Dexibuprofen biodegradable nanoparticles: one step closer towards a better ocular interaction study

Ocular inflammation is one of the most prevalent diseases in ophthalmology, which can affect various parts of the eye or the surrounding tissues. Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, are commonly used to treat ocular inflammation in the form of eye-drops. However, their bioavailability in ocular tissues is very low (less than 5%). Therefore, drug delivery systems such as biodegradable polymeric PLGA nanoparticles constitute a suitable alternative to topical eye administration, as they can improve ocular bioavailability and simultaneously reduce drug induced side effects. Moreover, their prolonged drug release can enhance patient treatment adherence as they require fewer administrations. Therefore, several formulations of PLGA based nanoparticles encapsulating dexibuprofen (active enantiomer of Ibuprofen) were prepared using the solvent displacement method employing different surfactants. The formulations have been characterized and their interactions with a customized lipid corneal membrane model were studied. Ex vivo permeation through ocular tissues and in vivo anti-inflammatory efficacy have also been studied.This work was supported by grant RTI2018-094120-B-I00 from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Regional Development Fund. ESL wants to acknowledge the Institute of Nanoscience and Nanotechnology (ART2018 project).info:eu-repo/semantics/publishedVersio

Predicting Alzheimer's disease CSF core biomarkers: a multimodal Machine Learning approach

IntroductionAlzheimer's disease (AD) is a progressive neurodegenerative disorder. Current core cerebrospinal fluid (CSF) AD biomarkers, widely employed for diagnosis, require a lumbar puncture to be performed, making them impractical as screening tools. Considering the role of sleep disturbances in AD, recent research suggests quantitative sleep electroencephalography features as potential non-invasive biomarkers of AD pathology. However, quantitative analysis of comprehensive polysomnography (PSG) signals remains relatively understudied. PSG is a non-invasive test enabling qualitative and quantitative analysis of a wide range of parameters, offering additional insights alongside other biomarkers. Machine Learning (ML) gained interest for its ability to discern intricate patterns within complex datasets, offering promise in AD neuropathology detection. Therefore, this study aims to evaluate the effectiveness of a multimodal ML approach in predicting core AD CSF biomarkers.MethodsMild-moderate AD patients were prospectively recruited for PSG, followed by testing of CSF and blood samples for biomarkers. PSG signals underwent preprocessing to extract non-linear, time domain and frequency domain statistics quantitative features. Multiple ML algorithms were trained using four subsets of input features: clinical variables (CLINVAR), conventional PSG parameters (SLEEPVAR), quantitative PSG signal features (PSGVAR) and a combination of all subsets (ALL). Cross-validation techniques were employed to evaluate model performance and ensure generalizability. Regression models were developed to determine the most effective variable combinations for explaining variance in the biomarkers.ResultsOn 49 subjects, Gradient Boosting Regressors achieved the best results in estimating biomarkers levels, using different loss functions for each biomarker: least absolute deviation (LAD) for the Aβ42, least squares (LS) for p-tau and Huber for t-tau. The ALL subset demonstrated the lowest training errors for all three biomarkers, albeit with varying test performance. Specifically, the SLEEPVAR subset yielded the best test performance in predicting Aβ42, while the ALL subset most accurately predicted p-tau and t-tau due to the lowest test errors.ConclusionsMultimodal ML can help predict the outcome of CSF biomarkers in early AD by utilizing non-invasive and economically feasible variables. The integration of computational models into medical practice offers a promising tool for the screening of patients at risk of AD, potentially guiding clinical decisions

Leigh syndrome is the main clinical characteristic of PTCD3 deficiency

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases

Altres ajuts: Generalitat de Catalunya, Departament de Salut; Generalitat de Catalunya, Departament d'Empresa i Coneixement i CERCA Program; Ministerio de Ciencia e Innovación; Instituto Nacional de Bioinformática; ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa; European Regional Development Fund (FEDER).Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%)

Clustering COVID-19 ARDS patients through the first days of ICU admission. An analysis of the CIBERESUCICOVID Cohort

Background Acute respiratory distress syndrome (ARDS) can be classified into sub-phenotypes according to different inflammatory/clinical status. Prognostic enrichment was achieved by grouping patients into hypoinflammatory or hyperinflammatory sub-phenotypes, even though the time of analysis may change the classification according to treatment response or disease evolution. We aimed to evaluate when patients can be clustered in more than 1 group, and how they may change the clustering of patients using data of baseline or day 3, and the prognosis of patients according to their evolution by changing or not the cluster.Methods Multicenter, observational prospective, and retrospective study of patients admitted due to ARDS related to COVID-19 infection in Spain. Patients were grouped according to a clustering mixed-type data algorithm (k-prototypes) using continuous and categorical readily available variables at baseline and day 3.Results Of 6205 patients, 3743 (60%) were included in the study. According to silhouette analysis, patients were grouped in two clusters. At baseline, 1402 (37%) patients were included in cluster 1 and 2341(63%) in cluster 2. On day 3, 1557(42%) patients were included in cluster 1 and 2086 (57%) in cluster 2. The patients included in cluster 2 were older and more frequently hypertensive and had a higher prevalence of shock, organ dysfunction, inflammatory biomarkers, and worst respiratory indexes at both time points. The 90-day mortality was higher in cluster 2 at both clustering processes (43.8% [n = 1025] versus 27.3% [n = 383] at baseline, and 49% [n = 1023] versus 20.6% [n = 321] on day 3). Four hundred and fifty-eight (33%) patients clustered in the first group were clustered in the second group on day 3. In contrast, 638 (27%) patients clustered in the second group were clustered in the first group on day 3.Conclusions During the first days, patients can be clustered into two groups and the process of clustering patients may change as they continue to evolve. This means that despite a vast majority of patients remaining in the same cluster, a minority reaching 33% of patients analyzed may be re-categorized into different clusters based on their progress. Such changes can significantly impact their prognosis

Implementació i desenvolupament d'estudis funcionals per demostrar la patogenicitat de variants de significat incert i identificar nous gens causants de malaltia

[cat] Les malalties metabòliques hereditàries (MMH) són un grup de patologies amb una gran heterogeneïtat clínica, bioquímica i genètica. Actualment se n’han descrit més de 1 800. En l’última dècada la implementació de les tècniques de seqüenciació massiva (seqüenciació de l’exoma complet, WES i seqüenciació del genoma complet, WGS) ha permès identificar la causa genètica de la malaltia en un gran nombre de pacients i descobrir un nombre important de nous gens associats a patologia. Malgrat això, aproximadament la meitat dels pacients roman sense rebre un diagnòstic genètic definitiu. Això es deu, en part, a limitacions tècniques pel que fa a la detecció i priorització de variants candidates, així com a l gran nombre de variants de significat incert (VUS) que s’identifiquen. Per aquests motius cal desenvolupar aproximacions addicionals que ajudin a prioritzar i interpretar l’impacte funcional de les variants identificades per poder-ne determinar la seva patogenicitat. En el context d’aquesta tesi doctoral, hem aplicat una estratègia multiòmica, basada en la combinació de WES/WGS amb la seqüenciació de l’RNA (RNA seq)seq), en una cohort de 65 pacients amb sospita de MMH. L’estratègia utilitzada ha permès orientar el diagnòstic i identificar gens causants de malaltia en un gran nombre de pacients. No obstant això, en molts casos s’ha requerit la realització d’estudis funcionals , la contribució dels quals ha estat essencial per demostrar l’impacte de les variants prioritzades i assolir un diagnòstic genètic definitiu. En concret, la tesi doctoral inclou cinc publicacions originals , quatre de les quals es centr en la caracterització funcional de noves variants genètiques en quatre gens implicats en funcions molt diverses : CCDC186 , PEX1 , PTCD3 i TFRC . En el primer treball hem identificat tres pacients amb variants en CCDC186 i confirmem que aquestes s’associen a un trastorn que presenta una característica combinació d’alteracions endocrines i del sistema nerviós central. En el segon article hem aplicat la combinació de WES, RNA seq i estudis funcionals per acabar amb l’odissea diagnòstica de més 30 anys en una pacient amb deficiència de PEX1 . En el tercer treball hem descrit tres pacients amb variants en PTCD3 , que codifica per una proteïna associada al ribosoma mitocondrial , la disfunció de la qual provoca un trastorn del metabolisme energètic mitocondrial. A més, hem consolidat que les variants en aquest gen s’associen amb l a síndrome de Leigh. En el quart treball hem descrit per primer cop que diferents variants en TFRC poden associar-se a diverses presentacions clíniques: immunodeficiència combinada, anèmia microcítica o cardiomiopatia hipertròfica. Estudis funcionals realitzats en models cel·lulars modificats genèticament mitjançant CRISPR/Cas9 han ampliat les bases fisiopatològiques de la deficiència de TFRC , tot mostrant que, en diferent mesura, les variants identificades en els pacients afecten la viabilitat cel·lular, l a regulació de l’autofàgia i l’homeòstasi del ferro i provoquen una alteració generalitzada de la funció mitocondrial. La cinquena publicació ha consistit en el desenvolupament d’un flux de treball basat en el sistema d’edició genètica CRISPR/Cas9 en cèl·lules HAP1 . L’estratègia proposada permet introduir les variants genètiques identificades en pacients i generar models cel·lulars models cel·lulars knockin per tal d’investigar les seves conseqüències funcionals. . En conclusió, aquesta tesi doctoral emfatitza la importància d’implementar els estudis de validació funcional, així com altres aproximacions òmiques més enllà del WES/WGS al flux diagnòstic de les malalties minoritàries. En aquest sentit, la incorporació de l’RNAl’RNA-seq ha contribuït de forma important al diagnòstic gràcies a la identificació de variants causants de patologia no prioritzades pel WES, així com generant evidències funcionals de l’impacte d’aquelles variants que afecten generant el processament del mRNA.. A més, els estudis funcionals realitzats en material biològic dels pacients o en models cel·lulars modificats genèticament han contribuït en avaluar l’impacte de les variants identificades, tot ajudant a dirigir el diagnòstic i en alguns casos a la presa de decisions decisions clíniques

Implementació i desenvolupament d'estudis funcionals per demostrar la patogenicitat de variants de significat incert i identificar nous gens causants de malaltia

Programa de Doctorat en Biomedicina[cat] Les malalties metabòliques hereditàries (MMH) són un grup de patologies amb una gran heterogeneïtat clínica, bioquímica i genètica. Actualment se n’han descrit més de 1 800. En l’última dècada la implementació de les tècniques de seqüenciació massiva (seqüenciació de l’exoma complet, WES i seqüenciació del genoma complet, WGS) ha permès identificar la causa genètica de la malaltia en un gran nombre de pacients i descobrir un nombre important de nous gens associats a patologia. Malgrat això, aproximadament la meitat dels pacients roman sense rebre un diagnòstic genètic definitiu. Això es deu, en part, a limitacions tècniques pel que fa a la detecció i priorització de variants candidates, així com a l gran nombre de variants de significat incert (VUS) que s’identifiquen. Per aquests motius cal desenvolupar aproximacions addicionals que ajudin a prioritzar i interpretar l’impacte funcional de les variants identificades per poder-ne determinar la seva patogenicitat. En el context d’aquesta tesi doctoral, hem aplicat una estratègia multiòmica, basada en la combinació de WES/WGS amb la seqüenciació de l’RNA (RNA seq)seq), en una cohort de 65 pacients amb sospita de MMH. L’estratègia utilitzada ha permès orientar el diagnòstic i identificar gens causants de malaltia en un gran nombre de pacients. No obstant això, en molts casos s’ha requerit la realització d’estudis funcionals , la contribució dels quals ha estat essencial per demostrar l’impacte de les variants prioritzades i assolir un diagnòstic genètic definitiu. En concret, la tesi doctoral inclou cinc publicacions originals , quatre de les quals es centr en la caracterització funcional de noves variants genètiques en quatre gens implicats en funcions molt diverses : CCDC186 , PEX1 , PTCD3 i TFRC . En el primer treball hem identificat tres pacients amb variants en CCDC186 i confirmem que aquestes s’associen a un trastorn que presenta una característica combinació d’alteracions endocrines i del sistema nerviós central. En el segon article hem aplicat la combinació de WES, RNA seq i estudis funcionals per acabar amb l’odissea diagnòstica de més 30 anys en una pacient amb deficiència de PEX1 . En el tercer treball hem descrit tres pacients amb variants en PTCD3 , que codifica per una proteïna associada al ribosoma mitocondrial , la disfunció de la qual provoca un trastorn del metabolisme energètic mitocondrial. A més, hem consolidat que les variants en aquest gen s’associen amb l a síndrome de Leigh. En el quart treball hem descrit per primer cop que diferents variants en TFRC poden associar-se a diverses presentacions clíniques: immunodeficiència combinada, anèmia microcítica o cardiomiopatia hipertròfica. Estudis funcionals realitzats en models cel·lulars modificats genèticament mitjançant CRISPR/Cas9 han ampliat les bases fisiopatològiques de la deficiència de TFRC , tot mostrant que, en diferent mesura, les variants identificades en els pacients afecten la viabilitat cel·lular, l a regulació de l’autofàgia i l’homeòstasi del ferro i provoquen una alteració generalitzada de la funció mitocondrial. La cinquena publicació ha consistit en el desenvolupament d’un flux de treball basat en el sistema d’edició genètica CRISPR/Cas9 en cèl·lules HAP1 . L’estratègia proposada permet introduir les variants genètiques identificades en pacients i generar models cel·lulars models cel·lulars knockin per tal d’investigar les seves conseqüències funcionals. . En conclusió, aquesta tesi doctoral emfatitza la importància d’implementar els estudis de validació funcional, així com altres aproximacions òmiques més enllà del WES/WGS al flux diagnòstic de les malalties minoritàries. En aquest sentit, la incorporació de l’RNAl’RNA-seq ha contribuït de forma important al diagnòstic gràcies a la identificació de variants causants de patologia no prioritzades pel WES, així com generant evidències funcionals de l’impacte d’aquelles variants que afecten generant el processament del mRNA.. A més, els estudis funcionals realitzats en material biològic dels pacients o en models cel·lulars modificats genèticament han contribuït en avaluar l’impacte de les variants identificades, tot ajudant a dirigir el diagnòstic i en alguns casos a la presa de decisions decisions clíniques

Novel customized age-dependent corneal membranes and interactions with biodegradable nanoparticles loaded with dexibuprofen

Ocular inflammation is one of the most prevalent diseases in ophthalmology and it is currently treated using eye drops of nonsteroidal antiinflammatory drugs such as dexibuprofen (DXI). However, their bioavailability is low and therefore, PLGA nanoparticles constitute a suitable approach to be administered as eyedrops. Therefore, DXI has been encapsulated into PLGA nanoparticles (DXI-NPs). Although the eye, and specifically the cornea, suffers from age-related changes in its composition, current medications are not focused on these variations. Therefore, to elucidate the interaction mechanism of DXI-NPs with the cornea in relation with age, two different corneal membrane models have been developed (corresponding to adult and elder population) using lipid monolayers, large and giant unilamellar vesicles. Interactions of both DXI and DXI-NPs were studied with these models by means of Langmuir balance technique, dipole potential, anisotropy and confocal microscopy. In addition, fluorescently labelled nanoparticles were administered to mice in order to corroborate these data obtained in vitro. It was observed that DXI-NPs interact with lipid membranes through an adhesion process, mainly in the rigid regions and afterwards DXI-NPs are internalized by a wrapping process. Furthermore, differences on the dipole potential caused by DXI-NPs in each corneal membrane have been obtained due to the increase of membrane rigidity on the ECMM. Additionally, it can be confirmed that DXI-NPs adhere to Lo phase and also inside the lipid membrane. Finally, in vitro and in vivo results corroborate that DXI-NPs are adhered to the more ordered phase. Finally, differences between interactions of DXI-NPs with the elder and adult corneal tissue were observed.The authors want to acknowledge the support of the Spanish Ministry under the project PID2021-122187NB-C32 and the support of the Generalitat of Catalonia (2017SGR1447). ESL wants to acknowledge the support of the Grants for the Requalification of the Spanish University System and the Institute of Nanoscience and Nanotechnology (ART2018 project). GE, acknowledges the financial support of the Generalitat de Catalunya for the PhD scholarship FI-SDUR (2020FISDU187). The authors also acknowledge the student Roberto Rocamora for his technical support. The authors would like to thank Manel Bosch Marimón from the Scientific and Technological Center of the University of Barcelona for his technical support.Peer reviewe

Obtenció de nanopartícules polimèriques per a l'administració ocular de fàrmacs i estudis d'interacció amb tècniques biofísiques

Ageing is a biological process that leads to a decrease in physiological functions, and is the main risk factor for most common diseases in developed countries. The process causes modifications in biological membranes, especially affecting some body structures, such as the ocular surface. Specifically, the lipids of the corneal ocular surface present modifications related to age, with increased epithelial permeability making the elderly more vulnerable to corneal injuries. Inflammation is one of the most common pathologies evident in ophthalmology, which can affect different eye structures. Non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen, are widely used to treat ocular inflammation. Therefore, a drug delivery system such as PLGA biodegradable polymeric nanoparticles is an interesting alternative, as it can improve drug bioavailability, reduce the number of administrations required, and increase therapeutic adherence. This work also encompassed the proposal of a methodology for the development of formulations of nanoparticles that contain encapsulated dexibuprofen, and the creation of a model of corneal membrane that would permit in vitro studies of interactions. Keywords: dexibuprofen, biological membranes, eye, nanoparticles

Diagnostic odyssey in an adult patient with ophthalmologic abnormalities and hearing loss: Contribution of RNA-seq to the diagnosis of a PEX1 deficiency.

Peroxisomal biogenesis disorders (PBDs) are a heterogeneous group of genetic diseases. Multiple peroxisomal pathways are impaired, and very long chain fatty acids (VLCFA) are the first line biomarkers for the diagnosis. The clinical presentation of PBDs may range from severe, lethal multisystemic disorders to milder, late-onset disease. The vast majority of PBDs belong to Zellweger Spectrum Disordes (ZSDs) and represents a continuum of overlapping clinical symptoms, with Zellweger syndrome being the most severe and Heimler syndrome the less severe disease. Mild clinical conditions frequently present normal or slight biochemical alterations, making the diagnosis of these patients challenging. In the present study we used a combined WES and RNA-seq strategy to diagnose a patient presenting with retinal dystrophy as the main clinical symptom. Results showed the patient was compound heterozygous for mutations in PEX1. VLCFA were normal, but retrospective analysis of lysosphosphatidylcholines (LPC) containing C22:0-C26:0 species was altered. This simple test could avoid the diagnostic odyssey of patients with mild phenotype, such as the individual described here, who was diagnosed very late in adult life. We provide functional data in cell line models that may explain the mild phenotype of the patient by demonstrating the hypomorphic nature of a deep intronic variant altering PEX1 mRNA processin