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Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy.
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Hígado/citología , Técnicas de Cultivo de Órganos , Animales , Inestabilidad Genómica , Hepatocitos/citología , Humanos , Ratones , Organoides/citologíaRESUMEN
Neddylation has been implicated in various cellular pathways and in the pathophysiology of numerous diseases. We identified four individuals with bi-allelic variants in NAE1, which encodes the neddylation E1 enzyme. Pathogenicity was supported by decreased NAE1 abundance and overlapping clinical and cellular phenotypes. To delineate how cellular consequences of NAE1 deficiency would lead to the clinical phenotype, we focused primarily on the rarest phenotypic features, based on the assumption that these would best reflect the pathophysiology at stake. Two of the rarest features, neuronal loss and lymphopenia worsening during infections, suggest that NAE1 is required during cellular stress caused by infections to protect against cell death. In support, we found that stressing the proteasome system with MG132-requiring upregulation of neddylation to restore proteasomal function and proteasomal stress-led to increased cell death in fibroblasts of individuals with NAE1 genetic variants. Additionally, we found decreased lymphocyte counts after CD3/CD28 stimulation and decreased NF-κB translocation in individuals with NAE1 variants. The rarest phenotypic feature-delayed closure of the ischiopubic rami-correlated with significant downregulation of RUN2X and SOX9 expression in transcriptomic data of fibroblasts. Both genes are involved in the pathophysiology of ischiopubic hypoplasia. Thus, we show that NAE1 plays a major role in (skeletal) development and cellular homeostasis during stress. Our approach suggests that a focus on rare phenotypic features is able to provide significant pathophysiological insights in diseases caused by mutations in genes with pleiotropic effects.
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Discapacidad Intelectual , Linfopenia , Humanos , Proteína NEDD8/genética , Proteína NEDD8/metabolismo , Transducción de Señal/genética , Discapacidad Intelectual/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Linfopenia/genéticaRESUMEN
Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.
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Discapacidad Intelectual , Megalencefalia , Trastornos del Neurodesarrollo , Animales , Humanos , Niño , Pez Cebra/genética , Pez Cebra/metabolismo , Caenorhabditis elegans/metabolismo , Trastornos del Neurodesarrollo/genética , Discapacidad Intelectual/genética , Fenotipo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Megalencefalia/genética , Discapacidades del Desarrollo/genética , Mutación Missense/genética , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.
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Cardiomiopatías , Síndromes Congénitos de Insuficiencia de la Médula Ósea , Errores Innatos del Metabolismo Lipídico , Lipidómica , Enfermedades Mitocondriales , Miopatías Mitocondriales , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades Musculares , Enfermedades del Sistema Nervioso , Rabdomiólisis , Humanos , Enfermedades Mitocondriales/diagnóstico , Carnitina , Cisteamina , LípidosRESUMEN
PURPOSE: Pediatric cholestasis is the phenotypic expression of clinically and genetically heterogeneous disorders of bile acid synthesis and flow. Although a growing number of monogenic causes of pediatric cholestasis have been identified, the majority of cases remain undiagnosed molecularly. METHODS: In a cohort of 299 pediatric participants (279 families) with intrahepatic cholestasis, we performed exome sequencing as a first-tier diagnostic test. RESULTS: A likely causal variant was identified in 135 families (48.56%). These comprise 135 families that harbor variants spanning 37 genes with established or tentative links to cholestasis. In addition, we propose a novel candidate gene (PSKH1) (HGNC:9529) in 4 families. PSKH1 was particularly compelling because of strong linkage in three consanguineous families who shared a novel hepatorenal ciliopathy phenotype. Two of the four families shared a founder homozygous variant while the third had a different homozygous variant in PSKH1. PSKH1 encodes a putative protein serine kinase of unknown function. Patient fibroblasts displayed abnormal cilia that are long and show abnormal transport. A homozygous Pskh1 mutant mouse faithfully recapitulated the human phenotype and displayed abnormally long cilia. The phenotype could be rationalized by the loss of catalytic activity observed for each recombinant PSKH1 variant using in vitro kinase assays. CONCLUSION: Our results support the use of genomics in the workup of pediatric cholestasis and reveal PSKH1-related hepatorenal ciliopathy as a novel candidate monogenic form.
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Infertility is a complex condition affecting millions of couples worldwide. The current definition of infertility, based on clinical criteria, fails to account for the molecular and cellular changes that may occur during the development of infertility. Recent advancements in sequencing technology and single-cell analysis offer new opportunities to gain a deeper understanding of these changes. The endometrium has a potential role in infertility and has been extensively studied to identify gene expression profiles associated with (impaired) endometrial receptivity. However, limited overlap among studies hampers the identification of relevant downstream pathways that could play a role in the development of endometrial-related infertility. To address these challenges, we propose sequencing the endometrial transcriptome of healthy and infertile women at the single-cell level to consistently identify molecular signatures. Establishing consensus on physiological patterns in endometrial samples can aid in identifying deviations in infertile patients. A similar strategy has been used with great success in cancer research. However, large collaborative initiatives, international uniform protocols of sample collection and processing are crucial to ensure reliability and reproducibility. Overall, the proposed approach holds promise for an objective and accurate classification of endometrial-based infertility and has the potential to improve diagnosis and treatment outcomes.
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Infertilidad Femenina , Femenino , Humanos , Infertilidad Femenina/diagnóstico , Infertilidad Femenina/genética , Infertilidad Femenina/metabolismo , Reproducibilidad de los Resultados , Endometrio/metabolismo , Transcriptoma , Resultado del Tratamiento , Implantación del Embrión/fisiologíaRESUMEN
BACKGROUND: Newborn screening (NBS) programmes identify a wide range of disease phenotypes, which raises the question whether early identification and treatment is beneficial for all. This study aims to answer this question for primary carnitine deficiency (PCD) taking into account that NBS for PCD identifies newborns with PCD and also until then undiagnosed mothers. METHODS: We investigated clinical, genetic (variants in SLC22A5 gene) and functional (carnitine transport activity in fibroblasts) characteristics of all referred individuals through NBS (newborns and mothers) and clinically diagnosed patients with PCD (not through NBS). Disease phenotype in newborns was predicted using data from PCD mothers and cases published in literature with identical SLC22A5 variants. RESULTS: PCD was confirmed in 19/131 referred newborns, 37/82 referred mothers and 5 clinically diagnosed patients. Severe symptoms were observed in all clinically diagnosed patients, 1 newborn and none of the mothers identified by NBS. PCD was classified as severe in all 5 clinically diagnosed patients, 3/19 newborns and 1/37 mothers; as benign in 8/19 newborns and 36/37 mothers and as unknown in 8/19 newborns. Carnitine transport activity completely separated severe phenotype from benign phenotype (median (range): 4.0% (3.5-5.0)] vs 26% (9.5-42.5), respectively). CONCLUSION: The majority of mothers and a significant proportion of newborns with PCD identified through NBS are likely to remain asymptomatic without early treatment. Conversely, a small proportion of newborns with predicted severe PCD could greatly benefit from early treatment. Genetic variants and carnitine transport activity can be used to distinguish between these groups.
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Carnitina , Tamizaje Neonatal , Femenino , Humanos , Recién Nacido , Estudios Retrospectivos , Miembro 5 de la Familia 22 de Transportadores de Solutos/genética , Mutación , Carnitina/genéticaRESUMEN
The endothelial cell lining creates an interface between circulating blood and adjoining tissue and forms one of the most critical barriers and targets for therapeutical intervention. Recent studies suggest that fucoidans, sulfated and fucose-rich polysaccharides from brown seaweed, show multiple promising biological effects, including anti-inflammatory properties. However, their biological activity is determined by chemical characteristics such as molecular weight, sulfation degree, and molecular structure, which vary depending on the source, species, and harvesting and isolation method. In this study, we investigated the impact of high molecular weight (HMW) fucoidan extract on endothelial cell activation and interaction with primary monocytes (MNCs) in lipopolysaccharide (LPS)-induced inflammation. Gentle enzyme-assisted extraction combined with fractionation by ion exchange chromatography resulted in well-defined and pure fucoidan fractions. FE_F3, with a molecular weight ranging from 110 to 800 kDa and a sulfate content of 39%, was chosen for further investigation of its anti-inflammatory potential. We observed that along with higher purity of fucoidan fractions, the inflammatory response in endothelial mono- and co-cultures with MNCs was reduced in a dose-dependent manner when testing two different concentrations. This was demonstrated by a decrease in IL-6 and ICAM-1 on gene and protein levels and a reduced gene expression of TLR-4, GSK3ß and NF-kB. Expression of selectins and, consequently, the adhesion of monocytes to the endothelial monolayer was reduced after fucoidan treatment. These data indicate that the anti-inflammatory effect of fucoidans increases with their purity and suggest that fucoidans might be useful in limiting the inflammatory response of endothelial cells in cases of LPS-induced bacterial infection.
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Células Endoteliales , Lipopolisacáridos , Lipopolisacáridos/farmacología , Peso Molecular , Polisacáridos/química , Antiinflamatorios , LeucocitosRESUMEN
BACKGROUND: The reimbursement of new technologies in inpatient care is not always linked to a requirement for evidence-based evaluation of patient benefit. In Germany, every new technology approved for market was until recently eligible for reimbursement in inpatient care unless explicitly excluded. The aim of this work was (1) to investigate the type of evidence that was available at the time of introduction of 25 innovative technologies and how this evidence evolved over time, and (2) to explore the relationship between clinical evidence and utilization for these technologies in German inpatient care. METHODS: This study combined different methods. A systematic search for evidence published between 2003 and 2017 was conducted in four bibliographic databases, clinical trial registries, resources for clinical guidelines, and health technology assessment-databases. Information was also collected on funding mechanisms and safety notices. Utilization was measured by hospital procedures captured in claims data. The body of evidence, funding and safety notices per technology were analyzed descriptively. The relationship between utilization and evidence was explored empirically using a multilevel regression analysis. RESULTS: The number of included publications per technology ranges from two to 498. For all technologies, non-comparative studies form the bulk of the evidence. The number of randomized controlled clinical trials per technology ranges from zero to 19. Some technologies were utilized for several years without an adequate evidence base. A relationship between evidence and utilization could be shown for several but not all technologies. CONCLUSIONS: This study reveals a mixed picture regarding the evidence available for new technologies, and the relationship between the development of evidence and the use of technologies over time. Although the influence of funding and safety notices requires further investigation, these results re-emphasize the need for strengthening market approval standards and HTA pathways as well as approaches such as coverage with evidence development.
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Pacientes Internos , Evaluación de la Tecnología Biomédica , Humanos , Bases de Datos Factuales , AlemaniaRESUMEN
Low serum levels of 1α, 25-dihydroxyvitamin D3 (VD3) are associated with a higher mortality in trauma patients with sepsis or ARDS. However, the molecular mechanisms behind this observation are not yet understood. VD3 is known to stimulate lung maturity, alveolar type II cell differentiation, or pulmonary surfactant synthesis and guides epithelial defense during infection. In this study, we investigated the impact of VD3 on the alveolar-capillary barrier in a co-culture model of alveolar epithelial cells and microvascular endothelial cells respectively in the individual cell types. After stimulation with bacterial LPS (lipopolysaccharide), gene expression of inflammatory cytokines, surfactant proteins, transport proteins, antimicrobial peptide, and doublecortin-like kinase 1 (DCLK1) were analyzed by real-time PCR, while corresponding proteins were evaluated by ELISA, immune-fluorescence, or Western blot. The effect of VD3 on the intracellular protein composition in H441 cells was analyzed by quantitative liquid chromatography-mass spectrometry-based proteomics. VD3 effectively protected the alveolar-capillary barrier against LPS treatment, as indicated by TEER measurement and morphological assessment. VD3 did not inhibit the IL-6 secretion by H441 and OEC but restricted the diffusion of IL-6 to the epithelial compartment. Further, VD3 could significantly suppress the surfactant protein A expression induced in the co-culture system by LPS treatment. VD3 induced high levels of the antimicrobial peptide LL-37, which counteracted effects by LPS and strengthened the barrier. Quantitative proteomics identified VD3-dependent protein abundance changes ranging from constitutional extracellular matrix components and surfactant-associated proteins to immune-regulatory molecules. DCLK1, as a newly described target molecule for VD3, was prominently stimulated by VD3 (10 nM) and seems to influence the alveolar-epithelial cell barrier and regeneration.
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Células Endoteliales , Interleucina-6 , Humanos , Lipopolisacáridos/farmacología , Proteínas Asociadas a Surfactante Pulmonar , Células Epiteliales Alveolares , Tensoactivos , Quinasas Similares a DoblecortinaRESUMEN
INTRODUCTION: Inborn-Errors of Metabolism (IEM) are genetic disorders resulting from mutations in genes encoding proteins involved in biochemical-metabolic pathways. However, some IEMs lack specific biochemical markers. Early incorporation of next-generation-sequencing (NGS) including whole exome sequencing (WES) into the diagnostic algorithm of IEMs herein provided, increases diagnostic accuracy, permits genetic counseling and improves therapeutic options. This is exemplified by diseases affecting aminoacyl-tRNA synthetases (ARSs), enzymes involved in protein translation. Recent studies showed that supplementing amino-acids to cell-culture and patients with ARSs deficiencies resulted in improvement of biochemical and clinical parameters, respectively.
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Errores Innatos del Metabolismo , Humanos , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/genética , Errores Innatos del Metabolismo/terapia , Mutación , Biomarcadores , Asesoramiento Genético , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
For liver transplantations, human leukocyte antigen (HLA) matching is not routinely performed because observed effects have been inconsistent. Nevertheless, long-term liver transplantation outcomes remain suboptimal. The availability of a more precise HLA-matching algorithm, Predicted Indirectly Recognizable HLA Epitopes II (PIRCHE-II), now enables robust assessment of the association between HLA matching and liver transplantation outcomes. We performed a single-center retrospective cohort study of 736 liver transplantation patients. Associations between PIRCHE-II and HLAMatchmaker scores and mortality, graft loss, acute and chronic rejection, ischemic cholangiopathy, and disease recurrence were evaluated with Cox proportional hazards models. Associations between PIRCHE-II with 1-year, 2-year, and 5-year outcomes and severity of acute rejection were assessed with logistic and linear regression analyses, respectively. Subgroup analyses were performed for autoimmune and nonautoimmune indications, and patients aged 30 years and younger, and older than 30 years. PIRCHE-II and HLAMatchmaker scores were not associated with any of the outcomes. However, patients who received transplants for autoimmune disease showed more acute rejection and graft loss, and these risks negatively associated with age. Rhesus mismatch more than doubled the risk of disease recurrence. Moreover, PIRCHE-II was inversely associated with graft loss in the subgroup of patients aged 30 years and younger with autoimmune indications. The absence of associations between PIRCHE-II and HLAMatchmaker scores and the studied outcomes refutes the need for HLA matching for liver (stem cell) transplantations for nonautoimmune disease. For autoimmune disease, the activated immune system seems to increase risks of acute rejection and graft loss. Our results may suggest the benefits of transplantations with rhesus matched but PIRCHE-II mismatched donor livers.
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Enfermedades Autoinmunes , Trasplante de Hígado , Algoritmos , Rechazo de Injerto/epidemiología , Supervivencia de Injerto , Antígenos HLA , Prueba de Histocompatibilidad , Humanos , Trasplante de Hígado/efectos adversos , Estudios RetrospectivosRESUMEN
Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is included in many newborn screening (NBS) programs. Acylcarnitine-based NBS for LCHADD not only identifies LCHADD, but also the other deficiencies of the mitochondrial trifunctional protein (MTP), a multi-enzyme complex involved in long-chain fatty acid ß-oxidation. Besides LCHAD, MTP harbors two additional enzyme activities: long-chain enoyl-CoA hydratase (LCEH) and long-chain ketoacyl-CoA thiolase (LCKAT). Deficiency of one or more MTP activities causes generalized MTP deficiency (MTPD), LCHADD, LCEH deficiency (not yet reported), or LCKAT deficiency (LCKATD). To gain insight in the outcomes of MTP-deficient patients diagnosed after the introduction of NBS for LCHADD in the Netherlands, a retrospective evaluation of genetic, biochemical, and clinical characteristics of MTP-deficient patients, identified since 2007, was carried out. Thirteen patients were identified: seven with LCHADD, five with MTPD, and one with LCKATD. All LCHADD patients (one missed by NBS, clinical diagnosis) and one MTPD patient (clinical diagnosis) were alive. Four MTPD patients and one LCKATD patient developed cardiomyopathy and died within 1 month and 13 months of life, respectively. Surviving patients did not develop symptomatic hypoglycemia, but experienced reversible cardiomyopathy and rhabdomyolysis. Five LCHADD patients developed subclinical neuropathy and/or retinopathy. In conclusion, patient outcomes were highly variable, stressing the need for accurate classification of and discrimination between the MTP deficiencies to improve insight in the yield of NBS for LCHADD. NBS allowed the prevention of symptomatic hypoglycemia, but current treatment options failed to treat cardiomyopathy and prevent long-term complications. Moreover, milder patients, who might benefit from NBS, were missed due to normal acylcarnitine profiles.
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Cardiomiopatías , Hipoglucemia , Errores Innatos del Metabolismo Lipídico , Rabdomiólisis , 3-Hidroxiacil-CoA Deshidrogenasas , Cardiomiopatías/diagnóstico , Cardiomiopatías/genética , Humanos , Recién Nacido , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Miopatías Mitocondriales , Proteína Trifuncional Mitocondrial/deficiencia , Biología Molecular , Tamizaje Neonatal , Enfermedades del Sistema Nervioso , Países Bajos , Estudios Retrospectivos , Rabdomiólisis/diagnóstico , Rabdomiólisis/genéticaRESUMEN
Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid ß-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. In conclusion, all patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition.
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Errores Innatos del Metabolismo Lipídico , Miopatías Mitocondriales , Enfermedades Musculares , 3-Hidroxiacil-CoA Deshidrogenasas , Adolescente , Cardiomiopatías , Niño , Preescolar , Coenzima A , Diagnóstico Tardío , Ácidos Grasos/metabolismo , Humanos , Errores Innatos del Metabolismo Lipídico/diagnóstico , Errores Innatos del Metabolismo Lipídico/genética , Errores Innatos del Metabolismo Lipídico/metabolismo , Miopatías Mitocondriales/diagnóstico , Miopatías Mitocondriales/genética , Proteína Trifuncional Mitocondrial/deficiencia , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/genética , Enfermedades del Sistema Nervioso , RabdomiólisisRESUMEN
Inborn errors of metabolism (IEMs) comprise a diverse group of individually rare monogenic disorders that affect metabolic pathways. Mutations lead to enzymatic deficiency or dysfunction, which results in intermediate metabolite accumulation or deficit leading to disease phenotypes. Currently, treatment options for many IEMs are insufficient. Rarity of individual IEMs hampers therapy development and phenotypic and genetic heterogeneity suggest beneficial effects of personalized approaches. Recently, cultures of patient-own liver-derived intrahepatic cholangiocyte organoids (ICOs) have been established. Since most metabolic genes are expressed in the liver, patient-derived ICOs represent exciting possibilities for in vitro modeling and personalized drug testing for IEMs. However, the exact application range of ICOs remains unclear. To address this, we examined which metabolic pathways can be studied with ICOs and what the potential and limitations of patient-derived ICOs are to model metabolic functions. We present functional assays in patient ICOs with defects in branched-chain amino acid metabolism (methylmalonic acidemia), copper metabolism (Wilson disease), and transporter defects (cystic fibrosis). We discuss the broad range of functional assays that can be applied to ICOs, but also address the limitations of these patient-specific cell models. In doing so, we aim to guide the selection of the appropriate cell model for studies of a specific disease or metabolic process.
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Errores Innatos del Metabolismo de los Aminoácidos , Organoides , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Humanos , Hígado/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Redes y Vías Metabólicas , Organoides/metabolismoRESUMEN
Fucoidans, sulfated polysaccharides from brown algae, possess multiple bioactivities in regard to osteogenesis, angiogenesis, and inflammation, all representing key molecular processes for successful bone regeneration. To utilize fucoidans in regenerative medicine, a delivery system is needed which temporarily immobilizes the polysaccharide at the injured site. Hydrogels have become increasingly interesting biomaterials for the support of bone regeneration. Their structural resemblance with the extracellular matrix, their flexible shape, and capacity to deliver bioactive compounds or stem cells into the affected tissue make them promising materials for the support of healing processes. Especially injectable hydrogels stand out due to their minimal invasive application. In the current study, we developed an injectable thermosensitive hydrogel for the delivery of fucoidan based on chitosan, collagen, and ß-glycerophosphate (ß-GP). Physicochemical parameters such as gelation time, gelation temperature, swelling capacity, pH, and internal microstructure were studied. Further, human bone-derived mesenchymal stem cells (MSC) and human outgrowth endothelial cells (OEC) were cultured on top (2D) or inside the hydrogels (3D) to assess the biocompatibility. We found that the sol-gel transition occurred after approximately 1 min at 37 °C. Fucoidan integration into the hydrogel had no or only a minor impact on the mentioned physicochemical parameters compared to hydrogels which did not contain fucoidan. Release assays showed that 60% and 80% of the fucoidan was released from the hydrogel after two and six days, respectively. The hydrogel was biocompatible with MSC and OEC with a limitation for OEC encapsulation. This study demonstrates the potential of thermosensitive chitosan-collagen hydrogels as a delivery system for fucoidan and MSC for the use in regenerative medicine.
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Quitosano , Hidrogeles , Quitosano/química , Colágeno/química , Células Endoteliales , Humanos , Hidrogeles/química , PolisacáridosRESUMEN
Nickel-titanium (NiTi) belongs to the group of shape-memory alloys (SMAs), which are characterized by flexibility and reversible deformability. Advanced techniques in 3D printing by selective laser-melting (SLM) process allow the manufacturing of complex patient-specific implants from SMAs. Osteosynthesis materials made of NiTi could be used for minimally invasive surgical approaches in oral- and maxillofacial surgery. However, the in vivo biocompatibility has not yet been fully investigated, especially in load-sharing and load-bearing implants. The aim of this study was to evaluate the in vivo biocompatibility of SLM-produced NiTi for intraosseous and subperiosteal applications. Test specimens were implanted into the frontonasal bone of ten miniature pigs. To assess peri-implant bone metabolism, fluorescent dye was administered after 2, 4, 6, 10, 12, and 14 weeks intraperitoneally. Specimens and the surrounding tissues were harvested after 8 and 16 weeks for histological analysis. While the NiTi implants presented a higher bone-to-implant contact ratio (BIC) after 8 than after 16 weeks (43.3 vs. 40.3%), the titanium implants had a significantly higher BIC after 16 weeks (33.6 vs. 67.7%). Histologically, no signs of peri-implant inflammation or foreign-body reaction were detectable. With respect to this preliminary study design, 3D-printed NiTi shows sufficient biocompatibility for intraosseous and subperiosteal implant placement.
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Rayos Láser , Níquel/efectos adversos , Prótesis e Implantes , Titanio/efectos adversos , Animales , Materiales Biocompatibles , Huesos , Ensayo de Materiales , Porcinos , Porcinos EnanosRESUMEN
Graphene oxide (GO) is a promising material for bone tissue engineering, but the validation of its molecular biological effects, especially in the context of clinically applied materials, is still limited. In this study, we compare the effects of graphene oxide framework structures (F-GO) and reduced graphene oxide-based framework structures (F-rGO) as scaffold material with a special focus on vascularization associated processes and mechanisms in the bone. Highly porous networks of zinc oxide tetrapods serving as sacrificial templates were used to create F-GO and F-rGO with porosities >99% consisting of hollow interconnected microtubes. Framework materials were seeded with human mesenchymal stem cells (MSC), and the cell response was evaluated by confocal laser scanning microscopy (CLSM), deoxyribonucleic acid (DNA) quantification, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and alkaline phosphatase activity (ALP) to define their impact on cellular adhesion, osteogenic differentiation, and secretion of vascular growth factors. F-GO based scaffolds improved adhesion and growth of MSC as indicated by CLSM and DNA quantification. Further, F-GO showed a better vascular endothelial growth factor (VEGF) binding capacity and improved cell growth as well as the formation of microvascular capillary-like structures in co-cultures with outgrowth endothelial cells (OEC). These results clearly favored non-reduced graphene oxide in the form of F-GO for bone regeneration applications. To study GO in the context of a clinically used implant material, we coated a commercially available xenograft (Bio-Oss® block) with GO and compared the growth of MSC in monoculture and in coculture with OEC to the native scaffold. We observed a significantly improved growth of MSC and formation of prevascular structures on coated Bio-Oss®, again associated with a higher VEGF binding capacity. We conclude that graphene oxide coating of this clinically used, but highly debiologized bone graft improves MSC cell adhesion and vascularization.
Asunto(s)
Grafito , Células Madre Mesenquimatosas , Adhesión Celular , Diferenciación Celular , ADN/metabolismo , Células Endoteliales , Grafito/química , Humanos , Células Madre Mesenquimatosas/metabolismo , Osteogénesis , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The genome of coronaviruses, including SARS-CoV-2, encodes for two proteases, a papain like (PLpro ) protease and the so-called main protease (Mpro ), a chymotrypsin-like cysteine protease, also named 3CLpro or non-structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that Mpro proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS-CoV, SARS-CoV-2, and hCoV-NL63 coronviruses, an LC-MS based N-terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS-CoV-2 Mpro , 445 cleavage events from more than 300 proteins are identified, while 151 and 331 Mpro derived cleavage events are identified for SARS-CoV and hCoV-NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406.
Asunto(s)
COVID-19/virología , Proteasas 3C de Coronavirus/metabolismo , Coronavirus Humano NL63/enzimología , Fragmentos de Péptidos/análisis , SARS-CoV-2/enzimología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Proteínas Virales/metabolismo , COVID-19/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Factor 4G Eucariótico de Iniciación/metabolismo , Interacciones Huésped-Patógeno , Humanos , Pulmón/metabolismo , Pulmón/virología , Especificidad por SustratoRESUMEN
Aminoacyl-tRNA synthetases (aaRS) are ubiquitously expressed enzymes responsible for ligating amino acids to their cognate tRNA molecules through an aminoacylation reaction. The resulting aminoacyl-tRNA is delivered to ribosome elongation factors to participate in protein synthesis. Seryl-tRNA synthetase (SARS1) is one of the cytosolic aaRSs and catalyzes serine attachment to tRNASer . SARS1 deficiency has already been associated with moderate intellectual disability, ataxia, muscle weakness, and seizure in one family. We describe here a new clinical presentation including developmental delay, central deafness, cardiomyopathy, and metabolic decompensation during fever leading to death, in a consanguineous Turkish family, with biallelic variants (c.638G>T, p.(Arg213Leu)) in SARS1. This missense variant was shown to lead to protein instability, resulting in reduced protein level and enzymatic activity. Our results describe a new clinical entity and expand the clinical and mutational spectrum of SARS1 and aaRS deficiencies.