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Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-ß-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a "one health approach".
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Doenças do Cão , Infecções por Proteus , Cães , Humanos , Animais , Antibacterianos/farmacologia , Proteus mirabilis/genética , Animais de Estimação/genética , Infecções por Proteus/veterinária , Infecções por Proteus/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Genômica , República da Coreia , Testes de Sensibilidade Microbiana/veterinária , Plasmídeos , Doenças do Cão/genéticaRESUMO
BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation in the nosocomial environment. CPKP carried by companion dogs, which are close to human beings, should be considered a common threat to public health. However, CPKP dissemination through companion animals is still under consideration of major diagnosis and surveillance systems. METHODS: Two CPKP isolates which were genotyped to harbor bla NDM-5-encoding IncX3 plasmids, were subjected to the whole-genome study. Whole bacterial DNA was isolated, sequenced, and assembled with Oxford Nanopore long reads and corrected with short reads from the Illumina NovaSeq 6000 platform. The whole-genome structure and positions of antimicrobial resistance (AMR) genes were identified and visualized using CGView. Worldwide datasets were downloaded from the NCBI GenBank database for whole-genome comparative analysis. The whole-genome phylogenetic analysis was constructed using the identified whole-chromosome SNP sites from K. pneumoniae HS11286. RESULTS: As a result of the whole-genome identification, 4 heterogenous plasmids and a single chromosome were identified, each carrying various AMR genes. Multiple novel structures were identified from the AMR genes, coupled with mobile gene elements (MGE). The comparative whole-genome epidemiology revealed that ST378 K. pneumoniae is a novel type of CPKP, carrying a higher prevalence of AMR genes. CONCLUSIONS: The characterized whole-genome analysis of this study shows the emergence of a novel type of CPKP strain carrying various AMR genes with variated genomic structures. The presented data in this study show the necessity to develop additional surveillance programs and control measures for a novel type of CPKP strain.
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Enterobacteriáceas Resistentes a Carbapenêmicos , Infecções por Klebsiella , Animais , Cães , Humanos , Klebsiella pneumoniae , Antibacterianos/farmacologia , Filogenia , Infecções por Klebsiella/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , beta-Lactamases/genética , Plasmídeos/genética , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Testes de Sensibilidade MicrobianaRESUMO
The circulation of carbapenemase-producing Escherichia coli (CPEC) in our society is a serious concern for vulnerable patients in nosocomial environments. However, the genomic epidemiology of the circulation of CPEC bacteria among companion animals remains largely unknown. In this study, epidemiological analysis was conducted using complete genome identification of CPEC ST410 isolates obtained from companion animals. To estimate the genomic distance and relatedness of the isolates, a total of 37 whole-genome datasets of E. coli ST410 strains were downloaded and comparatively analysed. As a result of the analysis, the genomic structure of the chromosomes and plasmids was identified, revealing the genomic positions of multiple resistance and virulence genes. The isolates in this study were grouped into the subclade H24/RxC, with fimH24, and substituted quinolone resistance-determining regions (QRDRs) and multiple beta-lactamases, including extended-spectrum ß-lactamase (ESBL) and carbapenemase. In addition, the in silico comparison of the whole-genome datasets revealed unidentified ST410 H24/Rx subgroups, including either high pathogenicity islands (HPIs) or H21 serotypes. Considering the genetic variations and resistance gene dissemination of the isolates carried by companion animals, future approaches for preventive measurement must include the "One Health" perspective for public health in our society.
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Escherichia coli , Genômica , Animais , Epidemiologia Molecular , Escherichia coli/genética , beta-Lactamases/genéticaRESUMO
Vasohibin-2 (VASH2), a homologue of vasohibin-1 (VASH1), is overexpressed in various cancer cells and promotes tumor progression. We therefore regard VASH2 as a molecular target for cancer treatment. Here we applied vaccine technology to develop a therapy against VASH2. We selected two amino acid sequences of VASH2 protein; the MTG and RRR peptides, which contain possible B cell epitopes. These sequences are identical between the human and murine VASH2 proteins and distinct from those of the VASH1 protein. We conjugated these peptides with the carrier protein keyhole limpet hemocyanin, mixed with an adjuvant, and injected subcutaneously twice at a 2-week interval in mice. Both vaccines increased antibodies against the antigen peptide; however, only the MTG peptide vaccine increased antibodies that recognized the recombinant VASH2 protein. When Lewis lung cancer (LLC) cells were subcutaneously inoculated, tumors isolated from mice immunized with the MTG peptide vaccine showed a significant decrease in the expression of epithelial-to-mesenchymal transition (EMT) markers. EMT is responsible for cancer cell invasion and metastasis. When the LLC cells were injected into the tail vein, the MTG peptide vaccine inhibited lung metastasis. Moreover, the MTG peptide vaccine inhibited the metastasis of pancreatic cancer cells to the liver in an orthotopic mouse model, and there was a significant inverse correlation between the ELISA titer and metastasis inhibition. Therefore, we propose that the MTG peptide vaccine is a novel anti-metastatic cancer treatment that targets VASH2 and can be applied even in the most malignant and highly metastatic pancreatic cancer.
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Neoplasias Pancreáticas , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Anticorpos , Fatores de Transcrição , Peptídeos , Vacinas de Subunidades Antigênicas , Proteínas de Ciclo Celular , Proteínas Angiogênicas/metabolismoRESUMO
KRAS is the most frequently mutated oncogene in human tumors, and its activating mutations represent important therapeutic targets. The combination of Cas9 and guide RNA from the CRISPR-Cas system recognizes a specific DNA sequence and makes a double-strand break, which enables editing of the relevant genes. Here, we harnessed CRISPR to specifically target mutant KRAS alleles in cancer cells. We screened guide RNAs using a reporter system and validated them in cancer cells after lentiviral delivery of Cas9 and guide RNA. The survival, proliferation, and tumorigenicity of cancer cells in vitro and the growth of tumors in vivo were determined after delivery of Cas9 and guide RNA. We identified guide RNAs that efficiently target mutant KRAS without significant alterations of the wild-type allele. Doxycycline-inducible expression of this guide RNA in KRAS-mutant cancer cells transduced with a lentiviral vector encoding Cas9 disrupted the mutant KRAS gene, leading to inhibition of cancer cell proliferation both in vitro and in vivo. Intra-tumoral injection of lentivirus and adeno-associated virus expressing Cas9 and sgRNA suppressed tumor growth in vivo, albeit incompletely, in immunodeficient mice. Expression of Cas9 and the guide RNA in cells containing wild-type KRAS did not alter cell survival or proliferation either in vitro and in vivo. Our study provides a proof-of-concept that CRISPR can be utilized to target driver mutations of cancers in vitro and in vivo.
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Tryptophanyl-tRNA synthetase (WRS) is one of the aminoacyl-tRNA synthetases (ARSs) that possesses noncanonical functions. Full-length WRS is released during bacterial infection and primes the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex to elicit innate immune responses. However, the role of WRS in viral infection remains unknown. Here, we show that full-length WRS is secreted by immune cells in the early phase of viral infection and functions as an antiviral cytokine. Treatment of cells with recombinant WRS protein promotes the production of inflammatory cytokines and type I interferons (IFNs) and curtails virus replication in THP-1 and Raw264.7 cells but not in TLR4-/- or MD2-/- bone marrow-derived macrophages (BMDMs). Intravenous and intranasal administration of recombinant WRS protein induces an innate immune response and blocks viral replication in vivo These findings suggest that secreted full-length WRS has a noncanonical role in inducing innate immune responses to viral infection as well as to bacterial infection.IMPORTANCE ARSs are essential enzymes in translation that link specific amino acids to their cognate tRNAs. In higher eukaryotes, some ARSs possess additional, noncanonical functions in the regulation of cell metabolism. Here, we report a novel noncanonical function of WRS in antiviral defense. WRS is rapidly secreted in response to viral infection and primes the innate immune response by inducing the secretion of proinflammatory cytokines and type I IFNs, resulting in the inhibition of virus replication both in vitro and in vivo Thus, we consider WRS to be a member of the antiviral innate immune response. The results of this study enhance our understanding of host defense systems and provide additional information on the noncanonical functions of ARSs.
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Infecções por Rhabdoviridae/imunologia , Triptofano-tRNA Ligase/genética , Triptofano-tRNA Ligase/metabolismo , Vesiculovirus/patogenicidade , Administração Intranasal , Administração Intravenosa , Animais , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Células HeLa , Humanos , Imunidade Inata , Interferon Tipo I/metabolismo , Camundongos , Células RAW 264.7 , Infecções por Rhabdoviridae/genética , Células THP-1 , Triptofano-tRNA Ligase/administração & dosagem , Vesiculovirus/imunologiaRESUMO
Rubicon is part of a Beclin-1-Vps34-containing autophagy complex. Rubicon induces antimicrobial responses upon Toll-like receptor (TLR) stimulation and functions as a feedback inhibitor to prevent unbalanced proinflammatory responses depending on dectin-1 signaling. However, the role played by Rubicon during antiviral immune responses, particularly the type I interferon (IFN) responses, remains largely unknown. Here, we report that Rubicon acts as a negative regulator for virus-triggered IFN signaling. Knockdown of Rubicon promoted type I interferon signaling and inhibited virus replication, while overexpression of Rubicon had the opposite effect. Rubicon specifically interacts with the interferon regulatory factor (IRF) association domain (IAD) of IRF3, and this interaction leads to inhibition of the dimerization of IRF3, which negatively regulates IFN-mediated antiviral response. Thus, our findings suggest the novel additional role of Rubicon as a negative regulator that inhibits the IFN signaling and cellular antiviral responses, providing a novel cellular mechanism of IRF3 inhibition.IMPORTANCE The type I IFN system is a critical innate immune response that protects organisms against virus infection. However, type I IFN signaling must be tightly regulated to avoid excessive production of IFNs. Hence, negative regulatory mechanisms for type I IFN signaling are important, and to date, several related molecules have been identified. Here, we show that Rubicon is a major negative regulator of type I IFN signaling, and unlike previous reports of cellular molecules that inhibit IRF3 activation via proteasomal degradation or dephosphorylation of IRF3, we show that Rubicon interacts with IRF3 and that ultimately this interaction leads to inhibition of the dimerization of IRF3. Thus, we identified a novel negative regulator of type I IFN signaling pathways and a novel cellular mechanism of IRF3 inhibition. The results of this study will increase our understanding of the role of negative-feedback mechanisms that regulate type I IFN signaling and maintain immune homeostasis.
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Vírus da Influenza A Subtipo H1N1/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon Tipo I/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Multimerização Proteica , Transdução de Sinais , Vesiculovirus/imunologia , Animais , Camundongos , Células RAW 264.7RESUMO
AIM: Hepatic fibrosis and angiogenesis occur in parallel during the progression of liver disease. Fibrosis promotes angiogenesis via inducing vascular endothelial growth factor (VEGF) from the activated hepatic stellate cells (HSCs). In turn, increased neovessel formation causes fibrosis, although the underlying molecular mechanism remains undetermined. In the current study, we aimed to address a role of endothelial cells (ECs) as a source of latent transforming growth factor (TGF)-ß, the precursor of the most fibrogenic cytokine TGF-ß. METHODS: After recombinant VEGF was administered to mice via the tail vein, hepatic angiogenesis and fibrogenesis were evaluated using immunohistochemical and biochemical analyses in addition to investigation of TGF-ß activation using primary cultured HSCs and liver sinusoidal ECs (LSECs). RESULTS: In addition to increased hepatic levels of CD31 expression, VEGF-treated mice showed increased α-smooth muscle actin (α-SMA) expression, hepatic contents of hydroxyproline, and latency associated protein degradation products, which reflects cell surface activation of TGF-ß via plasma kallikrein (PLK). Liberating the PLK-urokinase plasminogen activator receptor complex from the HSC surface by cleaving a tethering phosphatidylinositol linker with its specific phospholipase C inhibited the activating latent TGF-ß present in LSEC conditioned medium and subsequent HSC activation. CONCLUSION: Neovessel formation (angiogenesis) accelerates liver fibrosis at least in part via provision of latent TGF-ß that activated on the surface of HSCs by PLK, thereby resultant active TGF-ß stimulates the activation of HSCs.
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Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Neovascularização Patológica/complicações , Neovascularização Patológica/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Meios de Cultivo Condicionados/farmacologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Estreladas do Fígado , Calicreínas/sangue , Cirrose Hepática/sangue , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neovascularização Patológica/sangue , Fator A de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
To identify an economically viable waste management system for bioplastics, thermoplastic starch (TPS) and poly(butylene adipate-co-terephthalate) (PBAT) were anaerobically digested under hydrogen (H2)/carbon dioxide (CO2) and nitrogen (N2) gas-purged conditions to compare methane (CH4) production and biodegradation. Regardless of the type of bioplastics, CH4 production was consistently higher with H2/CO2 than with N2. The highest amount of CH4 was produced at 307.74 mL CH4/g volatile solids when TPS digested with H2/CO2. A stepwise increased in CH4 yield was observed, with a nominal initial increment followed by accelerated methanogenesis conversion as H2 was depleted. This may be attributed to a substantial shift in the microbial structure from hydrogenotrophic methanogen (Methanobacteriales and Methanomicrobiales) to heterotrophs (Spirochaetia). In contrast, no significant change was observed with PBAT, regardless of the type of purged gas. TPS was broken down into numerous derivatives, including volatile fatty acids. TPS produced more byproducts with H2/CO2 (i.e., 430) than with N2 (i.e., 320). In contrast, differential scanning calorimetry analysis on PBAT revealed an increase in crystallinity from 10.20 % to 12.31 % and 11.36 % in the H2/CO2- and N2-purged conditions, respectively, after 65 days of testing. PBAT surface modifications were characterized via Fourier transform infrared spectroscopy and scanning electron microscopy. The results suggest that the addition of H2/CO2 can enhance the CH4 yield and increase the breakdown rate of TPS more than that of PBAT. This study provides novel insights into the CH4 production potential of two bioplastics with different biodegradabilities in H2/CO2-mediated anaerobic digestion systems.
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Hidrogênio , Amido , Anaerobiose , Amido/química , Amido/metabolismo , Dióxido de Carbono , Bactérias/metabolismo , Metano/metabolismoRESUMO
Based on the current situation of Korean culture and society, the population of companion animals in South Korea is growing rapidly along with zoonotic risks. The current data regarding zoonotic infections in companion dogs reported in Korea is sparse. This study aims to investigate the seroprevalence of seven potential zoonotic pathogens in companion dogs in South Korea: Anaplasma phagocytophilum, Borrelia burgdoferi, Ehrlichia canis, Coxiella burnetii, Brucella canis, Leptospira spp. and canine influenza A virus. A total of 284 serum samples were collected from 2018 to 2021, and the immunoglobulin G (IgG) antibodies against 7 zoonotic pathogens were detected using enzyme-linked immunosorbent assays. Samples were divided into five groups and analysed based on age. IgG antibodies against six of the seven pathogens were detected. The highest seropositivity rate was detected for canine influenza A virus exposure (59.1%) for which the rates were the highest in dogs under 1 year old and declined with age. Positivity rates of the other pathogens were relatively low: 1.76% for Leptospira spp., 1.40% for A. phagocytophilum and E. canis, 1.06% for B. canis and 0.35% for B. burgdoferi. No antibodies against C. burnetii were detected in this study. The exposure of dogs in South Korea to six zoonotic pathogens was serologically confirmed, highlighting a potential risk for human infection. The zoonotic risk of companion dogs cannot be neglected, and implementation of One Health approach should be advocated to establish effective preventive measures.
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Anaplasma phagocytophilum , Animais de Estimação , Animais , Humanos , Cães , Estudos Soroepidemiológicos , República da Coreia/epidemiologia , Imunoglobulina GRESUMO
Lumpy skin disease (LSD) is one of the most important emerging transboundary diseases. Recently, LSD has emerged in many countries in the northern hemisphere. The LSD virus has a huge genome and is highly resistant to environmental conditions. The virus is also host-specific and large ruminants, such as cattle and domestic water buffalo, are particularly susceptible. In addition, wild ruminants can serve as potential reservoirs for spreading the LSD virus. The emergence might be related to climate change in various regions because LSD is an arthropod-borne infectious disease. This disease causes enormous economic losses, such as leather damage, decreased milk production, abortion, and death in infected ruminants. The economic importance of LSD in the bovine industry has forced countries to develop and implement control strategies against the disease. With the recent global spread and the economic impact, LSD will be discussed intensively. In addition, effective preventive measures are suggested based on the presence or absence of LSD outbreaks.
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Doenças dos Bovinos , Doenças Transmissíveis Emergentes , Doença Nodular Cutânea , Vírus da Doença Nodular Cutânea , Animais , Bovinos , Doença Nodular Cutânea/epidemiologia , Doença Nodular Cutânea/prevenção & controle , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/prevenção & controle , Doenças Transmissíveis Emergentes/veterinária , Vírus da Doença Nodular Cutânea/genética , Surtos de Doenças/veterinária , Doenças dos Bovinos/epidemiologiaRESUMO
The research was carried out to analyze the combined and mechanical properties of polypropylene (PP)/fly ash (FA)/waste stone powder (WSP) composite materials. PP, FA and WSP were mixed and prepared into PP100 (pure PP), PP90 (90 wt% PP + 5 wt% FA + 5 wt% WSP), PP80 (80 wt% PP + 10 wt% FA + 10 wt% WSP), PP70 (70 wt% PP + 15 wt% FA + 15 wt% WSP), PP60 (60 wt% PP + 20 wt% FA + 20 wt% WSP) and PP50 (50 wt% PP + 25 wt% FA + 25 wt% WSP) composite materials using an injection molding machine. The research results indicate that all PP/FA/WSP composite materials can be prepared through the injection molding process and there are no cracks or fractures found on the surface of the composite materials. The research results of thermogravimetric analysis are consistent with expectations, indicating that the preparation method of the composite materials in this study is reliable. Although the addition of FA and WSP powder cannot increase the tensile strength, it is very helpful to improve the bending strength and notched impact energy. Especially for notched impact energy, the addition of FA and WSP results in an increase in the notched impact energy of all PP/FA/WSP composite materials by 14.58-22.22%. This study provides a new direction for the reuse of various waste resources. Moreover, based on the excellent bending strength and notched impact energy, the PP/FA/WSP composite materials have great application potential in the composite plastic industry, artificial stone, floor tiles and other industries in the future.
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Canine brucellosis caused by Brucella canis infection occurs mainly in dogs, and is a zoonotic disease that also has the possibility of infection in humans. Many studies have been conducted to understand the immunopathological mechanism of B. canis infection. However, the precise immune mechanism remains to be elucidated because compared to other Brucella spp., B. canis has different immune evasion mechanisms. In this study, gene expression levels of Toll-like receptors (TLRs) and TLR-associated molecules and cytokine production were analyzed to figure out the roles of immune-related host factors in B. canis infection. Time-dependent gene expression of TLRs (1-10) and TLR-related molecules (TNF-α, IL-5, IL-23, CCL4, CD40 and NFκ-B) and release of Th1, Th2 and Th17-related cytokines (IFN-γ, IL-1ß, IL-4, IL-6, IL-10 and IL-17A) were investigated in DH82 canine macrophages infected with B. canis. Time-dependent induction of TLRs 3, 7 and 8 was observed, and TLR 7 had the highest expression level (p <0.05). The expression levels of all TLR-related genes were significantly increased after infection. In particular, the expression of the CCL4 and IL-23 genes was highly induced. The amounts of IL-1ß, IL-6 and IL-10 were significantly increased by B. canis infection, but the amounts of IL-4 and IL-17A were not. The production of IL-1ß and IL-6 was the highest at 24 hr after B. canis infection (p <0.05). This study demonstrates that TLRs 3, 7 and 8 are prominent sites of to immune response induction with the production of related cytokines and a nuclear factor in DH82 cells infected with B. canis. These results suggest a sequential immune mechanism of B. canis infection, involving TLRs, cytokines and their associated factors.
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Brucella canis , Brucelose , Doenças do Cão , Humanos , Cães , Animais , Citocinas/metabolismo , Brucella canis/genética , Interleucina-10/genética , Interleucina-17 , Interleucina-6/metabolismo , Interleucina-4/genética , Brucelose/veterinária , Macrófagos , Receptores Toll-Like/genética , Expressão Gênica , Receptores de Citocinas/genética , Interleucina-23RESUMO
Stem cell-based therapy has been highlighted as a potential avenue to promote tissue regeneration, where stimulation of stem cells to differentiate into the targeted cell type is essential. One of the factors that induce stem cells to differentiate is their surrounding microenvironment. In this study, the correlation between mild reductant and early osteogenic commitment was evaluated. A cell surface-reducing microenvironment significantly silenced the transforming growth factor (TGF)-ß signaling pathway of mesenchymal stem cells (MSCs), followed by increased focal adhesion and inhibition of cell membrane protein dimerization. Furthermore, in vivo transplantation of MSCs exposed to the reducing microenvironment resulted in an early osteogenic commitment and neobone formation. Thus, these results highlight the potential of cell surface-reducing microenvironment to influence early osteogenic commitment.
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Microambiente Celular , Osteogênese , Adesão Celular , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/citologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
Despite the immunogenicity of vaccines currently used in poultry, several pathogens, including avian influenza virus (AIV) and Newcastle disease virus (NDV), cause enormous economic losses to the global poultry industry. The efficacy of vaccines can be improved by the introduction of effective adjuvants. This study evaluated a novel water-in-oil emulsion adjuvant, CAvant® WO-60, which effectively enhanced both the immunogenicity of conserved influenza antigen sM2HA2 and inactivated whole H9N2 antigen (iH9N2). CAvant® WO-60 induced both humoral and cell-mediated immunity in mice and provided 100% protection from challenge with 10 LD50 of A/Aquatic bird/Korea/W81/2005 (H5N2) and A/Chicken/Korea/116/2004 (H9N2) AIV. Importantly, immunization of chickens with iH9N2 plus inactivated NDV LaSota (iNDV) bivalent inactivated vaccine emulsified in CAvant® WO-60 induced seroprotective levels of antigen-specific antibody responses. Taken together, these results suggested that CAvant® WO-60 is a promising adjuvant for poultry vaccines.
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We have reported that autophagy is crucial for clearance of amyloidogenic human IAPP (hIAPP) oligomer, suggesting that an autophagy enhancer could be a therapeutic modality against human diabetes with amyloid accumulation. Here, we show that a recently identified autophagy enhancer (MSL-7) reduces hIAPP oligomer accumulation in human induced pluripotent stem cell-derived ß-cells (hiPSC-ß-cells) and diminishes oligomer-mediated apoptosis of ß-cells. Protective effects of MSL-7 against hIAPP oligomer accumulation and hIAPP oligomer-mediated ß-cell death are significantly reduced in cells with knockout of MiTF/TFE family members such as Tfeb or Tfe3. MSL-7 improves glucose tolerance and ß-cell function of hIAPP+ mice on high-fat diet, accompanied by reduced hIAPP oligomer/amyloid accumulation and ß-cell apoptosis. Protective effects of MSL-7 against hIAPP oligomer-mediated ß-cell death and the development of diabetes are also significantly reduced by ß-cell-specific knockout of Tfeb. These results suggest that an autophagy enhancer could have therapeutic potential against human diabetes characterized by islet amyloid accumulation.
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Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Autofagia/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Polipeptídeo Amiloide das Ilhotas Pancreáticas/genética , Polipeptídeo Amiloide das Ilhotas Pancreáticas/metabolismo , Animais , Apoptose/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Técnicas de Inativação de Genes , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina , Macroautofagia/fisiologia , Camundongos , Camundongos TransgênicosRESUMO
Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.
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Infection of hepatitis B virus (HBV) increase the incidence of chronic liver disease and hepatocellular carcinoma (HCC). The hepatitis B viral x (HBx) protein encoded by the HBV genome contributes to the pathogenesis of HCC and thus, negative regulation of HBx is beneficial for the alleviation of the disease pathogenesis. MARCH5 is a mitochondrial E3 ubiquitin ligase and here, we show that high MARCH5 expression levels are correlated with improved survival in HCC patients. MARCH5 interacts with HBx protein mainly accumulated in mitochondria and targets it for degradation. The N-terminal RING domain of MARCH5 was required for the interaction with HBx, and MARCH5H43W lacking E3 ligase activity failed to reduce HBx protein levels. High expression of HBx results in the formation of protein aggregates in semi-denaturing detergent agarose gels and MARCH5 mediates the elimination of protein aggregates through the proteasome pathway. HBx-induced ROS production, mitophagy, and cyclooxygenase-2 gene expression were suppressed in the presence of high MARCH5 expression. These results suggest MARCH5 as a target for alleviating HBV-mediated liver disease.
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Carcinoma Hepatocelular/metabolismo , Vírus da Hepatite B/química , Hepatite B/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , Agregados Proteicos , Proteólise , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Células HEK293 , Células HeLa , Hepatite B/complicações , Hepatite B/virologia , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Agregação Patológica de Proteínas/metabolismo , Taxa de Sobrevida , Transfecção , Ubiquitina-Proteína Ligases/genéticaRESUMO
BACKGROUND: Tissue engineering is an interdisciplinary field that attempts to restore or regenerate tissues and organs through biomimetic fabrication of scaffolds with specific functionality. In recent years, graphene oxide (GO) is considered as promising biomaterial due to its nontoxicity, high dispersity, and hydrophilic interaction, and these characteristics are key to stimulating the interactions between substrates and cells. METHOD: In this study, GO substrates were fabricated via chemically immobilizing GO at 1.0 mg/ml on glass slides. Furthermore, we examined the osteogenic responses of murine mesenchymal-like stem cells, C3H10T1/2 cells, on GO substrates. RESULTS: C3H10T1/2 cells on GO substrates resulted in increased cell surface area, enhanced cellular adhesions, and instigated osteogenic differentiation. Furthermore, priming of C3H10T1/2 cells with chondrocyte-conditioned medium (CM) could further induce a synergistic effect of osteogenesis on GO substrates. CONCLUSIONS: All of these data suggest that GO substrate along with CM is suitable for upregulating osteogenic responses of mesenchymal stem cells.