LINE-1 retrotransposons contribute to mouse PV interneuron development.

Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.

Nomophobia and Alcohol, Tobacco, and Cannabis Consumption in Adolescents in Galicia.

This study aimed at exploring the association of nomophobia with alcohol, tobacco, and/or cannabis consumption among high school students. We carried out a cross-sectional study among high school and vocational training students in Galicia, Northwest Spain (N = 3,100). Collected data included nomophobia, sociodemographic variables, and alcohol, tobacco, and cannabis consumption. Nomophobia was measured using the validated Nomophobia Questionnaire. Adjusted odds ratios (ORs) and their 95 percent confidence intervals (CIs) were estimated using generalized linear mixed models. More than a quarter of the adolescents (27.7 percent) had nomophobia. We found an association between nomophobia and a high level of tobacco smoking in the last month in boys (OR = 2.16; 95 percent CI: 1.55-3.03). Nomophobia was also associated with higher odds of binge drinking in both genders (girls: OR = 1.86; 95 percent CI: 1.61-3.52; boys: OR = 2.29; 95 percent CI: 1.68-3.13) and with cannabis consumption in boys (OR = 1.74; 95 percent CI: 1.07-2.81). Our findings highlight the importance of a comprehensive investigation of the factors underlying alcohol, tobacco, and cannabis consumption in the adolescent population.

BRAF Inhibitors in Metastatic Colorectal Cancer and Mechanisms of Resistance: A Review of the Literature.

Metastatic colorectal cancer (mCRC) with mutated BRAF exhibits distinct biological and molecular features that set it apart from other subtypes of CRC. Current standard treatment for these tumors involves a combination of chemotherapy (CT) and VEGF inhibitors. Recently, targeted therapy against BRAF and immunotherapy (IT) for cases with microsatellite instability (MSI) have been integrated into clinical practice. While targeted therapy has shown promising results, resistance to treatment eventually develops in a significant portion of responsive patients. This article aims to review the available literature on mechanisms of resistance to BRAF inhibitors (BRAFis) and potential therapeutic strategies to overcome them.

Dendrimers: Exploring Their Wide Structural Variety and Applications.

Dendrimers constitute a distinctive category of synthetic materials that bear resemblance to proteins in various aspects, such as discrete structural organization, globular morphology, and nanoscale dimensions. Remarkably, these attributes coexist with the capacity for facile large-scale production. Due to these advantages, the realm of dendrimers has undergone substantial advancement since their inception in the 1980s. Numerous reviews have been dedicated to elucidating this subject comprehensively, delving into the properties and applications of quintessential dendrimer varieties like PAMAM, PPI, and others. Nevertheless, the contemporary landscape of dendrimers transcends these early paradigms, witnessing the emergence of a diverse array of novel dendritic architectures in recent years. In this review, we aim to present a comprehensive panorama of the expansive domain of dendrimers. As such, our focus lies in discussing the key attributes and applications of the predominant types of dendrimers existing today. We will commence with the conventional variants and progressively delve into the more pioneering ones, including Janus, supramolecular, shape-persistent, and rotaxane dendrimers.

Endogenous retroviruses can propagate TDP-43 proteinopathy.

How does neurodegeneration spread in the brain? Leveraging TDP-43 fly models of amyotrophic lateral sclerosis (ALS), Chang and Dubnau recently reported that the endogenous retrovirus (ERV) mdg4 can trigger and transmit TDP-43 proteinopathy in vivo. Their results suggest that human ERVs could be targeted to develop future ALS therapies.

Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2 mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36 h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5 days after) finally altering spermatogenesis. TNFα (0.5 and 1 µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

Calibración y mantenimiento de aparatos de láser de baja potencia: estudio descriptivo

Resumen: Introducción: El láser de baja potencia es una técnica utilizada en varias áreas de la salud: fisioterapia, odontología, otorrinolaringología y dermatología, entre otras. Para lograr los efectos terapéuticos la dosificación es fundamental y depende de los niveles de energía emitidos por los equipos. Objetivo: Conocer el estado de calibración y mantenimiento de los equipos de laserterapia de baja potencia, en servicios de fisioterapia u homólogos de instituciones de Montevideo, Uruguay. Método: Se realizó un estudio descriptivo, transversal en servicios de fisioterapia de Montevideo. Se analizaron 20 equipos de láser de baja potencia. Resultados: 7 de los aparatos evaluados se mantuvieron dentro de los límites adecuados de emisión. De los 13 láser fuera de los límites para entregar una emisión adecuada, 6 tenían un desajuste leve, 1 presentaba un desajuste moderado y 6 presentaban un desajuste severo con dosis nulas o que no generaban emisión. Del total de equipos evaluados solo 6 habían realizado una calibración previa y solo 2 de estos se encontraban dentro de los tiempos recomendados. Conclusión: Se observó un gran número de equipos de láser de baja potencia con desajustes en su emisión. En la mayoría de los casos no se lleva un correcto mantenimiento de los aparatos de láser de baja potencia. Se entiende relevante que los equipos sean calibrados para poder ofrecer una terapéutica segura y efectiva a los usuarios.

Resumo: Introdução: O laser de baixa potência é uma técnica utilizada em diversas áreas da saúde: fisioterapia, odontologia, otorrinolaringologia, dermatologia, dentre outras. Para obter efeitos terapêuticos, a dosagem é essencial e depende dos níveis de energia emitidos pelo equipamento. Objetivo: conhecer o estado de calibração e manutenção de equipamentos de laserterapia de baixa potência, em serviços de fisioterapia ou congêneres de instituições em Montevideu - Uruguai. Método: estudo descritivo, transversal, realizado em serviços de fisioterapia em Montevideo, Uruguai. 20 equipamentos de laser de baixa potência foram analisados. Resultados: 7 dos aparelhos avaliados permaneceram dentro dos limites de emissão adequados. Dos 13 lasers fora dos limites para entregar a emissão adequada, 6 tiveram uma leve incompatibilidade, 1 teve uma incompatibilidade moderada e 6 apresentaram uma incompatibilidade grave. Do total de equipamentos avaliados, apenas 6 haviam realizado uma calibragem prévia e apenas 2 destes estavam dentro do prazo recomendado (1 ano). Conclusão: observou-se um grande número de equipamentos de laser de baixa potência com desequilíbrios em sua emissão. Na maioria dos casos, os dispositivos a laser de baixa potência não recebem manutenção adequada. Acredita-se importante que os equipamentos sejam calibrados para oferecer uma terapia segura e eficaz aos usuários.

Abstract: Introduction: The low-power laser is a technique used in several areas of health: physiotherapy, dentistry, otorhinolaryngology and dermatology, among others. To achieve therapeutic effects, the dosage is essential and depends on the energy levels emitted by the device. Objective: To know the status of calibration and maintenance of low power laser therapy devices, in physiotherapy services or counterparts of institutions in Montevideo - Uruguay. Method: A descriptive, cross-sectional study was conducted in physiotherapy services in Montevideo, Uruguay. 20 low-power laser devices were analyzed. Results: 7 of the devices evaluated remained within the appropriate emission limits. Of the 13 lasers outside the limits to deliver adequate emission, 6 had a slight mismatch, 1 had a moderate mismatch, and 6 presented a severe mismatch. Among the total devices evaluated, only 6 had performed a previous calibration and only 2 of these were within the recommended times frames. Conclusion: A large number of low-power laser devices with imbalances in their emission was observed. In most cases, low-power laser devices are not properly maintained. Equipment should be timely calibrated in order to offer safe and effective therapy to users.

Element Content in Different Wheat Flours and Bread Varieties.

The most consumed cereal-based product worldwide is bread. "Caaveiro", an autochthonous variety with a recent growing interest, is one of the wheat varieties that fulfill the 25% local flour requirement in the PGI "Pan Galego" bread baking industry. The element content of the refined wheat flours used to make "Pan Galego" (''Caaveiro'', FCv; Castilla, FC; and a mixture of both, FM) was evaluated in ICP-MS. In addition, wholegrain flour (FWM) was included in the analysis. Loaves of bread were made with these flours (a, 100% FC; b, 100% FCv); and c, FM: 75% FC + 25% FCv) and their element content was analyzed. Wholegrain flour ranked the highest in almost all elements, highlighting the P (494.80 mg/100 g), while the FM and the FC presented the opposite behavior, with the highest Se values (14.4 and 15.8 mg/100 g, respectively). FCv was situated in an intermediate position regarding P, K, Mg, Mn, Zn, Fe and Na content, standing closer to FWM, although it presents the highest values for Cu (1076.3 µg/100 g). The differences observed in flour were maintained in bread. Hence, the local cultivar ''Caaveiro'' has an interesting nutritional profile from the point of view of the element content.

Distinctive features of the Gac-Rsm pathway in plant-associated Pseudomonas.

Productive plant-bacteria interactions, either beneficial or pathogenic, require that bacteria successfully sense, integrate and respond to continuously changing environmental and plant stimuli. They use complex signal transduction systems that control a vast array of genes and functions. The Gac-Rsm global regulatory pathway plays a key role in controlling fundamental aspects of the apparently different lifestyles of plant beneficial and phytopathogenic Pseudomonas as it coordinates adaptation and survival while either promoting plant health (biocontrol strains) or causing disease (pathogenic strains). Plant-interacting Pseudomonas stand out for possessing multiple Rsm proteins and Rsm RNAs, but the physiological significance of this redundancy is not yet clear. Strikingly, the components of the Gac-Rsm pathway and the controlled genes/pathways are similar, but the outcome of its regulation may be opposite. Therefore, identifying the target mRNAs bound by the Rsm proteins and their mode of action (repression or activation) is essential to explain the resulting phenotype. Some technical considerations to approach the study of this system are also given. Overall, several important features of the Gac-Rsm cascade are now understood in molecular detail, particularly in Pseudomonas protegens CHA0, but further questions remain to be solved in other plant-interacting Pseudomonas.

Exploring the expression and functionality of the rsm sRNAs in Pseudomonas syringae pv. tomato DC3000.

The Gac-rsm pathway is a global regulatory network that governs mayor lifestyle and metabolic changes in gamma-proteobacteria. In a previous study, we uncovered the role of CsrA proteins promoting growth and repressing motility, alginate production and virulence in the model phytopathogen Pseudomonas syringae pv. tomato (Pto) DC3000. Here, we focus on the expression and regulation of the rsm regulatory sRNAs, since Pto DC3000 exceptionally has seven variants (rsmX1-5, rsmY and rsmZ). The presented results offer further insights into the functioning of the complex Gac-rsm pathway and the interplay among its components. Overall, rsm expressions reach maximum levels at high cell densities, are unaffected by surface detection, and require GacA for full expression. The rsm levels of expression and GacA-dependence are determined by the sequences found in their -35/-10 promoter regions and GacA binding boxes, respectively. rsmX5 stands out for being the only rsm in Pto DC3000 whose high expression does not require GacA, constituting the main component of the total rsm pool in a gacA mutant. The deletion of rsmY and rsmZ had minor effects on Pto DC3000 motility and virulence phenotypes, indicating that rsmX1-5 can functionally replace them. On the other hand, rsmY or rsmZ overexpression in a gacA mutant did not revert its phenotype. Additionally, a negative feedback regulatory loop in which the CsrA3 protein promotes its own titration by increasing the levels of several rsm RNAs in a GacA-dependent manner has been disclosed as part of this work.

Prevention of adverse drug reactions in hospitalized older patients with multi-morbidity and polypharmacy: the SENATOR* randomized controlled clinical trial.

BACKGROUND: Multi-morbidity and polypharmacy increase the risk of non-trivial adverse drug reactions (ADRs) in older people during hospitalization. Despite this, there are no established interventions for hospital-acquired ADR prevention. METHODS: We undertook a pragmatic, multi-national, parallel arm prospective randomized open-label, blinded endpoint (PROBE) controlled trial enrolling patients at six European medical centres. We randomized 1,537 older medical and surgical patients with multi-morbidity and polypharmacy on admission in a 1:1 ratio to SENATOR software-guided medication optimization plus standard care (intervention, n = 772, mean number of daily medications = 9.34) or standard care alone (control, n = 765, mean number of daily medications = 9.23) using block randomization stratified by site and admission type. Attending clinicians in the intervention arm received SENATOR-generated advice at a single time point with recommendations they could choose to adopt or not. The primary endpoint was occurrence of probable or certain ADRs within 14 days of randomization. Secondary endpoints were primary endpoint derivatives; tertiary endpoints included all-cause mortality, re-hospitalization, composite healthcare utilization and health-related quality of life. RESULTS: For the primary endpoint, there was no difference between the intervention and control groups (24.5 vs. 24.8%; OR 0.98; 95% CI 0.77-1.24; P = 0.88). Similarly, with secondary and tertiary endpoints, there were no significant differences. Among attending clinicians in the intervention group, implementation of SENATOR software-generated medication advice points was poor (~15%). CONCLUSIONS: In this trial, uptake of software-generated medication advice to minimize ADRs was poor and did not reduce ADR incidence during index hospitalization.

Unraveling the Influence of Common von Willebrand factor variants on von Willebrand Disease Phenotype: An Exploratory Study on the Molecular and Clinical Profile of von Willebrand Disease in Spain Cohort.

The clinical diagnosis of von Willebrand disease (VWD), particularly type 1, can be complex because several genetic and environmental factors affect von Willebrand factor (VWF) plasma levels. An estimated 60% of the phenotypic variation is attributable to hereditary factors, with the ABO blood group locus being the most influential. However, recent studies provide strong evidence that nonsynonymous single nucleotide variants (SNVs) contribute to VWF and factor VIII phenotypic variability in healthy individuals. This study aims to investigate the role of common VWF SNVs on VWD phenotype by analyzing data from 219 unrelated patients included in the "Molecular and Clinical Profile of von Willebrand Disease in Spain project." To that end, generalized linear mixed-effects regression models were fitted, and additive and epistatic analyses, and haplotype studies were performed, considering five VWD-related measures (bleeding score, VWF:Ag, VWF:RCo, factor VIII:C, and VWF:CB). According to these analyses, homozygotes: for p.Thr789Ala(C) would be expected to show 39% higher VWF:Ag levels; p.Thr1381Ala(C), 27% lower VWF:Ag levels; and p.Gln852Arg(C), 52% lower VWF:RCo levels. Homozygotes for both p.Thr789Ala(C) and p.Gln852Arg(T) were predicted to show 185% higher VWF:CB activity, and carriers of two copies of the p.Thr1381Ala(T)/p.Gln852Arg(T) haplotype would present a 100% increase in VWF:RCo activity. These results indicate a substantial effect of common VWF variation on VWD phenotype. Although additional studies are needed to determine the true magnitude of the effects of SNVs on VWF, these findings provide new evidence regarding the contribution of common variants to VWD, which should be taken into account to enhance the accuracy of the diagnosis and classification of this condition. ClinicalTrials.gov identifier: NCT02869074.

He had it Comin': ERPs Reveal a Facilitation for the Processing of Misfortunes to Antisocial Characters.

Human sociality and prosociality rely on social and moral feelings of empathy, compassion, envy, schadenfreude, as well as on the preference for prosocial over antisocial others. We examined the neural underpinnings of the processing of lexical input designed to tap into these type of social feelings. Brainwave responses from 20 participants were measured as they read sentences comprising a randomly delivered ending outcome (fortunate or unfortunate) to social agents previously profiled as prosocial or antisocial individuals. Fortunate outcomes delivered to prosocial and antisocial agents aimed to tap into empathy and envy/annoying feelings, respectively, whereas unfortunate ones into compassion for prosocial agents and schadenfreude for antisocial ones. ERP modulations in early attention-capture (100-200 ms), semantic fit (400 ms), and late reanalysis processes (600 ms) were analyzed. According to the functional interpretation of each of these event-related electrophysiological effects, we conclude that: 1) a higher capture of attention is initially obtained in response to any type of outcome delivered to a prosocial versus an antisocial agent (frontal P2); 2) a facilitated semantic processing occurs for unfortunate outcomes delivered to antisocial agents (N400); and 3) regardless of the protagonist's social profile, an increased later reevaluation for overall unfortunate versus fortunate outcomes takes place (Late Positive Potential). Thus, neural online measures capture a stepwise unfolding impact of social factors during language comprehension, which include a facilitated processing of misfortunes when they happen to occur to antisocial peers (i.e., schadenfreude).

The inflammatory mediators TNFα and nitric oxide arrest spermatogonia GC-1 cell cycle.

During an inflammatory process of the testis, the network of somatic, immune, and germ cell interactions is altered leading to organ dysfunction. In testicular biopsies of infertile men, spermatogenesis impairment is associated with reduced spermatogonia proliferation, increased number of immune cells, and content of pro-inflammatory cytokines. TNFα-TNFR and nitric oxide (NO)-NO synthase systems are up-regulated in models of testicular damage and in human testis with maturation arrest. The purpose of this study was to test the hypothesis that TNFα-TNFR system and NO alter the function of spermatogonia in the inflamed testis. We studied the effect of TNFα and NO on GC-1 spermatogonia cell cycle progression and death by flow cytometry. GC-1 cells expressed TNFR1 and TNFR2 (immunofluorescence). TNFα (10 and 50 ng/ml) and DETA-Nonoate (0.5 and 2 mM), a NO releaser, increased the percentage of cells in S-phase of the cell cycle and reduced the percentage in G1, inducing also cell apoptosis. TNFα effect was not mediated by oxidative stress unlike NO, since the presence of N-acetyl-l-cysteine (2.5 and 5.0 mM) prevented NO induced cell cycle arrest and death. GC-1 spermatogonia overpass NO induced cell cycle arrest but no TNFα, since after removal of NO, spermatogonia progressed through the cell cycle. We propose TNFα and NO might contribute to impairment of spermatogenesis by preventing adequate functioning of the spermatogonia population. Our results showed that TNFα and NO impaired spermatogonia cell cycle, inducing GC-1 arrest in the S phase.

AmrZ and FleQ Co-regulate Cellulose Production in Pseudomonas syringae pv. Tomato DC3000.

Pseudomonas syringae pv. tomato DC3000 carries the wssABCDEFGHI operon for the synthesis of acetylated cellulose, whose production is stimulated by increasing the intracellular levels of the second messenger c-di-GMP. This enhances air-liquid biofilm formation and generates a wrinkly colony morphotype in solid media. In the present study we show that cellulose production is a complex process regulated at multiple levels and involving different players in this bacterium. Using different in vitro approaches, including Electrophoretic Mobility Shift Assay (EMSA) and footprint analysis, we demonstrated the interrelated role of two transcriptional regulators, AmrZ and FleQ, over cellulose production in Pto DC3000 and the influence of c-di-GMP in this process. Under physiological c-di-GMP levels, both regulators bind directly to adjacent regions at the wss promoter inhibiting its expression. However, just FleQ responds to c-di-GMP releasing from its wss operator site and converting from a repressor to an activator of cellulose production. The additive effect of the double amrZ/fleQ mutation on the expression of wss, together with the fact that they are not cross-regulated at the transcriptional level, suggest that FleQ and AmrZ behave as independent regulators, unlike what has been described in other Pseudomonas species. Furthermore, this dual co-regulation exerted by AmrZ and FleQ is not limited to cellulose production, but also affects other important phenotypes in Pto DC3000, such as motility and virulence.

Is Polymerase Chain Reaction in Neonatal Dried Blood Spots Reliable for the Diagnosis of Congenital Cytomegalovirus Infection?

BACKGROUND: Detection of cytomegalovirus (CMV) DNA by real-time polymerase chain reaction (rt-PCR) in dried blood spots (DBSs) collected for newborn screening has been assessed for retrospective diagnosis of congenital CMV (cCMV) infection, with variable results (sensitivities ranging from 34% to 100%). We aimed to assess the accuracy of this technique in Spain in a large patient series. METHODS: Ambispective, multicenter study including patients with confirmed cCMV from the Spanish Registry of cCMV patients. cCMV was established on the presence of CMV DNA in any body fluid, by positive culture findings or by molecular techniques during the first 2 weeks of life. Children in whom cCMV had been excluded were used as negative controls. Neonatal DBS samples were collected from both groups. The presence of CMV DNA was assessed by rt-PCR (RealStar CMV, Altona, Germany) in a central laboratory. RESULTS: One-hundred three patients and 81 controls from 10 hospitals were included. The performance of CMV DNA determination in DBS for the diagnosis of cCMV was as follows (95% confidence interval): sensitivity 0.56 (0.47-0.65), specificity 0.98 (0.91-0.99), positive likelihood ratio 22.81 (5.74-90.58) and negative likelihood ratio 0.45 (0.36-0.56). Sensitivity increased with the birth viral load (bVL) log category. In cCMV patients, lower bVL was the single variable associated with a negative DBS rt-PCR result (P = 0.017). CONCLUSIONS: The sensitivity of CMV rt-PCR in DBS in our series was low and correlated with the bVL. Thus, a negative DBS result would not rule out cCMV infection, especially in patients with a low viremia level at birth.

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.