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The rapid brain maturation in childhood and adolescence accompanies the development of socio-emotional functioning. However, it is unclear how the maturation of the neural activity drives the development of socio-emotional functioning and individual differences. This study aimed to reflect the age dependence of inter-individual differences in brain responses to socio-emotional scenarios and to develop naturalistic imaging indicators to assess the maturity of socio-emotional ability at the individual level. Using three independent naturalistic imaging datasets containing healthy participants (n = 111, 21 and 122), we found and validated that age-modulated inter-individual concordance of brain responses to socio-emotional movies in specific brain regions. The similarity of an individual's brain response to the average response of older participants was defined as response typicality, which predicted an individual's emotion regulation strategies in adolescence and theory of mind (ToM) in childhood. Its predictive power was not superseded by age, sex, cognitive performance or executive function. We further showed that the movie's valence and arousal ratings grounded the response typicality. The findings highlight that forming typical brain response patterns may be a neural phenotype underlying the maturation of socio-emotional ability. The proposed response typicality represents a neuroimaging approach to measure individuals' maturity of cognitive reappraisal and ToM.
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Encéfalo , Teoría de la Mente , Humanos , Niño , Adolescente , Encéfalo/diagnóstico por imagen , Emociones/fisiología , Habilidades Sociales , Función EjecutivaRESUMEN
Colorectal cancer is one of the malignant tumors that pose a serious threat to human health. A particularly bad prognosis might be expected for colorectal tumors with the unique molecular subtype BRAF V600E mutation. With the development of precision therapy, the advent of molecularly targeted therapies and immune checkpoint inhibitors has improved the outcome of intermediate to advanced colorectal cancer. However, the duration of drug benefit is usually short, and overall survival and progression-free survival remain suboptimal. Therefore, investigators are exploring more rational, safe, and effective drug combination regimens through clinical trials to provide longer survival for patients with such genetic mutations with metastatic colorectal cancer (mCRC). This article reviews the progress of clinical research on molecularly targeted drugs, immune checkpoint inhibitors, first-line chemotherapeutic agents, and different combination therapy regimens (including different targeted drug combinations, immune combination targeting, and chemotherapy combination targeting) for colorectal cancer patients with BRAF V600E mutation, which provides a reference for further in-depth clinical exploration of the treatment of colorectal cancer patients with BRAF V600E mutation.
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Neoplasias Colorrectales , Proteínas Proto-Oncogénicas B-raf , Humanos , Proteínas Proto-Oncogénicas B-raf/genética , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Pronóstico , MutaciónRESUMEN
Herein, we have constructed a magnetic graphene field-effect transistor biosensor (MGFETs) for highly sensitive detection of cardiac troponin I (CTNI). Graphene films transferred to ITO conductive glass as conductive channels. CTNI aptamer was immobilized onto the graphene film via 1-pyrene-butanoic acid succinimidyl ester (PBASE) to capture CTNI. Magnetic nanobeads (MBs) modified with CTNI antibody were added to the reaction chamber to form an aptamer/CTNI/antibody/magnetic nanobeads sandwich-type complex. We found that the magnetic force exerted on the complex leads to an impedance change of the graphene film. The reason for this result is that the magnetic field exerts an influence on the MBs, causing CTNI aptamer strand to bend, resulting in a change in the distance between the double conductive layers of the graphene film surface and the test solution. With periodic sampling integration, different concentrations of CTNI can be detected with high sensitivity. Due to the stringent recognition capability and high affinity between the CTNI aptamer and CTNI, MGFETs have the potential to detect various types of proteins. Furthermore, MGFETs also have the potential to be utilized for the detection of DNA or specific cells in the future.
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Hepatocellular carcinoma is a common gastrointestinal malignancy with a high mortality rate and limited treatment options. Molecularly targeted drugs combined with immune checkpoint inhibitors have shown unique advantages over single-agent applications, significantly prolonging patient survival. This paper reviews the research progress of molecular-targeted drugs combined with immune checkpoint inhibitors in the treatment of hepatocellular carcinoma and discusses the effectiveness and safety of the combination of the two drugs to provide a reference for the further application of molecular-targeted drugs combined with immune checkpoint inhibitors in clinical practice.
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Although the fabrication of controllable three-dimensional (3D) microstructures on substrates has been proposed as an effective solution for SERS, there remains a gap in the detection and manufacturability of 3D substrates with high performance. In this study, photolithography is adopted to obtain a pyramid-like array on a patterned sapphire substrate (PSS), with Al2O3 as the dielectric layer. In addition, silver nanoparticles (AgNPs) are used to decorate Au films to obtain mass-producible 3D SRES substrates. In the case of low fluorescence, the substrate realizes the coupling of localized surface plasmon polaritons (LSPs) and surface plasmon polaritons (SPPs), which is consistent with the simulation results obtained using the finite element method. The performance of the SERS substrate is evaluated using rhodamine 6G (R6G) and toluidine blue (TB) as probe molecules with detection limits of 10-11 M and 10-9 M, respectively. The substrate exhibits high hydrophobicity and excellent light-capturing capability. Moreover, it shows self-cleaning ability and long-term stability in practical applications. Allowing for the consistency of the composite substrate in the preparation process and the high reproducibility of the test results, it is considered to be promising for mass production.
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Arrayed resonant cavity with outstanding optical trapping ability have received increasing attention in surface-enhanced Raman spectroscopy (SERS). Here, a three-dimensional (3D) composite AgNPs-Al2O3/Au/inverted patterned sapphire substrate PMMA (IPSSPMMA) flexible resonant cavity system is theoretically and experimentally investigated as a flexible SERS sensor. With the help of an effective plasma coupling (localized surface plasmons (LSPs) and surface plasmon polaritons (SPPs)), as shown by the Finite Element Method, a resonant cavity between IPSSPMMA and a particle-film nanostructure is created. Moreover, the proposed fabrication scheme can be easily used for large-scale fabrication. To measure the performance of IPSSPMMA, Rhodamine 6â G (R6G) and Crystalline violet (CV) were used as probe molecules with limit of detection (LOD) of 6.01 × 10-12 M and 5.36 × 10-10 M, respectively, and enhancement factors (EF) of R6G up to 8.6 × 109. Besides, in-situ detection of CV on the surface of aquatic products with a LOD of 3.96 × 10-5 M, enables highly sensitive in-situ detection of surface analytes. The Raman performance and in-situ detection results demonstrate that the proposed flexible compositing resonant cavity system has the advantages of ultra-sensitivity, stability, uniformity, and reproducibility, and has great potential for applications in the food safety field.
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BACKGROUND: Social anxiety disorder (SAD) is a prevalent and impairing mental disorder among children and adolescents. The bed nucleus of the stria terminalis (BNST) plays a critical role in anxiety disorders, including valence surveillance and hypervigilance for potential threats. However, the role of BNST and its related functional network in children and adolescents with SAD has not been fully investigated. This study examined the aberration of BNST's functional connectivity and its age dependence in adolescents with SAD. METHODS: Using a sample of 75 SAD patients and 75 healthy controls (HCs) children aged 9-18 years old, we delineated the group-by-age interaction of BNST-seeded functional connectivity (FC) during resting state and movie-watching. The relationships between BNST-seeded FC and clinical scores were also examined. RESULTS: During movie viewing, the FC between the right BNST and the left amygdala, bilateral posterior cingulate cortex (PCC), bilateral superior temporal cortex, and right pericalcarine cortex showed a diagnostic group-by-age interaction. Compared to HCs, SAD patients showed a significant enhancement of the above FC at younger ages. Meanwhile, they showed an age-dependent decrease in FC between the right BNST and left amygdala. Furthermore, for SAD patients, FC between the right BNST and left amygdala during movie viewing was positively correlated with separation anxiety scores. CONCLUSIONS: The right BNST plays an essential role in the aberrant brain functioning in children and adolescents with SAD. The atypicality of BNST's FC has remarkable age dependence in SAD, suggesting an association of SAD with neurodevelopmental traits.
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Fobia Social , Núcleos Septales , Humanos , Adolescente , Niño , Núcleos Septales/diagnóstico por imagen , Imagen por Resonancia Magnética , Ansiedad , Amígdala del Cerebelo/diagnóstico por imagenRESUMEN
[This corrects the article DOI: 10.3389/fonc.2023.1197698.].
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Subcortical brain regions play essential roles in the pathology of social anxiety disorder (SAD). While adolescence is the peak period of SAD, the relationships between altered development of the subcortical regions during this period and SAD are still unclear. This study investigated the age-dependent alterations in structural co-variance among subcortical regions and between subcortical and cortical regions, aiming to reflect aberrant coordination during development in the adolescent with SAD. High-resolution T1-weighted images were obtained from 76 adolescents with SAD and 67 healthy controls (HC), ranging from 11 to 17.9 years. Symptom severity was evaluated with the Social Anxiety Scale for Children (SASC) and the Depression Self Rating Scale for Children (DSRS-C). Structural co-variance and sliding age-window analyses were used to detect age-dependent group differences in inter-regional coordination patterns among subcortical regions and between subcortical and cortical regions. The volume of the striatum significantly correlated with SAD symptom severity. The SAD group exhibited significantly enhanced structural co-variance among key regions of the striatum (putamen and caudate). While the co-variance decreased with age in healthy adolescents, the co-variance in SAD adolescents stayed high, leading to more apparent group differences in middle adolescence. Moreover, the striatum's mean structural co-variance with cortical regions decreased with age in HC but increased with age in SAD. Adolescents with SAD suffer aberrant developmental coordination among the key regions of the striatum and between the striatum and cortical regions. The degree of incoordination is age-dependent, which may represent a neurodevelopmental trait of SAD.
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Social anxiety disorder (SAD) is a common anxiety disorder in childhood and adolescence. Studies on SAD in adults have reported both structural and functional aberrancies of the brain at the group level. However, evidence has shown differences in anxiety-related brain abnormalities between adolescents and adults. Since children and adolescents can afford limited scan time, optimizing the scan tasks is essential for SAD research in children and adolescents. Thus, we need to address whether brain structure, resting-state fMRI, and naturalistic imaging enable individualized identification of SAD in children and adolescents, which measurement is more effective, and whether pooling multi-modal features can improve the identification of SAD. We comprehensively addressed these questions by building machine learning models based on parcel-wise brain features. We found that naturalistic fMRI yielded higher classification accuracy (69.17%) than the other modalities and the classification performance showed dependence on the contents of the movie. The classification models also identified contributing brain regions, some of which exhibited correlations with the symptoms scores of SAD. However, pooling brain features from the three modalities did not help enhance the classification accuracy. These results support the application of carefully designed naturalistic imaging in recognizing children and adolescents at risk of SAD.
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Imagen por Resonancia Magnética , Fobia Social , Adolescente , Adulto , Trastornos de Ansiedad/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Mapeo Encefálico/métodos , Niño , Humanos , Imagen por Resonancia Magnética/métodos , Fobia Social/diagnóstico por imagenRESUMEN
Adolescence is the peak period for the incidence of anxiety disorders. Recent findings have revealed the immaturity of neural networks underlying emotional regulation in this population. Brain vulnerability to anxiety in adolescence is related to the unsynchronised development of anxiety-relevant brain functional systems. However, our current knowledge on brain deficits in adolescent anxiety is mainly borrowed from studies on adults. Understanding adolescent-specific brain deficits is essential for developing biomarkers and brain-based therapies targeting adolescent anxiety. This article reviews and compares recent neuroimaging literature on anxiety-related brain structural and functional deficits between adolescent and adult populations, and proposes a model highlighting the differences between adolescence and adulthood in anxiety-related brain networks. This model emphasises that in adolescence the emotional control system tends to be hypoactivated, the fear conditioning system is immature, and the reward and stress response systems are hypersensitive. Furthermore, the striatum's functional links to the amygdala and the prefrontal cortex are strengthened, while the link between the prefrontal cortex and the amygdala is weakened in adolescence. This model helps to explain why adolescents are vulnerable to anxiety disorders and provides insights into potential brain-based approaches to intervene in adolescent anxiety disorders.
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OBJECTIVES: The objectives of this study were to investigate the correlations between the minimum effective volume (MEV) of lidocaine 1.5% for an ultrasound-guided popliteal sciatic nerve block and individual factors including the cross-sectional nerve area, sex, age, body mass index, and the depth of the sciatic nerve and to evaluate the safety of combined femoral and sciatic nerve blocks by monitoring the plasma concentration of local anesthetics. METHODS: Forty patients received combined single-shot femoral and continuous sciatic nerve blocks. The femoral nerve block was performed with an in-plane technique and 15 mL of lidocaine 1.5%. A continuous peripheral nerve block annular tube was positioned between the tibial and peroneal nerves inside the paraneural sheath. Thirty minutes after the femoral nerve block, a loading dose of 5 mL of lidocaine 1.5% was given to block the sciatic nerve after obtaining the maximum compound muscle action potential (CMAP) amplitude using nerve conduction studies. Additional lidocaine 1.5% was pumped at a rate of 30 mL/h through the indwelling annular tube if, after 8 minutes, the CMAP amplitude was still present. The CMAP amplitude monitored by the nerve conduction studies and pinprick tests were recorded every 2 minutes after the administration of lidocaine 1.5%. When the CMAP amplitude decreased to nearly 0 mV, this MEV was recorded. The influences of the cross-sectional area of the sciatic nerve, sex, age, body mass index, and the depth of the sciatic nerve on the MEV were analyzed using stepwise multiple linear regression. Blood samples were collected from 10 patients to evaluate the safety of combined femoral and sciatic nerve blocks by ultra-performance liquid chromatography-tandem mass spectrometry. Blood was drawn at 0 minutes before femoral nerve injection; 0 minutes before sciatic nerve injection; 8 minutes after sciatic nerve injection; and 0, 10, 20, 30, 45, 60, 75, 90, and 120 minutes after the pumping of lidocaine 1.5% stopped. RESULTS: A significant correlation was found between the MEV of lidocaine 1.5% and the cross-sectional area of the sciatic nerve (r=0.459), with a regression equation of the MEV (mL)=5.969+0.095×(the cross-sectional area of the sciatic nerve). The coefficient of determination was 0.211 (P<0.05). The MEV of lidocaine 1.5% for complete sciatic nerve blocks ranged from 7 to 15 mL. The maximum concentrations of lidocaine, monoethylglycinexylidide, and glycinexylidide were 1672.9 (227.6), 265.7 (32.7), and 42.2 (22.4) ng/mL, respectively. CONCLUSIONS: There is a positive correlation between the cross-sectional area of the sciatic nerve and the MEV. The regression equation can help to predict the MEV of lidocaine 1.5% for popliteal sciatic nerve blocks. The maximum concentrations of lidocaine and its metabolites did not approach toxic threshold limits in this study.
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Anestésicos Locales/administración & dosificación , Lidocaína/administración & dosificación , Bloqueo Nervioso , Relación Dosis-Respuesta a Droga , Nervio Femoral , Humanos , Estudios Prospectivos , Nervio CiáticoRESUMEN
OBJECTIVE: To investigate superiorities of a special needle-over-cannula adopting different location methods for continuous femoral nerve block (CFNB) for geriatric lower extremity joint arthroplasty. METHODS: 60 elderly patients intending to receive scheduled knee or hip replacement surgery were recruited and divided into 3 groups randomly. Group 1 (n=20) adopted fascial pop for continuous femoral nerve block and postoperative analgesia with indwelling cannula. Group 2 (n=20) adopted location guided by B ultrasound, and Group 3 (n=20) adopted fascial pop combined with B ultrasound. RESULTS: There was significant difference in the performing time of cannula indwelling on average between each two groups (P<0.01). There was no significant difference among three groups about visual analogue scale (VAS) score, Ramsay sedation score (RSS), incidence of nausea and vomit, or patient's satisfaction at 6, 12, 24 and 48 h. Infection at the puncture site, toxic reaction of local anesthetics and respiratory depression were absent during the cannula indwelling. All the patients did not receive any other analgesic, and the indwelling time of external cannula was 45.3 hours on average. There was only one patient in group 2 who felt mild pains in front of the thigh after removing the indwelling cannula. No stolidity or other abnormal symptom was found among the remaining patients. CONCLUSIONS: Shorter indwelling cannula time and higher success rate of single attempt placement suggest that fascial pop combined ultrasound guidance is worth for clinical recommendation.
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Type II citrullinaemia, also known as citrin deficiency, is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3. One of the clinical manifestations of type II citrullinaemia is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency (NICCD, OMIM# 605814). In this study, a 5-month-old female Chinese neonate diagnosed with type II citrullinaemia was examined. The diagnosis was based on biochemical and clinical findings, including organic acid profiling using a gas chromatography mass spectrometry (GC/MS), and the patient's parents were unaffected. Approximately 14 kb of the exon sequences of the SLC25A13 and two relative genes (ASS1 and FAH) from the proband and 100 case-unrelated controls were captured by array-based capture method followed by high-throughput next-generation sequencing. Two single-nucleotide mutations were detected in the proband, including the previous reported c.1177+1G>A mutation and a novel c.754 G>A mutation in the SLC25A13 gene. Sanger sequence results showed that the patient was a compound heterozygote for the two mutations. The novel mutation (c.754 G>A), which is predicted to affect the normal structure and function of citrin, is a candidate pathogenic mutation. Target sequence capture combined with high-throughput next-generation sequencing technologies is proven to be an effective method for molecular genetic testing of type II citrullinaemia.
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Proteínas de Unión al Calcio/deficiencia , Citrulinemia/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Mutación Missense , Transportadores de Anión Orgánico/deficiencia , Secuencia de Aminoácidos , Pueblo Asiatico/genética , Secuencia de Bases , Estudios de Casos y Controles , Análisis Mutacional de ADN/métodos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Datos de Secuencia MolecularRESUMEN
Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.
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Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Patología Molecular , Adulto , Preescolar , Variaciones en el Número de Copia de ADN/genética , Femenino , Tamización de Portadores Genéticos , Genética de Población , Voluntarios Sanos , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Distrofia Muscular de Duchenne/patología , Eliminación de SecuenciaRESUMEN
OBJECTIVE: To investigate the feasibility of a non-stimulation needle with an external indwelling cannula for upper-limb surgery and acute postoperative pain management. METHODS: 62 patients undergoing either scheduled or emergency upper-limb surgery received brachial plexus block of modified interscalene or axillary brachial and then postoperative patient-controlled analgesia (PCA) with local analgesics using a specially designed non-stimulation needle with an external indwelling cannula. The outcome measurements included anesthetic effect, acute or chronic complications, postoperative analgesic effect and patient's satisfaction. RESULTS: The success rate of anesthesia was 96.8%. The single attempt placement with the external indwelling cannula was achieved in 85.2% of patients with axillary brachial plexus block and 78.8% with modified interscalene brachial plexus block. The incidence of severe intoxication was 3.7% with axillary brachial plexus block and 3.0% with modified interscalene brachial plexus block. No hematoma at the injection site, Horner's syndrome, hoarseness or dyspnea was observed. Postoperative analgesic effect was achieved in 100% and activities were slightly lowered in 91.7%. The incidence of nausea and vomit was 8.3%; patient's satisfaction was 9.1 on a 10-point scale system. Infection, nerve injury and respiratory depression were absent during the catheter indwelling. The indwelling time of external indwelling cannula was 30.5 h on average. There was no nerve injury related complication after withdrawing the external indwelling catheter. CONCLUSIONS: Brachial plexus block using a non-stimulation needle with an external indwelling cannula has favorable intra-operative anesthetic benefit and provides an excellent postoperative analgesic outcome. The low incidence of complications and favorable patient's satisfaction suggest that non-stimulation needle with an external indwelling cannula is a useful and safe anesthetic tool in brachial nerve block and acute postoperative pain management.
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Analgesia Controlada por el Paciente/instrumentación , Anestésicos Locales/administración & dosificación , Plexo Braquial/efectos de los fármacos , Agujas , Bloqueo Nervioso/métodos , Manejo del Dolor/métodos , Dolor Postoperatorio/tratamiento farmacológico , Extremidad Superior/cirugía , Adolescente , Adulto , Anciano , Analgesia Controlada por el Paciente/métodos , Catéteres , Diseño de Equipo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Náusea/inducido químicamente , Bloqueo Nervioso/efectos adversos , Bloqueo Nervioso/instrumentación , Resultado del Tratamiento , Vómitos/inducido químicamente , Adulto JovenRESUMEN
Carboxylesterases (CES) metabolize esters. Two CES isoforms are expressed in human liver (CES1 and CES2) and liver extracts are used in reaction phenotyping studies to discern interindividual metabolic variation. We tested the hypothesis that an individual's CES phenotype can be characterized by reporter substrates/probes that interrogate native CES1 and CES2 activities in liver and immunoblotting methods. We obtained 25 livers and found that CES1 is the main hydrolytic enzyme. Moreover, although CES1 protein levels were similar, we observed large interindividual variation in bioresmethrin hydrolysis rates (17-fold), a pyrethroid metabolized by CES1 but not CES2. Bioresmethrin hydrolysis rates did not correlate with CES1 protein levels. In contrast, procaine hydrolysis rates, a drug metabolized by CES2 but not CES1, were much less variant (3-fold). Using activity-based fluorophosphonate probes (FP-biotin), which covalently reacts with active serine hydrolases, CES1 protein was the most active enzyme in the livers. Finally, using bioorthogonal probes and click chemistry methodology, the half-life of CES 1 and 2 in cultured HepG2 cells was estimated at 96 h. The cause of the differential CES1 activities is unknown, but the underlying factors will be important to understand because several carboxylic acid ester drugs and environmental toxicants are metabolized by this enzyme.
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Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Hígado/enzimología , Adolescente , Adulto , Anciano , Ácido Araquidónico/metabolismo , Biotransformación , Western Blotting , Femenino , Humanos , Hidrólisis , Inmunoprecipitación , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Dystrophin (DMD) gene is the largest gene containing 79 exons involving various mutation types and regions, and targeted next-generation sequencing (NGS) was employed in detecting DMD gene mutation in the present study. A literature-annotated disease nonsense mutation (c.10141C>T, NM_004006.1) in exon 70 that has been reported as Duchenne Muscular Dystrophy (DMD)-causing mutation was found in our two patients, the proband and his cousin. In the present study two main methods were used, the next-generation sequencing and the classic Sanger sequencing. The exon capture followed by HiSeq2000 sequencing was specifically used in this study. Combined applications of the next-generation sequencing platform and bioinformatics are proved to be effective methods for DMD diagnosis.
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Codón sin Sentido/fisiología , Distrofina/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Adulto , Pueblo Asiatico/genética , Niño , Codón sin Sentido/genética , Análisis Mutacional de ADN/métodos , Distrofina/análisis , Distrofina/química , Familia , Femenino , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Linaje , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína/genética , Literatura de Revisión como AsuntoRESUMEN
Carboxylesterases (CES) have important roles in pesticide and drug metabolism and contribute to the clearance of ester-containing xenobiotics in mammals. Tissues with the highest levels of CES expression are the liver and small intestine. In addition to xenobiotics, CES also harness their broad substrate specificity to hydrolyze endobiotics, such as cholesteryl esters and triacylglycerols. Here, we determined if two human CES isoforms, CES1 and CES2, hydrolyze the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide (AEA), and two prostaglandin glyceryl esters (PG-Gs), which are formed by COX-mediated oxygenation of 2AG. We show that recombinant CES1 and CES2 efficiently hydrolyze 2AG to arachidonic acid (AA) but not amide-containing AEA. Steady-state kinetic parameters for CES1- and CES2-mediated 2AG hydrolysis were, respectively, kcat, 59 and 43 min(-1); Km, 49 and 46 µM; and kcat/Km, 1.2 and 0.93 µM(-1) min(-1). kcat/Km values are comparable to published values for rat monoacylglycerol lipase (MAGL)-catalyzed 2AG hydrolysis. Furthermore, we show that CES1 and CES2 also efficiently hydrolyze PGE2-G and PGF2α-G. In addition, when cultured human THP1 macrophages were treated with exogenous 2AG or PG-G (10 µM, 1 h), significant quantities of AA or PGs were detected in the culture medium; however, the ability of macrophages to metabolize these compounds was inhibited (60-80%) following treatment with paraoxon, the toxic metabolite of the insecticide parathion. Incubation of THP1 cell lysates with small-molecule inhibitors targeting CES1 (thieno[3,2-e][1]benzothiophene-4,5-dione or JZL184) significantly reduced lipid glyceryl ester hydrolase activities (40-50% for 2AG and 80-95% for PG-Gs). Immunodepletion of CES1 also markedly reduced 2AG and PG-G hydrolase activities. These results suggested that CES1 is in part responsible for the hydrolysis of 2AG and PG-Gs in THP1 cells, although it did not rule out a role for other hydrolases, especially with regard to 2AG metabolism since a substantial portion of its hydrolysis was not inactivated by the inhibitors. An enzyme (Mr 31-32 kDa) of unknown function was detected by serine hydrolase activity profiling of THP1 cells and may be a candidate. Finally, the amounts of in situ generated 2AG and PG-Gs in macrophages were enhanced by treating the cells with bioactive metabolites of OP insecticides. Collectively, the results suggest that in addition to MAGL and fatty-acid amide hydrolase (FAAH), which have both been documented to terminate endocannabinoid signaling, CES may also have a role. Furthermore, since PG-Gs have been shown to possess biological activities in their own right, CES may represent an important enzyme class that regulates their in vivo levels.
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Carboxilesterasa/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Insecticidas/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Monocitos/efectos de los fármacos , Paraoxon/toxicidad , Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Carboxilesterasa/genética , Hidrolasas de Éster Carboxílico/genética , Células Cultivadas , Cromatografía Líquida de Alta Presión , Endocannabinoides , Glicéridos/metabolismo , Humanos , Hidrólisis , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Espectrometría de Masas , Monocitos/inmunología , Alcamidas Poliinsaturadas/metabolismo , Prostaglandinas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Two major isoforms of human carboxylesterases (CEs) are found in metabolically active tissues, CES1 and CES2. These hydrolytic enzymes are involved in xenobiotic and endobiotic metabolism. CES1 is abundantly expressed in human liver and monocytes/macrophages, including the THP1 cell line; CES2 is expressed in liver but not in monocytes/macrophages. The cholesteryl ester hydrolysis activity in human macrophages has been attributed to CES1. Here, we report the direct inhibitory effects of several endogenous oxysterols and fatty acids on the CE activity of THP1 monocytes/macrophages and recombinant human CES1 and CES2. Using THP1 whole-cell lysates we found: (1) 27-hydroxycholesterol (27-HC) is a potent inhibitor of carboxylesterase activity (IC50=33 nM); (2) 24(S),25-epoxycholesterol had moderate inhibitory activity (IC(50)=8.1 microM); and (3) cholesterol, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 25-hydroxycholesterol each had little inhibitory activity. 27-HC was a partially noncompetitive inhibitor of recombinant CES1 (K(iapp)=10 nM) and impaired intracellular CES1 activity following treatment of intact THP1 cells. In contrast, recombinant CES2 activity was not inhibited by 27-HC, suggesting isoform-selective inhibition by 27-HC. Furthermore, unsaturated fatty acids were better inhibitors of CES1 activity than saturated fatty acids, while CES2 activity was unaffected by any fatty acid. Arachidonic acid (AA) was the most potent fatty acid inhibitor of recombinant CES1 and acted by a noncompetitive mechanism (K(iapp)=1.7 microM); when not complexed to albumin, exogenous AA penetrated intact THP1 cells and inhibited CES1. Inhibition results are discussed in light of recent structural models for CES1 that describe ligand binding sites separate from the active site. In addition, oxysterol-mediated inhibition of CES1 activity was demonstrated by pretreatment of human liver homogenates or intact THP1 cells with exogenous 27-HC, which resulted in significantly reduced hydrolysis of the pyrethroid insecticide bioresmethrin, a CES1-specific xenobiotic substrate. Collectively, these findings suggest that CE activity of recombinant CES1, cell lysates, and intact cells can be impaired by naturally occurring lipids, which may compromise the ability of CES1 to both detoxify environmental pollutants and metabolize endogenous compounds in vivo.