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The biofilm-induced "relatively immune-compromised zone" creates an immunosuppressive microenvironment that is a significant contributor to refractory infections in orthopedic endophytes. Consequently, the manipulation of immune cells to co-inhibit or co-activate signaling represents a crucial strategy for the management of biofilm. This study reports the incorporation of Mn2+ into mesoporous dopamine nanoparticles (Mnp) containing the stimulator of interferon genes (STING) pathway activator cGAMP (Mncp), and outer wrapping by M1-like macrophage cell membrane (m-Mncp). The cell membrane enhances the material's targeting ability for biofilm, allowing it to accumulate locally at the infectious focus. Furthermore, m-Mncp mechanically disrupts the biofilm through photothermal therapy and induces antigen exposure through photodynamic therapy-generated reactive oxygen species (ROS). Importantly, the modulation of immunosuppression and immune activation results in the augmentation of antigen-presenting cells (APCs) and the commencement of antigen presentation, thereby inducing biofilm-specific humoral immunity and memory responses. Additionally, this approach effectively suppresses the activation of myeloid-derived suppressor cells (MDSCs) while simultaneously boosting the activity of T cells. Our study showcases the efficacy of utilizing m-Mncp immunotherapy in conjunction with photothermal and photodynamic therapy to effectively mitigate residual and recurrent infections following the extraction of infected implants. As such, this research presents a viable alternative to traditional antibiotic treatments for biofilm that are challenging to manage.
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Biopelículas , Indoles , Proteínas de la Membrana , Polímeros , Biopelículas/efectos de los fármacos , Polímeros/química , Animales , Indoles/química , Indoles/farmacología , Ratones , Proteínas de la Membrana/metabolismo , Nanopartículas/química , Fotoquimioterapia/métodos , Porosidad , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Femenino , Transducción de Señal/efectos de los fármacos , Terapia Fototérmica , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
As an interferon-stimulating factor protein, STING plays a role in the response and downstream liaison in antiviral natural immunity. Upon viral invasion, the immediate response of STING protein leads to a series of changes in downstream proteins, which ultimately leads to an antiviral immune response in the form of proinflammatory cytokines and type I interferons, thus triggering an innate immune response, an adaptive immune response in vivo, and long-term protection of the host. In the field of antiviral natural immunity, it is particularly important to rigorously and sequentially probe the dynamic changes in the antiviral natural immunity connector protein STING caused by the entire anti-inflammatory and anti-pathway mechanism and the differences in upstream and downstream proteins. Traditionally, proteomics technology has been validated by detecting proteins in a 2D platform, for which it is difficult to sensitively identify changes in the nature and abundance of target proteins. With the development of mass spectrometry (MS) technology, MS-based proteomics has made important contributions to characterizing the dynamic changes in the natural immune proteome induced by viral infections. MS analytical techniques have several advantages, such as high throughput, rapidity, sensitivity, accuracy, and automation. The most common techniques for detecting complex proteomes are liquid chromatography (LC) and mass spectrometry (MS). LC-MS (Liquid Chromatography-Mass Spectrometry), which combines the physical separation capability of LC and the mass analysis capability of MS, is a powerful technique mainly used for analyzing the proteome of cells, tissues, and body fluids. To explore the combination of traditional proteomics techniques such as Western blotting, Co-IP (co-Immunoprecipitation), and the latest LC-MS methods to probe the anti-inflammatory pathway and the differential changes in upstream and downstream proteins induced by the antiviral natural immune junction protein STING.
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Inmunidad Innata , Proteómica , Proteómica/métodos , Cromatografía Liquida/métodos , Humanos , Western Blotting/métodos , Espectrometría de Masas/métodos , Inmunoprecipitación/métodos , Animales , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/inmunología , Cromatografía Líquida con Espectrometría de MasasRESUMEN
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
Click chemistry, also known as "link chemistry," is an important molecular connection method that can achieve simple and efficient connections between specific small molecular groups at the molecular level. Click chemistry offers several advantages, including high efficiency, good selectivity, mild conditions, and few side reactions. These features make it a valuable tool for in-depth analysis of various protein posttranslational modifications (PTMs) caused by changes in cell metabolism during viral infection. This chapter considers the palmitoylation, carbonylation, and alkylation of STING and presents detailed information and experimental procedures for measuring PTMs using click chemistry.
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Química Clic , Procesamiento Proteico-Postraduccional , Química Clic/métodos , Humanos , Alquilación , Lipoilación , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Carbonilación ProteicaRESUMEN
Cryo-electron microscopy is a powerful methodology in structural biology and has been broadly used in high-resolution structure determination for challenging samples, which are not readily available for traditional techniques. In particular, the strength of super macro-complexes and the lack of a need for crystals for cryo-EM make this technique feasible for the structural study of complexes involved in antiviral innate immunity. This chapter presents detailed information and experimental procedures of Cryo-EM for determining the structures of the complexes using STING as an example. The procedures included a sample quality check, high-resolution data acquisition, and image processing for Cryo-EM 3D structure determination.
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Microscopía por Crioelectrón , Inmunidad Innata , Microscopía por Crioelectrón/métodos , Humanos , Proteínas de la Membrana/inmunología , Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodosRESUMEN
The immune resistance of tumor microenvironment (TME) causes immune checkpoint blockade therapy inefficient to hepatocellular carcinoma (HCC). Emerging strategies of using chemotherapy regimens to reverse the immune resistance provide the promise for promoting the efficiency of immune checkpoint inhibitors. The induction of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) in tumor cells evokes the adaptive immunity and remodels the immunosuppressive TME. In this study, we report that mitoxantrone (MIT, a chemotherapeutic drug) activates the cGAS-STING signaling pathway of HCC cells. We provide an approach to augment the efficacy of MIT using a signal transducer and activator of transcription 3 (STAT3) inhibitor called napabucasin (NAP). We prepare an aminoethyl anisamide (AEAA)-targeted polyethylene glycol (PEG)-modified poly (lactic-co-glycolic acid) (PLGA)-based nanocarrier for co-delivery of MIT and NAP. The resultant co-nanoformulation can elicit the cGAS-STING-based immune responses to reshape the immunoresistant TME in the mice orthotopically grafted with HCC. Consequently, the resultant co-nanoformulation can promote anti-PD-1 antibody for suppressing HCC development, generating long-term survival, and inhibiting tumor recurrence. This study reveals the potential of MIT to activate the cGAS-STING signaling pathway, and confirms the feasibility of nano co-delivery for MIT and NAP on achieving HCC chemo-immunotherapy.
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Carcinoma Hepatocelular , Inmunoterapia , Neoplasias Hepáticas , Proteínas de la Membrana , Mitoxantrona , Nucleotidiltransferasas , Factor de Transcripción STAT3 , Mitoxantrona/farmacología , Mitoxantrona/uso terapéutico , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/patología , Animales , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Humanos , Nucleotidiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Factor de Transcripción STAT3/metabolismo , Ratones , Inmunoterapia/métodos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Benzofuranos , NaftoquinonasRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fetal neuromuscular disorder characterized by the gradual deterioration of motor neurons. Semen Strychni pulveratum (SSP), a processed version of Semen Strychni (SS) powder, is widely used to treat ALS in China. Vomicine is one of the most primary components of SS. However, their pharmacological effects and mechanisms for ALS remain elusive. AIM OF THE STUDY: This study aimed to evaluate the neuroprotective and anti-neuroinflammatory effects of SSP and vomicine, as well as to explore their protective roles in ALS and the underlying mechanisms. MATERIALS AND METHODS: In vivo, 8-week-old hSOD1-WT mice and hSOD1-G93A mice were orally administered different concentrations of SSP (SSP-L = 5.46 mg/ml, SSP-M = 10.92 mg/ml or SSP-H = 16.38 mg/ml) once every other day for 8 weeks. A series of experiments, including body weight measurement, footprint tests, Hematoxylin & Eosin staining, and Nissl staining, were performed to evaluate the preventive effect of SSP. Immunofluorescence staining, western blotting, and RT-qPCR were subsequently performed to evaluate activation of the cGAS-STING-TBK1 pathway in the spinal cord. In vitro, hSOD1G93A NSC-34 cells were treated with vomicine to further explore the pharmacological mechanism of vomicine in the treatment of ALS via the cGAS-STING-TBK1 pathway. RESULTS: SSP improved motor function, body weight loss, gastrocnemius muscle atrophy, and motor neuron loss in the spine and cortex of hSOD1-G93A mice. Furthermore, the cGAS-STING-TBK1 pathway was activated in the spinal cord of hSOD1-G93A mice, with activation predominantly observed in neurons and microglia. However, the levels of cGAS, STING, and pTBK1 proteins and cGAS, IRF3, IL-6, and IL-1ß mRNA were reversed following intervention with SSP. Vomicine not only downregulated the levels of cGAS, TBK1, IL-6 and IFN-ß mRNA, but also the levels of cGAS and STING protein in hSOD1G93A NSC-34 cells. CONCLUSION: This study demonstrated that SSP and vomicine exert neuroprotective and anti-neuroinflammatory effects in the treatment of ALS. SSP and vomicine may reduce neuroinflammation by regulating the cGAS-STING-TBK1 pathway, and could thereby play a role in ALS treatment.
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Esclerosis Amiotrófica Lateral , Proteínas de la Membrana , Fármacos Neuroprotectores , Nucleotidiltransferasas , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Ratones , Proteínas de la Membrana/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nucleotidiltransferasas/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Transgénicos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Modelos Animales de EnfermedadRESUMEN
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
Migration of endothelial and many other cells requires spatiotemporal regulation of protrusive and contractile cytoskeletal rearrangements that drive local cell shape changes. Unexpectedly, the small GTPase Rho, a crucial regulator of cell movement, has been reported to be active in both local cell protrusions and retractions, raising the question of how Rho activity can coordinate cell migration. Here, we show that Rho activity is absent in local protrusions and active during retractions. During retractions, Rho rapidly activated ezrin-radixin-moesin proteins (ERMs) to increase actin-membrane attachment, and, with a delay, nonmuscle myosin 2 (NM2). Rho activity was excitable, with NM2 acting as a slow negative feedback regulator. Strikingly, inhibition of SLK/LOK kinases, through which Rho activates ERMs, caused elongated cell morphologies, impaired Rho-induced cell contractions, and reverted Rho-induced blebbing. Together, our study demonstrates that Rho activity drives retractions by sequentially enhancing ERM-mediated actin-membrane attachment for force transmission and NM2-dependent contractility.
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Actomiosina , Movimiento Celular , Forma de la Célula , Proteínas del Citoesqueleto , Actomiosina/metabolismo , Humanos , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de la Membrana/metabolismo , Actinas/metabolismo , Animales , Miosina Tipo II/metabolismoRESUMEN
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
Purpose: We assessed whether NICD1 expression, c-MYC expression, and P63 expression by immunohistochemistry (IHC) correlate with prognosis and high-risk clinicopathological features in lacrimal gland adenoid cystic carcinoma (ACC). Methods: Records of patients with lacrimal gland ACC who underwent surgery between 1998 to 2018 were reviewed. Clinicopathologic and treatment data were collected. Tumor tissues were subjected to light microscopy and IHC. Results: Of 43 patients treated during the study period, 21 had archived tumor tissue available and were included. The median age at diagnosis was 47 years, and 13 patients (62%) were male. Thirteen patients (62%) had T2 disease, and none had nodal or distant metastasis at diagnosis. Tumors were positive for NICD1 expression in eight cases (38%), c-MYC expression in eight (38%), and P63 expression in 11 (52%). Positive NICD1 expression was associated with predominantly solid (vs. cribriform/tubular) pattern (P < 0.001), treatment with orbital exenteration (vs. eye-sparing surgery) (P = 0.008), local recurrence (P = 0.047), and death (P = 0.012). Negative P63 expression was associated with predominantly solid pattern (P = 0.001), local recurrence (P = 0.012), distant metastasis (P = 0.001), and death (P = 0.035). A higher percentage of tumor cells staining for c-MYC was associated with presence of perineural invasion (P = 0.036). Positive NICD1 expression was associated with worse disease-free survival (hazard ratio, 6.27; 95% CI, 1.29-30.46), whereas positive P63 expression was associated with better disease-free survival (hazard ratio, 0.03; 95% CI, 0.0002-0.26). Conclusions: IHC for NICD1 and P63 should be considered in lacrimal gland ACC because of their prognostic value and potential as treatment targets.
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Biomarcadores de Tumor , Carcinoma Adenoide Quístico , Neoplasias del Ojo , Enfermedades del Aparato Lagrimal , Proteínas Proto-Oncogénicas c-myc , Receptor Notch1 , Humanos , Carcinoma Adenoide Quístico/metabolismo , Carcinoma Adenoide Quístico/patología , Carcinoma Adenoide Quístico/diagnóstico , Masculino , Persona de Mediana Edad , Femenino , Adulto , Pronóstico , Enfermedades del Aparato Lagrimal/metabolismo , Enfermedades del Aparato Lagrimal/patología , Enfermedades del Aparato Lagrimal/diagnóstico , Anciano , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias del Ojo/metabolismo , Neoplasias del Ojo/patología , Neoplasias del Ojo/diagnóstico , Estudios Retrospectivos , Biomarcadores de Tumor/metabolismo , Receptor Notch1/metabolismo , Inmunohistoquímica , Adulto Joven , Proteínas de la Membrana , Factores de Transcripción , Proteínas Supresoras de TumorRESUMEN
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
Intercellular protein-protein interactions (PPIs) have pivotal roles in biological functions and diseases. Membrane proteins are therefore a major class of drug targets. However, studying such intercellular PPIs is challenging because of the properties of membrane proteins. Current methods commonly use purified or modified proteins that are not physiologically relevant and hence might mischaracterize interactions occurring in vivo. Here, we describe Cell-Int: a cell interaction assay for studying plasma membrane PPIs. The interaction signal is measured through conjugate formation between two populations of cells each expressing either a ligand or a receptor. In these settings, membrane proteins are in their native environment thus being physiologically relevant. Cell-Int has been applied to the study of diverse protein partners, and enables to investigate the inhibitory potential of blocking antibodies, as well as the retargeting of fusion proteins for therapeutic development. The assay was also validated for screening applications and could serve as a platform for identifying new protein interactors.
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Comunicación Celular , Membrana Celular , Proteínas de la Membrana , Unión Proteica , Mapeo de Interacción de Proteínas , Humanos , Proteínas de la Membrana/metabolismo , Mapeo de Interacción de Proteínas/métodos , Membrana Celular/metabolismo , Animales , Células HEK293 , Bioensayo/métodosRESUMEN
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.