RESUMO
Arterial calcification is an important characteristic of cardiovascular disease. It has key parallels with skeletal mineralization; however, the underlying cellular mechanisms responsible are not fully understood. Mitochondrial dynamics regulate both bone and vascular function. In this study, we therefore examined mitochondrial function in vascular smooth muscle cell (VSMC) calcification. Phosphate (Pi)-induced VSMC calcification was associated with elongated mitochondria (1.6-fold increase, p < 0.001), increased mitochondrial reactive oxygen species (ROS) production (1.83-fold increase, p < 0.001) and reduced mitophagy (9.6-fold decrease, p < 0.01). An increase in protein expression of optic atrophy protein 1 (OPA1; 2.1-fold increase, p < 0.05) and a converse decrease in expression of dynamin-related protein 1 (DRP1; 1.5-fold decrease, p < 0.05), two crucial proteins required for the mitochondrial fusion and fission process, respectively, were noted. Furthermore, the phosphorylation of DRP1 Ser637 was increased in the cytoplasm of calcified VSMCs (5.50-fold increase), suppressing mitochondrial translocation of DRP1. Additionally, calcified VSMCs showed enhanced expression of p53 (2.5-fold increase, p < 0.05) and ß-galactosidase activity (1.8-fold increase, p < 0.001), the cellular senescence markers. siRNA-mediated p53 knockdown reduced calcium deposition (8.1-fold decrease, p < 0.01), mitochondrial length (3.0-fold decrease, p < 0.001) and ß-galactosidase activity (2.6-fold decrease, p < 0.001), with concomitant mitophagy induction (3.1-fold increase, p < 0.05). Reduced OPA1 (4.1-fold decrease, p < 0.05) and increased DRP1 protein expression (2.6-fold increase, p < 0.05) with decreased phosphorylation of DRP1 Ser637 (3.20-fold decrease, p < 0.001) was also observed upon p53 knockdown in calcifying VSMCs. In summary, we demonstrate that VSMC calcification promotes notable mitochondrial elongation and cellular senescence via DRP1 phosphorylation. Furthermore, our work indicates that p53-induced mitochondrial fusion underpins cellular senescence by reducing mitochondrial function.
Assuntos
Dinâmica Mitocondrial , Músculo Liso Vascular , Calcificação Vascular , Humanos , beta-Galactosidase/metabolismo , Células Cultivadas , Dinâmica Mitocondrial/genética , Dinâmica Mitocondrial/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/metabolismoRESUMO
The late gestational rise in glucocorticoids contributes to the structural and functional maturation of the perinatal heart. Here, we hypothesized that glucocorticoid action contributes to the metabolic switch in perinatal cardiomyocytes from carbohydrate to fatty acid oxidation. In primary mouse fetal cardiomyocytes, dexamethasone treatment induced expression of genes involved in fatty acid oxidation and increased mitochondrial oxidation of palmitate, dependent upon a glucocorticoid receptor (GR). Dexamethasone did not, however, induce mitophagy or alter the morphology of the mitochondrial network. In vivo, in neonatal mice, dexamethasone treatment induced cardiac expression of fatty acid oxidation genes. However, dexamethasone treatment of pregnant C57Bl/6 mice at embryonic day (E)13.5 or E16.5 failed to induce fatty acid oxidation genes in fetal hearts assessed 24 h later. Instead, at E17.5, fatty acid oxidation genes were downregulated by dexamethasone, as was GR itself. PGC-1α, required for glucocorticoid-induced maturation of primary mouse fetal cardiomyocytes in vitro, was also downregulated in fetal hearts at E17.5, 24 h after dexamethasone administration. Similarly, following a course of antenatal corticosteroids in a translational sheep model of preterm birth, both GR and PGC-1α were downregulated in heart. These data suggest that endogenous glucocorticoids support the perinatal switch to fatty acid oxidation in cardiomyocytes through changes in gene expression rather than gross changes in mitochondrial volume or mitochondrial turnover. Moreover, our data suggest that treatment with exogenous glucocorticoids may interfere with normal fetal heart maturation, possibly by downregulating GR. This has implications for clinical use of antenatal corticosteroids when preterm birth is considered a possibility. KEY POINTS: Glucocorticoids are steroid hormones that play a vital role in late pregnancy in maturing fetal organs, including the heart. In fetal cardiomyocytes in culture, glucocorticoids promote mitochondrial fatty acid oxidation, suggesting they facilitate the perinatal switch from carbohydrates to fatty acids as the predominant energy substrate. Administration of a synthetic glucocorticoid in late pregnancy in mice downregulates the glucocorticoid receptor and interferes with the normal increase in genes involved in fatty acid metabolism in the heart. In a sheep model of preterm birth, antenatal corticosteroids (synthetic glucocorticoid) downregulates the glucocorticoid receptor and the gene encoding PGC-1α, a master regulator of energy metabolism. These experiments suggest that administration of antenatal corticosteroids in anticipation of preterm delivery may interfere with fetal heart maturation by downregulating the ability to respond to glucocorticoids.
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Glucocorticoides , Nascimento Prematuro , Animais , Dexametasona/farmacologia , Ácidos Graxos , Feminino , Coração Fetal , Glucocorticoides/farmacologia , Camundongos , Miócitos Cardíacos , Gravidez , Receptores de Glucocorticoides/genética , OvinosRESUMO
BACKGROUND: IS26-flanked transposons played an increasingly important part in the mobilization and development of resistance determinants. Heterogeneous resistance-encoding plasmid clusters with polymorphic MDR regions (MRRs) conferred by IS26 in an individual Escherichia coli isolate have not yet been detected. OBJECTIVES: To characterize the complete sequence of a novel blaCTX-M-65- and fosA3-carrying IncZ-7 plasmid with dynamic MRRs from an E. coli isolate, and to depict the mechanism underlying the spread of resistance determinants and genetic polymorphisms. METHODS: The molecular characterization of a strain carrying blaCTX-M-65 and fosA3 was analysed by antimicrobial susceptibility testing and MLST. The transferability of a plasmid bearing blaCTX-M-65 and fosA3 was determined by conjugation assays, and the complete structure of the plasmid was obtained by Illumina, PacBio and conventional PCR mapping, respectively. The circular forms derived from IS26-flanked transposons were detected by reverse PCR and sequencing. RESULTS: A novel IncZ-7 plasmid pEC013 (â¼118kb) harbouring the blaCTX-M-65 and fosA3 genes was recovered from E. coli isolate EC013 belonging to D-ST117. The plasmid was found to have heterogeneous and dynamic MRRs in an individual strain and the IS26-flanked composite transposon-derived circular intermediates were identified and characterized in pEC013. CONCLUSIONS: The heterogeneous MRRs suggested that a single plasmid may actually be a cluster of plasmids with the same backbone but varied MRRs, reflecting the plasmid's heterogeneity and the survival benefits of having a response to antimicrobial-related threatening conditions in an individual strain.
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Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/classificação , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade MicrobianaRESUMO
Osteosarcoma is a common primary malignant bone tumor that occurs mainly in children and adolescents. Recent evidence has demonstrated that miR-34a is involved in the invasion and metastasis of osteosarcoma. This study aims to explore the effect of biological behavior of miR-34a on osteosarcoma. First, we collect osteosarcoma and adjacent specimens, and the relative expression of miR-34a and C-IAP2 messenger RNA was quantitated by real-time polymerase chain reaction. Furthermore, miR-34a stimulant is synthesized and transfected onto osteosarcoma MG-63 cells. The effect of overexpression of miR-34a on osteosarcoma was detected by colony-forming assay, Annexin V-fluorescein isothiocyanate Apoptosis Detection Kit I, Transwell assay, and animal experiment in vivo. Finally, the relative levels of C-IAP2 and Bcl-2 protein were checked by western blot, and the activity of caspase-3 and caspase-9 was tested by spectrophotometry assay. In conclusion, miR-34a was downregulated in osteosarcoma cells. And the expression of C-IAP2 and Bcl-2 protein was drastically inhibited, and the activities of caspase-3 and caspase-9 were significantly increased after transfecting miR-34a onto osteosarcoma MG-63 cells. And the overexpression of miR-34a can inhibit cell invasion and metastasis, promote cell apoptosis, and arrest cells in G0/G1 period. And the animal experiment in vivo demonstrated that the overexpression of miR-34a could significantly inhibit the growth of osteosarcoma in animal skin. Taken together, we indicated that miR-34a can inhibit tumor invasion and metastasis in osteosarcoma, and its mechanism may be partly related to downregulating the expression of C-IAP2 and Bcl-2 protein directly or indirectly.
Assuntos
Proteínas Inibidoras de Apoptose/genética , MicroRNAs/genética , Osteossarcoma/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Apoptose/genética , Caspase 3/genética , Caspase 9/genética , Movimento Celular/genética , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Camundongos , MicroRNAs/biossíntese , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study aimed to evaluate the effect of PRKCH rs2230500 genetic polymorphism on efficacy of amlodipine and telmisartan for patients with hypertension. A total of 136 essential hypertension (EH) patients were treated with amlodipine (70 patients) or telmisartan (66 patients), respectively. Genetic polymorphism was genotyped by Sanger sequencing. Both baseline and post-treatment blood pressure (BP) and heart rate were measured to evaluate the influence of genetic polymorphism on the antihypertensive response. No significant difference in the absolute decrease in diastolic blood pressure (DBP),systolic blood pressure (SBP), and mean arterial pressure (MAP) was observed among PRKCH rs2230500 genotypes after 4-week amlodipine or telmisartan therapy (p > 0.05). However, when compared with carriers or GG genotype, the antihypertensive effect of PRKCH rs2230500 GA/AA carriers was superior in telmisartan treatment group. PRKCH rs2230500 gene polymorphism is significantly related to the efficiency in telmisartan therapy (p = 0.02). The PRKCH rs2230500 may influence the antihypertensive efficacy of telmisartan in Chinese EH patients, and further studies are needed to confirm these findings.
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Anlodipino/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Benzimidazóis/uso terapêutico , Benzoatos/uso terapêutico , Hipertensão Essencial/tratamento farmacológico , Hipertensão Essencial/genética , Proteína Quinase C/genética , Adulto , Idoso , Pressão Arterial/efeitos dos fármacos , Pressão Arterial/genética , Povo Asiático , Feminino , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/genética , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , TelmisartanRESUMO
Di-(2-ethylhexyl) phthalate (DEHP) is associated with atherosclerosis-related cardiovascular disease complications, but we lack direct evidence of its unfavorable effect on atherogenesis. In this study, we aimed to clarify in vivo and in vitro the contribution of DEHP to the development of atherosclerosis and its underlying mechanisms. Apolipoprotein E-deficient (apoE(-/-)) mice chronically treated with DEHP for 4 weeks showed exacerbated hyperlipidemia, systemic inflammation, and atherosclerosis. In addition, DEHP promoted low-density lipoprotein (LDL) oxidation, which led to inflammation in endothelial cells as evidenced by increased protein expression of pro-inflammatory mediators. Furthermore, chronic DEHP treatment increased hepatic cholesterol accumulation by downregulating the protein expression of key regulators in cholesterol clearance including LDL receptor, cholesterol 7α-hydrolase, ATP-binding cassette transporter G5 and G8, and liver X receptor α. Moreover, the adiposity and inflammation of white adipose tissues were promoted in DEHP-treated apoE(-/-) mice. In conclusion, DEHP may disturb cholesterol homeostasis and deregulate the inflammatory response, thus leading to accelerated atherosclerosis.
Assuntos
Apolipoproteínas E/deficiência , Aterosclerose/induzido quimicamente , Dietilexilftalato/toxicidade , Plastificantes/toxicidade , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Apolipoproteínas E/genética , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/patologia , Linhagem Celular , Colesterol/sangue , Modelos Animais de Doenças , Progressão da Doença , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Predisposição Genética para Doença , Humanos , Mediadores da Inflamação/sangue , Lipoproteínas LDL/sangue , Fígado/efeitos dos fármacos , Fígado/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Knockout , Fenótipo , Medição de Risco , Fatores de TempoRESUMO
Long-term exposure to di-(2-ethylhexyl) phthalate (DEHP) is highly associated with carcinogenicity, fetotoxicity, psychological disorders and metabolic diseases, but the detrimental effects and mechanisms are not fully understood. We investigated the effect of exposing mouse mothers to DEHP, and the underlying mechanism, on blood pressure, obesity and cholesterol metabolism as well as psychological and learning behaviors in offspring. Tail-cuff plethysmography was used for blood pressure measurement; Western blot used was for phosphorylation and expression of protein; hematoxylin and eosin staining, Nissl staining and Golgi staining were used for histological examination. The serum levels of cholesterol, triglycerides and glucose were measured by blood biochemical analysis. Hepatic cholesterol and triglyceride levels were assessed by colorimetric assay kits. Offspring behaviors were evaluated by open-field activity, elevated plus maze, social preference test and Morris water maze. Maternal DEHP exposure deregulated the phosphorylation of endothelial nitric oxide synthase and upregulated angiotensin type 1 receptor in offspring, which led to increased blood pressure. It led to obesity in offspring by increasing the size of adipocytes in white adipose tissue and number of adipocytes in brown adipose tissue. It increased the serum level of cholesterol in offspring by decreasing the hepatic capacity for cholesterol clearance. The impaired social interaction ability induced by maternal DEHP exposure might be due to abnormal neuronal development. Collectively, our findings provide new evidence that maternal exposure to DEHP has a lasting effect on the physiological functions of the vascular system, adipose tissue and nerve system in offspring.
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Adiposidade/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Pressão Sanguínea/efeitos dos fármacos , Colesterol/sangue , Dietilexilftalato/toxicidade , Hipertensão/induzido quimicamente , Exposição Materna , Efeitos Tardios da Exposição Pré-Natal , Comportamento Social , Animais , Biomarcadores/sangue , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Encéfalo/fisiopatologia , Feminino , Hipertensão/fisiopatologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , GravidezRESUMO
Excess nitric oxide (NO) promotes the progression of atherosclerosis by increasing the oxidation of low-density lipoprotein (LDL) and inflammatory responses. However, little is known about the impact of NO and its underlying molecular mechanism on lipid metabolism of macrophage foam cells. In this study, Oil-red O staining, cholesterol and triglyceride assay, Dil-oxidized LDL (oxLDL) binding assay, cholesterol efflux assay, real-time RT-PCR and Western blot analysis were used for in vitro experiments. Apolipoprotein E-deficient (apoE(-/-) ) and apoE and inducible nitric oxide synthase-deficient (apoE(-/-) iNOS(-/-) ) mice were as our in vivo models. Treatment with S-nitroso-N-acetyl-D,L-penicillamine (SNAP), an NO donor, exacerbated oxLDL-induced cholesterol accumulation in macrophages, because of reduced efficacy of cholesterol efflux. In addition, SNAP decreased the protein level of ATP-binding cassette transporter A1 (ABCA1) without affecting scavenger receptor type A (SR-A), CD36, ABCG1, or SR-B1 levels. This SNAP-mediated downregulation of ABCA1 was mainly through the effect of NO but not peroxynitrite. Furthermore, the SNAP-downregulated ABCA1 was due to the decrease in the liver X receptor α (LXRα)-dependent transcriptional regulation. Moreover, genetic deletion of iNOS increased the serum capacity of reverse cholesterol efflux and protein expression of LXRα, ABCA1, and SR-BI in aortas and retarded atherosclerosis in apoE(-/-) mice. Our findings provide new insights in the pro-atherogenic effect of excess NO on cholesterol metabolism in macrophages.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Aterosclerose/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Óxido Nítrico/metabolismo , Receptores Nucleares Órfãos/metabolismo , Transportador 1 de Cassete de Ligação de ATP/biossíntese , Animais , Apolipoproteínas E/genética , Aterosclerose/induzido quimicamente , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Lipoproteínas LDL , Fígado/efeitos dos fármacos , Fígado/metabolismo , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/toxicidade , Óxido Nítrico Sintase Tipo II/metabolismo , Receptores Nucleares Órfãos/biossíntese , Oxirredução , S-Nitroso-N-Acetilpenicilamina/administração & dosagemRESUMO
BACKGROUND AND AIM: Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with unclear etiology and mechanism(s). Glycine N-methyltransferase (GNMT) plays a central role in inflammatory diseases such as hepatitis and atherosclerosis. However, little is known about the impact of GNMT and the involved mechanism in the pathogenesis of IBD. In the current study, we investigated the role of GNMT in the mouse model of dextran sulfate sodium (DSS)-induced colitis. METHODS: Protein expression was determined by Western blotting or immunohistochemistry. Histopathology was examined by hematoxylin and eosin staining. Levels of pro-inflammatory cytokines were evaluated by ELISA kits. RESULTS: GNMT was expressed in the epithelium of the colon under normal conditions, and with DSS treatment, its expression was predominant in infiltrated leukocytes of lesions. Mice with genetic deletion of GNMT (GNMT(-/-) ) showed increased susceptibility to DSS induction of colitis, as revealed by the progression of colitis. Additionally, severe colonic inflammation, including increased crypt loss, leukocyte infiltration, and hemorrhage, was greater with DSS treatment in GNMT(-/-) than wild-type mice. Furthermore, the expression of adhesion molecule and inflammatory mediators in the colon was significantly higher with DSS treatment in GNMT(-/-) than wild-type mice. Moreover, loss of GNMT decreased cell apoptosis in colitis lesions with DSS treatment. CONCLUSIONS: Collectively, our findings suggest that GNMT may be a crucial molecule in the pathogenesis of DSS-induced colitis. This finding may provide new information for a potential therapeutic target in treating IBD.
Assuntos
Colite Ulcerativa/genética , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/fisiologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Glicina N-Metiltransferase/metabolismo , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Terapia de Alvo MolecularRESUMO
TGF-ß (transforming growth factor-ß) signaling is involved in a myriad of cellular processes and its dysregulation has been implicated in many human diseases, including fibrosis and cancer. TGF-ß transcriptional responses are controlled by tail phosphorylation of transcription factors SMAD2 and SMAD3 (mothers against decapentaplegic homolog 2/3). Therefore, targeted dephosphorylation of phospho-SMAD3 could provide an innovative mechanism to block some TGF-ß-induced transcriptional responses, such as the transcription of SERPINE-1, which encodes plasminogen activator inhibitor 1 (PAI-1). Here, by developing and employing a bifunctional molecule, BDPIC (bromoTAG-dTAG proximity-inducing chimera), we redirected multiple phosphatases, tagged with bromoTAG, to dephosphorylate phospho-SMAD3, tagged with dTAG. Using CRISPR-Cas9 technology, we generated homozygous double knock-in A549 bromoTAG/bromoTAG PPM1H/ dTAG/dTAG SMAD3 cells, in which the BDPIC-induced proximity between bromoTAG-PPM1H and dTAG-SMAD3 led to a robust dephosphorylation of dTAG-SMAD3 and a significant decrease in SERPINE-1 transcription. Our work demonstrates targeted dephosphorylation of phospho-proteins as an exciting modality for rewiring cell signaling.
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Maize moldy and spoilage due to microbial growth is a significant challenge in grain storage. This study aimed to evaluate the effectiveness of a zinc oxide nanocomposite, ZnO@mSiO2, prepared in our previous research, in inhibiting mold growth and preserving maize cell quality. The results demonstrated that ZnO@mSiO2 could effectively inhibit the growth of dominant microorganism, Aspergillus flavus, Talaromyces variabilis, Penicillium citrinum and Fusarium graminearum, in maize storage. Aspergillus flavus was selected as the model fungus, ZnO@mSiO2 effectively disrupted fungal hyphae structure, leading to reduced hyphal mass and inhibited spore germination. The inhibitory effect of ZnO@mSiO2 on mold growth was concentration-dependent. However, the ZnO@mSiO2 at an appropriate concentration (not exceeding 3.0 g/kg) preserved the integrity of maize cell membranes and enhancing the antioxidant activity within maize cells. The findings highlight the potential of ZnO@mSiO2 as an effective protectant to inhibit mold growth and preserve maize quality during storage.
Assuntos
Óxido de Zinco , Óxido de Zinco/farmacologia , Zea mays/microbiologia , Aspergillus flavus , Fungos , Grão ComestívelRESUMO
PINK1, mutated in familial forms of Parkinson's disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions.
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Reversible phosphorylation of the transcription factor EB (TFEB) coordinates cellular responses to metabolic and other stresses. During nutrient replete and stressor-free conditions, phosphorylated TFEB is primarily localized to the cytoplasm. Stressor-mediated reduction of TFEB phosphorylation promotes its nuclear translocation and context-dependent transcriptional activity. In this study, we explored targeted dephosphorylation of TFEB as an approach to activate TFEB in the absence of nutrient deprivation or other cellular stress. Through an induction of proximity between TFEB and several phosphatases using the AdPhosphatase system, we demonstrate targeted dephosphorylation of TFEB in cells. Furthermore, by developing a heterobifunctional molecule BDPIC (bromoTAG-dTAG proximity-inducing chimera), we demonstrate targeted dephosphorylation of TFEB-dTAG through induced proximity to bromoTAG-PPP2CA. Targeted dephosphorylation of TFEB-dTAG by bromoTAG-PPP2CA with BDPIC at the endogenous levels is sufficient to induce nuclear translocation and some transcriptional activity of TFEB.
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The transient receptor potential vanilloid type 1 (TRPV1) is crucial in the pathogenesis of atherosclerosis; yet its role and underlying mechanism in the formation of macrophage foam cells remain unclear. Here, we show increased TRPV1 expression in the area of foamy macrophages in atherosclerotic aortas of apolipoprotein E-deficient mice. Exposure of mouse bone-marrow-derived macrophages to oxidized low-density lipoprotein (oxLDL) upregulated the expression of TRPV1. In addition, oxLDL activated TRPV1 and elicited calcium (Ca(2+)) influx, which were abrogated by the pharmacological TRPV1 antagonist capsazepine. Furthermore, oxLDL-induced lipid accumulation in macrophages was ameliorated by TRPV1 agonists but exacerbated by TRPV1 antagonist. Treatment with TRPV1 agonists did not affect the internalization of oxLDL but promoted cholesterol efflux by upregulating the efflux ATP-binding cassette (ABC) transporters ABCA1 and ABCG1. Moreover, the upregulation of ABC transporters was mainly through liver X receptor α-(LXRα-) dependent regulation of transcription. Moreover, the TNF-α-induced inflammatory response was alleviated by TRPV1 agonists but aggravated by the TRPV1 antagonist and LXR α siRNA in macrophages. Our data suggest that LXR α plays a pivotal role in TRPV1-activation-conferred protection against oxLDL-induced lipid accumulation and TNF-α-induced inflammation in macrophages.
Assuntos
Inflamação/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Western Blotting , Cálcio/metabolismo , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Células Cultivadas , Imunoprecipitação da Cromatina , Inflamação/induzido quimicamente , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Camundongos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , RNA Interferente Pequeno , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
A multi-drug resistant Escherichia coli C21 was isolated from a chicken in China. It was shown to be positive for the presence of the blaTEM-1, blaCTX-M-55 and rmtB genes by PCR. This strain was examined by phylogenetic grouping, conjugation experiments, plasmid analysis, PCR-based replicon typing and multi-locus sequence typed (MLST). The genetic environment of blaCTX-M-55 was investigated by PCR mapping. The strain belonged to phylogroup A, ST156. The blaCTX-M-55 and rmtB genes were found to be present in separate plasmids that belonged to the IncI1 and IncN families, respectively. These antibiotic-resistant plasmids could be transferred to the recipient strain alone or together. A new arrangement of ISEcp1Δ-IS1294-ΔISEcp1-blaCTX-M-55 -ORF477, in which the ISEcp1 element was disrupted by another IS1294 element, was identified initially. Conjugative transfer and IS elements found in this study could lead to the rapid dissemination of blaCTX-M-55 and rmtB among strains of Enterobacteriaceae, which could pose a threat to animal husbandry and public health.
Assuntos
Escherichia coli/genética , Metiltransferases/genética , beta-Lactamases/genética , Animais , Galinhas , China , Mapeamento Cromossômico , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/isolamento & purificação , Ordem dos Genes , Metiltransferases/metabolismo , Tipagem Molecular , Reação em Cadeia da Polimerase , beta-Lactamases/metabolismoRESUMO
To develop gastric floating erodible plug pulse capsules with compound Danshen as the model drug, in order to realize the pulse release of traditional Chinese medicines. Through the study on impermeable capsules, optimized prescriptions, drug-containing rapid-release tablets and prescription screening, and erodible plug prescription and process, we successfully prepared compounded Danshen pulse capsule, so as to provide a new dosage form for controlling and treating heart disease to better cater to clinical demands.
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Composição de Medicamentos/métodos , Medicamentos de Ervas Chinesas/química , Salvia miltiorrhiza/química , Cápsulas , Preparações de Ação Retardada , Medicamentos de Ervas Chinesas/uso terapêutico , Cardiopatias/tratamento farmacológico , PermeabilidadeRESUMO
Carbon dioxide (CO2) absorption and capture is an effective measure to achieve the "dual carbon" goal of carbon peak and carbon neutrality in China. Organic amine compounds are widely used in the industrial separation and recovery of CO2. Thus, the establishment of analytical methods for organic amine compounds is of great significance for the research and development of carbon capture and storage (CCS) technology and carbon capture, utilization and storage (CCUS) technology. In this study, a method was developed for the determination of nine organic amine compounds in CO2 absorption liquid by hydrophilic interaction liquid chromatography (HILIC)-electrostatic field orbitrap high resolution mass spectrometry. The sample was diluted with water and filtered through a 0.22 µm nylon membrane before sampling and analysis. An Accucore HILIC column (100 mm×2.1 mm, 2.6 µm) was used for separation at 30 â. Gradient elution was conducted using 90% acetonitrile aqueous solution containing 5 mmol/L ammonium formate and 0.1% formic acid as mobile phase A and 10% acetonitrile aqueous solution containing 5 mmol/L ammonium formate and 0.1% formic acid as mobile phase B. Determination was performed using an electrospray ion source (ESI) in the positive ion mode. The quantitative analysis was carried out by standard addition method. The chromatographic retention performance of different chromatographic columns and the influence of different mobile phases on the separation of the organic amine compounds were compared, and the method was validated. The results showed that the linear ranges of the nine organic amine compounds were 0.04-25000 ng/mL with the linear correlation coefficients (R2) greater than 0.9910. The limits of detection (LODs) of the method were in the range of 0.0004-0.0080 ng/mL, and the limits of quantification (LOQs) of the method were in the range of 0.0035-0.0400 ng/mL. The average recoveries of the method ranged from 85.30% to 104.26% with relative standard deviations (RSDs) of 0.04%-7.95% at the spiked levels of 1, 1.5 and 3 times sample concentration. The established method was applied to detect the absorption waste liquid of a cement plant, and nine organic amine compounds could be effectively detected. The stability of the actual sample was tested, and the RSDs were 0.10%-6.35% in 48 h at 4 â. The method is sensitive, rapid and accurate for the determination of the nine organic amine compounds in industrial waste water. It can provide reference for the detection of organic amine compounds, and provide strong technical support for the research and industrial application of CO2 capture technology.
RESUMO
The emergence and rapid spread of multidrug resistant (MDR) Gram-negative bacteria have posed a serious threat to global health and security. Because of the time-consuming, high cost and high risk of developing new antibiotics, a significant method is to use antibiotic adjuvants to revitalize the existing antibiotics. The purpose of the study is to research the traditional Chinese medicine baicalin with the function of inhibiting the efflux pump and EDTA whether their single or combination can increase the activity of colistin against colistin-resistant Salmonella in vitro and in vivo, and to explore its molecular mechanisms. In vitro antibacterial experiments, we have observed that baicalin and EDTA alone could enhance the antibacterial activity of colistin. At the same time, the combination of baicalin and EDTA also showed a stronger synergistic effect on colistin, reversing the colistin resistance of all Salmonella strains. Molecular docking and RT-PCR results showed that the combination of baicalin and EDTA not only affected the expression of mcr-1, but also was an effective inhibitor of MCR-1. In-depth synergistic mechanism analysis revealed that baicalin and EDTA enhanced colistin activity through multiple pathways, including accelerating the tricarboxylic acid cycle (TCA cycle), inhibiting the bacterial antioxidant system and lipopolysaccharide (LPS) modification, depriving multidrug efflux pump functions and attenuating bacterial virulence. In addition, the combinational therapy of colistin, baicalin and EDTA displayed an obvious reduction in bacterial loads cfus of liver and spleen compared with monotherapy and 2-drug combination therapy. In conclusion, our study indicates that the combination of baicalin and EDTA as a novel colistin adjuvant can provide a reliable basis for formulating the therapeutic regimen for colistin resistant bacterial infection.
Assuntos
Colistina , Proteínas de Escherichia coli , Animais , Antibacterianos/farmacologia , Colistina/farmacologia , Farmacorresistência Bacteriana Múltipla , Ácido Edético/farmacologia , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana/veterinária , Simulação de Acoplamento Molecular , SalmonellaRESUMO
Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.
Assuntos
Proteínas Quinases , Proteína Fosfatase 2 , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fosforilação , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Especificidade por Substrato , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
Mutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson's disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria ("mitophagy") by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.