RESUMO
Microplastics are ubiquitous in the natural environment, especially in waters, and their potential impact is also a key issue of concern. In this study, we used 1 µm, 1000 µg/L, polystyrene (PS-MPs) particles to analyze the effects after exposure for 14 and 28 days in rare minnow (Gobiocypris rarus). Results indicated that PS-MPs induce structural alterations in the intestinal tissue, including epithelial damage, villi damage and the inflammatory cell infiltration, while the changes were severer after exposure for 28 days. Polystyrene microplastics also significantly increased the activities of catalase (CAT, increased 142 % and 385 % in 14d and 28d), superoxide dismutase (SOD, increased 17.76 % and 23.43 % in the 14d and 28d) and the content of malondialdehyde (MDA, increased 14.5 % and 442 % in the 14d and 28d), glutathione (GSH, increased 146 % and 298 % in the 14d and 28d). The results not only showed the characterization of gut microbial communities in rare minnow, but also indicated that microbial diversity and composition were altered in gut of fish exposed to PS-MPs. In the control groups, Proteobacteria (31.36-54.54 %), Actinobacteriota (39.99-52.54 %), Fusobacteriota (1.43-1.78 %), Bacteriadota (0.31-0.57 %) were the four dominant bacterial phyla in the intestinal of rare minnow. After exposure to microplastics, In the gut microbiota, the proportion of Proteobacteria increased 9.27 % and 30 % with exposure time, while Actinobacteria decreased 37.89 % and significantly different after 28 days. In addition, metabolomic analysis suggested that exposure to PS-MPs induced alterations of metabolic profiles in rare minnow and differential metabolites were involved in energy metabolism, inflammatory responsible secretion, oxidative stress, nucleotide and its metabolomics. In conclusion, our findings suggest that long-term exposure to microplastics could induce intestinal inflammation, oxidative stress, microbiota dysbiosis and metabolic disorder in rare minnow, and the alterations and severity were exacerbated by prolonged exposure. This study has extended our cognition of the toxicity of polystyrene, and enriched theoretical data for exploring the toxicological mechanism of microplastics.
Assuntos
Cyprinidae , Poluentes Químicos da Água , Animais , Microplásticos/toxicidade , Plásticos/toxicidade , Plásticos/metabolismo , Poliestirenos/toxicidade , Poliestirenos/metabolismo , Disbiose/induzido quimicamente , Cyprinidae/metabolismo , Estresse Oxidativo , Glutationa/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
Microplastics are environmental contaminants and an emergent concern. Microplastics are abundant in freshwater and can cause biochemical stress in freshwater organisms. In the current study, rare minnows (Gobiocypris rarus) were exposed to 1µm polystyrene microplastics at 200 µg/L concentration. We observed various sublethal effects after four weeks of exposure but no mortality. Numerous cellular and tissue alterations were observed in the liver. Differential metabolites and differentially expressed genes between control and exposure groups were identified and mapped to pathways in the Kyoto Encyclopedia of Genes and Genomes. The combination of transcriptomic and metabolomic analyses revealed significantly varied metabolic pathways between the two groups. These pathways were involved in glucolipid, amino acid, and nucleotide metabolism. Results demonstrated that MP exposure induced immune reaction, oxidative stress, and disturbed glycolipid and energy metabolism. The current study provided novel insights into the molecular and metabolic mechanisms of microplastic ecotoxicology in rare minnow.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/metabolismo , Metaboloma , Microplásticos/toxicidade , Estresse Oxidativo , Transcriptoma , Poluentes Químicos da Água/toxicidade , Animais , Cyprinidae , Fígado/efeitos dos fármacosRESUMO
Enterovirus 71 (EV71) can cause hand-foot-and-mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remains poorly understood. Here, we report that EV71 induces degradation of gasdermin D (GSDMD), an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1 to 193 (GSDMD1-193). However, unlike the N-terminal fragment produced by caspase-1 cleavage, this fragment fails to trigger cell death or inhibit EV71 replication. Importantly, a T239D or F240D substitution abrogates the activity of GSDMD consisting of amino acids 1 to 275 (GSDMD1-275). This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response.IMPORTANCE Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated interleukin-1ß (IL-1ß) secretion. In this process, the N-terminal domain of p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection downregulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N-terminal fragment disrupted for inducing cell pyroptosis. Notably, GSDMD1-275 (p30) inhibits EV71 replication whereas GSDMD1-193 does not. These results reveal a new strategy for EV71 to evade the antiviral response.
Assuntos
Enterovirus Humano A/fisiologia , Interações Hospedeiro-Patógeno , Proteínas de Neoplasias/metabolismo , Piroptose , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Ligação a Fosfato , Ligação Proteica , ProteóliseRESUMO
UNLABELLED: Enterovirus 71 (EV71) causes hand, foot, and mouth disease in young children and infants. Severe infection with EV71 can lead to various neurological complications or fatal diseases. However, the mechanism of EV71 pathogenesis is poorly understood. Emerging evidence suggests that EV71 modulates type I interferon (IFN) and cytokine responses. Here, we show that EV71 disables components of the TAB2 complex through the 3C protein. When expressed in mammalian cells, EV71 3C interacts with TAB2 and TAK1, which inhibits NF-κB activation. Furthermore, 3C mediates cleavage of TAB2 and its partners, which requires the protease activity. H40D or C147S substitution in the 3C active sites abolishes its activity, whereas R84Q or V154S substitution in the RNA binding domain has no effect. The 3C protein targets TAB2 at Q113-S114, TAK1 at Q360-S361, TAB1 both at Q414-G415 and Q451-S452, and TAB3 at Q173-G174 and Q343-G344. Importantly, overexpression of TAB2 inhibits EV71 replication, whereas addition of cleaved fragments has no effect. Thus, an equilibrium between the TAB2 complex and EV71 3C represents a control point of viral infection. These results suggest that TAK1/TAB1/TAB2/TAB3 cleavage mediated by EV71 may be a mechanism to interfere with inflammatory responses. IMPORTANCE: The TAK1 complex plays a critical role in the activation of NF-κB and cytokine production. However, little is known about its connection to enterovirus 71 (EV71). We demonstrate that EV71 3C suppresses cytokine expression via cleavage of the TAK1 complex proteins. EV71 3C interacts with TAB2 and TAK1. Furthermore, overexpression of TAB2 inhibits EV71 replication, whereas addition of cleaved fragment has no effect. These results suggest that the interplay of EV71 and the TAK1 complex influences the outcome of viral infection.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cisteína Endopeptidases/metabolismo , Citocinas/antagonistas & inibidores , Enterovirus Humano A/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Virais/metabolismo , Proteases Virais 3C , Substituição de Aminoácidos , Linhagem Celular , Cisteína Endopeptidases/genética , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Humanos , Hidrólise , Evasão da Resposta Imune , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Virais/genéticaRESUMO
Enterovirus 71 (EV71) is the major causative pathogen of hand, foot, and mouth disease (HFMD). Its pathogenicity is not fully understood, but innate immune evasion is likely a key factor. Strategies to circumvent the initiation and effector phases of anti-viral innate immunity are well known; less well known is whether EV71 evades the signal transduction phase regulated by a sophisticated interplay of cellular and viral proteins. Here, we show that EV71 inhibits anti-viral type I interferon (IFN) responses by targeting the mitochondrial anti-viral signaling (MAVS) protein--a unique adaptor molecule activated upon retinoic acid induced gene-I (RIG-I) and melanoma differentiation associated gene (MDA-5) viral recognition receptor signaling--upstream of type I interferon production. MAVS was cleaved and released from mitochondria during EV71 infection. An in vitro cleavage assay demonstrated that the viral 2A protease (2A(pro)), but not the mutant 2A(pro) (2A(pro)-110) containing an inactivated catalytic site, cleaved MAVS. The Protease-Glo assay revealed that MAVS was cleaved at 3 residues between the proline-rich and transmembrane domains, and the resulting fragmentation effectively inactivated downstream signaling. In addition to MAVS cleavage, we found that EV71 infection also induced morphologic and functional changes to the mitochondria. The EV71 structural protein VP1 was detected on purified mitochondria, suggesting not only a novel role for mitochondria in the EV71 replication cycle but also an explanation of how EV71-derived 2A(pro) could approach MAVS. Taken together, our findings reveal a novel strategy employed by EV71 to escape host anti-viral innate immunity that complements the known EV71-mediated immune-evasion mechanisms.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais/farmacologia , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Interferon Tipo I/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Enterovirus Humano A/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Inibidores de Proteases/farmacologia , Infecções por Vírus de RNA , Rabdomiossarcoma , Transdução de SinaisRESUMO
Colorectal cancer (CRC) is a common type of gastrointestinal tract (GIT) cancer and poses an enormous threat to human health. Current strategies for metastatic colorectal cancer (mCRC) therapy primarily focus on chemotherapy, targeted therapy, immunotherapy, and radiotherapy; however, their adverse reactions and drug resistance limit their clinical application. Advances in nanotechnology have rendered lipid nanoparticles (LNPs) a promising nanomaterial-based drug delivery system for CRC therapy. LNPs can adapt to the biological characteristics of CRC by modifying their formulation, enabling the selective delivery of drugs to cancer tissues. They overcome the limitations of traditional therapies, such as poor water solubility, nonspecific biodistribution, and limited bioavailability. Herein, we review the composition and targeting strategies of LNPs for CRC therapy. Subsequently, the applications of these nanoparticles in CRC treatment including drug delivery, thermal therapy, and nucleic acid-based gene therapy are summarized with examples provided. The last section provides a glimpse into the advantages, current limitations, and prospects of LNPs in the treatment of CRC.
Assuntos
Neoplasias Colorretais , Nanopartículas , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/terapia , Nanopartículas/química , Lipídeos/química , Animais , Antineoplásicos/química , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Terapia Genética/métodos , Sistemas de Liberação de Medicamentos/métodos , LipossomosRESUMO
Combination therapy with the synergistic effect is an effective way in cancer chemotherapy. Herein, an antiangiogenic sorafenib (SOR) and hypoxia-activated prodrug tirapazamine (TPZ)-coencapsulated liposome (LipTPZ/SOR) is prepared for chemotherapy of hepatocellular carcinoma (HCC). SOR is a multi-target tyrosine kinase inhibitor that can inhibit tumor cell proliferation and angiogenesis. The antiangiogenesis effect of SOR can reduce oxygen supply and aggravate tumor hypoxia, which is able to activate hypoxia-sensitive prodrug TPZ, exhibiting the synergistic antitumor effect. LipTPZ/SOR at different molar ratios of TPZ and SOR can significantly inhibit the proliferation of hepatocellular carcinoma cells. The mole ratio of TPZ and SOR was optimized to 2:1, which exhibited the best synergetic antitumor effect. The synergistic antitumor mechanism of SOR and TPZ was also investigated in vivo. After treated with SOR, the number of vessels was decreased, and the degree of hypoxia was aggravated in tumor tissues. What is more, in the presence of SOR, TPZ could be activated to inhibit tumor growth. The combination of TPZ and SOR exhibited an excellent synergistic antitumor effect. This research not only provides an innovative strategy to aggravate tumor hypoxia to promote TPZ activation but also paints a blueprint about a new nanochemotherapy regimen for the synergistic chemotherapy of HCC, which has excellent biosafety and bright clinical application prospects.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Pró-Fármacos , Humanos , Tirapazamina/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Sorafenibe/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Lipossomos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Hipóxia/tratamento farmacológico , Pró-Fármacos/farmacologia , Linhagem Celular TumoralRESUMO
We found that immunosuppressive monocytic-myeloid-derived suppressor cells (M-MDSCs) were more likely to be recruited by glioblastoma (GBM) through adhesion molecules on GBM-associated endothelial cells upregulated post-chemoradiotherapy. These cells are continuously generated during tumor progression, entering tumors and expressing PD-L1 at a high level, allowing GBM to exhaust T cells and evade attack from the immune system, thereby facilitating GBM relapse. αLy-6C-LAMP is composed of (i) drug cores with slightly negative charges condensed by cationic protamine and plasmids encoding PD-L1 trap protein, (ii) pre-formulated cationic liposomes targeted to Ly-6C for encapsulating the drug cores, and (iii) a layer of red blood cell membrane on the surface for effectuating long-circulation. αLy-6C-LAMP persistently targets peripheral, especially splenic, M-MDSCs and delivers secretory PD-L1 trap plasmids, leveraging M-MDSCs to transport the plasmids crossing the blood-brain barrier (BBB), thus expressing PD-L1 trap protein in tumors to inhibit PD-1/PD-L1 pathway. Our proposed drug delivery strategy involving intermediaries presents an efficient cross-BBB drug delivery concept that incorporates live-cell targeting and long-circulating nanotechnology to address GBM recurrence.
Assuntos
Antígeno B7-H1 , Barreira Hematoencefálica , Neoplasias Encefálicas , Sistemas de Liberação de Medicamentos , Glioblastoma , Células Supressoras Mieloides , Recidiva Local de Neoplasia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Humanos , Células Supressoras Mieloides/efeitos dos fármacos , Linhagem Celular Tumoral , Recidiva Local de Neoplasia/prevenção & controle , Lipossomos , Camundongos Endogâmicos C57BL , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protaminas/química , Protaminas/administração & dosagem , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismoRESUMO
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene editing has attracted extensive attentions in various fields, however, its clinical application is hindered by the lack of effective and safe delivery system. Herein, we reported a cationic micelle nanoparticle composed of cholesterol-modified branched small molecular PEI (PEI-CHO) and biodegradable PEG-b-polycarbonate block copolymer (PEG-PC), denoted as PEG-PC/PEI-CHO/pCas9, for the CRISPR/Cas9 delivery to realize genomic editing in cancer. Specifically, PEI-CHO condensed pCas9 into nanocomplexes, which were further encapsulated into PEG-PC nanoparticles (PEG-PC/PEI-CHO/pCas9). PEG-PC/PEI-CHO/pCas9 had a PEG shell, protecting DNA from degradation by nucleases. Enhanced cellular uptake of PEG-PC/PEI-CHO/pCas9 nanoparticles was observed as compared to that mediated by Lipo2k/pCas9 nanoparticles, thus leading to significantly elevated transfection efficiency after escaping from endosomes via the proton sponge effect of PEI. In addition, the presence of PEG shell greatly improved biocompatibility, and significantly enhanced the in vivo tumor retention of pCas9 compared to PEI-CHO/pCas9. Notably, apparent downregulation of GFP expression could be achieved both in vitro and in vivo by using PEG-PC/PEI-CHO/pCas9-sgGFP nanoparticles. Furthermore, PEG-PC/PEI-CHO/pCas9-sgMcl1 induced effective apoptosis and tumor suppression in a HeLa tumor xenograft mouse model by downregulating Mcl1 expression. This work may provide an alternative paradigm for the efficient and safe genome editing in cancer.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Micelas , Nanopartículas , Edição de Genes/métodos , Nanopartículas/química , Sistemas CRISPR-Cas/genética , Animais , Humanos , Neoplasias/terapia , Neoplasias/genética , Camundongos Nus , Camundongos , Polietilenoglicóis/química , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Polímeros/químicaRESUMO
Hand, foot, and mouth disease (HFMD) is a common infectious disease in infants and children, especially those under five years of age. EV-A71 is a common pathogen that causes HFMD and the primary pathogen leading to severe or fatal HFMD, which is characterized by neurological complications. However, the underlying mechanisms of EV-A71 pathogenesis remain largely unknown. In this report, we used proteomic and phosphorylated proteomic methods to characterize the proteome and phosphoproteome profiles of EV-A71-infected human neuroblastoma SK-N-SH cells. More than 7744 host proteins and 10069 phosphorylation modification sites were successfully quantified. Among them, 974 proteins and 3648 phosphorylation modification sites were regulated significantly during EV-A71 infection. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that EV-A71 altered cell biological processes, including protein synthesis, RNA splicing and metabolism in SK-N-SH cells. Notably, based on the prediction of upregulated kinases during EV-A71 infection, we identified specific kinase inhibitors approved by the FDA, with ceralasertib, bosutinib, flavin mononucleotide, minocycline, pimasertib and acetylcysteine inhibiting EV-A71 infection. Finally, EV-A71 proteins were found to be phosphorylated during infection, with one site (S184 on 3D polymerase) observed to be crucial for viral replication because a S184A mutation knocked out viral replication. The results improve our understanding of the host response to EV-A71 infection of neuroblastoma cells and provide potential targets for developing anti-EV-A71 strategies.
Assuntos
Enterovirus Humano A , Infecções por Enterovirus , Enterovirus , Doença de Mão, Pé e Boca , Neuroblastoma , Criança , Lactente , Humanos , Proteômica , Enterovirus Humano A/fisiologia , Replicação Viral , Proteoma/farmacologia , Antivirais/farmacologiaRESUMO
A three-dimensional (3D) macroporous reduced graphene oxide/polypyrrole (rGO/Ppy) hydrogel assembled by bacterial cells was fabricated and applied for microbial fuel cells. By taking the advantage of electroactive cell-induced bioreduction of graphene oxide and in-situ polymerization of Ppy, a facile self-assembly by Shewanella oneidensis MR-1and in-situ polymerization approach for 3D rGO/Ppy hydrogel preparation was developed. This facile one-step self-assembly process enabled the embedding of living electroactive cells inside the hydrogel electrode, which showed an interconnected 3D macroporous structures with high conductivity and biocompatibility. Electrochemical analysis indicated that the self-assembly of cell-embedding rGO/Ppy hydrogel enhanced the electrochemical activity of the bioelectrode and reduced the electron charge transfer resistance between the cells and the electrode. Impressively, extremely high power output of 3366 ± 42 mW m-2 was achieved from the MFC with cell-embedding rGO/Ppy hydrogel rGO/Ppy, which was 8.6 times of that delivered from the MFC with bare electrode. Further analysis indicated that the increased cell loading by the hydrogel and improved electrochemical activity by the rGO/Ppy composite would be the underlying mechanism for this performance improvement. This study provided a facile approach to fabricate the biocompatible and electrochemical active 3D nanocomposites for MFC, which would also be promising for performance optimization of various bioelectrochemical systems.
Assuntos
Fontes de Energia Bioelétrica , Fontes de Energia Bioelétrica/microbiologia , Polímeros/química , Pirróis/química , Hidrogéis , EletrodosRESUMO
Enterovirus 71 (EV71) and Coxsackie A16 (CVA16) are two major causative agents of hand, foot, and mouth disease (HFMD) in young children. However, the mechanisms regulating the replication and pathogenesis of EV71/CVA16 remain incompletely understood. We performed a genome-wide CRISPR-Cas9 knockout screen and identified Ragulator as a mediator of EV71-induced apoptosis and pyroptosis. The Ragulator-Rag complex is required for EV71 and CVA16 replication. Upon infection, the Ragulator-Rag complex recruits viral 3D protein to the lysosomal surface through the interaction between 3D and RagB. Disruption of the lysosome-tethered Ragulator-Rag-3D complex significantly impairs the replication of EV71/CVA16. We discovered a novel EV71 inhibitor, ZHSI-1, which interacts with 3D and significantly reduces the lysosomal tethering of 3D. ZHSI-1 treatment significantly represses replication of EV71/CVA16 as well as virus-induced pyroptosis associated with viral pathogenesis. Importantly, ZHSI-1 treatment effectively protects against EV71 infection in neonatal and young mice. Thus, our study indicates that targeting lysosome-tethered Ragulator-Rag-3D may be an effective therapeutic strategy for HFMD.
Assuntos
Enterovirus Humano A , Doença de Mão, Pé e Boca , Proteínas não Estruturais Virais , Animais , Camundongos , Apoptose , Sistemas CRISPR-Cas , Enterovirus Humano A/genética , Lisossomos , Piroptose , Proteínas não Estruturais Virais/genética , Replicação Viral , Doença de Mão, Pé e Boca/virologia , Modelos Animais de DoençasRESUMO
Enterovirus 71 (EV71) causes hand-foot-and-mouth disease and neurological complications in young children. Although the underlying mechanisms remain obscure, impaired or aberrant immunity is thought to play a role. In infected cells, EV71 suppresses type I interferon responses mediated by retinoid acid-inducible gene I (RIG-I). This involves the EV71 3C protein, which disrupts the formation of a functional RIG-I complex. In the present study, we report that EV71 inhibits the induction of innate immunity by Toll-like receptor 3 (TLR3) via a distinct mechanism. In HeLa cells stimulated with poly(I · C), EV71 inactivates interferon regulatory factor 3 and drastically suppresses interferon-stimulated gene expression. Notably, EV71 specifically downregulates a TRIF, TIR domain-containing adaptor inducing beta interferon (IFN-ß). When expressed alone in mammalian cells, EV71 3C is capable of exhibiting these activities. EV71 3C associates with and induces TRIF cleavage in the presence of Z-VAD-FMK, a caspase inhibitor. TRIF cleavage depends on its amino acid pair Q312-S313, which resembles a proteolytic site of picornavirus 3C proteases. Further, site-specific 3C mutants with a defective protease activity bind TRIF but fail to mediate TRIF cleavage. Consequently, these 3C mutants are unable to inhibit NF-κB and IFN-ß promoter activation. TRIF cleavage mediated by EV71 may be a mechanism to impair type I IFN production in response to Toll-like receptor 3 (TLR3) activation.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/imunologia , Enterovirus Humano A/patogenicidade , Interações Hospedeiro-Patógeno , Receptor 3 Toll-Like/imunologia , Proteínas Virais/metabolismo , Proteases Virais 3C , Cisteína Endopeptidases/genética , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Virais/genéticaRESUMO
EV71 is the primary pathogenic cause of hand-foot-mouth disease (HFMD), but an effective antiviral drug currently is unavailable. Rupintrivir, an inhibitor against human rhinovirus (HRV), has potent antiviral activities against EV71. We determined the high-resolution crystal structures of the EV71 3C(pro)/rupintrivir complex, showing that although rupintrivir interacts with EV71 3C(pro) similarly to HRV 3C(pro), the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C(pro). Our structures reveal that EV71 3C(pro) possesses a surface-recessive S2' pocket that is not present in HRV 3C(pro) that contributes to the additional substrate binding affinity. Combined with mutagenic studies, we demonstrated that catalytic Glu71 is irreplaceable for maintaining the overall architecture of the active site and, most importantly, the productive conformation of catalytic His40. We discovered the role of a previously uncharacterized residue, Arg39 of EV71 3C(pro), that can neutralize the negative charge of Glu71, which may subsequently assist deprotonation of His40 during proteolysis.
Assuntos
Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/química , Isoxazóis/química , Isoxazóis/metabolismo , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteases Virais 3C , Sequência de Aminoácidos , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Enterovirus Humano A/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fenilalanina/análogos & derivados , Ligação Proteica , Estrutura Terciária de Proteína , Valina/análogos & derivados , Proteínas Virais/genéticaRESUMO
OBJECTIVES: Three-dimensional radiological anatomic characteristics of condyle trabeculae was obtained quantitatively based on a volume-of-interest (VOI) analysis. METHODS: Nine human mandibular condyle specimens were scanned by micro-computed tomography (micro-CT). A total of 34 VOIs were selected from each condyle specimen, which were divided into six layers and four parts to analyze the morphological characteristics of trabeculae based on cylindrical VOIs with a diameter and height of 2 mm. One-way analysis of variance was used to compare the regional differences of morphological parameters among each layer and part. RESULTS: Values for bone mineral density, bone volume/total volume, trabecular thickness, and trabecular bone number were greater in the anterior part compared with the posterior part; and the lateral part was larger than the medial part in the first, second, and third layers, while the medial part was larger in the fourth and fifth layers; these values in the first and sixth layers were much larger, while those in the third and fourth layers were smaller. Bone surface area/bone volume, trabecular spacing, and trabecular bone pattern factor were larger in the posterior part than in the anterior part; and the lateral part was larger than the medial part in the fourth and fifth layers, while the medial part was larger in the first and second layers. CONCLUSIONS: The morphological distribution of VOIs was anisotropic within trabecular bone of human mandibular condyles. The upper and lower ends of trabecular bone were much more compact, with higher bone density, trabecular thickness, and trabecular number than in the middle layers.
Assuntos
Osso Esponjoso , Côndilo Mandibular , Anisotropia , Densidade Óssea , Osso Esponjoso/diagnóstico por imagem , Humanos , Côndilo Mandibular/diagnóstico por imagem , Microtomografia por Raio-X/métodosRESUMO
BACKGROUND: Loss of motor function in the trapezius muscle is one complication of radical neck dissection after cutting the accessory nerve (AN) during surgery. Nerve repair is an effective method to restore trapezius muscle function, and includes neurolysis, direct suture, and nerve grafting. The suprascapular nerve (SCN) and AN are next to each other in position. The function of the AN and SCN in shoulder elevation and abduction movement is synergistic. SCN might be considered by surgeons for AN reanimation. AIM: To obtain anatomical and clinical data for partial suprascapular nerve-to-AN transfer. METHODS: Ten sides of cadavers perfused with formalin were obtained from the Department of Human Anatomy, Histology and Embryology, Peking University Health Science Center. The SCN (n = 10) and AN (n = 10) were carefully dissected in the posterior triangle of the neck, and the trapezius muscle was dissected to fully display the accessory nerve. The length of the SCN from the origin of the brachial plexus (a point) to the scapular notch (b point) and the distance of the SCN from the origin point (a point) to the point (c point) where the AN entered the border of the trapezius muscle were measured. The length and branches of the AN in the trapezius muscle were measured. A female patient aged 55 years underwent surgery for partial SCN to AN transfer at Department of Oral and Maxillofacial Surgery, Peking University School and Hospital of Stomatology. The patient suffered from recurrent upper gingival cancer. Radical neck dissection was performed on the right side, and the right AN was removed at the intersection between the nerve and the posterior border of the SCM muscle. One-third of the diameter of the SCN was cut off, and combined epineurial and perineurial sutures were applied between the distal end of the cut-off fascicles of the SCN and the proximal end of the AN without tension. Both subjective and objective evaluations were performed before, three months after, and nine months after surgery. For the subjective evaluation, the questionnaire included the Neck Dissection Impairment Index (NDII) and the Constant Shoulder Scale. Electromyography was used for the objective examination. Data were analyzed using t tests with SPSS 19.0 software to determine the relationship between the length of the SCN and the linear distance. A P value of < 0.05 was considered as statistically significant. RESULTS: The whole length of the AN in the trapezius muscle was 16.89 cm. The average numbers of branches distributed in the descending, horizontal and ascending portions were 3.8, 2.6 and 2.2, respectively. The diameter of the AN was 1.94 mm at the anterior border of the trapezius. The length of the suprascapular nerve from the origin of the brachial plexus to the scapular notch was longer than the distance of the suprascapular nerve from the origin point to the point where the accessory nerve entered the upper edge of the trapezius muscle. The amplitude of trapezius muscle electromyography indicated that both the horizontal and ascending portions of the trapezius muscle on the right side had better function than the left side nine months after surgery. The results showed that the right-sided supraspinatus and infraspinatus muscles did not lose more function than the left side. CONCLUSION: Based on anatomical data and clinical application, partial suprascapular nerve-to-AN transfer could be achieved and may improve innervation of the affected trapezius muscle after radical neck dissection.
RESUMO
Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-alpha/beta exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-beta, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-beta activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.
Assuntos
RNA Helicases DEAD-box/antagonistas & inibidores , Enterovirus Humano A/patogenicidade , Tolerância Imunológica , Fator Regulador 3 de Interferon/biossíntese , Interferon Tipo I/biossíntese , Proteínas Virais/imunologia , Fatores de Virulência/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Substituição de Aminoácidos , Proteínas Reguladoras de Apoptose , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Enterovirus Humano A/imunologia , Humanos , Evasão da Resposta Imune , Fator Regulador 3 de Interferon/imunologia , Interferon Tipo I/imunologia , Mutação de Sentido Incorreto , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas de Ligação a RNA , Receptores Imunológicos , Fatores de Transcrição/biossíntese , Fator de Necrose Tumoral alfa/biossíntese , Proteínas Virais/genética , Proteínas Virais/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Incorporating metal elements into polymers is a feasible means to fabricate new materials with multiple functionalities. In this work, a metal coordinated cationic polymer (MCCP) was developed. Ferric ions were incorporated into the polyethyleneimine-ß-cyclodextrin (PC) polymer chain via coordination to produce a zipped-up polymer with a micro-ordered and macro-disordered topological structure. By varying the metal concentration, a tunable superstructure could be formed on the nano-templates via the "zipping" effect. In addition, the physicochemical properties of the assembly of MCCPs and nucleic acids were tailored by tuning the composition of the metal ions and polymers. The loading efficiency of Rhodamine-B by MCCPs was enhanced. The in vitro and in vivo results showed that the hybrid materials could be adjusted to deliver nucleic acids or small molecules with good performance and acquired the capacity of generating reactive oxygen species in tumor cells. Thus, the tunable and multifunctional MCCP system has great potential in nanomedicine and biomaterial science.
Assuntos
Neoplasias do Colo/metabolismo , Complexos de Coordenação/química , Compostos Férricos/química , Nanomedicina , Polímeros/química , Animais , Cátions/química , Células Cultivadas , Neoplasias do Colo/diagnóstico por imagem , Corantes Fluorescentes/química , Humanos , Camundongos , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/metabolismo , Imagem Óptica , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Rodaminas/química , Propriedades de SuperfícieRESUMO
Cytomembrane-derived nanoplatforms are an effective biomimetic strategy in cancer therapy. To improve their functionality and expandability for enhanced vaccination, a eukaryotic-prokaryotic vesicle (EPV) nanoplatform is designed and constructed by fusing melanoma cytomembrane vesicles (CMVs) and attenuated Salmonella outer membrane vesicles (OMVs). Inheriting the virtues of the parent components, the EPV integrates melanoma antigens with natural adjuvants for robust immunotherapy and can be readily functionalized with complementary therapeutics. In vivo prophylactic testing reveals that the EPV nanoformulation can be utilized as a prevention vaccine to stimulate the immune system and trigger the antitumor immune response, combating tumorigenesis. In the melanoma model, the poly(lactic-co-glycolic acid)-indocyanine green (ICG) moiety (PI)-implanted EPV (PI@EPV) in conjunction with localized photothermal therapy with durable immune inhibition shows synergetic antitumor effects as a therapeutic vaccine. The eukaryotic-prokaryotic fusion strategy provides new perspectives for the design of tumor-immunogenic, self-adjuvanting, and expandable vaccine platforms.
Assuntos
Melanoma/prevenção & controle , Nanomedicina/métodos , Fototerapia , Salmonella/química , Vacinação/métodos , Animais , Vacinas Anticâncer/química , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Verde de Indocianina/química , Melanoma/patologia , Camundongos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/químicaRESUMO
Enteroviruses (EVs) have emerged a substantial threat to public health. EVs infection range from mild to severe disease, including mild respiratory illness, diarrhea, poliomyelitis, hand, foot, and mouth disease, aseptic meningitis, and encephalitis. In the Asia-Pacific region, for example, one of the best studied enterovirus 71 (EV71) has been associated with pandemics of hand, foot, and mouth disease (HFMD) in children, particularly those under the age of five. Serious HFMD cases are associated with neurological complications, such as aseptic meningitis, acute flaccid paralysis, brainstem encephalitis, and have been associated with as many as 1000s of deaths in children and infants from 2008 to 2017, in China. More than 90% of laboratory confirmed deaths due to HMFD are associated with EV71. However, little is known about the pathogenesis of EVs. Studies have reported that EVs-infected patients with severe complications show elevated serum concentrations of IL-1ß. The secretion of IL-1ß is mediated by NLRP3 inflammasome during EV71 and CVB3 infection. Enteroviruses 2B and 3D proteins play an important role in activation of NLRP3 inflammasome, while 3C and 2A play important roles in antagonizing the activation of NLRP3 and the secretion of IL-1ß. In this review, we summarize current knowledge regarding the molecular mechanisms that underlie the activation and regulation of the NLRP3 inflammasome, particularly how viral proteins regulate NLRP3 inflammasome activation. These insights into the relationship between the NLRP3 inflammasome and the pathogenesis of EVs infection may ultimately inform the development of novel antiviral drugs.