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N6-methyladenosine (m6A) is the most prevalent RNA modification and is associated with various biological processes. Proteins that function as readers and writers of m6A modifications have been shown to play critical roles in human malignancies. Here, we identified KH-type splicing regulatory protein (KHSRP) as an m6A binding protein that contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). High KHSRP levels were detected in PDAC and predicted poor patient survival. KHSRP deficiency suppressed PDAC growth and metastasis in vivo. Mechanistically, KHSRP recognized and stabilized FAK pathway mRNAs, including MET, ITGAV and ITGB1, in an m6A-dependent manner, which led to activation of downstream FAK signaling that promoted PDAC progression. Targeting KHSRP with a PROTAC showed promising tumor suppressive effects in mouse models, leading to prolonged survival. Together, these findings indicate that KHSRP mediates FAK pathway activation in an m6A-dependent manner to support PDAC growth and metastasis, highlighting the potential of KHSRP as a therapeutic target in pancreatic cancer.
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Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.
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Virus del Síndrome Respiratorio y Reproductivo Porcino , ARN Mensajero , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Síndrome Respiratorio y de la Reproducción Porcina/genética , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Macrófagos Alveolares/virología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/metabolismo , Interacciones Huésped-Patógeno/genética , Interferón gamma/genética , Interferón gamma/metabolismo , Interferón gamma/inmunología , Transcripción GenéticaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) is a pathogen that causes severe abortions in sows and high piglet mortality, resulting in huge economic losses to the pig industry worldwide. The emerging and novel PRRSV isolates are clinically and biologically important, as there are likely recombination and pathogenic differences among PRRSV genomes. Furthermore, the NADC34-like strain has become a major epidemic strain in some parts of China, but the characterization and pathogenicity of the latest strain in Inner Mongolia have not been reported in detail. In this study, an NADC34-like strain (CHNMGKL1-2304) from Tongliao City, Inner Mongolia was successfully isolated and characterized, and confirmed the pathogenicity in pigs. The phylogenetic tree showed that this strain belonged to sublineage 1.5 and had high homology with the strain JS2021NADC34. There is no recombination between CHNMGKL1-2304 and any other domestic strains. Animal experiments show that the CHNMGKL1-2304 strain is moderately virulent to piglets, which show persistent fever, weight loss and high morbidity but no mortality. The presence of PRRSV nucleic acids was detected in both blood, tissues, nasal and fecal swabs. In addition, obvious pathological changes and positive signals were observed in lung, lymph node, liver and spleen tissues when subjected to hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). This report can provide a basis for epidemiological investigations and subsequent studies of PRRSV.
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Genoma Viral , Filogenia , Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Virus del Síndrome Respiratorio y Reproductivo Porcino/clasificación , China , Síndrome Respiratorio y de la Reproducción Porcina/virología , Síndrome Respiratorio y de la Reproducción Porcina/patología , Virulencia , Evolución MolecularRESUMEN
The selective catalytic oxidation (SCO) is an effective method for removing slipped high-concentration ammonia from NH3-fueled engine exhaust gas. Herein a novel bifunctional catalyst was synthesized by mechanically mixing sulfated Ce/ZrO2 (Ce/ZrO2-S) with a small fraction of Pt/Al2O3 (Pt 0.1 wt.%) for SCO of NH3. As expected, the introduction of a small amount of Pt/Al2O3 significantly improved NH3 conversion ability of Ce/ZrO2-S catalysts toward low-temperature direction. When the mass ratio of Pt/Al2O3 to Ce/ZrO2-S was 7.5% (the corresponding mixed catalyst was denoted as P@CZS-7.5), T90 temperature was 312 °C. More importantly, P@CZS-7.5 catalyst exhibited a much better N2 selectivity (> 96%) in a wide temperature range (320 ~ 450 °C). H2-TPR results revealed that the addition of a trace amount of Pt/Al2O3 significantly led to a distinct shift of reduction temperature peak toward low-temperature direction, thereby greatly improved the low-temperature redox performance of mixed catalysts. Furthermore, NH3-TPD and BET results showed that P@CZS-7.5 catalyst exhibited a similar NH3 adsorption capacity to Ce/ZrO2-S catalyst, while the former had a relatively higher specific surface area than the latter. It was considered as a crucial factor for P@CZS-7.5 catalyst maintaining superior N2 selectivity in high-concentration NH3 (5000 ppm) removal processes. In situ DRIFTS results indicated that P@CZS-7.5 catalyst followed the internal selective catalytic reduction (i-SCR) mechanism in NH3-SCO reactions.
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Óxido de Aluminio , Amoníaco , Oxidación-Reducción , Amoníaco/química , Catálisis , Óxido de Aluminio/química , Platino (Metal)/química , Circonio/químicaRESUMEN
The relevance of biopterin metabolism in resistance to immune checkpoint blockade (ICB) therapy remains unknown. We demonstrate that the deficiency of quinoid dihydropteridine reductase (QDPR), a critical enzyme regulating biopterin metabolism, causes metabolite dihydrobiopterin (BH2) accumulation and decreases the ratio of tetrahydrobiopterin (BH4) to BH2 in pancreatic ductal adenocarcinomas (PDACs). The reduced BH4/BH2 ratio leads to an increase in reactive oxygen species (ROS) generation and a decrease in the distribution of H3K27me3 at CXCL1 promoter. Consequently, myeloid-derived suppressor cells are recruited to tumor microenvironment via CXCR2 causing resistance to ICB therapy. We discovered that BH4 supplementation is capable to restore the BH4/BH2 ratio, enhance anti-tumor immunity, and overcome ICB resistance in QDPR-deficient PDACs. Tumors with lower QDPR expression show decreased responsiveness to ICB therapy. These findings offer a novel strategy for selecting patient and combining therapies to improve the effectiveness of ICB therapy in PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/metabolismo , Humanos , Animales , Ratones , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Microambiente Tumoral , Línea Celular Tumoral , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/farmacología , Ratones Endogámicos C57BL , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Femenino , Masculino , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Metastasis is the major cause of cancer death. However, what types of heterogenous cancer cells in primary tumour and how they metastasise to the target organs remain largely undiscovered. DESIGN: We performed single-cell RNA sequencing and spatial transcriptomic analysis in primary colorectal cancer (CRC) and metastases in the liver (lCRC) or ovary (oCRC). We also conducted immunofluorescence staining and functional experiments to examine the mechanism. RESULTS: Integrative analyses of epithelial cells reveal a stem-like cell cluster with high protein tyrosine phosphatase receptor type O (PTPRO) and achaete scute-like 2 (ASCL2) expression as the metastatic culprit. This cell cluster comprising distinct subpopulations shows distinct liver or ovary metastatic preference. Population 1 (P1) cells with high delta-like ligand 4 (DLL4) and MAF bZIP transcription factor A (MAFA) expression are enriched in primary CRC and oCRC, thus may be associated with ovarian metastasis. P3 cells having a similar expression pattern as cholangiocytes are found mainly in primary CRC and lCRC, presuming to be likely the culprits that specifically metastasise to the liver. Stem-like cells interacted with cancer-associated fibroblasts and endothelial cells via the DLL4-NOTCH signalling pathway to metastasise from primary CRC to the ovary. In the oCRC microenvironment, myofibroblasts provide cancer cells with glutamine and perform a metabolic reprogramming, which may be essential for cancer cells to localise and develop in the ovary. CONCLUSION: We uncover a mechanism for organ-specific CRC metastasis.
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Neoplasias Colorrectales , Neoplasias Hepáticas , Femenino , Humanos , Neoplasias Colorrectales/patología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Neoplasias Hepáticas/patología , Perfilación de la Expresión Génica , Transducción de Señal/genética , Regulación Neoplásica de la Expresión Génica , Metástasis de la Neoplasia/genética , Microambiente Tumoral/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismoRESUMEN
The biological functions of noncoding RNA N6-methyladenosine (m6A) modification remain poorly understood. In the present study, we depict the landscape of super-enhancer RNA (seRNA) m6A modification in pancreatic ductal adenocarcinoma (PDAC) and reveal a regulatory axis of m6A seRNA, H3K4me3 modification, chromatin accessibility and oncogene transcription. We demonstrate the cofilin family protein CFL1, overexpressed in PDAC, as a METTL3 cofactor that helps seRNA m6A methylation formation. The increased seRNA m6As are recognized by the reader YTHDC2, which recruits H3K4 methyltransferase MLL1 to promote H3K4me3 modification cotranscriptionally. Super-enhancers with a high level of H3K4me3 augment chromatin accessibility and facilitate oncogene transcription. Collectively, these results shed light on a CFL1-METTL3-seRNA m6A-YTHDC2/MLL1 axis that plays a role in the epigenetic regulation of local chromatin state and gene expression, which strengthens our knowledge about the functions of super-enhancers and their transcripts.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Cromatina/genética , ARN , Epigénesis Genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Oncogenes/genética , Metiltransferasas/genéticaRESUMEN
N6-methyladenosine (m6A) modification of gene transcripts plays critical roles in cancer. Here we report transcriptomic m6A profiling in 98 tissue samples from 65 individuals with pancreatic ductal adenocarcinoma (PDAC). We identify 17,996 m6A peaks with 195 hyper-methylated and 93 hypo-methylated in PDAC compared with adjacent normal tissues. The differential m6A modifications distinguish two PDAC subtypes with different prognosis outcomes. The formation of the two subtypes is driven by a newly identified m6A regulator CSTF2 that co-transcriptionally regulates m6A installation through slowing the RNA Pol II elongation rate during gene transcription. We find that most of the CSTF2-regulated m6As have positive effects on the RNA level of host genes, and CSTF2-regulated m6As are mainly recognized by IGF2BP2, an m6A reader that stabilizes mRNAs. These results provide a promising PDAC subtyping strategy and potential therapeutic targets for precision medicine of PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , ARN Mensajero/genética , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Regulación Neoplásica de la Expresión Génica , Proteínas de Unión al ARN/genética , Neoplasias PancreáticasRESUMEN
The therapeutic options for treating pancreatic ductal adenocarcinoma (PDAC) are limited, and resistance to gemcitabine, a cornerstone of PDAC chemotherapy regimens, remains a major challenge. N6-methyladenosine (m6A) is a prevalent modification in mRNA that has been linked to diverse biological processes in human diseases. Herein, by characterizing the global m6A profile in a panel of gemcitabine-sensitive and gemcitabine-insensitive PDAC cells, we identified a key role for elevated m6A modification of the master G0-G1 regulator FZR1 in regulating gemcitabine sensitivity. Targeting FZR1 m6A modification augmented the response to gemcitabine treatment in gemcitabine-resistant PDAC cells both in vitro and in vivo. Mechanistically, GEMIN5 was identified as a novel m6A mediator that specifically bound to m6A-modified FZR1 and recruited the eIF3 translation initiation complex to accelerate FZR1 translation. FZR1 upregulation maintained the G0-G1 quiescent state and suppressed gemcitabine sensitivity in PDAC cells. Clinical analysis further demonstrated that both high levels of FZR1 m6A modification and FZR1 protein corresponded to poor response to gemcitabine. These findings reveal the critical function of m6A modification in regulating gemcitabine sensitivity in PDAC and identify the FZR1-GEMIN5 axis as a potential target to enhance gemcitabine response. SIGNIFICANCE: Increased FZR1 translation induced by m6A modification engenders a gemcitabine-resistant phenotype by inducing a quiescent state and confers a targetable vulnerability to improve treatment response in PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas Cdh1 , Línea Celular Tumoral , Gemcitabina/farmacología , Gemcitabina/uso terapéutico , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , ARN Mensajero/genética , Neoplasias PancreáticasRESUMEN
Porcine Reproductive and Respiratory Syndrome (PRRS) threats the swine industry seriously. The spread of live vaccine virus leads to the emergence of recombinant virus, which brings biosafety problems. The replication-deficient virus as a vaccine candidate would avoid this problem. In the present study, the recombinant lentiviral plasmid pLV-EF1α-EGFP-2A-ORF4 was co-transfected with lentivirus in HEK293FT cells. The transfection mixture was harvested and transduced into Marc-145 to screen a cell line stably expressing the PRRSV ORF4 with puromycin. The cell line Marc-145-GP4 was confirmed with PCR, RT-PCR, IFA, and Western blotting using a monoclonal antibody against Glycoprotein 4 (GP4) of PRRSV. To obtain a replication-deficient PRRSV, Western blotting the recombinant plasmid pNM09-ΔORF4 was constructed by Overlap PCR and DNA recombinant technology with the pNM09 as a backbone plasmid. The pNM09-ΔORF4 was transfected into Marc-145-GP4 with electroporation after transcription in vitro. The replication-deficient virus was rescued on Marc-145-GP4 cells with trans-complementation of ORF4 gene and verified by RT-PCR and IFA. The results indicated that a cell line Marc-145-GP4 stably expressed PRRSV ORF4 was obtained. The recombinant GP4 was successfully expressed and obtained a monoclonal antibody Anti-A-GP4-70, which can specifically react with the virus. Finally, the replication-deficient virus rNM09-ΔORF4 can be rescued with low titer and could only reproduce on the Marc-145-GP4 cells. Unfortunately, the rNM09-ΔORF4 showed too low virus replication titer to determine it. This study lays the foundation for the rapid detection of PRRS and the functional study of GP4 and provides experience for replication-deficient PRRSV.
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Síndrome Respiratorio y de la Reproducción Porcina , Virus del Síndrome Respiratorio y Reproductivo Porcino , Enfermedades de los Porcinos , Animales , Porcinos , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Carga Viral/veterinaria , Línea Celular , Glicoproteínas , Anticuerpos Monoclonales , Replicación Viral/genéticaRESUMEN
Infectious bovine rhinotracheitis (IBR) is caused by Bovine herpesvirus type 1 (BoHV-1), which seriously threatens the global cattle industry. Only vaccination to improve immunity is the most direct and effective means to prevent IBR. Attempts are being made to use subunit vaccines, deleted or recombinant viral vaccines to reduce or eradicate IBR. For investigating the immunological characteristics of glycoprotein B subunit vaccine in pattern animal guinea pigs, the partial glycoprotein B (gB) of BoHV-1 with dominant antigenic characteristic was selected. A recombinant prokaryotic expression vector pET-32a-gB with the truncated gB gene was constructed, expressed, identified and the purified proteins were used to immunize guinea pigs. The immune effect of the subunit vaccine was assessed by monitoring clinical symptoms, viral load, antibody secretion, and histopathological changes. The results indicated that guinea pigs immunized with the gB subunit vaccine produced high levels of anti-gB antibodies and virus-neutralizing antibodies. The gB subunit vaccine significantly reduced viral shedding and lung tissue damage after IBRV challenge. The animals inoculated the gB subunit vaccine also had less virus reactivation. Its protective effect on viral shedding and tissue damage was similar to that of inactivated BoHV-1 vaccine. This work is a proof-of-concept study of subunit vaccine-induced protection against BoHV-1. And it is expected to be a candidate vaccine for the prevention of IBR.
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Herpesvirus Bovino 1 , Rinotraqueítis Infecciosa Bovina , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Bovinos , Cobayas , Herpesvirus Bovino 1/genética , Rinotraqueítis Infecciosa Bovina/prevención & control , Vacunas de Productos Inactivados , Vacunas de Subunidad/genética , Vacunas Virales/genéticaRESUMEN
Since porcine reproductive and respiratory syndrome virus (PRRSV) was first described in China in 1996, several genetically distinct strains of PRRSV have emerged with varying pathogenicity and severity, thereby making the prevention and control of PRRS more difficult in China and worldwide. Between 2017 and 2021, the detection rate of NADC34-like strain in China increased. To date, NADC34-like strains have spread to 10 Chinese provinces and have thus developed different degrees of pathogenicity and mortality. In this review, we summarize the history of NADC34-like strains in China and clarify the prevalence, genomic characteristics, restriction fragment length polymorphisms, recombination, pathogenicity, and vaccine status of this strain in China. In so doing, this study aims to provide a basis for the further development of prevention and control measures targeting the NADC34-like strain.
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Bovine respiratory disease complex (BRDC) is a comprehensive disease in cattle caused by various viral and bacterial infections. Among them, bovine herpesvirus type I (BoHV-1) and bovine viral diarrhea virus (BVDV) play important roles and have caused huge financial losses for the cattle industry worldwide. At present, vaccines against BRDC include trivalent attenuated BoHV-1, BVDV-1, and BVDV-2 live vaccines, BoHV-1 live attenuated vaccines, and BoHV-1/BVDV bivalent live attenuated vaccines, which have limitations in terms of their safety and efficacy. To solve these problems, we optimized the codon of the BVDV-1 E2 gene, added the signal peptide sequence of the BoHV-1 gD gene, expressed double BVDV-1 E2 glycoproteins in tandem at the BoHV-1 gE gene site, and constructed a BoHV-1 genetics-engineered vectored vaccine with gE gene deletion, named BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE. This study compared the protective effects in BoHV-1, BoHV-1 ΔgE, BoHV-1 gE/E2-Linker-E2+, and BVDV-1 inactivated antigen immunized guinea pigs and calves. The results showed that BoHV-1 gE/E2-Linker-E2+ could successfully induce guinea pigs and calves to produce specific neutralizing antibodies against BVDV-1. In addition, after BoHV-1 and BVDV-1 challenges, BoHV-1 gE/E2-Linker-E2+ can produce a specific neutralizing antibody response against BoHV-1 and BVDV-1 infections. Calves immunized with this type of virus can be distinguished as either vaccinated animals (gE-) or naturally infected animals (gE+). In summary, our data suggest that BoHV-1 gE/E2-Linker-E2+ and BoHV-1 ΔgE have great potential to prevent BVDV-1 or BoHV-1 infection.
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Virus de la Diarrea Viral Bovina Tipo 1 , Virus de la Diarrea Viral Bovina , Herpesvirus Bovino 1 , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Bovinos , Diarrea , Virus de la Diarrea Viral Bovina Tipo 1/genética , Virus de la Diarrea Viral Bovina/genética , Cobayas , Herpesvirus Bovino 1/genética , Vacunas Atenuadas/genética , Vacunas Combinadas , Vacunas Virales/genéticaRESUMEN
Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5' and 3' ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3' end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP+ or EGFP- will lay the foundation for research on BoHV-1 and vaccine development in the future.
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N6-methyladenosine (m6A) is the most prevalent RNA modification, and the effect of its dysregulation on esophageal squamous cell carcinoma (ESCC) development remains unclear. Here, by performing transcriptome-wide m6A sequencing in 16 ESCC tissue samples, we identified the key roles of m6A in TNFRSF1A (also known as TNFR1)-mediated MAPK and NF-κB activation in ESCC. Mechanistically, a functional protein involved in m6A methylation, ATXN2, is identified that augments the translation of TNFRSF1A by binding to m6A-modified TNFRSF1A mRNA. Upregulation of the TNFRSF1A protein level, a vital upstream switch for TNFRSF1A-mediated signaling events, activates the NF-κB and MAPK pathways and thus promotes ESCC development. Furthermore, TNFRSF1A m6A modifications and protein levels are upregulated in ESCC, and high levels of TNFRSF1A m6A and protein are correlated with poor ESCC patient survival. These results collectively indicate that the m6A-TNFRSF1A axis is critical for ESCC development and thus may serve as a potential druggable target.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Ataxina-2/genética , Ataxina-2/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , FN-kappa B/metabolismo , ARN Mensajero/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genéticaRESUMEN
The enhancer of zeste homolog 2 (EZH2) is a methylated modification enzyme of Histone H3-Lys 27. The high expression of EZH2 in cells is closely related to the progression, invasion, and metastasis of neoplasm. Therefore, this target is gradually becoming one of the research hot spots of tumor pathogenesis, and the inhibitors of the EZH2 enzyme are expected to become new antitumor drugs. This study used a series of virtual screening technologies to calculate the affinity between the compounds obtained from the ZINC15 database and the target protein EZH2, the stability of the ligand-receptor complex. This experiment also predicted the toxicity and absorption, distribution, metabolism, and excretion (ADME) properties of the candidate drugs in order to obtain compounds with excellent pharmacological properties. Finally, the ligand-receptor complex under in vivo situation was estimated by molecular dynamics simulation to observe whether the complex could exist steadily in the body. The experimental results showed that the two natural compounds ZINC000004217536 and ZINC000003938642 could bind tightly to EZH2, and the ligand-receptor complex could exist stably in vivo. Moreover, these two compounds were calculated to be nontoxic. They also had a high degree of intestinal absorption and high bioavailability. In vitro experiments confirmed that drug ZINC000003938642 could inhibit the proliferation and migration of osteosarcoma, which could serve as potential lead compounds. Therefore, the discovery of these two natural products had broad prospects in the development of EZH2 inhibitors, providing new clues for the treatment or adjuvant treatment of tumors.
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OBJECTIVES: This study aimed to identify novel targets in the carcinogenesis, therapy and prognosis of osteosarcoma from genomic level, together with screening ideal lead compounds with potential inhibition regarding MMP-9. METHODS: Gene expression profiles from GSE12865, GSE14359, GSE33382, GSE36001 and GSE99671 were obtained respectively from GEO database. Differentially expressed genes were identified, and functional enrichment analysis, such as GO, KEGG, GSEA, PPI were performed to make a comprehensive understanding of the hub genes. Next, a series of high-precision computational techniques were conducted to screen potential lead compounds targeting MMP9, including virtual screening, ADME, toxicity prediction, and accurate docking analysis. RESULTS: 10 genes, MMP9, CD74, SPP1, CXCL12, TYROBP, FCER1G, HCLS1, ARHGDIB, LAPTM5 and IGF1R were identified as hub genes in the initiation of osteosarcoma. Machine learning, multivariate Cox analysis, ssGSEA and survival analysis demonstrated that these genes had values in prognosis, immune-correlation and targeted treatment. Tow novel compounds, ZINC000072131515 and ZINC000004228235, were screened as potential inhibitor regarding MMP9, and they could bind to MMP9 with favorable interaction energy and high binding affinity. Meanwhile, they were precited to be efficient and safe drugs with low-ames mutagenicity, none weight evidence of carcinogenicity, as well as non-toxic with liver. CONCLUSIONS: This study revealed the significance of 10-gene signature in the development of osteosarcoma. Besides, drug candidates identified in this study provided a solid basis on MMP9 inhibitors' development.
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Recent studies have reported that host proteins regulate Rabies virus (RABV) infection via distinct mechanisms. The abnormal neural function caused by RABV infection is related to the abnormal synaptic signal transmission in which the RABV glycoprotein (G) is involved. In the present study, two recombinant Rabies viruses (rRABVs), namely rSAD-SAD-Flag-G and rSAD-CVS-Flag-G, were established and rescued based on rSAD and verified by indirect fluorescence assay (IFA), and western blotting (WB). To investigate how the G protein interacts with synaptosomal-associated protein 25 (SNAP25), primary neuronal cells (PNC) of embryonic mice were cultured and infected with rRABVs. Immunoprecipitation (IP) and LC-MS/MS analysis of glycoprotein-binding proteins, which were flag tagged, were carried out to determine the interaction of G protein and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNARE) complex in PNC. G protein and the SNARE member SNAP25 were co-expressed in HEK293 cells or primary neuronal cells to investigate their colocalization. Knockdown of SNAP25 with small interfering RNA (siRNA) was conducted on mNA cells, and rRABV replication was observed by IFA, qRT-PCR, and virus titration. The results indicated that rRABVs were successfully rescued and grew well in PNC. Flag-tag IP and confocal microscopy demonstrated that SNAP25 works together with G protein and colocalizes with G on the cytomembrane of HEK293 cells. The downregulation of SNAP25, using RNA interference, resulted in a significant decrease in the number of viral mRNAs, viral proteins, and virus particles. Furthermore, the regression of SNAP25 did not affect the initial infection of the virus but reduced the infectivity of progeny virions.
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Interacciones Microbiota-Huesped , Fusión de Membrana , Neuronas/virología , Virus de la Rabia/fisiología , Proteínas SNARE/genética , Proteína 25 Asociada a Sinaptosomas/genética , Animales , Células Cultivadas , Regulación de la Expresión Génica , Células HEK293 , Humanos , Ratones , ARN Interferente Pequeño , Proteínas SNARE/metabolismoRESUMEN
The habenula is a nuclear complex composed of the lateral habenula (LHb) and medial habenula (MHb), two distinct structures. Much progress has been made to emphasize the role of the LHb in the pathogenesis of depression. In contrast, relatively less research has focused on the MHb. However, in recent years, the role of the MHb has begun to gain increasing attention. The MHb connects to the interpeduncular nucleus (IPN) both morphologically and functionally. The MHb-IPN pathway plays an important role in regulating higher brain functions, including cognition, reward, and decision making. It indicates a role of the MHb in the pathogenesis of depression. Thus, we investigated the role of the MHb-IPN pathway in depression. MHb metabolic activity was increased in the chronic unpredictable mild stress (CUMS)-exposed rat model of depression. MHb lesions in the CUMS-exposed rats reversed anhedonia-like behavior, as observed in the sucrose preference test, and significantly downregulated the elevated metabolic activity of the IPN. Substance P (SP)-containing neurons of the MHb were found to innervate the IPN and to be the main source of SP in the IPN. SP content of IPN tissue of the CUMS-exposed rats was increased and MHb lesions reversed this change. In the in vitro experiment, firing rate recordings showed that SP perfusion increased the activity of IPN neurons. Our results suggest that hyperactivity of the MHb-IPN circuit is involved in the anhedonia-like behavior of depression, and that SP mediates the effect of the MHb on IPN neurons.