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PURPOSE: This study aimed to investigate the relationship between spinal-pelvic parameters and recurrence of lumbar disc herniation (rLDH) after percutaneous endoscopic lumbar discectomy (PELD) through a retrospective case-control study. METHODS: Patients who underwent PELD for single-segment LDH at our hospital were included in this study. The relationship between sagittal balance parameters of the spine and recurrence was analysed through correlation analysis, and ROC curves were plotted. The baseline characteristics, sagittal balance parameters of the spine and radiological parameters of the case and control groups were compared, and the relationship between sagittal balance parameters of the spine and recurrence of rLDH after PELD was determined through univariate and multivariate logistic regression analysis. RESULTS: Correlation analysis showed that PI and ∆PI-LL were negatively correlated with grouping (r = -0.090 and -0.120, respectively, P = 0.001 and 0.038). ROC curve analysis showed that the area under the curve (ROC-AUC) for predicting rLDH based on PI was 0.65 (CI95% = 0.598, 0.720), with a cut-off of 50.26°. The ROC-AUC for predicting rLDH based on ∆PI-LL was 0.56 (CI95% = 0.503, 0.634), with a cut-off of 28.21°. Multivariate logistic regression analysis showed that smoking status (OR = 2.667, P = 0.008), PI ≤ 50.26 (OR = 2.161, P = 0.009), ∆PI-LL ≤ 28.21 (OR = 3.185, P = 0.001) and presence of Modic changes (OR = 4.218, P = 0.001) were independent risk factors, while high DH (OR = 0.788, P = 0.001) was a protective factor. CONCLUSION: PI < 50.26 and ∆PI-LL < 28.21 were risk factors for recurrence of lumbar disc herniation after spinal endoscopic surgery and had some predictive value for post-operative recurrence.
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Discectomía Percutánea , Desplazamiento del Disco Intervertebral , Humanos , Estudios de Casos y Controles , Estudios Retrospectivos , Desplazamiento del Disco Intervertebral/cirugía , Vértebras Lumbares/diagnóstico por imagen , Vértebras Lumbares/cirugíaRESUMEN
Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.
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Ésteres del Colesterol , Subunidades gamma de la Proteína de Unión al GTP , Gotas Lipídicas , Animales , Femenino , Masculino , Ratones , Ésteres del Colesterol/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Gotas Lipídicas/metabolismo , Proteínas/metabolismo , Triglicéridos/metabolismoRESUMEN
The scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein (HDL) receptor, is a membrane glycoprotein that mediates selective uptake of HDL-cholesterol and cholesterol ester (CE) into cells. SR-B1 is subject to posttranslational regulation; however, the underlying mechanisms still remain obscure. Here, we identified a novel SR-B1-interacting protein, GIPC1 (GAIP-interacting protein, C terminus 1) that interacts with SR-B1 and stabilizes SR-B1 by negative regulation of its proteasomal and lysosomal degradation pathways. The physiological interaction between SR-B1 and GIPC1 was supported by co-immunoprecipitation of wild-type and mutant GIPC1 constructs in SR-B1 ± GIPC1 overexpressing cells, in native liver cells, and in mouse liver tissues. Overexpression of GIPC1 increased endogenous SR-B1 protein levels, subsequently increasing selective HDL-cholesterol/CE uptake and cellular triglyceride (TG) and total cholesterol (TC) levels, whereas silencing of GIPC1 in the mouse liver was associated with blunted hepatic SR-B1 levels, elevated plasma TG and TC, and attenuated hepatic TG and TC content. A positive correlation was identified between GIPC1 and SR-B1 expression, and both expressions of GIPC1 and SR-B1 from human liver samples were inversely correlated with body mass index (BMI) from human subjects. We therefore conclude that GIPC1 plays a key role in the stability and function of SR-B1 and can also effectively regulate hepatic lipid and cholesterol metabolism. These findings expand our knowledge of the regulatory roles of GIPC1 and suggest that GIPC1 exerts a major effect on cell surface receptors such as SR-B1 and its associated hepatic lipid and cholesterol metabolic processes.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD36/química , Colesterol/metabolismo , Hígado/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Transporte Biológico , Antígenos CD36/genética , Antígenos CD36/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Estabilidad ProteicaRESUMEN
Apart from its role in inflammation and immunity, chemerin is also involved in white adipocyte biology. To study the role of chemerin in adipocyte metabolism, we examined the function of chemerin in brown adipose tissue. Brown and white adipocyte precursors were differentiated into adipocytes in the presence of Chemerin siRNA. Chemerin-deficient (Chem-/- ) mice were compared to wild-type mice when fed a high-fat diet. Chemerin is expressed during brown adipocyte differentiation and knock down of chemerin mRNA results in decreased brown adipocyte differentiation with reduced fatty acid uptake in brown adipocytes. Chem-/- mice are leaner than wild-type mice but gain more weight when challenged with high-fat diet feeding, resulting in a larger increase in fat deposition. Chem-/- mice develop insulin resistance when on a high-fat diet or due to age. Brown adipose depots in Chem-/- mice weigh more than in wild-type mice, but with decreased mitochondrial content and function. Compared to wild-type mice, male Chem-/- mice have decreased oxygen consumption, CO2 production, energy expenditure, and a lower respiratory exchange ratio. Additionally, body temperature of Chem-/- mice is lower than that of wild-type mice. These results revealed that chemerin is expressed during brown adipocyte differentiation and has a pivotal role in energy metabolism through brown adipose tissue thermogenesis.
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Tejido Adiposo Pardo/patología , Envejecimiento/patología , Quimiocinas/fisiología , Dieta Alta en Grasa , Metabolismo Energético , Hiperinsulinismo/patología , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intercelular/fisiología , Tejido Adiposo Pardo/metabolismo , Animales , Femenino , Hiperinsulinismo/etiología , Hiperinsulinismo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Consumo de Oxígeno , TermogénesisRESUMEN
The scavenger receptor, class B type 1 (SR-B1), is a multiligand membrane receptor protein that functions as a physiologically relevant high-density lipoprotein (HDL) receptor whose primary role is to mediate selective uptake or influx of HDL-derived cholesteryl esters into cells and tissues. SR-B1 also facilitates the efflux of cholesterol from peripheral tissues, including macrophages, back to liver. As a regulator of plasma membrane cholesterol content, SR-B1 promotes the uptake of lipid soluble vitamins as well as viral entry into host cells. These collective functions of SR-B1 ultimately affect programmed cell death, female fertility, platelet function, vasculature inflammation, and diet-induced atherosclerosis and myocardial infarction. SR-B1 has also been identified as a potential marker for cancer diagnosis and prognosis. Finally, the SR-B1-linked selective HDL-cholesteryl ester uptake pathway is now being evaluated as a gateway for the delivery of therapeutic and diagnostic agents. In this review, we focus on the regulation and functional significance of SR-B1 in mediating cholesterol movement into and out of cells.
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Antígenos CD36/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Receptores de Lipoproteína/metabolismo , Animales , Transporte Biológico , HumanosRESUMEN
ABSTRACT: We explored the protective effect of spironolactone on cardiac function in the patients undergoing coronary artery bypass grafting (CABG) by determining serum hypoxia-inducible factor-1α (HIF-1α) before and after CABG. We used the propensity score matching method retrospectively to select 174 patients undergoing CABG in our hospital from March 2018 to December 2019. Of the 174 patients, 87 patients taking spironolactone for more than 3 months before CABG were used as a test group and other 87 patients who were not taking spironolactone as a control group. In all patients, serum HIF-1α and troponin I levels were determined before as well as 24 hours and 7 days after CABG, serum N-terminal probrain natriuretic peptide (NT-proBNP) level was determined before as well as 12, 24, and 36 hours after CABG, and electrocardiographic monitoring was performed within 36 hours after CABG. The results indicated that there were no significant differences in the HIF-1α level between the test group and the control group before and 7 days after CABG, but the HIF-1α level was significantly lower in the test group than that in the control group 24 hours after CABG (P < 0.01). The 2 groups were not significantly different in the troponin I level at any time point. There was no significant difference in the serum NT-proBNP level between the test group and the control group before CABG, but NT-proBNP (BNP) levels were all significantly lower in the test group than those in the control group at postoperative 12, 24, and 36 hour time points (all P <0.05). The incidence of postoperative atrial fibrillation was also significantly lower in the test group than that in the control group (P = 0.035). Spironolactone protects cardiac function probably by improving myocardial hypoxia and inhibiting myocardial remodeling.
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Puente de Arteria Coronaria , Estenosis Coronaria/cirugía , Subunidad alfa del Factor 1 Inducible por Hipoxia/sangre , Antagonistas de Receptores de Mineralocorticoides/uso terapéutico , Espironolactona/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Fibrilación Atrial/etiología , Fibrilación Atrial/prevención & control , Biomarcadores/sangre , Puente de Arteria Coronaria/efectos adversos , Estenosis Coronaria/sangre , Estenosis Coronaria/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antagonistas de Receptores de Mineralocorticoides/efectos adversos , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Estudios Retrospectivos , Factores de Riesgo , Espironolactona/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Troponina I/sangreRESUMEN
OBJECTIVE: During the coronavirus disease 2019 (COVID-19) pandemic, exploring insulin resistance and beta-cell activity is important for understanding COVID-19âassociated new-onset diabetes. We assessed insulin sensitivity and fasting insulin secretion in patients with COVID-19 without diabetes on admission and at 3 and 6 months after discharge. METHODS: This 6-month prospective study assessed data from the records of 64 patients without diabetes diagnosed with COVID-19 at Wenzhou Central Hospital, China. Each patient was followed up at 3 and 6 months after discharge. Repeated measures analysis of variance was used to investigate differences in multiple measurements of the same variable at different times. Linear regression analysis was performed to analyze the contributor for changes in the triglyceride-glucose (TyG) index. RESULTS: Fasting C-peptide levels in patients at baseline were lower than the normal range. Compared with the baseline results, patients had significantly elevated fasting C-peptide levels (0.35 ± 0.24 vs 2.36 ± 0.98 vs 2.52 ± 1.11 µg/L; P < .001), homeostasis model assessment for beta-cell function (0.42, interquartile range [IQR] 0.36-0.62 vs 2.54, IQR 1.95-3.42 vs 2.90, IQR 2.02-4.23; P < .001), and TyG indices (8.57 ± 0.47 vs 8.73 ± 0.60 vs 8.82 ± 0.62; P = .006) and decreased fasting glucose levels (5.84 ± 1.21 vs 4.95 ± 0.76 vs 5.40 ± 0.68 mmol/L; P = .003) at the 3- and 6-month follow-up. Male gender, age, interferon-alfa treatment during hospitalization, and changes in total cholesterol and high-density lipoprotein levels were significantly associated with changes in the TyG index. CONCLUSION: Our study provided the first evidence that COVID-19 may increase the risk of insulin resistance in patients without diabetes.
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COVID-19 , Diabetes Mellitus , Resistencia a la Insulina , Adulto , Glucemia , Humanos , Insulina , Masculino , Estudios Prospectivos , SARS-CoV-2 , TriglicéridosRESUMEN
Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.
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Sistemas de Transporte de Aminoácidos/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Adenosina Trifosfato/metabolismo , Adulto , Sistemas de Transporte de Aminoácidos/deficiencia , Sistemas de Transporte de Aminoácidos/genética , Animales , Transporte Biológico , Línea Celular , AMP Cíclico/metabolismo , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Proteínas de Transporte de Membrana/genética , Ratones , ARN Mensajero/genética , Adulto JovenRESUMEN
Cholesterol is an important component of plasma membranes (PMs) and the precursor of all steroid hormones. In steroidogenic tissues, upon hormone stimulation, there is a rapid transfer of cholesterol to the mitochondria, which is the site of the initial step in steroidogenesis. In the current study, we examined PM cholesterol trafficking for steroidogenesis. In a mitochondrial reconstitution assay, adrenal PMs supported steroidogenesis in the absence of additional transport proteins. Depletion of cholesterol in PMs by 50% eliminated the membranes' ability to support steroidogenesis in vitro and reduced steroid production in intact Y1 adrenocortical cells. Syntaxin (STX)-5 and α-soluble N-ethylmaleimide-sensitive factor attachment protein (α-SNAP) are enriched in adrenal PMs, and adrenocorticotropic hormone treatment of rats recruited STX5 and α-SNAP to adrenal PMs and mitochondria. Immunodepletion of STX5 and α-SNAP from PMs decreased steroidogenesis supported by PMs in vitro. Protease digestion of PMs decreased, whereas recombinant STX5 or α-SNAP restored, the PMs' ability to support steroidogenesis. Knockdown of either STX5 or α-SNAP in Y1 cells decreased stimulated steroidogenesis. These results indicate that STX5 and α-SNAP facilitate cholesterol trafficking from PMs to mitochondria for adrenal steroid synthesis and underscore the importance of vesicular trafficking of PM cholesterol for steroidogenesis.-Deng, B., Shen, W.-J., Dong, D., Azhar, S., Kraemer, F. B. Plasma membrane cholesterol trafficking in steroidogenesis.
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Lípidos de la Membrana/metabolismo , Esteroides/biosíntesis , Animales , Transporte Biológico , Células Cultivadas , Gotas Lipídicas/metabolismo , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Proteínas SNARE/metabolismoRESUMEN
Cholesterol is required for maintenance of plasma membrane fluidity and integrity and for many cellular functions. Cellular cholesterol can be obtained from lipoproteins in a selective pathway of HDL-cholesteryl ester (CE) uptake without parallel apolipoprotein uptake. Scavenger receptor B type 1 (SR-B1) is a cell surface HDL receptor that mediates HDL-CE uptake. It is most abundantly expressed in liver, where it provides cholesterol for bile acid synthesis, and in steroidogenic tissues, where it delivers cholesterol needed for storage or steroidogenesis in rodents. SR-B1 transcription is regulated by trophic hormones in the adrenal gland, ovary, and testis; in the liver and elsewhere, SR-B1 is subject to posttranscriptional and posttranslational regulation. SR-B1 operates in several metabolic processes and contributes to pathogenesis of atherosclerosis, inflammation, hepatitis C virus infection, and other conditions. Here, we summarize characteristics of the selective uptake pathway and involvement of microvillar channels as facilitators of selective HDL-CE uptake. We also present the potential mechanisms of SR-B1-mediated selective cholesterol transport; the transcriptional, posttranscriptional, and posttranslational regulation of SR-B1; and the impact of gene variants on expression and function of human SR-B1. A better understanding of this unique pathway and SR-B1's role may yield improved therapies for a wide variety of conditions.
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Antígenos CD36/metabolismo , Colesterol/metabolismo , Regulación de la Expresión Génica , Secuencia de Aminoácidos , Animales , Antígenos CD36/química , Antígenos CD36/genética , Humanos , Polimorfismo Genético , Transporte de ProteínasRESUMEN
To determine the effects of nordihydroguaiaretic acid (NDGA) on metabolic and molecular changes in response to feeding a typical American fast food or Western diet, mice were fed an American lifestyle-induced obesity syndrome (ALIOS) diet and subjected to metabolic analysis. Male C57BL/6J mice were randomly assigned to the ALIOS diet, the ALIOS diet supplemented with NDGA (NDGA+ALIOS), or a control diet and were maintained on the specific diet for 8 weeks. Mice fed the ALIOS diet showed increased body, liver, and epididymal fat pad weight as well as increased plasma alanine transaminase (ALT) and aspartate aminotransferase (AST) levels (a measure of liver injury) and liver triglyceride content. Coadministration of NDGA normalized body and epididymal fat pad weight, ALT and AST levels, and liver triglycerides. NDGA treatment also improved insulin sensitivity but not glucose intolerance in mice fed the ALIOS diet. In mice fed the NDGA+ALIOS diet, NDGA supplementation induced peroxisome proliferator-activated receptor α (PPARα; the master regulator of fatty acid oxidation) and mRNA levels of carnitine palmitoyltransferases Cpt1c and Cpt2, key genes involved in fatty acid oxidation, compared with the ALIOS diet. NDGA significantly reduced liver endoplasmic reticulum (ER) stress response C/EBP homologous protein, compared with chow or the ALIOS diet, and also ameliorated ALIOS diet-induced elevation of apoptosis signaling protein, caspase 3. Likewise, NDGA downregulated the ALIOS diet-induced mRNA levels of Pparg, fatty acid synthase Fasn, and diacylglycerol acyltransferase Dgat2 NDGA treatment of ALIOS-fed mice upregulated the hepatic expression of antioxidant enzymes, glutathione peroxidase 4, and peroxiredoxin 3 proteins. In conclusion, we provide evidence that NDGA improves metabolic dysregulation by simultaneously modulating the PPARα transcription factor and key genes involved in fatty acid oxidation, key antioxidant and lipogenic enzymes, and apoptosis and ER stress signaling pathways.
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Dieta Occidental/efectos adversos , Larrea/química , Estilo de Vida , Masoprocol/farmacología , Obesidad/metabolismo , Obesidad/prevención & control , Adipogénesis/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ácidos Grasos/metabolismo , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/inducido químicamente , Obesidad/patología , Oxidación-Reducción/efectos de los fármacos , PPAR alfa/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
MOTIVATION: Extensive efforts have been devoted to understanding the antigenic peptides binding to MHC class I and II molecules since they play a fundamental role in controlling immune responses and due their involvement in vaccination, transplantation, and autoimmunity. The genes coding for the MHC molecules are highly polymorphic, and it is difficult to build computational models for MHC molecules with few know binders. On the other hand, previous studies demonstrated that some MHC molecules share overlapping peptide binding repertoires and attempted to group them into supertypes. Herein, we present a framework of the utility of supertype clustering to gain more information about the data to improve the prediction accuracy of class II MHC-peptide binding. RESULTS: We developed a new method, called superMHC, for class II MHC-peptide binding prediction, including three MHC isotypes of HLA-DR, HLA-DP, and HLA-DQ, by using supertype clustering in conjunction with RLS regression. The supertypes were identified by using a novel repertoire dissimilarity index to quantify the difference in MHC binding specificities. The superMHC method achieves the state-of-the-art performance and is demonstrated to predict binding affinities to a series of MHC molecules with few binders accurately. These results have implications for understanding receptor-ligand interactions involved in MHC-peptide binding.
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Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos/metabolismo , Sitios de Unión , Análisis por Conglomerados , Biología Computacional/métodos , Antígenos HLA-DP/química , Antígenos HLA-DP/metabolismo , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Unión ProteicaRESUMEN
RNA-protein interactions (RPIs) have critical roles in numerous fundamental biological processes, such as post-transcriptional gene regulation, viral assembly, cellular defence and protein synthesis. As the number of available RNA-protein binding experimental data has increased rapidly due to high-throughput sequencing methods, it is now possible to measure and understand RNA-protein interactions by computational methods. In this study, we integrate a sequence-based derived kernel with regularized least squares to perform prediction. The derived kernel exploits the contextual information around an amino acid or a nucleic acid as well as the repetitive conserved motif information. We propose a novel machine learning method, called RPiRLS to predict the interaction between any RNA and protein of known sequences. For the RPiRLS classifier, each protein sequence comprises up to 20 diverse amino acids but for the RPiRLS-7G classifier, each protein sequence is represented by using 7-letter reduced alphabets based on their physiochemical properties. We evaluated both methods on a number of benchmark data sets and compared their performances with two newly developed and state-of-the-art methods, RPI-Pred and IPMiner. On the non-redundant benchmark test sets extracted from the PRIDB, the RPiRLS method outperformed RPI-Pred and IPMiner in terms of accuracy, specificity and sensitivity. Further, RPiRLS achieved an accuracy of 92% on the prediction of lncRNA-protein interactions. The proposed method can also be extended to construct RNA-protein interaction networks. The RPiRLS web server is freely available at http://bmc.med.stu.edu.cn/RPiRLS.
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Biología Computacional/métodos , Proteínas de Unión al ARN/química , ARN/química , Programas Informáticos , Algoritmos , Secuencia de Aminoácidos , Área Bajo la Curva , Bases de Datos Genéticas , Unión Proteica , Reproducibilidad de los Resultados , Flujo de TrabajoRESUMEN
Scavenger receptor class B type 1 (SR-B1), an HDL receptor plays a crucial role in cholesterol metabolism in the liver, steroidogenic tissues, and vascular cells including macrophages. SR-B1 is subject to regulation at the transcription, posttranscription and posttranslational levels. We previously provided evidence that PDZ domain containing NHERF1 and NHERF2 regulate SR-B1 protein levels post-transcriptionally, although the underlying mechanism(s) by which NHERF1 and NHERF2 regulate SR-B1 protein levels is not well understood. In this study, we demonstrate that SR-B1 is degraded intracellularly via ubiquitin-proteasome pathway and that SR-B1 can be ubiquitinated at K500 and K508 residues. Overexpression of NHERF1 or NHERF2 enhanced SR-B1 ubiquitination and degradation. NHERF1 and NHERF2 promote SR-B1 ubiquitination at sites K508 and K500, respectively. These results suggest that NHERF1 and NHERF2 down-regulated SR-B1 at least in part via the ubiquitin/proteasome pathway.
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Fosfoproteínas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores Depuradores de Clase B/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Animales , Células CHO , Células Cultivadas , Cricetulus , Estabilidad Proteica , Ratas , UbiquitinaciónRESUMEN
BACKGROUND: Steroidogenesis is a complex, multi-steps biological process in which, cholesterol precursor is converted to steroids in a tissue specific and tropic hormone dependent manner. Given that steroidogenesis is achieved by coordinated functioning of multiple tissue specific enzymes, many steroids intermediates/metabolites are generated during this process. Both the steroid products as well as major lipoprotein cholesterol donor, high-density lipoprotein 3 (hHDL3) have the potential to negatively regulate steroidogenesis via increased oxidative stress/reactive oxygen species (ROS) generation. METHODS: In the current study, we examined the effects of treatment of a mouse model of steroidogenesis, Y1-BS1 adrenocortical tumor cells with pregnenolone, 22(R)-Hydroxycholesterol [22(R)-diol] or hHDL3 on ROS production, phosphorylation status of p38 MAPK and cAMP response element-binding protein (CREB), CREB transcriptional activity and mRNA expression of StAR, CPY11A1/P450scc and antioxidant enzymes, superoxide dismutases [Cu,ZnSOD (SOD1), MnSOD (SOD2)], catalase (CAT) and glutathione peroxidase 1 (GPX1). We also detected the steroid product in p38 MAPK inhibitor treated Y1 cells by HPLC-MS / MS. RESULTS: Treatment of Y1 cells with H2O2 greatly enhanced the phosphorylation of both p38 MAPK and CREB protein. Likewise, treatment of cells with pregnenolone, 22(R) diol or hHDL3 increased ROS production measured with the oxidation-sensitive fluorescent probe 2',7'-Dichlorofluorescin diacetate (DCFH-DA). Under identical experimental conditions, treatment of cells with these agents also increased the phosphorylation of p38 MAPK and CREB. This increased CREB phosphorylation however, was associated with its decreased transcriptional activity. The stimulatory effects of pregnenolone, 22(R)-diol and hHDL3 on CREB phosphorylation was abolished by a specific p38 MAPK inhibitor, SB203580. Pregnenolone, and 22(R) diol but not hHDL3 upregulated the mRNA expression of SOD1, SOD2 and GPX1, while down-regulated the mRNA levels of StAR and CYP11A1. The p38 inhibitor SB203580 could increase the steroid production in HDL3, 22(R)-diol or pregnenolone treated cells. CONCLUSION: Our data demonstrate induction of a ROS/p38 MAPK -mediated feedback inhibitory pathway by oxy-cholesterol and steroid intermediates and products attenuates steroidogenesis via inhibition of CREB transcriptional activity.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Transducción de Señal , Esteroides/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/metabolismo , Neoplasias de la Corteza Suprarrenal/patología , Animales , Western Blotting , Catalasa/genética , Catalasa/metabolismo , Línea Celular Tumoral , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/genética , Enzima de Desdoblamiento de la Cadena Lateral del Colesterol/metabolismo , Retroalimentación Fisiológica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/genética , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/farmacología , Hidroxicolesteroles/farmacología , Ratones , Oxidantes/farmacología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Pregnenolona/farmacología , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Glutatión Peroxidasa GPX1RESUMEN
Lipid droplets (LDs) in steroidogenic tissues have a cholesteryl ester (CE) core surrounded by a phospholipid monolayer that is coated with associated proteins. Compared with other tissues, they tend to be smaller in size and more numerous in numbers. These LDs are enriched with PLIN1c, PLIN2 and PLIN3. Both CIDE A and B are found in mouse ovary. Free cholesterol (FC) released upon hormone stimulation from LDs is the preferred source of cholesterol substrate for steroidogenesis, and HSL is the major neutral cholesterol esterase mediating the conversion of CEs to FC. Through the interaction of HSL with vimentin and StAR, FC is translocated to mitochondria for steroid hormone production. Proteomic analyses of LDs isolated from loaded primary ovarian granulosa cells, mouse MLTC-1 Leydig tumor cells and mouse testes revealed LD associated proteins that are actively involved in modulating lipid homeostasis along with a number of steroidogenic enzymes. Microscopy analysis confirmed the localization of many of these proteins to LDs. These studies broaden the role of LDs to include being a platform for functional steroidogenic enzyme activity or as a port for transferring steroidogenic enzymes and/or steroid intermediates, in addition to being a storage depot for CEs.
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Colesterol/metabolismo , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos/fisiología , Mitocondrias/metabolismo , Esteroides/metabolismo , Animales , Humanos , Proteómica/métodosRESUMEN
Salt-inducible kinase 1 (SIK1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMPK family of kinases. SIK1 expression is rapidly induced in Y1 adrenal cells in response to ACTH via the cAMP-PKA signaling cascade, and it has been suggested that an increased level of SIK1 expression inhibits adrenal steroidogenesis by repressing the cAMP-dependent transcription of steroidogenic proteins, CYP11A1 and StAR, by attenuating CREB transcriptional activity. Here we show that SIK1 stimulates adrenal steroidogenesis by modulating the selective HDL-CE transport activity of SR-B1. Overexpression of SIK1 increases cAMP-stimulated and SR-B1-mediated selective HDL-BODIPY-CE uptake in cell lines without impacting SR-B1 protein levels, whereas knockdown of SIK1 attenuated cAMP-stimulated selective HDL-BODIPY-CE uptake. SIK1 forms a complex with SR-B1 by interacting with its cytoplasmic C-terminal domain, and in vitro kinase activity measurements indicate that SIK1 can phosphorylate the C-terminal domain of SR-B1. Among potential phosphorylation sites, SIK1-catalyzed phosphorylation of Ser496 is critical for SIK1 stimulation of the selective CE transport activity of SR-B1. Mutational studies further demonstrated that both the intact catalytic activity of SIK1 and its PKA-catalyzed phosphorylation are essential for SIK1 stimulation of SR-B1 activity. Finally, overexpression of SIK1 caused time-dependent increases in SR-B1-mediated and HDL-supported steroid production in Y1 cells; however, these effects were lost with knockdown of SR-B1. Taken together, these studies establish a role for SIK1 in the positive regulation of selective HDL-CE transport function of SR-B1 and steroidogenesis and suggest a potential mechanism for SIK1 signaling in modulating SR-B1-mediated selective CE uptake and associated steroidogenesis.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Depuradores de Clase B/metabolismo , Glándulas Suprarrenales/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico Activo , Línea Celular , Ésteres del Colesterol/metabolismo , Técnicas de Silenciamiento del Gen , Lipoproteínas HDL/metabolismo , Masculino , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/genéticaRESUMEN
Insulin resistance (IR) underlies metabolic disease. Visceral, but not subcutaneous, white adipose tissue (WAT) has been linked to the development of IR, potentially due to differences in regulatory protein abundance. Here we investigate how protein levels are changed in IR in different WAT depots by developing a targeted proteomics approach to quantitatively compare the abundance of 42 nuclear proteins in subcutaneous and visceral WAT from a commonly used insulin-resistant mouse model, Lepr(db/db), and from C57BL/6J control mice. The most differentially expressed proteins were important in adipogenesis, as confirmed by siRNA-mediated depletion experiments, suggesting a defect in adipogenesis in visceral, but not subcutaneous, insulin-resistant WAT. Furthermore, differentiation of visceral, but not subcutaneous, insulin-resistant stromal vascular cells (SVCs) was impaired. In an in vitro approach to understand the cause of this impaired differentiation, we compared insulin-resistant visceral SVCs to preadipocyte cell culture models made insulin resistant by different stimuli. The insulin-resistant visceral SVC protein abundance profile correlated most with preadipocyte cell culture cells treated with both palmitate and TNFα. Together, our study introduces a method to simultaneously measure and quantitatively compare nuclear protein expression patterns in primary adipose tissue and adipocyte cell cultures, which we show can reveal relationships between differentiation and disease states of different adipocyte tissue types.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Resistencia a la Insulina , Proteínas Nucleares/metabolismo , Tejido Adiposo Blanco/patología , Animales , Línea Celular , Dieta Alta en Grasa/efectos adversos , Masculino , Espectrometría de Masas , Ratones Endogámicos C57BL , Ratones ObesosRESUMEN
In this work, we developed a sensitive and efficient ratiometric electrochemical method for lipopolysaccharide (LPS) detection using Cu-based metal-organic frameworks (Cu-MOFs) as a catalyst and target-triggered quadratic cycles for signal amplification. First, in the presence of target LPS, the conformation change of the specifically designed hairpin probes 1 (HP1) triggered the target cyclic-induced polymerization to produce the output DNA with the aid of phi29 DNA polymerase (phi29). Then, the obtained output DNA hybridized with ferrocene-labeled hairpin probes 2 (Fc-HP2, which were immobilized on the electrode) to generate a nicking endonuclease (N.BstNBI) cleavage site. Thus, with N.BstNBI, the original signal molecules of Fc left from the electrode, and the single-stranded capture-probe-modified sensing interface was obtained. At this time, signal probes conducted by Au-nanoparticles-functionalized Cu-MOFs and labeled hairpin probes 3 (HP3/AuNPs/Cu-MOFs) were hybridized with capture probes for hairpin assembly. Herein, AuNPs/Cu-MOFs were not only used as nanocarriers for immobilizing HP3 but also acted as electroactive materials for signal reporting. With the proposed target-triggered quadratic cycles, the cleavage sites of Fc-HP2 were cut, and capture probes were obtained to hybridize with HP3/AuNPs/Cu-MOFs, which caused the signal decrease of Fc. Then Cu-MOFs were closed to the electrode for the signal increase of Cu-MOFs. Furthermore, when glucose was present in the detection solution, AuNPs/Cu-MOFs catalyzed the oxidation of glucose to realize the enzyme-free signal amplification. By measuring the peak currents ratio of the Cu-MOFs and Fc, the proposed aptasenor for LPS detection showed a low detection limit (0.33 fg/mL) and a wide linear range from 1.0 fg/mL to 100 ng/mL with high accuracy and sensitivity. This ratiometric electrochemical approach is expected to be a valuable strategy for detection of other analytes.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles , Cobre/química , Técnicas Electroquímicas , Hidrazinas/química , Lipopolisacáridos/análisis , Compuestos Organometálicos/química , Catálisis , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
In a number of biological studies, the raw gene expression data are not usually published due to different causes, such as data privacy and patent rights. Instead, significant gene lists with fold change values are usually provided in most studies. However, due to variations in data sources and profiling conditions, only a small number of common significant genes could be found among similar studies. Moreover, traditional gene set based analyses that consider these genes have not taken into account the fold change values, which may be important to distinguish between the different levels of significance of the genes. Human embryonic stem cell derived cardiomyocytes (hESC-CM) is a good representative of this category. hESC-CMs, with its role as a potentially unlimited source of human heart cells for regenerative medicine, have attracted the attentions of biological and medical researchers. Because of the difficulty of acquiring data and the resulting expenses, there are only a few related hESC-CM studies and few hESC-CM gene expression data are provided. In view of these challenges, we propose a new Gene Set Enrichment Ensemble (GSEE) approach to perform gene set based analysis on individual studies based on significant up-regulated gene lists with fold change data only. Our approach provides both explicit and implicit ways to utilize the fold change data, in order to make full use of scarce data. We validate our approach with hESC-CM data and fetal heart data, respectively. Experimental results on significant gene lists from different studies illustrate the effectiveness of our proposed approach.