RESUMEN
The αß T cell receptor (TCR), in association with the CD3γε-CD3δε-CD3ζζ signalling hexamer, is the primary determinant of T cell development and activation, and of immune responses to foreign antigens. The mechanism of assembly of the TCR-CD3 complex remains unknown. Here we report a cryo-electron microscopy structure of human TCRαß in complex with the CD3 hexamer at 3.7 Å resolution. The structure contains the complete extracellular domains and all the transmembrane helices of TCR-CD3. The octameric TCR-CD3 complex is assembled with 1:1:1:1 stoichiometry of TCRαß:CD3γε:CD3δε:CD3ζζ. Assembly of the extracellular domains of TCR-CD3 is mediated by the constant domains and connecting peptides of TCRαß that pack against CD3γε-CD3δε, forming a trimer-like structure proximal to the plasma membrane. The transmembrane segment of the CD3 complex adopts a barrel-like structure formed by interaction of the two transmembrane helices of CD3ζζ with those of CD3γε and CD3δε. Insertion of the transmembrane helices of TCRαß into the barrel-like structure via both hydrophobic and ionic interactions results in transmembrane assembly of the TCR-CD3 complex. Together, our data reveal the structural basis for TCR-CD3 complex assembly, providing clues to TCR triggering and a foundation for rational design of immunotherapies that target the complex.
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Modelos Moleculares , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , Estructura Cuaternaria de Proteína , Complejo Receptor-CD3 del Antígeno de Linfocito T/metabolismoRESUMEN
A significant body of research has documented the profound changes in global atmospheric conditions during the COVID-19 pandemic. However, there is still an inadequate comprehensive comparison and assessment of countries before, during, and after the pandemic. Variations in restriction policies, human behaviors, and national traits lead to significant differences in how restriction policies affect atmospheric pollution. This study focuses on NO2, a pollutant with high temporal sensitivity, and utilizes the Oxford COVID-19 policy stringency index along with demographic information. Through spatial-temporal mapping, we analyzed NO2 emission fluctuations and calculated the emission changes in each country. Drawing from this analysis, we explored the relationships among these factors and found that over the span of 2019-2022, across 193 countries, global NO2 emissions displayed a distinct trajectory: initially decreasing, subsequently rebounding, and eventually fluctuating. Most countries exhibited seasonal variations in NO2 emissions. Additionally, the study uncovered a correlation between the stringency of COVID-19 policies and the reduction in NO2 emissions: as policies became stricter, emissions significantly decreased in most countries. In contrast, in countries with lower population densities, stricter policies paradoxically led to an increase in emissions. These findings underscore the importance of considering demographic factors and geographical context in the formulation and implementation of environmental policies.
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COVID-19 , Monitoreo del Ambiente , Dióxido de Nitrógeno , Dióxido de Nitrógeno/análisis , Humanos , Monitoreo del Ambiente/métodos , Contaminación del Aire/análisis , Contaminantes Atmosféricos/análisis , SARS-CoV-2 , Tecnología de Sensores Remotos , Pandemias , Política AmbientalRESUMEN
The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 Å crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Firmicutes/enzimología , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Sistemas CRISPR-Cas , Cristalografía por Rayos X , Ingeniería Genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Estabilidad del ARN , ARN Bacteriano/genética , ARN Guía de Kinetoplastida/química , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Especificidad por SustratoRESUMEN
Tau is a microtubule-associated protein involved in regulation of assembly and spatial organization of microtubule in neurons. However, in pathological conditions, tau monomers assemble into amyloid filaments characterized by the cross-ß structures in a number of neurodegenerative diseases known as tauopathies. In this review, we summarize recent progression on the characterization of structures of tau monomer and filament, as well as the dynamic liquid droplet assembly. Our aim is to reveal how post-translational modifications, amino acid mutations, and interacting molecules modulate the conformational ensemble of tau monomer, and how they accelerate or inhibit tau assembly into aggregates. Structure-based aggregation inhibitor design is also discussed in the context of dynamics and heterogeneity of tau structures.
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Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Procesamiento Proteico-Postraduccional , Tauopatías/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Amiloide/química , Amiloide/genética , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Proteínas Amiloidogénicas/metabolismo , Animales , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Agregación Patológica de Proteínas , Tauopatías/genética , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
The human immunodeficiency virus (HIV)-1 protein Vif has a central role in the neutralization of host innate defences by hijacking cellular proteasomal degradation pathways to subvert the antiviral activity of host restriction factors; however, the underlying mechanism by which Vif achieves this remains unclear. Here we report a crystal structure of the Vif-CBF-ß-CUL5-ELOB-ELOC complex. The structure reveals that Vif, by means of two domains, organizes formation of the pentameric complex by interacting with CBF-ß, CUL5 and ELOC. The larger domain (α/ß domain) of Vif binds to the same side of CBF-ß as RUNX1, indicating that Vif and RUNX1 are exclusive for CBF-ß binding. Interactions of the smaller domain (α-domain) of Vif with ELOC and CUL5 are cooperative and mimic those of SOCS2 with the latter two proteins. A unique zinc-finger motif of Vif, which is located between the two Vif domains, makes no contacts with the other proteins but stabilizes the conformation of the α-domain, which may be important for Vif-CUL5 interaction. Together, our data reveal the structural basis for Vif hijacking of the CBF-ß and CUL5 E3 ligase complex, laying a foundation for rational design of novel anti-HIV drugs.
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Subunidad beta del Factor de Unión al Sitio Principal/química , Subunidad beta del Factor de Unión al Sitio Principal/metabolismo , Proteínas Cullin/química , Proteínas Cullin/metabolismo , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/química , Productos del Gen vif del Virus de la Inmunodeficiencia Humana/metabolismo , Secuencia de Aminoácidos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Cristalografía por Rayos X , Elonguina , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Supresoras de la Señalización de Citocinas , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Cyclization of the polypeptide backbone has proven to be a powerful strategy for enhancing protein stability for fundamental research and pharmaceutical application. The use of such an approach is restricted by how well a targeted polypeptide can be efficiently ligated. Recently, an Asx-specific peptide ligase identified from a tropical cyclotide-producing plant and named butelase 1 exhibited excellent cyclization kinetics that cannot be matched by other known ligases, including intein, PATG, PCY1, and sortase A. In this work, we aimed to examine whether butelase 1 facilitated protein conformational stability for structural investigation. First, we successfully expressed recombinant butelase 1 (rBTase) in the yeast Pichia pastoris. Next, rBTase was shown to be highly efficient in the cyclization of the p53-binding domain (N-terminal domain) of murine double minute X (N-MdmX), an important target for designing anticancer drugs. The cyclized N-MdmX (cMdmX) exhibited increased conformational stability and improved interaction with the ligand compared with those of noncyclized N-MdmX. Importantly, the thermal melting process was completely reversible, contrary to noncyclized N-MdmX, and the melting temperature ( Tm) of cMdmX was increased to 47 from 43 °C. This stable conformation of cMdmX was further confirmed by 15N-1H heteronuclear single-quantum coherence nuclear magnetic resonance (NMR) spectroscopy. The complex of cMdmX and the ligand was tested for protein crystallization, and several promising findings were revealed. Therefore, our work not only provides a recombinant version of butelase 1 but also suggests a conventional approach for preparing stable protein samples for both protein crystallization and NMR structural investigation.
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Fabaceae/enzimología , Ligasas/química , Proteínas Proto-Oncogénicas/química , Secuencia de Aminoácidos , Animales , Cristalización/métodos , Cristalografía por Rayos X/métodos , Ciclización , Ratones , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive subtype of ALL characterized by its high heterogeneity and unfavorable clinical features. Despite improved insights in genetic and epigenetic landscapes of T-ALL, the molecular mechanisms that drive malignant T-cell development remain unclear. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in B-cell malignancies, but little is known about its function and regulatory network in T-ALL. Here we found extremely low levels of BACH2 in T-ALL clinical samples and cell lines compared to normal T cells. Overexpression of BACH2 in T-ALL cells not only induced cell growth retardation but also inhibited cancer progression and infiltration in xenografts. Further RNA sequencing (RNA-seq) analysis revealed significant alterations in regulation of defense and immune responses in T-ALL cells upon BACH2 overexpression. Strikingly, CD28 and CD40LG, two essential stimulatory molecules on T cells, were for the first time identified as novel downstream targets repressed by BACH2 in T-ALL cells. Interestingly, both CD28 and CD40LG were indispensable for T-ALL survival, since largely or completely silencing CD28 and CD40LG led to rapid cell death, whereas partial knockdown of them resulted in cell-cycle arrest and enhanced apoptosis. More importantly, BACH2-mediated CD28 and CD40LG signals contributed to cell migration and dissemination of T-ALL cells to the bone marrow, thus adding a new layer to the BACH2-mediated tumor immunoregulation in T-cell malignancies.
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Antígenos CD28 , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfocitos/metabolismo , Linfocitos T/metabolismoRESUMEN
Phage therapy offers a promising approach to combat the growing threat of antimicrobial resistance. Yet, key questions remain regarding dosage, administration routes, combination therapy, and the causes of therapeutic failure. In this study, we focused on a novel lytic phage, ФAb4B, which specifically targeted the Acinetobacter baumannii strains with KL160 capsular polysaccharide, including the pan-drug resistant A. baumannii YQ4. ФAb4B exhibited the ability to effectively inhibit biofilm formation and eradicate mature biofilms independently of dosage. Additionally, it demonstrated a wide spectrum of antibiotic-phage synergy and did not show any cytotoxic or haemolytic effects. Continuous phage injections, both intraperitoneally and intravenously over 7 d, showed no acute toxicity in vivo. Importantly, phage therapy significantly improved neutrophil counts, outperforming ciprofloxacin. However, excessive phage injections suppressed neutrophil levels. The combinatorial treatment of phage-ciprofloxacin rescued 91% of the mice, a superior outcome compared to phage alone (67%). The efficacy of the combinatorial treatment was independent of phage dosage. Notably, prophylactic administration of the combinatorial regimen provided no protection, but even when combined with a delayed therapeutic regimen, it saved all the mice. Bacterial resistance to the phage was not a contributing factor to treatment failure. Our preclinical study systematically describes the lytic phage's effectiveness in both in vitro and in vivo settings, filling in crucial details about phage treatment against bacteriemia caused by A. baumannii, which will provide a robust foundation for the future of phage therapy.
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Infecciones por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Bacteriófagos , Biopelículas , Ciprofloxacina , Farmacorresistencia Bacteriana Múltiple , Terapia de Fagos , Acinetobacter baumannii/virología , Acinetobacter baumannii/efectos de los fármacos , Terapia de Fagos/métodos , Infecciones por Acinetobacter/terapia , Infecciones por Acinetobacter/microbiología , Animales , Biopelículas/efectos de los fármacos , Bacteriófagos/fisiología , Ratones , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Ciprofloxacina/uso terapéutico , Ciprofloxacina/farmacología , Modelos Animales de Enfermedad , Femenino , Ratones Endogámicos BALB CRESUMEN
Porcine epidemic diarrhea virus (PEDV) is the cause of an economically important swine disease. Previous studies suggested that PEDV does not elicit a robust IFN response, but the mechanism(s) used to evade or block this innate immune response was not known. In this study, we found that PEDV infection blocked synthetic dsRNA-induced IFN-ß production by interfering with the activation of interferon regulatory factor 3 (IRF3). We identified PEDV replicase encoded papain-like protease 2 (PLP2) as an IFN antagonist that depends on catalytic activity for its function. We show that levels of ubiquitinated proteins are reduced during PEDV infection and that PEDV PLP2 has deubiquitinase (DUB) activity that recognizes and processes both K-48 and K-63 linked polyubiquitin chains. Furthermore, we found that PEDV PLP2 strongly inhibits RIG-I- and STING-activated IFN expression and that PEDV PLP2 can be co-immunoprecipitated with and deubiquitinates RIG-I and STING, the key components of the signalling pathway for IFN expression. These results show that PEDV infection suppresses production of IFN-ß and provides evidence indicating that the PEDV papain-like protease 2 acts as a viral DUB to interfere with the RIG-I- and STING-mediated signalling pathway.
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Regulación de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Interferón beta/metabolismo , Papaína/metabolismo , Virus de la Diarrea Epidémica Porcina/patogenicidad , Ubiquitina/metabolismo , Animales , Chlorocebus aethiops , Proteasas Similares a la Papaína de Coronavirus , Endopeptidasas/metabolismo , Células HEK293 , Humanos , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Interferón beta/genética , Interferón beta/inmunología , Papaína/genética , Papaína/farmacología , Virus de la Diarrea Epidémica Porcina/inmunología , Transducción de Señal , Porcinos , Células VeroRESUMEN
Since bacteriophages (phages) were firstly reported at the beginning of the 20th century, the study on them experiences booming-fading-emerging with discovery and overuse of antibiotics. Although they are the hotspots for therapy of antibiotic-resistant strains nowadays, natural phage applications encounter some challenges such as limited host range and bacterial resistance to phages. Synthetic biology, one of the most dramatic directions in the recent 20-years study of microbiology, has generated numerous methods and tools and has contributed a lot to understanding phage evolution, engineering modification, and controlling phage-bacteria interactions. In order to better modify and apply phages by using synthetic biology techniques in the future, in this review, we comprehensively introduce various strategies on engineering or modification of phage genome and rebooting of recombinant phages, summarize the recent researches and potential directions of phage synthetic biology, and outline the current application of engineered phages in practice.
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Seminoma is the most common malignant solid tumor in 14 to 44 year-old men. However, its molecular features and tumor microenvironment (TME) is largely unexplored. Here, we perform a series of studies via genomics profiling (single cell multi-omics and spatial transcriptomics) and functional examination using seminoma samples and a seminoma cell line. We identify key gene expression programs share between seminoma and primordial germ cells, and further characterize the functions of TFAP2C in promoting tumor invasion and migration. We also identify 15 immune cell subtypes in TME, and find that subtypes with exhaustion features were located closer to the tumor region through combined spatial transcriptome analysis. Furthermore, we identify key pathways and genes that may facilitate seminoma disseminating beyond the seminiferous tubules. These findings advance our knowledge of seminoma tumorigenesis and produce a multi-omics atlas of in situ human seminoma microenvironment, which could help discover potential therapy targets for seminoma.
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Neoplasias de Células Germinales y Embrionarias , Seminoma , Neoplasias Testiculares , Masculino , Humanos , Adolescente , Adulto Joven , Adulto , Seminoma/genética , Seminoma/metabolismo , Seminoma/patología , Multiómica , Neoplasias de Células Germinales y Embrionarias/genética , Neoplasias Testiculares/metabolismo , Microambiente Tumoral/genéticaRESUMEN
Activating transcription factor 3 (ATF3) is a stress-inducible gene reported with anti-inflammatory response effects against bacterial infections. This study focuses on the function of ATF3 in alveolar epithelial type II cells (A549) following Mycobacterium tuberculosis (MTB) infection. First, RT-qPCR results detected reduced ATF3 expression in broncho-alveolar lavage fluid (BALF) of MTB-infected patients, whereas the ATF3 level was upregulated in A549 cells at early stages after MTB infection but decreased later. The binding relationship between ATF3 and TIMP metallopeptidase inhibitor 2 (TIMP2) promoter was predicted via bioinformatic prediction and validated by ChIP and luciferase assays. ATF3 bound to TIMP2 promoter for transcriptional activation. Overexpression of ATF3 or TIMP2 enhanced autophagy activity, elevated p62 levels and the LC3BII/LC3BI ratio, and decreased IL-6 and TNF-α levels in A549 cells. The ATF3/TIMP2 axis suppressed the NF-κB pathway to alleviate inflammatory responses in A549 cells. Mice were exposed to MTB aerosol for in vivo experiments. Increased ATF3 expression was correlated with increased autophagy activity, clearance of bacteria as well as inflammation resolution in mouse lung tissues. In conclusion, this study demonstrates that ATF3 promotes cell autophagy and suppresses inflammatory response in MTB-infected A549 cells via TIMP2 activation and NF-κB suppression.
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Factor de Transcripción Activador 3 , Células Epiteliales Alveolares , Inflamación , Mycobacterium tuberculosis , Tuberculosis , Factor de Transcripción Activador 3/genética , Factor de Transcripción Activador 3/metabolismo , Células Epiteliales Alveolares/metabolismo , Animales , Inflamación/genética , Inflamación/metabolismo , Ratones , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , FN-kappa B/metabolismo , Tuberculosis/genética , Tuberculosis/metabolismoRESUMEN
Overexpressed Mdm2 and its 7homolog MdmX impair p53 activity in many cancers. Small molecules mimicking a p53 peptide can effectively inhibit Mdm2 but not MdmX. Here, we show a strategy for improving lead compounds for Mdm2 and MdmX inhibition based on the multivalency of the p53 peptide. Crystal structures of MdmX complexed with nutlin-3a, a strong Mdm2 inhibitor but a weak one for MdmX, reveal that nutlin-3a fits into the ligand binding pocket of MdmX mimicking the p53 peptide. However, due to distinct flexibility around the MdmX ligand binding pocket, the structures are missing many important intermolecular interactions that exist in the MdmX/p53 peptide and Mdm2/nultin-3a complexes. By targeting these flexible regions, we identify allosteric and additive fragments that enhance the binding affinity of nutlin-3a for MdmX, leading to potent Mdm2/MdmX inhibitors with anticancer activity. Our work provides a practical approach to drug design for signal transduction therapy.
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Antineoplásicos , Proteínas de Ciclo Celular , Neoplasias , Proteínas Proto-Oncogénicas , Proteína p53 Supresora de Tumor , Antineoplásicos/farmacología , Proteínas de Ciclo Celular/metabolismo , Humanos , Ligandos , Neoplasias/metabolismo , Péptidos/metabolismo , Péptidos/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Automated, image based high-content screening is a fundamental tool for discovery in biological science. Modern robotic fluorescence microscopes are able to capture thousands of images from massively parallel experiments such as RNA interference (RNAi) or small-molecule screens. As such, efficient computational methods are required for automatic cellular phenotype identification capable of dealing with large image data sets. In this paper we investigated an efficient method for the extraction of quantitative features from images by combining second order statistics, or Haralick features, with curvelet transform. A random subspace based classifier ensemble with multiple layer perceptron (MLP) as the base classifier was then exploited for classification. Haralick features estimate image properties related to second-order statistics based on the grey level co-occurrence matrix (GLCM), which has been extensively used for various image processing applications. The curvelet transform has a more sparse representation of the image than wavelet, thus offering a description with higher time frequency resolution and high degree of directionality and anisotropy, which is particularly appropriate for many images rich with edges and curves. A combined feature description from Haralick feature and curvelet transform can further increase the accuracy of classification by taking their complementary information. We then investigate the applicability of the random subspace (RS) ensemble method for phenotype classification based on microscopy images. A base classifier is trained with a RS sampled subset of the original feature set and the ensemble assigns a class label by majority voting. RESULTS: Experimental results on the phenotype recognition from three benchmarking image sets including HeLa, CHO and RNAi show the effectiveness of the proposed approach. The combined feature is better than any individual one in the classification accuracy. The ensemble model produces better classification performance compared to the component neural networks trained. For the three images sets HeLa, CHO and RNAi, the Random Subspace Ensembles offers the classification rates 91.20%, 98.86% and 91.03% respectively, which compares sharply with the published result 84%, 93% and 82% from a multi-purpose image classifier WND-CHARM which applied wavelet transforms and other feature extraction methods. We investigated the problem of estimation of ensemble parameters and found that satisfactory performance improvement could be brought by a relative medium dimensionality of feature subsets and small ensemble size. CONCLUSIONS: The characteristics of curvelet transform of being multiscale and multidirectional suit the description of microscopy images very well. It is empirically demonstrated that the curvelet-based feature is clearly preferred to wavelet-based feature for bioimage descriptions. The random subspace ensemble of MLPs is much better than a number of commonly applied multi-class classifiers in the investigated application of phenotype recognition.
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Procesamiento de Imagen Asistido por Computador/métodos , Algoritmos , Animales , Inteligencia Artificial , Células CHO , Cricetinae , Cricetulus , Drosophila melanogaster/citología , Células HeLa , Humanos , Microscopía Fluorescente , Redes Neurales de la Computación , Reconocimiento de Normas Patrones Automatizadas/métodos , Análisis de OndículasRESUMEN
BACKGROUND: The importance of microRNAs (miRs) has been documented in infections. This study estimated the role of miR-340-5p in Mycobacterium tuberculosis (Mtb)-infected alveolar type II cells. METHODS: The microarray of GEO database was analyzed to find the differentially expressed miRs caused by Mtb infection, and miR-340-5p was selected as the research object. The effects of Mtb infection on A549 cells were studied by MTT, CFU, EdU, flow cytometry and ELISA assays. miR-340-5p expression was altered in Mtb-infected A549 cells. The downstream target of miR-340-5p was found by bioinformatics analysis and verified by the rescue experiment. The pathways regulated by miR-340-5p and its target gene were further studied. RESULTS: Mtb infection suppressed the activity of A549 cells and promoted the release of inflammatory factors. Mtb infection inhibited miR-340-5p expression. Overexpression of miR-340-5p enhanced the resistance of A549 cells to Mtb infection. Moreover, miR-340-5p targeted TMED7. Overexpression of TMED7 reversed the protective effect of miR-340-5p on Mtb-infected A549 cells. miR-340-5p inhibited the activation of NF-κB by targeting TMED7. CONCLUSION: miR-340-5p inhibits the activation of NF-κB by targeting TMED7, thus alleviating the injury of A549 cells caused by Mtb infection. This study may offer a novel approach to Mtb infection.
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Antimicrobial resistance (AMR) is a global crisis for human public health which threatens the effective prevention and control of ever-increasing infectious diseases. The advent of pandrug-resistant bacteria makes most, if not all, available antibiotics invalid. Meanwhile, the pipeline of novel antibiotics development stagnates, which prompts scientists and pharmacists to develop unconventional antimicrobials. Bacteriophage-derived endolysins are cell wall hydrolases which could hydrolyze the peptidoglycan layer from within and outside of bacterial pathogens. With high specificity, rapid action, high efficiency, and low risk of resistance development, endolysins are believed to be among the best alternative therapeutic agents to treat multidrug resistant (MDR) bacteria. As of now, endolysins have been applied to diverse aspects. In this review, we comprehensively introduce the structures and activities of endolysins and summarize the latest application progress of recombinant endolysins in the fields of medical treatment, pathogen diagnosis, food safety, and agriculture.
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The present study aimed to investigate the regulatory mechanism of chemokine (C-X-C motif) receptor 4 (CXCR4) on endothelial progenitor cells (EPCs) through the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway under hypoxic conditions. Mononuclear cells were isolated from the bone marrow (BM) of young Sprague-Dawley (SD) rats. Bone marrow-derived endothelial progenitor cells (BM-EPCs) were characterized by using Dil-labeled acetylated low-density lipoprotein (Dil-ac-LDL) and fluorescein isothiocyanate-labeled UEA (FITC-UEA-1). Phenotype identification of BM-EPCs was based on red cytoplasm and green cytomembrane. Flow cytometry was employed to examine the markers CD14, CD34, and KDR. Expression level of the EPC-specific surface marker CD14 was found to be negative, while the expression level of CD34 and KDR was positive. In addition, CXCR4 was stably overexpressed in BM-EPCs after transfection with adenovirus-CXCR4. Cell proliferation, migration and apoptosis abilities were measured through the application of CCK-8, followed by Transwell and flow cytometry assays. The expression level of CXCR4, PI3K and Akt was determined by reverse transcription-quantitative PCR and western blotting assays. Functional experiments demonstrated that hypoxia inhibited BM-EPC proliferation and migration, while accelerating BM-EPC apoptosis. Additionally, CXCR4 was found to promote proliferation and migration, and suppress apoptosis in BM-EPCs with or without hypoxia treatment. Evidence also demonstrated that CXCR4 markedly upregulated the expression levels of PI3K and Akt. Furthermore, PI3K inhibitor (LY294002) and CXCR4 inhibitor (AMD3100) effectively inhibited the proliferation, migration and resistance to apoptosis of CXCR4-mediated BM-EPCs under hypoxic conditions.
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The global Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has affected more than eight million people. There is an urgent need to investigate how the adaptive immunity is established in COVID-19 patients. In this study, we profiled adaptive immune cells of PBMCs from recovered COVID-19 patients with varying disease severity using single-cell RNA and TCR/BCR V(D)J sequencing. The sequencing data revealed SARS-CoV-2-specific shuffling of adaptive immune repertories and COVID-19-induced remodeling of peripheral lymphocytes. Characterization of variations in the peripheral T and B cells from the COVID-19 patients revealed a positive correlation of humoral immune response and T-cell immune memory with disease severity. Sequencing and functional data revealed SARS-CoV-2-specific T-cell immune memory in the convalescent COVID-19 patients. Furthermore, we also identified novel antigens that are responsive in the convalescent patients. Altogether, our study reveals adaptive immune repertories underlying pathogenesis and recovery in severe versus mild COVID-19 patients, providing valuable information for potential vaccine and therapeutic development against SARS-CoV-2 infection.
Asunto(s)
Linfocitos B/inmunología , Betacoronavirus/patogenicidad , Infecciones por Coronavirus/inmunología , Inmunidad Celular , Inmunidad Humoral , Neumonía Viral/inmunología , Linfocitos T/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Linfocitos B/clasificación , Linfocitos B/virología , Betacoronavirus/inmunología , COVID-19 , Estudios de Casos y Controles , China , Convalecencia , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/patología , Infecciones por Coronavirus/virología , Progresión de la Enfermedad , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno/inmunología , Humanos , Memoria Inmunológica , Pandemias , Neumonía Viral/genética , Neumonía Viral/patología , Neumonía Viral/virología , Receptores de Antígenos de Linfocitos B/clasificación , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Receptores de Antígenos de Linfocitos T/clasificación , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Análisis de la Célula Individual , Linfocitos T/clasificación , Linfocitos T/virologíaRESUMEN
In preclinical and phase I and II clinical studies, 2'-deoxy-2'-ß-fluoro-4'-azidocytidine (FNC) displays a potent and long-lasting inhibition of HIV-1 infection. To investigate its mechanism of action, we compared it with the well-documented lamivudine (3TC). Pharmacokinetic studies revealed that the intracellular retention of FNC triphosphate in peripheral blood mononuclear cells was markedly longer than that of the 3TC triphosphate. FNC selectively enters and is retained in HIV target cells, where it exerts long-lasting prevention of HIV-1 infection. In addition to inhibition of HIV-1 reverse transcription, FNC also restores A3G expression in CD4+ T cells in FNC-treated HIV-1 patients. FNC binds to the Vif-E3 ubiquitin ligase complex, enabling A3G to avoid Vif-induced ubiquitination and degradation. These data reveal the mechanisms underlying the superior anti-HIV potency and long-lasting action of FNC. Our results also suggest a potential clinical application of FNC as a long-lasting pre-exposure prophylactic agent capable of preventing HIV infection.