RESUMO
The endosomal sorting complex required for transport (ESCRT) is a conserved protein machine mediating membrane remodeling and scission. In the context of viral infection, different components of the ESCRT-III complex, which serve as the core machinery to catalyze membrane fission, are involved in diverse viruses' entry, replication, and/or budding. However, the interplay between ESCRT-III and viral factors in the virus life cycle, especially for that of large enveloped DNA viruses, is largely unknown. Recently, the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 were determined for entry and/or egress of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we identified the final three ESCRT-III components Chm7, Ist1, and Vps2A of Spodoptera frugiperda. Overexpression of the dominant-negative forms of these proteins or RNAi downregulation of their transcripts significantly reduced infectious budded viruses (BVs) production of AcMNPV. Quantitative PCR together with confocal and transmission electron microscopy analysis revealed that these proteins were required for internalization and trafficking of BV during entry and egress of nucleocapsids. In infected Sf9 cells, nine ESCRT-III components were distributed on the nuclear envelope and plasma membrane, and except for Chm7, the other components were also localized to the intranuclear ring zone. Y2H and BiFC analysis revealed that 42 out of 64 BV-related proteins including 35 BV structural proteins and 7 non-BV structural proteins interacted with single or multiple ESCRT-III components. By further mapping the interactome of 64 BV-related proteins, we established the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress.IMPORTANCEFrom archaea to eukaryotes, the endosomal sorting complex required for transport (ESCRT)-III complex is hijacked by many enveloped and nonenveloped DNA or RNA viruses for efficient replication. However, the mechanism of ESCRT-III recruitment, especially for that of large enveloped DNA viruses, remains elusive. Recently, we found the ESCRT-III components Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60 are necessary for the entry and/or egress of budded viruses (BVs) of Autographa californica multiple nucleopolyhedrovirus. Here, we demonstrated that the other three ESCRT-III components Chm7, Ist1, and Vps2A play similar roles in BV infection. By determining the subcellular localization of ESCRT-III components in infected cells and mapping the interaction of nine ESCRT-III components and 64 BV-related proteins, we built the interaction networks of ESCRT-III and the viral protein complexes involved in BV entry and egress. These studies provide a fundamental basis for understanding the mechanism of the ESCRT-mediated membrane remodeling for replication of baculoviruses.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Interações entre Hospedeiro e Microrganismos , Nucleopoliedrovírus , Spodoptera , Proteínas Virais , Internalização do Vírus , Liberação de Vírus , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/ultraestrutura , Nucleopoliedrovírus/metabolismo , Nucleopoliedrovírus/fisiologia , Nucleopoliedrovírus/ultraestrutura , Spodoptera/citologia , Spodoptera/metabolismo , Spodoptera/ultraestrutura , Spodoptera/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Replicação Viral , Transporte Biológico , Células Sf9RESUMO
The general anesthesia associated with long-term cognitive impairment has been causing the concern of the whole society. In particular, repeated anesthetic exposures may affect executive function, processing speed, and fine motor skills, which all directly depended on the functions of oligodendrocytes, myelin, and axons. However, the underlying mechanisms are still largely unknown. To investigate the spatial and temporal alterations in oligodendrocytes in the corpus callosum (CC) and hippocampus following repeated sevoflurane exposures (3%, for 2 h) from postnatal day 6 (P6) to P8, we used immunofluorescence, Western blot, and a battery of behavioral tests. As previously stated, we confirmed that early anesthetic exposures hampered both cognitive and motor performance during puberty in the rotarod and banes tests. Intriguingly, we discovered that the proliferation of oligodendrocyte progenitor cells (OPCs) was immediately enhanced after general anesthesia in the CC and hippocampus from P8 to P32. From P8 through P15, the overall oligodendrocyte population remained constant. However, along with the structural myelin abnormalities, the matured oligodendrocytes statistically reduced in the CC (from P15) and hippocampus (from P32). Administration of clemastine, which could induce OPC differentiation and myelin formation, significantly increased matured oligodendrocytes and promoted myelination and cognition. Collectively, we first demonstrated the bi-directional influence of early sevoflurane exposures on oligodendrocyte maturation and proliferation, which contributes to the cognitive impairment induced by general anesthesia. These findings illustrated the dynamic changes in oligodendrocytes in the developing brain following anesthetic exposures, as well as possible therapeutic strategies for multiple general anesthesia associated cognitive impairment.
Assuntos
Oligodendroglia , Maturidade Sexual , Animais , Camundongos , Sevoflurano/efeitos adversos , Animais Recém-Nascidos , Bainha de MielinaRESUMO
As a major air pollutant, PM2.5 can induce apoptosis of nerve cells, causing impairment of the learning and memory capabilities of humans and animals. Ferroptosis is a newly discovered way of programmed cell death. It is unclear whether the neurotoxicity induced by PM2.5 is related to the ferroptosis of nerve cells. In this study, we observed the changes in ferroptosis hallmarks of SH-SY5Y cells after exposure to various doses (40, 80, and 160 µg/mL PM2.5) for 24 h, exposure to 40 µg/mL PM2.5 for various times (24, 48, and 72 h), as well as exposure to various components (Po, organic extracts; Pw, water-soluble extracts; Pc, carbon core component). The results showed that PM2.5 reduced the cell viability, the content of GSH, and the activity of GSH-PX and SOD in SH-SY5Y cells with exposure dose and duration increasing. On the other hand, PM2.5 increased the content of iron, MDA, and the level of lipid ROS in SH-SY5Y cells with exposure dose and duration increasing. Additionally, PM2.5 reduced the expression levels of HO-1, NRF2, SLC7A11, and GPX4. The ferroptosis inhibitors Fer-1 and DFO significantly increase the cells viabilities and significantly reversed the changes of other above ferroptosis hallmarks. We also observed the different effects on ferroptosis hallmarks in the SH-SY5Y cells exposed to PM2.5 (160 µg/mL) and its various components (organic extracts, water-soluble extracts, and carbon core) for 24 h. We found that only the organic extracts shared similar results with PM2.5 (160 µg/mL). This study demonstrated that PM2.5 induced ferroptosis of SH-SY5Y cells, and organic extracts might be the primary component that caused ferroptosis.
Assuntos
Ferroptose , Material Particulado , Animais , Humanos , Linhagem Celular Tumoral , Ferroptose/efeitos dos fármacos , Ferro/toxicidade , Material Particulado/toxicidade , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: Candida albicans has been regarded as the most predominant oral fungal pathogen and the main cause of denture stomatitis. This study aimed to investigate C. albicans adherence to three types of denture base polymers: heat-cured polymethylmethacrylate (PMMA), CAD-CAM milled and 3D-printed. The efficacy of four common disinfection techniques, glutaraldehyde, brushing, microwave irradiation, and Polident overnight tablets, were also examined. MATERIAL AND METHODS: Sixty blocks of pink acrylic specimens were fabricated from each polymer group. To investigate the C. albicans adherence, as well as the efficacy of different disinfection techniques on removing the yeast from the different materials, specimens were cultured within the fungal culture overnight followed by disinfection. The adhered C. albicans on the materials were then obtained by vortexing in phosphate buffered saline (PBS), and the numbers of the yeast in the suspensions were evaluated by measuring the optical density and/or colony-forming units on agar plates. Data were expressed as mean ± SEM (standard error of the mean). Statistical differences were evaluated by one-way analysis of variance (ANOVA) followed by the post hoc Tukey HSD tests. RESULTS: Significant differences in C. albicans adherence to the three polymers were noted. CAD-CAM milled and heat-cured PMMA showed significantly less C. albicans adherence compared with 3D printed PMMA. No significant difference was noted between milled and heat-cured PMMA. In the disinfection test, microwave irradiation, mechanical brushing, and Polident tablets were found to be effective in removing fungal attachment on the different denture materials, while glutaraldehyde was found to be the least effective. CONCLUSION: C. albicans adherence to the polymers varies greatly based on the types of PMMA. 3D-printed had the highest fungal biofilm attachment. Microwave irradiation, mechanical brushing, and Polident overnight tablets had comparable results in removing C. albicans from all types of PMMA, while glutaraldehyde was not as effective.
Assuntos
Candida albicans , Polimetil Metacrilato , Desinfecção/métodos , Temperatura Alta , Teste de Materiais , Polímeros , Desenho Assistido por Computador , Impressão Tridimensional , Bases de Dentadura , Propriedades de SuperfícieRESUMO
Different from holometabolous insects, the hemipteran species such as pea aphid Acyrthosiphon pisum exhibit reduced immune responses with the absence of the genes coding for antimicrobial peptide (AMP), immune deficiency (IMD), peptidoglycan recognition proteins (PGRPs), and other immune-related molecules. Prior studies have proved that phenoloxidase (PO)-mediated melanization, hemocyte-mediated phagocytosis, and reactive oxygen species (ROS) participate in pea aphid defense against bacterial infection. Also, the conserved signaling, Jun N-terminal kinase (JNK) pathway, has been suggested to be involved in pea aphid immune defense. However, the precise role of the JNK signaling, its interplay with other immune responses and its regulation in pea aphid are largely unknown. In this study, using in vitro biochemical assays and in vivo bioassays, we demonstrated that the JNK pathway regulated hemolymph PO activity, hydrogen peroxide concentration and hemocyte phagocytosis in bacteria infected pea aphids, suggesting that the JNK pathway plays a central role in regulating immune responses in pea aphid. We further revealed the JNK pathway is regulated by microRNA-184 in response to bacterial infection. It is possible that in common the JNK pathway plays a key role in immune system of hemipteran insects and microRNA-184 regulates the JNK pathway in animals.
Assuntos
Afídeos/imunologia , Proteínas de Insetos/imunologia , MAP Quinase Quinase 4/imunologia , MicroRNAs/imunologia , Transdução de Sinais/imunologia , Animais , Afídeos/genética , Proteínas de Insetos/genética , MAP Quinase Quinase 4/genética , MicroRNAs/genética , Fagocitose/genética , Transdução de Sinais/genéticaRESUMO
Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 is a class III viral fusion protein that mediates low-pH-triggered membrane fusion during virus entry. Although the structure of GP64 in a postfusion conformation has been solved, its prefusion structure and the mechanism of how the protein refolds to execute fusion are unknown. In its postfusion structure, GP64 is composed of five domains (domains I to V). Domain IV (amino acids [aa] 374 to 407) contains two loops (loop 1 and loop 2) that form a hydrophobic pocket at the membrane-distal end of the molecule. To determine the roles of domain IV, we used alanine-scanning mutagenesis to replace each of the individual residues and the contact-forming residues within domain IV and evaluate their contributions to GP64-mediated membrane fusion and virus infection. In many cases, replacement of a single amino acid had no significant impact on GP64. However, replacement of R392 or disruption of the N381-N385, N384-Y388, N385-W393, or K389-W393 contact resulted in poor cell surface expression and fusion loss of the modified GP64, whereas replacement of E390 or G391 or disruption of the N381-K389, N381-Q401, or N381-I403 contact reduced the cell surface expression level of the constructs and the ability of GP64 to mediate fusion pore expansion. In contrast, replacement of N407 or disruption of contact D404-S406 appeared to restrict fusion pore expansion without affecting expression. Combined with the finding that these constructs remain in the prefusion conformation or have a dramatically less efficient transition from the prefusion to the postfusion state under acidic conditions, we proposed that domain IV is necessary for refolding of GP64 during membrane fusion.IMPORTANCE Baculovirus GP64 is grouped with rhabdovirus G, herpesvirus gB, and thogotovirus glycoproteins as a class III viral fusion protein. In their postfusion structures, these proteins contain five domains (domains I to V). Distinct from domain IV of rhabdovirus G and herpesvirus gB proteins, which is composed of ß-sheets, domain IV of GP64 is a loop region; the same domain in thogotovirus glycoproteins has not been solved. In addition, domain IV is proximal to domain I (fusion domain) in prefusion structures of vesicular stomatitis virus (VSV) G and human cytomegalovirus (HCMV) gB but resides at the domain I-distal end of the molecule in a postfusion conformation. In this study, we identified that highly conserved residues and contacts within domain IV of AcMNPV GP64 are necessary for low-pH-triggered conformational change and fusion pore expansion. Our results highlight the roles of domain IV of class III viral fusion proteins in refolding during membrane fusion.
Assuntos
Fusão de Membrana/fisiologia , Nucleopoliedrovírus/fisiologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Baculoviridae , Linhagem Celular , Membrana Celular , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Modelos Moleculares , Conformação Molecular , Domínios Proteicos , Análise de Sequência de Proteína , Thogotovirus , Vesiculovirus , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genética , Internalização do VírusRESUMO
BACKGROUND AND OBJECTIVE: Excessive osteoclast activity is a major characteristic of pathogenic bone loss in inflammatory bone diseases including periodontitis. However, beyond the knowledge that osteoclasts are differentiated from the monocyte/macrophage lineage and share common ancestry with macrophages and DC, the nature and function of osteoclast precursors are not completely understood. Furthermore, little is known about how osteoclast precursors respond to bacterial infection in vivo. We have previously demonstrated in vitro that the periodontal pathogen Porphyromonas gingivalis (Pg) plays a biphasic role on the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated the in vivo effect of Pg infection on the regulation of osteoclast precursors, using a mouse calvarial infection model. METHODS AND RESULTS: C57BL/6 wild-type and the myeloid differentiation factor 88 knockout (MyD88-/- ) mice were infected with Pg by calvarial injection. Local and systemic bone loss, and the number and function of CD11b+ c-fms+ cells from bone marrow and spleen were analyzed. Our results show that Pg infection induces localized inflammatory infiltration and osteoclastogenesis, as well as increased number and osteoclastogenic potential of CD11b+ c-fms+ osteoclast precursors in the bone marrow and periphery. We also show that CD11b+ c-fms+ RANK+ and CD11b+ c-fms+ RANK- are precursors with similar osteoclastogenic and pro-inflammatory potentials. In addition, CD11b+ c-fms+ cells exhibit an antigen-specific T-cell immune-suppressive activity, which are increased with Pg infection. Moreover, we demonstrate that MyD88 is involved in the regulation of osteoclast precursors upon Pg infection. CONCLUSIONS: In this study, we demonstrate an enhanced dual function of osteoclast precursors following calvarial Pg infection. Based on our findings, we propose the following model: Pg infection increases a pool of precursor cells that can be shunted toward osteoclast formation at the infection/inflammation sites, while at the same time dampening host immune responses, which is beneficial for the persistence of infection and maintenance of the characteristic chronic nature of periodontitis. Understanding the nature, function, and regulation of osteoclast precursors will be helpful for identifying therapeutic interventions to aid in the control and prevention of inflammatory bone loss diseases including periodontitis.
Assuntos
Infecções por Bacteroidaceae/patologia , Osteoclastos/citologia , Crânio/microbiologia , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/microbiologia , Porphyromonas gingivalis , Ligante RANKRESUMO
The endosomal sorting complex required for transport (ESCRT) machinery is necessary for budding of many enveloped viruses. Recently, it was demonstrated that Vps4, the key regulator for recycling of the ESCRT-III complex, is required for efficient infection by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, ESCRT assembly, regulation, and function are complex, and little is known regarding the details of participation of specific ESCRT complexes in AcMNPV infection. In this study, the core components of ESCRT-I (Tsg101 and Vps28) and ESCRT-III (Vps2B, Vps20, Vps24, Snf7, Vps46, and Vps60) were cloned from Spodoptera frugiperda Using a viral complementation system and RNA interference (RNAi) assays, we found that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. In cells knocking down or overexpressing dominant negative (DN) forms of the components of ESCRT-I and ESCRT-III complexes, entering virions were partially trapped within the cytosol. To examine only egress, cells were transfected with the double-stranded RNA (dsRNA) targeting an individual ESCRT-I or ESCRT-III gene and viral bacmid DNA or viral bacmid DNA that expressed DN forms of ESCRT-I and ESCRT-III components. We found that ESCRT-III components (but not ESCRT-I components) are required for efficient nuclear egress of progeny nucleocapsids. In addition, we found that several baculovirus core or conserved proteins (Ac11, Ac76, Ac78, GP41, Ac93, Ac103, Ac142, and Ac146) interact with Vps4 and components of ESCRT-III. We propose that these viral proteins may form an "egress complex" that is involved in recruiting ESCRT-III components to a virus egress domain on the nuclear membrane.IMPORTANCE The ESCRT system is hijacked by many enveloped viruses to mediate budding and release. Recently, it was found that Vps4, the key regulator of the cellular ESCRT machinery, is necessary for efficient entry and egress of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, little is known about the roles of specific ESCRT complexes in AcMNPV infection. In this study, we demonstrated that ESCRT-I and ESCRT-III complexes are required for efficient entry of AcMNPV into insect cells. The components of ESCRT-III (but not ESCRT-I) are also necessary for efficient nuclear egress of progeny nucleocapsids. Several baculovirus core or conserved proteins were found to interact with Vps4 and components of ESCRT-III, and these interactions may suggest the formation of an "egress complex" involved in the nuclear release or transport of viral nucleocapsids.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Nucleopoliedrovírus/fisiologia , Vírion/fisiologia , Liberação de Vírus , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Citosol/virologia , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Teste de Complementação Genética , Insetos/citologia , Insetos/virologia , Nucleocapsídeo/metabolismo , RNA de Cadeia Dupla , Spodoptera/genética , Proteínas Virais/metabolismo , Montagem de Vírus , Replicação ViralRESUMO
In eukaryotic cells, the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) proteins comprise the minimal machinery that triggers fusion of transport vesicles with their target membranes. Comparative studies revealed that genes encoding the components of the SNARE system are highly conserved in yeast, insect, and human genomes. Upon infection of insect cells by the virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the transcript levels of most SNARE genes initially were upregulated. We found that overexpression of dominant-negative (DN) forms of NSF or knockdown of the expression of NSF, the key regulator of the SNARE system, significantly affected infectious AcMNPV production. In cells expressing DN NSF, entering virions were trapped in the cytoplasm or transported to the nucleus with low efficiency. The presence of DN NSF also moderately reduced trafficking of the viral envelope glycoprotein GP64 to the plasma membrane but dramatically inhibited production of infectious budded virions (BV). Transmission electron microscopy analysis of infections in cells expressing DN NSF revealed that progeny nucleocapsids were retained in a perinuclear space surrounded by inner and outer nuclear membranes. Several baculovirus conserved (core) proteins (Ac76, Ac78, GP41, Ac93, and Ac103) that are important for infectious budded virion production were found to associate with NSF, and NSF was detected within the assembled BV. Together, these data indicate that the cellular SNARE system is involved in AcMNPV infection and that NSF is required for efficient entry and nuclear egress of budded virions of AcMNPV.IMPORTANCE Little is known regarding the complex interplay between cellular factors and baculoviruses during viral entry and egress. Here, we examined the cellular SNARE system, which mediates the fusion of vesicles in healthy cells, and its relation to baculovirus infection. Using a DN approach and RNA interference knockdown, we demonstrated that a general disruption of the SNARE machinery significantly inhibited the production of infectious BV of AcMNPV. The presence of a DN NSF protein resulted in low-efficiency entry of BV and the retention of progeny nucleocapsids in the perinuclear space during egress. Combined with these effects, we also found that several conserved (core) baculovirus proteins closely associate with NSF, and these results suggest their involvement in the egress of BV. Our findings are the first to demonstrate that the SNARE system is required for efficient entry of BV and nuclear egress of progeny nucleocapsids of baculoviruses.
Assuntos
Proteínas Sensíveis a N-Etilmaleimida/genética , Proteínas Sensíveis a N-Etilmaleimida/metabolismo , Nucleopoliedrovírus/fisiologia , Proteínas SNARE/metabolismo , Internalização do Vírus , Liberação de Vírus , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/virologia , Citoplasma/virologia , Humanos , Microscopia Eletrônica de Transmissão , Proteínas Sensíveis a N-Etilmaleimida/deficiência , Nucleocapsídeo/metabolismo , Nucleopoliedrovírus/ultraestrutura , Interferência de RNA , Proteínas SNARE/genética , Células Sf9 , Spodoptera/citologia , Spodoptera/virologia , Proteínas do Envelope Viral/metabolismo , Vírion , Montagem de Vírus , Leveduras/metabolismoRESUMO
The metal-organic framework (MOF) MIL-101Cr was readily encapsulated by a very thin shell (around 30 nm) of hydrophobic mesoporous silica, which replicates the irregular shape of the MOF nanocrystals. Such a silica shell facilitates the diffusion of hydrophobic reactants with enhancement of the catalytic activity of the MOF and significantly improved catalytic stability of the MOF in the oxidation of indene.
RESUMO
This study focuses on the protective effect of germacrone on human umbilical vein endothelial cells(HUVECs) damaged by H2O2-induced oxidative stress and its possible mechanisms. The oxidative damage model was established by using 500 µmolâ¢L⻹ H2O2 to treat HUVECs for 3 hours, and then protected with different concentrations of germacrone for 24 hours. The effect of germacrone on cell viability of HUVECs damaged by H2O2 was detected by MTT. The contents of PGI2, TXB2, ET-1, t-PA, PAI-1, TNF-α and IL-6 were detected by ELISA. The content of NO was detected by using nitrate reductase method. Colorimetry was used to detect NOS and GSH-Px. The contents of MDA, SOD and LDH were detected by TBA, WST-1 and microplate respectively. Apoptosis was observed by Hoechst 33258 fluorescent staining. The mRNA expressions of Bax, Bcl-2 and Caspase-3 in cells were detected by RT-PCR. The results showed that the cell damage rate was 52% after treated with 500 µmolâ¢L⻹ H2O2 for 3 hours. The cell activity was increasing with the rise of germacrone concentration within the range of 20-200 molâ¢L⻹. Compared with normal group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were lower after damaged with H2O2. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were increased. Compared with model group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were increased after treated with germacrone. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were lower after treated with germacrone. According to Hoechst 33258 fluorescence staining, compared with normal group, the cell membrane and the nucleus showed strong dense blue fluorescence, and the number of cells significantly decreased in model group. Compared with model group, blue fluorescence intensity decreased in drug group. The above findings demonstrate that germacrone may improve the effect on HUVECs damaged by H2O2-induced oxidative stress by resisting oxidation and inhibiting cell apoptosis.
Assuntos
Apoptose , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Estresse Oxidativo , Sesquiterpenos de Germacrano/farmacologia , Células Cultivadas , Humanos , Peróxido de HidrogênioRESUMO
UNLABELLED: Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ~156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ~60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure. IMPORTANCE: Baculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.
Assuntos
Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Lepidópteros/virologia , Nucleopoliedrovírus/fisiologia , Animais , Lepidópteros/genética , Dados de Sequência Molecular , Nucleopoliedrovírus/crescimento & desenvolvimento , Análise de Sequência de DNA , Fatores de TempoRESUMO
AIM: To investigate the effects of co-delivering IL-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pCIA-P, which encodes the surface protein antigen PAc of Streptococcus mutans. METHODS: Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pCIA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA (sIgA) were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-γ and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method. RESULTS: Marked expression of IL-6 was found in COS-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and sIgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-γ from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pCIA-P and pCI vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats. CONCLUSION: Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.
Assuntos
Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Cárie Dentária/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Superfície/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Células COS , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Feminino , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Imunização/métodos , Imunoglobulina A Secretora/imunologia , Interferon gama/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Plasmídeos/imunologia , Ratos , Ratos Wistar , Saliva/imunologia , Streptococcus mutans/imunologiaRESUMO
In a tritrophic context of plant-insect-entomopathogen, plants play important roles in modulating the interaction of insects and their pathogenic viruses. Currently, the influence of plants on the transmission of insect viruses has been mainly studied on baculoviruses and some RNA viruses, whereas the impact of plants on other insect viruses is largely unknown. Here, we identified a new densovirus infecting the green peach aphid Myzus persicae and tested whether and how host plants influence the transmission of the aphid densovirus. The complete single-stranded DNA genome of the virus, M. persicae densovirus 2, is 5 727 nt and contains inverted terminal repeats. Transcription and phylogenetic analysis indicated that the virus was distinct from other a few identified aphid densoviruses. The virus abundance was detected highly in the intestinal tract of aphids, compared with the lower level of it in other tissues including head, embryo, and epidermis. Cabbage and pepper plants had no obvious effect on the vertical transmission and saliva-mediated horizontal transmission of the virus. However, the honeydew-mediated horizontal transmission among aphids highly depended on host plants (65% on cabbages versus 17% on peppers). Although the virus concentration in the honeydew produced by aphids between 2 plants was similar, the honeydew production of the infected aphids reared on peppers was dramatically reduced. Taken together, our results provide evidence that plants influence the horizontal transmission of a new densovirus in an aphid population by modulating honeydew secretion of aphids, suggesting plants may manipulate the spread of an aphid-pathogenic densovirus in nature.
Assuntos
Afídeos , Densovirus , Animais , Afídeos/genética , FilogeniaRESUMO
Membrane budding is essential for the egress of many enveloped viruses, and this process shares similarities with the biogenesis of multivesicular bodies (MVBs). In eukaryotic cells, the budding of intraluminal vesicles (IVLs) is mediated by the endosomal sorting complex required for transport (ESCRT) machinery and some viruses require ESCRT machinery components or functions to bud from host cells. Baculoviruses, such as Autographa californica multiple nucleopolyhedrovirus (AcMNPV), enter host cells by clathrin-mediated endocytosis. Viral DNA replication and nucleocapsid assembly occur within the nucleus. Some progeny nucleocapsids are subsequently trafficked to, and bud from, the plasma membrane, forming budded virions (BV). To determine whether the host ESCRT machinery is important or necessary for AcMNPV replication, we cloned a cDNA of Spodoptera frugiperda VPS4, a key regulator for disassembly and recycling of ESCRT III. We then examined viral infection and budding in the presence of wild-type (WT) or dominant negative (DN) forms of VPS4. First, we used a viral complementation system, in combination with fluorescent tags, to examine the effects of transiently expressed WT or DN VPS4 on viral entry. We found that dominant negative VPS4 substantially inhibited virus entry. Entering virus was observed within aberrant compartments containing the DN VPS4 protein. We next used recombinant bacmids expressing WT or DN VPS4 proteins to examine virus egress. We found that production of infectious AcMNPV BV was substantially reduced by expression of DN VPS4 but not by WT VPS4. Together, these results indicate that a functional VPS4 is necessary for efficient AcMNPV BV entry into, and egress from, insect cells.
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Proteínas de Insetos/metabolismo , Nucleopoliedrovírus/fisiologia , Spodoptera/metabolismo , Spodoptera/virologia , Vírion/fisiologia , Internalização do Vírus , Liberação de Vírus , Sequência de Aminoácidos , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Insetos/genética , Dados de Sequência Molecular , Nucleopoliedrovírus/genética , Alinhamento de Sequência , Spodoptera/genética , Vírion/genética , Replicação ViralRESUMO
In oxides, the substitution of non-oxide anions (F(-),S(2-),N(3-) and so on) for oxide introduces many properties, but the least commonly encountered substitution is where the hydride anion (H(-)) replaces oxygen to form an oxyhydride. Only a handful of oxyhydrides have been reported, mainly with electropositive main group elements or as layered cobalt oxides with unusually low oxidation states. Here, we present an oxyhydride of the perhaps most well-known perovskite, BaTiO(3), as an O(2-)/H(-) solid solution with hydride concentrations up to 20% of the anion sites. BaTiO(3-x)H(x) is electronically conducting, and stable in air and water at ambient conditions. Furthermore, the hydride species is exchangeable with hydrogen gas at 400 °C. Such an exchange implies diffusion of hydride, and interesting diffusion mechanisms specific to hydrogen may be at play. Moreover, such a labile anion in an oxide framework should be useful in further expanding the mixed-anion chemistry of the solid state.
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Mammalian cells generate ATP through mitochondrial respiration and glycolysis. Mitochondria not only play a key role in cell energy metabolism but also in cell cycle regulation. As a neurotoxic pollutant, benzo(a)pyrene (BaP) can trigger neuronal oxidative damage and apoptosis. However, the features of BaP-induced energy metabolism disturbance in SH-SY5Y cells has rarely been addressed. This study aimed to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indications of respiratory activities and glycolytic. SH-SY5Y cells were treated with BaP to establish a cytotoxicity model, and butylated hydroxy anisole (BHA) was used to alleviate the damages induced by BaP. Using the Seahorse Extracellular Flux analyzer (XFp), we found that BaP significantly reduced basal respiration, ATP-linked OCR in SH-SY5Y cells with dose- and time-dependent. BHA supplementation recovered the mitochondrial respiration, synchronously attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers, then rescued BaP-induced cell apoptosis. But long-term exposure to BaP or exposure to a high dosage of BaP could decrease OCR associated with maximal respiratory, spare capacity, and glycolysis metabolism. At the same time, the damage to cells is also more severe with the rate of apoptosis and mitochondrial membrane potential (ΔΨm) loss rising sharply, which were not entirely reversed by BHA. This study provides energy metabolism-related, indicative biomarkers of cytotoxicity induced by BaP, which might provide information for early prevention and intervention.
Assuntos
Benzo(a)pireno , Mitocôndrias , Neuroblastoma , Humanos , Trifosfato de Adenosina/metabolismo , Benzo(a)pireno/toxicidade , Glicólise , Mitocôndrias/metabolismo , Neuroblastoma/metabolismo , RespiraçãoRESUMO
The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein mediates membrane fusion during entry. Fusion results from a low-pH-triggered conformational change in GP64 and subsequent interactions with the membrane bilayers. The low-pH sensor and trigger of the conformational change are not known, but histidine residues are implicated because the pK(a) of histidine is near the threshold for triggering fusion by GP64. We used alanine substitutions to examine the roles of all individual and selected clusters of GP64 histidine residues in triggering and mediating fusion by GP64. Three histidine residues (H152, H155, and H156), located in fusion loop 2, were identified as important for membrane fusion. These three histidine residues were important for efficient pore expansion but were not required for the pH-triggered conformational change. In contrast, a cluster of three histidine residues (H245, H304, and H430) located near the base of the central coiled coil was identified as a putative sensor for low pH. Three alanine substitutions in cluster H245/H304/H430 resulted in dramatically reduced membrane fusion and the apparent loss of the prefusion conformation at neutral pH. Thus, the H245/H304/H430 cluster of histidines may function or participate as a pH sensor by stabilizing the prefusion structure of GP64.
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Membrana Celular/metabolismo , Células Gigantes/metabolismo , Histidina/genética , Fusão de Membrana , Spodoptera/virologia , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Eritrócitos , Histidina/química , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação/genética , Nucleopoliedrovírus/patogenicidade , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Ovinos , Proteínas Virais de Fusão/genética , Replicação ViralRESUMO
Aphids are common agricultural pests with a wide range of hosts from agriculture to forestry plants. As known, aphids also serve as the major vectors to transmit plant viruses. Although numerous studies have focused on interactions between aphids and plant viruses, little is known about the aphid viruses, i.e., the insect viruses that are infectious to aphids. In the past four decades, several aphid viruses have been identified in diverse aphid species. In this review, we present a brief view of the aphid pathogenic viruses from several aspects, including classification of aphid viruses and characters of the viral genome, integration of viral sequences in host genomes, infection symptoms and influence on aphids, as well as host range and transmission modes. Taken together, these studies have increased our understanding of the rarely known aphid viruses, and will potentially contribute to the development of new strategies for controlling aphid populations.
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As a widespread environmental pollutant, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-induced neurotoxicity has received increasing attention. Studies have shown that BPDE-induced neurodegeneration is due partly to neuronal apoptosis. Unlike apoptosis, ferroptosis is a non-apoptotic form of programmed cell death, but its specific role in the neurotoxicity of BPDE remains unclear. In this work, we investigated the ferroptosis in BPDE-induced cell death in human neuroblastoma cell line SH-SY5Y using a specific pharmacological inhibitor. Lipid peroxides, malondialdehyde production, glutathione / glutathione peroxidase activity, superoxide dismutase activity, and iron content were evaluated. Consistent with previous studies, our data showed that 0.5 µM BPDE poisoning for 24 hr could induce cell apoptosis and that cell survival could be improved by using apoptosis inhibitors. But with prolonged exposure time (72 hr) or increased exposure dose (1.0 µM), we have elucidated and validated that BPDE triggered ferroptosis in human SH-SY5Y cells. We also revealed that suppression of ferroptosis by specific inhibitors, ferrostatin-1 and deferoxamine, significantly rescued the phenotypes of ferroptosis induced by BPDE. BPDE downregulated Nrf2 and its target genes related to redox regulation, GPX4 and SLC7A11, but upregulated HO-1. Our results first demonstrated that BPDE caused cytotoxic effects on cell death via apoptosis and ferroptosis. Most notably, long-term environmental exposure to BPDE becomes a concern due to ferroptosis. Redox imbalance is controlled by the Nrf2, SLC7A11, and HO-1, through which lipid peroxides and ferrous ion accumulation cause ferroptosis after BPDE treatment. These findings highlight that targeting ferroptosis could serve as an effective protective strategy for neurotoxicity of BPDE.