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A novel colorimetric platform was designed for the determination of S. aureus by utilizing a dual-recognition strategy, where wheat germ agglutinin (WGA)-functionalized magnetic beads were served as separation elements to capture and enrich S. aureus efficiently from the matrix. Horseradish peroxidase (HRP) labeled chicken anti-protein A IgY (HRP-IgY) was used to label the captured S. aureus. A chicken IgY was introduced as a signal tracer to bind with staphylococcal protein A (SPA) on the surface of S. aureus, which can circumvent the interference from protein G-producing Streptococcus. Subsequently, the colorimetric signal was achieved by an HRP-catalyzed reaction, which was amplified by HRP-IgY bound by approximately 80,000 SPA molecules on one S. aureus. The entire detection process could be accomplished within 90 min. Under optimal conditions, the linear response of different S. aureus concentrations ranged from 7.8 × 102 to 2.0 × 105 CFU/mL and the limit of detection reached down to 3.9 × 102 CFU/mL. Some common non-target bacteria yielded negative results, indicating the excellent specificity of the method. The developed strategy was successfully applied to the determination of S. aureus in various types of samples with satisfactory recoveries. Therefore, the novel dual-recognition strategy possessed the advantages of high sensitivity, specificity, and low cost and exhibited considerable potential as a promising tool to defend public health.
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Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Aglutininas del Germen de Trigo , Colorimetría/métodos , Inmunoglobulinas , Infecciones Estafilocócicas/diagnóstico , Infecciones Estafilocócicas/microbiología , Peroxidasa de Rábano Silvestre/metabolismoRESUMEN
OBJECTIVES: This study aims to investigate clinical outcomes, imaging changes, and age differences with regard to temporomandibular joint disc condylar complex with anterior disc displacement without reduction (ADDWoR). METHODS: A total of 37 patients (45 lateral joints) with ADDWoR who were admitted to The First Affiliated Hospital of Zheng Zhou University from January 2016 to June 2023 were selected. The patients were composed of 4 males and 33 females and had an average age of 23.5 years. The average course of the disease was 14.4 months. Clinical and magnetic resonance imaging (MRI) data were collected at the end of initial diagnosis and follow-up, and the length and thickness of the articular disc, the angle of the disc condyle, and the height of the condyle were measured. The statistical significance of the changes was assessed using SPSS 25.0 software package. RESULTS: At the end of follow-up, disc displacement in three patients (three lateral joints) was healed. Approximately 48.4% of the patients felt that limitation of mandibular movement was not alleviated; 58.3% of patients reported that pain during mouth opening was not reduced; 54.5% reported pain while chewing; 33.3% of the patients showed facial deviation, and only one showed remission. The mean disk-condyle angle increased from 61.63° to 67.81°. The average length of articular disc shortened from 8.20 mm to 7.27 mm, and the height of the condyle significantly decreased from 23.17 mm to 22.76 mm (P<0.05). The absorption ratio of the condyle increased, and no significant differences in the changes of joint soft and hard tissues between the adolescent and adult groups (P>0.05). CONCLUSIONS: In different age groups of patients with ADDWoR, clinical symptoms cannot be completely relieved. The disc is anteriorly displaced and shortens, condylar height decreases, and secondary facial asymmetry and mandibular retraction occur.
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Luxaciones Articulares , Trastornos de la Articulación Temporomandibular , Masculino , Adulto , Femenino , Adolescente , Humanos , Adulto Joven , Disco de la Articulación Temporomandibular , Cóndilo Mandibular , Imagen por Resonancia Magnética/métodos , Dolor/complicaciones , Dolor/patología , Articulación Temporomandibular/patologíaRESUMEN
The role of follicular T helper (Tfh) cells in humoral response has been considered essential in recent years. Understanding how Tfh cells control complex humoral immunity is critical to developing strategies to improve the efficacy of vaccines against SARS-CoV-2 and other emerging pathogens. However, the immunologic mechanism of Tfh cells in SARS-CoV-2 receptor binding domain (RBD) vaccine strategy is limited. In this study, we expressed and purified recombinant SARS-CoV-2 RBD protein in Drosophila S2 cells for the first time and explored the mechanism of Tfh cells induced by RBD vaccine in humoral immune response. We mapped the dynamic of Tfh cell in lymph node and spleen following RBD vaccination and revealed the relationship between Tfh cells and humoral immune response induced by SARS-CoV-2 RBD vaccine through correlation analysis, blocking of IL-21 signaling pathway, and co-culture of Tfh with memory B cells. Recombinant RBD protein elicited a predominant Tfh1 and Tfh1-17 subset response and strong GC responses in spleen and lymph nodes, especially to enhanced vaccination. IL-21 secreted by Tfh cells affected the development and differentiation of B cells and played a key role in the humoral immune response. These observations will help us further understand the mechanism of protective immune response induced by COVID-19 vaccine and has guiding significance for the development of vaccines against newly emerging mutants.
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In atherosclerosis, macrophages derived from blood monocytes contribute to non-resolving inflammation, which subsequently primes necrotic core formation, and ultimately triggers acute thrombotic vascular disease. Nevertheless, little is known about how inflammatory cells, especially the macrophages fuel atherosclerosis. CD68, a unique class D scavenger receptor (SRD) family member, is specifically expressed in monocytes/macrophages and remarkably up-regulated upon oxidized low-density lipoprotein (ox-LDL) stimulation. Nonetheless, whether and how myeloid-specific CD68 affects atherosclerosis remains to be defined. To determine the essential in vivo role and mechanism linking CD68 to atherosclerosis, we engineered global and myeloid-specific CD68-deficient mice on an ApoE-null background. On Western diet, both the mice with global and the myeloid-restricted deletion of CD68 on ApoE-null background attenuated atherosclerosis, accompanied by diminished immune/inflammatory cell burden and necrotic core content, but increased smooth muscle cell content in atherosclerotic plaques. In vitro experiments revealed that CD68 deficiency in macrophages resulted in attenuated ox-LDL-induced macrophage apoptosis. Additionally, CD68 deficiency suppressed ROS production, while removal of ROS can markedly reversed this effect. We further showed that CD68 deficiency affected apoptosis through inactivation of the mitogen-activated protein kinase (MAPK) pathway. Our findings establish CD68 as a macrophage lineage-specific regulator of "ROS-MAPK-apoptosis" axis, thus providing a previously unknown mechanism for the prominence of CD68 as a risk factor for coronary artery disease. Its therapeutic inhibition may provide a potent lever to alleviate the cardiovascular disease.
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Aterosclerosis , Proteínas Quinasas Activadas por Mitógenos , Animales , Ratones , Apolipoproteínas E/genética , Apoptosis , Aterosclerosis/metabolismo , Necrosis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Hydrogen sulfide (H2 S) is a redox gasotransmitter. It has been shown that H2 S has a key role in host antiviral defense by inhibiting interleukin production and S-sulfhydrating Keap1 lead to Nrf2/ARE pathway activation. However, it is yet unclear whether H2 S can play an antiviral role by regulating autophagy. In this study, we found that exogenous H2 S decreased the expression of human T-cell leukemia virus type-1 (HTLV-1) protein and HTLV-1 induced autophagosomes accumulation. Transmission electron microscope assays indicated that autophagosomes accumulation decreased after H2 S administration. HTLV-1-transformed T-cell lines had a high level of CSE (H2 S endogenous enzyme) which could be induced in Hela by HTLV-1 infection. Immunoblot demonstrated that overexpression of CSE inhibited HTLV-1 protein expression and autophagy. And we got the opposite after CSE knockdown. Meanwhile, H2 S could not restrain the autophagy when ATG4B had a mutant at its site of 89. In a word, these results suggested that H2 S modulated HTLV-1 protein expression via ATG4B. Therefore, our findings suggested a new mechanism by which H2 S defended against virus infection.
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Virus Linfotrópico T Tipo 1 Humano , Sulfuro de Hidrógeno , Leucemia de Células T , Humanos , Virus Linfotrópico T Tipo 1 Humano/fisiología , Sulfuro de Hidrógeno/farmacología , Sulfuro de Hidrógeno/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Antivirales/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Cisteína Endopeptidasas/metabolismoRESUMEN
BACKGROUND: Adaptive immune response has been thought to play a key role in SARS-CoV-2 infection. The role of B cells, CD4+T, and CD8+T cells are different in vaccine-induced immune response, thus it is imperative to explore the functions and kinetics of adaptive immune response. We collected blood samples from unvaccinated and vaccinated individuals. To assess the mechanisms contributing to protective immunity of CoronaVac vaccines, we mapped the kinetics and durability of humoral and cellular immune responses after primary and boost vaccination with CoronaVac vaccine in different timepoints. MATERIALS AND METHODS: We separate PBMC and plasma from blood samples. The differentiation and function of RBD-spcific CD4+T and CD8+T cells were analyzed by flow cytometry and ELISA. Antibodies response was analyzed by ELISA. ELISPOT analysis was perfomed to detected the RBD-spcific memory B cells. CBA analysis was performed to detected the cytokine immune profiles. Graphpad prism 8 and Origin 2021 were used for statistical analysis. RESULTS: Vaccine-induced CD4+T cell responses to RBD were more prominent than CD8+T cell responses, and characterized by a predominant Th1 and weak Th17 helper response. CoronaVac vaccine triggered predominant IgG1 antibody response and effectively recalled specific antibodies to RBD protein after booster vaccination. Robust antigen-specific memory B cells were detected (p < 0.0001) following booster vaccination and maintained at 6 months (p < 0.0001) following primary vaccination. Vaccine-induced CD4+T cells correlated with CD8+T cells (r = 0.7147, 0.3258, p < 0.0001, p = 0.04), memory B cell responses (r = 0.7083, p < 0.0001), and IgG and IgA (r = 0.6168, 0.5519, p = 0.0006, 0.003) after vaccination. In addition, vaccine induced a broader and complex cytokine pattern in plasma at early stage. CONCLUSION: Taken together, these results highlight the potential role of B cell and T cell responses in vaccine-induced long-term immunity.
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COVID-19 , SARS-CoV-2 , Humanos , Leucocitos Mononucleares , COVID-19/prevención & control , Vacunación , Citocinas , Ensayo de Immunospot Ligado a Enzimas , Inmunidad , Anticuerpos AntiviralesRESUMEN
Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment. Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.
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Antígeno CD11b , Melanoma , Fagocitosis , Animales , Antígenos de Neoplasias , Antígeno CD11b/genética , Línea Celular , Línea Celular Tumoral , Colorantes Fluorescentes , Melanoma/genética , Ratones , Mutación Puntual , Microambiente TumoralRESUMEN
Although increasing evidence links the gut microbiota with the development of colorectal cancer, the molecular mechanisms for microbiota regulation of tumorigenesis are not fully understood. Here, we found that a member of the TNFα-induced protein 8 (TNFAIP8) family called TIPE2 (TNFAIP8-like 2) was significantly upregulated in murine intestinal tumors and in human colorectal cancer, and colorectal cancer with high expression of Tipe2 mRNA associated with reduced survival time of patients. Consistent with these findings, TIPE2 deficiency significantly inhibited the development of colorectal cancer in mice treated with azoxymethane/dextran sodium sulfate and in Apcmin/+ mice. TIPE2 deficiency attenuated the severity of colitis by successfully resolving and restricting colonic inflammation and protected colonic myeloid cells from death during colitis. Transplantation of TIPE2-deficient bone marrow into wild-type mice successfully dampened the latter's tumorigenic phenotype, indicating a hematopoietic-specific role for TIPE2. Mechanistically, restricting the expansion of Enterobacteriaceae/Escherichia coli (E. coli) decreased intestinal inflammation and reduced the incidence of colonic tumors. Collectively, these data suggest that hematopoietic TIPE2 regulates intestinal antitumor immunity by regulation of gut microbiota. TIPE2 may represent a new therapeutic target for treating colorectal cancer.
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Colitis , Neoplasias del Colon , Microbioma Gastrointestinal , Animales , Carcinogénesis/genética , Colitis/inducido químicamente , Escherichia coli/metabolismo , Humanos , Inflamación/complicaciones , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Autophagy is an intracellular degradation process that maintains cellular homeostasis. It is essential for protecting organisms from environmental stress. Autophagy can help the host to eliminate invading pathogens, including bacteria, viruses, fungi, and parasites. However, pathogens have evolved multiple strategies to interfere with autophagic signaling pathways or inhibit the fusion of autophagosomes with lysosomes to form autolysosomes. Moreover, host cell matrix degradation by different types of autophagy can be used for the proliferation and reproduction of pathogens. Thus, determining the roles and mechanisms of autophagy during pathogen infections will promote understanding of the mechanisms of pathogen|â|host interactions and provide new strategies for the treatment of infectious diseases.
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Autofagia , Interacciones Huésped-Patógeno , Bacterias , Lisosomas , Transducción de SeñalRESUMEN
BACKGROUND AND AIMS: Atherosclerosis, a progressive inflammatory disease characterized by elevated inflammation and lipid accumulation in the aortic endothelium, arises in part from the infiltration of inflammatory cells into the vascular wall. However, it is not fully defined how inflammatory cells, especially macrophages, affect the pathogenesis of atherosclerosis. Schlafen4 (Slfn4) mRNA is remarkably upregulated upon ox-LDL stimulation in macrophages. Nonetheless, the role of Slfn4 in foam cell formation remains unclear. METHODS: To determine whether and how Slfn4 regulates lesion macrophage function during atherosclerosisï¼we engineered ApoE-/-Slfn4-/- double-deficient mice on an ApoE-/- background and evaluated the deficiency of Slfn4 expression in atherosclerotic lesion formation in vivo. RESULTS: Our results demonstrate that total absence of SLFN4 and the bone marrow-restricted deletion of Slfn4 in ApoE-/- mice remarkably diminish inflammatory cell numbers within arterial plaques as well as limit development of atherosclerosis in moderate hypercholesterolemia condition. This is linked to a marked reduction in the expression of proinflammatory cytokines, the generation of the reactive oxygen species (ROS) and the apoptosis of cells. Furthermore, the activation of MAPKs and apoptosis signaling pathways is compromised in the absence of Slfn4. CONCLUSIONS: These findings demonstrate a novel role of Slfn4 in modulating vascular inflammation and atherosclerosis, highlighting a new target for the related diseases.
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OBJECTIVE: The aim of this study was to investigate the role and molecular regulatory mechanisms of baicalin in oral squamous cell carcinoma (OSCC) progression. DESIGN: Gene expression in OSCC cells was detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR). OSCC cell viability, migration, invasion and stemness were measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, Transwell, and sphere formation assays. The target genes of miR-106b-5p were predicted using bioinformatic tools. The interaction between microRNA-miR-106b-5p (miR-106b-5p) and disabled homolog 2 (DAB2) was confirmed by a luciferase reporter assay. TOP/FOP-Flash reporter assay and western blot analysis were used to analyze the activity of the Wnt/ß-catenin pathway. RESULTS: Baicalin inhibited OSCC cell viability, migration, invasion, and stemness. Baicalin downregulated miR-106b-5p expression. In addition, MiR-106b-5p upregulation reversed the effects of baicalin on OSCC cells. As a target gene of miR-106b-5p, DAB2 was negatively regulated by miR-106b-5p and upregulated by baicalin in OSCC cells. MiR-106b-5p activated Wnt/ß-catenin pathway in OSCC cells by inhibiting DAB2. Baicalin suppressed Wnt/ß-catenin pathway by upregulating DAB2. In rescue assays, miR-106b-5p overexpression-induced promotion of OSCC cellular processes was attenuated by DAB2 upregulation. CONCLUSIONS: Baicalin exerts anti-tumor effects in OSCC by inhibiting the miR-106b-5p-Wnt/ß-catenin pathway via upregulating DAB2.
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Carcinoma de Células Escamosas , Flavonoides/farmacología , MicroARNs , Neoplasias de la Boca , Vía de Señalización Wnt , Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/genética , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
Exposure of mucosal epithelial cells to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 is known to disrupt epithelial cell junctions by impairing stathmin-mediated microtubule depolymerization. However, the pathological significance of this process and its underlying molecular mechanism remain unclear. Here we show that treatment of epithelial cells with pseudotyped HIV-1 viral particles or recombinant gp120 protein results in the activation of protein kinase G 1 (PKG1). Examination of epithelial cells by immunofluorescence microscopy reveals that PKG1 activation mediates the epithelial barrier damage upon HIV-1 exposure. Immunoprecipitation experiments show that PKG1 interacts with stathmin and phosphorylates stathmin at serine 63 in the presence of gp120. Immunoprecipitation and immunofluorescence microscopy further demonstrate that PKG1-mediated phosphorylation of stathmin promotes its autophagic degradation by enhancing the interaction between stathmin and the autophagy adaptor protein p62. Collectively, these results suggest that HIV-1 exposure exploits the PKG1/stathmin axis to affect the microtubule cytoskeleton and thereby perturbs epithelial cell junctions. Our findings reveal a novel molecular mechanism by which exposure to HIV-1 increases epithelial permeability, which has implications for the development of effective strategies to prevent mucosal HIV-1 transmission.
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Permeabilidad de la Membrana Celular , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Células Epiteliales/patología , VIH-1/fisiología , Microtúbulos/metabolismo , Estatmina/metabolismo , Movimiento Celular , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Células Epiteliales/metabolismo , Células Epiteliales/virología , Infecciones por VIH/virología , Humanos , Microtúbulos/virología , Fosforilación , Estatmina/genéticaRESUMEN
The ongoing pandemic of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. Like other coronaviruses, the SARS-CoV-2 genome encodes spike (S) glycoproteins, which protrude from the surface of mature virions. The S glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. Surface location of the S glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. In the light of its crucial roles in viral infection and adaptive immunity, the S protein is the focus of most vaccine strategies as well as therapeutic interventions. In this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the SARS-CoV-2 S glycoprotein, aiming to provide valuable insights into the design and development of the S protein-based vaccines as well as therapeutics.
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Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/prevención & control , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/inmunología , Anticuerpos Neutralizantes/inmunología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Neumonía Viral/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
To develop a specific method for the detection of S. aureus, chicken anti-protein A IgY was adopted for specifically capturing S. aureus, depending on the specific recognition of staphylococcal protein A (SPA) by chicken anti-protein A IgY, which can eliminate the interference from protein G-producing Streptococcus. HRP labeled IgG, Fc region of which has a high affinity towards SPA, was paired with IgY for the colorimeter analysis of the system. By optimizing the system, a super-low detection limit of 11â¯CFU of S. aureus in 100⯵L PBS without enrichment, with a linear range from 5.0â¯×â¯102â¯CFUâ¯mL-1 to 5.0â¯×â¯104â¯CFUâ¯mL-1 was obtained. The entire assay was accomplished in less than 90â¯min and no cross-reactivity with the other tested bacterial species was observed. Moreover, the developed assay has been applied for the detection of S. aureus in three different types of real samples (sodium chloride injection, apple juice and human urine) with satisfactory results. To the best of our knowledge, it is the first time to report using chicken anti-protein A IgY and any IgG to detect S. aureus based on the dual-recognition mode of SPA. The novel method opened up a way for monitoring S. aureus in food samples with high sensitivity, specificity and simple operation.
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Técnicas Biosensibles/métodos , Colorimetría/métodos , Inmunoglobulinas/química , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/aislamiento & purificación , Animales , Pollos , Análisis de los Alimentos/métodos , Jugos de Frutas y Vegetales/microbiología , Humanos , Inmunoglobulina G/química , Límite de Detección , Infecciones Estafilocócicas/orinaRESUMEN
Numerous crystal structures of HIV gp120 have been reported, alone or with receptor CD4 and cognate antibodies; however, no sole gp120/CD4 complex without stabilization by an antibody is available. Here, we report a crystal structure of the gp120/CD4 complex without the aid of an antibody from HIV-1 CRF07_BC, a strain circulating in China. Interestingly, in addition to the canonical binding surface, a second interacting interface was identified. A mutagenesis study on critical residues revealed that the stability of this interface is important for the efficiency of Env-mediated membrane fusion. Furthermore, we found that a broad neutralizing antibody, ibalizumab, which targets CD4 in the absence of gp120, occupies the same binding surface as the second interface identified here on gp120. Therefore, we identified the possibility of the involvement of a second gp120-CD4 interaction interface during viral entry, and also provided a reasonable explanation for the broad activity of neutralizing antibody ibalizumab.
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Antígenos CD4/química , Proteína gp120 de Envoltorio del VIH/química , VIH-1/metabolismo , Dominios Proteicos , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/farmacología , Antígenos CD4/metabolismo , Células COS , Línea Celular , Línea Celular Tumoral , Chlorocebus aethiops , Cristalografía por Rayos X , Células HEK293 , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , VIH-1/genética , Células HeLa , Humanos , Simulación de Dinámica Molecular , Unión Proteica/efectos de los fármacos , Internalización del Virus/efectos de los fármacosRESUMEN
The protein elicitor MoHrip2, which was extracted from Magnaporthe oryzae as an exocrine protein, triggers the tobacco immune system and enhances blast resistance in rice. However, the detailed mechanisms by which MoHrip2 acts as an elicitor remain unclear. Here, we investigated the structure of MoHrip2 to elucidate its functions based on molecular structure. The three-dimensional structure of MoHrip2 was obtained. Overall, the crystal structure formed a ß-barrel structure and showed high similarity to the pathogenesis-related (PR) thaumatin superfamily protein thaumatin-like xylanase inhibitor (TL-XI). To investigate the functional regions responsible for MoHrip2 elicitor activities, the full length and eight truncated proteins were expressed in Escherichia coli and were evaluated for elicitor activity in tobacco. Biological function analysis showed that MoHrip2 triggered the defense system against Botrytis cinerea in tobacco. Moreover, only MoHrip2M14 and other fragments containing the 14 amino acids residues in the middle region of the protein showed the elicitor activity of inducing a hypersensitive response and resistance related pathways, which were similar to that of full-length MoHrip2. These results revealed that the central 14 amino acid residues were essential for anti-pathogenic activity.