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1.
Rev Cardiovasc Med ; 24(12): 347, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39077074

RESUMO

Background: Dilated cardiomyopathy (DCM) has a poor prognosis and high mortality. The relationship between the deformation capacity of the biatrial and biventricular regions in patients with DCM remains unclear. Methods: This retrospective study used cardiovascular magnetic resonance (CMR) to assess patient enrollment between September 2020 to May 2022. Feature tracking (FT) was used to evaluate biventricular global radial strain (GRS), global circumferential strain (GCS) and global longitudinal strain (GLS). Fast long-axis method was used to evaluate biatrial GLS by analyzing balanced steady-state free precession cine images. The median follow-up period was 362 days (interquartile range: 234 to 500 days). DCM patients were divided into two groups based on the occurrence or non-occurrence of major adverse cardiac event (MACE). The primary endpoint was defined as all-cause death, heart transplantation, and adverse ventricular arrhythmia. The secondary end point included hospitalizations due to heart failure. Cox regression analysis was utilized for variables and Kaplan-Meier survival was utilized for clinical outcomes. Results: There were 124 DCM patients (52.82 ± 12.59 years, 67.74% male) and 53 healthy volunteers (53.17 ± 14.67 years, 52.83% male) recruited in this study. Biventricular GRS, GCS, GLS, and biatrial GLS were significantly impaired in the DCM group compared with the healthy group. In receiver-operating characteristic curve, biatrial GLS and biventricular GRS, GCS, and GLS showed significant prognostic value in predicting MACEs (all p < 0.05). In multivariate Cox regression analysis, left ventricular (LV) GLS offered a significant and independent prognostic value surpassing other CMR parameters in predicting MACE. In Kaplan-Meier analysis, patients with a LV GLS > -4.81% had a significantly higher rate of MACE (Log-rank p < 0.001). Conclusions: LV GLS was independently associated with MACEs in DCM patients by using FT and fast long-axis method derived from CMR. Comprehensive CMR examination including biatrial and biventricular functions should be systematically performed, to understand disease characteristics, as well as improve the risk stratification and therapeutic management for patients with DCM.

2.
Eur J Nucl Med Mol Imaging ; 50(2): 465-474, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36171409

RESUMO

PURPOSE: We aimed to evaluate whether [68 Ga]Ga-FAPI-04 PET/CT could characterize the early stages of cardiac fibrosis in pressure overload heart failure. METHODS: Sprague-Dawley rats underwent abdominal aortic constriction (AAC) (n = 12) and sham surgery (n = 10). All rats were scanned with [68 Ga]Ga-FAPI-04 PET/CT at 2, 4, and 8 weeks after surgery. The expression of fibroblast activation protein (FAP) in the myocardium was detected by immunohistochemistry. [68 Ga]Ga-FAPI-04 PET signal and FAP expression were compared between two groups. RESULTS: Compared with the sham group, the AAC group presented with decreased ejection fraction (EF) and fractional shortening (FS) and increased left ventricular internal dimensions in diastole (LVIDd) and systole (LVIDs) at 4 and 8 weeks (all p < 0.01). The AAC group showed higher [68 Ga]Ga-FAPI-04 accumulation in the heart than the sham group at 2, 4, and 8 weeks, and FAPI increased significantly from 2 to 8 weeks (all p < 0.001). Immunohistochemistry confirmed the higher density of the FAP+ area in the AAC group. The intensity of the [68 Ga]Ga-FAPI-04 correlated with the density of the FAP+ area (p < 0.001). The expression of the [68 Ga]Ga-FAPI-04 at 4 weeks correlated with the deterioration of cardiac function at 8 weeks (EF: R = - 0.87; FS: R = - 0.72; LVIDd: R = 0.77; LVIDs: R = 0.79; all p < 0.001). The AAC group also showed an increased [68 Ga]Ga-FAPI-04 signal in the liver, peaking at 4 weeks and then declining. Cardiac and liver PET signals correlated at 4 weeks in the AAC group (R = 0.69, p = 0.0010), suggesting an early fibrotic link between organs. A combination of the [68 Ga]Ga-FAPI-04 intensity in the heart and liver at 4 weeks better predicted the deterioration of cardiac function at 8 weeks. CONCLUSIONS: The activated fibroblasts in the heart and liver after pressure overload can be monitored by [68 Ga]Ga-FAPI-04 PET/CT, which reveals an early fibrotic link in cardio-liver interactions and could better predict nonischemic heart failure prognosis.


Assuntos
Insuficiência Cardíaca , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Animais , Ratos , Fibroblastos , Radioisótopos de Gálio , Insuficiência Cardíaca/diagnóstico por imagem , Imagem Molecular , Ratos Sprague-Dawley
3.
Cell Biol Int ; 47(10): 1749-1759, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37431269

RESUMO

Calcific aortic valve disease (CAVD) is the most common valvular heart disease, with an increasing prevalence due to an aging population. The pathobiology of CAVD is a multifaceted and actively regulated process, but the detailed mechanisms have not been elucidated. The present study aims to identify the differentially expressed genes (DEGs) in calcified aortic valve tissues, and to analyze the correlation between DEGs and clinical features in CAVD patients. The DEGs were screened by microarray in normal and CAVD groups (n = 2 for each group), and confirmed by quantitative real-time polymerase chain reaction in normal (n = 12) and calcified aortic valve tissues (n = 34). A total of 1048 DEGs were identified in calcified aortic valve tissues, including 227 upregulated mRNAs and 821 downregulated mRNAs. Based on multiple bioinformatic analyses, three 60S ribosomal subunit components (RPL15, RPL18, and RPL18A), and two 40S ribosomal subunit components (RPS15 and RPS21) were identified as the top 5 hub genes in the protein-protein interaction network of DEGs. The expression of RPL15 and RPL18 was also found significantly decreased in calcified aortic valve tissues (both p < .01), and negatively correlated with the osteogenic differentiation marker OPN in CAVD patients (both p < .01). Moreover, inhibition of RPL15 or RPL18 exacerbated the calcification of valve interstitial cells under osteogenic induction conditions. The present study proved that decreased expression of RPL15 and RPL18 was closely associated with aortic valve calcification, which provided valuable clues to find therapeutic targets for CAVD.


Assuntos
Estenose da Valva Aórtica , Valva Aórtica , Idoso , Humanos , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Células Cultivadas , Osteogênese/genética
4.
Cell Mol Life Sci ; 79(3): 146, 2022 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-35190902

RESUMO

Calcific aortic valve disease (CAVD) is a common valve disease characterized by the fibro-calcific remodeling of the aortic valves, which is an actively regulated process involving osteogenic differentiation of valvular interstitial cells (VICs). MicroRNA (miRNA) is an essential regulator in diverse biological processes in cells. The present study aimed to explore the role and mechanism of miR-22 in the osteogenic differentiation of VICs. The expression profile of osteogenesis-related miRNAs was first detected in aortic valve tissue from CAVD patients (n = 33) and healthy controls (n = 12). miR-22 was highly expressed in calcified valve tissues (P < 0.01), and the expression was positively correlated with the expression of OPN (rs = 0.820, P < 0.01) and Runx2 (rs = 0.563, P < 0.01) in VICs isolated from mild or moderately calcified valves. The sustained high expression of miR-22 was also validated in an in-vitro VICs osteogenic model. Adenovirus-mediated gain-of-function and loss-of-function experiments were then performed. Overexpression of miR-22 significantly accelerated the calcification process of VICs, manifested by significant increases in calcium deposition, alkaline phosphate activity, and expression of osteoblastic differentiation markers. Conversely, inhibition of miR-22 significantly negated the calcification process. Subsequently, calcium-binding protein 39 (CAB39) was identified as a target of miR-22. Overexpression of miR-22 significantly reduced the expression of CAB39 in VICs, leading to decreased catalytic activity of the CAB39-LKB1-STRAD complex, which, in turn, exacerbated changes in the AMPK-mTOR signaling pathway, and ultimately accelerated the calcification process. In addition, ROS generation and autophagic activity during VIC calcification were also regulated by miR-22/CAB39 pathway. These results indicate that miR-22 is an important accelerator of the osteogenic differentiation of VICs, and a potential therapeutic target in CAVD.


Assuntos
Estenose da Valva Aórtica/patologia , Valva Aórtica/patologia , Calcinose/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , MicroRNAs/genética , Osteogênese , Idoso , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/metabolismo , Calcinose/genética , Calcinose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
5.
Metab Eng ; 70: 129-142, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35085780

RESUMO

Ergothioneine (ERG) is an unusual sulfur-containing amino acid. It is a potent antioxidant, which shows great potential for ameliorating neurodegenerative and cardiovascular diseases. L-ergothioneine is rare in nature, with mushrooms being the primary dietary source. The chemical synthesis process is complex and expensive. Alternatively, ERG can be produced by fermentation of recombinant microorganisms engineered for ERG overproduction. Here, we describe the engineering of S. cerevisiae for high-level ergothioneine production on minimal medium with glucose as the only carbon source. To this end, metabolic engineering targets in different layers of the amino acid metabolism were selected based on literature and tested. Out of 28 targets, nine were found to improve ERG production significantly by 10%-51%. These targets were then sequentially implemented to generate an ergothioneine-overproducing yeast strain capable of producing 106.2 ± 2.6 mg/L ERG in small-scale cultivations. Transporter engineering identified that the native Aqr1 transporter was capable of increasing the ERG production in a yeast strain with two copies of the ERG biosynthesis pathway, but not in the strain that was further engineered for improved precursor supply. Medium optimization indicated that additional supplementation of pantothenate improved the strain's productivity further and that no supplementation of amino acid precursors was necessary. Finally, the engineered strain produced 2.39 ± 0.08 g/L ERG in 160 h (productivity of 14.95 ± 0.49 mg/L/h) in a controlled fed-batch fermentation without supplementation of amino acids. This study paves the way for the low-cost fermentation-based production of ergothioneine.


Assuntos
Ergotioneína , Meios de Cultura/metabolismo , Ergotioneína/genética , Fermentação , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
Eur J Clin Invest ; 52(9): e13804, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35506324

RESUMO

BACKGROUND: Vascular aging is an important risk factor for various cardiovascular diseases. Transcription factor krüppel-like factor 4 (KLF4) could regulate the phenotypic transformation of the vascular smooth muscle cell (VSMC) in the pathogenesis of aortic diseases. The present study aimed to explore the role and mechanism of KLF4 in angiotensin II (Ang II)-induced VSMC senescence. METHODS: The VSMC senescence mouse model was induced by sustained release of Ang II (1.0 µg/kg/min) for 4 weeks. The premature senescent VSMCs were induced by Ang II (0.1 µmol/L) for 72 h. Cellular senescence was measured by senescence-associated ß-galactosidase (SA-ß-gal) activity and p53/p16 expression. The autophagic activity was evaluated by autophagic flux and autophagic marker expression. RESULTS: The expression of KLF4 was extremely increased in abdominal aorta tissues after 1-week Ang II stimulation (p < .01) but began to decrease in later periods. Decreased expression of KLF4 was also detected in premature senescent VSMCs. Overexpression of KLF4 could enhance the antisenescence ability of VSMCs. Significantly decreased amounts of SA-ß-gal-positive cells and lower p53/p16 expression were detected in KLF4-overexpressing VSMCs (p < .01). Next, telomerase reverse transcriptase (TERT) was identified as a direct downstream target of KLF4 in VSMCs. Overexpression of KLF4 in VSMCs prevented the decreased expression of TERT under Ang II stimulation condition, which could in turn, contribute to the enhanced autophagic activity, and ultimately to the improved antisenescence ability of VSMCs. CONCLUSIONS: Our results demonstrated that overexpression of KLF4 prevented Ang II-induced VSMC senescence by promoting TERT-mediated autophagy. These findings provided novel potential targets for the prevention and therapy of vascular aging.


Assuntos
Angiotensina II , Autofagia , Fator 4 Semelhante a Kruppel , Músculo Liso Vascular , Angiotensina II/farmacologia , Animais , Células Cultivadas , Senescência Celular , Fator 4 Semelhante a Kruppel/metabolismo , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Supressora de Tumor p53
7.
Biotechnol Bioeng ; 119(2): 376-387, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34786710

RESUMO

Cis,cis-muconic acid (CCM) is a promising polymer building block. CCM can be made by whole-cell bioconversion of lignin hydrolysates or de novo biosynthesis from sugar feedstocks using engineered microorganisms. At present, however, there is no established process for large-scale CCM production. In this study, we developed an integrated process for manufacturing CCM from glucose by yeast fermentation. We systematically engineered the CCM-producing Saccharomyces cerevisiae strain by rewiring the shikimate pathway flux and enhancing phosphoenolpyruvate supply. The engineered strain ST10209 accumulated less biomass but produced 1.4 g/L CCM (70 mg CCM per g glucose) in microplate assay, 71% more than the previously engineered strain ST8943. The strain ST10209 produced 22.5 g/L CCM in a 2 L fermenter with a productivity of 0.19 g/L/h, compared to 0.14 g/L/h achieved by ST8943 in our previous report under the same fermentation conditions. The fermentation process was demonstrated at pilot scale in 10 and 50 L steel tanks. In 10 L fermenter, ST10209 produced 20.8 g/L CCM with a CCM yield of 0.1 g/g glucose and a productivity of 0.21 g/L/h, representing the highest to-date CCM yield and productivity. We developed a CCM recovery and purification process by treating the fermentation broth with activated carbon at low pH and low temperature, achieving an overall CCM recovery yield of 66.3% and 95.4% purity. In summary, we report an integrated CCM production process employing engineered S. cerevisiae yeast.


Assuntos
Engenharia Metabólica/métodos , Saccharomyces cerevisiae , Ácido Sórbico/análogos & derivados , Fermentação , Glucose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Sórbico/química , Ácido Sórbico/isolamento & purificação , Ácido Sórbico/metabolismo
8.
Cell Mol Biol Lett ; 27(1): 85, 2022 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-36209049

RESUMO

BACKGROUND: Diabetic cardiomyopathy (DCM) results from pathological changes in cardiac structure and function caused by diabetes. Excessive oxidative stress is an important feature of DCM pathogenesis. MicroRNAs (miRNAs) are key regulators of oxidative stress in the cardiovascular system. In the present study, we screened for the expression of oxidative stress-responsive miRNAs in the development of DCM. Furthermore, we aimed to explore the mechanism and therapeutic potential of miR-92a-2-5p in preventing diabetes-induced myocardial damage. METHODS: An experimental type 2 diabetic (T2DM) rat model was induced using a high-fat diet and low-dose streptozotocin (30 mg/kg). Oxidative stress injury in cardiomyocytes was induced by high glucose (33 mmol/L). Oxidative stress-responsive miRNAs were screened by quantitative real-time PCR. Intervention with miR-92a-2-5p was accomplished by tail vein injection of agomiR in vivo or adenovirus transfection in vitro. RESULTS: The expression of miR-92a-2-5p in the heart tissues was significantly decreased in the T2DM group. Decreased miR-92a-2-5p expression was also detected in high glucose-stimulated cardiomyocytes. Overexpression of miR-92a-2-5p attenuated cardiomyocyte oxidative stress injury, as demonstrated by increased glutathione level, and reduced reactive oxygen species accumulation, malondialdehyde and apoptosis levels. MAPK interacting serine/threonine kinase 2 (MKNK2) was verified as a novel target of miR-92a-2-5p. Overexpression of miR-92a-2-5p in cardiomyocytes significantly inhibited MKNK2 expression, leading to decreased phosphorylation of p38-MAPK signaling, which, in turn, ameliorated cardiomyocyte oxidative stress injury. Additionally, diabetes-induced myocardial damage was significantly alleviated by the injection of miR-92a-2-5p agomiR, which manifested as a significant improvement in myocardial remodeling and function. CONCLUSIONS: miR-92a-2-5p plays an important role in cardiac oxidative stress, and may serve as a therapeutic target in DCM.


Assuntos
Diabetes Mellitus Tipo 2 , Cardiomiopatias Diabéticas , MicroRNAs , Animais , Apoptose , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/patologia , Glucose/metabolismo , Glutationa/metabolismo , Malondialdeído/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Proteínas Serina-Treonina Quinases , Ratos , Espécies Reativas de Oxigênio/metabolismo , Serina/metabolismo , Estreptozocina/metabolismo
9.
Proc Natl Acad Sci U S A ; 116(19): 9324-9332, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31000602

RESUMO

The cellular machinery that supports protein synthesis and secretion lies at the foundation of cell factory-centered protein production. Due to the complexity of such cellular machinery, the challenge in generating a superior cell factory is to fully exploit the production potential by finding beneficial targets for optimized strains, which ideally could be used for improved secretion of other proteins. We focused on an approach in the yeast Saccharomyces cerevisiae that allows for attenuation of gene expression, using RNAi combined with high-throughput microfluidic single-cell screening for cells with improved protein secretion. Using direct experimental validation or enrichment analysis-assisted characterization of systematically introduced RNAi perturbations, we could identify targets that improve protein secretion. We found that genes with functions in cellular metabolism (YDC1, AAD4, ADE8, and SDH1), protein modification and degradation (VPS73, KTR2, CNL1, and SSA1), and cell cycle (CDC39), can all impact recombinant protein production when expressed at differentially down-regulated levels. By establishing a workflow that incorporates Cas9-mediated recombineering, we demonstrated how we could tune the expression of the identified gene targets for further improved protein production for specific proteins. Our findings offer a high throughput and semirational platform design, which will improve not only the production of a desired protein but even more importantly, shed additional light on connections between protein production and other cellular processes.


Assuntos
Interferência de RNA , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Genoma Fúngico , Microfluídica , Proteínas Recombinantes/genética , Recombinação Genética , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
Pharm Biol ; 60(1): 17-24, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34846265

RESUMO

CONTEXT: Vascular calcification is a major complication of chronic renal failure, which has been identified as an active process partly driven by osteogenic transition of vascular smooth muscle cells (VSMCs). Aspirin could prevent cardiomyocyte damage by inducing heat shock response. OBJECTIVE: This study investigates the effect of aspirin on alleviating VSMC calcification. MATERIALS AND METHODS: An in vitro VSMC calcification model was established by 10-day calcification induction in osteogenic medium. VSMCs were grouped as following: control group (normal medium), calcified group (osteogenic medium) and treated group (osteogenic medium with 1 or 4 mmol/L aspirin). VSMC calcification was evaluated by calcified nodules formation, intracellular calcium concentration and osteoblastic marker (OPN and Runx2) expression. RESULTS: After 10-day culture, the intracellular calcium concentration in calcified group was significantly higher than that in control group (1.16 ± 0.04 vs. 0.14 ± 0.01 µg/mg, p < 0.01), but significantly reduced in 1 mmol/L aspirin treated group (0.74 ± 0.05 µg/mg, p < 0.01), and 4 mmol/L aspirin treated group (0.93 ± 0.03 µg/mg, p < 0.01). The elevated expression of OPN and Runx2 induced by osteogenic medium was significantly relieved after 1 or 4 mmol/L aspirin treatment. The expression of HSF1, HSP70 and HSP90 was decreased in calcification-induced VSMCs, but significantly increased after treatment of aspirin. Furthermore, inhibition of HSP70 (or HSP90) by small-molecule inhibitor or small interfering RNA could partially abolish the anti-calcification effect of aspirin, proved by the changes of intracellular calcium concentration and osteoblastic marker expression. DISCUSSION AND CONCLUSIONS: Aspirin could relieve the calcification of VSMCs partially through HSP70- or HSP90-mediated heat shock response. These findings expanded the understanding of aspirin pharmacology, and imply that local induction expression of HSPs might be a potential therapeutic strategy for the prevention and therapy of vascular calcification.


Assuntos
Aspirina/farmacologia , Resposta ao Choque Térmico/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Calcificação Vascular/tratamento farmacológico , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Inibidores da Agregação Plaquetária/farmacologia , Ratos , Ratos Sprague-Dawley
11.
J Cell Physiol ; 236(7): 5421-5431, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33372301

RESUMO

The excessive proliferation and migration of vascular smooth muscle cells (VSMCs) play vital roles in neointimal hyperplasia and vascular restenosis. In the present study, we aimed to investigate the function and mechanism of octamer-binding transcription factor 4 (OCT4, a key transcription factor for maintaining stem cells in de-differentiated state) on neointima formation in response to vascular injury. Quantitative reverse-transcription polymerase chain reaction and western blot results displayed a significant increase of OCT4 levels in injured carotid arteries. Immunohistochemistry and immunofluorescence assays confirmed that the increased OCT4 expression was primarily localized in α-SMA-positive VSMCs from neointima, and colocalized with PCNA in the nuclei of VSMCs. Adenovirus-mediated OCT4 overexpression in injured carotid arteries exacerbated intimal thickening, while OCT4 knockdown significantly inhibited intimal thickening. In-vitro experiments confirmed that the increased OCT4 expression in VMSCs could be induced by platelet-derived growth factor-BB (PDGF-BB) in a time-dependent manner. Overexpression of OCT4 greatly promoted VSMCs proliferation and migration, while OCT4 knockdown significantly retarded the PDGF-BB-induced excessive proliferation and migration of VSMCs. Bioinformatics analysis, dual-luciferase reporter assay, and chromatin immunoprecipitation assay confirmed that OCT4 could upregulate matrix metalloproteinases 2 (MMP2) expression through promoting its transcription. Moreover, knockdown of MMP2 significantly attenuated OCT4-mediated VSMCs proliferation and migration. These results indicated that OCT4 facilitated neointimal formation in response to vascular injury by MMP2-mediated VSMCs proliferation and migration, and targeting OCT4 in VSMCs might be a novel therapeutic strategy for vascular restenosis.


Assuntos
Lesões das Artérias Carótidas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Músculo Liso Vascular/metabolismo , Neointima/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo
12.
Metab Eng ; 64: 52-63, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33465478

RESUMO

Synthetic biology enables the production of small molecules by recombinant microbes for pharma, food, and materials applications. The secretion of products reduces the cost of separation and purification, but it is challenging to engineer due to the limited understanding of the transporter proteins' functions. Here we describe a method for genome-wide transporter disruption that, in combination with a metabolite biosensor, enables the identification of transporters impacting the production of a given target metabolite in yeast Saccharomyces cerevisiae. We applied the method to study the transport of xenobiotic compounds, cis,cis-muconic acid (CCM), protocatechuic acid (PCA), and betaxanthins. We found 22 transporters that influenced the production of CCM or PCA. The transporter of the 12-spanner drug:H(+) antiporter (DHA1) family Tpo2p was further confirmed to import CCM and PCA in Xenopus expression assays. We also identified three transporter proteins (Qdr1p, Qdr2p, and Apl1p) involved in betaxanthins transport. In summary, the described method enables high-throughput transporter identification for small molecules in cell factories.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Antiporters , Engenharia Metabólica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ácido Sórbico , Biologia Sintética
13.
Cardiovasc Drugs Ther ; 35(1): 103-111, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33044585

RESUMO

PURPOSE: Vein graft failure (VGF) is an important limitation for coronary artery bypass graft (CABG) surgery. Inhibition of the excessive proliferation and migration of venous smooth muscle cells (SMCs) is an effective strategy to alleviate VGF during the CABG perioperative period. In the present study, we aimed to explore the role and potential mechanism of all-trans retinoic acid (ATRA) on preventing vein grafts stenosis. METHODS: The autogenous vein grafts model was established in the right jugular artery of rabbits. Immunohistochemistry staining and western blot assays were used to detected the protein expression, while real-time PCR assay was applied for mRNAs expression detection. The interaction between proteins was identified by co-immunoprecipitation assay. The Cell Counting Kit-8 and wound-healing assays were used to investigate the role of ATRA on human umbilical vein smooth muscle cells (HUVSMCs) function. Cell cycle progression was identified by flow cytometry assay. RESULTS: Vein graft stenosis and SMCs hyperproliferation were confirmed in vein grafts by histological and Ki-67 immunohistochemistry assays. Treatment of ATRA (10 mg/kg/day) significantly mitigated the stenosis extent of vein grafts, demonstrated by the decreased thickness of intima-media, and decreased Ki-67 expression. ATRA could repress the PDGF-bb-induced excessive proliferation and migration of HUVSMCs, which was mediated by Rb-E2F dependent cell cycle inhibition. Meanwhile, ATRA could reduce the interaction between KLF5 and RARα, thereby inhibiting the function of cis-elements of KLF5. KLF5-induced inducible nitric oxide synthase (iNOS) expression activation could be significantly inhibited by ATRA. CONCLUSIONS: These results suggested that ATRA treatment may represent an effective prevention and therapy avenue for VGF.


Assuntos
Constrição Patológica/tratamento farmacológico , Fatores de Transcrição Kruppel-Like/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Técnicas de Cultura de Células , Ponte de Artéria Coronária/efeitos adversos , Humanos , Antígeno Ki-67/imunologia , Masculino , Coelhos
14.
Proc Natl Acad Sci U S A ; 115(47): E11025-E11032, 2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30397111

RESUMO

Baker's yeast Saccharomyces cerevisiae is one of the most important and widely used cell factories for recombinant protein production. Many strategies have been applied to engineer this yeast for improving its protein production capacity, but productivity is still relatively low, and with increasing market demand, it is important to identify new gene targets, especially targets that have synergistic effects with previously identified targets. Despite improved protein production, previous studies rarely focused on processes associated with intracellular protein retention. Here we identified genetic modifications involved in the secretory and trafficking pathways, the histone deacetylase complex, and carbohydrate metabolic processes as targets for improving protein secretion in yeast. Especially modifications on the endosome-to-Golgi trafficking was found to effectively reduce protein retention besides increasing protein secretion. Through combinatorial genetic manipulations of several of the newly identified gene targets, we enhanced the protein production capacity of yeast by more than fivefold, and the best engineered strains could produce 2.5 g/L of a fungal α-amylase with less than 10% of the recombinant protein retained within the cells, using fed-batch cultivation.


Assuntos
Engenharia Metabólica/métodos , Biossíntese de Proteínas/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Via Secretória/fisiologia , alfa-Amilases/biossíntese , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Histona Desacetilases/genética , Transporte Proteico/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , Via Secretória/genética
15.
Toxicol Ind Health ; 37(7): 391-397, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34047658

RESUMO

Di-2-ethylhexyl phthalate (DEHP) is a type of plasticizer widely used in industry. It is well-known for its toxic effects to endocrine and reproductive systems and has been detected in amniotic fluid and placenta. In the present study, we explored the effects of DEHP on heart development by using zebrafish as a model organism. DEHP (0.02 pg) was injected into the yolk sac of zebrafish embryos at the one-cell stage. No significant difference was found in embryonic lethality between control and DEHP groups at 1-day postfertilization (dpf), but mortality significantly increased in DEHP groups at 2 and 3 dpf. The average heart rate was significantly reduced in the surviving DEHP-treated zebrafish larvae at 3 and 4 dpf. In addition, massive pericardial edema was found in DEHP-treated zebrafish (12.6 ± 1.5%), which was significantly higher than that of the control group. Serious heart looping disorder was also observed in DEHP-treated larvae, mainly manifested with an elongated atrial-ventricular distance. Moreover, the expression of heart development transcription factors was affected by DEHP injection. Real-time polymerase chain reaction confirmed that five transcription factors (hand2, tp53, mef2c, esr1, and tbx18) were significantly downregulated in the DEHP group at 2 dpf, and three transcription factors (zic3, tcf21, and gata4) were significantly upregulated. Our results emphasize the need for the development of a nontoxic plasticizer to prevent possible deleterious effects on humans and other life-forms.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/efeitos dos fármacos , Dietilexilftalato/toxicidade , Embrião não Mamífero/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/crescimento & desenvolvimento , Glicoproteínas de Membrana/efeitos dos fármacos , Proteínas de Peixe-Zebra/efeitos dos fármacos , Peixe-Zebra/crescimento & desenvolvimento , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Humanos , Exposição Ocupacional/efeitos adversos , Organogênese/efeitos dos fármacos , Plastificantes/toxicidade , Poluentes Químicos da Água/toxicidade
16.
Metab Eng ; 62: 51-61, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32818629

RESUMO

Resveratrol is a plant secondary metabolite with multiple health-beneficial properties. Microbial production of resveratrol in model microorganisms requires extensive engineering to reach commercially viable levels. Here, we explored the potential of the non-conventional yeast Yarrowia lipolytica to produce resveratrol and several other shikimate pathway-derived metabolites (p-coumaric acid, cis,cis-muconic acid, and salicylic acid). The Y. lipolytica strain expressing a heterologous pathway produced 52.1 ± 1.2 mg/L resveratrol in a small-scale cultivation. The titer increased to 409.0 ± 1.2 mg/L when the strain was further engineered with feedback-insensitive alleles of the key genes in the shikimate pathway and with five additional copies of the heterologous biosynthetic genes. In controlled fed-batch bioreactor, the strain produced 12.4 ± 0.3 g/L resveratrol, the highest reported titer to date for de novo resveratrol production, with a yield on glucose of 54.4 ± 1.6 mg/g and a productivity of 0.14 ± 0.01 g/L/h. The study showed that Y. lipolytica is an attractive host organism for the production of resveratrol and possibly other shikimate-pathway derived metabolites.


Assuntos
Yarrowia , Reatores Biológicos , Engenharia Metabólica , Resveratrol , Ácido Chiquímico , Yarrowia/genética
17.
Metab Eng ; 61: 427-436, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-31404648

RESUMO

Lactone flavors with fruity, milky, coconut, and other aromas are widely used in the food and fragrance industries. Lactones are produced by chemical synthesis or by biotransformation of plant-sourced hydroxy fatty acids. We established a novel method to produce flavor lactones from abundant non-hydroxylated fatty acids using yeast cell factories. Oleaginous yeast Yarrowia lipolytica was engineered to perform hydroxylation of fatty acids and chain-shortening via ß-oxidation to preferentially twelve or ten carbons. The strains could produce γ-dodecalactone from oleic acid and δ-decalactone from linoleic acid. Through metabolic engineering, the titer was improved 4-fold, and the final strain produced 282 mg/L γ-dodecalactone in a fed-batch bioreactor. The study paves the way for the production of lactones by fermentation of abundant fatty feedstocks.


Assuntos
4-Butirolactona/análogos & derivados , Técnicas de Cultura Celular por Lotes , Ácido Linoleico/metabolismo , Ácido Oleico/metabolismo , Yarrowia , 4-Butirolactona/biossíntese , 4-Butirolactona/genética , Yarrowia/genética , Yarrowia/metabolismo
18.
BMC Cardiovasc Disord ; 20(1): 435, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028234

RESUMO

BACKGROUND: To evaluate the coronary plaque characteristics of coronary arteries using computed tomography angiography (CTA) in order to assess the risk of coronary artery disease and the relevance of high sensitivity C reactive protein (hs-CRP) in patients with Diabetes Mellitus (DM). METHODS: The clinical data of 400 DM patients and 400 non-DM patients from January 2017 to December 2019 were collected, including the results of coronaryCTA. The plasma hs-CRP level of the two groups were divided into three groups: CRP ≤ 1, 1 < CRP ≤ 2, CRP > 2. The correlation of the degree of stenosis, the number of plaques, the nature of plaques and hs-CRP value between the two groups was evaluated. RESULTS: Compared with non-DM patients, the incidence of coronary artery plaques and lumen stenosis in DM patients was more higher than that in non-DM patients. DM patients were more likely to have more diseased vessels, especially diffuse vascular disease (12.00% vs 1.75%; P < 0.001). Subjects with high hs-CRP levels were more likely to have any plaque compared with individuals showing normal hs-CRP levels (p<0.01). There was no statistical significance in non calcified plaque with high level of hs-CRP, but the occurrence of plaque types in DM group was statistically significant compared with other hs-CRP levels in non DM group. Subjects with high hs-CRP were observed to be at increased risk for the presence of calcified plaque and severe narrowing in the unadjusted values. CONCLUSIONS: Coronary CTA combined with hs-CRP can accurately detect the characteristics of coronary artery stenosis and plaque in DM patients, which has an important clinical value in the risk assessment of coronary heart disease in DM patients.


Assuntos
Proteína C-Reativa/análise , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Doença da Artéria Coronariana/diagnóstico por imagem , Estenose Coronária/diagnóstico por imagem , Diabetes Mellitus/sangue , Tomografia Computadorizada Multidetectores , Placa Aterosclerótica , Adulto , Idoso , Biomarcadores/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/epidemiologia , Estenose Coronária/sangue , Estenose Coronária/epidemiologia , Estudos Transversais , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Feminino , Fatores de Risco de Doenças Cardíacas , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Regulação para Cima
19.
Heart Lung Circ ; 29(11): 1725-1732, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32224088

RESUMO

BACKGROUND: In recent years, obese patients presenting with acute thoracic aortic dissection have not been uncommon and there are often pulmonary complications among them. Whether a higher body mass index (BMI) is associated with more pulmonary complications or even a higher mortality rate has yet to be determined. This study aimed to evaluate the effects of higher BMI on pulmonary complications and other surgical outcomes. METHODS: A total of 404 patients who underwent acute thoracic aortic dissection surgery were retrospectively studied. They were divided into three groups based on their BMI: normal weight (BMI 18.5 to <25 kg/m2, n=173), overweight (BMI 25 to <30 kg/m2, n=145) and obese (BMI ≥30 kg/m2, n=86). Clinical data were collected and analysed among groups. RESULTS: No statistical significance was detected among the groups for postoperative complications, in-hospital mortality and hospital or ICU stay, except for prolonged intubation, the proportion of which was highest in the obese group followed by the overweight and normal groups (40.7% vs 29% vs 11%, respectively; p<0.001). Furthermore, logistic regression analysis showed that postoperative renal failure (OR=16.984) and cardiopulmonary bypass time (OR=1.013) were independent risk factors for in-hospital mortality, while higher BMI (OR=7.148 for BMI ≥25 and 18.967 for BMI ≥30), transfused red blood cells (OR=1.004), and postoperative renal failure (OR=7.386) were independent risk factors for prolonged ventilation (p<0.05). CONCLUSION: Body mass index had no effect on in-hospital mortality but may be closely correlated with prolonged intubation for patients undergoing aortic dissection surgery. This finding suggests that these patients should receive more aggressive pulmonary management.


Assuntos
Aneurisma da Aorta Torácica/cirurgia , Dissecção Aórtica/cirurgia , Índice de Massa Corporal , Intubação Intratraqueal/métodos , Procedimentos Cirúrgicos Vasculares/métodos , Doença Aguda , Dissecção Aórtica/mortalidade , Dissecção Aórtica/fisiopatologia , Aneurisma da Aorta Torácica/mortalidade , Aneurisma da Aorta Torácica/fisiopatologia , China/epidemiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Taxa de Sobrevida/tendências , Fatores de Tempo
20.
J Cell Biochem ; 120(10): 18278-18287, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31144399

RESUMO

Hyperglycemia in diabetic patients would cause cardiomyocytes oxidative stress and apoptosis due to the excessive reactive oxygen species (ROS) accumulation, leading to progressive deterioration of cardiac structure and function. Long noncoding RNAs (lncRNAs) play essential roles on controlling oxidative stress and apoptotic activity. In the present study, RNA sequencing was used to detect the differentially expressed lncRNAs during high glucose-induced cardiomyocytes oxidative stress and apoptosis. A total of 306/400 lncRNAs were identified as differentially expressed, including 156/198 lncRNAs with increased expression and 150/202 lncRNAs with decreased expression at 24 hours/48 hours after high-glucose stimulation respectively. Among these dysregulated lncRNAs, 45 lncRNAs were consistently differentially expressed in cardiomyocytes at both two time points after high-glucose stimulation. Twenty lncRNAs were upregulated and 25 lncRNAs were downregulated at both 24 hours and 48 hours, respectively. The top three upregulated lncRNAs, NONRATT029805.2, NONRATT007560.2, and NONRATT002486.2 were selected for functional studies to determine the role in oxidative stress-related apoptosis. The results showed that inhibition of non-ratt007560.2 could abate the formation of ROS and reduce apoptosis, suggesting NONRATT007560.2 might play critical roles in the development of cardiomyopathy. The dysregulated lncRNAs might participate in regulating cardiomyocytes oxidative stress and apoptosis. These findings would be important theoretical and experimental basis for investigation on diabetic cardiomyopathy pathogenesis.


Assuntos
Apoptose/efeitos dos fármacos , Perfilação da Expressão Gênica , Glucose/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo , RNA Longo não Codificante/genética , Animais , Animais Recém-Nascidos , Apoptose/genética , Células Cultivadas , Cardiomiopatias Diabéticas/diagnóstico , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Humanos , Miócitos Cardíacos/metabolismo , Interferência de RNA , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Regulação para Cima/efeitos dos fármacos
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