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BACKGROUND: Early diagnosis of thyroid cancer is hampered by the inability of fine-needle aspiration biopsy (FNAB) to accurately classify â¼30% of cases while preoperative cancer staging detects lymph nodal involvement in only half of cases. Liquid biopsy may present an accurate, non-invasive alternative for preoperative thyroid nodule assessment. Thyrotropin receptor (TSHR) mRNA, a surrogate marker for circulating cancer cells (CTC), may be an option for early detection of malignancy from peripheral blood, but requires methodological improvements. We aimed to investigate if TSHR mRNA can be detected in low sample volumes by employing an ultrasensitive method - droplet digital PCR (ddPCR). METHODS: Less than 5 mL of blood was collected from 47 patients with thyroid nodules (25 benign and 22 malignant). RNA was isolated from the fraction of mononuclear cells where CTCs segregate. Samples were analysed for the presence of TSHR mRNA by ddPCR. RESULTS: Thyrotropin receptor mRNA was detectable in 4 mL sample volumes, with the test having good specificity (80%) but modest diagnostic accuracy (68.1%). Combining TSHR mRNA with ultrasound features and FNAB diagnosis, the test reaches high rule-out performances (sensitivity = 90% and NPV = 88.2%). Strikingly, TSHR mRNA correctly classified all samples with thyroid capsule invasion, lymph node metastasis and extrathyroidal extension. If aggressiveness is defined using these parameters, TSHR mRNA test reaches 100% sensitivity and 100% NPV for detecting high-risk cases. CONCLUSIONS: Employing ddPCR for TSHR mRNA improves its measurement by enabling detection in sample volumes common for laboratory testing. The test displays high prognostic performance, showing potential in preoperative risk assessment.
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Saprolegnia parasitica causes saprolegniosis, a disease responsible for significant economic losses in aquaculture and declines of fish populations in the wild, but the knowledge of its distribution and prevalence in the environment is limited. We developed a fast, sensitive and specific S. parasitica droplet digital PCR (ddPCR) assay and demonstrated its applicability for the detection and quantification of the pathogen in environmental samples: swab DNA collected from the host (trout skin, surface of eggs) and environmental DNA extracted from water. The developed assay was used to assess how abiotic (i.e. physico-chemical parameters of the water) and biotic (health status of the host) factors influence the S. parasitica load in the environment. The pathogen load in water samples was positively correlated with some site-specific abiotic parameters such as electrical conductivity (EC) and calcium, while fluorides were negatively correlated, suggesting that physico-chemical parameters are important for determining S. parasitica load in natural waters. Furthermore, skin swabs of injured trout had significantly higher pathogen load than swabs collected from healthy fish, confirming that S. parasitica is a widespread opportunistic pathogen. Our results provide new insights into various environmental factors that influence the distribution and abundance of S. parasitica.
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ADN Ambiental , Enfermedades de los Peces , Saprolegnia , Animales , Acuicultura , Calcio , Enfermedades de los Peces/epidemiología , Fluoruros , Saprolegnia/genética , Trucha/genética , AguaRESUMEN
Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality worldwide. Defects in trophoblast invasion, differentiation of extravillous trophoblasts and spiral artery remodeling are key factors in PE development. Currently there are no predictive biomarkers clinically available for PE. Recent technological advancements empowered transcriptome exploration and led to the discovery of numerous non-coding RNA species of which microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are the most investigated. They are implicated in the regulation of numerous cellular functions, and as such are being extensively explored as potential biomarkers for various diseases. Altered expression of numerous lncRNAs and miRNAs in placenta has been related to pathophysiological processes that occur in preeclampsia. In the following text we offer summary of the latest knowledge of the molecular mechanism by which lnRNAs and miRNAs (focusing on the chromosome 19 miRNA cluster (C19MC)) contribute to pathophysiology of PE development and their potential utility as biomarkers of PE, with special focus on sample selection and techniques for the quantification of lncRNAs and miRNAs in maternal circulation.
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MicroARN Circulante/sangre , Preeclampsia/sangre , Preeclampsia/diagnóstico , ARN Largo no Codificante/sangre , Biomarcadores/sangre , Diferenciación Celular/genética , Movimiento Celular/genética , Proliferación Celular/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 19/metabolismo , MicroARN Circulante/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Embarazo , ARN Largo no Codificante/genética , Transcriptoma , Trofoblastos/metabolismoRESUMEN
Osteoarthritis is a common cause of disability worldwide. Although commonly referred to as a disease of the joint cartilage, osteoarthritis affects all joint tissues equally. The pathogenesis of this degenerative process is not completely understood; however, a low-grade inflammation leading to an imbalance between anabolic and katabolic processes is a well-established factor. The complex network of cytokines regulating these processes and cell communication has a central role in the development and progression of osteoarthritis. Concentrations of both proinflammatory and anti-inflammatory cytokines were found to be altered depending on the osteoarthritis stage and activity. In this review, we analyzed individual cytokines involved in the immune processes with an emphasis on their function in osteoarthritis.
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Quimiocinas/metabolismo , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Osteoartritis/etiología , Osteoartritis/metabolismo , Animales , Biomarcadores , Humanos , Mediadores de Inflamación/metabolismo , Osteoartritis/patologíaRESUMEN
Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups-patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management.
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Ácidos Nucleicos Libres de Células/sangre , Neoplasias Colorrectales/sangre , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/genética , ADN Tumoral Circulante , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Biopsia Líquida , Masculino , Persona de Mediana Edad , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/sangre , Proteínas Proto-Oncogénicas p21(ras)/sangreRESUMEN
INTRODUCTION: MicroRNAs have a significant role in the pathogenesis of preeclampsia. Circulating microRNAs could represent a potential biomarker for preeclampsia. The aim of this study was to evaluate plasma miR210-3p and miR518b in preeclampsia and healthy pregnancy for the first time by digital droplet PCR (ddPCR). METHODS: Thirty-six pregnant women (seventeen healthy pregnancies, nineteen preeclampsia patients) were involved from the Clinic for Gynaecology and Obstetrics "Narodni front" in Belgrade, Serbia. Plasma miR210-3p, miR518b and cel-miR-39 as a spike-in control were measured by ddPCR. RESULTS: MiR518b was significantly elevated in preeclampsia compared to a healthy pregnancy (P = 0.034; 0.302(0.217-0.421) vs. 0.171(0.110-0.266)). MiR210-3p showed no significant difference between the two groups (P = 0.951). The adjustment of miR518b was made for a gestational age and smoking status and the difference between the preeclampsia and healthy pregnancy group was more significant (P = 0.026; 0.300(0.216-0.419) vs. 0.172(0.121-0.245)). Plasma miR-518b was significantly higher in the group of preeclampsia patients with proteinuria above the 75th percentile for the group (P = 0.033), in women who smoked (P = 0.039), and was positively related to uric acid in preeclampsia (P = 0.018, r = 0.536). Plasma miR518b was able to significantly discriminate between preeclampsia and healthy pregnancy, yielding AUC of 0.712 (95%CI:0.539-0.891), P = 0.028. CONCLUSIONS: In this study plasma microRNA were measured for the first time in preeclampsia and healthy pregnancies with ddPCR. Placenta-specific miR-518b could serve as a potential biomarker for discriminating preeclampsia and healthy pregnancy, which should be confirmed on a larger study population. This study has failed to confirm the same potential for miR210-3p.
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MicroARN Circulante/sangre , MicroARNs/sangre , Placenta , Preeclampsia/sangre , Adulto , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Edad Gestacional , Humanos , Persona de Mediana Edad , Embarazo , Adulto JovenRESUMEN
Circadian rhythms regulate a plethora of physiological processes. Perturbations of the rhythm can result in pathologies which are frequently studied in inbred mouse strains. We show that the genotype of mouse lines defines the circadian gene expression patterns. Expression of majority of core clock and output metabolic genes are phase delayed in the C56BL/6J line compared to 129S2 in the adrenal glands and the liver. Circadian amplitudes are generally higher in the 129S2 line. Experiments in dark - dark (DD) and light - dark conditions (LD), exome sequencing and data mining proposed that mouse lines differ in single nucleotide variants in the binding regions of clock related transcription factors in open chromatin regions. A possible mechanisms of differential circadian expression could be the entrainment and transmission of the light signal to peripheral organs. This is supported by the genotype effect in adrenal glands that is largest under LD, and by the high number of single nucleotide variants in the Receptor, Kinase and G-protein coupled receptor Panther molecular function categories. Different phenotypes of the two mouse lines and changed amino acid sequence of the Period 2 protein possibly contribute further to the observed differences in circadian gene expression.
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Glándulas Suprarrenales/metabolismo , Ritmo Circadiano/genética , Hígado/metabolismo , Ratones de la Cepa 129/genética , Ratones Endogámicos C57BL/genética , Animales , Minería de Datos , Genotipo , Luz , Masculino , Ratones de la Cepa 129/metabolismo , Ratones Endogámicos C57BL/metabolismo , Polimorfismo de Nucleótido Simple , Especificidad de la Especie , Factores de Transcripción/genética , Secuenciación del ExomaRESUMEN
Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.
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Colesterol/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , Lanosterol/metabolismo , Animales , Modulador del Elemento de Respuesta al AMP Cíclico/deficiencia , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Sistema Enzimático del Citocromo P-450/genética , Cromatografía de Gases y Espectrometría de Masas , Humanos , Lanosterol/química , Masculino , Ratones , Ratones Noqueados , Modelos Teóricos , Oxidación-Reducción , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Esteroles/análisis , Esteroles/metabolismo , Testículo/metabolismoRESUMEN
Circadian clocks are endogenous oscillators driving daily rhythms in physiology. The cell-autonomous clock is governed by an interlocked network of transcriptional feedback loops. Hundreds of clock-controlled genes (CCGs) regulate tissue specific functions. Transcriptome studies reveal that different organs (e.g. liver, heart, adrenal gland) feature substantially varying sets of CCGs with different peak phase distributions. To study the phase variability of CCGs in mammalian peripheral tissues, we develop a core clock model for mouse liver and adrenal gland based on expression profiles and known cis-regulatory sites. 'Modulation factors' associated with E-boxes, ROR-elements, and D-boxes can explain variable rhythms of CCGs, which is demonstrated for differential regulation of cytochromes P450 and 12 h harmonics. By varying model parameters we explore how tissue-specific peak phase distributions can be generated. The central role of E-boxes and ROR-elements is confirmed by analysing ChIP-seq data of BMAL1 and REV-ERB transcription factors.
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Proteínas CLOCK/fisiología , Relojes Circadianos , Regulación de la Expresión Génica , Animales , Ratones Endogámicos C57BL , Especificidad de Órganos , Fotoperiodo , Transcripción GenéticaRESUMEN
The biochemical basis of the mammalian circadian clock can be described by transcriptional-translational feedback loops with a period of about 24 h. Crucial endogenous factors are under circadian control (i.e., body temperature, blood pressure, hormone secretion and metabolite levels). Also, drug metabolism, including phases I-III and the drug-responsive nuclear receptors, is controlled by the clock. Disturbances in circadian rhythm in humans can lead to pathologies, which is exemplified by increased cancer risk in long-term shift workers. On the other hand, best tolerability of drugs with minimum side effects can be achieved if the timing of drug treatment is synchronized with the patients' individual clock. The aim of this review is to underline the importance of accepting the individuals' endogenous clock which can contribute to personalized, patient-friendly optimization of drug therapies.
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Ritmo Circadiano , Sistema Enzimático del Citocromo P-450/fisiología , Inactivación Metabólica/fisiología , Animales , Artritis Reumatoide/tratamiento farmacológico , Asma/tratamiento farmacológico , Enfermedades Cardiovasculares/tratamiento farmacológico , Cronoterapia de Medicamentos , Regulación Enzimológica de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/fisiología , Úlcera Gástrica/tratamiento farmacológicoRESUMEN
Non-alcoholic fatty liver disease (NAFLD) is the most predominant liver disease worldwide and hepatic manifestation of the metabolic syndrome. Its histology spectrum ranges from steatosis, to steatohepatitis (NASH) that can further progress to cirrhosis and hepatocellular carcinoma (HCC). The increasing incidence of NAFLD has contributed to rising numbers of HCC occurrences. NAFLD progression is governed by genetic susceptibility, environmental factors, lifestyle and features of the metabolic syndrome, many of which overlap with HCC. Gene expression profiling and genome wide association studies have identified novel disease pathways and polymorphisms in genes that may be potential biomarkers of NAFLD progression. However, the multifactorial nature of NAFLD and the limited number of sufficiently powered studies are among the current limitations for validated biomarkers of clinical utility. Further studies incorporating the links between circadian regulation and hepatic metabolism might represent an additional direction in the search for predictive biomarkers of liver disease progression and treatment outcomes.
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Carcinoma Hepatocelular/genética , Hígado Graso/genética , Genómica , Animales , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/patología , Progresión de la Enfermedad , Hígado Graso/metabolismo , Hígado Graso/patología , Estudios de Asociación Genética , Humanos , Estilo de Vida , Cirrosis Hepática/complicaciones , Cirrosis Hepática/epidemiología , Cirrosis Hepática/patología , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Enfermedad del Hígado Graso no AlcohólicoRESUMEN
Light, restricted feeding, and hormonal inputs may operate as time givers (zeitgebers) for the circadian clock within peripheral organs through the activation of tissue-specific signaling cascades. cAMP signaling through CREM (cAMP-responsive element modulator) and its variant ICER (inducible cAMP early repressor) is linked to the circadian regulation of pineal melatonin synthesis, although little is known about its influence in other organs. We performed experiments in the absence of light and feeding-time cues to test which core clock genes are controlled by CREM/ICER in the liver and adrenal gland. In vivo, Crem loss-of-function mutation resulted in fine-tuning of all measured adrenal clock genes (Per1/2/3, Cry1/2, Bmal1, and Rev-erbα), whereas only Per1 and Cry1 were affected in the liver. Icer expression was circadian in the adrenal gland, with peak gene expression at zeitgeber 12 and the highest protein levels at zeitgeber â¼20. The expression of both Icer and Per1 genes responded to cAMP stimuli in an immediate-early fashion. In immortal cells, forskolin induced expression of Per1 after 2 h, and de novo protein synthesis led to Per1 attenuation. We show that the de novo synthesized protein responsible for Per1 attenuation is ICER. Indeed, Per1 expression is up-regulated in cells ectopically expressing antisense Icer, and mobility shift experiments identified ICER binding to cAMP-responsive elements of the Per1 promoter. We propose that ICER acts as a noise filter for different signals that could affect transcription in the adrenal gland. Because ICER is an immediate-early repressor, the circadian nature of adrenal Icer expression could serve a role in a time-dependent gating mechanism.
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Glándulas Suprarrenales/metabolismo , Relojes Circadianos/fisiología , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/fisiología , Hígado/metabolismo , Proteínas Circadianas Period/biosíntesis , Glándulas Suprarrenales/citología , Animales , Línea Celular Tumoral , Criptocromos/genética , Criptocromos/metabolismo , AMP Cíclico/genética , AMP Cíclico/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Hígado/citología , Ratones , Ratones Noqueados , Especificidad de Órganos/fisiología , Proteínas Circadianas Period/genética , Biosíntesis de Proteínas/fisiologíaRESUMEN
The mammalian circadian clock is driven by cell-autonomous transcriptional feedback loops that involve E-boxes, D-boxes, and ROR-elements. In peripheral organs, circadian rhythms are additionally affected by systemic factors. We show that intrinsic combinatorial gene regulation governs the liver clock. With a temporal resolution of 2 h, we measured the expression of 21 clock genes in mouse liver under constant darkness and equinoctial light-dark cycles. Based on these data and known transcription factor binding sites, we develop a six-variable gene regulatory network. The transcriptional feedback loops are represented by equations with time-delayed variables, which substantially simplifies modelling of intermediate protein dynamics. Our model accurately reproduces measured phases, amplitudes, and waveforms of clock genes. Analysis of the network reveals properties of the clock: overcritical delays generate oscillations; synergy of inhibition and activation enhances amplitudes; and combinatorial modulation of transcription controls the phases. The agreement of measurements and simulations suggests that the intrinsic gene regulatory network primarily determines the circadian clock in liver, whereas systemic cues such as light-dark cycles serve to fine-tune the rhythms.
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Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Ritmo Circadiano , Redes Reguladoras de Genes , Hígado/metabolismo , Factores de Transcripción ARNTL/genética , Animales , Relojes Circadianos , Criptocromos/genética , Proteínas de Unión al ADN/genética , Elementos E-Box , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Modelos Genéticos , Proteínas Circadianas Period/genética , Fotoperiodo , Factores de Transcripción/genéticaRESUMEN
The essential role of the Crem gene in normal sperm development is widely accepted and is confirmed by azoospermia in male mice lacking the Crem gene. The exact number of genes affected by Crem absence is not known, however a large difference has been observed recently between the estimated number of differentially expressed genes found in Crem knock-out (KO) mice compared to the number of gene loci bound by CREM. We therefore re-examined global gene expression in male mice lacking the Crem gene using whole genome transcriptome analysis with Affymetrix microarrays and compared the lists of differentially expressed genes from Crem-/- mice to a dataset of genes where binding of CREM was determined by Chip-seq. We determined the global effect of CREM on spermatogenesis as well as distinguished between primary and secondary effects of the CREM absence. We demonstrated that the absence of Crem deregulates over 4700 genes in KO testis. Among them are 101 genes associated with spermatogenesis 41 of which are bound by CREM and are deregulated in Crem KO testis. Absence of several of these genes in mouse models has proven their importance for normal spermatogenesis and male fertility. Our study showed that the absence of Crem plays a more important role on different aspects of spermatogenesis as estimated previously, with its impact ranging from apoptosis induction to deregulation of major circadian clock genes, steroidogenesis and the cell-cell junction dynamics. Several new genes important for normal spermatogenesis and fertility are down-regulated in KO testis and are therefore possible novel targets of CREM.
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Modulador del Elemento de Respuesta al AMP Cíclico/fisiología , Regulación de la Expresión Génica , Testículo/metabolismo , Animales , Apoptosis , Transporte Biológico , Modulador del Elemento de Respuesta al AMP Cíclico/biosíntesis , Fertilización , Perfilación de la Expresión Génica , Homocigoto , Masculino , Melatonina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa/métodos , Espermátides/metabolismo , Espermatogénesis , Espermatozoides/metabolismoRESUMEN
The cytochrome P450 (CYP) genes Cyp51, Cyp11a1, Cyp17a1, Cyb11b1, Cyp11b2 and Cyp21a1 are involved in the adrenal production of corticosteroids, whose circulating levels are circadian. cAMP signaling plays an important role in adrenal steroidogenesis. By using cAMP responsive element modulator (Crem) knockout mice, we show that CREM isoforms contribute to circadian expression of steroidogenic CYPs in the mouse adrenal gland. Most striking was the CREM-dependent hypomethylation of the Cyp17a1 promoter at zeitgeber time 12, which resulted in higher Cyp17a1 mRNA and protein expression in the knockout adrenal glands. The data indicate that products of the Crem gene control the epigenetic repression of Cyp17 in mouse adrenal glands.
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Glándulas Suprarrenales/metabolismo , Modulador del Elemento de Respuesta al AMP Cíclico/metabolismo , Esteroide 17-alfa-Hidroxilasa/genética , Animales , Ritmo Circadiano , Corticosterona/sangre , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Metilación de ADN , Epigénesis Genética/fisiología , Regulación Enzimológica de la Expresión Génica , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas , Esteroide 17-alfa-Hidroxilasa/biosíntesisRESUMEN
Circadian rhythms affect the total cholesterol levels in humans and animals, although their effect on cholesterol synthesis remain poorly understood. Here, we show for the first time that intermediates of the post-squalene portion of cholesterol synthesis also follow a circadian rhythm in the mouse liver. We used Crem-knock-out mice to investigate the effects of cAMP response element modulator (CREM) isoforms on cholesterol synthesis over time, as compared to wild-type mice. Multiple linear regression and cosinor statistical analysis were carried out on data obtained from 166 liver samples of mice, and the 24-h profiles were modelled across genotype, gender and zeitgeber time for lanosterol, 24,25-dihydrolanosterol, testis meiosis-activating sterol, and 7-dehydrocholesterol, along with cholesterol. The levels of these sterols were higher in female mice compared to males, although the genotype/gender factors showed no effects on the circadian oscillation of these sterols, except for 24,25-dihydrolanosterol. This study also highlights the importance of the statistical methods, where time, genotype and gender are the studied variables.
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Colesterol/biosíntesis , Ritmo Circadiano , Hígado/metabolismo , Escualeno/metabolismo , Animales , Modulador del Elemento de Respuesta al AMP Cíclico/genética , Interpretación Estadística de Datos , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
BACKGROUND: Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. RESULTS: In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. CONCLUSIONS: Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.
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Ritmo Circadiano/genética , Expresión Génica , Ratones Endogámicos/genética , Reacción en Cadena de la Polimerasa , Animales , Humanos , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa/normasRESUMEN
We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14alpha-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited approximately 10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-d1 is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme.