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BACKGROUND: To find a useful hypoxia non-invasive biomarker for evaluating early treatment response and prognosis to definitive chemoradiotherapy (dCRT) in patients with esophageal squamous cell carcinoma (ESCC), using blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI). METHODS: The R2* values were obtained pre- and 2-3 weeks post-dCRT in 28 patients with ESCC using BOLD MRI. Independent samples t-test (normality) or Mann-Whitney U test (non-normality) was used to compare the differences of R2*-related parameters between the complete response (CR) and the non-CR groups. Diagnostic performance of parameters in predicting response was tested with receiver operating characteristic (ROC) curve analysis. The 3-year overall survival (OS) was evaluated using Kaplan Meier curve, log rank test, and Cox proportional hazards regression analysis. RESULTS: The post-R2*, ∆R2*, and ∆%R2* in the CR group were significantly higher than those in the non-CR group (P = 0.002, 0.003, and 0.006, respectively). The R2*-related parameters showed good prediction of tumor response, with AUC ranging from 0.813 to 0.829. The 3-year OS rate in patients with ∆R2* >-7.54 s- 1 or CR were significantly longer than those with ∆R2* ≤ -7.54 s- 1 (72.37% vs. 0.00%; Hazard ratio, HR = 0.196; 95% confidence interval, 95% CI = 0.047-0.807; P = 0.024) or non-CR (76.47% vs. 29.27%; HR = 0.238, 95% CI = 0.059-0.963; P = 0.044). CONCLUSIONS: The preliminary results demonstrated that the R2* value might be a useful hypoxia non-invasive biomarker for assessing response and prognosis of ESCC treated with dCRT. BOLD MRI might be used as a potential tool for evaluating tumor oxygenation metabolism, which is routinely applied in clinical practice and beneficial to clinical decision-making. A large sample size was needed for further follow-up studies to confirm the findings.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Humanos , Carcinoma de Células Escamosas de Esófago/terapia , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Neoplasias Esofágicas/diagnóstico por imagen , Neoplasias Esofágicas/terapia , Pronóstico , Quimioradioterapia/métodos , Biomarcadores , Imagen por Resonancia Magnética/métodos , Hipoxia/tratamiento farmacológico , Estudios RetrospectivosRESUMEN
Among hematological malignancies, multiple myeloma (MM) represents the leading indication of autologous hematopoietic stem cell transplantation (auto-HCT). Auto-HCT is predominantly performed with peripheral blood stem cells (PBSCs), and the mobilization and collection of PBSCs are essential steps for auto-HCT. Despite the improved success of conventional methods with the incorporation of novel agents for PBSC mobilization in MM, mobilization failure is still a concern. The current review comprehensively summarizes various mobilization strategies for mobilizing PBSCs in MM patients and the evolution of these strategies over time. Moreover, existing evidence substantiates that the mobilization regimen used may be an important determinant of graft content. However, limited data are available on the effects of graft characteristics in patient outcomes other than hematopoietic engraftment. In this review, we discussed the effect of graft characteristics on clinical outcomes, mobilization failure, factors predictive of poor mobilization, and potential mobilization regimens for such patients.
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Neoplasias Hematológicas , Trasplante de Células Madre Hematopoyéticas , Mieloma Múltiple , Humanos , Movilización de Célula Madre Hematopoyética/métodos , Mieloma Múltiple/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Trasplante Autólogo , Factor Estimulante de Colonias de GranulocitosRESUMEN
BACKGROUND: Phosphorus commonly reduces lipid deposition in the vertebrates. However, the underlying mechanisms involved in the process remain unclear. METHODS: Yellow catfish were given three experimental diets with dietary phosphate levels of 3.22, 6.47 and 7.99 g Pi kg- 1, respectively, for 8 weeks. The contents of triglyceride, non-esterified free fatty acids, adenosine triphosphate, nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide, enzymatic activities, mRNA and protein expression were determined in the intestinal tissues. Hematoxylin and eosin, Oil Red O staining, and transmission electron microscope were performed for intestinal tissues. Primary intestinal epithelial cells were isolated from yellow catfish intestine. Western blot analysis, Immunoprecipitation assays, Immunofluorescence staining, and RNA extraction and quantitative real-time PCR were decided. Luciferase reporter assays and electrophoretic mobility shift assay were used to evaluate the function of Sirt3, PPARα and Lcad promoters. RESULTS: High dietary phosphate intake activated intestinal phosphate absorption and excretion, and reduced lipid deposition through increasing lipolysis in the intestine. Moreover, phosphate incubation increased the mRNA and protein expression of krüppel like factor 4 (klf4), silent mating-type information regulation 2 homolog 3 (sirt3), peroxisome proliferator activated receptor alpha (pparα) and long chain acyl-CoA dehydrogenase (lcad) in the intestinal epithelial cells (IECs), and klf4 knockdown attenuated the phosphate-induced increase of protein levels of Sirt3, Pparα and Lcad. Further investigation found that Klf4 overexpression increased the activity of sirt3 and pparα promoters, which in turn reduced the acetylation and protein level of Lcad. CONCLUSION: Dietary Pi excess induced lipid degradation by the activation of the Klf4-Sirt3/Pparα-Lcad pathway in the intestine and primary IECs. Video Abstract.
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Sirtuina 3 , Animales , Lípidos , Lipólisis , Oxidación-Reducción , PPAR alfa/metabolismo , ARN Mensajero/metabolismo , Sirtuina 3/genética , BagresRESUMEN
Excessive phosphorus (Pi) contributes to eutrophication in an aquatic environment, which threatens human and fish health. However, the mechanisms by which Pi overload influences aquatic animals remain largely unexplored. In the present study, Pi supplementation increased the Pi content, inhibited lipid accumulation and lipogenesis, and stimulated lipolysis in the liver. Pi supplementation increased the phosphorylation of glycogen synthase kinase-3 ß (GSK3ß) at serine 9 (S9) but inhibited the phosphorylation of GSK3α at tyrosine 279 (Y279), GSK3ß at tyrosine 216 (Y216), and peroxisome proliferator-activated receptor α (PPARα) at serine 84 (S84) and threonine 265 (T265). Pi supplementation also upregulated PPARα protein expression and stimulated its transcriptional activity, thereby inducing lipolysis. Pi suppressed GSK3ß activity and prevented GSK3ß, but not GSK3α, from interacting with PPARα, which in turn alleviated PPARα phosphorylation. GSK3ß-induced phosphorylation of PPARα was dependent on GSK3ß S9 dephosphorylation rather than Y216 phosphorylation. Mechanistically, underphosphorylation of PPARα mediated Pi-induced lipid degradation through transcriptionally activating adipose triglyceride lipase (atgl) and very long-chain-specific acyl-CoA dehydrogenase (acadvl). Collectively, our findings uncovered a new mechanism by which Pi facilitates lipolysis via the GSK3ß-PPARα pathway and highlighted the importance of S84 and T265 phosphorylation in PPARα action.
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Lipólisis , PPAR alfa , Animales , Humanos , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Lípidos , Hígado/metabolismo , Fosforilación , PPAR alfa/metabolismo , PecesRESUMEN
PURPOSE: There is a lack of studies that systematically evaluate the clinical factors of PICC-RVT such as treatment, tumor stage, metastasis, and chemotherapy drugs in cancer patients. This study, therefore, aims to evaluate the clinical factors of catheter-related venous thrombosis in cancer patients with indwelling PICC to provide a basis for the clinical prevention and reduction of thrombosis. METHODS: Relevant studies were retrieved from major databases (PubMed, Embase, Web of Science, China National Knowledge Infrastructure (CNKI), WanFang Data, and China Biology Medicine disc (CMB)) and searched from their earliest available dates until July 2022. If two or more studies had the same outcome, a meta-analysis using RevMan 5.4.1 was performed. This systematic review was registered at PROSPERO (number CRD42022358426). RESULTS: A total of 19 articles involving 19,824 patients were included for quantitative analysis. Meta-analysis of these studies indicated that a history of chemotherapy, tumor type, tumor stage, presence or absence of metastasis, and use of fluorouracil, etoposide, platinum drugs, and taxane were all risk factors for PICC catheter thrombosis in cancer patients. CONCLUSION: In clinical PICC catheter thrombosis prevention, patients with the above characteristics should be watched more closely than other patients, as they have a higher risk for PICC catheter thrombosis. Based on the present evidence at hand, radiotherapy cannot be considered to be related to the formation of PICC-RVT in cancer patients.
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Catéteres , Neoplasias , Humanos , China , Bases de Datos Factuales , Etopósido , Neoplasias/tratamiento farmacológicoRESUMEN
Drug resistance in fungal pathogens has risen steadily over the past decades due to long-term azole therapy or triazole usage in agriculture. Modification of the drug target protein to prevent drug binding is a major recognized route to induce drug resistance. However, mechanisms for nondrug target-induced resistance remain only loosely defined. Here, we explore the molecular mechanisms of multidrug resistance resulted from an efficient adaptation strategy for survival in drug environments in the human pathogen Aspergillus fumigatus We show that mutants conferring multidrug resistance are linked with mitochondrial dysfunction induced by defects in heme A biosynthesis. Comparison of the gene expression profiles between the drug-resistant mutants and the parental wild-type strain shows that multidrug-resistant transporters, chitin synthases, and calcium-signaling-related genes are significantly up-regulated, while scavenging mitochondrial reactive oxygen species (ROS)-related genes are significantly down-regulated. The up-regulated-expression genes share consensus calcium-dependent serine threonine phosphatase-dependent response elements (the binding sites of calcium-signaling transcription factor CrzA). Accordingly, drug-resistant mutants show enhanced cytosolic Ca2+ transients and persistent nuclear localization of CrzA. In comparison, calcium chelators significantly restore drug susceptibility and increase azole efficacy either in laboratory-derived or in clinic-isolated A. fumigatus strains. Thus, the mitochondrial dysfunction as a fitness cost can trigger calcium signaling and, therefore, globally up-regulate a series of embedding calcineurin-dependent-response-element genes, leading to antifungal resistance. These findings illuminate how fitness cost affects drug resistance and suggest that disruption of calcium signaling might be a promising therapeutic strategy to fight against nondrug target-induced drug resistance.
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Aspergillus fumigatus/efectos de los fármacos , Señalización del Calcio/fisiología , Farmacorresistencia Fúngica/efectos de los fármacos , Farmacorresistencia Fúngica/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Transferasas Alquil y Aril/genética , Antifúngicos/farmacología , Aspergillus fumigatus/genética , Quelantes del Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Quitina Sintasa/genética , Farmacorresistencia Fúngica/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Hemo/análogos & derivados , Hemo/biosíntesis , Humanos , Proteínas de la Membrana/genética , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Fosfoproteínas Fosfatasas/metabolismo , Especies Reactivas de Oxígeno , Factores de Transcripción/metabolismo , Triazoles/farmacologíaRESUMEN
Due to many special characteristics, zinc oxide nanoparticles (ZnO NPs) are widely used all over the world, leading to their wide distribution in the environment. However, the toxicities and mechanisms of environmental ZnO NP-induced changes of physiological processes and metabolism remain largely unknown. Here, we found that addition of dietary ZnO NPs disturbed hepatic Zn metabolism, increased hepatic Zn and lipid accumulation, downregulated lipolysis, induced oxidative stress, and activated mitophagy; N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, Zn2+ ions chelator) alleviated high ZnO NP-induced Zn and lipid accumulation, oxidative stress, and mitophagy. Mechanistically, the suppression of mitochondrial oxidative stress attenuated ZnO NP-activated mitophagy and ZnO NP-induced lipotoxicity. Taken together, our study elucidated that mitochondrial oxidative stress mediated ZnO NP-induced mitophagy and lipotoxicity; ZnO NPs could be dissociated to free Zn2+ ions, which partially contributed to ZnO NP-induced changes in oxidative stress, mitophagy, and lipid metabolism. Our study provides novel insights into the impacts and mechanism of ZnO NPs as harmful substances inducing lipotoxicity of aquatic organisms, and accordingly, metabolism-relevant parameters will be useful for the risk assessment of nanoparticle materials in the environment.
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Nanopartículas del Metal , Nanopartículas , Óxido de Zinc , Animales , Agua Dulce , Lípidos , Nanopartículas del Metal/toxicidad , Mitocondrias/metabolismo , Mitofagia , Nanopartículas/toxicidad , Estrés Oxidativo , Óxido de Zinc/toxicidadRESUMEN
The mitochondrial unfolded protein response (UPRmt) is known as a conservative mechanism in response to mitochondrial dysfunction. Thus, based on UPRmt, this study was conducted to determine the mechanism of a high-fat diet (HFD) inducing mitochondrial dysfunction and its role in stimulating hepatic lipid dysregulation. The choline-activated alleviating effect was also evaluated. In vivo, yellow catfish were fed three diets (control, HFD, and HFD + choline diet) for 10 weeks. In vitro, hepatocytes isolated from yellow catfish and the HepG2 cell line were cultured and incubated with fatty acid (FA) for 48 h. (1) HFD-induced mitochondrial dysfunction via SIRT3/mtHSP70-mediated UPRmt. HFD inhibited the subcellular localization of SIRT3 into the mitochondrion, resulting in the up-regulating of mtHSP70 acetylation via lysine residues 493 and 507. The mtHSP70 acetylation promoted the stability of mtHSP70, which then led to the UPRmt and further mitochondrial dysfunction. (2) SIRT3/mtHSP70-mediated UPRmt regulated HFD/FA-induced hepatic lipid dysregulation. SIRT3/mtHSP70-mediated UPRmt reduced FA ß-oxidation via mitochondrial dysfunction and then led to lipid dysregulation. Additionally, the mtHSP70-ACOX1 interaction was confirmed. (3) Choline alleviated HFD-induced UPRmt via up-regulating the localization of SIRT3 into the mitochondrion, which in turn led to the subsequent ameliorating effect on HFD-induced hepatic lipid dysregulation. Through SIRT3-mediated mtHSP70 deacetylation, dietary choline alleviates HFD-induced hepatic lipid dysregulation via UPRmt. This provides the first proof of acetylation regulating UPRmt and the crosstalk between UPRmt and FA ß-oxidation.
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Sirtuina 3 , Colina/metabolismo , Colina/farmacología , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Hígado/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismoRESUMEN
Aplastic anemia (AA) is a T cell immune-mediated autoimmune disease. Overactivated CD8+ T cells play a leading role in the pathogenesis of AA, which may be due to disbalance in costimulatory and coinhibitory signals in T cells. In this study, we firstly investigated the expression of OX40, 4-1BB, GITR, ICOS, CTLA-4, LAG-3, and TIM-3 on CD8+ T cells from untreated patients with AA and healthy individuals (HIs) by flow cytometry. Moreover, we further analyzed the phenotype and functional characteristics of CD8+GITR+ T cells to more fully assess the T cell activation dysfunction in AA. We for the first time demonstrated significantly decreased percentage of CD8+GITR+ T cells in AA, and CD8+GITR+CTLA-4+ T cells were significantly higher in patients with AA compared with HIs. Conversely, the percentage of CD8+GITR+granzyme B+ and CD8+GITR+perforin+ T cells in AA patients was significantly reduced. Our preliminary data illustrate that the CD8+GITR+ T cell population might negatively regulate overactive T cell activation in AA.
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Anemia Aplásica/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores de Superficie Celular/inmunología , Adulto , Antígenos CD/inmunología , Antígeno CTLA-4/inmunología , Femenino , Humanos , Masculino , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
In this study, the mechanisms of environmentally relevant doses of Cu and Zn mixtures influencing lipid deposition and metabolism were investigated in freshwater teleost yellow catfish Pelteobagrus fulvidraco (2 months old, 4.95 (t0.01 g, mean ± SEM). Our study indicated that waterborne Cu exposure increased lipid content, while Zn activated lipophagic flux and alleviated Cu-induced lipid accumulation. Yellow catfish hepatocytes treated with Zn or Zn + Cu activated autophagy-specific lipophagy, decreased lipid storage, and increased nonesterified fatty acid (NEFA) release, suggesting a causal relationship between lipophagy and lipid droplet (LD) breakdown under Zn and Zn + Cu conditions. Our further investigation found that Beclin1 deacetylation by sirtuin 1 (SIRT1) was required for Zn- and Zn + Cu-induced lipophagy and lipolysis, and lysine residues 427 and 434 were key sites for Beclin1 deacetylation. Taken together, these findings show that the Zn-induced deacetylation of Beclin1 promotes lipophagy as an important pathway to alleviate Cu-induced lipid accumulation in fish, which reveals a previously unidentified mechanism for understanding the antagonistic effects of Cu and Zn on metabolism at their environmentally relevant concentrations. Our results highlight the importance of combined exposure when the biological effects of heavy metals are evaluated during environmental risk assessments.
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Beclina-1/metabolismo , Bagres , Cobre/toxicidad , Metabolismo de los Lípidos , Zinc/metabolismo , Animales , Autofagia , Hígado/metabolismoRESUMEN
Besides their function as a physical barrier against pathogens, ß-defensins possess the ability to induce direct or indirect chemotaxis in leukocytes of mammals. However little is known about the ability of defensins to guide the migration of macrophages in fish. The objective of our study was to investigate whether ß-defensin 1 (maBD1) can recruit leukocytes (specifically macrophages) in vivo and in vitro in a farmed cyprinid fish Megalobrama amblycephala. The M. amblycephala ß-defensin 1 (maBD1) gene was amplified from the head-kidney transcriptome. Synthetic maBD1 polypeptide (as well as its N-terminus half, but not the C-terminus half) was capable of inducing the migration of leukocytes (specifically macrophages) at concentrations from 26.0⯵g/mL to 52.0⯵g/mL in head kidney tissue in vitro. When injected intraperitoneally in vivo, the number of leukocytes in the peritoneal cavity was in positive correlation with the maBD1 concentration. maBD1 also induced the expression of two proinflammatory cytokines (IL-1beta and TNF-alpha) in spleen, head and body kidney, and hepatopancreas. These results strongly indicate that BD1 has a chemoattractant capacity for macrophages, as well as the ability to modulate the expression of proinflammatory cytokines in fish.
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Quimiotaxis/inmunología , Cyprinidae/inmunología , Proteínas de Peces/farmacología , Macrófagos/inmunología , beta-Defensinas/farmacología , Animales , Quimiotaxis/efectos de los fármacos , Macrófagos/efectos de los fármacos , Distribución AleatoriaRESUMEN
Columnaris disease (CD) caused by Flavobacterium columnare (F. columnare) is lack of knowledge on effective treatment measures. Bacterial pathogens require iron as an essential nutrient to infect the host. While hepcidin acts as a master regulator in iron metabolism, its contribution to host defense is emerging as complex and multifaceted. In vitro, recombinant Ctenopharyngodon idellus (C. idellus) hepcidin (CiHep) and synthetic CiHep both showed the ability to increase the expression of hepcidin and ferritin in C. idellus kidney cells, especially the recombinant CiHep. In vivo, recombinant CiHep improved the survival rate of C. idellus challenged with F. columnare. In addition, the fish fed diet containing recombinant CiHep (group H-1) had a higher survival rate than other pretreatment groups. The study showed that recombinant CiHep regulated iron metabolism causing iron redistribution, decreasing serum iron levels and increasing iron accumulation in the hepatopancreas. Moreover, the expression of iron-related genes was upregulated in various degrees at a different time except for group H-1. Immune-related genes were also evaluated, showing higher expression in the groups pretreated with CiHep at an early stage of infection. Of note, a clear upregulation of more immune genes occurred in the groups pretreated with recombinant CiHep than that pretreated with synthetic CiHep in the late stage of infection. In conclusion, the recombinant CiHep has a protective effect on the host response to bacterial pathogens. We speculate that hepcidin protects C. idellus against F. columnare infection via regulating the iron distribution and immune gene expression.
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Carpas/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Hepcidinas/inmunología , Inmunidad Innata/genética , Hierro/metabolismo , Secuencia de Aminoácidos , Animales , Carpas/genética , Carpas/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Infecciones por Flavobacteriaceae/inmunología , Infecciones por Flavobacteriaceae/veterinaria , Flavobacterium/fisiología , Perfilación de la Expresión Génica , Hepcidinas/genética , Hepcidinas/metabolismoRESUMEN
Macrophages are very versatile immune cells, with the characteristics of a proinflammatory phenotype in response to pathogen-associated molecular patterns. However, the specific activation marker genes of macrophages have not been systematically investigated in teleosts. In this work, leukocytes (WBC) were isolated using the Percoll gradient method. Macrophages were enriched by the adherent culture of WBC, then stimulated with lipopolysaccharide (LPS). Macrophages were identified by morphological features, functional activity and authorized cytokine expression. Subsequently, we collected samples, constructed and sequenced transcriptomic libraries including WBC, resting macrophage (Mø) and activated macrophage (M(LPS)) groups. We gained a total of 20.36 Gb of clean data including 149.24 million reads with an average length of 146 bp. Transcriptome analysis showed 708 differential genes between WBC and Mø, 83 differentially expressed genes between Mø and M(LPS). Combined with RT-qPCR, we proposed that four novel cell surface marker genes (CD22-like, CD63, CD48 and CD276) and two chemokines (CXCL-like and CCL39.3) would be emerging potential marker genes of macrophage in grass carp. Furthermore, CD69, CD180, CD27, XCL32a.2 and CXCL8a genes can be used as marker genes to confirm whether macrophages are activated. Transcriptome profiling reveals novel molecules associated with macrophages in C. Idella, which may represent a potential target for macrophages activation.
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Carpas/genética , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Activación de Macrófagos/genética , Macrófagos/metabolismo , Animales , Biomarcadores/metabolismo , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Ontología de Genes , Riñón Cefálico/citología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Anotación de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estallido Respiratorio/efectos de los fármacosRESUMEN
The rapid and global emergence of azole resistance in the human pathogen Aspergillus fumigatus has drawn attention. Thus, a thorough understanding of its mechanisms of drug resistance requires extensive exploration. In this study, we found that the loss of the putative calcium-dependent protein-encoding gene algA causes an increased frequency of azole-resistant A. fumigatus isolates. In contrast to previously identified azole-resistant isolates related to cyp51A mutations, only one isolate carries a point mutation in cyp51A (F219L mutation) among 105 independent stable azole-resistant isolates. Through next-generation sequencing (NGS), we successfully identified a new mutation (R243Q substitution) conferring azole resistance in the putative A. fumigatus farnesyltransferase Cox10 (AfCox10) (AFUB_065450). High-performance liquid chromatography (HPLC) analysis verified that the decreased absorption of itraconazole in related Afcox10 mutants is the primary reason for itraconazole resistance. Moreover, a complementation experiment by reengineering the mutation in a parental wild-type background strain demonstrated that both the F219L and R243Q mutations contribute to itraconazole resistance in an algA-independent manner. These data collectively suggest that the loss of algA results in an increased frequency of azole-resistant isolates with a non-cyp51A mutation. Our findings indicate that there are many unexplored non-cyp51A mutations conferring azole resistance in A. fumigatus and that algA defects make it possible to isolate drug-resistant alleles. In addition, our study suggests that genome-wide sequencing combined with alignment comparison analysis is an efficient approach to identify the contribution of single nucleotide polymorphism (SNP) diversity to drug resistance.
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Aspergillus fumigatus/efectos de los fármacos , Farmacorresistencia Fúngica/genética , Farnesiltransferasa/genética , Proteínas Fúngicas/genética , Mutación , Antifúngicos/farmacología , Aspergillus fumigatus/genética , Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Farnesiltransferasa/metabolismo , Proteínas Fúngicas/metabolismo , Expresión Génica , Prueba de Complementación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Itraconazol/farmacología , Polimorfismo de Nucleótido SimpleRESUMEN
Filamentous fungi have a dominant nonhomologous-end joining (NHEJ) DNA repair pathway, which results in the majority of transformed progenies having random heterologous insertion mutagenesis. Thus, lack of a versatile genome-editing tool prevents us from carrying out precise genome editing to explore the mechanism of pathogenesis. Moreover, clinical isolates that have a wild-type ku80 background without any selection nutrition marker especially suffer from low homologous integration efficiency. In this study, we have established a highly efficient CRISPR mutagenesis system to carry out precise and efficient in-frame integration with or without marker insertion with approximately 95-100% accuracy via very short (approximately 35-bp) homology arms in a process referred to as microhomology-mediated end joining (MMEJ). Based on this system, we have successfully achieved an efficient and precise integration of an exogenous GFP tag at the predicted site without marker insertion and edited a conidial melanin gene pksP and a catalytic subunit of calcineurin gene cnaA at multiple predicted sites with or without selection marker insertion. Moreover, we found that MMEJ-mediated CRISPR-Cas9 mutagenesis is independent of the ku80 pathway, indicating that this system can function as a powerful and versatile genome-editing tool in clinical Aspergillus isolates.
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Aspergillus fumigatus/genética , Sistemas CRISPR-Cas , Mutagénesis Insercional/métodos , Secuencia de Aminoácidos , Codón , Proteínas Fúngicas/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Polimerasa III/genéticaRESUMEN
BACKGROUND/AIMS: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin lymphoma, has significant prognostic heterogeneity. This study aimed to generate a prognostic prediction model based on autophagy-related genes for DLBCL patients. METHODS: Utilizing bioinformatics techniques, we analyzed the clinical information and transcriptome data of DLBCL patients from the Gene Expression Omnibus (GEO) database. Through unsupervised clustering, we identified new autophagy-related molecular subtypes and pinpointed differentially expressed genes (DEGs) between these subtypes. Based on these DEGs, a prognostic model was constructed using Cox and Lasso regression. The effectiveness, accuracy, and clinical utility of this prognostic model were assessed using numerous independent validation cohorts, survival analyses, receiver operating characteristic (ROC) curves, multivariate Cox regression analysis, nomograms, and calibration curves. Moreover, functional analysis, immune cell infiltration, and drug sensitivity analysis were performed. RESULTS: DLBCL patients with different clinical characterizations (age, molecular subtypes, ECOG scores, and stages) showed different expression features of autophagy-related genes. The prediction model was constructed based on the eight autophagy-related genes (ADD3, IGFBP3, TPM1, LYZ, AFDN, DNAJC10, GLIS3, and CCDC102A). The prognostic nomogram for overall survival of DLBCL patients incorporated risk level, stage, ECOG scores, and molecular subtypes, showing excellent agreement between observed and predicted outcomes. Differences were noted in the proportions of immune cells (native B cells, Treg cells, CD8+ T cell, CD4+ memory activated T cells, gamma delta T cells, macrophages M1, and resting mast cells) between high-risk and low-risk groups. LYZ and ADD3 exhibited correlations with drug resistance to most chemotherapeutic drugs. CONCLUSIONS: This study established a novel prognostic assessment model based on the expression profile of autophagy-related genes and clinical characteristics of DLBCL patients, explored immune infiltration and predicted drug resistance, which may guide precise and individualized immunochemotherapy regimens.
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Linfoma de Células B Grandes Difuso , Humanos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Linfoma de Células B Grandes Difuso/genética , Autofagia/genética , Probabilidad , Linfocitos B , Resistencia a Medicamentos , Pronóstico , Proteínas de Unión a CalmodulinaRESUMEN
Aims: Studies demonstrated that oxidized fish oil (OFO) promoted oxidative stress and induced mitochondrial dysfunction and lipotoxicity, which attenuated beneficial effects of fish oil supplements in the treatment of nonalcoholic fatty liver disease (NAFLD). The current study was performed on yellow catfish, a good model to study NAFLD, and its hepatocytes to explore whether selenium (Se) could alleviate OFO-induced lipotoxicity via the inhibition of oxidative stress and determine its potential mechanism. Results: The analysis of triglycerides content, oxidative stress parameters, and histological and transmission electronic microscopy observation showed that high dietary Se supplementation alleviated OFO-induced lipotoxicity, oxidative stress, and mitochondrial injury and dysfunction. RNA-sequencing and immunoblotting analysis indicated that high dietary Se reduced OFO-induced decline of peroxisome-proliferator-activated receptor alpha (Pparα) and ubiquitin-specific protease 4 (Usp4) protein expression. High Se supplementation also alleviated OFO-induced reduction of thioredoxin reductase 2 (txnrd2) messenger RNA (mRNA) expression level and activity. The txnrd2 knockdown experiments revealed that txnrd2 mediated Se- and oxidized eicosapentaenoic acid (oxEPA)-induced changes of mitochondrial reactive oxygen species (mtROS) and further altered Usp4 mediated-deubiquitination and stabilization of Pparα, which, in turn, modulated mitochondrial fatty acid ß-oxidation and metabolism. Mechanistically, Usp4 deubiquitinated Pparα and ubiquitin-proteasome-mediated Pparα degradation contributed to oxidative stress-induced mitochondrial dysfunction. Innovation: These findings uncovered a previously unknown mechanism by which Se and OFO interacted to affect lipid metabolism via the Txnrd2-mtROS-Usp4-Pparα pathway, which provides the new target for NAFLD prevention and treatment. Conclusion: Se ameliorated OFO-induced lipotoxicity via the inhibition of mitochondrial oxidative stress, remodeling of Usp4-mediated deubiquitination, and stabilization of Pparα. Antioxid. Redox Signal. 40, 433-452.
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Enfermedades Mitocondriales , Enfermedad del Hígado Graso no Alcohólico , Selenio , Humanos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Hígado/metabolismo , Aceites de Pescado/farmacología , Aceites de Pescado/metabolismo , Selenio/farmacología , Selenio/metabolismo , PPAR alfa/genética , Oxidorreductasas/metabolismo , Estrés Oxidativo , Enfermedades Mitocondriales/metabolismoRESUMEN
PURPOSE: Leukaemia remains a major contributor to global mortality, representing a significant health risk for a substantial number of cancer patients. Despite notable advancements in the field, existing treatments frequently exhibit limited efficacy or recurrence. Here, we explored the potential of abolishing HVEM (herpes virus entry mediator, TNFRSF14) expression in tumours as an effective approach to treat acute lymphoblastic leukaemia (ALL) and prevent its recurrence. METHODS: The clinical correlations between HVEM and leukaemia were revealed by public data analysis. HVEM knockout (KO) murine T cell lymphoblastic leukaemia cell line EL4 were generated using CRISPR-Cas9 technology, and syngeneic subcutaneous tumour models were established to investigate the in vivo function of HVEM. Immunohistochemistry (IHC), RNA-seq and flow cytometry were used to analyse the tumour immune microenvironment (TIME) and tumour draining lymph nodes (dLNs). Immune functions were investigated by depletion of immune subsets in vivo and T cell functional assays in vitro. The HVEM mutant EL4 cell lines were constructed to investigate the functional domain responsible for immune escape. RESULTS: According to public databases, HVEM is highly expressed in patients with ALL and acute myeloid leukemia (AML) and is negatively correlated with patient prognosis. Genetic deletion of HVEM in EL4 cells markedly inhibited tumour progression and prolonged the survival of tumour-bearing mice. Our experiments proved that HVEM exerted its immunosuppressive effect by inhibiting antitumour function of CD8+ T cell through CRD1 domain both in vivo and in vitro. Additionally, we identified a combination therapy capable of completely eradicating ALL tumours, which induces immune memory toward tumour protection. CONCLUSIONS: Our study reveals the potential mechanisms by which HVEM facilitates ALL progression, and highlights HVEM as a promising target for clinical applications in relapsed ALL therapy.
RESUMEN
Sulfamethoxazole (SMZ) and zinc (Zn) are widespread harmful materials in aquatic ecosystems and cause toxic effects to aquatic animals under their individual exposure. Although they often co-exist in aquatic environments, little is known about their joint effects and mechanism influencing aquatic animals. Herein, SMZ induced mitochondrial and lysosomal dysfunction, inhibited autophagy flux, and induced lipotoxicity. However, SMZ-induced changes of these physiological and metabolic processes above were reversed by Zn exposure, indicating the antagonism between Zn and SMZ. SOD1-knockdown abrogated the reversing effects of Zn on mitochondria dysfunction and autophagy flux blockage induced by SMZ, suggesting that SOD1 was essential for Zn to reverse SMZ-induced mitochondria dysfunction and autophagy impairment. Our further investigation found that Zn regulated STAT3 translocation to lysosomes and mitochondria to attenuate SMZ-induced lipotoxicity, and SOD1 was required for these processes. Mechanistically, STAT3 was associated with ATP6V1 A in a coiled-coil domain-dependent manner, and pS710-STAT3-and pY753-STAT3-independent manners. Moreover, SMZ suppressed autophagic degradation of damaged mitochondria via inhibiting interaction between STAT3 and ATP6V1 A and increasing pS710-STAT3 level; SMZ impaired mitochondrial ß-oxidation via decreasing pY753-STAT3 level and STAT3 mitochondrial localization. Zn reversed these SMZ-induced effects to alleviate SMZ-induced lipotoxicity. Taken together, our data showed that SMZ impaired mitochondrial ß-oxidation and lysosomal acidification via the downregulation of SOD1, leading to lipotoxicity, and that Zn reversed SMZ-induced changes of these important biological processes and attenuated SMZ-induced lipotoxicity. Thus, our study identified previously unidentified mechanisms for the antagonistic mechanisms of Zn and SMZ on aquatic animals, which provided novel insights into the environmental risk assessments of the joint exposure between heavy metals and antibiotics in the aquatic organisms.
Asunto(s)
Sulfametoxazol , Zinc , Animales , Zinc/metabolismo , Sulfametoxazol/toxicidad , Sulfametoxazol/metabolismo , Ecosistema , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/farmacología , Ácidos/metabolismo , Lisosomas/metabolismo , Agua Dulce , Mitocondrias/metabolismoRESUMEN
Purpose: Pulmonary artery hypertension (PAH) is a common complication of chronic obstructive pulmonary disease and obstructive sleep apnea/hypopnea syndrome worldwide. Pulmonary vascular alterations associated with PAH have multifactorial causes, in which endothelial cells play an important role. Autophagy is closely related to endothelial cell injury and the development of PAH. PIF1 is a multifunctional helicase crucial for cell survival. The present study investigated the effect of PIF1 on autophagy and apoptosis in human pulmonary artery endothelial cells (HPAECs) under chronic hypoxia stress. Methods: Chronic hypoxia Gene expression profiling chip-assays identified the PIF1 gene as differentially expressed, which was verified by RT-qPCR analysis. Electron microscopy, immunofluorescence, and Western blotting were used to analyze autophagy and the expression of LC3 and P62. Apoptosis was analyzed using flow cytometry. Results: Our study found that chronic hypoxia induces autophagy in HPAECs, and apoptosis was exacerbated by inhibiting autophagy. Levels of the DNA helicase PIF1 were increased in HPAECs after chronic hypoxia. PIF1 knockdown inhibited autophagy and promoted the apoptosis of HPAECs under chronic hypoxia stress. Conclusion: Based on these findings, we conclude that PIF1 inhibits the apoptosis of HPAECs by accelerating the autophagy pathway. Therefore, PIF1 plays a crucial role in HPAEC dysfunction in chronic hypoxia-induced PAH and may be a potential target for the treatment of PAH.