RESUMEN
Beyond its role as the bearer of genetic material, DNA also plays a crucial role in the activation phase of innate immunity. Pathogen recognition receptors (PRRs) and their homologs, pathogen-associated molecular patterns (PAMPs), form the foundation for driving innate immune activation and the induction of immune responses during infection. In the context of DNA viruses or bacterial infections, specific DNA sequences are recognized and bound by DNA sensors, marking the DNA as a PAMP for host recognition and subsequent activation of innate immunity. The primary DNA sensor pathway known to date is cGAS-STING, which can induce Type I interferons (IFN) and innate immune responses against viruses and bacteria. Additionally, the cGAS-STING pathway has been identified to mediate functions in autophagy and senescence. Herein, we introduce methods for using DNA PAMPs as molecular tools to study the role of cGAS-STING and its signaling pathway in regulating innate immunity, both in vitro and in vivo.
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ADN , Inmunidad Innata , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , ADN/metabolismo , ADN/genética , Animales , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , RatonesRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Shuangdan Jiedu Decoction (SJD) is a formula composed of six Chinese herbs with heat-removing and detoxifying, antibacterial, and anti-inflammatory effects, which is clinically used in the therapy of various inflammatory diseases of the lungs including COVID-19, but the therapeutic material basis of its action as well as its molecular mechanism are still unclear. AIM OF THE STUDY: The study attempted to determine the therapeutic effect of SJD on LPS-induced acute lung injury (ALI), as well as to investigate its mechanism of action and assess its therapeutic potential for the cure of inflammation-related diseases in the clinical setting. MATERIALS AND METHODS: We established an ALI model by tracheal drip LPS, and after the administration of SJD, we collected the bronchoalveolar lavage fluid (BALF) and lung tissues of mice and examined the expression of inflammatory factors in them. In addition, we evaluated the effects of SJD on the cyclic guanosine monophosphate-adenosine monophosphate synthase -stimulator of interferon genes (cGAS-STING) and inflammasome by immunoblotting and real-time quantitative polymerase chain reaction (RT-qPCR). RESULTS: We demonstrated that SJD was effective in alleviating LPS-induced ALI by suppressing the levels of pro-inflammatory cytokines in the BALF, improving the level of lung histopathology and the number of neutrophils, as well as decreasing the inflammatory factor-associated gene expression. Importantly, we found that SJD could inhibit multiple stimulus-driven activation of cGAS-STING and inflammasome. Further studies showed that the Chinese herbal medicines in SJD had no influence on the cGAS-STING pathway and inflammasome alone at the formulated dose. By increasing the concentration of these herbs, we observed inhibitory effects on the cGAS-STING pathway and inflammasome, and the effect exerted was maximal when the six herbs were combined, indicating that the synergistic effects among these herbs plays a crucial role in the anti-inflammatory effects of SJD. CONCLUSIONS: Our research demonstrated that SJD has a favorable protective effect against ALI, and its mechanism of effect may be associated with the synergistic effect exerted between six Chinese medicines to inhibit the cGAS-STING and inflammasome abnormal activation. These results are favorable for the wide application of SJD in the clinic as well as for the development of drugs for ALI from herbal formulas.
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Lesión Pulmonar Aguda , Medicamentos Herbarios Chinos , Inflamasomas , Lipopolisacáridos , Proteínas de la Membrana , Nucleotidiltransferasas , Transducción de Señal , Animales , Lesión Pulmonar Aguda/tratamiento farmacológico , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/metabolismo , Lipopolisacáridos/toxicidad , Medicamentos Herbarios Chinos/farmacología , Medicamentos Herbarios Chinos/uso terapéutico , Nucleotidiltransferasas/metabolismo , Inflamasomas/metabolismo , Inflamasomas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Endogámicos C57BL , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Modelos Animales de Enfermedad , Pulmón/efectos de los fármacos , Pulmón/patología , Pulmón/metabolismo , Líquido del Lavado Bronquioalveolar/citologíaRESUMEN
Mitophagy selectively eliminates damaged or dysfunctional mitochondria, playing a crucial role in maintaining mitochondrial quality control. However, it remains unclear whether mitophagy can be fully activated and how it evolves after SCI. Our RNA-seq analysis of animal samples from sham and 1, 3, 5, and 7 days post-SCI indicated that mitophagy was indeed inhibited during the acute and subacute early stages. In vitro experiments showed that this inhibition was closely related to excessive production of reactive oxygen species (ROS) and the downregulation of BNIP3. Excessive ROS led to the blockage of mitophagy flux, accompanied by further mitochondrial dysfunction and increased neuronal apoptosis. Fortunately, ligustilide (LIG) was found to have the ability to reverse the oxidative stress-induced downregulation of BNIP3 and enhance mitophagy through BNIP3-LC3 interaction, alleviating mitochondrial dysfunction and ultimately reducing neuronal apoptosis. Further animal experiments demonstrated that LIG alleviated oxidative stress and mitophagy inhibition, rescued neuronal apoptosis, and promoted tissue repair, ultimately leading to improved motor function. In summary, this study elucidated the state of mitophagy inhibition following SCI and its potential mechanisms, and confirmed the effects of LIG-enhanced mitophagy through BNIP3-LC3, providing new therapeutic targets and strategies for repairing SCI.
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4-Butirolactona , Apoptosis , Proteínas de la Membrana , Mitofagia , Neuronas , Estrés Oxidativo , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Neuronas/metabolismo , Traumatismos de la Médula Espinal/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacología , Ratas , Especies Reactivas de Oxígeno/metabolismo , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Asociadas a MicrotúbulosRESUMEN
BACKGROUND/AIMS: Tumor response to radiation is thought to depend on the direct killing of tumor cells. Our laboratory has called this into question. Firstly, we showed that the biology of the host, specifically the endothelial expression of acid sphingomyelinase (ASMase), was critical in determining tumor radiocurability. Secondly, we have shown that the immune system can enhance radiation response by allowing a complete tumor control in hemi-irradiated tumors. In this paper, we focus on the integration of these two findings. METHODS: We used Lewis Lung Carcinoma (LLC) cells, injected in the flank of either: (i) ASMase knockout or (ii) WT of matched background (sv129xBl/6) or (iii) C57Bl/6 mice. Radiation therapy (RT) was delivered to 50% or 100% of the LLC tumor volume. Tumor response, immune infiltration (CD8+ T cells), ICAM-1, and STING activation were measured. Radiotherapy was also combined with methyl-cyclodextrin, to inhibit the ASMase-mediated formation of ceramide-enriched lipid rafts. RESULTS: We recapitulated our previous finding, namely that tumor hemi-irradiation was sufficient for tumor control in the LLC/C57Bl/6 model. However, in ASMase KO mice hemi-irradiation was ineffective. Likewise, pharmacological inhibition of ASMase significantly reduced the tumor response to hemi-irradiation. Further, we demonstrated elevated ICAM-1 expression, increased levels of CD8+ T cells, ICAM-1, and STING activation in tumors growing in C57Bl/6 mice, as well as the ASMase WT strain. However, no such changes were seen in tumors growing in ASMase KO mice. CONCLUSION: ASMase and ceramide generation are necessary to mediate a radiation-induced anti-tumor immune response via STING activation.
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Linfocitos T CD8-positivos , Carcinoma Pulmonar de Lewis , Molécula 1 de Adhesión Intercelular , Ratones Endogámicos C57BL , Ratones Noqueados , Esfingomielina Fosfodiesterasa , Animales , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielina Fosfodiesterasa/genética , Carcinoma Pulmonar de Lewis/inmunología , Carcinoma Pulmonar de Lewis/patología , Carcinoma Pulmonar de Lewis/radioterapia , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/metabolismo , Ratones , Molécula 1 de Adhesión Intercelular/metabolismo , Molécula 1 de Adhesión Intercelular/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de la radiación , Linfocitos T CD8-positivos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ceramidas/metabolismo , Microdominios de Membrana/metabolismo , Línea Celular TumoralRESUMEN
Background: Cancer-associated fibroblasts (CAFs) are the key components of the immune barrier in liver cancer. Therefore, gaining a deeper understanding of the heterogeneity and intercellular communication of CAFs holds utmost importance in boosting immunotherapy effectiveness and improving clinical outcomes. Methods: A comprehensive analysis by combing single-cell, bulk, and spatial transcriptome profiling with multiplexed immunofluorescence was conducted to unravel the complexities of CAFs in liver cancer. Results: Through an integrated approach involving 235 liver cancer scRNA-seq samples encompassing over 1.2 million cells, we found that CAFs were particularly increased in hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). FAP + fibroblasts were identified as the dominant subtype of CAFs, and which were mainly involved in extracellular matrix organization and angiogenesis. These CAFs were enriched in the tumor boundary of HCC, but diffusely scattered within ICC. The DAB2 + and SPP1 + tumor-associated macrophages (TAMs) reinforce the function of FAP + CAFs through signals such as TGF-ß, PDGF, and ADM. Notably, the interaction between DAB2 + TAMs and FAP + CAFs promoted the formation of immune barrier and correlated with poorer patient survival, non-response to immunotherapy in HCC. High FAP and DAB2 immunohistochemical scores predicted shorter survival and higher serum AFP concentration in a local clinical cohort of 90 HCC patients. Furthermore, this communication pattern might be applicable to other solid malignancies as well. Conclusions: The interaction between DAB2 + TAMs and FAP + CAFs appears crucial in shaping the immune barrier. Strategies aimed at disrupting this communication or inhibiting the functions of FAP + CAFs could potentially enhance immunotherapy effectiveness and improve clinical outcomes.
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Fibroblastos Asociados al Cáncer , Carcinoma Hepatocelular , Neoplasias Hepáticas , Microambiente Tumoral , Humanos , Neoplasias Hepáticas/terapia , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/inmunología , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Hepatocelular/terapia , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/inmunología , Microambiente Tumoral/inmunología , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Colangiocarcinoma/terapia , Colangiocarcinoma/patología , Colangiocarcinoma/inmunología , Colangiocarcinoma/metabolismo , Inmunoterapia/métodos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Masculino , Femenino , EndopeptidasasRESUMEN
Background: Identification of the unknown pathogenic factor driving atherosclerosis not only enhances the development of disease biomarkers but also facilitates the discovery of new therapeutic targets, thus contributing to the improved management of coronary artery disease (CAD). We aimed to identify causative protein biomarkers in CAD etiology based on proteomics and 2-sample Mendelian randomization (MR) design. Methods: Serum samples from 33 first-onset CAD patients and 31 non-CAD controls were collected and detected using protein array. Differentially expressed analyses were used to identify candidate proteins for causal inference. We used 2-sample MR to detect the causal associations between the candidate proteins and CAD. Network MR was performed to explore whether metabolic risk factors for CAD mediated the risk of identified protein. Vascular expression of candidate protein in situ was also detected. Results: Among the differentially expressed proteins identified utilizing proteomics, we found that circulating Golgi protein 73 (GP73) was causally associated with incident CAD and other atherosclerotic events sharing similar etiology. Network MR approach showed low-density lipoprotein cholesterol and glycated hemoglobin serve as mediators in the causal pathway, transmitting 42.1% and 8.7% effects from GP73 to CAD, respectively. Apart from the circulating form of GP73, both mouse model and human specimens imply that vascular GP73 expression was also upregulated in atherosclerotic lesions and concomitant with markers of macrophage and phenotypic switching of vascular smooth muscle cells (VSMCs). Conclusions: Our study supported GP73 as a biomarker and causative for CAD. GP73 may involve in CAD pathogenesis mainly via dyslipidemia and hyperglycemia, which may enrich the etiological information and suggest future research direction on CAD.
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Biomarcadores , Enfermedad de la Arteria Coronaria , Proteínas de la Membrana , Análisis de la Aleatorización Mendeliana , Proteómica , Humanos , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Ratones , Animales , Proteínas de la Membrana/genética , Proteínas de la Membrana/sangre , Masculino , Femenino , Biomarcadores/sangre , Persona de Mediana Edad , LDL-Colesterol/sangre , Hemoglobina Glucada/análisis , Hemoglobina Glucada/metabolismo , Estudios de Casos y Controles , Aterosclerosis/sangre , Aterosclerosis/genéticaRESUMEN
BACKGROUND: Most primary cutaneous melanomas have pathogenesis driven by ultraviolet exposure and genetic mutations, whereas acral lentiginous melanoma (ALM) and metastatic melanoma are much less, if at all, linked with the former. Thus, we evaluated both ultraviolet related and non-ultraviolet related melanomas. Mutations in the MUC16 and TTN genes commonly occur concurrently in these melanoma patients, but their combined prognostic significance stratified by gender and cancer subtype remains unclear. METHODS: The cBioPortal database was queried for melanoma studies and returned 16 independent studies. Data from 2447 melanoma patients were utilized including those with ALM, cutaneous melanoma (CM), and melanoma of unknown primary (MUP). Patients were grouped based on the presence or absence of MUC16 and TTN mutations. Univariate Cox regression and Student's t-tests were used to analyze hazard ratios and total mutation count comparisons, respectively. RESULTS: TTN mutations, either alone or concurrently with MUC16 mutations, significantly correlated with worse prognosis overall, in both genders, and in CM patients. ALM patients with both mutations had better prognoses than CM patients, while ALM patients with neither mutation had worse prognosis than CM patients. For MUP patients, only MUC16 mutations correlated with worse prognosis. ALM patients with neither MUC16 nor TTN mutations had significantly more total mutations than MUP patients, followed by CM patients. CONCLUSION: TTN mutations are a potential marker of poor prognosis in melanoma, which is amplified in the presence of concurrent MUC16 mutations. ALM patients with neither gene mutations had worse prognosis, suggesting a protective effect of having both MUC16 and TTN mutations. Only MUC16 mutations conferred a worse prognosis for MUP patients. Comprehensive genetic profiling in melanoma patients may facilitate personalized treatment strategies to optimize patient outcomes.
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Conectina , Melanoma , Mutación , Neoplasias Cutáneas , Humanos , Melanoma/genética , Melanoma/mortalidad , Melanoma/patología , Femenino , Masculino , Pronóstico , Estudios Retrospectivos , Persona de Mediana Edad , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/mortalidad , Neoplasias Cutáneas/patología , Conectina/genética , Antígeno Ca-125 , Factores Sexuales , Proteínas de la Membrana/genética , Anciano , Biomarcadores de Tumor/genética , AdultoRESUMEN
Arsenic is a toxic metal-like element widely used in the pesticide, preservative and semiconductor industries. However, accumulation of arsenic through the food chain can cause serious damage to animal and human health. However, the toxic mechanism of arsenic-induced hepatotoxicity in chickens is not clear, and the present study aimed to investigate the potential role of cGAS-STING and NF-κB pathways on inflammatory injury in chicken liver. In this study, 75 white-feathered broilers were divided into a control group, a low-dose arsenic group (4 mg/kg) and a high-dose arsenic group (8 mg/kg) to investigate the toxic effects of arsenic on chicken liver. In this study, we found that pathological changes such as inflammatory cell infiltration and vesicular degeneration occurred in the liver when exposed to ATO. Crucially, exposure to ATO triggered the cGAS-STING pathway and markedly raised the levels of mRNA and protein expression of cGAS, STING, TBK1, and IRF7. The type I interferon response was also triggered. Simultaneously, STING induced the activation of the conventional NF-κB signaling pathway and stimulated the expression of genes associated with inflammation, such as IL-6, TNF-α and IL-1ß. In summary, the induction of inflammatory responses via cGAS-STING and NF-κB signaling pathways under high ATO exposure provides new ideas for further studies on the toxicological mechanisms of arsenic.
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Trióxido de Arsénico , Pollos , Inmunidad Innata , Hígado , FN-kappa B , Nucleotidiltransferasas , Transducción de Señal , Animales , Trióxido de Arsénico/toxicidad , FN-kappa B/metabolismo , Inmunidad Innata/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Hígado/inmunología , Transducción de Señal/efectos de los fármacos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Inflamación/inducido químicamente , Enfermedad Hepática Inducida por Sustancias y Drogas , Proteínas Aviares/metabolismo , Proteínas Aviares/genéticaRESUMEN
BACKGROUND: Genome instability (GI) is a hallmark of esophageal squamous cell carcinoma (ESCC) while factors affecting GI remain unclear. METHODS: Here, we aimed to characterize genomic events representing specific mechanisms of GI based on 201 ESCC samples and validated our findings at the patient, single-cell and cancer cell-line levels, including a newly generated multi-omics dataset of the trial NCT04006041. RESULTS: A two-gene (AHNAK and AHNAK2) mutation signature was identified to define the "AHNAK1/2-mutant" cancer subtype. Single-cell-assisted multi-omics analysis showed that this subtype had a higher neoantigen load, active antigen presentation, and proficient CD8 + T cell infiltrations, which were validated at pan-cancer levels. Mechanistically, AHNAK1/2-mutant ESCC was characterized by impaired response of TGF-ß and the inefficient alternative end-join repair (Alt-EJ) that might promote GI. Knockdown of AHNAK in ESCC cell lines resulted in more Alt-EJ events and increased sensitivities to cisplatin. Furthermore, this two-gene signature accurately predicted better responses to DNA-damaging therapy in various clinical settings (HR ≈ 0.25). The two-gene signature predicted higher pCR rates in ESCCs receiving neoadjuvant immunotherapy-involved treatment. Finally, a molecular classification scheme was built and outperformed established molecular typing models in the prognosis stratification of ESCC patients. CONCLUSION: Our study extended our understanding of the AHNAK family in promoting GI and selecting treatment responders of ESCC.
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Neoplasias Esofágicas , Inmunoterapia , Proteínas de la Membrana , Proteínas de Neoplasias , Factor de Crecimiento Transformador beta , Animales , Femenino , Humanos , Masculino , Ratones , Línea Celular Tumoral , Proteínas del Citoesqueleto , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Neoplasias Esofágicas/inmunología , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/terapia , Carcinoma de Células Escamosas de Esófago/inmunología , Carcinoma de Células Escamosas de Esófago/patología , Inmunoterapia/métodos , Proteínas de la Membrana/genética , Mutación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Pronóstico , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The structural components of the thymus are essential for guiding T cell development, but a thorough spatial view is still absent. Here we develop the TSO-his tool, designed to integrate multimodal data from single-cell and spatial transcriptomics to decipher the intricate structure of human thymus. Specifically, we characterize dynamic changes in cell types and critical markers, identifying ELOVL4 as a mediator of CD4+ T cell positive selection in the cortex. Utilizing the mapping function of TSO-his, we reconstruct thymic spatial architecture at single-cell resolution and recapitulates classical cell types and their essential co-localization for T cell development; additionally, previously unknown co-localization relationships such as that of CD8αα with memory B cells and monocytes are identified. Incorporating VDJ sequencing data, we also delineate distinct intermediate thymocyte states during αß T cell development. Overall, these insights enhance our understanding of thymic biology and may inform therapeutic interventions targeting T cell-mediated immune responses.
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Análisis de la Célula Individual , Timocitos , Timo , Transcriptoma , Humanos , Timocitos/metabolismo , Timocitos/citología , Análisis de la Célula Individual/métodos , Timo/citología , Timo/metabolismo , Timo/inmunología , Perfilación de la Expresión Génica/métodos , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , MultiómicaRESUMEN
G9a is a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9), which is involved in the regulation of gene expression. We had previously reported that G9a is expressed in developing tendons in vivo and in vitro and that G9a-deficient tenocytes show impaired proliferation and differentiation in vitro. In this study, we investigated the functions of G9a in tendon development in vivo by using G9a conditional knockout (G9a cKO) mice. We crossed Sox9Cre/+ mice with G9afl/fl mice to generate G9afl/fl; Sox9Cre/+ mice. The G9a cKO mice showed hypoplastic tendon formation at 3 weeks of age. Bromodeoxyuridine labeling on embryonic day 16.5 (E16.5) revealed decreased cell proliferation in the tenocytes of G9a cKO mice. Immunohistochemical analysis revealed decreased expression levels of G9a and its substrate, H3K9me2, in the vertebral tendons of G9a cKO mice. The tendon tissue of the vertebrae and limbs of G9a cKO mice showed reduced expression of a tendon marker, tenomodulin (Tnmd), and col1a1 genes, suggesting that tenocyte differentiation was suppressed. Overexpression of G9a resulted in enhancement of Tnmd and col1a1 expression in tenocytes in vitro. These results suggest that G9a regulates the proliferation and differentiation of tendon progenitor cells during tendon development. Thus, our results suggest that G9a plays an essential role in tendon development.
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Diferenciación Celular , Proliferación Celular , N-Metiltransferasa de Histona-Lisina , Ratones Noqueados , Tendones , Animales , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Tendones/metabolismo , Tendones/embriología , Ratones , Tenocitos/metabolismo , Histonas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción SOX9/metabolismo , Factor de Transcripción SOX9/genéticaRESUMEN
TMEM106B is an endolysosomal transmembrane protein not only associated with multiple neurological disorders including frontotemporal dementia, Alzheimer's disease, and hypomyelinating leukodystrophy but also potentially involved in COVID-19. Additionally, recent studies have identified amyloid fibrils of C-terminal TMEM106B in both aged healthy and neurodegenerative brains. However, so far little is known about physiological functions of TMEM106B in the endolysosome and how TMEM106B is involved in a wide range of human conditions at molecular levels. Here, we performed lipidomic analysis of the brain of TMEM106B-deficient mice. We found that TMEM106B deficiency significantly decreases levels of two major classes of myelin lipids, galactosylceramide and its sulfated derivative sulfatide. Subsequent co-immunoprecipitation assay showed that TMEM106B physically interacts with galactosylceramidase. We also found that galactosylceramidase activity was significantly increased in TMEM106B-deficient brains. Thus, our results suggest that TMEM106B interacts with galactosylceramidase to regulate myelin lipid metabolism and have implications for TMEM106B-associated diseases.
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Galactosilceramidasa , Metabolismo de los Lípidos , Lisosomas , Proteínas de la Membrana , Ratones Noqueados , Vaina de Mielina , Proteínas del Tejido Nervioso , Animales , Ratones , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Lisosomas/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Vaina de Mielina/metabolismo , Galactosilceramidasa/metabolismo , Galactosilceramidasa/genética , Encéfalo/metabolismo , Ratones Endogámicos C57BL , Sulfoglicoesfingolípidos/metabolismo , Células HEK293RESUMEN
Lidamycin (LDM) has been confirmed to have a strong anti-pancreatic cancer effect and can affect the mitochondrial function of pancreatic cancer cells. Mitofusin-2 (Mfn2) is located in the outer membrane of mitochondria, and Mfn2 is currently believed to play a role in cancer inhibition in pancreatic cancer. In order to explore whether the anti-pancreatic cancer effect of LDM is related to Mfn2-mediated mitophagy, Bioinformatics and in vitro cell experiments are used for experimental research. The experimental results demonstrated that Mfn2 is correlated with mitochondrial autophagy in pancreatic cancer. Lidamycin can increase the expression of Mfn2 in pancreatic cancer and affect the process of EMT, affect the level of reactive oxygen species and mitochondrial membrane potential, and increase the expression of mitochondrial autophagy marker proteins BNIP3L and Beclin1. These results demonstrate that Mfn2 affects mitophagy in pancreatic cancer cells by regulating the expression of Mfn2.
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GTP Fosfohidrolasas , Proteínas de la Membrana , Proteínas Mitocondriales , Mitofagia , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Mitofagia/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Línea Celular Tumoral , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Aminoglicósidos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Beclina-1/metabolismo , Beclina-1/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de TumorRESUMEN
Neuroligin-2 (Nlgn2) is a key synaptic adhesion protein at virtually all GABAergic synapses, which recruits GABAARs by promoting assembly of the postsynaptic gephyrin scaffold. Intriguingly, loss of Nlgn2 differentially affects subsets of GABAergic synapses, indicating that synapse-specific interactors and redundancies define its function, but the nature of these interactions remain poorly understood. Here we investigated how Nlgn2 function in hippocampal area CA1 is modulated by two proposed interaction partners, MDGA1 and MDGA2. We show that loss of MDGA1 expression, but not heterozygous deletion of MDGA2, ameliorates the abnormal cytosolic gephyrin aggregation, the reduction in inhibitory synaptic transmission and the exacerbated anxiety-related behaviour characterizing Nlgn2 knockout (KO) mice. Additionally, combined Nlgn2 and MDGA1 deletion causes an exacerbated layer-specific loss of gephyrin puncta. Given that both Nlgn2 and the MDGA1 have been correlated with many psychiatric disorders, our data support the notion that cytosolic gephyrin aggregation may represent an interesting target for novel therapeutic strategies.
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Proteínas Portadoras , Moléculas de Adhesión Celular Neuronal , Proteínas de la Membrana , Ratones Noqueados , Receptores de GABA-A , Sinapsis , Animales , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Ratones , Proteínas Portadoras/metabolismo , Proteínas Portadoras/genética , Sinapsis/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-A/genética , Citosol/metabolismo , Masculino , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Transmisión Sináptica , Ratones Endogámicos C57BL , Región CA1 Hipocampal/metabolismoRESUMEN
Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8, ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8/9/15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8/15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8/9/15high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8/9/15high patient samples. High ADAM8/9/15 expression correlated with high Ki67 and high ADAM8/15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8/9/15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8/9/15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.
Asunto(s)
Proteínas ADAM , Proliferación Celular , Progresión de la Enfermedad , Proteínas de la Membrana , Mieloma Múltiple , Transducción de Señal , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Mieloma Múltiple/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Masculino , Femenino , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Persona de Mediana Edad , Biomarcadores de Tumor , AncianoRESUMEN
Reactive astrocytes play critical roles in the occurrence of various neurological diseases such as multiple sclerosis. Activation of astrocytes is often accompanied by a glycolysis-dominant metabolic switch. However, the role and molecular mechanism of metabolic reprogramming in activation of astrocytes have not been clarified. Here, we found that PKM2, a rate-limiting enzyme of glycolysis, displayed nuclear translocation in astrocytes of EAE (experimental autoimmune encephalomyelitis) mice, an animal model of multiple sclerosis. Prevention of PKM2 nuclear import by DASA-58 significantly reduced the activation of mice primary astrocytes, which was observed by decreased proliferation, glycolysis and secretion of inflammatory cytokines. Most importantly, we identified the ubiquitination-mediated regulation of PKM2 nuclear import by ubiquitin ligase TRIM21. TRIM21 interacted with PKM2, promoted its nuclear translocation and stimulated its nuclear activity to phosphorylate STAT3, NF-κB and interact with c-myc. Further single-cell RNA sequencing and immunofluorescence staining demonstrated that TRIM21 expression was upregulated in astrocytes of EAE. TRIM21 overexpressing in mice primary astrocytes enhanced PKM2-dependent glycolysis and proliferation, which could be reversed by DASA-58. Moreover, intracerebroventricular injection of a lentiviral vector to knockdown TRIM21 in astrocytes or intraperitoneal injection of TEPP-46, which inhibit the nuclear translocation of PKM2, effectively decreased disease severity, CNS inflammation and demyelination in EAE. Collectively, our study provides novel insights into the pathological function of nuclear glycolytic enzyme PKM2 and ubiquitination-mediated regulatory mechanism that are involved in astrocyte activation. Targeting this axis may be a potential therapeutic strategy for the treatment of astrocyte-involved neurological disease.
Asunto(s)
Astrocitos , Encefalomielitis Autoinmune Experimental , Ribonucleoproteínas , Regulación hacia Arriba , Animales , Astrocitos/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/genética , Ratones , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hormonas Tiroideas/metabolismo , Hormonas Tiroideas/genética , Proteínas de Unión a Hormona Tiroide , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ratones Endogámicos C57BL , Piruvato Quinasa/metabolismo , Piruvato Quinasa/genética , Transporte Activo de Núcleo Celular , Femenino , Glucólisis , Ubiquitinación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Núcleo Celular/metabolismoRESUMEN
Rationale: Parkin (an E3 ubiquitin protein ligase) is an important regulator of mitophagy. However, the role of Parkin in viral myocarditis (VMC) remains unclear. Methods: Coxsackievirus B3 (CVB3) infection was induced in mice to create VMC. Cardiac function and inflammatory response were evaluated by echocardiography, histological assessment, and molecular analyses. AAV9 (adeno-associated virus 9), transmission electron microscopy (TEM) and western blotting were used to investigate the mechanisms by which Parkin regulates mitophagy and cardiac inflammation. Results: Our data indicated that Parkin- and BNIP3 (BCL2 interacting protein 3 like)-mediated mitophagy was activated in VMC mice and neonatal rat cardiac myocytes (NRCMs) infected with CVB3, which blocked autophagic flux by inhibiting autophagosome-lysosome fusion. Parkin silencing aggravated mortality and accelerated the development of cardiac dysfunction in CVB3-treated mice. While silencing of Parkin did not significantly increase inflammatory response through activating NF-κB pathway and production of inflammatory cytokines post-VMC, the mitophagy activity were reduced, which stimulated the accumulation of damaged mitochondria. Moreover, Parkin silencing exacerbated VMC-induced apoptosis. We consistently found that Parkin knockdown disrupted mitophagy activity and inflammatory response in NRCMs. Conclusion: This study elucidated the important role of Parkin in maintaining cardiac function and inflammatory response by regulating mitophagy activity and the NF-κB pathway during acute VMC. Although the functional impact of mitophagy remains unclear, our findings suggest that Parkin silencing may accelerate VMC development.
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Infecciones por Coxsackievirus , Mitofagia , Miocarditis , Miocitos Cardíacos , Ubiquitina-Proteína Ligasas , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Miocarditis/virología , Miocarditis/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Masculino , Ratas , Enterovirus Humano B/fisiología , Apoptosis , Modelos Animales de Enfermedad , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , FN-kappa B/metabolismo , Ratones Endogámicos BALB CRESUMEN
Hearing loss affects around 5% of the global population. Two preliminary studies have described genetic variants in sporadic individuals with hearing loss from Pakistan. Here we extend these studies to determine the spectrum of variants in a cohort of individuals with no previous history of hearing loss. Individuals with hearing loss born to consanguineous couples were identified from special schools. Audiograms were assessed. DNA from participants negative for GJB2 pathogenic variants was subjected to exome sequencing. Data were filtered to include variants with frequencies < 0.01 in the public databases. The effects of the missense variants on respective amino acids were analyzed by using PyMol software. Among the 44 participants, hearing loss was moderate for two individuals; 14 exhibited moderately-severe hearing loss while 25 had a severe degree of hearing loss. Hearing loss was reported to have been progressive in four participants and was currently profound in three participants. Variants were unambiguously identified in 17 genes, of which the majority affected SLC26A4. CDH23, MYO15A and OTOF were other significant contributors. Deleterious variants detected in two genes suggest new associations for hearing loss. Molecular characterization of hearing loss in our cohort revealed high genetic heterogeneity with a 75% diagnostic rate.
Asunto(s)
Secuenciación del Exoma , Pérdida Auditiva , Transportadores de Sulfato , Humanos , Masculino , Niño , Femenino , Pérdida Auditiva/genética , Transportadores de Sulfato/genética , Adolescente , Proteínas Relacionadas con las Cadherinas , Cadherinas/genética , Proteínas de la Membrana/genética , Conexina 26/genética , Predisposición Genética a la Enfermedad , Pakistán , Consanguinidad , Conexinas/genética , Mutación Missense , MiosinasRESUMEN
Objective: The objective of the study is to investigate whether quercetin ameliorates Alzheimer's disease (AD)-like pathology in APP/PS1 double transgenic mice and its hypothesized mechanism, contributing to the comprehension of AD pathogenesis. Methods: A total of 30 APP/PS1 transgenic mice were randomized into model group (APP/PS1), quercetin group (APP/PS1+Q), and donepezil hydrochloride group (APP/PS1+DON). Simultaneously, there were 10 C57 mice of the same age served as a control group. Three months posttreatment, the effects of quercetin on AD mice were evaluated using the Morris water maze (MWM) test, Y maze experiment, immunohistochemistry, immunofluorescence, and western blotting. Results: Results from the water maze and Y maze indicated that quercetin significantly improved cognitive impairment in APP/PS1 transgenic AD mice. Additionally, serum enzyme-linked immunosorbent assay (ELISA) results demonstrated that quercetin elevated MDA, superoxide dismutase (SOD), CAT, GSH, acetylcholine (ACh), and acetylcholinesterase (AChE) levels in AD mice. Hematoxylin-eosin (HE) staining, Nissl staining, and hippocampal tissue thioflavine staining revealed that quercetin reduced neuronal damage and Aß protein accumulation in AD mice. Western blot validated protein expression in the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 pathway associated with oxidative stress and apoptosis, confirming quercetin's potential molecular mechanism of enhancing AD mouse cognition. Furthermore, western blot findings indicate that quercetin significantly alters protein expression in the Keap1/Nrf2/HO-1 pathway. Moreover, molecular docking analysis suggests that Keap1, NQO1, HO-1, caspase-3, Bcl-2, and Bax proteins in the Keap1/Nrf2/HO-1 pathway may be potential regulatory targets of quercetin. These findings will provide a molecular basis for quercetin's clinical application in AD treatment. Conclusion: Quercetin can improve cognitive impairment and AD-like pathology in APP/PS1 double transgenic mice, potentially related to quercetin's activation of the Keap1/Nrf2/HO-1 pathway and reduction of cell apoptosis.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Apoptosis , Encéfalo , Disfunción Cognitiva , Modelos Animales de Enfermedad , Hemo-Oxigenasa 1 , Proteína 1 Asociada A ECH Tipo Kelch , Ratones Transgénicos , Factor 2 Relacionado con NF-E2 , Estrés Oxidativo , Quercetina , Animales , Quercetina/farmacología , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/efectos de los fármacos , Factor 2 Relacionado con NF-E2/genética , Estrés Oxidativo/efectos de los fármacos , Ratones , Disfunción Cognitiva/tratamiento farmacológico , Disfunción Cognitiva/metabolismo , Hemo-Oxigenasa 1/metabolismo , Apoptosis/efectos de los fármacos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Transducción de Señal/efectos de los fármacos , Presenilina-1/genética , Presenilina-1/metabolismo , Masculino , Ratones Endogámicos C57BL , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Antioxidantes/farmacología , Antioxidantes/metabolismoRESUMEN
Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) is a major candidate for the blood-stage malaria vaccine. Genetic polymorphisms of global pfama-1suggest that the genetic diversity of the gene can disturb effective vaccine development targeting this antigen. This study was conducted to explore the genetic diversity and gene structure of pfama-1 among P. falciparum isolates collected in the Khyber Pakhtunkhwa (KP) province of Pakistan. A total of 19 full-length pfama-1 sequences were obtained from KP-Pakistan P. falciparum isolates, and genetic polymorphism and natural selection were investigated. KP-Pakistan pfama-1 exhibited genetic diversity, wherein 58 amino acid changes were identified, most of which were located in ectodomains, and domains I, II, and III. The amino acid changes commonly found in the ectodomain of global pfama-1 were also detected in KP-Pakistan pfama-1. Interestingly, 13 novel amino acid changes not reported in the global population were identified in KP-Pakistan pfama-1. KP-Pakistan pfama-1 shared similar levels of genetic diversity with global pfama-1. Evidence of natural selection and recombination events were also detected in KP-Pakistan pfama-1.