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Chiral analysis is of high interest in many fields such as chemistry, pharmaceuticals and metabolomics. Mass spectrometry and ion mobility spectrometry are useful analytical tools, although they cannot be used as stand-alone methods. Here, we propose an efficient strategy for the enantiomer characterization of amino acids (AAs) using non-covalent copper complexes. A single ion mobility monitoring (SIM2) method was applied on a TIMS-ToF mass spectrometer to maximize the detection and mobility separation of isomers. Almost all of the 19 pairs of proteinogenic AA enantiomers could be separated with at least one combination with the chiral references L-Phe and L-Pro. Furthermore, we extended the targeted SIM2 method by stitching several mobility ranges, in order to be able to analyze complex mixtures in a single acquisition while maintaining high mobility resolution. Most of the enantiomeric pairs of AAs separated with the SIM2 method were also detected with this approach. The SIM2 stitching method thus opens the way to a more comprehensive chiral analysis with TIMS-ToF instruments.
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Aminoácidos , Espectrometria de Mobilidade Iônica , Espectrometria de Mobilidade Iônica/métodos , Estereoisomerismo , Aminoácidos/química , Aminoácidos/análise , Espectrometria de Massas/métodos , Cobre/química , Cobre/análise , Ensaios de Triagem em Larga Escala/métodosRESUMO
AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.
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Artrite Reumatoide , Armadilhas Extracelulares , Periodontite , Humanos , Desiminases de Arginina em Proteínas/genética , Artrite Reumatoide/genética , Periodontite/genética , Neutrófilos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
PURPOSE: Successful microlaryngeal surgery relies on an adequate laryngeal exposure. Recognizing the likelihood of challenging exposure prior to microlaryngeal surgery may assist in selecting the appropriate surgical approach and even prompt consideration of alternative treatment options. We aim to apply the mini-Laryngoscore, a preoperative assessment tool, to our study population and incorporate novel variables to optimize the prediction model. METHODS: This single-center prospective cohort study included 80 consecutive patients undergoing elective microlaryngeal surgery, from January 1, to June 30, 2023. Each patient underwent a presurgical evaluation of 15 parameters and an intraoperative scoring of the anterior commissure visualization. These parameters were assessed for their association with difficult laryngeal exposure, using multiple logistic regression analysis. We created a novel prediction model for DLE and compared it with the existing model, the mini-Laryngoscore. RESULTS: Out of 80 patients, 24 (30%) patients had difficult laryngeal exposure, including 3 cases (3.8%) in which visualization of the anterior commissure was not possible. A large neck diameter (OR, 1.4; CI 1.1-1.9) and the presence of upper teeth (OR, 8.9; CI 1.3-62.8) were independent risk factors for a difficult laryngeal exposure, while a larger interincisors gap was the only independent protector factor (OR, 0.3; CI 0.1-0.8). The logistic regression model combining these three independent risk factors displayed a high discriminative value AUC = 0.89 (CI 0.81-0.97). The predictive performance of the mini-Laryngoscore was 0.73 (CI 0.62-0.85). CONCLUSION: Combining two parameters from the mini-Laryngoscore (upper jaw dental state and interincisors gap distance) with neck circumference measurement can accurately predict the risk of difficult laryngeal exposure.
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Laringoscopia , Laringe , Humanos , Estudos Prospectivos , Microcirurgia , Laringe/cirurgia , Pescoço/cirurgiaRESUMO
PURPOSE: Since the beginning of 2023, ChatGPT emerged as a hot topic in healthcare research. The potential to be a valuable tool in clinical practice is compelling, particularly in improving clinical decision support by helping physicians to make clinical decisions based on the best medical knowledge available. We aim to investigate ChatGPT's ability to identify, diagnose and manage patients with otorhinolaryngology-related symptoms. METHODS: A prospective, cross-sectional study was designed based on an idea suggested by ChatGPT to assess the level of agreement between ChatGPT and five otorhinolaryngologists (ENTs) in 20 reality-inspired clinical cases. The clinical cases were presented to the chatbot on two different occasions (ChatGPT-1 and ChatGPT-2) to assess its temporal stability. RESULTS: The mean score of ChatGPT-1 was 4.4 (SD 1.2; min 1, max 5) and of ChatGPT-2 was 4.15 (SD 1.3; min 1, max 5), while the ENTs mean score was 4.91 (SD 0.3; min 3, max 5). The Mann-Whitney U test revealed a statistically significant difference (p < 0.001) between both ChatGPT's and the ENTs's score. ChatGPT-1 and ChatGPT-2 gave different answers in five occasions. CONCLUSIONS: Artificial intelligence will be an important instrument in clinical decision-making in the near future and ChatGPT is the most promising chatbot so far. Despite needing further development to be used with safety, there is room for improvement and potential to aid otorhinolaryngology residents and specialists in making the most correct decision for the patient.
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Otolaringologia , Cirurgiões , Humanos , Inteligência Artificial , Estudos Transversais , Estudos Prospectivos , Tomada de Decisão ClínicaRESUMO
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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Doenças por Armazenamento dos Lisossomos , Mucopolissacaridose II , Humanos , Células-Tronco , Linhagem Celular , Dente Decíduo , Lisossomos , Polpa Dentária , Diferenciação Celular/fisiologia , Proliferação de CélulasRESUMO
Metabolomics has become increasingly popular in recent years for many applications ranging from clinical diagnosis, human health to biotechnological questioning. Despite technological advances, metabolomic studies are still currently limited by the difficulty of identifying all metabolites, a class of compounds with great chemical diversity. Although lengthy chromatographic analyses are often used to obtain comprehensive data, many isobar and isomer metabolites still remain unresolved, which is a critical point for the compound identification. Currently, ion mobility spectrometry is being explored in metabolomics as a way to improve metabolome coverage, analysis throughput and isomer separation. In this review, all the steps of a typical workflow for untargeted metabolomics are discussed considering the use of an ion mobility instrument. An overview of metabolomics is first presented followed by a brief description of ion mobility instrumentation. The ion mobility potential for complex mixture analysis is discussed regarding its coupling with a mass spectrometer alone, providing gas-phase separation before mass analysis as well as its combination with different separation platforms (conventional hyphenation but also multidimensional ion mobility couplings), offering multidimensional separation. Various instrumental and analytical conditions for improving the ion mobility separation are also described. Finally, data mining, including software packages and visualization approaches, as well as the construction of ion mobility databases for the metabolite identification are examined.
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Espectrometria de Mobilidade Iônica , Metabolômica , Humanos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodos , Fluxo de TrabalhoRESUMO
Niemann-Pick type C1 (NPC1) is a fatal inherited disease, caused by pathogenic variants in NPC1 gene, which leads to intracellular accumulation of non-esterified cholesterol and glycosphingolipids. This accumulation leads to a wide range of clinical manifestations, including neurological and cognitive impairment as well as psychiatric disorders. The pathophysiology of cerebral damage involves loss of Purkinje cells, synaptic disturbance, and demyelination. Miglustat, a reversible inhibitor of glucosylceramide synthase, is an approved treatment for NPC1 and can slow neurological damage. The aim of this study was to assess the levels of peripheric neurodegeneration biomarkers of NPC1 patients, namely brain-derived neurotrophic factor (BDNF), platelet-derived growth factors (PDGF-AA and PDGF-AB/BB), neural cell adhesion molecule (NCAM), PAI-1 Total and Cathepsin-D, as well as the levels of cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), a biomarker for NPC1. Molecular analysis of the NPC1 patients under study was performed by next generation sequencing (NGS) in cultured fibroblasts. We observed that NPC1 patients treated with miglustat have a significant decrease in PAI-1 total and PDGF-AA concentrations, and no alteration in BDNF, NCAM, PDGF-AB/BB and Cathepsin D. We also found that NPC1 patients treated with miglustat have normalized levels of 3ß,5α,6ß-triol. The molecular analysis showed four described mutations, and for two patients was not possible to identify the second mutated allele. Our results indicate that the decrease of PAI-1 and PDGF-AA in NPC1 patients could be involved in the pathophysiology of this disease. This is the first work to analyze those plasmatic markers of neurodegenerative processes in NPC1 patients.
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Fator Neurotrófico Derivado do Encéfalo , Doença de Niemann-Pick Tipo C , Humanos , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/patologia , Inibidor 1 de Ativador de Plasminogênio , Fator de Crescimento Derivado de Plaquetas , Biomarcadores , BecaplerminaRESUMO
Epidemiological studies aim to assess associations between diseases and risk factors. Such investigations involve a large sample size and require powerful analytical methods to measure the effects of risk factors, resulting in a long analysis time. In this study, chemical exposure markers were detected as the main variables strongly affecting two components coming from a principal component analysis (PCA) exploration of the metabolomic data generated from urinary samples collected on a cohort of about 500 individuals using direct introduction coupled with a Fourier-transform ion cyclotron resonance instrument. The assignment of their chemical identity was first achieved based on their isotopic fine structures detected at very high resolution (Rp > 900,000). Their identification as dimethylbiguanide and sotalol was obtained at level 1, thanks to the available authentic chemical standards, tandem mass spectrometry (MS/MS) experiments, and collision cross section measurements. Epidemiological data confirmed that the subjects discriminated by PCA had declared to be prescribed these drugs for either type II diabetes or cardiac arrhythmia. Concentrations of these drugs in urine samples of interest were also estimated by rapid quantification using an external standard calibration method, direct introduction, and MS/MS experiments. Regression analyses showed a good correlation between the estimated drug concentrations and the scores of individuals distributed on these specific PCs. The detection of these chemical exposure markers proved the potential of the proposed high-throughput approach without any prior drug exposure knowledge as a powerful emerging tool for rapid and large-scale phenotyping of subjects enrolled in epidemiological studies to rapidly characterize the chemical exposome and adherence to medical prescriptions.
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OBJECTIVES: To assess the influence of estrogen deficiency on tooth eruption rate (TER) and gene expression of estrogen receptor alpha and beta (ERα and ERß) in the odontogenic region of teeth with continuous formation in a rat model. MATERIALS AND METHODS: Ovariectomies (OVX; n = 25) and sham surgeries (SHAM; n = 25) were performed in female Wistar rats when animals were 25 days old. The TER of the lower incisors, both in impeded (hyperfunction condition) and unimpeded (trimmed incisal edge-hypofunction condition) conditions, was evaluated using standardized digital photographs acquired every 48-72 h for 3 weeks (35th-53rd day of life), using a camera coupled to a stereomicroscope. Quantitative real-time PCR was performed to evaluate the relative gene expression of ERα and ERß in the odontogenic region. RESULTS: The OVX group showed a significant reduction in TER when compared to the SHAM group, only in the impeded condition (p = 0.03). There was no statistically significant difference between the groups in ERα gene expression (p = 0.33). ERß showed a significantly higher gene expression in the OVX group (p ≤ 0.05). CONCLUSIONS: Estrogen deficiency decreases TER in teeth under impeded condition. Estrogen deficiency also increases ERß gene expression in the odontogenic region of teeth with continuous formation. CLINICAL RELEVANCE: Hormonal disturbances affecting estrogen levels can cause alterations in dental formation and teeth eruption.
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Anormalidades Dentárias , Erupção Dentária , Ratos , Animais , Feminino , Humanos , Erupção Dentária/fisiologia , Ratos Wistar , Receptor alfa de Estrogênio , Incisivo , Receptor beta de Estrogênio/genética , Estrogênios , Receptores de Estrogênio , OvariectomiaRESUMO
Splicing of pre-mRNA is a crucial regulatory stage in the pathway of gene expression. The majority of human genes that encode proteins undergo alternative pre-mRNA splicing and mutations that affect splicing are more prevalent than previously thought. Targeting aberrant RNA(s) may thus provide an opportunity to correct faulty splicing and potentially treat numerous genetic disorders. To that purpose, the use of engineered U1 snRNA (either modified U1 snRNAs or exon-specific U1s-ExSpeU1s) has been applied as a potentially therapeutic strategy to correct splicing mutations, particularly those affecting the 5' splice-site (5'ss). Here we review and summarize a vast panoply of studies that used either modified U1 snRNAs or ExSpeU1s to mediate gene therapeutic correction of splicing defects underlying a considerable number of genetic diseases. We also focus on the pre-clinical validation of these therapeutic approaches both in vitro and in vivo, and summarize the main obstacles that need to be overcome to allow for their successful translation to clinic practice in the future.
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Precursores de RNA , Splicing de RNA , Humanos , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Mutação , Processamento AlternativoRESUMO
INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .
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Metabolômica , Metadados , Curadoria de Dados/métodos , Espectrometria de Massas/métodos , Metaboloma , Metabolômica/métodosRESUMO
During orthodontic tooth movement (OTM), there is the release of cytokines in the gingival crevicular fluid (GCF) that are supposed to participate in the bone remodeling. This systematic review aimed at assessing the effects of photobiomodulation (PBM) on the levels of these cytokines during OTM. This systematic review according to Cochrane Collaboration guidelines aimed to answer the clinical question following the PICOS strategy. The broad search in the literature was performed before 05 April, 2021 in six electronic databases (Pubmed, Web of Science, Scopus, Embase, Cochrane, Biblioteca Virtual de Saúde) and supplemented by the grey literature. The risk of bias of randomized and non-randomized clinical trials was evaluated by two tools: RoB 2 and ROBINS-I. Mean and standard deviation of cytokine levels was extracted to meta-analysis, and the GRADE system was applied to assess the quality of the evidence. Nine studies were included in this review. Low-level laser therapy (LLLT) was the photobiomodulation type used in most of the studies (n = 8). The wavelengths used varied from 618 to 980 nm and also differed concerning the light emission pattern. LLLT increased the levels of IL-1ß, IL-8, OPN, and PGE2, but not TNF-α1, TGF-ß1. The levels of IL6, RANKL, and OPG presented conflicting results. LLLT was statistically associated with an increase of IL-1ß levels (standard mean difference [SMD] = 1.99; 95% confidence interval = 0.66 to 3.33; p < 0.001) with low certainty of evidence. LLLT may increase the levels of IL-1ß and other cytokines; however, the results should be interpreted with caution due to protocol variations between studies, and the few studies added in the meta-analysis.
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Citocinas , Líquido do Sulco Gengival , Imunomodulação , Terapia com Luz de Baixa Intensidade , Técnicas de Movimentação Dentária , Citocinas/análise , Líquido do Sulco Gengival/química , Humanos , Terapia com Luz de Baixa Intensidade/métodos , Técnicas de Movimentação Dentária/métodosRESUMO
The optical and electrochemical properties of a series of polyoxometalate (POM) oxoclusters decorated with two bodipy (boron-dipyrromethene) light-harvesting units were examined. Evaluated here in this polyanionic donor-acceptor system is the effect of the solvent and associated counterions on the intramolecular photoinduced electron transfer. The results show that both solvents and counterions have a major impact upon the energy of the charge-transfer state by modifying the solvation shell around the POMs. This modification leads to a significantly shorter charge separation time in the case of smaller counterion and slower charge recombination in a less polar solvent. These results were rationalized in terms of Marcus theory and show that solvent and counterion both affect the driving force for photoinduced electron transfer and the reorganization energy. This was corroborated with theoretical investigations combining DFT and molecular dynamics simulations.
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RATIONALE: Isomer metabolites are involved in metabolic pathways, and their characterization is essential but remains challenging even using high-performance analytical platforms. The addition of ion mobility prior to mass analysis can help to separate isomers. Here, the ability of a recently developed trapped ion mobility spectrometry system to separate metabolite isomers was examined. METHODS: Three pairs of estrogen isomers were studied as a model of isomeric metabolites under both negative and positive electrospray ionization (ESI) modes using a commercial trapped ion mobility spectrometry-TOF mass spectrometer. The standard metabolites were also spiked into human urine to evaluate the efficiency of trapped ion mobility spectrometry to separate isomers in complex mixtures. RESULTS: The estradiol glucuronide isomers (E2 ß-3G and E2 ß-17G) could be distinguished as deprotonated species, while the estradiol epimers (E2 ß and E2 α) and the methoxyestradiol isomers (2-MeO-E2 ß and 4-MeO-E2 ß) were separated as lithiated adducts in positive ionization mode. When performing analyses in the urine matrix, no alteration in the ion mobility resolving power was observed and the measured collision cross section (CCS) values varied by less than 1.0%. CONCLUSIONS: The trapped ion mobility spectrometry-TOF mass spectrometer enabled the separation of the metabolite isomers with very small differences in CCS values (ΔCCS% = 2%). It is shown to be an effective tool for the rapid characterization of isomers in complex matrices.
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Estrogênios , Espectrometria de Massas/métodos , Estrogênios/química , Estrogênios/isolamento & purificação , Estrogênios/urina , Humanos , IsomerismoRESUMO
More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.
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Doenças do Sistema Nervoso Central/complicações , Doenças do Sistema Nervoso Central/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Doenças por Armazenamento dos Lisossomos/complicações , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Nanopartículas/química , RNA Interferente Pequeno/administração & dosagem , Animais , Encéfalo/metabolismo , Doenças do Sistema Nervoso Central/genética , Doenças do Sistema Nervoso Central/metabolismo , Estabilidade de Medicamentos , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Lysosomal storage diseases (LSDs) are a heterogeneous group of genetic disorders with variable degrees of severity and a broad phenotypic spectrum, which may overlap with a number of other conditions. While individually rare, as a group LSDs affect a significant number of patients, placing an important burden on affected individuals and their families but also on national health care systems worldwide. Here, we present our results on the use of an in-house customized next-generation sequencing (NGS) panel of genes related to lysosome function as a first-line molecular test for the diagnosis of LSDs. Ultimately, our goal is to provide a fast and effective tool to screen for virtually all LSDs in a single run, thus contributing to decrease the diagnostic odyssey, accelerating the time to diagnosis. Our study enrolled a group of 23 patients with variable degrees of clinical and/or biochemical suspicion of LSD. Briefly, NGS analysis data workflow, followed by segregation analysis allowed the characterization of approximately 41% of the analyzed patients and the identification of 10 different pathogenic variants, underlying nine LSDs. Importantly, four of those variants were novel, and, when applicable, their effect over protein structure was evaluated through in silico analysis. One of the novel pathogenic variants was identified in the GM2A gene, which is associated with an ultra-rare (or misdiagnosed) LSD, the AB variant of GM2 Gangliosidosis. Overall, this case series highlights not only the major advantages of NGS-based diagnostic approaches but also, to some extent, its limitations ultimately promoting a reflection on the role of targeted panels as a primary tool for the prompt characterization of LSD patients.
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Marcadores Genéticos , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Lisossomos/patologia , Saúde Global , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/genética , Análise de Sequência de DNARESUMO
Mutations in the GNPTAB and GNPTG genes cause mucolipidosis (ML) type II, type III alpha/beta, and type III gamma, which are autosomal recessively inherited lysosomal storage disorders. GNPTAB and GNPTG encode the α/ß-precursor and the γ-subunit of N-acetylglucosamine (GlcNAc)-1-phosphotransferase, respectively, the key enzyme for the generation of mannose 6-phosphate targeting signals on lysosomal enzymes. Defective GlcNAc-1-phosphotransferase results in missorting of lysosomal enzymes and accumulation of non-degradable macromolecules in lysosomes, strongly impairing cellular function. MLII-affected patients have coarse facial features, cessation of statural growth and neuromotor development, severe skeletal abnormalities, organomegaly, and cardiorespiratory insufficiency leading to death in early childhood. MLIII alpha/beta and MLIII gamma are attenuated forms of the disease. Since the identification of the GNPTAB and GNPTG genes, 564 individuals affected by MLII or MLIII have been described in the literature. In this report, we provide an overview on 258 and 50 mutations in GNPTAB and GNPTG, respectively, including 58 novel GNPTAB and seven novel GNPTG variants. Comprehensive functional studies of GNPTAB missense mutations did not only gain insights into the composition and function of the GlcNAc-1-phosphotransferase, but also helped to define genotype-phenotype correlations to predict the clinical outcome in patients.
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Mucolipidoses/genética , Mutação , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Éxons , Humanos , Íntrons , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/classificação , Doenças por Armazenamento dos Lisossomos do Sistema Nervoso/genética , Mucolipidoses/classificação , Fenótipo , Prognóstico , Domínios Proteicos , Transferases (Outros Grupos de Fosfato Substituídos)/químicaRESUMO
In recent years, the RNA molecule became one of the most promising targets for therapeutic intervention. Currently, a large number of RNA-based therapeutics are being investigated both at the basic research level and in late-stage clinical trials. Some of them are even already approved for treatment. RNA-based approaches can act at pre-mRNA level (by splicing modulation/correction using antisense oligonucleotides or U1snRNA vectors), at mRNA level (inhibiting gene expression by siRNAs and antisense oligonucleotides) or at DNA level (by editing mutated sequences through the use of CRISPR/Cas). Other RNA approaches include the delivery of in vitro transcribed (IVT) mRNA or the use of oligonucleotides aptamers. Here we review these approaches and their translation into clinics trying to give a brief overview also on the difficulties to its application as well as the research that is being done to overcome them.
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Terapia Genética , Oligonucleotídeos Antissenso , Oligonucleotídeos , Terapia Genética/tendências , Humanos , Splicing de RNA , RNA Mensageiro , RNA Interferente PequenoRESUMO
Due to the presence of pollutants in the environment and food, the assessment of human exposure is required. This necessitates high-throughput approaches enabling large-scale analysis and, as a consequence, the use of high-performance analytical instruments to obtain highly informative metabolomic profiles. In this study, direct introduction mass spectrometry (DIMS) was performed using a Fourier transform ion cyclotron resonance (FT-ICR) instrument equipped with a dynamically harmonized cell. Data quality was evaluated based on mass resolving power (RP), mass measurement accuracy, and ion intensity drifts from the repeated injections of quality control sample (QC) along the analytical process. The large DIMS data size entails the use of bioinformatic tools for the automatic selection of common ions found in all QC injections and for robustness assessment and correction of eventual technical drifts. RP values greater than 106 and mass measurement accuracy of lower than 1 ppm were obtained using broadband mode resulting in the detection of isotopic fine structure. Hence, a very accurate relative isotopic mass defect (RΔm) value was calculated. This reduces significantly the number of elemental composition (EC) candidates and greatly improves compound annotation. A very satisfactory estimate of repeatability of both peak intensity and mass measurement was demonstrated. Although, a non negligible ion intensity drift was observed for negative ion mode data, a normalization procedure was easily applied to correct this phenomenon. This study illustrates the performance and robustness of the dynamically harmonized FT-ICR cell to perform large-scale high-throughput metabolomic analyses in routine conditions. Graphical abstract Analytical performance of FT-ICR instrument equipped with a dynamically harmonized cell.
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Espectrometria de Massas/métodos , Metabolômica/métodos , Urinálise/métodos , Ciclotrons , Confiabilidade dos Dados , Análise de Fourier , Humanos , Espectrometria de Massas/instrumentação , Metabolômica/instrumentação , Urinálise/instrumentaçãoRESUMO
Gas-phase fragmentation of single strand DNA-peptide noncovalent complexes is investigated in positive and negative electrospray ionization modes.Collision-induced dissociation experiments, performed on the positively charged noncovalent complex precursor ions, have confirmed the trend previously observed in negative ion mode, i.e. a high stability of noncovalent complexes containing very basic peptidic residues (i.e. R > K) and acidic nucleotide units (i.e. Thy units), certainly incoming from the existence of salt bridge interactions. Independent of the ion polarity, stable noncovalent complex precursor ions were found to dissociate preferentially through covalent bond cleavages of the partners without disrupting noncovalent interactions. The resulting DNA fragment ions were found to be still noncovalently linked to the peptides. Additionally, the losses of an internal nucleic fragment producing "three-body" noncovalent fragment ions were also observed in both ion polarities, demonstrating the spectacular salt bridge interaction stability. The identical fragmentation patterns (regardless of the relative fragment ion abundances) observed in both polarities have shown a common location of salt bridge interaction certainly preserved from solution. Nonetheless, most abundant noncovalent fragment ions (and particularly three-body ones) are observed from positively charged noncovalent complexes. Therefore, we assume that, independent of the preexisting salt bridge interaction and zwitterion structures, multiple covalent bond cleavages from single-stranded DNA/peptide complexes rely on an excess of positive charges in both electrospray ionization ion polarities.