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The high transmissibility of SARS-CoV-2 Omicron subvariants was generally ascribed to immune escape. It remained unclear whether the emerging variants have gradually acquired replicative fitness in human respiratory epithelial cells. We sought to evaluate the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited a dramatically increased replicative capacity and infectivity than B.1.1.529 and an ancestral strain wildtype (WT) in human nasal and airway organoids. BA.5 spike pseudovirus showed a significantly higher entry efficiency than that carrying WT or B.1.1.529 spike. Notably, we observed prominent syncytium formation in BA.5-infected nasal and airway organoids, albeit elusive in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of spike in nasal organoids. Moreover, BA.5 replicated modestly in alveolar organoids, with a significantly lower titer than B.1.1.529 and WT. Collectively, the higher entry efficiency and fusogenic activity of BA.5 spike potentiated viral spread through syncytium formation in the human airway epithelium, leading to enhanced replicative fitness and immune evasion, whereas the attenuated replicative capacity of BA.5 in the alveolar organoids may account for its benign clinical manifestation.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/genética , Nariz , Organoides , Glicoproteína da Espícula de Coronavírus/genética , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
Inhibition of twisting intramolecular charge transfer (TICT) is one of the most attractive methods for fluorescence-on analysis, whereas it remains enigmatic whether the fluorescence in a TICT-based probe could be thoroughly lightened. Here, for maximizing the fluorescence-on signal of the TICT-based probe, we develop a model by employing chemical reaction to directly cleave the linkage between the rotational electron donor and acceptor with a predisposed fluorescent signal close to zero. To validate this assumption, a nonfluorescent probe with barrierless rotation is successfully achieved by grafting acryloyl with -CâC- recognition sites onto coumarin, and 7-hydroxycoumarin with bright blue fluorescence could be released within 3 s upon probing KMnO4 with an amount as low as 0.95 nM and 6.6 pg. We believe that the present strategy could not only deepen the insights of photochemistry but also facilitate the development of a theranostic drug delivery system, energy conversion, pollution control, and health risk reduction.
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Corantes , Fluorescência , Fotoquímica , RotaçãoRESUMO
Reported herein is a highly active and durable hydrogen evolution reaction (HER) electrocatalyst, which is constructed following a tandem interface strategy and functional in alkaline and even neutral medium (pH ≈ 7). The ternary composite material, consisting of conductive nickel foam (NF) substrate, Ni3 S2 -MoS2 heterostructure, and TiO2 coating, is synthesized by the hydrothermal method and atomic layer deposition (ALD) technique. Representative results include: (1) versatile characterizations confirm the proposed composite structure and strong electronic interactions among comprised sulfide and oxide species; (2) the material outperforms commercial Pt/C by recording an overpotential of 115 mV and a Tafel slope of 67 mV dec-1 under neutral conditions. A long-term stability in alkaline electrolytes up to 200 h and impressive overall water splitting behavior (1.56 V @ 10 mA cm-2 ) are documented; (3) implementation of ALD oxide tandem layer is crucial to realize the design concept with superior HER performance by modulating a variety of heterointerface and intermediates electronic structure.
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Foamy macrophages containing prominent cytoplasmic lipid droplets (LDs) are found in a variety of infectious diseases. However, their role in Streptococcus uberis-induced mastitis is unknown. Herein, we report that S. uberis infection enhances the fatty acid synthesis pathway in macrophages, resulting in a sharp increase in LD levels, accompanied by a significantly enhanced inflammatory response. This process is mediated by the involvement of fatty acid binding protein 4 (FABP4), a subtype of the fatty acid-binding protein family that plays critical roles in metabolism and inflammation. In addition, FABP4 siRNA inhibitor cell models showed that the deposition of LDs decreased, and the mRNA expression of Tnf, Il1b and Il6 was significantly downregulated after gene silencing. As a result, the bacterial load in macrophages increased. Taken together, these data demonstrate that macrophage LD formation is a host-driven component of the immune response to S. uberis. FABP4 contributes to promoting inflammation via LDs, which should be considered a new target for drug development to treat infections.
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Doenças dos Bovinos , Mastite Bovina , Infecções Estreptocócicas , Feminino , Animais , Bovinos , Gotículas Lipídicas/metabolismo , Macrófagos/microbiologia , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Inflamação/metabolismo , Inflamação/veterinária , Infecções Estreptocócicas/veterinária , Mastite Bovina/microbiologia , Doenças dos Bovinos/metabolismoRESUMO
Mammary gland-derived Escherichia coli is an important pathogen causing dairy cow mastitis. Mammary gland mucosal immunity against infectious E. coli mainly depends on recognition of pathogen-associated molecular patterns by innate receptors. Stimulator of interferon (IFN) gene (STING) has recently been the dominant mediator in reacting to bacterial intrusion and preventing inflammatory disorders. In this study, we first proved that the diguanylate cyclase YeaJ relieves mouse mammary gland pathological damage by changing E. coli phenotypic and host STING-dependent innate immunity responses. YeaJ decreases mammary gland circular vacuoles, bleeding, and degeneration in mice. In addition, YeaJ participates in STING-IRF3 signaling to regulate inflammation in vivo. In vitro, YeaJ decreases damage to macrophages (RAW264.7) but not to mouse mammary epithelial cells (EpH4-Ev). Consistent with the results in mouse mammary glands, YeaJ significantly activates the STING/TBK1/IRF3 pathway in RAW264.7 macrophages as well. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study highlights a novel role for YeaJ in E. coli infection, which provides a better understanding of host-bacterium interactions and potential prophylactic strategies for infections. IMPORTANCE E. coli is the etiological agent of environmental mastitis in dairy cows, which causes massive financial losses worldwide. However, the pathophysiological role of YeaJ in the interaction between E. coli and host remains unclear. We found that YeaJ significantly influences various biological characteristics and suppresses severe inflammatory response as well as greater damage. YeaJ alleviates damage to macrophages (RAW264.7) and mouse mammary gland. Moreover, these effects of YeaJ are achieved at least partial by mediating the STING-IRF3 signaling pathway. In conclusion, the deletion of yeaJ facilitates E. coli NJ17 escape from STING-dependent innate immunity recognition in vitro and in vivo. This study is the basis for further research to better understand host-bacterium interactions and provides potential prophylactic strategies for infections.
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Células Epiteliais/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/imunologia , Escherichia coli/metabolismo , Macrófagos/microbiologia , Fósforo-Oxigênio Liases/metabolismo , Animais , Biofilmes/crescimento & desenvolvimento , Adesão Celular , Proteínas de Escherichia coli/genética , Feminino , Regulação Bacteriana da Expressão Gênica/imunologia , Glândulas Mamárias Animais/citologia , Camundongos , Movimento , Mutação , Fósforo-Oxigênio Liases/genética , Células RAW 264.7RESUMO
Metabolic alterations occur in pathogenic infections, but the role of lipid metabolism in the progression of bacterial mastitis is unclear. Cross talk between lipid droplets (LDs) and invading bacteria occurs, and targeting of de novo lipogenesis inhibits pathogen reproduction. In this study, we investigate the role(s) of lipid metabolism in mammary cells during Streptococcus uberis infection. Our results indicate that S. uberis induces the synthesis of fatty acids and production of LDs. Importantly, taurine reduces fatty acid synthesis, the abundance of LDs and the in vitro bacterial load of S. uberis These changes are mediated, at least partly, by the E3 ubiquitin ligase IDOL, which is associated with the degradation of low-density lipoprotein receptors (LDLRs). We have identified a critical role for IDOL-mediated fatty acid synthesis in bacterial infection, and we suggest that taurine may be an effective prophylactic or therapeutic strategy for preventing S. uberis mastitis.
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Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus/efeitos dos fármacos , Taurina/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Proteólise , Receptores de LDLRESUMO
Reported herein is comprehensive study of a highly active and stable cobalt catalyst for overall water splitting. This composite SFCNF/Co1- x S@CoN, consisting of S-doped flexible carbon nanofiber (SFCNF) matrix, Co1- x S nanoparticles, and CoN coatings, is prepared by integration of electrospinning and atomic layer deposition (ALD) technique. Representative results include the following: 1) ultrathin CoN layer is deposited by ALD on the surface of flexible substrate without any sacrifice of SFCNF and Co1- x S; 2) the composite exhibits strong electrocatalytic activity in both acidic and basic solutions. The overpotentials of hydrogen evolution reaction (HER) and oxygen evolution reaction (OER) are 20 and 180 mV, respectively, at a current density of 10 mA cm-2 in basic medium. A small Tafel slope of 54.4 mV dec-1 is observed in 0.5 m H2 SO4 electrolyte; 3) tested as overall water splitting electrode, the composite records a current density of 10 mA cm-2 at a relative low cell voltage of 1.58 V and long-term stability for 20 h at a current density of up to 50 mA cm-2 . The superior performance for overall water splitting is probably attributed to the synergistic effect of Co1- x S and ALD CoN. Specifically, implementation of ALD can be extended to innovate nanostructured materials for overall water splitting and even other renewable energy aspects.
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With potentially high lithium (Li) exchange capacity and long cycle ability, Ti-based oxides of H2TiO3 and H4Ti5O12 are considered to be promising Li-ion sieve (LIS) materials applied for Li resource extraction in the liquid phase. However, the LISs usually demonstrate unsatisfactory Li exchange performance under the approximately neutral condition without the strong impetus derived from the rapid combination between OH- in the surrounding solution and H+ ionized from LIS. Herein, a hybrid of H2TiO3/H4Ti5O12 with rich phase boundaries is constructed via a facile one-step solid-state method. Owing to the different Fermi energy levels of the two phases, the electrons are transferred at the phase interface between H2TiO3 and H4Ti5O12, developing an internal electric field (IEF). The built IEF provides an extra driving force to boost the solid-phase Li+ transport, hence enhancing the Li extraction kinetics. Threrfore, the H2TiO3/H4Ti5O12 hybrid exhibits outstanding Li exchange performance of 42.43 and 20.50 mg g-1 under alkaline and neutral conditions, corresponding to the hightest Li extraction rate of 5.30 and 2.05 mg g-1 h-1 reported so far. Our work offers an innovative strategy to promote the Li exchange performance of LIS especially under neutral conditions.
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The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.
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COVID-19 , Humanos , SARS-CoV-2 , Pulmão , OrganoidesRESUMO
A robust in vitro model of the human respiratory epithelium, including the alveolar and the airway epithelium, is essential for understanding the biology and pathology of the human respiratory system. We previously described a protocol to derive human lung organoids from primary lung tissues. We now describe a protocol to induce bidirectional differentiation to generate mature alveolar or airway organoids. The lung organoids are consecutively expanded for over one year with high stability, while the differentiated alveolar and airway organoids morphologically and functionally simulate the human alveolar and airway epithelium to a near-physiological level. Thus, we establish a robust organoid culture system of the entire human respiratory epithelium, the first two-phase bipotential organoid culture system that enables long-term expansion and bidirectional differentiation of respiratory epithelial cells. The long-term expandable lung organoids and differentiated organoids generate a stable and renewable source of respiratory epithelial cells, enabling scientists to reconstruct and expand the human respiratory epithelium in culture dishes. The respiratory organoid system provides a unique and physiologically active in vitro model of the human respiratory epithelium for various applications, including studying respiratory viral infection, disease modeling, drug screening, and pre-clinical testing. Graphical abstract.
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Reported herein is the atomic layer deposition (ALD) of novel ternary ZnCoxOy films possessing p-type semiconducting behavior. The preparation comprises of optimized ZnO and Co3O4 deposition in sub-cycles using the commercially available precursors cyclopentadienylcobalt dicarbonyl (CpCo(CO)2), diethylzinc (DEZ) and ozone (O3). A systematic exploration of the film's microstructure, crystallinity, optical properties and electrical properties was conducted and revealed an association with Zn/Co stoichiometry. The noteworthy results include the following: (1) by adjusting the sub-cycle of ZnO/ Co3O4 to 1/10, a spinel structured ZnCoxOy film was grown at 150 °C, with it exhibiting a smooth surface, good crystallinity and high purity; (2) the material transmittance and bandgap decreased as the Co element concentration increased; (3) the ZnCoxOy film is more stable than its p-type analog Co3O4 film; and (4) upon p-n diode fabrication, the ZnCoxOy film demonstrated good rectification behaviors as well as very low and stable reverse leakage in forward and reverse-biased voltages, respectively. Its application in thin film transistors and flexible or transparent semiconductor devices is highly suggested.
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The lack of a robust in vitro model of the human respiratory epithelium hinders the understanding of the biology and pathology of the respiratory system. We describe a defined protocol to derive human lung organoids from adult stem cells in the lung tissue and induce proximal differentiation to generate mature airway organoids. The lung organoids are then consecutively expanded for over 1 year with high stability, while the differentiated airway organoids are used to morphologically and functionally simulate human airway epithelium to a near-physiological level. Thus, we establish a robust organoid model of the human airway epithelium. The long-term expansion of lung organoids and differentiated airway organoids generates a stable and renewable source, enabling scientists to reconstruct and expand the human airway epithelial cells in culture dishes. The human lung organoid system provides a unique and physiologically active in vitro model for various applications, including studying virus-host interaction, drug testing, and disease modeling.
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Pulmão , Organoides , Adulto , Diferenciação Celular , Células Epiteliais , Humanos , TóraxRESUMO
The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.
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COVID-19 , Mucosa Nasal , Organoides , SARS-CoV-2 , COVID-19/virologia , Humanos , Mucosa Nasal/virologia , Organoides/virologia , SARS-CoV-2/classificação , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Técnicas de Cultura de TecidosRESUMO
Horseshoe bats host numerous SARS-related coronaviruses without overt disease signs. Bat intestinal organoids, a unique model of bat intestinal epithelium, allow direct comparison with human intestinal organoids. We sought to unravel the cellular mechanism(s) underlying bat tolerance of coronaviruses by comparing the innate immunity in bat and human organoids. We optimized the culture medium, which enabled a consecutive passage of bat intestinal organoids for over one year. Basal expression levels of IFNs and IFN-stimulated genes were higher in bat organoids than in their human counterparts. Notably, bat organoids mounted a more rapid, robust and prolonged antiviral defense than human organoids upon Poly(I:C) stimulation. TLR3 and RLR might be the conserved pathways mediating antiviral response in bat and human intestinal organoids. The susceptibility of bat organoids to a bat coronavirus CoV-HKU4, but resistance to EV-71, an enterovirus of exclusive human origin, indicated that bat organoids adequately recapitulated the authentic susceptibility of bats to certain viruses. Importantly, TLR3/RLR inhibition in bat organoids significantly boosted viral growth in the early phase after SARS-CoV-2 or CoV-HKU4 infection. Collectively, the higher basal expression of antiviral genes, especially more rapid and robust induction of innate immune response, empowered bat cells to curtail virus propagation in the early phase of infection.
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COVID-19 , Quirópteros , Viroses , Animais , Humanos , Quirópteros/genética , Antivirais/farmacologia , Receptor 3 Toll-Like/genética , SARS-CoV-2 , Organoides , Terapia de ImunossupressãoRESUMO
The airways and alveoli of the human respiratory tract are lined by two distinct types of epithelium, which are the primary targets of respiratory viruses. We previously established long-term expanding human lung epithelial organoids from lung tissues and developed a 'proximal' differentiation protocol to generate mucociliary airway organoids. However, a respiratory organoid system with bipotential of the airway and alveolar differentiation remains elusive. Here we defined a 'distal' differentiation approach to generate alveolar organoids from the same source for the derivation of airway organoids. The alveolar organoids consisting of type I and type II alveolar epithelial cells (AT1 and AT2, respectively) functionally simulate the alveolar epithelium. AT2 cells maintained in lung organoids serve as progenitor cells from which alveolar organoids derive. Moreover, alveolar organoids sustain a productive SARS-CoV-2 infection, albeit a lower replicative fitness was observed compared to that in airway organoids. We further optimized 2-dimensional (2D) airway organoids. Upon differentiation under a slightly acidic pH, the 2D airway organoids exhibit enhanced viral replication, representing an optimal in vitro correlate of respiratory epithelium for modeling the high infectivity of SARS-CoV-2. Notably, the higher infectivity and replicative fitness of the Omicron variant than an ancestral strain were accurately recapitulated in these optimized airway organoids. In conclusion, we have established a bipotential organoid culture system able to reproducibly expand the entire human respiratory epithelium in vitro for modeling respiratory diseases, including COVID-19.
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Streptococcus uberis (S. uberis) is an important pathogen causing mastitis, which causes continuous inflammation and dysfunction of mammary glands and leads to enormous economic losses. Most research on infection continues to be microbial metabolism-centric, and many overlook the fact that pathogens require energy from host. Mouse is a common animal model for studying bovine mastitis. In this perspective, we uncover metabolic reprogramming during host immune responses is associated with infection-driven inflammation, particularly when caused by intracellular bacteria. Taurine, a metabolic regulator, has been shown to effectively ameliorate metabolic diseases. We evaluated the role of taurine in the metabolic regulation of S. uberis-induced mastitis. Metabolic profiling indicates that S. uberis exposure triggers inflammation and metabolic dysfunction of mammary glands and mammary epithelial cells (the main functional cells in mammary glands). Challenge with S. uberis upregulates glycolysis and oxidative phosphorylation in MECs. Pretreatment with taurine restores metabolic homeostasis, reverses metabolic dysfunction by decrease of lipid, amino acid and especially energy disturbance in the infectious context, and alleviates excessive inflammatory responses. These outcomes depend on taurine-mediated activation of the AMPK-mTOR pathway, which inhibits the over activation of inflammatory responses and alleviates cellular damage. Thus, metabolic homeostasis is essential for reducing inflammation. Metabolic modulation can be used as a prophylactic strategy against mastitis.
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Metabolismo Energético/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Mastite/prevenção & controle , Infecções Estreptocócicas/prevenção & controle , Streptococcus/patogenicidade , Taurina/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Glândulas Mamárias Animais/imunologia , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/microbiologia , Mastite/imunologia , Mastite/metabolismo , Mastite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Transdução de Sinais , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/microbiologia , Streptococcus/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Mammary gland-derived Escherichia coli (E. coli) is an important pathogen causing dairy cow mastitis. YdiV, with EAL-like domains, inhibits flagellum biogenesis and motility and affects c-di-GMP (eubacterial signaling molecule) concentration changes in bacteria. However, the pathophysiological role of ydiV in host-pathogen cross-talk still needs to be elucidated. In this study, firstly constructed the ydiV mutant (NJ17ΔydiV) and ydiV complementary (cNJ17ΔydiV) E. coli strains to infect mouse mammary epithelial cells (EpH4-Ev) and macrophages (RAW264.7), as well as mouse mammary glands, respectively. Then biological characteristics, adaptor molecules in related signaling pathways, proinflammatory cytokines and the extent of host cell damage was evaluated. Compared with E. coli NJ17 infected mice, the bacterial load in the mammary gland of NJ17ΔydiV was significantly lower and the extent of the damage was alleviated. Notably, the deletion of ydiV significantly aggravated cell damage in RAW264.7 cells and compared with the wild-type strain, NJ17ΔydiV significantly activated the STING/TBK1/IRF3 pathway in macrophages. In EpH4-Ev cells, although STING did not sense E. coli NJ17 invasion, IRF3 was activated by the NJ17ΔydiV strain. Taken together, ydiV deletion significantly affects a variety of biological characteristics and induces severe cell damage, while the STING/TBK1/IRF3 pathway actively participated in pathogen elimination in the host. This study highlights a new role for ydiV in E. coli infection and provides a foundation for further studies to better understand host-bacteria interactions and potential prophylactic strategies for infectious diseases.
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Proteínas de Transporte/metabolismo , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Evasão da Resposta Imune/genética , Animais , Carga Bacteriana , Proteínas de Transporte/genética , Linhagem Celular , Células Epiteliais/imunologia , Células Epiteliais/virologia , Proteínas de Escherichia coli/genética , Feminino , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Fator Regulador 3 de Interferon/imunologia , Glândulas Mamárias Humanas/citologia , Glândulas Mamárias Humanas/virologia , Proteínas de Membrana/imunologia , Camundongos , Mutação , Proteínas Serina-Treonina Quinases/imunologia , Células RAW 264.7RESUMO
Mammary epithelial cells (MECs) play an important role against Streptococcus uberis infection which is one of the main causes of bovine mastitis and a potential threat to human health. Toll-like receptors (TLRs) and their mediated signaling pathways are critical in both innate and infection responses, yet their roles in anti-S. uberis infection in MECs remains poorly defined. In this work we investigated the regulatory mechanisms of TLR2 in inflammatory responses, where WT and TLR2-/- mice were euthanized at 15-18 days gestation, and mammary gland tissues were collected aseptically. The mouse MECs (MMECs) were isolated by combined digestion with type I collagenase, hyaluronidase and trypsin. We challenged MMECs with S. uberis and quantified antioxidant capacity as well as reactive oxygen species (ROS), proinflammatory cytokines and cell damage at different times. The loss of TLR2 function in MMECs results in more serious cell damage, increased cell adhesion, and significantly decreased ROS and mitochondrial ROS (mROS) with bactericidal function in response to S. uberis infection. Moreover, it was observed that the antioxidant capacity declined, and the production of TLR2-mediated cytokines (except CXC ligand 15) also were reduced. We demonstrated that TLR2 can mediate cellular anti-infective processes in MMECs by regulating the production of ROS and mROS and the secretion of cytokines. The results suggest an unpredicted role of TLR2 in MMECs in response to S. uberis infection.
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Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/patologia , Infecções Estreptocócicas/imunologia , Streptococcus/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Apoptose , Células Cultivadas , Células Epiteliais/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estresse Oxidativo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/genéticaRESUMO
Mastitis caused by Streptococcus uberis (S. uberis) is a common and difficult-to-cure clinical disease in dairy cows. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2-/- and TLR4-/- mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage, and bacterial count. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host in controlling the secretion of inflammatory factors and the elimination of intracellular S. uberis. Our results demonstrated that TLR2-mediated mROS has a significant effect on S. uberis-induced host defense responses in mammary glands as well as in MECs.
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Mastite/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Masculino , Glândulas Mamárias Animais/microbiologia , Glândulas Mamárias Animais/patologia , Mastite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Organismos Livres de Patógenos Específicos , Infecções Estreptocócicas/microbiologia , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Multilayered hard coatings with a CrN matrix and an Al2O3, TiO2, or nanolaminate-Al2O3/TiO2 sealing layer were designed by a hybrid deposition process combined with physical vapor deposition (PVD) and atomic layer deposition (ALD). The strategy was to utilize ALD thin films as pinhole-free barriers to seal the intrinsic defects to protect the CrN matrix. The influences of the different sealing layers added in the coatings on the microstructure, surface roughness, and corrosion behaviors were investigated. The results indicated that the sealing layer added by ALD significantly decreased the average grain size and improved the corrosion resistance of the CrN coatings. The insertion of the nanolaminate-Al2O3/TiO2 sealing layers resulted in a further increase in corrosion resistance, which was attributed to the synergistic effect of Al2O3 and TiO2, both acting as excellent passivation barriers to the diffusion of corrosive substances.