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Fish cells, such as grass carp (Ctenopharyngodon idella) kidney (CIK) cells, are harder to transfect than mammalian cells. There is a need for an efficient gene delivery system for fish cells. Here, we used CIK cell line as a model to develop a strategy to enhance RNA and plasmid DNA transfection efficiency using a nanocarrier generated from α-lactalbumin (α-NC). α-NC absorbed nucleic acid cargo efficiently and exhibited low cytotoxicity. Plasmid transfection was more efficient with α-NC than with liposomal transfection reagents. We used α-NC to co-transfect Tol2 transposase mRNA and a plasmid containing Cas9 and GFP, generating a stable transgenic CIK cell line. Genome and RNA sequencing revealed that the Cas9 and GFP fragments were successfully inserted into the genome of CIK cells and efficiently transcribed. In this study, we established an efficient transfection system for fish cells using α-NC, simplifying the process of generating stable transgenic fish cell lines.
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DNA methylation and chromatin accessibility play important roles in gene expression, but their function in subgenome expression dominance remains largely unknown. We conducted comprehensive analyses of the transcriptome, DNA methylation, and chromatin accessibility in liver and muscle tissues of allotetraploid common carp, aiming to reveal the function of epigenetic modifications in subgenome expression dominance. A noteworthy overlap in differential expressed genes (DEGs) as well as their functions was observed across the two subgenomes. In the promoter and gene body, the DNA methylation level of the B subgenome was significantly different than that of the A subgenome. Nevertheless, differences in DNA methylation did not align with changes in homoeologous biased expression across liver and muscle tissues. Moreover, the B subgenome exhibited a higher prevalence of open chromatin regions and greater chromatin accessibility, in comparison to the A subgenome. The expression levels of genes located proximally to open chromatin regions were significantly higher than others. Genes with higher chromatin accessibility in the B subgenome exhibited significantly elevated expression levels compared to the A subgenome. Contrastingly, genes without accessibility exhibited similar expression levels in both subgenomes. This study contributes to understanding the regulation of subgenome expression dominance in allotetraploid common carp.
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Carpas , Metilação de DNA , Animais , Carpas/genética , Genoma de Planta , Cromatina/genética , Poliploidia , Regulação da Expressão Gênica de PlantasRESUMO
Female common carp grow faster than male individuals, implying that rearing females could be more profitable in aquaculture. Non-coding RNAs (ncRNAs) serve as versatile regulators with multiple functions in diverse biological processes. However, the roles of ncRNAs in the sex differentiation of common carp are less studied. In this study, we investigated the expression profiles of ncRNAs, including miRNAs, lncRNAs, and circRNAs, in the gonads to comprehend the roles of ncRNAs in sex differentiation in common carp. A substantial number of differentially expressed (DE) ncRNAs in ovaries and testes were identified. Some miRNAs, notably miR-205, miR-214, and miR-460-5p, might modulate hormone synthesis and thus maintain sex. A novel miRNA, novel_158, was predicted to suppress the expression of foxl3. DE lncRNAs were associated with oocyte meiosis, GnRH signaling pathways, and steroid biosynthesis, while DE circRNA target genes were enriched in the WNT signaling pathway and MAPK signaling pathway. We also analyzed ncRNA-mRNA interactions to shed light on the crosstalk between competing endogenous RNAs (ceRNAs), which is the critical mechanism by which lncRNAs and circRNAs function. Some lncRNAs and circRNAs may be able to competitively bind novel_313, a new miRNA, and thus regulate hsd17ß3. Our research will provide a valuable resource for understanding the genetic basis of gonadal differentiation and development in common carp.
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Carpas , MicroRNAs , RNA Longo não Codificante , Humanos , Animais , Masculino , Feminino , MicroRNAs/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ovário/metabolismo , Carpas/genética , Carpas/metabolismo , Testículo/metabolismo , Perfilação da Expressão Gênica , RNA não TraduzidoRESUMO
The selective corrosion of NiTi alloys was studied using density functional theory calculations, and the dissolution trends of the NiTi-B2 and NiTi-B19' phases in the initial oxidation stage were compared to predict their corrosion difference. The dissolution process of Ni and Ti was simulated by creating Ni or Ti vacancies on the unoxidized and oxidized NiTi alloy surfaces. The results show that the surface vacancy formation energy of Ti vacancies is higher than that of Ni vacancies, indicating that Ti is more difficult to dissolve than Ni. Furthermore, oxidation promotes and impedes the dissolution of Ni and Ti, respectively. This study improves the fundamental understanding of the corrosion mechanism of NiTi alloys.
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Epigenetic modifications are critical in precisely regulating gene expression. The common carp (Cyprinus carpio) is an economically important fish species, and females exhibit faster growth rates than males. However, the studies related to epigenetic modifications in the common carp gonads are limited. In this study, we conducted the Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) and Bisulfite sequencing (BS-seq) to explore the roles of epigenetic modifications in the common carp gonads. We identified 84,207 more accessible regions and 77,922 less accessible regions in ovaries compared to testes, and some sex-biased genes showed differential chromatin accessibility in their promoter regions, such as sox9a and zp3. Motif enrichment analysis showed that transcription factors (TFs) associated with embryonic development and cell proliferation were heavily enriched in ovaries, and the TFs Foxl2 and SF1 were only identified in ovaries. We also analyzed the possible regulations between chromatin accessibility and gene expression. By BS-seq, we identified 2087 promoter differentially methylated genes (promoter-DMGs) and 5264 gene body differentially methylated genes (genebody-DMGs) in CG contexts. These genebody-DMGs were significantly enriched in the Wnt signaling pathway, TGF-beta signaling pathway, and GnRH signaling pathway, indicating that methylation in gene body regions could play an essential role in sex maintenance, just like methylation in promoter regions. Combined with transcriptomes, we revealed that the expression of dmrtb1-like, spag6, and fels was negatively correlated with their methylation levels in promoter regions. Our study on the epigenetic modifications of gonads contributes to elucidating the molecular mechanism of sex differentiation and sex maintenance in the common carp.
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Carpas , Cromatina , Feminino , Animais , Masculino , Cromatina/genética , Metilação de DNA , Carpas/genética , Epigênese Genética , GônadasRESUMO
BACKGROUND: Clopidogrel in combination with aspirin after acute coronary syndromes (ACS) reduces recurrent ischaemic events compared to aspirin alone. Further reductions in events have been demonstrated when clopidogrel is replaced by ticagrelor or prasugrel albeit with increases in bleeding. There are few studies documenting the patterns of use of P2Y12 inhibitors or their association with outcomes in the Australian population. AIMS: To describe the patterns of use of each P2Y12 inhibitor and to determine the associations between initial P2Y12 inhibitor use and outcomes. METHODS: Data were extracted from Cooperative National Registry of ACS, Guideline Adherence and Clinical Events (CONCORDANCE)-a prospective database of patients presenting to 43 sites across Australia with ACS. Patients were stratified based on first antiplatelet agent received. Baseline clinical characteristics were compared between these patient groups and hospital investigations, management as well as in-hospital and 12 months outcomes (death, a composite of cardiac-related death, myocardial infarction, and stroke, and major bleeding) were compared between the three treatment cohorts after adjustment for differences in baseline characteristics. RESULTS: Mean ages of the clopidogrel (n=7,537), ticagrelor (n=1,878), and prasugrel (n=347) cohorts were 65, 63, and 58 yrs respectively (p<0.0001), the mean Global Registry of Acute Coronary Events (GRACE) risk scores were 107, 104, and 102 (p=0.0016). The ticagrelor and prasugrel cohorts were more likely to receive percutaneous coronary intervention (PCI) (clopidogrel 52%, ticagrelor 66%, prasugrel 88%, p<0.0001), and evidence based medications (≥4 guideline indicated medications: clopidogrel 76%, ticagrelor 82%, prasugrel 93%, p<0.0001). Patients treated with ticagrelor and prasugrel were less likely to experience in-hospital death (clopidogrel 2.5%, ticagrelor 1.4%, prasugrel 1.2%, p=0.05), major adverse cardiac events (MACE) (clopidogrel 5.1%, ticagrelor 3.0%, prasugrel 3.5% [p=0.01]), or bleeding (clopidogrel 8.4%, ticagrelor 4.6%, prasugrel 7.5% [p<0.001]) compared to clopidogrel. These differences were no longer apparent after multivariable adjustment. There was no difference in outcomes between cohorts at 12 months. CONCLUSIONS: In Australia, ticagrelor and prasugrel are used in younger patients who are more likely to undergo percutaneous coronary intervention (PCI) and receive evidence based therapy. Patients receiving clopidogrel were more likely to experience in hospital ischaemic or bleeding events but this was explained by their higher baseline risk. Selection of therapy was not associated with any difference in outcomes at 12-month follow-up, but our findings suggest there is room for improvement towards guideline-driven usage of P2Y12 inhibitors.
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Síndrome Coronariana Aguda , Intervenção Coronária Percutânea , Síndrome Coronariana Aguda/tratamento farmacológico , Aspirina/uso terapêutico , Austrália/epidemiologia , Clopidogrel/uso terapêutico , Hemorragia/induzido quimicamente , Mortalidade Hospitalar , Humanos , Inibidores da Agregação Plaquetária/uso terapêutico , Cloridrato de Prasugrel/uso terapêutico , Antagonistas do Receptor Purinérgico P2Y/uso terapêutico , Ticagrelor/uso terapêutico , Resultado do TratamentoRESUMO
Primordial germ cells (PGCs) play an important role in sexual fate determination and gonadal development in gonochoristic fish, such as zebrafish and medaka. However, little is known about the function of PGCs in hermaphroditic fish. Rice field eel (Monopterus albus), a protogynous hermaphroditic fish, is an economically valuable aquaculture species. We eliminated PGCs in rice field eels during embryogenesis via morpholino-mediated knockdown dead end (dnd). The PGCs-depleted gonads developed into testis-like structures with Sertoli cells and Leydig cells. The gene expression pattern of 15-month-old PGCs-depleted gonads showed that male-biased genes, dmrt1, sox9a, gsdf, and amh, were significantly higher than that of the WT, whereas female-biased genes, foxl2 and cyp19a1a, were significantly decreased. These results indicate that PGCs are essential for ovarian differentiation in rice field eel, and PGCs-depleted gonads develop into sterile males without undergoing the female and intersex stages. Our study is the first to identify the role of PGCs in sex differentiation in rice field eel, a protogynous hermaphrodite teleost. And it is of great significance in rice field eel for discovering the underlying mechanism of sex differentiation and establishing sex control technology.
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Smegmamorpha , Peixe-Zebra , Animais , Enguias/genética , Enguias/metabolismo , Feminino , Células Germinativas , Masculino , Processos de Determinação Sexual/genética , Diferenciação Sexual/genéticaRESUMO
Cellulose nanocrystal (CNC)/poly(lactic acid) (PLA) in situ nanocomposite scaffolds were fabricated by in situ polymerization of lactic acid and CNC which was directly utilized as aqueous suspension, followed by a process of thermally induced phase separation. The CNC/PLA in situ nanocomposite porous scaffolds were characterized by mechanical test, protein adsorption, hemolysis test, in vitro degradation measurement, TEM, FTIR, SEM and WAXD. Compared to the PLA scaffold, the CNC/PLA in situ nanocomposite scaffolds showed a greatly increased compression modulus, an improved hemocompatibility and protein adsorption capacity. The inclusion of CNCs boosted the in vitro degradation of the in situ nanocomposite porous scaffolds and facilitated the deposition of Ca2+, CO32-, PO43- ions in simulated body fluid. Furthermore, cell cultures were carried out on the CNC/PLA in situ nanocomposite porous scaffolds. In comparison with the PLA scaffold, the in situ nanocomposite scaffolds improved cell attachment and enhanced cell proliferation, denoting low cytotoxicity and good cytocompatibility. It can therefore be concluded that such scaffolds with excellent mechanical property, biocompatibility, biomineralization capacity and bioactivity hold great potential for bone tissue engineering.
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Celulose/química , Nanocompostos/química , Nanopartículas/química , Poliésteres/química , Engenharia Tecidual , Alicerces Teciduais/química , Linhagem Celular Tumoral , Sobrevivência Celular , Hemólise , Humanos , Peso Molecular , Nanocompostos/ultraestrutura , Nanopartículas/ultraestrutura , Porosidade , Proteínas/química , Análise EspectralRESUMO
The rice field eel, Monopterus albus, is a protogynous hermaphrodite fish, in which the gonads are initially female ovaries which then transform into male testes. The exact mechanisms governing sex reversal in the rice field eel are unknown. In this study, a novel alternative splicing variant of GnRH2 (GnRH2-SV), retaining the second intron, was discovered in the gonad of the rice field eel. Compared to GnRH2, GnRH2-SV may give rise to a novel truncated GnRH2-associated peptide (New GAP2). The normal transcript of GnRH2 was primarily expressed in the brain, and could also be detected in the liver, spleen, ovary, and testis. However, GnRH2-SV was only expressed in the ovary and testis. During sex reversal, GnRH2 expression levels increased significantly at late stages; however, expression levels of GnRH2-SV were lower in ovary than in ovotestis and testis. We also examined the effect of three peptides (GnRHa, GAP2, and New GAP2) on gonadal sex differentiation during the third stage of ovarian development of the rice field eel. Compared to the control group, the expression of amh increased significantly following incubation with each of the three peptides. However, only New GAP2 stimulated the expression of sox9a1 mRNA in vitro. After intraperitoneal injection of GAP2, the expression of amh, foxl2, and cyp19a1a increased significantly after 12â¯h; the concentration of serum 11-KT was also significantly increased at the 12â¯h time point. Treatment with New GAP2 significantly increased the expression of amh, dmrt1a, and sox9a1, and also increased the concentration of serum 11-KT. After treated with GnRHa, the expression of amh, dmrt1a, sox9a1, cyp19a1a, and foxl2 increased significantly, as did the level of serum E2. These results indicated that both GAP2 and New GAP2 play a crucial role in inducing expression changes of sex-differentiation related genes, and may be involved in the gonadal development and sex reversal in the rice field eel.