RESUMO
TWIST1 induces epithelial-to-mesenchymal transition (EMT) to drive cancer metastasis. It is yet unclear what determines TWIST1 functions to activate or repress transcription. We found that the TWIST1 N-terminus antagonizes TWIST1-regulated gene expression, cancer growth and metastasis. TWIST1 interacts with both the NuRD complex and the NuA4/TIP60 complex (TIP60-Com) via its N-terminus. Non-acetylated TWIST1-K73/76 selectively interacts with and recruits NuRD to repress epithelial target gene transcription. Diacetylated TWIST1-acK73/76 binds BRD8, a component of TIP60-Com that also binds histone H4-acK5/8, to recruit TIP60-Com to activate mesenchymal target genes and MYC. Knockdown of BRD8 abolishes TWIST1 and TIP60-Com interaction and TIP60-Com recruitment to TWIST1-activated genes, resulting in decreasing TWIST1-activated target gene expression and cancer metastasis. Both TWIST1/NuRD and TWIST1/TIP60-Com complexes are required for TWIST1 to promote EMT, proliferation, and metastasis at full capacity. Therefore, the diacetylation status of TWIST1-K73/76 dictates whether TWIST1 interacts either with NuRD to repress epithelial genes, or with TIP60-Com to activate mesenchymal genes and MYC. Since BRD8 is essential for TWIST1-acK73/76 and TIP60-Com interaction, targeting BRD8 could be a means to inhibit TWIST1-activated gene expression.
Assuntos
Neoplasias , Humanos , Neoplasias/genética , Transição Epitelial-Mesenquimal/genética , Proteínas Nucleares/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Matrine (MT) is a kind of alkaloid extracted from Sophora and is a promising substitute for chemical nematicides and botanical pesticides. The present study utilized sodium alginate (SA), zeolite imidazole salt skeleton (ZIF), and MT as raw materials to prepare a pH-response-release nematicide through the electrostatic spray technique. Zinc metal-organic framework (ZIF-8) was initially synthesized, followed by the successful loading of MT. Subsequently, the electrostatic spray process was employed to encapsulate it in SA, resulting in the formation of MT/ZIF-8@SA microcapsules. The efficiency of encapsulation and drug loadings can reach 79.93 and 26.83%, respectively. Soybean cyst nematode (SCN) is one of the important pests that harm crops; acetic acid produced by plant roots and CO2 produced by root respiration causing a decrease in the pH of the surrounding environment, which is most attractive to the SCN when the pH is between 4.5 and 5.4. MT/ZIF-8@SA releases the loaded MT in response to acetic acid produced by roots and acidic oxides produced by root respiration. The rate of release was 37.67% higher at pH 5.25 compared with pH 8.60. The control efficiency can reach 89.08% under greenhouse conditions. The above results demonstrate that the prepared MT/ZIF-8@SA not only exhibited excellent efficacy but also demonstrated a pH-responsive release of the nematicide.
RESUMO
HER2-positive (HER2+) breast cancers (BrCs) contain approximately equal numbers of ERα+HER2+ and ERα-HER2+ cases. An enduring obstacle is the unclear cell lineage-related characteristics of these BrCs. Although ERα+HER2+ BrCs could lose ERα to become ERα-HER2+ BrCs, direct evidence is missing. To investigate ERα dependencies and their implications during BrC growth and metastasis, we generated ERαCreRFP-T mice that produce an RFP-marked ERα+ mammary gland epithelial cell (MGEC) lineage. RCAS virus-mediated expression of Erbb2, a rodent Her2 homolog, first produced comparable numbers of ERα+RFP+Erbb2+ and ERα-RFP-Erbb2+ MGECs. Early hyperplasia developed mostly from ERα+RFP+Erbb2+ cells and ERα-RFP-Erbb2+ cells in these lesions were rare. The subsequently developed ductal carcinomas in situ had 64% slow-proliferating ERα+RFP+Erbb2+ cells, 15% fast-proliferating ERα-RFP+Erbb2+ cells derived from ERα+RFP+Erbb2+ cells, and 20% fast-proliferating ERα-RFP-Erbb2+ cells. The advanced tumors had mostly ERα-RFP+Erbb2+ and ERα-RFP-Erbb2+ cells and only a very small population of ERα+RFP+Erbb2+ cells. In ERα-RFP+Erbb2+ cells, GATA3 and FoxA1 decreased expression and ERα promoter regions became methylated, consistent with the loss of ERα expression. Lung metastases consisted of mostly ERα-RFP+Erbb2+ cells, a few ERα-RFP-Erbb2+ cells, and no ERα+RFP+Erbb2+ cells. The high metastatic capacity of ERα-RFP+Erbb2+ cells was associated with ERK1/2 activation. These results show that the slow-proliferating, nonmetastatic ERα+RFP+Erbb2+ cells progressively lose ERα during tumorigenesis to become fast-proliferating, highly metastatic ERα-RFP+Erbb2+ cells. The ERα-Erbb2+ BrCs with an ERα+ origin are more aggressive than those ERα-Erbb2+ BrCs with an ERα- origin, and thus, they should be distinguished and treated differently in the future.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Receptor alfa de Estrogênio/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/genética , Receptor ErbB-2/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/secundário , Linhagem Celular Tumoral , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Transformação Celular Neoplásica , Receptor alfa de Estrogênio/metabolismo , Feminino , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Regiões Promotoras Genéticas , Receptor ErbB-2/metabolismo , Transdução de Sinais , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Fucoidan has an anti-obesity effect. However, there are few studies on its mechanism. In this study, we investigated the in vitro and in silico inhibitory properties of fucoidan against pancreatic lipase for the first time. We examined the changes in composition, structure, and pancreatic lipase inhibition of fucoidan during in vitro digestion. RESULTS: Simulated saliva-gastrointestinal digestion resulted in a slight decrease in the molecular weight of fucoidan but no significant changes in the monosaccharide composition, sulfate content, and functional groups. Moreover, the digestion process significantly increased the inhibition of pancreatic lipase by fucoidan. The study on the type of inhibition showed that the inhibition of pancreatic lipase by fucoidan belonged to mixed inhibition with competitive inhibition. Molecular docking analysis showed that fucoidan could bind to the active site of pancreatic lipase. CONCLUSION: This study indicates that fucoidan can be a potential functional food for anti-obesity. © 2024 Society of Chemical Industry.
Assuntos
Lipase , Pâncreas , Polissacarídeos , Simulação de Acoplamento Molecular , Pâncreas/metabolismo , Lipase/química , DigestãoRESUMO
The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica (TcA) in Escherichia coli (E. coli) as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. KEY POINTS: ⢠T. californica AChE was expressed solubly in prokaryotic system. ⢠The biochemical properties of free/immobilized enzyme were characterized. ⢠The sensitivity of enzyme to insecticides was evaluated in vitro and in silico.
Assuntos
Inseticidas , Inseticidas/farmacologia , Acetilcolinesterase/genética , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Triclorfon , Simulação de Acoplamento Molecular , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
In order to reduce the use of fungicide and ensure food safety, it is necessary to develop fungicide with low toxicity and high efficiency to reduce residues. Azoxystrobin (AZOX), which is derived from mushrooms, is an excellent choice. However, conventional AZOX release is difficult to regulate. In this paper, a pH-responsive fungicide delivery system for the preparation of AZOX by impregnation method was reported. The Zinc metal-organic framework/Biomass charcoal (ZIF-8/BC) support was first prepared, and subsequently, the AZOX-ZIF-8/BC nano fungicide was prepared by adsorption of AZOX onto ZIF-8/BC by dipping. Gray mold, caused by Botrytis cinerea, is one of the most important crop diseases worldwide. AZOX-ZIF-8/BC could respond to oxalic acid produced by Botrytis cinerea to release loaded AZOX. When pH = 4.8, it was 48.42% faster than when pH = 8.2. The loading of AZOX on ZIF-8/BC was 19.83%. In vitro and pot experiments showed that AZOX-ZIF-8/BC had significant fungicidal activity, and 300 mg/L concentration of AZOX-ZIF-8-BC could be considered as a safe and effective control of Botrytis cinerea. The above results indicated that the prepared AZOX-ZIF-8/BC not only exhibited good drug efficacy but also demonstrated pH-responsive fungicide release.
Assuntos
Fungicidas Industriais , Estruturas Metalorgânicas , Solanum lycopersicum , Fungicidas Industriais/farmacologia , Carvão Vegetal/farmacologia , Estruturas Metalorgânicas/farmacologia , Zinco/farmacologia , Biomassa , Doenças das Plantas/prevenção & controle , BotrytisRESUMO
Water-responsive (WR) materials, due to their controllable mechanical response to humidity without energy actuation, have attracted lots of attention to the development of smart actuators. WR material-based smart actuators can transform natural humidity to a required mechanical motion and have been widely used in various fields, such as soft robots, micro-generators, smart building materials, and textiles. In this paper, the development of smart actuators based on different WR materials has been reviewed systematically. First, the properties of different biological WR materials and the corresponding actuators are summarized, including plant materials, animal materials, and microorganism materials. Additionally, various synthetic WR materials and their related applications in smart actuators have also been introduced in detail, including hydrophilic polymers, graphene oxide, carbon nanotubes, and other synthetic materials. Finally, the challenges of the WR actuator are analyzed from the three perspectives of actuator design, control methods, and compatibility, and the potential solutions are also discussed. This paper may be useful for the development of not only soft actuators that are based on WR materials, but also smart materials applied to renewable energy.
Assuntos
Nanotubos de Carbono , Água , Animais , Interações Hidrofóbicas e Hidrofílicas , PolímerosRESUMO
BACKGROUND: To investigate the eye movement functions in children with amblyopia and recovered amblyopia by a binocular eye-tracking paradigm. METHODS: Eye movements of 135 pediatric subjects (age range: 4-14 years), including 45 amblyopic children, 45 recovered amblyopic children and 45 age-similar normal controls, were recorded under binocular viewing with corrected refractive errors (if any). The deviation of gaze positions relative to the target location was recorded as the mean from both eyes. Main outcome measures included fixation deviations (degree) along horizontal and vertical axes in the sustained fixation test (Fix-X, Fix-Y) and visually guided saccade test (Sac-X, Sac-Y), which were compared across the three groups and between each two groups. RESULTS: All the four deviations were significantly larger in the amblyopia group compared to the other two groups, indicating increased inaccuracy of sustained and post-saccadic fixations in amblyopia. However, there was no significant difference in deviations between recovered amblyopic children and normal controls. Repeated measures showed similar results overall and within each group. Mild to moderate amblyopes and severe amblyopes did not differ in the four deviations. No significant interaction was found between subject groups and clinical characteristics (age, refractive status, and anisometropia). CONCLUSION: Amblyopic children have poor eye movement functions with increased inaccuracy of sustained and post-saccadic fixations, which appear to be restored in children with recovered amblyopia. Binocular assessment of eye movements provides valuable indicators of functional recovery in amblyopia.
Assuntos
Ambliopia , Anisometropia , Adolescente , Criança , Pré-Escolar , Movimentos Oculares , Humanos , Movimentos Sacádicos , Acuidade VisualRESUMO
To meet the ever-growing human demands for food, fuel, and fiber, agricultural activities have dramatically altered the global carbon (C) and nitrogen (N) cycles. These biogeochemical cycles along with water, phosphorus, and sulfur cycles are fundamental features of life on Earth. Human alteration of the global N cycle has had both positive and negative outcomes. To efficiently feed a growing population, crop-livestock production systems have been developed, however, these systems also contribute significantly to environmental pollution and global climate change. Management of agricultural waste (AW) and the application of N fertilizers are central to the issues of greenhouse gas (GHG) emissions and nutrient runoff that contributes to the eutrophication of water bodies. If managed properly, AW can provide nutrients for plants and contribute to the conservation of soil health. In order to achieve the long-term conservation of agricultural production systems, it is important to promote the proper recycling of AW in agroecosystems and to minimize the reliance on chemical N fertilizers. Composting is one of the sustainable and effective approaches for recycling AW in agriculture. However, the conventional composting process is dilatory and produces compost with low N content compared to chemical N fertilizers. For this reason, comprehensive research is required to improve the composting process and the N content of the soil organic amendments. This work aims to explore the beneficial effects of the integrated application of biochar and specific C and N cycling microorganisms to the composting process and the quality of the composted products. In pursuit of replacing chemical N fertilizers with bio/organic fertilizers, we further discussed the power of the combined application of compost, biochar, and N-fixing bacteria in agricultural production systems. The knowledge of smart integration of AW and microorganisms in agriculture could solve the main agricultural and environmental problems associated with human-induced flows of C and N. Building upon the knowledge disseminated in review to further extensive research will pave the way for better management of agricultural production systems and sustainable C and N cycling in agriculture.
Assuntos
Carbono , Compostagem , Agricultura , Fertilizantes/análise , Humanos , Nitrogênio/análise , SoloRESUMO
Estrogen and its cognate receptor, ERα, regulate cell proliferation, differentiation, and carcinogenesis in the endometrium by controlling gene transcription. ERα requires co-activators to mediate transcription via mechanisms that are largely uncharacterized. Herein, using growth-regulating estrogen receptor binding 1 (GREB1) as an ERα target gene in Ishikawa cells, we demonstrate that nuclear receptor co-activator 6 (NCOA6) is essential for estradiol (E2)/ERα-activated GREB1 transcription. We found that NCOA6 associates with the GREB1 promoter and enhancer in an E2-independent manner and that NCOA6 knockout reduces chromatin looping, enhancer-promoter interactions, and basal GREB1 expression in the absence of E2. In the presence of E2, ERα bound the GREB1 enhancer and also associated with its promoter, and p300, myeloid/lymphoid or mixed-lineage leukemia protein 4 (MLL4), and RNA polymerase II were recruited to the GREB1 enhancer and promoter. Consequently, the levels of the histone modifications H3K4me1/3, H3K9ac, and H3K27ac were significantly increased; enhancer and promoter regions were transcribed; and GREB1 mRNA was robustly transcribed. NCOA6 knockout reduced ERα recruitment and abolished all of the aforementioned E2-induced events, making GREB1 completely insensitive to E2 induction. We also found that GREB1-deficient Ishikawa cells are much more resistant to chemotherapy and that human endometrial cancers with low GREB1 expression predict poor overall survival. These results indicate that NCOA6 has an essential role in ERα-mediated transcription by increasing enhancer-promoter interactions through chromatin looping and by recruiting RNA polymerase II and the histone-code modifiers p300 and MLL4. Moreover, GREB1 loss may predict chemoresistance of endometrial cancer.
Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/fisiologia , Estrogênios/farmacologia , Proteínas de Neoplasias/fisiologia , Coativadores de Receptor Nuclear/fisiologia , Regiões Promotoras Genéticas , Antineoplásicos/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Proteína p300 Associada a E1A/fisiologia , Receptor alfa de Estrogênio/genética , Feminino , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/fisiologia , Histonas/química , Humanos , Proteína de Leucina Linfoide-Mieloide/fisiologiaRESUMO
Blakeslea trispora is an industrial fungal species used for large-scale production of carotenoids. However, B. trispora light-regulated physiological processes, such as carotenoid biosynthesis and phototropism, are not fully understood. In this study, we isolated and characterized three photoreceptor genes, btwc-1a, btwc-1b, and btwc-1c, in B. trispora Bioinformatics analyses of these genes and their protein sequences revealed that the functional domains (PAS/LOV [Per-ARNT-Sim/light-oxygen-voltage] domain and zinc finger structure) of the proteins have significant homology to those of other fungal blue-light regulator proteins expressed by Mucor circinelloides and Neurospora crassa The photoreceptor proteins were synthesized by heterologous expression in Escherichia coli The chromogenic groups consisting of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were detected to accompany BTWC-1 proteins by using high-performance liquid chromatography (HPLC) and fluorescence spectrometry, demonstrating that the proteins may be photosensitive. The absorbance changes of the purified BTWC-1 proteins seen under dark and light conditions indicated that they were light responsive and underwent a characteristic photocycle by light induction. Site-directed mutagenesis of the cysteine residual (Cys) in BTWC-1 did not affect the normal expression of the protein in E. coli but did lead to the loss of photocycle response, indicating that Cys represents a flavin-binding domain for photon detection. We then analyzed the functions of BTWC-1 proteins by complementing btwc-1a, btwc-1b, and btwc-1c into the counterpart knockout strains of M. circinelloides for each mcwc-1 gene. Transformation of the btwc-1a complement into mcwc-1a knockout strains restored the positive phototropism, while the addition of btwc-1c complement remedied the deficiency of carotene biosynthesis in the mcwc-1c knockout strains under conditions of illumination. These results indicate that btwc-1a and btwc-1c are involved in phototropism and light-inducible carotenogenesis. Thus, btwc-1 genes share a conserved flavin-binding domain and act as photoreceptors for control of different light transduction pathways in B. trisporaIMPORTANCE Studies have confirmed that light-regulated carotenogenesis is prevalent in filamentous fungi, especially in mucorales. However, few investigations have been done to understand photoinduced synthesis of carotenoids and related mechanisms in B. trispora, a well-known industrial microbial strains. In the present study, three photoreceptor genes in B. trispora were cloned, expressed, and characterized by bioinformatics and photoreception analyses, and then in vivo functional analyses of these genes were constructed in M. circinelloides The results of this study will lead to a better understanding of photoreception and light-regulated carotenoid synthesis and other physiological responses in B. trispora.
Assuntos
Proteínas Fúngicas/genética , Mucorales/genética , Fotorreceptores Microbianos/genética , Sequência de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Mucorales/metabolismo , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Alinhamento de SequênciaRESUMO
As an ideal carotenoid producer, Blakeslea trispora has gained much attention due to its large biomass and high production of ß-carotene and lycopene. However, carotenogenesis regulation in B. trispora still needs to be clarified, as few investigations have been conducted at the molecular level in B. trispora In this study, a gene homologous to carotenogenesis regulatory gene (crgA) was cloned from the mating type (-) of B. trispora, and the deduced CrgA protein was analyzed for its primary structure and domains. To clarify the crgA-mediated regulation in B. trispora, we used the strategies of gene knockout and complementation to investigate the effect of crgA expression on the phenotype of B. trispora In contrast to the wild-type strain, the crgA null mutant (ΔcrgA) was defective in sporulation but accumulated much more ß-carotene (31.2% improvement at the end) accompanied by enhanced transcription of three structural genes (hmgR, carB, and carRA) for carotenoids throughout the culture time. When the wild-type copy of crgA was complemented into the crgA null mutant, sporulation, transcription of structural genes, and carotenoid production were restored to those of the wild-type strain. A gas chromatography-mass spectrometry (GC-MS)-based metabolomic approach and multivariate statistical analyses were performed to investigate the intracellular metabolite profiles. The reduced levels of tricarboxylic acid (TCA) cycle components and some amino acids and enhanced levels of glycolysis intermediates and fatty acids indicate that more metabolic flux was driven into the mevalonate (MVA) pathway; thus, the increase of precursors and fat content contributes to the accumulation of carotenoids.IMPORTANCE The zygomycete Blakeslea trispora is an important strain for the production of carotenoids on a large scale. However, the regulation mechanism of carotenoid biosynthesis is still not well understood in this filamentous fungus. In the present study, we sought to investigate how crgA influences the expression of structural genes for carotenoids, carotenoid biosynthesis, and other anabolic phenotypes. This will lead to a better understanding of the global regulation mechanism of carotenoid biosynthesis and facilitate engineering this strain in the future for enhanced production of carotenoids.
Assuntos
Carbono/metabolismo , Carotenoides/metabolismo , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Mucorales/genética , Proteínas Fúngicas/metabolismo , Mucorales/metabolismoRESUMO
Over the past 30 years, the ever-rising demands of the modern and growing population have led to the rapid development of agricultural and industrial sectors worldwide. However, this expansion has exposed the environment to various pollutants including heavy metal (HM)s. Almost all HMs are serious toxicants and can pose serious health risks to living organisms in addition to their bioaccumulative and non-biodegradable nature. Different techniques have been developed to restore the ecological functions of the HM-contaminated soil (HMCS). However, the major downfalls of the commonly used remediation technologies are the generation of secondary wastes, high operating costs, and high energy consumption. Phytoremediation is a prominent approach that is more innocuous than the existing remediation approaches. Some microbes-plant interactions enhance the bioremediation process, with heavy metal resistant-plant growth promoting bacteria (HMRPGPB) being widely used to assist phytoremediation of HMs. However, the most common of all major microbial assisted-phytoremediation disturbances is that the HM-contaminated soil is generally deficient in nutrients and cannot sustain the rapid growth of the applied HMRPGPB. In this case, biochar has recently been approved as a potential carrier of microbial agents. The biochar-HMRPGPB-plant association could provide a promising green approach to remediate HM-polluted sites. Therefore, this review addresses the mechanisms through which biochar and HMRPGPB can enhance phytoremediation. This knowledge of biochar-HMRPGPB-plant interactions is significant with respect to sustainable management of the HM-polluted environment in terms of both ecology and economy, and it offers the possibility of further development of new green technologies.
Assuntos
Bactérias/metabolismo , Biodegradação Ambiental , Carvão Vegetal/farmacologia , Embriófitas/metabolismo , Metais Pesados/metabolismo , Poluentes do Solo/metabolismo , Desenvolvimento Vegetal/efeitos dos fármacos , Desenvolvimento Vegetal/fisiologia , Solo/químicaRESUMO
BACKGROUND: The mechanism of blood vessel formation and degeneration still remains unclear. Transforming growth factor-ß1 (TGF-ß1) signaling is a critical pathway in this progression and can induce multiple biological effects. Osteopontin (OPN) is involved in mineral metabolism and the inflammatory response associated with vascular calcification. METHODS: To identify the relationship between TGF-ß signaling pathway and OPN, we stimulated human vascular endothelial cells (HVECs) and human aortic endothelial cells (HAECs) using various concentration of TGF-ß1 in vitro. RESULTS: As assessed by flow cytometry and western blots, apoptosis levels were significantly increased with TGF-ß1 treatment. We also demonstrated that OPN increased in vitro with TGF-ß signaling by western blot and quantitative real time polymerase chain reaction (qRT-PCR) analyses. The inhibitory phosphorylation of endothelial nitric-oxide synthase (eNOS) (Thr495) was also up-regulated by TGF-ß signaling. Meanwhile, the anti-inflammatory factor Nrf2 and the activating phosphorylation of eNOS (Ser1177) were down-regulated. CONCLUSIONS: Taken together, our findings demonstrate that TGF-ß signaling can induce the expression of OPN, which may play an important role in the dysfunction of the vascular wall.
Assuntos
Células Endoteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Osteopontina/genética , Fator de Crescimento Transformador beta1/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Osteopontina/metabolismo , Fosforilação/efeitos dos fármacos , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Smad3/genética , Proteína Smad3/metabolismoRESUMO
The aim of this study was to investigate the regulation mechanism of aquaporin 9 (AQP9) gene on inflammatory response and cardiac function in rats with myocardial infarction (MI) through extracellular signal-regulated kinase1/2 (ERK1/2) pathway. The constructed rats models of MI were randomly divided into 6 groups: control group (sham operation group, MI modeling sham operation), model group (MI modeling), NC group (MI modeling, tail vein injection of AQP9 negative control sequence vector), AQP9 shRNA group (MI modeling, tail vein injection of AQP9 shRNA plasmid vector), U0126 group (MI modeling, tail vein injection of ERK signaling pathway inhibitor), and AQP9 shRNA + U0126 group. The hemodynamics and cardiac function of rats in each group were detected on the seventh day of modeling. The levels of AQP9 and inflammatory factors [tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10)] in peripheral blood of rats were detected by ELISA method. qRT-PCR and western blot were used to detect the mRNA and protein expression of AQP9, ERK1/2, B-cell lymphoma-2 (Bcl-2), Bcl-associated x (Bax) in the myocardial tissue of rats. TTC and TUNEL staining were used to observe myocardial infarct size and apoptosis of myocardial cells in each group. Compared with control group, the levels of heart rate, left ventricular end-diastolic pressure, TNF-α, and IL-6 were increased in each group of rats with MI (all p < 0.05), while the levels of systolic blood pressure, diastolic blood pressure, mean arterial pressure, left ventricular systolic pressure, and IL-10 were significantly decreased (all p < 0.05). The mRNA and protein expression levels of AQP9, ERK1/2 phosphorylation and Bax were significantly increased, as well as the myocardial infarct size, apoptosis index of myocardial tissue (all p < 0.05), the mRNA and protein expression levels of Bcl-2 were significantly decreased (all p < 0.05). The AQP9 gene knock-down or exogenous administration of the ERK1/2 inhibitor U0126 could improve the above indexes. However, the combination of AQP9 gene knock-down and U0126 showed no further effect. Silencing AQP9 gene can inhibit the activation of ERK1/2 signaling pathway, attenuate the inflammatory response in rats with MI, inhibit apoptosis of myocardial cells, and improve cardiac function.
Assuntos
Aquaporinas/genética , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Contração Miocárdica/fisiologia , Infarto do Miocárdio/genética , Miocárdio/metabolismo , Animais , Apoptose , Aquaporinas/biossíntese , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Masculino , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , RNA/genética , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
BACKGROUND: Although ductal carcinoma in situ (DCIS) is a non-invasive breast cancer, many DCIS lesions may progress to invasive cancer and the genes and pathways responsible for its progression are largely unknown. FGFR1 plays an important role in cell proliferation, differentiation and carcinogenesis. The purpose of this study is to examine the roles of FGFR1 signaling in gene expression, cell proliferation, tumor growth and progression in a non-invasive DCIS model. METHODS: DCIS.COM cells were transfected with an empty vector to generate DCIS-Ctrl cells. DCIS-iFGFR1 cells were transfected with an AP20187-inducible iFGFR1 vector to generate DCIS-iFGFR1 cells. iFGFR1 consists of the v-Src myristoylation membrane-targeting sequence, FGFR1 cytoplasmic domain and the AP20187-inducible FKBP12 dimerization domain, which simulates FGFR1 signaling. The CRISPR/Cas9 system was employed to knockout ERK1, ERK2 or TNFAIP3 in DCIS-iFGFR1 cells. Established cell lines were treated with/without AP20187 and with/without FGFR1, MEK, or ERK1/2 inhibitor. The effects of these treatments were determined by Western blot, RNA-Seq, real-time RT-PCR, cell proliferation, mammosphere growth, xenograft tumor growth, and tumor histopathological assays. RESULTS: Activation of iFGFR1 signaling in DCIS-iFGFR1 cells enhanced ERK1/2 activities, induced partial epithelial-to-mesenchymal transition (EMT) and increased cell proliferation. Activation of iFGFR1 signaling promoted DCIS growth and progression to invasive cancer derived from DCIS-iFGFR1 cells in mice. Activation of iFGFR1 signaling also altered expression levels of 946 genes involved in cell proliferation, migration, cancer pathways, and other molecular and cellular functions. TNFAIP3, a ubiquitin-editing enzyme, is upregulated by iFGFR1 signaling in a FGFR1 kinase activity and in an ERK2-dependent manner. Importantly, TNFAIP3 knockout not only inhibited the AP20187-induced proliferation and tumor growth of DCIS-iFGFR1 cells, but also further reduced baseline proliferation and tumor growth of DCIS-iFGFR1 cells without AP20187 treatment. CONCLUSIONS: Activation of iFGFR1 promotes ERK1/2 activity, EMT, cell proliferation, tumor growth, DCIS progression to invasive cancer, and altered the gene expression profile of DCIS-iFGFR1 cells. Activation of iFGFR1 upregulated TNFAIP3 in an ERK2-dependent manner and TNFAIP3 is required for iFGFR1 activation-promoted DCIS.COM cell proliferation, mammosphere growth, tumor growth and progression. These results suggest that TNFAIP3 may be a potential target for inhibiting DCIS growth and progression promoted by FGFR1 signaling.
Assuntos
Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/patologia , Transformação Celular Neoplásica/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Mama/citologia , Mama/patologia , Neoplasias da Mama/genética , Sistemas CRISPR-Cas , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Células Epiteliais , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica/patologia , Esferoides Celulares , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVE: To explore an efficient use of crude glycerol for the production of a highly thermostable ß-mannanase (ReTMan26) by Pichia pastoris X33. RESULTS: Cell growth was significantly inhibited by 4 and 6% (w/v) crude glycerol in 250 ml shake-flasks and in 5 l bioreactor batch cultures, respectively, but not affected by pure glycerol at the same concentrations. For further study, the impact of various impurities in crude glycerol on the cell growth of, and ReTMan26 production by, Pichia pastoris was investigated. Salts and methanol did not exert an inhibitory effect, but ≥ 0.2% and 0.3% (w/v) soap in shake-flask and bioreactor cultures, respectively, inhibited fermentation. Under identical conditions, the biomass and ReTMan26 activity produced by high-cell-density fermentation using 5% crude glycerol (glycerol at 80%, w/w) were slightly higher than those using 4% (w/v) pure glycerol. CONCLUSIONS: Non-pretreated ≤ 5% (w/v) crude glycerol could be effectively utilized for industrial production of ReTMan26, and the total production costs using crude glycerol were ~ 4.2% lower than those using pure glycerol.
Assuntos
Biocombustíveis , Carbono/metabolismo , Glicerol/metabolismo , Pichia/crescimento & desenvolvimento , Proteínas Recombinantes/biossíntese , beta-Manosidase/biossíntese , Biomassa , Reatores Biológicos/microbiologia , Meios de Cultura/química , Fermentação , Metanol/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Sais/metabolismo , beta-Manosidase/genéticaRESUMO
The effect of H2O2 concentration on the change of H-bonds at a water/quartz interface was systematically examined by surface-specific sum-frequency generation (SFG) spectroscopy. Molecular dynamics (MD) simulation was further utilized to interpret the specific molecular dynamics as well as the configuration and evolution of water and H2O2 molecules at the interface. The results from this study demonstrated the important role of surface H-bonds on determination of the stability of adsorbed H2O2 at solvated, silica, xerogel surfaces. It was revealed that prior to reaching the surface saturation with H2O2 molecules (less than 20% in bulk solution), multiple H-bonds were formed with silanols at relatively short interactive distances. These H-bonds proved to be strong enough to enable the overall stability of adsorbed H2O2. However, once saturated, the H2O2 molecules would be adsorbed at longer distances away from the surface, and could easily migrate to the bulk solution; therefore, in this case, the bonds failed to support stable H2O2 adsorption. These new findings explained the detailed molecular mechanism of the relationship between H2O2 concentration and H2O2 stability in H2O2-silica xerogels. This solves the current challenge of effective H2O2 storage, and provides fundamental insight for predicting the adsorption behavior of H2O2 at the silica surface.
RESUMO
BACKGROUND: Cervical cancer is one of the most common cancers in women worldwide. Emerging evidence suggests that kin17 is a tumor-promoting protein in some types of solid tumors. However, whether kin17 contributes to cervical cancer carcinogenesis remains unknown. METHODS: Kin17 expression in clinical samples from Guangdong Women and Children's Hospital and Health Institute was detected by immunohistochemical staining. A series of functional experiments including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay, 5-bromo-2'-deoxyuridine assay, colony formation, transwell assay, flow cytometry of apoptosis, and cell cycle were performed to explore the roles of kin17 in cervical cancer cells HeLa. RESULTS: In this study, we showed for the first time that the expression of kin17 was significantly increased in clinical cervical cancer samples, and associated with tumor differentiation, lymph node metastasis, and ki-67 expression in a clinicopathologic characteristics review. Furthermore, silence of kin17 in HeLa cells inhibited cell proliferation, clone formation, cell cycle progression, migration, and invasion, and also promoted cell apoptosis. CONCLUSION: Our findings demonstrate that kin17 is closely related to the cell proliferation and invasion of cervical cancer and could be a novel diagnostic and therapeutic target for cervical cancer management. The underlying mechanisms should be elucidated in future research.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a RNA/biossíntese , Neoplasias do Colo do Útero/metabolismo , Adulto , Apoptose/fisiologia , Biomarcadores Tumorais/genética , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/genética , Feminino , Células HeLa , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Invasividade Neoplásica , Proteínas de Ligação a RNA/genética , Transfecção , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/metabolismo , Displasia do Colo do Útero/patologiaRESUMO
Primary biliary cirrhosis (PBC) is an autoimmune chronic liver disease with worldwide increasing morbidity. However, the etiology of PBC is still unclear. Recently, the epithelial-mesenchymal transition (EMT) and interleukin-17A (IL-17A), a pro-inflammatory cytokine, were proposed to be involved in the pathogenesis of PBC. Therefore, in this study, we aimed to clarify the roles of IL-17A and/or EMT in the onset of PBC. The results showed that the median serum IL-17A level was significantly higher in 29 PBC patients (average course of 40.69 months) than that of 11 healthy controls. The intrahepatic biliary epithelial cells (IBECs), the major target of destruction in PBC, underwent EMT in PBC patients. The immunohistochemical analysis revealed that the protein levels of IL-17A receptor were increased in IBECs and the IL-17A protein was accumulated around the IBECs in the PBC patients. These results imply that the IL-17A-mediated signaling and EMT of intrahepatic biliary epithelial cells (IBEC-EMT) are key pathogenic processes of PBC. To study the association between IL-17A and IBECs-EMT, we then examined if IL-17A induced EMT using a human cell line of IBECs (HIBECs). After the treatment with IL-17A for 48 h, HIBECs changed into bipolar cells with a fibroblastic morphology. Additionally, the results of real-time PCR and Western blot analyses demonstrated that IL-17A up-regulated the expression of a mesenchymal marker vimentin and down-regulated the expression of an epithelial marker E-cadherin in HIBECs in the dose- and time-dependent manners. These results suggest that IL-17A may play an important role in the IBECs-EMT.