding pathogenesis of BA.
Assuntos
Atresia Biliar , Metaloproteinase 7 da Matriz , Curva ROC , Humanos , Atresia Biliar/sangue , Atresia Biliar/diagnóstico , Metaloproteinase 7 da Matriz/sangue , Lactente , Masculino , Feminino , Recém-Nascido , Reprodutibilidade dos Testes , Estudos Retrospectivos , Diagnóstico Diferencial , Pré-Escolar , Colestase/sangue , Colestase/diagnóstico , Estudos ProspectivosRESUMO
Variants in voltage-gated sodium channel (VGSC) genes are implicated in seizures, epilepsy, and neurodevelopmental disorders, constituting a significant aspect of hereditary epilepsy in the Chinese population. Through retrospective analysis utilizing next-generation sequencing (NGS), we examined the genotypes and phenotypes of VGSC-related epilepsy cases from a cohort of 691 epilepsy subjects. Our findings revealed that 5.1% of subjects harbored VGSC variants, specifically 22 with SCN1A, 9 with SCN2A, 1 with SCN8A, and 3 with SCN1B variants; no SCN3A variants were detected. Among these, 14 variants were previously reported, while 21 were newly identified. SCN1A variant carriers predominantly presented with Dravet Syndrome (DS) and Genetic Epilepsy with Febrile Seizures Plus (GEFS + ), featuring a heightened sensitivity to fever-induced seizures. Statistically significant disparities emerged between the SCN1A-DS and SCN1A-GEFS+ groups concerning seizure onset and genetic diagnosis age, incidence of status epilepticus, mental retardation, anti-seizure medication (ASM) responsiveness, and familial history. Notably, subjects with SCN1A variants affecting the protein's pore region experienced more frequent cluster seizures. All SCN2A variants were of de novo origin, and 88.9% of individuals with SCN2A variations exhibited cluster seizures. This research reveals a significant association between variations in VGSC-related genes and the clinical phenotype diversity of epilepsy subjects in China, emphasizing the pivotal role of NGS screening in establishing accurate disease diagnoses and guiding the selection of ASM.
Assuntos
Epilepsia , Genótipo , Canal de Sódio Disparado por Voltagem NAV1.1 , Canal de Sódio Disparado por Voltagem NAV1.2 , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Adulto Jovem , China/epidemiologia , População do Leste Asiático/genética , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/epidemiologia , Epilepsia/genética , Epilepsia/epidemiologia , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Canal de Sódio Disparado por Voltagem NAV1.2/genética , Fenótipo , Estudos Retrospectivos , Convulsões Febris/genética , Convulsões Febris/epidemiologiaRESUMO
The complexity of microvascular circulation has led to the development of advanced imaging techniques and biomimetic models. This study developed a multifaceted microfluidic-based microdevice as an in vitro model of microvasculature to replicate important geometric and functional features of in vivo perfusion in mice. The microfluidic device consisted of a microchannel for blood perfusion, mirroring the natural hierarchical branching vascular structures found in mice. Additionally, the device incorporated a steady gradient of oxygen (O2) which diffused through the polydimethylsiloxane (PDMS) layer, allowing for dynamic blood oxygenation. The assembled multi-layered microdevice was accompanied by a dual-modal imaging system that combined laser speckle contrast imaging (LSCI) and intrinsic signal optical imaging (ISOI) to visualize full-field blood flow distributions and blood O2 profiles. By closely reproducing in vivo blood perfusion and oxygenation conditions, this microvasculature model, in conjunction with numerical simulation results, can provide quantitative information on physiologically relevant hemodynamics and key O2 transport parameters that are not directly measurable in traditional animal studies.
Assuntos
Hemodinâmica , Microfluídica , Camundongos , Animais , Oxigênio , MicrovasosRESUMO
BACKGROUND: Although it has been established that elevated blood pressure and its variability worsen outcomes in spontaneous intracerebral hemorrhage, antihypertensives use during the acute phase still lacks robust evidence. A blood pressure-lowering regimen using remifentanil and dexmedetomidine might be a reasonable therapeutic option given their analgesic and antisympathetic effects. The objective of this superiority trial was to validate the efficacy and safety of this blood pressure-lowering strategy that uses remifentanil and dexmedetomidine in patients with acute intracerebral hemorrhage. METHODS: In this multicenter, prospective, single-blinded, superiority randomized controlled trial, patients with intracerebral hemorrhage and systolic blood pressure (SBP) 150 mmHg or greater were randomly allocated to the intervention group (a preset protocol with a standard guideline management using remifentanil and dexmedetomidine) or the control group (standard guideline-based management) to receive blood pressure-lowering treatment. The primary outcome was the SBP control rate (less than 140 mmHg) at 1 h posttreatment initiation. Secondary outcomes included blood pressure variability, neurologic function, and clinical outcomes. RESULTS: A total of 338 patients were allocated to the intervention (n = 167) or control group (n = 171). The SBP control rate at 1 h posttreatment initiation in the intervention group was higher than that in controls (101 of 161, 62.7% vs. 66 of 166, 39.8%; difference, 23.2%; 95% CI, 12.4 to 34.1%; P < 0.001). Analysis of secondary outcomes indicated that patients in the intervention group could effectively reduce agitation while achieving lighter sedation, but no improvement in clinical outcomes was observed. Regarding safety, the incidence of bradycardia and respiratory depression was higher in the intervention group. CONCLUSIONS: Among intracerebral hemorrhage patients with a SBP 150 mmHg or greater, a preset protocol using a remifentanil and dexmedetomidine-based standard guideline management significantly increased the SBP control rate at 1 h posttreatment compared with the standard guideline-based management.
Assuntos
Anti-Hipertensivos , Pressão Sanguínea , Hemorragia Cerebral , Dexmedetomidina , Remifentanil , Humanos , Dexmedetomidina/uso terapêutico , Dexmedetomidina/administração & dosagem , Remifentanil/administração & dosagem , Remifentanil/uso terapêutico , Masculino , Feminino , Estudos Prospectivos , Hemorragia Cerebral/tratamento farmacológico , Idoso , Pessoa de Meia-Idade , Método Simples-Cego , Pressão Sanguínea/efeitos dos fármacos , Anti-Hipertensivos/uso terapêutico , Anti-Hipertensivos/administração & dosagem , Resultado do Tratamento , Hipnóticos e Sedativos/uso terapêuticoRESUMO
CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce "marker-free" genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.
Assuntos
Edição de Genes , Malária , Humanos , Sistemas CRISPR-Cas , Plasmodium falciparum/genética , DNA , Malária/genéticaRESUMO
Sleep deprivation (SD) has been associated with a plethora of severe pathophysiological syndromes, including gut damage, which recently has been elucidated as an outcome of the accumulation of reactive oxygen species (ROS). However, the spatiotemporal analysis conducted in this study has intriguingly shown that specific events cause harmful damage to the gut, particularly to goblet cells, before the accumulation of lethal ROS. Transcriptomic and metabolomic analyses have identified significant enrichment of metabolites related to ferroptosis in mice suffering from SD. Further analysis revealed that melatonin could rescue the ferroptotic damage in mice by suppressing lipid peroxidation associated with ALOX15 signaling. ALOX15 knockout protected the mice from the serious damage caused by SD-associated ferroptosis. These findings suggest that melatonin and ferroptosis could be targets to prevent devastating gut damage in animals exposed to SD. To sum up, this study is the first report that proposes a noncanonical modulation in SD-induced gut damage via ferroptosis with a clearly elucidated mechanism and highlights the active role of melatonin as a potential target to maximally sustain the state during SD.
Assuntos
Ferroptose , Melatonina , Camundongos Knockout , Privação do Sono , Animais , Camundongos , Melatonina/metabolismo , Melatonina/farmacologia , Privação do Sono/metabolismo , Masculino , Espécies Reativas de Oxigênio/metabolismo , Camundongos Endogâmicos C57BL , Peroxidação de Lipídeos , Araquidonato 15-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 12-LipoxigenaseRESUMO
Irisin, a secreted myokine generated by fibronectin type III domain-containing protein 5, has recently shown the potential to alleviate inflammation. Cholecystokinin-octapeptide (CCK-8) is closely associated with the inflammatory factor TNF-α, a central cytokine in inflammatory reactions. However, the interactions between irisin and CCK-8 in regulating TNF-α production and the underlying mechanism have not yet been elucidated. In the present study, irisin treatment inhibited the basal and the CCK-8-induced TNF-α production in vivo. Additionally, neutralizing circulating irisin using an irisin antiserum significantly augmented the CCK-8-induced stimulation of TNF-α levels. Moreover, the incubation of head kidney cells with irisin or CCK-8 has opposite effects on TNF-α secretion. Notably, irisin treatment inhibited basal and CCK-8-stimulated TNF-α release and gene transcription in head kidney cells. Mechanistically, the inhibitory actions of irisin on basal and CCK-8-induced TNF-α production could be negated by co-administered with the selective integrin αVß5 inhibitor cilengitide. In addition, the inhibitory effect of irisin on basal and CCK-8-triggered TNF-α production could be abolished by the inhibition of the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, irisin impeded CCK-8-induced phosphorylation and degradation of IκBα, simultaneously inhibiting NF-κB phosphorylation, preventing its translocation into the nucleus, and suppressing its DNA-binding activity induced by CCK-8. Collectively, these results suggest that the inhibitory effect of irisin on TNF-α production caused by CCK-8 is mediated via the integrin αVß5-NF-κB signaling pathways in tilapia.
Assuntos
Ciclídeos , NF-kappa B , Animais , NF-kappa B/metabolismo , Sincalida/efeitos adversos , Fator de Necrose Tumoral alfa/farmacologia , Fibronectinas/genética , Ciclídeos/metabolismo , Transdução de Sinais , Inflamação/induzido quimicamenteRESUMO
GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.
Assuntos
DNA Helicases , RNA Helicases , Humanos , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas com Motivo de Reconhecimento de RNA , Regiões 5' não Traduzidas , Corpos de Inclusão Intranuclear , Ribossomos , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Eutrophication impacts freshwater ecosystems and biodiversity across the world. While temporal monitoring has shown changes in the nutrient inputs in many areas, how spatial and temporal beta diversity change along the eutrophication gradient under a changing context remains unclear. In this regard, analyses based on time series spanning multiple years are particularly scarce. We sampled benthic macroinvertebrates in 32 sites across three lake habitat types (MACROPHYTE, OPEN WATER, PHYTOPLANKTON) along the eutrophication gradient of Lake Taihu in four seasons from 2007 to 2019. Our purpose was to identify the relative contributions of spatial and temporal dissimilarity (i.e., inter-annual dissimilarity and seasonal dissimilarity) to overall benthic biodiversity. We also examined spatio-temporal patterns in community assembly mechanisms and how associated variation in benthic macroinvertebrate communities responded to nutrient indicators. Results showed that eutrophication caused macroinvertebrate community homogenization both along spatial and temporal gradients. Though spatial variability dominated the variation of species richness, abundance and community dissimilarity, seasons within years dissimilarity, inter-annual dissimilarity and seasonal dissimilarity were much more sensitive to eutrophication. Moreover, eutrophication inhibited a strong environmental control in benthic macroinvertebrate community assembly, including a dominant role of deterministic process in the spatial variation of macroinvertebrate communities and transition from stochastic to deterministic process in the temporal assembly of macroinvertebrate communities along the eutrophication gradient. In addition, some sites in PHYTOPLANKTON habitats showed similar spatial dissimilarity and spatial SES as sites in MACROPHYTE habitats, and the decreased spatial dissimilarity of three habitats implying that lake ecosystem recovery projects have achieved their goal at least to a certain degree.
Assuntos
Ecossistema , Lagos , Monitoramento Ambiental , Biodiversidade , Fitoplâncton , Eutrofização , ChinaRESUMO
Promoting green and low-carbon development has become the consensus of the policymakers and the academic, with green transformation of enterprises being the top priority. This paper adopts the difference-in-difference model to investigate the effect of green credit policy on green transition in China, by utilizing the "Green Credit Guidelines" (2012 Guidelines) policy as a quasi-natural experiment. Using panel data from publicly listed companies in China, an empirical investigation is conducted, we explain the dependent variable from two dimensions: economic performance and environmental performance, leading to the following results. First, the green credit policy affects the economic performance and environmental performance of treated firms positively, and the robust tests confirm the reliability of this primary conclusion. Second, the indirect impact of green credit policy on green transition can be explained through two mediating mechanism channels including internal capacity building and external market attention. In addition, the proposal of "Dual Carbon Targets" makes the impact a slight change. Finally, heterogeneous test also shows that the implementation effect of green credit policy is better in non-state-owned enterprises with high political relevance. These findings are providing valuable insights to promote green transition by designing more effective green credit policies.