RESUMEN
Macrophages play a pivotal role in the healing of diabetic ulcers. The sustained elevation of glucose levels damages the insulin signaling pathway in macrophages, leading to dysfunctional macrophages that struggle to transition from pro-inflammatory (M1) to reparative (M2) states. Therefore, modulating macrophage inflammatory responses via the insulin pathway holds promise for diabetic ulcer treatment. Additionally, the presence of biofilm impedes drug penetration, and the resulting immunosuppressive microenvironment exacerbates the persistent infiltration of pro-inflammatory M1 macrophages. Therefore, we designed an array of dissolvable microneedle (denoted as NPF@MN) loaded with self-assembled nanoparticles that could deliver NPF nanoparticles, acid-sensitive NPF-releasing Protocatechualdehyde (PA) with hypoglycemic and insulin-like effects, regulating macrophage polarization to an anti-inflammatory M2 phenotype. Additionally, this study extensively examined the mechanism by which NPF@MN accelerates the healing of diabetic ulcers through the activation of the insulin signaling pathway. Through RNA-seq and GSEA analysis, we identified a reduction in the expression of pathway-related factors such as IR, IRS-1, IRS-2, and SHC. Our work presents an innovative therapeutic approach targeting the insulin pathway in diabetic ulcers and underscores its translational potential for clinical management.
Asunto(s)
Biopelículas , Insulina , Macrófagos , Agujas , Transducción de Señal , Cicatrización de Heridas , Animales , Cicatrización de Heridas/efectos de los fármacos , Insulina/metabolismo , Ratones , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Biopelículas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Masculino , Antiinflamatorios/farmacología , Diabetes Mellitus Experimental , Nanopartículas/química , Células RAW 264.7 , Ratones Endogámicos C57BLRESUMEN
The mechanical properties of nanoparticles play a crucial role in regulating nanobiointeractions, influencing processes such as blood circulation, tumor accumulation/penetration, and internalization into cancer cells. Consequently, they have a significant impact on drug delivery and therapeutic efficacy. However, it remains unclear whether and how macrophages alter their biological function in response to nanoparticle elasticity. Here, we report on the nano-mechanical biological effects resulting from the interactions between elastic silica nanoparticles (SNs) and macrophages. The SNs with variational elasticity Young's moduli ranging from 81 to 837 MPa were synthesized, and it was demonstrated that M2 [tumor-associated macrophages (TAMs)] could be repolarized to M1 by the soft SNs. Additionally, our findings revealed that cell endocytosis, membrane tension, the curvature protein Baiap2, and the cytoskeleton were all influenced by the elasticity of SNs. Moreover, the mechanically sensitive protein Piezo1 on the cell membrane was activated, leading to calcium ion influx, activation of the NF-κB pathway, and the initiation of an inflammatory response. In vivo experiments demonstrated that the softest 81 MPa SNs enhanced tumor penetration and accumulation and repolarized TAMs in intratumoral hypoxic regions, ultimately resulting in a significant inhibition of tumor growth. Taken together, this study has established a cellular feedback mechanism in response to nanoparticle elasticity, which induces plasma membrane deformation and subsequent activation of mechanosensitive signals. This provides a distinctive "nano-mechanical immunoengineering" strategy for reprogramming TAMs to enhance cancer immunotherapy.
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Canales Iónicos , Nanopartículas , Macrófagos Asociados a Tumores , Animales , Nanopartículas/química , Ratones , Canales Iónicos/metabolismo , Canales Iónicos/química , Macrófagos Asociados a Tumores/metabolismo , Macrófagos Asociados a Tumores/inmunología , Dióxido de Silicio/química , Elasticidad , Células RAW 264.7 , Humanos , Ratones Endogámicos BALB CRESUMEN
Starvation is one of the main stresses for fish due to food shortage, the evasion of predators, and intraspecific competition. This research evaluated the impact of brief fasting periods on reactive oxygen species (ROS) levels, antioxidant response, mRNA expression of antioxidants, autophagy-related signaling genes, and autophagosome development in the muscle tissue of rice flower carp. Following a three-day fasting period, the levels of ROS and MDA rose. Additionally, after 3 d of fasting, there was a notable upregulation of NRF2 and significant increases in the levels of GSH and the activities of enzymes such as SOD, CAT, GST, GR, and GPX, while the expression of the autophagy marker gene LC3B did not change (p < 0.05). After 7 d of fasting, the content of the ROS, the activity of SOD and GR, and the GSH content reached the maximum (p < 0.05). Concurrently, there was a significant rise in the quantity of autophagosomes. An RT-qPCR analysis revealed that seven d of starvation significantly elevated the mRNA expression of genes associated with the initiation and expansion of autophagosome membranes, vesicle recycling, and cargo recruitment, including ULK1, BECLIN1, LC3B, ATG3, ATG4B, ATG4C, ATG5, ATG9, and P62. After feeding resumed for 3 d, the mRNA level of BECLIN1, ATG3, ATG4B, ATG4C, ATG5, LC3B, and P62 still remained at a high level. The LC3II protein reached its highest level. All autophagy-related gene expression decreased in the 7-day resumed feeding group. Our data implied that short-term fasting can cause oxidative stress and disrupt the antioxidant system first and then induce autophagy in the muscles of rice flower carp. These findings shed light on how fasting affects muscle homeostasis in fish. ROS-induced autophagy of the skeletal muscle may confer the resistance of rice flower carp to short-term fasting.
Asunto(s)
Autofagia , Carpas , Ayuno , Músculo Esquelético , Especies Reactivas de Oxígeno , Animales , Carpas/genética , Carpas/metabolismo , Autofagia/genética , Especies Reactivas de Oxígeno/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Estrés Oxidativo , Antioxidantes/metabolismoRESUMEN
DNA methyltransferases (Dnmts) are responsible for DNA methylation which influences patterns of gene expression and plays a crucial role in response to environmental changes. In this study, 7 LcDnmt genes were identified in the genome of large yellow croaker (Larimichthys crocea). The comprehensive analysis was conducted on gene structure, protein and location site of LcDnmts. LcDnmt proteins belonged to three groups (Dnmt1, Dnmt2, and Dnmt3) according to their conserved domains and phylogenetic analysis. Although Dnmt3 can be further divided into three sub groups (Dnmt3a, Dnmt3b, and Dnmt3l), there is no Dnmnt3l member in the large yellow croaker. Phylogenetic analysis revealed that the Dnmt family was highly conserved in teleosts. Expression patterns derived from the RNA-seq, qRT-PCR and Western blot analysis revealed that 2 LcDnmt genes (LcDnmt1 and LcDnmt3a2) significantly regulated under salinity stress in the liver, which was found to be dominantly expressed in the intestine and brain, respectively. These two genes may play an important role in the salinity stress of large yellow croaker and represent candidates for future functional analysis. Our results revealed the conservation of Dnmts during evolution and indicated a potential role of Dnmts in epigenetic regulation of response to salinity stress.
Asunto(s)
Metilación de ADN , Perciformes , Animales , Metilación de ADN/genética , Filogenia , Epigénesis Genética , Estrés Salino , ADN/metabolismo , Perciformes/genética , Perciformes/metabolismo , Proteínas de Peces/químicaRESUMEN
Cuproptosis is an emerging programmed cell death, displaying great potential in cancer treatment. However, intracellular copper content to induce cuproptosis is unmet, which mainly ascribes to the intracellular pumping out equilibrium mechanism by copper exporter ATP7A and ATP7B. Therefore, it is necessary to break such export balance mechanisms for desired cuproptosis. Mediated by diethyldithiocarbamate (DTC) coordination, herein a strategy to efficiently assemble copper ions into polydopamine nanostructure (PDA-DTC/Cu) for reprogramming copper metabolism of tumor is developed. The deposited Cu2+ can effectively trigger the aggregation of lipoylated proteins to induce cuproptosis of tumor cells. Beyond elevating intracellular copper accumulation, PDA-DTC/Cu enables to break the balance of copper metabolism by disrupting mitochondrial function and restricting the adenosine triphosphate (ATP) energy supply, thus catalytically inhibiting the expressions of ATP7A and ATP7B of tumor cells to enhance cuproptosis. Meanwhile, the killed tumor cells can induce immunogenic cell death (ICD) to stimulate the immune response. Besides, PDA-DTC/Cu NPs can promote the repolarization of tumor-associated macrophages (TAMs ) to relieve the tumor immunosuppressive microenvironment (TIME). Collectively, PDA-DTC/Cu presented a promising "one stone two birds" strategy to realize copper accumulation and inhibit copper export simultaneously to enhance cuproptosis for 4T1 murine breast cancer immunotherapy.
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Cobre , Inmunoterapia , Indoles , Nanoestructuras , Polímeros , Cobre/química , Polímeros/química , Animales , Inmunoterapia/métodos , Indoles/química , Indoles/farmacología , Ratones , Nanoestructuras/química , Línea Celular Tumoral , Humanos , Catálisis , Femenino , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismoRESUMEN
Repolarizing the tumor-associated macrophages (TAMs) towards the antitumoral M1-like phenotype has been a promising approach for cancer immunotherapy. However, the anti-cancer immune response is severely limited mainly by the repolarized M1-like macrophages belatedly returning to the M2-like phenotype (i.e., negative feedback). Inspired by nitric oxide (NO) effectively preventing repolarization of inflammatory macrophages in inflammatory diseases, herein, we develop an arginine assembly, as NO nano-donor for NO generation to prevent the negative feedback of the macrophage repolarization. The strategy is to first apply reversible tagging of hydrophobic terephthalaldehyde to create an arginine nano-assembly, and then load a toll-like receptor 7/8 agonist resiquimod (R848) (R848@Arg). Through this strategy, a high loading efficiency of 40 % for the arginine and repolarization characteristics for TAMs can be achieved. Upon the macrophage repolarization by R848, NO can be intracellularly generated from the released arginine by the upregulated inducible nitric oxide synthase. Mechanistically, NO effectively prevented the negative feedback of the repolarized macrophage by mitochondrial dysfunction via blocking oxidative phosphorylation. Notably, R848@Arg significantly increased the tumor inhibition ratio by 3.13-fold as compared to the free R848 by maintaining the M1-like phenotype infiltrating into tumor. The Arg-assembly as NO nano-donor provides a promising method for effective repolarization of macrophages.
Asunto(s)
Enfermedades Mitocondriales , Neoplasias , Humanos , Donantes de Óxido Nítrico , Retroalimentación , Macrófagos , Neoplasias/patología , Adyuvantes Inmunológicos/farmacología , Óxido Nítrico/farmacología , Inmunoterapia/métodos , Enfermedades Mitocondriales/patología , Microambiente TumoralRESUMEN
Transforming macrophages into the anti-inflammatory M2 phenotype could markedly strengthen inflammatory bowel disease (IBD) treatment, which is considered as a promising strategy. However, the high ferroptosis sensitivity of M2 macrophages, which decreases their activity, is a major stumbling block to this strategy. Therefore, promoting M2 polarization while simultaneously inhibiting ferroptosis to tackle this challenge is indispensable. Herein, a calciumcarbonate (CaCO3) mineralized liposome encapsulating a ferroptosis inhibitor (Fer-1) was developed (CaCO3@Lipo@Fer-1, CLF). The CaCO3 mineralized coating shields the liposomes to prevent the release of Fer-1 in circulation, while releasing Ca2+ in the acidic-inflammatory environment. This released Ca2+ promotes M2 polarization through the CaSR/AKT/ß-catenin pathway. The subsequently released Fer-1 effectively upregulates GSH and GPX4, scavenges reactive oxygen species, and inhibits ferroptosis in M2 macrophages. In vivo, CLF improved the targeting efficiency of IBD lesions (about 4.17-fold) through the epithelial enhanced permeability and retention (eEPR) effect and enhanced IBD therapy by increasing the M2/M1 macrophage ratio and inhibiting ferroptosis. We demonstrate that the synergistic regulation of macrophage polarization and ferroptosis sensitivity by this mineralized nanoinhibitor is a viable strategy for IBD therapy.
Asunto(s)
Ferroptosis , Enfermedades Inflamatorias del Intestino , Humanos , Macrófagos/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Antiinflamatorios/farmacología , FenotipoRESUMEN
Cancer is a leading cause of death worldwide, with an increasing mortality rate over the past years. The early detection of cancer contributes to early diagnosis and subsequent treatment. How to detect early cancer has become one of the hot research directions of cancer. Tumor biomarkers, biochemical parameters for reflecting cancer occurrence and progression have caused much attention in cancer early detection. Due to high sensitivity, convenience and low cost, biosensors have been largely developed to detect tumor biomarkers. This review describes the application of various biosensors in detecting tumor markers. Firstly, several typical tumor makers, such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), squamous cell carcinoma antigen (SCCA), carbohydrate, antigen19-9 (CA19-9) and tumor suppressor p53 (TP53), which may be helpful for early cancer detection in the clinic, are briefly described. Then, various biosensors, mainly focusing on electrochemical biosensors, optical biosensors, photoelectrochemical biosensors, piezoelectric biosensors and aptamer sensors, are discussed. Specifically, the operation principles of biosensors, nanomaterials used in biosensors and the application of biosensors in tumor marker detection have been comprehensively reviewed and provided. Lastly, the challenges and prospects for developing effective biosensors for early cancer diagnosis are discussed.
Asunto(s)
Técnicas Biosensibles , Nanoestructuras , Neoplasias , Masculino , Humanos , Biomarcadores de Tumor , Detección Precoz del Cáncer , Neoplasias/diagnóstico , BiomarcadoresRESUMEN
Salinity is an important environmental factor that affects the yield and quality of large yellow croaker (Larimichthys crocea) during aquaculture. Here, whole-genome bisulfite sequencing (WGBS), RNA-seq, bisulfite sequencing PCR (BSP), quantitative real-time PCR (qPCR), and dual luciferase reporter gene detection technologies were used to analyze the DNA methylation characteristics and patterns of the liver genome, the expression and methylation levels of important immune genes in large yellow croaker in response to salinity stress. The results of WGBS showed that the cytosine methylation of CG type was dominant, CpGIsland and repeat regions were important regions where DNA methylation occurred, and the DNA methylation in upstream 2k (2000bp upstream of the promoter) and repeat regions had different changes in the liver tissue of large yellow croaker in the response to the 12, 24, 36 salinity stress of 4 w (weeks). In the combined analysis of WGBS and transcriptome, the complement and coagulation cascade pathways were significantly enriched, in which the complement-related genes C7, C3, C5, C4, C1R, MASP1, and CD59 were mainly changed in response to salinity stress. In the studied area of MASP1 gene promoter, the methylation levels of many CpG sites as well as total cytosine were strongly negatively correlated with mRNA expression level. Methylation function analysis of MASP1 promoter further proved that DNA methylation could inhibit the activity of MASP1 promoter, indicating that salinity may affect the expressions of complement-related genes by DNA methylation of gene promoter region.
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Perciformes , Animales , Complemento C7/genética , Proteínas del Sistema Complemento/genética , Citosina/metabolismo , Metilación de ADN , Proteínas de Peces , Hígado/metabolismo , ARN Mensajero/metabolismo , Estrés Salino , SulfitosRESUMEN
MicroRNAs (miRNAs) play an important role in regulating gene expression, and myostatin (MSTN) has been widely recognized as a key gene for muscle growth and development. Through high-throughput sequencing to study the effects of starvation on miRNA transcriptomes in Larimichthys crocea muscle tissue, we found that the expression of miR-2014, miR-1231 and miR-1470 were significantly different between fasting and normal feeding Larimichthys crocea. Bioinformatics analysis predicted that miR-2014, miR-1231 and miR-1470 target MSTN mRNA 3'UTR. To verify the accuracy of predictions, we constructed double luciferase plasmids containing MSTN 3'UTR and confirmed that miR-2014-5p and miR-1231-5p can inhibit MSTN expression by targeting MSTN 3'UTR using double luciferase experiments, while miR-1470 is not involved in regulation. Subsequent site-directed mutation experiments reflected the specificity of the target sequence. In addition, quantitative PCR experiments revealed that miR-2014-5p and miR-1231-5p may participate in the regulation of MSTN expression in fasting and refeeding period, respectively. These results implied that miRNA may take part in muscle growth regulation during starvation. It provides some insights into the molecular regulation mechanism of MSTN in response to starvation stress in fish.
Asunto(s)
Proteínas de Peces/genética , MicroARNs/genética , Músculos/citología , Miostatina/metabolismo , Perciformes/crecimiento & desarrollo , Perciformes/genética , Animales , Biología Computacional/métodos , Proteínas de Peces/metabolismo , Regulación del Desarrollo de la Expresión Génica , Músculos/metabolismo , Miostatina/genética , Perciformes/metabolismo , Análisis de Secuencia de ARN/métodos , InaniciónRESUMEN
Salinity is a major ecological factor in the marine environment, and extremely important for the survival, development, and growth of fish. In this study, gill transcriptomes were examined by high-throughput sequencing at three different salinities (12 ppt as low salinity, 22 ppt as control salinity, and 32 ppt as high salinity) in an importantly economical fish silvery pomfret. A total of 187 genes were differentially expressed, including 111 up-regulated and 76 down-regulated transcripts in low-salinity treatment group and 107 genes differentially expressed, including 74 up-regulated and 33 down-regulated transcripts in high-salinity treatment group compared with the control group, respectively. Some pathways including NOD-like receptor signaling pathway, cytokine-cytokine receptor interaction, Toll-like receptor pathway, cardiac muscle contraction, and vascular smooth muscle contraction were significantly enriched. qPCR analysis further confirmed that mRNA expression levels of immune (HSP90A, IL-1ß, TNFα, TLR2, IP-10, MIG, CCL19, and IL-11) and ion transport-related genes (WNK2, NPY2R, CFTR, and SLC4A2) significantly changed under salinity stress. Low salinity stress caused more intensive expression changes of immune-related genes than high salinity. These results imply that salinity stress may affect immune function in addition to regulating osmotic pressure in silvery pomfret.
Asunto(s)
Peces/metabolismo , Branquias/metabolismo , Estrés Salino/fisiología , Transcriptoma , Animales , Secuencia de Bases , Regulación hacia Abajo , Peces/genética , Peces/inmunología , Regulación de la Expresión Génica , Branquias/inmunología , Anotación de Secuencia Molecular , Proteínas NLR/inmunología , Presión Osmótica , ARN Mensajero/metabolismo , Distribución Aleatoria , Receptores de Citocinas/inmunología , Estrés Salino/genética , Estrés Salino/inmunología , Transducción de Señal , Receptores Toll-Like/metabolismo , Regulación hacia ArribaRESUMEN
Lead (Pb) is a nonessential heavy metal that can be bioconcentrated to highly toxic levels in the environment. To understand the genes and toxicity/detoxification pathways of juvenile largemouth bass, liver transcriptomes were investigated in this fish after acute 96â¯h Pb exposure (Pb nitrate 0, 17.8, or 100â¯mg/L). Acute Pb exposure induced an immune response and apoptosis pathway activation in the liver. A number of transcripts related to complement and coagulation cascades were significantly increased. Up- and downregulated genes were significantly enriched in numerous pathways, including the natural killer cell-mediated cytotoxicity pathway, the Jak-STAT and P53 signaling pathways, cancer and apoptosis. These genes included Bidï¼Bcl-2, JNK, and PI3K (17.8â¯mg/L) and PI3K, AKT, PPARδ, RAS, MMPs, c-Jun p53, and PD-L1 (100â¯mg/L). Comprehensive analysis of liver transcriptomic data revealed numerous pathways associated with the immune system and carcinogenesis, especially pathways related to apoptosis and systemic lupus erythematosus.
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Lubina/genética , Redes Reguladoras de Genes , Plomo/toxicidad , Hígado/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Apoptosis , Lubina/crecimiento & desarrollo , Lubina/inmunología , Lubina/metabolismo , Plomo/análisis , Hígado/metabolismo , Transcriptoma/efectos de los fármacos , Contaminantes Químicos del Agua/análisisRESUMEN
As molecular chaperones, heat shock proteins (HSPs) play essential roles in cells in response to stress conditions. Recent studies about immune functions of HSPs in fish have also been reported. In this study, based on the reported cDNA sequences of the four HSP genes, HSP70, HSC70, HSP90α and HSP90ß, the temporal expression patterns of the four genes during embryonic development of dojo loach(Misgurnus anguillicaudatus) was assayed with qRT-PCR. All of the four genes were ubiquitously expressed in all detected embryonic developmental stages. Among of them, HSP70, HSC70 and HSP90ß were highly expressed in the organ formation stage, while HSP90α was the highest expressed in myotome formation stage. Further, the immune responses of the four HSP genes were assayed when loach were infected with three different pathogens, bacterium (Flavobacterium cloumnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia). All of the four genes were differentially expressed in four tissues such as skin, gills, spleen and kidney in response to the pathogenic invasion, but both HSP70 and HSP90α expressions were dramatically up-regulated. Further, the cellular responses of the loach skinand gill tissues were observed, in which the number of the skin goblet cells were significantly increased, and the gill lamellae became shorter and wider after infected. Thus, our work indicated that the HSPs may directly or indirectly involved in immune defense in fish, at least in the loach.
Asunto(s)
Cipriniformes/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/inmunología , Animales , Bacterias/patogenicidad , Cipriniformes/embriología , Cipriniformes/inmunología , Femenino , Enfermedades de los Peces/microbiología , Enfermedades de los Peces/parasitología , Hongos/patogenicidad , Perfilación de la Expresión Génica , Masculino , Parásitos/patogenicidadRESUMEN
DNA methylation is susceptible to various environmental factors such as salinity, temperature and nutritional conditions, and can affect gene function, organ metabolism, body growth and development. In order to explore the effect of starvation on growth-related genes in large yellow croaker (Larimichthys crocea), we studied methylation of the global DNA and growth-related genes (MSTN1,MSTN2,IGF1,IGF2) and the corresponding mRNA expressions, using ELISA-based technique, bisulfite sequencing PCR (BSP) technique and quantitative Real-time PCR (qRT-PCR) respectively. The results showed that the global DNA methylation levels were significantly different (pâ¯<0.05) between the experimental group and the control group at starvation 14d, 21d in muscle and at starvation 7d, 14d, 28d, and re-feeding 7d in liver. The CpG islands of MSTN1, MSTN2, IGF1 and IGF2 were enriched in exons rather than promoters. The proximal promoter of MSTN1 and IGF1 and the exon1 of MSTN2 had almost no methylation at all treatment stages. The methylation status in MSTN1 exon 1 and IGF2 exon 2 varied from different starvation time, and started to have significant differences on starvation 7d (pâ¯<0.05) both in liver and muscle. In the liver there was a strong positive correlation between IGF2 exon 2 methylation and global DNA methylation (râ¯=â¯0.7558). The mRNA expression levels of these growth-related genes were significantly different at starvation 14d (pâ¯<0.05), but did not have significant correlation with the methylation of these exons. The results implied that exon methylation of these growth-related genes might affect post-transcriptional process.
Asunto(s)
Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Hígado/metabolismo , Músculo Esquelético/metabolismo , Perciformes/fisiología , Inanición/veterinaria , Fenómenos Fisiológicos Nutricionales de los Animales , Animales , Acuicultura , China , Islas de CpG , Exones , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Hígado/crecimiento & desarrollo , Músculo Esquelético/crecimiento & desarrollo , Miostatina/genética , Miostatina/metabolismo , Especificidad de Órganos , Perciformes/crecimiento & desarrollo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Distribución Aleatoria , Somatomedinas/genética , Somatomedinas/metabolismo , Inanición/metabolismo , Factores de TiempoRESUMEN
In this study, we firstly determined the complete mitochondrial DNA sequence of the hybrid of Acanthopagrus schlegelii (â)×Pagrus major (â) using the next-generation sequencing and Polymerase Chain Reaction-based method (PCR). The total length of the hybrid mitochondrial genome was identical to the female parent as 16,649 bp in length, which contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and one displacement loop locus (a control region). The overall nucleotide composition is: 28.0%A, 27.9%T, 27.9%C, and 16.2%G, with a total A + T content of 45.9%. This study discovered the 99.8% sequence identity between the hybrid and its female parent, which confirmed the maternal inheritance pattern followed by the mitochondrial genome of the hybrid. The complete mitochondrial genome sequence of this hybrid sea bream may provide a valuable and useful resource for population genetic study and monitoring, and as well as for further conservation effort on this species.
RESUMEN
To investigate the effects of fasting on lipid metabolism in spotted seabass muscle and liver tissues, we analyzed mRNA levels and enzyme activities of lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and fatty acid synthetase (FAS), and the relationship among fat content, mRNA level, and enzyme activity during fasting of 35 days. The results showed that expressions of all the three genes were ubiquitous. During the fasting experiment, the hepatosomatic index (HSI) and fat content of muscle and liver tissues significantly decreased before 5 days of fasting (P < 0.05). mRNA levels of LPL increased significantly after 5 days of fasting in liver and 7 days in muscle. Abundance of HSL transcripts increased significantly after 14 days of fasting in both muscle and liver. The activities of LPL and HSL presented a trend that increased firstly, decreased subsequently, and then raised again with the prolonged fasting experiment (P < 0.05). However, activities and mRNA levels of FAS decreased significantly after 1 day of fasting in both muscle and liver. Moreover, activities and mRNA levels of FAS showed a moderate correlation in muscle. These results suggested that FAS had a sooner response to fasting than LPL and HSL in both muscle and liver tissues. LPL and HSL played important roles in lipolysis mainly by increasing enzyme activities in the early stage of fasting and mRNA levels in the later stage of fasting in both muscle and liver. Our results also provided useful information on regulating muscle fat content by fasting.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Peces/fisiología , Privación de Alimentos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Lipoproteína Lipasa/metabolismo , Esterol Esterasa/metabolismo , Animales , Clonación Molecular , Ácido Graso Sintasas/genética , Lipoproteína Lipasa/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Esterol Esterasa/genéticaRESUMEN
Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H2O2. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe2+, Cu2+, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.
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Arcidae/metabolismo , Hemocitos/metabolismo , Hemoglobinas/metabolismo , Peroxidasa/metabolismo , Secuencia de Aminoácidos , Animales , Peróxido de Hidrógeno/química , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Follistatin is a secreted glycoprotein, which involved in numerous physiological activities as an antagonist of the transforming growth factor-ß superfamily. However, little is known about the regulation mechanism of follistatin in fish. In this study we cloned and analyzed part of the 5' flanking region of the follistatin gene in Larimichthys crocea. Sequence analysis revealed several putative binding sites for transcription factors including activator protein 1 (AP1), myogenic differentiation factor (MyoD), stimulating protein 1 (SP1), and sex determining gene on the mammalian Y chromosome (SRY) in the cloned fragment. Transcriptional activities of two fragments (485 and 261 bp) truncated from follistatin upstream region were examined in vitro, using transient transfection in Ctenopharyngodon idella kidney (CIK) and Rattus norvegicus skeletal muscle myoblast (L6) cells. The result showed that the promoter activity correlated positively with the length of truncated fragments in both CIK and L6 cells. To study the regulation of follistatin expression in L. crocea, we cloned MyoD and SRY-box 8 (Sox8) genes and examined their action on the follistatin promoter by co-transfection in CIK and L6 cells. The results showed MyoD and Sox8 could suppress the activities of follistatin promoter at different levels in CIK and L6 cells.
Asunto(s)
Región de Flanqueo 5' , Proteínas de Peces/genética , Folistatina/genética , Perciformes/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Clonación Molecular , Proteínas de Peces/metabolismo , Folistatina/metabolismo , Expresión Génica , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Mioblastos/citología , Mioblastos/metabolismo , Perciformes/metabolismo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
The Cichlid fishes have played an important role in evolutionary biology, population studies and aquaculture industry with East African species representing a model suited for studying adaptive radiation and speciation for cichlid genome projects in which closely related genomes are fast emerging presenting questions on phenotype-genotype relations. The complete mitochondrial genomes presented here are for two closely related but eco-morphologically distinct Lake Victoria basin cichlids, Oreochromis variabilis, an endangered native species and Tilapia zilli, an invasive species, both of which are important economic fishes in local areas. The complete mitochondrial genomes determined for O. variabilis and T. zilli are 16 626 and 16,619 bp, respectively. Both the mitogenomes contain 13 protein-coding genes, 22 tRNAs, 2 rRNAs and a non-coding control region, which are typical of vertebrate mitogenomes. Phylogenetic analyses of the two species revealed that though both lie within family Cichlidae, they are remotely related.
Asunto(s)
ADN Mitocondrial , Genoma Mitocondrial , Genómica , Filogenia , Tilapia/clasificación , Tilapia/genética , Animales , Composición de Base , Genes Mitocondriales , Tamaño del Genoma , Sistemas de Lectura Abierta , Análisis de Secuencia de ADN , Secuenciación Completa del GenomaRESUMEN
The large yellow croaker (Larimichthys crocea) is an economically important fish species in Chinese mariculture industry. To understand the molecular basis underlying the response to fasting, Illumina HiSeqTM 2000 was used to analyze the liver transcriptome of fasting large yellow croakers. A total of 54,933,550 clean reads were obtained and assembled into 110,364 contigs. Annotation to the NCBI database identified a total of 38,728 unigenes, of which 19,654 were classified into Gene Ontology and 22,683 were found in Kyoto Encyclopedia of Genes and Genomes (KEGG). Comparative analysis of the expression profiles between fasting fish and normal-feeding fish identified a total of 7,623 differentially expressed genes (P < 0.05), including 2,500 upregulated genes and 5,123 downregulated genes. Dramatic differences were observed in the genes involved in metabolic pathways such as fat digestion and absorption, citrate cycle, and glycolysis/gluconeogenesis, and the similar results were also found in the transcriptome of skeletal muscle. Further qPCR analysis confirmed that the genes encoding the factors involved in those pathways significantly changed in terms of expression levels. The results of the present study provide insights into the molecular mechanisms underlying the metabolic response of the large yellow croaker to fasting as well as identified areas that require further investigation.