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Abstract Background In patients without a family history, Duchenne muscular dystrophy (DMD) is typically diagnosed at around 4–5 years of age. It is important to diagnose DMD during infancy or toddler stage in order to have timely access to treatment, opportunities for reproductive options, prevention of potential fatal reactions to inhaled anesthetics, awareness of a child’s abilities needed for good parenting, and opportunities for enrolment in clinical trials. Method We aimed to develop a short risk assessment tool based on developmental milestones that may contribute to the early detection of boys with DMD in primary care. As part of the case-control 4D-DMD study (Detection by Developmental Delay in Dutch boys with DMD), data on developmental milestones, symptoms and therapies for 76 boys with DMD and 12,414 boys from a control group were extracted from the health records of youth health care services and questionnaires. Multiple imputation, diagnostic validity and pooled backward logistic regression analyses with DMD (yes/no) as the dependent variable and attainment of 26 milestones until 36 months of age (yes/no) as the independent variable were performed. Descriptive statistics on symptoms and therapies were provided. Results A tool with seven milestones assessed at specific ages between 12 and 36 months resulted in a sensitivity of 79% (95CI:67–88%), a specificity of 95.8% (95%CI:95.3–96.2), and a positive predictive value of 1:268 boys. Boys with DMD often had symptoms (e.g. 43% had calf muscle pseudohypertrophy) and were referred to therapy (e.g. 59% for physical therapy) before diagnosis. Discussion This tool followed by the examination of other DMD-related symptoms could be used by youth health care professionals during day-to-day health assessments in the general population to flag children who require further action. Conclusions The majority of boys (79%) with DMD can be identified between 12 and 36 months of age with this tool. It increases the initial a priori risk of DMD from 1 in 5,000 to approximately 1 in 268 boys. We expect that other neuromuscular disorders and disabilities can also be found with this tool.

Background: Minimizing medication waste through the redispensing of oral anticancer drugs (OADs) that were unused by patients provides economic and environmental benefits, but this is not yet universally implemented in clinical care. Objective(S): To identify barriers and facilitators to the implementation of redispensing unused OADs in clinical care. Methods: A multicentre intervention study following a hybrid effectiveness-implementation type I design was conducted, consisting of semi-structured interviews with key stakeholders involved in the redispensing program: pharmacy employees, prescribing clinicians in oncology and haematology, patients who participated in redispensing and patients who declined trial participation. Questions encompassed experiences and suggestions for future implementation. The Consolidated Framework for Implementation Research (CFIR) guided data collection and categorisation of identified barriers and facilitators through thematic analysis. Results: In total, 35 interviews were conducted, identifying 15 themes encompassing barriers and facilitators, reflecting all CFIR domains. Facilitators encompassed: 1) convenient process requiring an acceptable time-investment; 2) support from project leaders and implementation champions; 3) being well-motivated by personal values and societal impact; 4) feeling ensured of medication quality upon redispensing; 5) endorsement by healthcare providers for patient participation; 6) clear and personal patient communication; 7) good visibility of intervention successes; and 8) implementation well supported through a collaborative network. Barriers encompassed: 1) unclear target population; 2) redispensing legally prohibited; 3) absence of financial compensation for pharmacies; 4) complexity arising from two parallel work processes; 5) widespread communication on adjustments within local teams challenging; 6) patient's low receptiveness due to burden of oncology treatment; and 7) lack of familiarization among pharmacy technicians. Conclusions: Facilitators for implementation of redispensing unused drugs mainly related to people's values, motivation, and societal demand, whereas barriers mainly encompassed practical issues, including knowledge, time, financial resources, and legal conditions. Strategies emphasizing the benefits of redispensing and further streamlining process compatibility could support implementation.

AbstractBackground The charity foundation Association Soutien Enfants Togo started a child health care (CHC) centre in Togo that was modelled after the Dutch high-quality CHC system to improve child health.Aim To describe health care data of children who visited the centre.Subjects and methods Data were routinely collected between October 2010-July 2017. Outcomes were completed vaccinations, growth, development, lifestyle, physical examination, and laboratory testing results.Results In total, 8,809 children aged 0–24 years were available. Half (47.5%) of children aged 0–4 years did not receive all eligible free vaccinations from the government. The proportions of stunted children (all) or with a developmental delay (0–4 years) were 10.1% and 9.5%, respectively. In total, 40–50% of all children did not wash their hands with soap after toilet or before eating, or did not use clean drinking water. Furthermore, 5.1–6.6% had insufficient vision, high eye pressure or hearing loss. Sickle cell disease was detected in 5.3%.Conclusion A large group of children in need of prevention and early treatment were detected, informed and treated by the centre. Further research is needed to confirm if this strategy can improve children’s health in Sub-Saharan Africa. Our data are available for further research.

Glucosinolates (GSLs) are a prototypical group of bioactive compounds found in the Brassicaceae family that promote human health and plant defense. The GSL-myrosinase system can be induced to release multiple bioactive products when plants are subjected to mechanical damage, environmental stress, or pathogen infection. While many GSLs promote human health, some cause deleterious effects when ingested. To engineer Brassicaceae crops with lower levels of harmful GSLs without sacrificing health-promoting GSLs requires a complete understanding of the origin and advances in GSL modification. Extensive early domestication studies were conducted using classic breeding and plant nutrition. More recently, genetic modification of specific groups of GSLs or levels of GSLs in specific tissues has been partially successful. However, efforts have fallen short of delivering a reduction in potentially harmful GSLs without concomitant losses to health-promoting effects and plant defense. The latest work has been to synthetically express GSL biosynthesis pathways in non-host crops or microbial species. However, yields have been far from economically sustainable. This review discusses key advances made in GSL modification that are promising for the precise modification of GSL content and composition for optimal plant defense and human health.

Background and purpose: The Drug Rediscovery Protocol (DRUP) is a Dutch, pan-cancer, nonrandomized clinical trial that aims to investigate the efficacy and safety of targeted and immunotherapies outside their registered indication in patients with advanced or metastatic cancer. Patients: Patients with advanced or metastatic cancer are eligible when there are no standard of care treatment options left and the tumor possesses a molecular genomic variant for which commercially available anticancer treatment is accessible off-label in DRUP. Clinical benefit is the study’s primary endpoint, characterized by a confirmed objective response or stable disease after at least 16 weeks of treatment. Results: More than 2,500 patients have undergone evaluation, of which over 1,500 have started treatment in DRUP. The overall clinical benefit rate (CBR) remains 33%. The nivolumab cohort for patients with microsatellite instable metastatic tumors proved highly successful with a CBR of 63%, while palbociclib or ribociclib in patients with tumors harboring CDK4/6 pathway alterations showed limited efficacy, with a CBR of 15%. The formation of two European initiatives (PCM4EU and PRIME-ROSE) strives to accelerate implementation and enhance data collection to broaden equitable access to anticancer treatments and gather more evidence. Conclusion: DRUP persists in improving patients access to off-label targeted or immunotherapy in the Netherlands and beyond. The expansion of DRUP-like clinical trials across Europe provides countless opportunities for broadening the horizon of precision oncology.

This study provides insight into the total duration of litigation from the perspective of litigants. It examines commercial court cases with claims between 1 million and 5 million EUR in the Netherlands. It focuses on cases in which first instance judgments are appealed. In many of these cases other related procedures ensue. Using detailed data of the case administration system of the courts in the Netherlands, cases are followed as they wind their way through the court system. The results show that court statistics do not reflect the time taken for disposition from the litigant’s perspective. A substantial number of high value cases are not concluded within a reasonable time and there is a wide spread of the time taken for disposition. For prospective litigants, it is not easy to predict the duration of litigation. The ensuing uncertainty makes it harder for them to assert their rights.

Summary: Background: The DRUG Access Protocol provides patients with cancer access to registered anti-cancer drugs that are awaiting reimbursement in the Netherlands and simultaneously collects prospective real-world data (RWD). Here, we present RWD from PD-1 blocker cemiplimab in patients with locally advanced or metastatic cutaneous squamous cell carcinoma (laCSCC; mCSCC). Methods: Patients with laCSCC or mCSCC received cemiplimab 350 mg fixed dose every three weeks. Primary endpoints were objective clinical benefit rate (CBR), defined as objective response (OR) or stable disease (SD) at 16 weeks, physician-assessed CBR, defined as clinician’s documentation of improved disease or SD based on evaluation of all available clinical parameters at 16 weeks, objective response rate (ORR), and safety, defined as grade ≥ 3 treatment related adverse events (TRAEs) occurring up to 30 days after last drug administration. Secondary endpoints included duration of response (DoR), progression-free survival (PFS), and overall survival (OS). Findings: Between February 2021 and December 2022, 151 patients started treatment. Objective and physician-assessed CBR were 54.3% (95% CI, 46.0–62.4) and 59.6% (95% CI, 51.3–67.5), respectively. ORR was 35.1% (95% CI, 27.5–43.3). After a median follow-up of 15.2 months, median DoR was not reached. Median PFS and OS were 12.2 (95% CI, 7.0-not reached) and 24.2 months (95% CI, 18.8-not reached), respectively. Sixty-eight TRAEs occurred in 29.8% of patients. Most commonly reported TRAE was a kidney transplant rejection (9.5%). Interpretation: Cemiplimab proved highly effective and safe in this real-world cohort of patients with laCSCC or mCSCC, confirming its therapeutic value in the treatment of advanced CSCC in daily clinical practice. Funding: The DRUG Access Protocol is supported by all participating pharmaceutical companies: Bayer, Janssen, Lilly, Merck, Novartis, Roche, and Sanofi.

Abstract Climatic changes can cause unpredictability in flood regimes that traditional flood risk management (FRM) approaches may struggle with. Therefore, flood resilience is seen as a supplementation to these approaches, putting a larger emphasis on flood acceptance and minimising consequences. An (emergent) group contributing towards flood resilience is civil society. This paper examines how civil society contributions can be furthered and guided in the Netherlands as well as exploring potential limitations in doing so. To achieve this, England is used as a good practice example due to a more developed and defined role for civil society being present here. Data were collected on both actual (England and the Netherlands) and potential (The Netherlands) civil society contributions. These were compared to identify potential avenues for Dutch civil society contributions to flood resilience that can be further investigated. The research shows that the most promising avenues are improving advocacy from citizens, improving local flood awareness and developing relationships between FRM authorities and existing citizen groups that can be harnessed and mobilised to support flood resilience. Additionally, the research also provides insights into potential limitations for transferring resilience approaches from one context to another beyond the cases discussed in this publication.

Summary: Background: Smoking impacts DNA methylation, but data are lacking on smoking-related differential methylation by sex or dietary intake, recent smoking cessation (

Introduction Pulmonary fibrosis is a severe disease which can be familial. A genetic cause can only be found in ∼40% of families. Searching for shared novel genetic variants may aid the discovery of new genetic causes of disease. Methods Whole-exome sequencing was performed in 152 unrelated patients with a suspected genetic cause of pulmonary fibrosis from the St Antonius interstitial lung disease biobank. Variants of interest were selected by filtering for novel, potentially deleterious variants that were present in at least three unrelated pulmonary fibrosis patients. Results The novel c.586G>A p.(E196K) variant in the ZCCHC8 gene was observed in three unrelated patients: two familial patients and one sporadic patient, who was later genealogically linked to one of the families. The variant was identified in nine additional relatives with pulmonary fibrosis and other telomere-related phenotypes, such as pulmonary arterial venous malformations, emphysema, myelodysplastic syndrome, acute myeloid leukaemia and dyskeratosis congenita. One family showed incomplete segregation, with absence of the variant in one pulmonary fibrosis patient who carried a PARN variant. The majority of ZCCHC8 variant carriers showed short telomeres in blood. ZCCHC8 protein was located in different lung cell types, including alveolar type 2 (AT2) pneumocytes, the culprit cells in pulmonary fibrosis. AT2 cells showed telomere shortening and increased DNA damage, which was comparable to patients with sporadic pulmonary fibrosis and those with pulmonary fibrosis carrying a telomere-related gene variant, respectively. Discussion The ZCCHC8 c.586G>A variant confirms the involvement of ZCCHC8 in pulmonary fibrosis and short-telomere syndromes and underlines the importance of including the ZCCHC8 gene in diagnostic gene panels for these diseases.

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vδ2+ T cells, while Vδ1+ T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vδ1+ and Vδ2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-γ, TNF-α and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p < 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p < 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

This article argues for a gradient approach to word order, which treats word order preferences, both within and across languages, as a continuous variable. Word order variability should be regarded as a basic assumption, rather than as something exceptional. Although this approach follows naturally from the emergentist usage-based view of language, we argue that it can be beneficial for all frameworks and linguistic domains, including language acquisition, processing, typology, language contact, language evolution and change, and formal approaches. Gradient approaches have been very fruitful in some domains, such as language processing, but their potential is not fully realized yet. This may be due to practical reasons. We discuss the most pressing methodological challenges in corpus-based and experimental research of word order and propose some practical solutions.

Background: Clinical and experimental data on the cardiac effects of acute hypernatremia are scarce and inconsistent. We aimed to determine and understand the effects of different levels of acute hypernatremia on the human ventricular action potential. Methods: We performed computer simulations using two different, very comprehensive models of the electrical activity of a single human ventricular cardiomyocyte, i.e., the Tomek–Rodriguez model following the O’Hara–Rudy dynamic (ORd) model and the Bartolucci–Passini–Severi model as published in 2020 (known as the ToR-ORd and BPS2020 models, respectively). Mild to extreme levels of hypernatremia were introduced into each model based on experimental data on the effects of hypernatremia on cell volume and individual ion currents. Results: In both models, we observed an increase in the intracellular sodium and potassium concentrations, an increase in the peak amplitude of the intracellular calcium concentration, a hyperpolarization of the resting membrane potential, a prolongation of the action potential, an increase in the maximum upstroke velocity, and an increase in the threshold stimulus current at all levels of hypernatremia and all stimulus rates tested. The magnitude of all of these effects was relatively small in the case of mild to severe hypernatremia but substantial in the case of extreme hypernatremia. The effects on the action potential were related to an increase in the sodium–potassium pump current, an increase in the sodium–calcium exchange current, a decrease in the rapid and slow delayed rectifier potassium currents, and an increase in the fast and late sodium currents. Conclusions: The effects of mild to severe hypernatremia on the electrical activity of human ventricular cardiomyocytes are relatively small. In the case of extreme hypernatremia, the effects are more pronounced, especially regarding the increase in threshold stimulus current.

A sodium current (INa) reduction occurs in the setting of many acquired and inherited conditions and is associated with cardiac conduction slowing and increased arrhythmia risks. The sodium channel blocker mexiletine has been shown to restore the trafficking of mutant sodium channels to the membrane. However, these studies were mostly performed in heterologous expression systems using high mexiletine concentrations. Moreover, the chronic effects on INa in a non-diseased cardiomyocyte environment remain unknown. In this paper, we investigated the chronic and acute effects of a therapeutic dose of mexiletine on INa and the action potential (AP) characteristics in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of a healthy individual. Control hiPSC-CMs were incubated for 48 h with 10 µM mexiletine or vehicle. Following the wash-out of mexiletine, patch clamp analysis and immunocytochemistry experiments were performed. The incubation of hiPSC-CMs for 48 h with mexiletine (followed by wash-out) induced a significant increase in peak INa of ~75%, without any significant change in the voltage dependence of (in)activation. This was accompanied by a significant increase in AP upstroke velocity, without changes in other AP parameters. The immunocytochemistry experiments showed a significant increase in membrane Nav1.5 fluorescence following a 48 h incubation with mexiletine. The acute re-exposure of hiPSC-CMs to 10 µM mexiletine resulted in a small but significant increase in AP duration, without changes in AP upstroke velocity, peak INa density, or the INa voltage dependence of (in)activation. Importantly, the increase in the peak INa density and resulting AP upstroke velocity induced by chronic mexiletine incubation was not counteracted by the acute re-administration of the drug. In conclusion, the chronic administration of a clinically relevant concentration of mexiletine increases INa density in non-diseased hiPSC-CMs, likely by enhancing the membrane trafficking of sodium channels. Our findings identify mexiletine as a potential therapeutic strategy to enhance and/or restore INa and cardiac conduction.

Human-induced stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool for studying development, pharmacology, and (inherited) arrhythmias. Unfortunately, hiPSC-CMs are depolarized and spontaneously active, even the working cardiomyocyte subtypes such as atrial- and ventricular-like hiPSC-CMs, in contrast to the situation in the atria and ventricles of adult human hearts. Great efforts have been made, using many different strategies, to generate more mature, quiescent hiPSC-CMs with more close-to-physiological resting membrane potentials, but despite promising results, it is still difficult to obtain hiPSC-CMs with such properties. The dynamic clamp technique allows to inject a current with characteristics of the inward rectifier potassium current (IK1), computed in real time according to the actual membrane potential, into patch-clamped hiPSC-CMs during action potential measurements. This results in quiescent hiPSC-CMs with a close-to-physiological resting membrane potential. As a result, action potential measurements can be performed with normal ion channel availability, which is particularly important for the physiological functioning of the cardiac SCN5A-encoded fast sodium current (INa). We performed in vitro and in silico experiments to assess the beneficial effects of the dynamic clamp technique in dissecting the functional consequences of the SCN5A-1795insD+/− mutation. In two separate sets of patch-clamp experiments on control hiPSC-CMs and on hiPSC-CMs with mutations in ACADVL and GNB5, we assessed the value of dynamic clamp in detecting delayed afterdepolarizations and in investigating factors that modulate the resting membrane potential. We conclude that the dynamic clamp technique has highly beneficial effects in all of the aforementioned settings and should be widely used in patch-clamp studies on hiPSC-CMs while waiting for the ultimate fully mature hiPSC-CMs.