RESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.
Asunto(s)
Coinfección , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Anticuerpos Neutralizantes , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Vacunación , Vacunas Atenuadas/genéticaRESUMEN
Many cytokines produced by Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells have been shown to participate in the pathogenesis of KSHV. Determination of the exact role of cytokines in Kaposi's sarcoma (KS) pathogenesis is limited, however, by the difficulty to manipulate the target genes in human endothelial cells. In this study, we sought to elucidate the role of cytokines in KSHV-infected human immortalized endothelial cell line (HuARLT cells) by knockout (KO) of the corresponding target genes using the CRISPR/Cas9 system. The cytokine production profile of KSHV-infected HuARLT cells was analyzed using a protein array, and several cytokines were found to be highly upregulated following KSHV infection. This study focused on CXCL1, which was investigated by knocked out in HuARLT cells. KSHV-infected CXCL1 KO cells underwent increased cell death compared to KSHV-infected wild-type (WT) cells and mock-infected CXCL1 KO cells. Lytic replication was not observed in KSHV-infected WT nor CXCL1 KO cells. Phosphorylation of STAT3 was significantly suppressed in KSHV-infected CXCL1 KO cells. Additionally, inhibitors of STAT3 and CXCL1 induced cell death in KSHV-infected endothelial cells. Our results show that CXCL1 production is required for the survival of KSHV-infected endothelial cells, and the CXCL1 to STAT3 phosphorylation signaling pathway may be a therapeutic target for KS.
Asunto(s)
Herpesvirus Humano 8 , Sarcoma de Kaposi , Humanos , Herpesvirus Humano 8/fisiología , Células Endoteliales , Fosforilación , Citocinas/metabolismo , Quimiocina CXCL1/genética , Quimiocina CXCL1/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Dengue virus, which belongs to the Flaviviridae family, can induce a range of symptoms from mild to severe, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. While infectious cloning technology is a useful tool for understanding viral pathogenesis and symptoms, it exhibits limitations when constructing the entire Flavivirus genome. The instability and toxicity of the genome to bacteria make its full-length construction in bacterial vectors a time-consuming and laborious process. To address these challenges, we employed the modified infectious subgenomic amplicon (ISA) method in this study, which can potentially be a superior tool for reverse genetic studies on the dengue virus. Using ISA, we generated recombinant dengue viruses de novo and validated their robust replication in both human and insect cell lines, which was comparable to that of the original strains. Moreover, the efficiency of ISA in genetically modifying the dengue virus was elucidated by successfully inserting the gene for green fluorescence protein into the genome of dengue virus serotype 4. Overall, this study highlighted the effectiveness of ISA for genetically engineering the dengue virus and provided a technical basis for a convenient reverse genetics system that could expedite investigations into the dengue virus.
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Virus del Dengue , Dengue , Flaviviridae , Flavivirus , Humanos , Virus del Dengue/genética , Genética Inversa/métodos , Flavivirus/genética , Flaviviridae/genética , Replicación Viral/genéticaRESUMEN
Klotho is a protein that plays different functions in female fertility. We have previously reported that klotho protein supplementation during in vitro maturation improves porcine embryo development, while klotho knockout for somatic cell cloning completely blocks full-term pregnancy in vivo. However, the effects of the microinjection of klotho protein or klotho knockdown dual vector in porcine embryos at different time points and the specific molecular mechanisms remain largely unknown. In this study, we injected the preassembled cas9 + sgRNA dual vector, for klotho knockdown, into the cytoplasm of the germinal vesicle stage of oocytes and into porcine embryos after 6-h parthenogenetic activation. Similarly, the klotho protein was inserted into the cytoplasm of germinal vesicle stage oocytes and porcine embryos after 6-h parthenogenetic activation. Compared with the controls, the microinjection of klotho dual vector markedly decreased the blastocyst formation rates in germinal vesicle stage oocytes and activated embryos. However, the efficiency of blastocyst formation when klotho protein was inserted before in vitro maturation was significantly higher than that after klotho protein insertion into parthenogenetically activated embryos. These results indicated that klotho knockdown may impair embryo development into blastocyst irrespective of injection timing. In addition, klotho protein injection timing in pig embryos may be an important factor for regulating embryo development.
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Oocitos , ARN Guía de Sistemas CRISPR-Cas , Embarazo , Animales , Femenino , Porcinos , Oocitos/fisiología , Blastocisto , Desarrollo Embrionario/genética , PartenogénesisRESUMEN
Several studies have examined exosomes derived from porcine follicular fluid (FF), but few have reported their application in controlled experiments. The main concern in the field of embryology may be that controlled conditions, such as using a defined medium intermittently, cause poor results in mammalian oocyte maturation and embryo development. The first reason is the absence of the FF, which copes with the majority of the processes emerging in oocytes and embryos. Therefore, we added exosomes derived from porcine FF to the maturation medium of porcine oocytes. For morphological assessment, cumulus cell expansion and subsequent embryonic development were evaluated. Moreover, several stainings, such as glutathione (GSH) and reactive oxygen species (ROS), fatty acid, ATP, and mitochondrial activity, as well as evaluations of gene expression and protein analysis, were used for the functional verification of exosomes. When the oocytes were treated with exosomes, the lipid metabolism and cell survival of the oocytes were fully recovered, as well as morphological evaluations compared to the porcine FF-excluded defined medium. Therefore, controlled experiments may produce reliable data if the exosomes are treated with the desired amounts, and we suggest applying FF-derived exosomes to promote experimental data when performing controlled experiments in embryology.
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Exosomas , Líquido Folicular , Embarazo , Femenino , Porcinos , Animales , Líquido Folicular/metabolismo , Antioxidantes/metabolismo , Exosomas/metabolismo , Oocitos/metabolismo , Desarrollo Embrionario , Glutatión/metabolismo , Lípidos , Técnicas de Maduración In Vitro de los Oocitos , Mamíferos/metabolismoRESUMEN
Multiple host proteins affect the gene expression of Kaposi's sarcoma-associated herpesvirus (KSHV) during latent and lytic replication. High-mobility group box 1 (HMGB1) serves as a highly conserved chromosomal protein inside the cell and a prototypical damage-associated molecular pattern molecule outside the cell. HMGB1 has been shown to play a pathogenic role in viral infectious diseases and to regulate the lytic replication of KSHV. However, its functional effects on the KSHV life cycle in KSHV-infected cells have not been fully elucidated. Here, we explored the role of intracellular and extracellular HMGB1 in KSHV virion production by employing CRISPR/Cas9-mediated HMGB1 knockout in the KSHV-producing iSLK BAC16 cell line. Intracellular HMGB1 formed complexes with various proteins, and the abundance of HMGB1-interacting proteins changed during latent and lytic replication. Moreover, extracellular HMGB1 was found to enhance lytic replication by phosphorylating JNK. Of note, the expression of viral genes was attenuated during lytic replication in HMGB1 knockout iSLK BAC16 cells, with significantly decreased production of infectious virions compared to that of wild-type cells. Collectively, our results demonstrate that HMGB1 is an important cellular cofactor that affects the generation of infectious KSHV progeny during lytic replication. IMPORTANCE The high-mobility group box 1 (HMGB1) protein has many intra- and extracellular biological functions with an intricate role in various diseases. In certain viral infections, HMGB1 affects the viral life cycle and pathogenesis. In this study, we explored the effects of HMGB1 knockout on the production of Kaposi's sarcoma-associated herpesvirus (KSHV). HMGB1 knockout decreased virion production in KSHV-producing cells by decreasing the expression of viral genes. The processes by which HMGB1 affects KSHV production may occur inside or outside infected cells. For instance, several cellular and viral proteins interacted with intracellular HMGB1 in a nucleosomal complex, whereas extracellular HMGB1 induced JNK phosphorylation, thereby enhancing lytic replication. Our results suggest that both intracellular and extracellular HMGB1 are necessary for efficient KSHV replication. Thus, HMGB1 may represent an effective therapeutic target for the regulation of KSHV production.
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Regulación Viral de la Expresión Génica , Proteína HMGB1/metabolismo , Herpesvirus Humano 8/fisiología , Virión/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Proteína HMGB1/genética , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Transducción de Señal , Proteínas Virales/genética , Activación Viral , Replicación ViralRESUMEN
Exposure to particulate matter (PM) is becoming a major global health issue. The amount and time of exposure to PM are known to be closely associated with cardiovascular diseases. However, the mechanism through which PM affects the vascular system is still not clear. Endothelial cells line the interior surface of blood vessels and actively interact with plasma proteins, including the complement system. Unregulated complement activation caused by invaders, such as pollutants, may promote endothelial inflammation. In the present study, we sought to investigate whether urban PM (UPM) acts on the endothelial environment via the complement system. UPM-treated human endothelial cells with normal human serum showed the deposition of membrane attack complexes (MACs) on the cell surface via the alternative pathway of the complement system. Despite the formation of MACs, cell death was not observed, and cell proliferation was increased in UPM-mediated complement activation. Furthermore, complement activation on endothelial cells stimulated the production of inflammation-related proteins. Our results revealed that UPM could activate the complement system in human endothelial cells and that complement activation regulated inflammatory reaction in microenvironment. These findings provide clues with regard to the role of the complement system in pathophysiologic events of vascular disease elicited by air pollution.
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Contaminantes Atmosféricos/efectos adversos , Activación de Complemento , Proteínas del Sistema Complemento/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Vasos Sanguíneos/patología , Muerte Celular , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citocinas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Material Particulado/efectos adversos , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
The demyelinating diseases of the central nervous system involve myelin abnormalities, oligodendrocyte damage, and consequent glia activation. Neurotoxicant cuprizone (CPZ) was used to establish a mouse model of demyelination. However, the effects of CPZ on microRNA (miRNA) expression and behavior have not been clearly reported. We analyzed the behavior of mice administered a diet containing 0.2% CPZ for 6 weeks, followed by 6 weeks of recovery. Rotarod analysis demonstrated that the treated group had poorer motor coordination than control animals. This effect was reversed after 6 weeks of CPZ withdrawal. Open-field tests showed that CPZ-treated mice exhibited significantly increased anxiety and decreased exploratory behavior. CPZ-induced demyelination was observed to be alleviated after 4 weeks of CPZ treatment, according to luxol fast blue (LFB) staining and myelin basic protein (MBP) expression. miRNA expression profiling showed that the expression of 240 miRNAs was significantly changed in CPZ-fed mice compared with controls. Furthermore, miR-155-5p and miR-20a-5p upregulations enhanced NgR induction through Smad 2 and Smad 4 suppression in demyelination. Taken together, our results demonstrate that CPZ-mediated demyelination induces behavioral deficits with apparent alterations in miRNA expression, suggesting that differences in miRNA expression in vivo may be new potential therapeutic targets for remyelination.
Asunto(s)
Cuprizona/efectos adversos , Enfermedades Desmielinizantes/psicología , Conducta Exploratoria/efectos de los fármacos , Redes Reguladoras de Genes/efectos de los fármacos , Animales , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/genética , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , MicroARNs/efectos de los fármacos , MicroARNs/genética , Prueba de Desempeño de Rotación con Aceleración ConstanteRESUMEN
Periodontitis is a common disease characterized by chronic inflammation and tissue destruction of gums. Human periodontal ligament stem cells (PDLSCs), derived from the periodontium, have stem cell properties similar to those of mesenchymal stem cells. PDLSCs possess not only the potential to differentiate into other tissues, but also immunomodulatory abilities. Macrophages play a critical role in periodontal disease, but little is known regarding the role of PDLSCs in macrophage modulation during inflammation. In this study, we investigated the effect of PDLSCs on the macrophage cell line. While the conditioned media from PDLSCs under normal culture conditions did not affect macrophage polarization, the lipopolysaccharide (LPS)-preconditioned PDLSCs induced significant changes in M1 polarization. Extracellular vesicles (EVs) isolated from the conditioned media of LPS-preconditioned PDLSCs induced strong M1 polarization of macrophages. Additionally, the M1 polarization was abolished by DNase I treatment of EVs. Therefore, the LPS-stimulated PDLSCs induce M1 polarization of macrophages through EVs, suggesting that the EVs from PDLSCs might be a potential therapeutic target for inflammation in the periodontium.
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Polaridad Celular/efectos de los fármacos , Vesículas Extracelulares/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/citología , Ligamento Periodontal/citología , Células Madre/citología , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Desoxirribonucleasa I/metabolismo , Vesículas Extracelulares/efectos de los fármacos , Humanos , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Solubilidad , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Células THP-1RESUMEN
It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-ß1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-ß1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of ß-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling.
Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Exosomas/efectos de los fármacos , MicroARNs/genética , Factor de Crecimiento Transformador beta1/farmacología , beta Catenina/genética , Células A549 , Comunicación Autocrina , Transición Epitelial-Mesenquimal/genética , Exosomas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/metabolismo , Transducción de Señal , beta Catenina/metabolismoRESUMEN
During evolution, herpesviruses have developed numerous, and often very ingenious, strategies to counteract efficient host immunity. Specifically, Kaposi's sarcoma-associated herpesvirus (KSHV) eludes host immunity by undergoing a dormant stage, called latency wherein it expresses a minimal number of viral proteins to evade host immune activation. Here, we show that during latency, KSHV hijacks the complement pathway to promote cell survival. We detected strong deposition of complement membrane attack complex C5b-9 and the complement component C3 activated product C3b on Kaposi's sarcoma spindle tumor cells, and on human endothelial cells latently infected by KSHV, TIME-KSHV and TIVE-LTC, but not on their respective uninfected control cells, TIME and TIVE. We further showed that complement activation in latently KSHV-infected cells was mediated by the alternative complement pathway through down-regulation of cell surface complement regulatory proteins CD55 and CD59. Interestingly, complement activation caused minimal cell death but promoted the survival of latently KSHV-infected cells grown in medium depleted of growth factors. We found that complement activation increased STAT3 tyrosine phosphorylation (Y705) of KSHV-infected cells, which was required for the enhanced cell survival. Furthermore, overexpression of either CD55 or CD59 in latently KSHV-infected cells was sufficient to inhibit complement activation, prevent STAT3 Y705 phosphorylation and abolish the enhanced survival of cells cultured in growth factor-depleted condition. Together, these results demonstrate a novel mechanism by which an oncogenic virus subverts and exploits the host innate immune system to promote viral persistent infection.
Asunto(s)
Apoptosis/inmunología , Complemento C3b/metabolismo , Complemento C5b/metabolismo , Herpesvirus Humano 8/fisiología , Sarcoma de Kaposi/virología , Latencia del Virus , Western Blotting , Proliferación Celular , Células Cultivadas , Complemento C3b/genética , Complemento C5b/genética , Endotelio Vascular/inmunología , Endotelio Vascular/patología , Endotelio Vascular/virología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Células Endoteliales de la Vena Umbilical Humana/inmunología , Células Endoteliales de la Vena Umbilical Humana/patología , Células Endoteliales de la Vena Umbilical Humana/virología , Humanos , Inflamación/inmunología , Inflamación/patología , Inflamación/virología , Neovascularización Patológica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Sarcoma de Kaposi/inmunología , Sarcoma de Kaposi/patología , Proteínas Virales/genética , Proteínas Virales/inmunología , Proteínas Virales/metabolismoRESUMEN
Existing evidence suggests a possible role of viruses in human bladder cancer development. Recently, Kaposi's sarcoma-associated herpesvirus (KSHV) was reported to be the most frequently detected virus in bladder cancer tissue from Croatian patients on screening with the Lawrence Livermore Microbial Detection Array. In the current study, to investigate the functional roles of KSHV in bladder cancer, five bladder cancer cell lines were infected with KSHV and their tumour progression-associated changes investigated. Four KSHV-infected bladder cancer cell lines were established; two invasive bladder cancer cell lines showed higher proliferation rates than uninfected cells. Additionally, these KSHV-infected invasive bladder cancer cells showed a greater number of colonies, which were also significantly larger than those of uninfected cells, in a soft agar colony formation assay. cDNA microarray analysis showed that various genes associated with cell proliferation and cancer development were upregulated in these KSHV-infected bladder cancer cells. Taken together, we suggest that KSHV infection affects the proliferation of a subset of invasive bladder cancer cells and may therefore play a role in their oncogenic progression. Further studies are required to elucidate the exact mechanism used by KSHV to promote bladder cancer progression.
Asunto(s)
Proliferación Celular/fisiología , Infecciones por Herpesviridae/patología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/patogenicidad , Sarcoma de Kaposi/virología , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/virología , Línea Celular , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Tumorales CultivadasRESUMEN
Despite high prevalence of upper urinary tract calculi (UUTC), there are few studies regarding patterns of care in Asian populations. We investigated treatment patterns and time trends in patients with newly diagnosed UUTC in Korea using the National Health Insurance database that includes de-identified claims from a random 2% sample of the entire population (> 1 million people). A total of 14,282 patients who received active treatments, including shock wave lithotripsy (SWL), ureteroscopic surgery (URS), percutaneous nephrolithotomy (PNL), and uretero/pyelolithotomy (UPL), for newly diagnosed UUTC between 2003 and 2013 were included. The number of primary and all treated cases of UUTC significantly (43% and 103.3%, respectively) increased over the 10-year period. While patients undergoing SWL, URS, PNL, and UPL as primary treatment increased by 43.7%, 31.9%, 87.5%, and 0%, respectively, the relative proportion undergoing each treatment remained constant over the 10 years (SWL > 90%, URS 4.5% to 7.8%, PNL 0.4% to 1.0%, and UPL < 0.4%, respectively). Multinomial logistic regression analysis showed that age > 40 years (compared to age < 30 years) was significantly associated with URS, PNL, and UPL, rather than SWL, while patients living in urban or suburban/rural areas (compared to metropolitan) were significantly less likely to undergo URS and PNL. In summary, the majority of Korean patients underwent SWL as primary treatment for UUTC, and the predominant use of SWL remained steady over a 10-year period in Korea. Our results will be valuable in examining treatment patterns and time trends in Korean UUTC patients.
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Cálculos Urinarios/terapia , Adulto , Anciano , Bases de Datos Factuales , Supervivencia sin Enfermedad , Femenino , Humanos , Litotricia/tendencias , Masculino , Persona de Mediana Edad , Nefrostomía Percutánea/tendencias , Oportunidad Relativa , República de Corea/epidemiología , Factores Sexuales , Factores Socioeconómicos , Resultado del Tratamiento , Cálculos Urinarios/epidemiología , Cálculos Urinarios/cirugíaRESUMEN
Kaposi's sarcoma (KS) is a vascular tumor, and KS spindle cells express endothelial-cell-specific markers. Generally, it is believed that KS originates from endothelial cells. However, as various mesodermal-derived tissue markers are also expressed in KS spindle cells, the exact origin of KS still needs to be elucidated. Here, Kaposi's sarcoma-associated herpesvirus (KSHV) was used to infect human mesenchymal stem cells derived from bone marrow (hMSC-bm), and we investigated the angiogenic properties of these cells, which are one of the most important pathologic features of KS. KSHV-infected hMSC-bm showed latent infection and increased tube formation activity in vitro. In addition, the expression of endothelial-cell-specific markers and a growth factor that affects the angiogenesis of endothelial cells was induced in KSHV-infected cells. This study suggests that human mesenchymal stem cells might have important roles in KS pathogenesis.
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Herpesvirus Humano 8/crecimiento & desarrollo , Células Madre Mesenquimatosas/virología , Neovascularización Fisiológica , Biomarcadores/análisis , Células Cultivadas , HumanosRESUMEN
Extracellular vesicles (EVs) contain a variety of biomolecules and provide information about the cells that produce them. EVs from cancer cells found in urine can be used as biomarkers to detect cancer, enabling early diagnosis and treatment. The potential of alpha-2-macroglobulin (A2M) and clusterin (CLU) as novel diagnostic urinary EV (uEV) biomarkers for bladder cancer (BC) was demonstrated previously. To validate the diagnostic value of these proteins in uEVs in a large BC cohort, urine handling conditions before uEV isolation should be optimized during sample transportation from medical centers. In this study, we analyzed the uEV protein quantity, EV particle number, and uEV-A2M/CLU after urine storage at 20°C and 4°C for 0-6 days, each. A2M and CLU levels in uEVs were relatively stable when stored at 4°C for a maximum of three days and at 20°C for up to 24 h, with minimal impact on analysis results. Interestingly, pre-processing to remove debris and cells by centrifugation and filtration of urine did not show any beneficial effects on the preservation of protein biomarkers of uEVs during storage. Here, the importance of optimizing shipping conditions to minimize the impact of pre-analytical handling on the uEVs protein biomarkers was emphasized. These findings provide insights for the development of clinical protocols that use uEVs for diagnostic purposes.
Asunto(s)
Líquidos Corporales , Vesículas Extracelulares , alfa 2-Macroglobulinas Asociadas al Embarazo , Neoplasias de la Vejiga Urinaria , Humanos , Embarazo , Femenino , Neoplasias de la Vejiga Urinaria/diagnóstico , Vejiga Urinaria , Factores de TranscripciónRESUMEN
Tripartite motif (TRIM) proteins are a large family of E3 ubiquitin ligases implicated in antiviral defense systems, tumorigenesis, and protein quality control. TRIM proteins contribute to protein quality control by regulating the ubiquitin-proteasome system, endoplasmic reticulum-associated degradation, and macroautophagy/autophagy. However, the detailed mechanisms through which various TRIM proteins regulate downstream events have not yet been fully elucidated. Herein, we identified a novel function of TRIM22 in the regulation of autophagy. TRIM22 promotes autophagosome-lysosome fusion by mediating the association of GABARAP family proteins with PLEKHM1, thereby inducing the autophagic clearance of protein aggregates, independent of its E3 ubiquitin ligase activity. Furthermore, a TRIM22 variant associated with early-onset familial Alzheimer disease interferes with autophagosome-lysosome fusion and autophagic clearance. These findings suggest TRIM22 as a critical autophagic regulator that orchestrates autophagosome-lysosome fusion by scaffolding autophagy-related proteins, thus representing a potential therapeutic target in neurodegenerative diseases.Abbreviations: AD: Alzheimer disease; ADAOO: AD age of onset; AICD: APP intracellular domain; APP: amyloid beta precursor protein; BSA: bovine serum albumin; cDNAs: complementary DNAs; CQ: chloroquine; CTF: carboxyl-terminal fragment; EBSS: Earle's balanced salt solution; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; HA: hemagglutinin; HOPS: homotypic fusion and protein sorting; IFN: interferon; IL1A/IL-1α: interleukin 1 alpha; KO: knockout; MTORC1: mechanistic target of rapamycin kinase complex 1; NFKBIA/IκBα: NFKB inhibitor alpha; NFE2L2/NRF2: NFE2 like bZIP transcription factor; PBS: phosphate-buffered saline; PI3K: class I phosphoinositide 3-kinase; PLA: proximity ligation assay; PLEKHM1: pleckstrin homology and RUN domain containing M1; PSEN1: presenilin 1; SEM: standard errors of the means; SNAREs: soluble N-ethylmaleimide-sensitive factor attachment protein receptors; SNCA: synuclein alpha; SNP: single nucleotide polymorphism; TBS: tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TRIM: tripartite motif; ULK1: unc-51 like autophagy activating kinase 1; WT: wild-type.
RESUMEN
High-mobility group box 1 (HMGB1) is a protein that binds to DNA and participates in various cellular processes, including DNA repair, transcription, and inflammation. It is also associated with cancer progression and therapeutic resistance. Despite its known role in promoting tumor growth and immune evasion in the tumor microenvironment, the contribution of HMGB1 to the development of Kaposi's sarcoma (KS) is not well understood. We investigated the effect of HMGB1 on KS pathogenesis using immortalized human endothelial cells infected with Kaposi's sarcoma-associated human herpes virus (KSHV). Our results showed that a higher amount of HMGB1 was detected in the supernatant of KSHV-infected cells compared to that of mock-infected cells, indicating that KSHV infection induced the secretion of HMGB1 in human endothelial cells. By generating HMGB1 knockout clones from immortalized human endothelial cells using CRISPR/Cas9, we elucidated the role of HMGB1 in KSHV-infected endothelial cells. Our findings indicate that the absence of HMGB1 did not induce lytic replication in KSHV-infected cells, but the cell viability of KSHV-infected cells was decreased in both 2D and 3D cultures. Through the antibody array for cytokines and growth factors, CXCL5, PDGF-AA, G-CSF, Emmprin, IL-17A, and VEGF were found to be suppressed in HMGB1 KO KSHV-infected cells compared to the KSHV-infected wild-type control. Mechanistically, phosphorylation of p38 would be associated with transcriptional regulation of CXCL5, PDGF-A and VEGF. These observations suggest that HMGB1 may play a critical role in KS pathogenesis by regulating cytokine and growth factor secretion and emphasize its potential as a therapeutic target for KS by modulating the tumor microenvironment.
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Codon pair deoptimization (CPD) attenuated type I porcine reproductive and respiratory syndrome virus (PRRSV). Infectious clones covering the full genome of a Korean type I PRRSV (E38) were synthesized, and CPD induced nine synonymous mutants of NSP1 (n = 1) and ORF7 (n = 8). In a trial to rescue live viruses from infectious clones, only four clones with mutations at nt 177 downstream of ORF7 were rescued, which showed a substantial decrease in cellular replication ability. The rescue-failed clones had two common mutation sites with a high minimum free energy and significantly modified RNA secondary structure relative to the original virus. In infected pigs, CPD viruses demonstrated significantly lower replication ability and pathogenicity than the original virus. However, immune response level induced by the attenuated viruses and the original virus was similar. This is the first study to demonstrate that type I PRRSV virulence can be attenuated through CPD application to ORF7.
Asunto(s)
Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Vacunas Virales , Virus , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Replicación Viral/genética , Codón , Mutación , Virus/genética , Inmunidad , Síndrome Respiratorio y de la Reproducción Porcina/genética , Vacunas Virales/genéticaRESUMEN
OBJECTIVE: Kaposi's sarcoma-associated herpesvirus (KSHV) is classified as a gamma-herpesvirus and it causes Kaposi's sarcoma in patients infected with the human immunodeficiency virus (HIV). Decoy receptor 3 (DcR3) is known as a decoy receptor for Fas ligand, LIGHT and TL1A and it can neutralize the biological effect of TL1A by inhibiting the TL1A-DR3 interaction in human endothelial cells. The present study examined the expression of DcR3 in human endothelial cells and its effect during the early stages of KSHV infection. METHODS: The expression of DcR3 was assessed using real-time RT-PCR and ELISA in human umbilical cord vein endothelial cells (HUVECs) infected with KSHV. Cell proliferation and apoptosis of KSHV-infected HUVECs were assessed after treatment of infected cells with an anti-DcR3 antibody or recombinant human TL1A. RESULTS: DcR3 expression was induced during the early phase of KSHV infection. Inhibition of DcR3 with anti-DcR3 antibodies or recombinant human TL1A-induced apoptosis in KSHV-infected HUVECs. CONCLUSION: The expression of DcR3 plays an important role in the prevention of apoptosis in HUVECs during the early phases of KSHV infection, thus ensuring the successful establishment and maintenance of the viral infection.