RESUMEN
Epidemic obesity is the most important risk factor for prediabetes and type 2 diabetes (T2D) in youth as it is in adults. Obesity shares pathophysiological mechanisms with T2D and is likely to share part of the genetic background. We aimed to test if weighted genetic risk scores (GRSs) for T2D, fasting glucose (FG) and fasting insulin (FI) predict glycaemic traits and if there is a causal relationship between obesity and impaired glucose metabolism in children and adolescents. Genotyping of 42 SNPs established by genome-wide association studies for T2D, FG and FI was performed in 1660 Italian youths aged between 2 and 19 years. We defined GRS for T2D, FG and FI and tested their effects on glycaemic traits, including FG, FI, indices of insulin resistance/beta cell function and body mass index (BMI). We evaluated causal relationships between obesity and FG/FI using one-sample Mendelian randomization analyses in both directions. GRS-FG was associated with FG (beta = 0.075 mmol/l, SE = 0.011, P = 1.58 × 10-11) and beta cell function (beta = -0.041, SE = 0.0090 P = 5.13 × 10-6). GRS-T2D also demonstrated an association with beta cell function (beta = -0.020, SE = 0.021 P = 0.030). We detected a causal effect of increased BMI on levels of FI in Italian youths (beta = 0.31 ln (pmol/l), 95%CI [0.078, 0.54], P = 0.0085), while there was no effect of FG/FI levels on BMI. Our results demonstrate that the glycaemic and T2D risk genetic variants contribute to higher FG and FI levels and decreased beta cell function in children and adolescents. The causal effects of adiposity on increased insulin resistance are detectable from childhood age.
Asunto(s)
Diabetes Mellitus Tipo 2 , Resistencia a la Insulina , Adolescente , Adulto , Glucemia/metabolismo , Niño , Preescolar , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Estudio de Asociación del Genoma Completo , Glucosa , Homeostasis , Humanos , Insulina/metabolismo , Resistencia a la Insulina/genética , Obesidad/epidemiología , Obesidad/genética , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Adulto JovenRESUMEN
BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), a chronic liver disease in children, ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). We investigated the role of Angiopoietin-2 (Ang-2) as a biomarker for pediatric NAFLD-related liver damage. METHODS: We assessed the plasma levels of Ang-2 and cytokeratin-18 (CK18) fragments and their association with histologic activity in 76 children with NAFLD and 28 controls. RESULTS: The mean plasma levels of Ang-2 and CK18 were higher in children with NAFLD than in age-matched controls (Ang-2 155.4 ± 72.5 vs 7.5 ± 2.3 ng/mL, p < 0.001; CK18 390.4 ± 145.6 vs 193.9 ± 30.8 IU/L, p < 0.001). Ang-2 was significantly increased (p < 0.0001) in children with NASH (N = 41) while CK18 was significantly increased (p = 0.002) in children with fibrosis (N = 47). Ang-2 levels accurately predicted NASH (AUROC 0.911; 95% CI 0.844-0.979; p < 0.0001), while CK18 predicted both NASH (AUROC 0.827; 95% CI 0.735-0.919; p < 0.0001) and fibrosis (AUROC 0.724; 95% CI 0.611-0.837; p = 0.001). Ang-2 and CK18 in combination were good predictors of NASH with a sensitivity of 71.4% and a specificity of 100%. CONCLUSIONS: In conclusion, our data suggested Ang-2 as a suitable biomarker of NASH in the pediatric population. However, our findings need external validation in other cohorts. IMPACT: Several circulating factors have been extensively studied as potential biomarkers for NASH. Angiopoietin-2 circulating levels are increased in children with NAFLD and are associated with NASH. Angiopoietin-2 levels are more efficient than CK18 levels at assessing the most severe form of disease, and the combining of these two biomarkers reached a positive predictive value of 100% for NASH.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Proteínas de Transporte Vesicular/sangre , Angiopoyetina 2 , Biomarcadores , Niño , Humanos , Hígado/patología , Cirrosis Hepática/complicaciones , Enfermedad del Hígado Graso no Alcohólico/diagnóstico , Valor Predictivo de las PruebasRESUMEN
Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker ß-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.
Asunto(s)
Antígenos de Neoplasias/genética , Proteínas Ribosómicas/genética , Neoplasias Gástricas/genética , Ubiquitina/genética , Ubiquitinas/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Factores de Transcripción del Choque Térmico/antagonistas & inhibidores , Factores de Transcripción del Choque Térmico/genética , Humanos , Metástasis de la Neoplasia , ARN Mensajero , ARN Interferente Pequeño/genética , Factor de Transcripción Sp1/antagonistas & inhibidores , Factor de Transcripción Sp1/genética , Neoplasias Gástricas/patologíaRESUMEN
Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1; enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.
Asunto(s)
Liraglutida/farmacología , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Fármacos Neuroprotectores/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis , Línea Celular Tumoral , Desoxiglucosa/toxicidad , Silenciador del Gen , Humanos , Insulina/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Piruvaldehído/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Prolyl isomerases (Peptidylprolyl isomerase, PPIases) are enzymes that catalyze the isomerization between the cis/trans Pro conformations. Three subclasses belong to the class: FKBP (FK506 binding protein family), Cyclophilin and Parvulin family (Pin1 and Par14). Among Prolyl isomerases, Pin1 presents as distinctive feature, the ability of binding to the motif pSer/pThr-Pro that is phosphorylated by kinases. Modulation of Pin1 is implicated in cellular processes such as mitosis, differentiation and metabolism: The enzyme is dysregulated in many diverse pathological conditions, i.e., cancer progression, neurodegenerative (i.e., Alzheimer's diseases, AD) and metabolic disorders (i.e., type 2 diabetes, T2D). Indeed, Pin1 KO mice develop a complex phenotype of premature aging, cognitive impairment in elderly mice and neuronal degeneration resembling that of the AD in humans. In addition, since the molecule modulates glucose homeostasis in the brain and peripherally, Pin1 KO mice are resistant to diet-induced obesity, insulin resistance, peripheral glucose intolerance and diabetic vascular dysfunction. In this review, we revise first critically the role of Pin1 in neuronal development and differentiation and then focus on the in vivo studies that demonstrate its pivotal role in neurodegenerative processes and glucose homeostasis. We discuss evidence that enables us to speculate about the role of Pin1 as molecular link in the pathogenesis of type 3 diabetes i.e., the clinical association of dementia/AD and T2D.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus/genética , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Isomerasa de Peptidilprolil/genética , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Ciclofilinas/genética , Diabetes Mellitus/patología , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/patología , Humanos , Neuronas/enzimología , Neuronas/patología , Isomerasa de Peptidilprolil/clasificación , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
The mitochondrial Elongation Factor Tu (EF-Tu), encoded by the TUFM gene, is a highly conserved GTPase, which is part of the mitochondrial protein translation machinery. In its activated form it delivers the aminoacyl-tRNAs to the A site of the mitochondrial ribosome. We report here on a baby girl with severe infantile macrocystic leukodystrophy with micropolygyria and a combined defect of complexes I and IV in muscle biopsy, caused by a novel mutation identified in TUFM. Using human mutant cells and the yeast model, we demonstrate the pathological role of the novel variant. Moreover, results of a molecular modeling study suggest that the mutant is inactive in mitochondrial polypeptide chain elongation, probably as a consequence of its reduced ability to bind mitochondrial aa-tRNAs. Four patients have so far been described with mutations in TUFM, and, following the first description of the disease in a single patient, we describe similar clinical and neuroradiological features in an additional patient.
Asunto(s)
Secuencia de Bases , ADN Mitocondrial/genética , Leucoencefalopatías/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Extensión de la Cadena Peptídica de Translación , Factor Tu de Elongación Peptídica/genética , Eliminación de Secuencia , ADN Mitocondrial/metabolismo , Femenino , Humanos , Leucoencefalopatías/metabolismo , Masculino , Mitocondrias/patología , Proteínas Mitocondriales/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neuroblastoma/metabolismo , Neuronas/metabolismo , Diferenciación Celular/fisiología , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Humanos , Proteínas de la Membrana/metabolismo , Neuroblastoma/tratamiento farmacológico , Neuronas/citología , Neuronas/efectos de los fármacos , Tretinoina/farmacologíaRESUMEN
Epigenetics plays a pivotal role in regulating gene expression in development, in response to cellular stress or in disease states, in virtually all cell types. MicroRNAs (miRNAs) are short, non-coding RNA molecules that mediate RNA silencing and regulate gene expression. miRNAs were discovered in 1993 and have been extensively studied ever since. They can be expressed in a tissue-specific manner and play a crucial role in tissue development and many biological processes. miRNAs are responsible for changes in the cell epigenome because of their ability to modulate gene expression post-transcriptionally. Recently, numerous studies have shown that miRNAs and other epigenetic factors can regulate each other or cooperate in regulating several biological processes. On the one hand, the expression of some miRNAs is silenced by DNA methylation, and histone modifications have been demonstrated to modulate miRNA expression in many cell types or disease states. On the other hand, miRNAs can directly target epigenetic factors, such as DNA methyltransferases or histone deacetylases, thus regulating chromatin structure. Moreover, several studies have reported coordinated actions between miRNAs and other epigenetic mechanisms to reinforce the regulation of gene expression. This paper reviews multiple interactions between miRNAs and epigenetic factors in skeletal muscle development and in response to stimuli or disease.
Asunto(s)
Adaptación Biológica , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/genética , Desarrollo de Músculos/genética , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Acetilación , Animales , Metilación de ADN , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Humanos , MetilaciónRESUMEN
BACKGROUND: The encephalomyopathic mtDNA depletion syndrome with methylmalonic aciduria is associated with deficiency of succinate-CoA ligase, caused by mutations in SUCLA2 or SUCLG1. We report here 25 new patients with succinate-CoA ligase deficiency, and review the clinical and molecular findings in these and 46 previously reported patients. PATIENTS AND RESULTS: Of the 71 patients, 50 had SUCLA2 mutations and 21 had SUCLG1 mutations. In the newly-reported 20 SUCLA2 patients we found 16 different mutations, of which nine were novel: two large gene deletions, a 1 bp duplication, two 1 bp deletions, a 3 bp insertion, a nonsense mutation and two missense mutations. In the newly-reported SUCLG1 patients, five missense mutations were identified, of which two were novel. The median onset of symptoms was two months for patients with SUCLA2 mutations and at birth for SUCLG1 patients. Median survival was 20 years for SUCLA2 and 20 months for SUCLG1. Notable clinical differences between the two groups were hepatopathy, found in 38% of SUCLG1 cases but not in SUCLA2 cases, and hypertrophic cardiomyopathy which was not reported in SUCLA2 patients, but documented in 14% of cases with SUCLG1 mutations. Long survival, to age 20 years or older, was reported in 12% of SUCLA2 and in 10% of SUCLG1 patients. The most frequent abnormality on neuroimaging was basal ganglia involvement, found in 69% of SUCLA2 and 80% of SUCLG1 patients. Analysis of respiratory chain enzyme activities in muscle generally showed a combined deficiency of complexes I and IV, but normal histological and biochemical findings in muscle did not preclude a diagnosis of succinate-CoA ligase deficiency. In five patients, the urinary excretion of methylmalonic acid was only marginally elevated, whereas elevated plasma methylmalonic acid was consistently found. CONCLUSIONS: To our knowledge, this is the largest study of patients with SUCLA2 and SUCLG1 deficiency. The most important findings were a significantly longer survival in patients with SUCLA2 mutations compared to SUCLG1 mutations and a trend towards longer survival in patients with missense mutations compared to loss-of-function mutations. Hypertrophic cardiomyopathy and liver involvement was exclusively found in patients with SUCLG1 mutations, whereas epilepsy was much more frequent in patients with SUCLA2 mutations compared to patients with SUCLG1 mutations. The mutation analysis revealed a number of novel mutations, including a homozygous deletion of the entire SUCLA2 gene, and we found evidence of two founder mutations in the Scandinavian population, in addition to the known SUCLA2 founder mutation in the Faroe Islands.
Asunto(s)
Codón sin Sentido/genética , Enfermedades Mitocondriales/genética , Mutación Missense/genética , Succinato-CoA Ligasas/genética , Adolescente , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/genética , Secuencia de Aminoácidos , Niño , Preescolar , Análisis Mutacional de ADN/métodos , ADN Mitocondrial/genética , Femenino , Genotipo , Humanos , Lactante , Recién Nacido , Masculino , Ácido Metilmalónico/metabolismo , Encefalomiopatías Mitocondriales/genética , Fenotipo , Adulto JovenRESUMEN
Leber's hereditary optic neuropathy (LHON) is associated with mitochondrial DNA (mtDNA) ND mutations that are mostly homoplasmic. However, these mutations are not sufficient to explain the peculiar features of penetrance and the tissue-specific expression of the disease and are believed to be causative in association with unknown environmental or other genetic factors. Discerning between clear-cut pathogenetic variants, such as those that appear to be heteroplasmic, and less penetrant variants, such as the homoplasmic, remains a challenging issue that we have addressed here using next-generation sequencing approach. We set up a protocol to quantify MTND5 heteroplasmy levels in a family in which the proband manifests a LHON phenotype. Furthermore, to study this mtDNA haplotype, we applied the cybridization protocol. The results demonstrate that the mutations are mostly homoplasmic, whereas the suspected heteroplasmic feature of the observed mutations is due to the co-amplification of Nuclear mitochondrial Sequences.
Asunto(s)
Núcleo Celular/genética , ADN Mitocondrial/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mitocondrias/genética , Mutación/genética , Atrofia Óptica Hereditaria de Leber/genética , Adenosina Trifosfato/biosíntesis , Adolescente , Adulto , Complejo I de Transporte de Electrón/genética , Metabolismo Energético , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Persona de Mediana Edad , Proteínas Mitocondriales/genética , Linaje , Temperatura , Adulto JovenRESUMEN
Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder caused by biallelic mutations in the Ataxia Telangiectasia-mutated gene. A-T shows a complex phenotype ranging from early-onset progressive neurodegeneration to immunodeficiencies, high incidence of infections, and tumors. Unfortunately, no therapy is up to now available for treating this condition. Recently, the short term treatment of ataxia-telangiectasia patients with glucocorticoids was shown to improve their neurological symptoms and possibly reverse cerebellar atrophy. Thus, corticosteroids represent an attractive approach for the treatment of this neurodegenerative disease. However, the molecular mechanism involved in glucocorticoid action in A-T is yet unknown. The aim of our work is to construct cDNA libraries containing those genes which are transactivated by the glucocorticoid analogue, dexamethasone, in A-T human cells. For this purpose, suppression subtractive hybridization has been performed on ATM-null lymphoblastoid cell transcriptome extracted following drug administration. Annotation of whole genes contained in the libraries has been obtained by coupling subtractive hybridization with microarray analysis. Positive transcripts have been validated by quantitative PCR. Through in silico analyses, identified genes have been classified on the basis of the pathway in which they are involved, being able to address signaling required for dexamethasone action. Most of the induced transcripts are involved in metabolic processes and regulation of cellular processes. Our results can help to unravel the mechanism of glucocorticoid action in the reversion of A-T phenotype. Moreover, the induction of a specific region of the ATM transcript has been identified as putative biomarker predictive of dexamethasone efficacy on ataxic patients.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Dexametasona/farmacología , Activación Transcripcional/efectos de los fármacos , Ataxia Telangiectasia/patología , Células Cultivadas , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Hibridación de Ácido Nucleico , Reacción en Cadena en Tiempo Real de la Polimerasa , Técnica de SustracciónRESUMEN
Ataxia telangiectasia (AT) is a rare genetic disease, still incurable, resulting from biallelic mutations in the ataxia telangiectasia-mutated (ATM) gene. Recently, short term treatment with glucocorticoid analogues improved neurological symptoms characteristic of this syndrome. Nevertheless, the molecular mechanism involved in glucocorticoid action in AT patients is not yet known. Here we describe, for the first time in mammalian cells, a short direct repeat-mediated noncanonical splicing event induced by dexamethasone, which leads to the skipping of mutations upstream of nucleotide residue 8450 of ATM coding sequence. The resulting transcript provides an alternative ORF translated in a new ATM variant with the complete kinase domain. This miniATM variant was also highlighted in lymphoblastoid cell lines from AT patients and was shown to be likely active. In conclusion, dexamethasone treatment may partly restore ATM activity in ataxia telangiectasia cells by a new molecular mechanism that overcomes most of the mutations so far described within this gene.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Dexametasona/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Alelos , Empalme Alternativo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Codón , Cicloheximida/farmacología , Proteínas Fúngicas , Variación Genética , Genotipo , Glucocorticoides/metabolismo , Células HeLa , Humanos , Linfocitos/metabolismo , Modelos Genéticos , Mutación , Sistemas de Lectura Abierta , Fosfotransferasas/metabolismo , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Endonucleasas Específicas del ADN y ARN con un Solo FilamentoRESUMEN
Recessive mutations in the mitochondrial arginyl-transfer RNA synthetase (RARS2) gene have been associated with early onset encephalopathy with signs of oxidative phosphorylation defects classified as pontocerebellar hypoplasia 6. We describe clinical, neuroimaging and molecular features on five patients from three unrelated families who displayed mutations in RARS2. All patients rapidly developed a neonatal or early-infantile epileptic encephalopathy with intractable seizures. The long-term follow-up revealed a virtual absence of psychomotor development, progressive microcephaly, and feeding difficulties. Mitochondrial respiratory chain enzymes in muscle and fibroblasts were normal in two. Blood and CSF lactate was abnormally elevated in all five patients at early stages while appearing only occasionally abnormal with the progression of the disease. Cerebellar vermis hypoplasia with normal aspect of the cerebral and cerebellar hemispheres appeared within the first months of life at brain MRI. In three patients follow-up neuroimaging revealed a progressive pontocerebellar and cerebral cortical atrophy. Molecular investigations of RARS2 disclosed the c.25A>G/p.I9V and the c.1586+3A>T in family A, the c.734G>A/p.R245Q and the c.1406G>A/p.R469H in family B, and the c.721T>A/p.W241R and c.35A>G/p.Q12R in family C. Functional complementation studies in Saccharomyces cerevisiae showed that mutation MSR1-R531H (equivalent to human p.R469H) abolished respiration whereas the MSR1-R306Q strain (corresponding to p.R245Q) displayed a reduced growth on non-fermentable YPG medium. Although mutations functionally disrupted yeast we found a relatively well preserved arginine aminoacylation of mitochondrial tRNA. Clinical and neuroimaging findings are important clues to raise suspicion and to reach diagnostic accuracy for RARS2 mutations considering that biochemical abnormalities may be absent in muscle biopsy.
Asunto(s)
Arginino-ARNt Ligasa/genética , Mutación , Atrofias Olivopontocerebelosas/enzimología , Atrofias Olivopontocerebelosas/genética , Cerebelo/enzimología , Cerebelo/patología , Cerebelo/fisiología , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Recién Nacido , Discapacidad Intelectual/sangre , Discapacidad Intelectual/líquido cefalorraquídeo , Discapacidad Intelectual/genética , Ácido Láctico/sangre , Ácido Láctico/líquido cefalorraquídeo , Síndrome de Lennox-Gastaut , Imagen por Resonancia Magnética/métodos , Masculino , Microcefalia/sangre , Microcefalia/líquido cefalorraquídeo , Microcefalia/genética , Mitocondrias/genética , Neuroimagen/métodos , Atrofias Olivopontocerebelosas/diagnóstico , Atrofias Olivopontocerebelosas/metabolismo , Trastornos Psicomotores/genética , Convulsiones/sangre , Convulsiones/líquido cefalorraquídeo , Convulsiones/genética , Espasmos Infantiles/sangre , Espasmos Infantiles/líquido cefalorraquídeo , Espasmos Infantiles/genéticaRESUMEN
INTRODUCTION: We have previously described the preclinical developments in enzyme-loaded red blood cells to be used in the treatment of several rare diseases, as well as in chronic conditions. AREA COVERED: Since our previous publication we have seen further progress in the previously discussed approaches and, interestingly enough, in additional new studies that further strengthen the idea that red blood cell-based therapeutics may have unique advantages over conventional enzyme replacement therapies in terms of efficacy and safety. Here we highlight these investigations and compare, when possible, the reported results versus the current therapeutic approaches. EXPERT OPINION: The continuous increase in the number of new potential applications and the progress from the encapsulation of a single enzyme to the engineering of an entire metabolic pathway open the field to unexpected developments and confirm the role of red blood cells as cellular bioreactors that can be conveniently manipulated to acquire useful therapeutic metabolic abilities. Positioning of these new approaches versus newly approved drugs is essential for the successful transition of this technology from the preclinical to the clinical stage and hopefully to final approval.
Asunto(s)
Sistemas de Liberación de Medicamentos , EritrocitosRESUMEN
BACKGROUND: During pregnancy, the balance between pro-inflammatory and anti-inflammatory responses is essential for ensuring healthy outcomes. Dietary Fatty acids may modulate inflammation. METHODS: We investigated the association between dietary fatty acids as profiled on red blood cells membranes and a few pro- and anti-inflammatory cytokines, including the adipokines leptin and adiponectin at ~38 weeks in 250 healthy women. RESULTS: We found a number of associations, including, but not limited to those of adiponectin with C22:3/C22:4 (coeff -1.44; p = 0.008), C18:1 c13/c14 (coeff 1.4; p = 0.02); endotoxin with C20:1 (coeff -0.9; p = 0.03), C22:0 (coeff -0.4; p = 0.05); MCP-1 with C16:0 (coeff 0.8; p = 0.04); and ICAM-1 with C14:0 (coeff -86.8; p = 0.045). Several cytokines including leptin were associated with maternal body weight (coeff 0.9; p = 2.31 × 10-5), smoking habits (i.e., ICAM-1 coeff 133.3; p = 0.09), or gestational diabetes (i.e., ICAM-1 coeff 688; p = 0.06). CONCLUSIONS: In a general cohort of pregnant women, the intake of fatty acids influenced the balance between pro- and anti-inflammatory molecules together with weight gain, smoking habits, and gestational diabetes.
Asunto(s)
Diabetes Gestacional , Leptina , Femenino , Humanos , Embarazo , Molécula 1 de Adhesión Intercelular , Adiponectina , Ácidos Grasos , CitocinasRESUMEN
PURPOSE: Gastric cancers (GC) display histological and molecular differences. This heterogeneity has limited the development of new therapeutic strategies which requires the identification of the molecular players involved in GC pathogenesis and the investigation of their responsiveness to drugs. Several proteasome subunits have been identified as prognostic markers in GC and their role studied by gene knockdown. However, proteasomes are multi-subunit protein complexes co-existing in multiple forms with distinct activity/specificity and ability to change in response to inhibitors. Information on the role of different proteasome particles in cancer and their relevance as therapeutic targets is limited. METHODS: Based on this evidence, subunit assembly into proteasome complexes and activity were investigated by native PAGE followed by immunoblotting, and by using fluorogenic substrates, respectively. RESULTS: Here we show that GC cell lines with epithelial and/or diffuse Lauren's histotype express different levels of immunoproteasome subunits and equal amounts of constitutive counterparts. Immunoproteasome subunits were highly expressed and preferentially assembled into 19S capped complexes in diffuse-type cells, where most of the activity was catalyzed by the 26S and 30S particles. In epithelial cells, activity appeared equally distributed between 19S- and 11S-capped proteolytic particles. This proteasome pattern was associated with higher resistance of diffuse-type cells to proteasome inhibition. Immunoproteasome inhibition by ONX 0914 did not influence cell viability but affected metastatic cell migration. CONCLUSIONS: These results suggest that pharmacological inhibition of the immunoproteasome may be useful in treating metastatic gastric cancers.
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Complejo de la Endopetidasa Proteasomal , Neoplasias Gástricas , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Citoplasma/metabolismo , Línea CelularRESUMEN
Mammalian complex V (F1F0-ATP synthase or ATPase) uses the proton gradient to generate ATP during oxidative phosphorylation and requires several helper proteins, including TMEM70, to form the holoenzyme in a stepwise process in which nuclear DNA is combined with mitochondrial DNA-encoded subunits. We report the clinical and molecular findings in three patients presenting lactic acidosis, 3-methylglutaconic aciduria, and hypertrophic cardiomyopathy. All three showed an isolated defect of fully assembled ATP synthase in association with a "common" (c.317-2A > G) and a new (c.628A > C/p.T210P) variant in TMEM70. Interestingly, one of the patients also showed nitric oxide-responsive pulmonary arterial hypertension, a finding never before associated with TMEM70 deficiency. In addition to widening the clinical and mutational spectrum of defective ATP synthase, our study also suggests that mutant TMEM70 associates in high molecular weight complexes (470-550 kDa) when expressed in Hela cells and exerts a direct action in ATP synthase biogenesis and assembly, mediating the incorporation of F1 moieties.
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Adenosina Trifosfatasas/biosíntesis , Proteínas Portadoras/biosíntesis , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/deficiencia , Mutación , Acidosis Láctica/genética , Acidosis Láctica/metabolismo , Secuencia de Bases , Cardiomiopatía Dilatada/genética , Cardiomiopatía Hipertrófica/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Ataxia Cerebelosa/genética , Niño , Preescolar , Hipertensión Pulmonar Primaria Familiar , Femenino , Fibroblastos/metabolismo , Células HeLa , Humanos , Hipertensión Pulmonar/genética , Lactante , Errores Innatos del Metabolismo/genética , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Piel/metabolismoRESUMEN
The resistance and the birth of new intrinsic and multidrug-resistant pathogenic species like C. auris is creating great concern in the antifungal world. Given the limited drug arsenal and the lack of effectiveness of the available compounds, there is an urgent need for innovative approaches. The murine mAb 2G8 was humanized and engineered in silico to develop a single-chain fragment variable (hscFv) antibody against ß-1,3-glucans which was then expressed in E. coli. Among the recombinant proteins developed, a soluble candidate with high stability and affinity was obtained. This selected protein is VL-linker-VH oriented, and it is characterized by the presence of two ubiquitin monomers at the N-terminus and a His tag at the C-terminus. This construct, Ub2-hscFv-His, guaranteed stability, solubility, efficient purification and satisfactory recovery of the recombinant product. HscFv can bind ß-1,3-glucans both as coated antigens and on C. auris and C. albicans cells similarly to its murine parental and showed long stability and retention of binding ability when stored at 4°, -20° and -80° C. Furthermore, it was efficient in enhancing the antifungal activity of drugs caspofungin and amphotericin B against C. auris. The use of biological drugs as antifungals is limited; here we present a promising hscFv which has the potential to be useful in combination with currently available antifungal drugs.
Asunto(s)
Antifúngicos , Micosis , Ratones , Animales , Antifúngicos/farmacología , Escherichia coli , Anfotericina B , Glucanos , Pruebas de Sensibilidad MicrobianaRESUMEN
Objective: Hyaluronic acid (HA) and N-terminal propeptide of type III procollagen (PIIINP) are two non-invasive biomarkers of liver fibrosis in non-alcoholic fatty liver disease (NAFLD). We examined the relationships of plasma levels of HA and PIIINP with kidney function in children with NAFLD. Methods: Plasma HA and PIIINP levels were measured using two commercially available enzyme-linked immunosorbent assay kits in a cohort of 106 Caucasian overweight or obese children with biopsy-proven NAFLD. Glomerular filtration rate (eGFR) was estimated using the Bedside Schwartz equation. Genotyping for the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 variant was performed using an allelic discrimination assay. Results: Children with fibrosis F2 had significantly higher plasma PIIINP and HA levels than those with F0 or F1 fibrosis. Liver fibrosis was positively associated with plasma HA and PIIINP, as well as with the presence of the risk allele G of PNPLA3 rs738409 variant, and negatively with eGFR. Moreover, eGFR showed significant inverse associations with HA and PIIINP levels, as well as the presence of G of PNPLA3 rs738409, and liver fibrosis stage. Notably, our multivariable regression models showed that higher plasma PIIINP (standardized beta coefficient: -0.206, P = 0.011) and HA levels (standardized beta coefficient: -0.531, P < 0.0001) were associated with lower eGFR values, even after adjustment for age, sex, systolic blood pressure, PNPLA3 rs738409 genotype, and any stage of liver fibrosis. Conclusions: Higher levels of HA and PIIINP were associated with lower eGFR values in Caucasian children with biopsy-proven NAFLD, independently of PNPLA3 rs738409 genotype and other potential confounding factors.
RESUMEN
The SARS-CoV-2 pandemic has caused approximately 6.3 million deaths, mainly due to the acute respiratory distress syndrome or multi-organ failure that characterizes COVID-19 acute disease. Post-acute COVID-19 syndrome, also known as long-COVID, is a condition characterized by a complex of symptoms that affects 10-20% of the individuals who have recovered from the infection. Scientific and clinical evidence demonstrates that long-COVID can develop in both adults and children. It has been hypothesized that multi-organ effects of long-COVID could be associated with the persistence of virus RNA/proteins in host cells, but the real mechanism remains to be elucidated. Therefore, we sought to determine the effects of the exogenous expression of the papain-like protease (PLpro) domain of the non-structural protein (NSP3) of SARS-CoV-2 in polarized human airway (Calu-3), intestinal (Caco-2), and liver (HepG2) epithelial cells, and to evaluate the ability of the natural antioxidant hydroxytyrosol (HXT) in neutralizing these effects. Our results demonstrated that PLpro was able to induce a cascade of inflammatory genes and proteins (mainly associated with the interferon pathway) and increase the apoptotic rate and expression of several oxidative stress markers in all evaluated epithelial cells. Noteably, the treatment with 10 µM HXT reverted PL-pro-dependent effects almost completely. This study provides the first evidence that SARS-CoV-2 PLpro remaining in host cells after viral clearance may contribute to the pathogenetic mechanisms of long-COVID. These effects may be counteracted by natural antioxidants. Further clinical and experimental studies are necessary to confirm this hypothesis.