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Among the many lysosomal storage disorders (LSDs) that would benefit from the establishment of novel cell models, either patient-derived or genetically engineered, is mucopolysaccharidosis type II (MPS II). Here, we present our results on the establishment and characterization of two MPS II patient-derived stem cell line(s) from deciduous baby teeth. To the best of our knowledge, this is the first time a stem cell population has been isolated from LSD patient samples obtained from the dental pulp. Taking into account our results on the molecular and biochemical characterization of those cells and the fact that they exhibit visible and measurable disease phenotypes, we consider these cells may qualify as a valuable disease model, which may be useful for both pathophysiological assessments and in vitro screenings. Ultimately, we believe that patient-derived dental pulp stem cells (DPSCs), particularly those isolated from human exfoliated deciduous teeth (SHEDs), may represent a feasible alternative to induced pluripotent stem cells (iPSCs) in many labs with standard cell culture conditions and limited (human and economic) resources.
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Enfermedades por Almacenamiento Lisosomal , Mucopolisacaridosis II , Humanos , Células Madre , Línea Celular , Diente Primario , Lisosomas , Pulpa Dental , Diferenciación Celular/fisiología , Proliferación CelularRESUMEN
INTRODUCTION: When it comes to disease modeling, countless models are available for Lysosomal Storage Diseases (LSD). Historically, two major approaches are well-established: in vitro assessments are performed in patient fibroblasts, while in vivo pre-clinical studies are performed in mouse models. Still, both platforms have a series of drawbacks. Thus, we implemented two alternative and innovative protocols to mimic a particular sub-group of LSDs, the Mucopolysaccharidoses both in vitro and in vivo. METHODS: The first one relies on a non-invasive approach using dental pulp stem cells from deciduous teeth (SHEDs). SHEDs are multipotent neuronal precursors that can easily be collected. The second uses a state-of-the-art gene editing technology (CRISPR/Cas9) to generate zebrafish disease models. RESULTS: Even though this is an ongoing project, we have already established and characterized two MPS II and one MPS VI SHED cell models. These cells self-maintain through several passages and can give rise to a variety of cells including neurons. Furthermore, all MPS-associated sub-cellular phenotypes we have assessed so far are easily observable in these cells. Regarding our zebrafish models, we have successfully knocked down both naglu and hgsnat and the first results we got from the behavioral analysis are promising ones, as we can observe altered activity and sleep patterns in the genetically modified fish. For this particular approach we chose MPS III forms as our target disorders, since their neurological features (hyperactivity, seizures and motor impairment) and lifespan decrease would be easily recognizable in zebrafish. CONCLUSION: Now that these methods are well-established in our lab, their potential is immense. On one hand, the newly developed models will be of ultimate value to understand the mechanisms underlying MPS sub-cellular pathology, which have to be further elucidated. On the other hand, they will constitute an optimal platform for drug testing in house. Also noteworthy, our models will be published as lab resources and made available for the whole LSD community.
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BACKGROUND: Fabry disease (FD) is a treatable cause of hypertrophic cardiomyopathy (HCM). We aimed to determine the independent predictors of FD and to define a clinically useful strategy to discriminate FD among HCM. METHODS: Multicenter study including 780 patients with the ESC definition of HCM. FD screening was performed by enzymatic assay in males and genetic testing in females. Multivariate regression analysis identified independent predictors of FD in HCM. A discriminant function analysis defined a score based on the weighted combination of these predictors. RESULTS: FD was found in 37 of 780 patients with HCM (4.7%): 31 with p.F113L mutation due to a founder effect; and 6 with other variants (p.C94S; p.M96V; p.G183V; p.E203X; p.M290I; p.R356Q/p.G360R). FD prevalence in HCM adjusted for the founder effect was 0.9%. Symmetric HCM (OR 3.464, CI95% 1.151-10.430), basal inferolateral late gadolinium enhancement (LGE) (OR 10.677, CI95% 3.633-31.380), bifascicular block (OR 10.909, CI95% 2.377-50.059) and ST-segment depression (OR 4.401, CI95% 1.431-13.533) were independent predictors of FD in HCM. The score ID FABRY-HCM [-0.729â¯+â¯(2.781xBifascicular block)â¯+â¯(0.590xST depression)â¯+â¯(0.831xSymmetric HCM)â¯+â¯(2.130xbasal inferolateral LGE)] had a negative predictive value of 95.8% for FD, with a cut-off of 1.0, meaning that, in the absence of both bifascicular block and basal inferolateral LGE, FD is a less probable cause of HCM, being more appropriate to perform HCM gene panel than targeted FD screening. CONCLUSION: FD prevalence in HCM was 0.9%. Bifascicular block and basal inferolateral LGE were the most powerful predictors of FD in HCM. In their absence, HCM gene panel is the most appropriate step in etiological study of HCM.
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Cardiomiopatía Hipertrófica/etiología , Enfermedad de Fabry/complicaciones , Enfermedad de Fabry/diagnóstico , Adulto , Anciano , Enfermedad de Fabry/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , LinajeRESUMEN
Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.
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Exones , Fibroblastos/metabolismo , Mucolipidosis/terapia , Mutación , Oligonucleótidos Antisentido/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores , Secuencia de Aminoácidos , Estudios de Casos y Controles , Humanos , Mucolipidosis/genética , Mucolipidosis/patología , Fenotipo , Homología de Secuencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
BACKGROUND: Knowledge on clinical profiles of late-onset phenotypes of Fabry disease (FD) is essential to better define their natural history. Our study aims to demonstrate a founder effect of FD due to the GLA gene mutation c.337T>C (p.F113L) in the Portuguese region of Guimarães; and to characterize the clinical profile of this late-onset phenotype in a large cohort of genetically related adult patients, living in the same region. METHODS AND RESULTS: FD screening was performed in 150 adult patients with hypertrophic cardiomyopathy (HCM) and found 25 Fabry patients (16.6%). The p.F113L mutation was found in 21 of them, leading to a genealogy study and haplotype analysis of the p.F113L patients. Genealogy research revealed a 12-generation family tree with a common ancestor to p.F113L patients, suggesting a founder effect that was supported by haplotype findings. Pedigree analysis was performed and 120 consecutive p.F113L patients underwent a predefined diagnostic evaluation of FD multiorgan involvement. This late-onset phenotype was characterized by common and/or potentially severe cardiac manifestations (left ventricular hypertrophy 40.8%, atrial fibrillation 5%, non-sustained ventricular tachycardia 12.5%, atrioventricular block 18.3%, bifascicular block 13.4%). Extracardiac manifestations included albuminuria>30â¯mg/24â¯h 36.1%, chronic kidney disease≥G3 7.6%, brain white matter lesions 54.4%, stroke 3.3%, sensorineural deafness 44.5%, cornea verticillata 13.9%. Plasma lyso-GB3 was undetectable in females, regardless of clinical manifestations. CONCLUSION: A founder effect of FD due to p.F113L mutation was documented by genealogy and genetics in a Portuguese region. In this late-onset phenotype, although cardiac manifestations carry the highest prognostic impact, extracardiac involvement is common.
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Enfermedad de Fabry/genética , Efecto Fundador , Mutación , Fenotipo , alfa-Galactosidasa/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Cardiomiopatía Hipertrófica/complicaciones , Estudios de Cohortes , Femenino , Humanos , Enfermedades de Inicio Tardío , Masculino , Persona de Mediana Edad , Portugal , Adulto JovenRESUMEN
BACKGROUND: Sporadic Parkinson's disease (PD) patients have lower α-galactosidase A (α-GAL A) enzymatic activity and Fabry disease (FD) patients potentially carry an increased risk of PD. OBJECTIVE: Determination of PD prevalence in FD and clinical, biochemical and vascular neuroimaging description of FD pedigrees with concomitant PD. METHODS: Clinical screening for PD in 229 FD patients belonging to 31 families, harbouring GLA gene mutation p.F113L, and subsequent pedigree analysis. Gender-stratified comparison of FD+/PD+ patients with their family members with FD but without PD (FD+/PD-) regarding Mainz scores, plasma & leukocytes α-GAL A enzymatic activity, urinary Gb3 and plasma Lyso-Gb3, vascular brain neuroimaging. RESULTS: Prevalence of PD in FD was 1.3% (3/229) (3% in patients aged ≥50 years). Three FD patients, one female (73 years old) (P1) and two males (60 and 65 years old) (P2 and P3), three different pedigrees, presented akinetic-rigid PD, with weak response to levodopa (16% - 36%), and dopaminergic deficiency on 18F-DOPA PET. No pathogenic mutations were found in a PD gene panel. FD+/PD+ patients had worse clinical severity of FD (above upper 75% IQR in Mainz scores), and cortico-subcortical white matter/small vessel lesions. P3 patient was under enzyme therapy, started 1 year before PD diagnosis. P2-P3 patients had higher leucocyte α-GAL A activity (2,2-3 vs.1,0 (median)(nmol/h/mg)). CONCLUSION: We have shown a high prevalence of PD in a late-onset phenotype of FD, presenting high cerebrovascular burden and weak response to levodopa. Further studies will untangle how much of this PD phenotype is due to Gb3 deposition versus cerebrovascular lesions in the nigro-striatal network.
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Encéfalo/diagnóstico por imagen , Enfermedad de Fabry , Glucolípidos/metabolismo , Leucocitos/enzimología , Enfermedad de Parkinson , Esfingolípidos/metabolismo , alfa-Galactosidasa/metabolismo , Adulto , Anciano , Estudios de Cohortes , Comorbilidad , Enfermedad de Fabry/diagnóstico por imagen , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/epidemiología , Enfermedad de Fabry/fisiopatología , Femenino , Glucolípidos/sangre , Glucolípidos/orina , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Neuroimagen , Enfermedad de Parkinson/diagnóstico por imagen , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/fisiopatología , Linaje , Fenotipo , Prevalencia , Esfingolípidos/sangre , Esfingolípidos/orina , alfa-Galactosidasa/sangre , alfa-Galactosidasa/genéticaRESUMEN
Several diseases are caused by inherited defects in lysosomes, the so-called lysosomal storage disorders (LSDs). In some of these LSDs, tissue macrophages transform into prominent storage cells, as is the case in Gaucher disease. Here, macrophages become the characteristic Gaucher cells filled with lysosomes laden with glucosylceramide, because of their impaired enzymatic degradation. Biomarkers of Gaucher cells were actively searched, particularly after the development of costly therapies based on enzyme supplementation and substrate reduction. Proteins selectively expressed by storage macrophages and secreted into the circulation were identified, among which glycoprotein non-metastatic protein B (GPNMB). This review focusses on the emerging potential of GPNMB as a biomarker of stressed macrophages in LSDs as well as in acquired pathologies accompanied by an excessive lysosomal substrate load in macrophages.
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Biomarcadores/metabolismo , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Espumosas/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Enfermedades por Almacenamiento Lisosomal/patología , Lisosomas/metabolismo , Glicoproteínas de Membrana/química , Enfermedades Metabólicas/metabolismo , Enfermedades Metabólicas/patología , Células Mieloides/metabolismo , FagocitosisRESUMEN
INTRODUCTION: Mutations in the GBA gene, encoding for the lysosomal enzyme glucocerebrosidase, are associated with Gaucher disease. Alterations in plasma sphingolipids have been reported in Gaucher, and similarly in brain extracts in Lewy body disease. As GBA mutations are prevalent risk factors for Parkinson's disease and overlap of molecular pathways are presumable, here we assessed the lipid profiles in Parkinson's patients with and without GBA mutations. METHODS: We sequenced all GBA exons in 415 Parkinson's patients, previously genotyped for LRRK2. 64 patients (29 GBA positive vs. 35 non-GBA-carriers including 18 LRRK2 positive and 17 non-mutated) were analyzed for chitotriosidase activity and for the concentration of 40 lipid classes using HPLC-MS. RESULTS: 29/415 patients (6.9%) carried 8 different GBA mutations associated with Gaucher or Parkinson's, including one novel mutation. Chitotriosidase activity was similar across the genetic groups, while the levels of key lipids were altered in GBA mutation carriers: Monohexosylceramide, Ceramide and Sphingomyelin were elevated; while Phosphatidic acid (PA), Phosphatidylethanolamine (PE), Plasmalogen phosphatidylethanolamine (PEp) and Acyl Phosphatidylglycerol (AcylPG) were decreased. CONCLUSION: The results suggest an important role for these lipids in GBA mediated Parkinson's disease and assist in the identification of common pathways between Gaucher and Parkinson's. Ultimately, our findings may lead to the identification of novel biomarkers for individuals at increased risk of developing Parkinson's disease.
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Biomarcadores/sangre , Lípidos/sangre , Enfermedad de Parkinson/sangre , Anciano , Femenino , Glucosilceramidasa/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Enfermedad de Parkinson/genéticaRESUMEN
We report on the clinical, biochemical, and genetic findings of a large family with the classical phenotype of Fabry disease due to the novel nonsense mutation c.607G>T (p.E203X) of the GLA gene, which occurs in the active site of the α-galactosidase A enzyme. This report highlights that (i) Fabry disease diagnosis should be considered in all cases of unexplained left ventricular hypertrophy (LVH), even in its milder forms; (ii) a complete evaluation of patients with unexplained LVH is important to find diagnostic red flags of treatable causes of LVH, such as Fabry disease; (iii) cascade family screening is paramount to the earlier diagnosis and treatment of other affected family members; and (iv) the Fabry disease phenotype is highly variable in heterozygote females, even within the same family.
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Enfermedad de Fabry/genética , Enfermedad de Fabry/fisiopatología , Hipertrofia Ventricular Izquierda/diagnóstico por imagen , alfa-Galactosidasa/genética , Adulto , Codón sin Sentido , Ecocardiografía , Femenino , Heterocigoto , Humanos , Hipertrofia Ventricular Izquierda/etiología , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Factores Sexuales , Adulto JovenRESUMEN
Gaucher disease is caused by inherited deficiency of lysosomal glucocerebrosidase. Proteome analysis of laser-dissected splenic Gaucher cells revealed increased amounts of glycoprotein nonmetastatic melanoma protein B (gpNMB). Plasma gpNMB was also elevated, correlating with chitotriosidase and CCL18, which are established markers for human Gaucher cells. In Gaucher mice, gpNMB is also produced by Gaucher cells. Correction of glucocerebrosidase deficiency in mice by gene transfer or pharmacological substrate reduction reverses gpNMB abnormalities. In conclusion, gpNMB acts as a marker for glucosylceramide-laden macrophages in man and mouse and gpNMB should be considered as candidate biomarker for Gaucher disease in treatment monitoring.
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The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.
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Colesterol/metabolismo , beta-Glucosidasa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Femenino , Enfermedad de Gaucher/metabolismo , Glicosilación , Humanos , Masculino , Ratones , Enfermedades de Niemann-Pick/metabolismo , Células RAW 264.7RESUMEN
In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LCMS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed.
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Enfermedad de Niemann-Pick Tipo C/metabolismo , Animales , Estudios de Casos y Controles , Femenino , Glicoesfingolípidos/metabolismo , Riñón/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Bazo/metabolismo , Esteroles/sangreRESUMEN
Heterologous protein production is a key technology for biotechnological, health sciences and many other research fields. Various approaches have been developed for its optimization, but the research emphasis has been on optimization of protein yield rather than protein quality. In this study, we have established a workflow for synthetic gene optimization for heterologous protein expression that combines bioinformatics, laboratory experiments, mass spectrometry and statistical analysis. Two gene primary structure analysis platforms, Anaconda and EuGene, and multivariate optimization methods were employed to re-design the Plasmodium falciparum lysyl-tRNA synthetase gene for optimal expression in Escherichia coli. Synthetic genes were expressed from common vectors, and amino acid mis-incorporations in the expressed proteins were detected and quantified using mass spectrometry. The association between the identified amino acid mis-incorporations and 23 gene variables was then analysed. The synthetic genes yielded significantly higher levels of protein relative to the wild-type gene, but 71 amino acid mis-incorporation sites were observed along the whole protein and across the synthetic genes that were statistically associated with specific codons and protein secondary structures. The optimization method that led to production of the most accurate protein was based on a multivariate approach that combined variables that are known to influence mRNA translation.
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Biotecnología/métodos , Biología Computacional/métodos , Espectrometría de Masas/métodos , Proteínas Recombinantes/biosíntesis , Interpretación Estadística de Datos , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/genéticaRESUMEN
Mutations within the lysosomal enzyme ß-glucocerebrosidase (GC) result in Gaucher disease and represent a major risk factor for developing Parkinson disease (PD). Loss of GC activity leads to accumulation of its substrate glucosylceramide and α-synuclein. Since lysosomal activity of GC is tightly linked to expression of its trafficking receptor, the lysosomal integral membrane protein type-2 (LIMP-2), we studied α-synuclein metabolism in LIMP-2-deficient mice. These mice showed an α-synuclein dosage-dependent phenotype, including severe neurological impairments and premature death. In LIMP-2-deficient brains a significant reduction in GC activity led to lipid storage, disturbed autophagic/lysosomal function, and α-synuclein accumulation mediating neurotoxicity of dopaminergic (DA) neurons, apoptotic cell death, and inflammation. Heterologous expression of LIMP-2 accelerated clearance of overexpressed α-synuclein, possibly through increasing lysosomal GC activity. In surviving DA neurons of human PD midbrain, LIMP-2 levels were increased, probably to compensate for lysosomal GC deficiency. Therefore, we suggest that manipulating LIMP-2 expression to increase lysosomal GC activity is a promising strategy for the treatment of synucleinopathies.
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Glucosilceramidasa/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/enzimología , Tronco Encefálico/patología , Tronco Encefálico/ultraestructura , Gliosis/complicaciones , Gliosis/patología , Humanos , Lípidos/química , Proteínas de Membrana de los Lisosomas/deficiencia , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/patología , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Neurotoxinas/toxicidadRESUMEN
Lysosomal integral membrane protein-2 (LIMP2) mediates trafficking of glucocerebrosidase (GBA) to lysosomes. Deficiency of LIMP2 causes action myoclonus-renal failure syndrome (AMRF). LIMP2-deficient fibroblasts virtually lack GBA like the cells of patients with Gaucher disease (GD), a lysosomal storage disorder caused by mutations in the GBA gene. While GD is characterized by the presence of glucosylceramide-laden macrophages, AMRF patients do not show these. We studied the fate of GBA in relation to LIMP2 deficiency by employing recently designed activity-based probes labeling active GBA molecules. We demonstrate that GBA is almost absent in lysosomes of AMRF fibroblasts. However, white blood cells contain considerable amounts of residual enzyme. Consequently, AMRF patients do not acquire lipid-laden macrophages and do not show increased plasma levels of macrophage markers, such as chitotriosidase, in contrast to GD patients. We next investigated the consequences of LIMP2 deficiency with respect to plasma glycosphingolipid levels. Plasma glucosylceramide concentration was normal in the AMRF patients investigated as well as in LIMP2-deficient mice. However, a marked increase in the sphingoid base, glucosylsphingosine, was observed in AMRF patients and LIMP2-deficient mice. Our results suggest that combined measurements of chitotriosidase and glucosylsphingosine can be used for convenient differential laboratory diagnosis of GD and AMRF.
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Epilepsias Mioclónicas Progresivas/diagnóstico , Animales , Células Cultivadas , Pruebas de Enzimas , Fibroblastos/enzimología , Técnica del Anticuerpo Fluorescente , Colorantes Fluorescentes/química , Glucosilceramidasa/metabolismo , Glucosilceramidas/metabolismo , Humanos , Leucocitos/enzimología , Proteínas de Membrana de los Lisosomas/deficiencia , Macrófagos/enzimología , Ratones , Epilepsias Mioclónicas Progresivas/enzimología , Psicosina/análogos & derivados , Psicosina/metabolismo , Receptores Depuradores/deficienciaRESUMEN
Triggered by the sequencing of the human genome, personalized medicine has been one of the fastest growing research areas in the last decade. Multiple software and hardware technologies have been developed by several projects, culminating in the exponential growth of genetic data. Considering the technological developments in this field, it is now fairly easy and inexpensive to obtain genetic profiles for unique individuals, such as those performed by several genetic analysis companies. The availability of computational tools that simplify genetic data analysis and the disclosure of biomedical evidences are of utmost importance. We present Variobox, a desktop tool to annotate, analyze, and compare human genes. Variobox obtains variant annotation data from WAVe, protein metadata annotations from Protein Data Bank, and sequences are obtained from Locus Reference Genomic or RefSeq databases. To explore the data, Variobox provides an advanced sequence visualization that enables agile navigation through genetic regions. DNA sequencing data can be compared with reference sequences retrieved from LRG or RefSeq records, identifying and automatically annotating new potential variants. These features and data, ranging from patient sequences to HGVS-compliant variant descriptions, are combined in an intuitive interface to analyze genes and variants. Variobox is a Java application, available at http://bioinformatics.ua.pt/variobox.
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Biología Computacional/métodos , Bases de Datos Genéticas , Variación Genética , Genoma Humano , Anotación de Secuencia Molecular , Secuencia de Aminoácidos , Secuencia de Bases , Humanos , Medicina de Precisión , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
Secondary structure of messenger RNA plays an important role in the bio-synthesis of proteins. Its negative impact on translation can reduce the yield of protein by slowing or blocking the initiation and movement of ribosomes along the mRNA, becoming a major factor in the regulation of gene expression. Several algorithms can predict the formation of secondary structures by calculating the minimum free energy of RNA sequences, or perform the inverse process of obtaining an RNA sequence for a given structure. However, there is still no approach to redesign an mRNA to achieve minimal secondary structure without affecting the amino acid sequence. Here we present the first strategy to optimize mRNA secondary structures, to increase (or decrease) the minimum free energy of a nucleotide sequence, without changing its resulting polypeptide, in a time-efficient manner, through a simplistic approximation to hairpin formation. Our data show that this approach can efficiently increase the minimum free energy by >40%, strongly reducing the strength of secondary structures. Applications of this technique range from multi-objective optimization of genes by controlling minimum free energy together with CAI and other gene expression variables, to optimization of secondary structures at the genomic level.
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Algoritmos , ARN Mensajero/química , Animales , Drosophila melanogaster/genética , Conformación de Ácido NucleicoRESUMEN
UNLABELLED: Numerous software applications exist to deal with synthetic gene design, granting the field of heterologous expression a significant support. However, their dispersion requires the access to different tools and online services in order to complete one single project. Analyzing codon usage, calculating codon adaptation index (CAI), aligning orthologs and optimizing genes are just a few examples. A software application, EuGene, was developed for the optimization of multiple gene synthetic design algorithms. In a seamless automatic form, EuGene calculates or retrieves genome data on codon usage (relative synonymous codon usage and CAI), codon context (CPS and codon pair bias), GC content, hidden stop codons, repetitions, deleterious sites, protein primary, secondary and tertiary structures, gene orthologs, species housekeeping genes, performs alignments and identifies genes and genomes. The main function of EuGene is analyzing and redesigning gene sequences using multi-objective optimization techniques that maximize the coding features of the resulting sequence. AVAILABILITY: EuGene is freely available for non-commercial use, at http://bioinformatics.ua.pt/eugene.