Neurofibromatosis type 1 families with first-degree relatives harbouring distinct NF1 pathogenic variants. Genetic counselling and familial diagnosis: what should be offered?

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder caused by pathogenic variants in NF1 Recently, NF1 testing has been included as a clinical criterion for NF1 diagnosis. Additionally, preconception genetic counselling in patients with NF1 focuses on a 50% risk of transmitting the familial variant as the risk of having a sporadic NF1 is considered the same as the general population. METHODS: 829 individuals, 583 NF1 sporadic cases and 246 patients with NF1 with documented family history, underwent genetic testing for NF1. Genotyping and segregation analysis of NF1 familial variants was determined by microsatellite analysis and NF1 sequencing. RESULTS: The mutational analysis of NF1 in 154 families with two or more affected cases studied showed the co-occurrence of two different NF1 germline pathogenic variants in four families. The estimated mutation rate in those families was 3.89×10-3, 20 times higher than the NF1 mutation rate (~2×10-4) (p=0.0008). Furthermore, the co-occurrence of two different NF1 germline pathogenic variants in these families was 1:39, 60 times the frequency of sporadic NF1 (1:2500) (p=0.003). In all cases, the de novo NF1 pathogenic variant was present in a descendant of an affected male. In two cases, variants were detected in the inherited paternal wild-type allele. CONCLUSIONS: Our results, together with previous cases reported, suggest that the offspring of male patients with NF1 could have an increased risk of experiencing de novo NF1 pathogenic variants. This observation, if confirmed in additional cohorts, could have relevant implications for NF1 genetic counselling, family planning and NF1 genetic testing.

Phospho-RNA-Seq Highlights Specific Small RNA Profiles in Plasma Extracellular Vesicles.

Small RNAs (sRNAs) are bioactive molecules that can be detected in biofluids, reflecting physiological and pathological states. In plasma, sRNAs are found within extracellular vesicles (EVs) and in extravesicular compartments, offering potential sources of highly sensitive biomarkers. Deep sequencing strategies to profile sRNAs favor the detection of microRNAs (miRNAs), the best-known class of sRNAs. Phospho-RNA-seq, through the enzymatic treatment of sRNAs with T4 polynucleotide kinase (T4-PNK), has been recently developed to increase the detection of thousands of previously inaccessible RNAs. In this study, we investigated the value of phospho-RNA-seq on both the EVs and extravesicular plasma subfractions. Phospho-RNA-seq increased the proportion of sRNAs used for alignment and highlighted the diversity of the sRNA transcriptome. Unsupervised clustering analysis using sRNA counts matrices correctly classified the EVs and extravesicular samples only in the T4-PNK treated samples, indicating that phospho-RNA-seq stresses the features of sRNAs in each plasma subfraction. Furthermore, T4-PNK treatment emphasized specific miRNA variants differing in the 5'-end (5'-isomiRs) and certain types of tRNA fragments in each plasma fraction. Phospho-RNA-seq increased the number of tissue-specific messenger RNA (mRNA) fragments in the EVs compared with the extravesicular fraction, suggesting that phospho-RNA-seq favors the discovery of tissue-specific sRNAs in EVs. Overall, the present data emphasizes the value of phospho-RNA-seq in uncovering RNA-based biomarkers in EVs.

Huntington's disease brain-derived small RNAs recapitulate associated neuropathology in mice.

Progressive motor alterations and selective death of striatal medium spiny neurons (MSNs) are key pathological hallmarks of Huntington's disease (HD), a neurodegenerative condition caused by a CAG trinucleotide repeat expansion in the coding region of the huntingtin (HTT) gene. Most research has focused on the pathogenic effects of the resultant protein product(s); however, growing evidence indicates that expanded CAG repeats within mutant HTT mRNA and derived small CAG repeat RNAs (sCAG) participate in HD pathophysiology. The individual contribution of protein versus RNA toxicity to HD pathophysiology remains largely uncharacterized and the role of other classes of small RNAs (sRNA) that are strongly perturbed in HD is uncertain. Here, we demonstrate that sRNA produced in the putamen of HD patients (HD-sRNA-PT) are sufficient to induce HD pathology in vivo. Mice injected with HD-sRNA-PT show motor abnormalities, decreased levels of striatal HD-related proteins, disruption of the indirect pathway, and strong transcriptional abnormalities, paralleling human HD pathology. Importantly, we show that the specific blockage of sCAG mitigates HD-sRNA-PT neurotoxicity only to a limited extent. This observation prompted us to identify other sRNA species enriched in HD putamen with neurotoxic potential. We detected high levels of tRNA fragments (tRFs) in HD putamen, and we validated the neurotoxic potential of an Alanine derived tRF in vitro. These results highlight that HD-sRNA-PT are neurotoxic, and suggest that multiple sRNA species contribute to striatal dysfunction and general transcriptomic changes, favoring therapeutic strategies based on the blockage of sRNA-mediated toxicity.

[Diet quality in a population aged over 65 and related socioeconomic factors]. / Calidad de la dieta en mayores de 65 años y factores socioeconómicos relacionados.

INTRODUCTION: The consumption of certain foods and healthy eating habits are related to preventing and suffering from a number of chronic diseases. These foods tend to be included in food pyramids, such as that proposed by the Spanish Society for Community Nutrition. One method of analysing diet quality is the use of indices that assess the frequency of consumption of different food groups. AIM: To analyse diet quality in a Spanish population of persons aged over 65years using the Healthy Eating Index and to determine how sociodemographic factors affect scores on the index. DESIGN: A cross-sectional, descriptive study was conducted on the diet followed at home by Spanish population aged over 65years, using the Healthy Eating Index and taking information from the 2014 European Health Interview Survey in Spain. Multiple linear regression analysis was used to determine the socioeconomic factors associated with diet quality. RESULTS: Of the study population, 89.6% need to make changes in their diet, while only 8.2% follow a healthy diet. Suffering from chronic diseases, overweight and occasional physical exercise were associated with higher scores on the Healthy Eating Index. CONCLUSION: Most of the population aged 65years or over need to make changes in their dietary patterns. Those with potential health risks are more careful about their diet. These findings confirm the need to promote guidelines for healthy eating in the non-clinical population.

Machine Learning-Assisted Raman Spectroscopy for pH and Lactate Sensing in Body Fluids.

This study presents the combination of Raman spectroscopy with machine learning algorithms as a prospective diagnostic tool capable of detecting and monitoring relevant variations of pH and lactate as recognized biomarkers of several pathologies. The applicability of the method proposed here is tested both in vitro and ex vivo. In a first step, Raman spectra of aqueous solutions are evaluated for the identification of characteristic patterns resulting from changes in pH or in the concentration of lactate. The method is further validated with blood and plasma samples. Principal component analysis is used to highlight the relevant features that differentiate the Raman spectra regarding their pH and concentration of lactate. Partial least squares regression models are developed to capture and model the spectral variability of the Raman spectra. The performance of these predictive regression models is demonstrated by clinically accurate predictions of pH and lactate from unknown samples in the physiologically relevant range. These results prove the potential of our method to develop a noninvasive technology, based on Raman spectroscopy, for continuous monitoring of pH and lactate in vivo.

Proteomic signature of circulating extracellular vesicles in dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) remains a major cause of heart failure and carries a poor prognosis despite important advances in recent years. Better disease characterization using novel molecular techniques is needed to refine its progression. This study explored the proteomic signature of plasma-derived extracellular vesicles (EVs) obtained from DCM patients and healthy controls using size-exclusion chromatography (SEC). EV-enriched fractions were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Raw data obtained from LC-MS/MS were analyzed against the Uniprot human database using MaxQuant software. Additional analyses using Perseus software were based on the Intensity-Based Absolute Quantification (iBAQ) values from MaxQuant analyses. A total of 90.07 ± 21 proteins (227 different proteins) in the DCM group and 96.52 ± 17.91 proteins (183 different proteins) in the control group were identified. A total of 176 proteins (74.6%) were shared by controls and DCM patients, whereas 51 proteins were exclusive for the DCM group and 7 proteins were exclusive for the control group. Fibrinogen (α, ß and γ chain), serotransferrin, α-1-antitrypsin, and a variety of apolipoprotein family members (C-I, C-III, D, H or ß-2-glycoprotein, and J or clusterin) were clustered in SEC-EVs derived from DCM patients relative to controls (p < 0.05). Regarding Gene Ontology analysis, response to stress and protein activation-related proteins were enriched in DCM-EVs compared with controls. Thus, the present study reports the distinct proteomic signature of circulating DCM-EVs compared with control-EVs. Furthermore, we confirm that SEC obtains highly purified EV fractions from peripheral blood samples for subsequent use in determining disease-specific proteomic signatures.

Extracellular vesicles do not contribute to higher circulating levels of soluble LRP1 in idiopathic dilated cardiomyopathy.

Idiopathic dilated cardiomyopathy (IDCM) is a frequent cause of heart transplantation. Potentially valuable blood markers are being sought, and low-density lipoprotein receptor-related protein 1 (LRP1) has been linked to the underlying molecular basis of the disease. This study compared circulating levels of soluble LRP1 (sLRP1) in IDCM patients and healthy controls and elucidated whether sLRP1 is exported out of the myocardium through extracellular vesicles (EVs) to gain a better understanding of the pathogenesis of the disease. LRP1 α chain expression was analysed in samples collected from the left ventricles of explanted hearts using immunohistochemistry. sLRP1 concentrations were determined in platelet-free plasma by enzyme-linked immunosorbent assay. Plasma-derived EVs were extracted by size-exclusion chromatography (SEC) and characterized by nanoparticle tracking analysis and cryo-transmission electron microscopy. The distributions of vesicular (CD9, CD81) and myocardial (caveolin-3) proteins and LRP1 α chain were assessed in SEC fractions by flow cytometry. LRP1 α chain was preferably localized to blood vessels in IDCM compared to control myocardium. Circulating sLRP1 was increased in IDCM patients. CD9- and CD81-positive fractions enriched with membrane vesicles with the expected size and morphology were isolated from both groups. The LRP1 α chain was not present in these SEC fractions, which were also positive for caveolin-3. The increase in circulating sLRP1 in IDCM patients may be clinically valuable. Although EVs do not contribute to higher sLRP1 levels in IDCM, a comprehensive analysis of EV content would provide further insights into the search for novel blood markers.

GBA Mutations Are Associated With Earlier Onset and Male Sex in Dementia With Lewy Bodies.

BACKGROUND: Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are Lewy body diseases characterized by similar pathological features. Several studies have shown a relation between alterations in the glucocerebrosidase gene (GBA) and the development of LB diseases. Here, we explored the role of GBA mutations in Spanish DLB patients. METHODS: GBA mRNA sequences were analyzed in a neuropathological (50 DLB, 43 PD, and 34 control brains) and in a clinical cohort (47 DLB patients and 131 unaffected individuals). RESULTS: Sixteen GBA mutation carriers were identified, 5 of which were brains with pure DLB. The most common mutation, E326K, was strongly associated with pure DLB and PD with dementia. GBA mutations were overrepresented in men and associated with earlier DLB onset. CONCLUSIONS: GBA mutations are also an important risk factor for DLB development in the Spanish population, are associated with earlier disease onset, and are more prevalent in men. © 2015 International Parkinson and Movement Disorder Society.

Decontamination of dental implant surface in peri-implantitis treatment: a literature review.

Etiological treatment of peri-implantitis aims to reduce the bacterial load within the peri-implant pocket and decontaminate the implant surface in order to promote osseointegration. The aim of this literature review was to evaluate the efficacy of different methods of implant surface decontamination. A search was conducted using the PubMed (Medline) database, which identified 36 articles including in vivo and in vitro studies, and reviews of different decontamination systems (chemical, mechanical, laser and photodynamic therapies). There is sufficient consensus that, for the treatment of peri-implant infections, the mechanical removal of biofilm from the implant surface should be supplemented by chemical decontamination with surgical access. However, more long-term research is needed to confirm this and to establish treatment protocols responding to different implant characterics.

Neuron-derived extracellular vesicles contain synaptic proteins, promote spine formation, activate TrkB-mediated signalling and preserve neuronal complexity.

Extracellular vesicles (EVs) play an important role in intercellular communication as carriers of signalling molecules such as bioactive miRNAs, proteins and lipids. EVs are key players in the functioning of the central nervous system (CNS) by influencing synaptic events and modulating recipient neurons. However, the specific role of neuron-to-neuron communication via EVs is still not well understood. Here, we provide evidence that primary neurons uptake neuron-derived EVs in the soma, dendrites, and even in the dendritic spines, and carry synaptic proteins. Neuron-derived EVs increased spine density and promoted the phosphorylation of Akt and ribosomal protein S6 (RPS6), via TrkB-signalling, without impairing the neuronal network activity. Strikingly, EVs exerted a trophic effect on challenged nutrient-deprived neurons. Altogether, our results place EVs in the spotlight for synaptic plasticity modulation as well as a possible therapeutic tool to fight neurodegeneration.

RNA-based analysis of two SMARCB1 mutations associated with familial schwannomatosis with meningiomas.

Germline mutations in the SMARCB1 gene cause familial schwannomatosis, a condition characterized by the presence of multiple schwannomas, although mutations in SMARCB1 have also been associated with rhadboid tumor predisposition syndrome 1 (RTPS1). Both schwannomatosis and RTPS1 are autosomal dominant conditions that predispose individuals to develop distinct types of tumors. We clinically and genetically characterized two families with schwannomatosis associated with SMARCB1 mutations. Eight affected members of these families developed different numbers of schwannomas and/or meningiomas at distinct ages, evidence that meningiomas are variably expressed in this condition. We identified two germline mutations in SMARCB1 associated with the familial disease, c.233-1G>A and the novel c.207_208dupTA mutation, which both proved to affect the main SMARCB1 isoforms at the RNA level distinctly. Interestingly, the c.207_208dupTA mutation had no effect on the coding sequence, pre-mRNA splicing or the level of expression of the SMARCB1 isoform 2. Furthermore, SMARCB1 isoforms harboring a premature termination codon were largely eliminated via the nonsense-mediated mRNA decay pathway. Our results highlight the importance of RNA-based studies to characterize SMARCB1 germline mutations in order to determine their impact on protein expression and gain further insight into the genetic basis of conditions associated with SMARCB1 mutations.

Identification of Hypoxia-Ischemia by Chemometrics Considering Systemic Changes of Physiology.

Perinatal asphyxia represents a major medical disorder and is related to around a fourth of all neonatal deaths worldwide. Specific thresholds for lactate or pH levels define the gold standard for detecting hypoxic-ischemic events as physiological abnormalities. In contrast to current gold standard, we analyze the systemic picture, represented by the whole set of biochemical parameters from blood gas analysis, by multiparametric machine learning algorithms. In a swine model with 22 objects, we investigate the impact of neonatal hypoxic-ischemic encephalopathy on 18 individual physiological parameters. In a first approach, we study the statistical significance of individual parameters by univariate analysis methods. In a second approach, we take the most relevant parameters as input for the development of predictive models by different hybrid and non-hybrid classification algorithms. The predictive power of our multiparametric models outperforms by far the limited performance of pH and lactate as reliable indicators, despite strong correlation with hypoxic-ischemic events. We have been able to detect hypoxic-ischemic events even one hour after the episode, with accuracies close to 100% in contrast to pH or lactate-based diagnosis with 62% and 78%, respectively. By all machine learning algorithms, lactate is recognized as the main contributor due to its longer-term evidence of hypoxia-ischemia episodes. However, substantial improvement of the diagnosis is achieved by predictions based on a systemic picture of different physiological parameters. Our results prove the potential applicability of our method as a support tool for decision-making that will allow obstetricians to identify hypoxic-ischemic episodes more accurately during labor.

Enterovirus co-infections and onychomadesis after hand, foot, and mouth disease, Spain, 2008.

Hand, foot, and mouth disease (HFMD), a common disease caused by enteroviruses (EVs), usually affects children. Clustered and sporadic HFMD cases, followed by onychomadesis (nail shedding), occurred during summer and fall 2008 in Valencia, Spain. Fecal samples from onychomadesis patients, who did or did not have previous HFMD, and from healthy children exposed to onychomadesis patients tested positive for EV. The complete viral protein 1 capsid gene sequence was obtained for typing and phylogenetic analysis. Two EV serotypes, coxsackievirus A10 and coxsackievirus B1 (CVB1), were mainly detected as a monoinfection or co-infection in a childcare center where an onychomadesis outbreak occurred. On the basis of our results, and detection of CVB1 in 2 other contemporary onychomadesis outbreaks in childcare centers in Spain, we propose that mixed infection of an EV serotype that causes HFMD, plus the serotype CVB1, could explain the emergence after HFMD of onychomadesis, a rare and late complication.

Link between alginate reaction front propagation and general reaction diffusion theory.

We provide a common theoretical framework reuniting specific models for the Ca(2+)-alginate system and general reaction diffusion theory along with experimental validation on a microfluidic chip. As a starting point, we use a set of nonlinear, partial differential equations that are traditionally solved numerically: the Mikkelsen-Elgsaeter model. Applying the traveling-wave hypothesis as a major simplification, we obtain an analytical solution. The solution indicates that the fundamental properties of the alginate reaction front are governed by a single dimensionless parameter λ. For small λ values, a large depletion zone accompanies the reaction front. For large λ values, the alginate reacts before having the time to diffuse significantly. We show that the λ parameter is of general importance beyond the alginate model system, as it can be used to classify known solutions for second-order reaction diffusion schemes, along with the novel solution presented here. For experimental validation, we develop a microchip model system, in which the alginate gel formation can be carried out in a highly controlled, essentially 1D environment. The use of a filter barrier enables us to rapidly renew the CaCl(2) solution, while maintaining flow speeds lower than 1 µm/s for the alginate compartment. This allows one to impose an exactly known bulk CaCl(2) concentration and diffusion resistance. This experimental model system, taken together with the theoretical development, enables the determination of the entire set of physicochemical parameters governing the alginate reaction front in a single experiment.

Hepatitis C virus sequences from different patients confirm the existence and transmissibility of subtype 2q, a rare subtype circulating in the metropolitan area of Barcelona, Spain.

The hepatitis C virus (HCV) has been classified into six genotypes and more than 70 subtypes with distinct geographical and epidemiological distributions. While 18 genotype 2 subtypes have been proposed, only 5 have had their complete sequence determined. The aim of this study was to characterize HCV isolates from three patients from the Barcelona metropolitan area of Spain for whom commercial genotyping methods provided discordant results. Full-length genome sequencing was carried out for 2 of the 3 patients; for the third patient only partial NS5B sequences could be obtained. The generated sequences were subjected to phylogenetic, recombination, and identity analyses. Sequences covering most of the HCV genome (9398 and 9566 nt in length) were obtained and showed a 90.3% identity to each other at the nucleotide level, while both sequences differed by 17.5-22.6% from the other fully sequenced genotype 2 subtypes. No evidence of recombination was found. The NS5B phylogenetic tree showed that sequences from the three patients cluster together with the only representative sequence of the provisionally designed 2q subtype, which also corresponds to a patient from Barcelona. Phylogenetic analysis of the full coding sequence showed that subtype 2q was more closely related to subtype 2k. The results obtained in this study suggest that subtype 2q now meets the requirements for confirmed designation status according to consensus criteria for HCV classification and nomenclature, and its epidemiological value is ensured as it has spread among several patients in the Barcelona metropolitan area.

Pseudomonas aeruginosa Susceptibility in Spain: Antimicrobial Activity and Resistance Suppression Evaluation by PK/PD Analysis.

Pseudomonas aeruginosa remains one of the major causes of healthcare-associated infection in Europe; in 2019, 12.5% of invasive isolates of P. aeruginosa in Spain presented combined resistance to ≥3 antimicrobial groups. The Spanish nationwide survey on P. aeruginosa antimicrobial resistance mechanisms and molecular epidemiology was published in 2019. Based on the information from this survey, the objective of this work was to analyze the overall antimicrobial activity of the antipseudomonal antibiotics considering pharmacokinetic/pharmacodynamic (PK/PD) analysis. The role of PK/PD to prevent or minimize resistance emergence was also evaluated. A 10,000-subject Monte Carlo simulation was executed to calculate the probability of target attainment (PTA) and the cumulative fraction of response (CFR) considering the minimum inhibitory concentration (MIC) distribution of bacteria isolated in ICU or medical wards, and distinguishing between sample types (respiratory and non-respiratory). Ceftazidime/avibactam followed by ceftolozane/tazobactam and colistin, categorized as the Reserve by the Access, Watch, Reserve (AWaRe) classification of the World Health Organization, were the most active antimicrobials, with differences depending on the admission service, sample type, and dose regimen. Discrepancies between EUCAST-susceptibility breakpoints for P. aeruginosa and those estimated by PK/PD analysis were detected. Only standard doses of ceftazidime/avibactam and ceftolozane/tazobactam provided drug concentrations associated with resistance suppression.

Platelet miRNA Biosignature Discriminates between Dementia with Lewy Bodies and Alzheimer's Disease.

Dementia with Lewy bodies (DLB) is one of the most common causes of degenerative dementia, after Alzheimer's disease (AD), and presents pathological and clinical overlap with both AD and Parkinson's disease (PD). Consequently, only one in three DLB cases is diagnosed correctly. Platelets, previously related to neurodegeneration, contain microRNAs (miRNAs) whose analysis may provide disease biomarkers. Here, we profiled the whole platelet miRNA transcriptome from DLB patients and healthy controls. Differentially expressed miRNAs were further validated in three consecutive studies from 2017 to 2019 enrolling 162 individuals, including DLB, AD, and PD patients, and healthy controls. Results comprised a seven-miRNA biosignature, showing the highest diagnostic potential for the differentiation between DLB and AD. Additionally, compared to controls, two miRNAs were down-regulated in DLB, four miRNAs were up-regulated in AD, and two miRNAs were down-regulated in PD. Predictive target analysis identified three disease-specific clusters of pathways as a result of platelet-miRNA deregulation. Our cross-sectional study assesses the identification of a novel, highly specific and sensitive platelet-associated miRNA-based biosignature, which distinguishes DLB from AD.

[Empirically Supported Psychological Treatments for Children and Adolescents: State of the Art].

Empirically Supported Psychological Treatments for Children and Adolescents: State of the Art. BACKGROUND: The empirical evidence accumulated on the efficacy, effectiveness, and efficiency of psychotherapeutic treatments in children and adolescents calls for an update. The main goal of this paper objective was to carry out a selective review of empirically supported psychological treatments for a variety of common psychological disorders and problems in childhood and adolescence. METHOD: A review was carried out of the psychological treatments for different psychological disorders and problems in social-emotional or behavioral adjustment in the child-adolescent population according to the Spanish National Health System (Clinical Practice Guidelines) levels of evidence and degrees of recommendation. RESULTS: The findings suggest that psychological treatments have empirical support for addressing a wide range of psychological problems in these developmental stages. The degree of empirical support ranges from low to high depending on the phenomenon analyzed. The review suggests unequal progress in the different fields of intervention. CONCLUSIONS: From this update, psychologists will be able to make informed decisions when implementing those empirically supported treatments to address the problems that occur in childhood and adolescence.

A unified approach to dielectric single cell analysis: impedance and dielectrophoretic force spectroscopy.

In this review we present a unified approach for single cell dielectric spectroscopy. Impedance spectroscopy and dielectrophoretic cell sorting, current microtechnologies applied in electrical analysis of single cells are discussed based on their closely related physical principles. In addition, examples of microfluidic devices will be presented: a microfabricated flow cytometer for single cell discrimination based on impedance analysis and a miniaturized continuous dielectrophoretic cell sorter, both using the concept of liquid electrodes. Using the experimental results obtained from both microdevices, we give a comparative overview over the dielectrophoretic sorting and impedance spectroscopy.

Optimization of microfluidic single cell trapping for long-term on-chip culture.

The poor efficiency of microfluidic single cell trapping is currently restricting the full potential of state-of-the-art single cell analyses. Using fluid dynamics simulations in combination with particle image velocimetry to systematically optimize trap architectures, we present a microfluidic chip with enhanced single cell trapping and on-chip culture performance. Upon optimization of trap geometries, we measured trapping efficiencies of up to 97%. Our device also enables the stable, relatively long-term culture of individual non-adherent mammalian cells in high-throughput without a significant decrease in cell viability. As a first application of this platform we demonstrate the automated separation of the two daughter cells generated upon single cell division. The reliable trapping and re-trapping of mammalian cells should for example provide the fundament for novel types of investigations in stem cell and tumour cell biology, which depend on reliable tracking of genealogical relationships such as in stem cell lineage tracking.