NDUFS1 is a critical component of mitochondrial respiratory chain Complex I (CI). Pathogenic variants of NDUFS1 can cause Leigh syndrome (LS), a severe pediatric mitochondrial disorder. To model NDUFS1-linked LS, we generated an iPSC line with homozygous missense mutations in exon 8 using CRISPR/Cas9. The cell line demonstrated typical morphology, expression of iPSC markers, ability to differentiate into all three germ layers, and genomic integrity. This model will enable the study of LS caused by CI in an isogenic context.

Abstract Background Brugada syndrome is a complex cardiac channelopathy characterised by significant genotype-phenotype heterogeneity¹. Whilst SCN5A is established as the primary gene², the underlying genetic architecture explaining clinical variability remains incompletely understood³,4. Purpose To characterise the genetic landscape of patients with suspected Brugada syndrome, analysing variant distribution across different electrocardiographic patterns using novel methodological approaches. Methods We characterised 284 consecutive patients with suspected Brugada syndrome referred between 2008-2025. Patients (191 males, 67.3%; median age 45.5 years) were stratified into four ECG pattern groups: non-diagnostic (n=49, 17.3%), drug-induced (n=95, 33.5%), spontaneous intermittent (n=103, 36.3%), and spontaneous persistent (n=37, 13.0%). Multi-tiered genetic testing included Sanger sequencing (n=74), NGS cardio panel (n=158), and whole exome sequencing (n=52). Variants were classified according to ACMG/AMP guidelines5. Following our previously validated approach6, we sub-classified variants of uncertain significance with strong pathogenic evidence for exploratory analysis. Results Overall genetic yield was 29.2% (83/284 patients), with SCN5A variants in 49 patients (17.3%). Using standard ACMG criteria, we observed a significant gradient from 0% in non-diagnostic to 8.1% in spontaneous persistent patterns (p=0.04). Enhanced criteria strengthened this gradient: 2.0% to 24.3% respectively (p=0.02). NGS analysis revealed ion channel genes in 31% of variants, structural proteins 13.3%, other genes 10%. Ion channel genes maintained similar gradient trends though not significant (p=0.072), whilst structural proteins and other genes showed uniform distribution. Clinical correlations included higher male prevalence in spontaneous intermittent patterns (76.7% vs 56.8%, p=0.03) and more frequent palpitations (36.9% vs 16.2%, p=0.02). Electrophysiological studies showed low-voltage areas more frequent in spontaneous intermittent patterns (61.9%, p=0.001). Conclusions This study introduces novel methodological innovations including four-class ECG pattern stratification and enhanced VUS subclassification that significantly improve genotype-phenotype correlation analysis. Sodium channel genetics represents the primary determinant of Brugada syndrome phenotypic complexity, with ion channels maintaining gradient trends unlike other gene categories. These approaches advance personalised risk stratification and support refined variant reclassification in cardiac channelopathies.

Background:Preterm birth is a critical period for brain development, as extrauterine factors can impair growth and myelination, thereby increasing the risk of neurodevelopmental impairment. Adequate nutrition and rapid weight gain are associated with better cognitive outcomes, and the choice of lipid emulsion during parenteral nutrition may influence these results. SMOFlipid®, enriched with omega-3 long-chain polyunsaturated fatty acids, could reduce inflammation and oxidative stress, potentially lowering bronchopulmonary dysplasia (BPD) risk. This study compared brain maturation at term-equivalent age (TEA) using MRI and neurodevelopment at 2 years in infants receiving either SMOFlipid® or Intralipid®. Methods:In this single-center retrospective observational cohort study, we included all very low birth weight (VLBW) infants admitted to the NICU of our institution between 2017 and 2021. Infants who underwent brain MRI at term-equivalent age and completed neurodevelopmental assessment at 2 years were included, and those with severe brain lesions were excluded. Patients were categorized into two groups based on the lipid emulsion administered during parenteral nutrition. The primary outcome was neurodevelopment at 24 months of corrected age (CA). The secondary outcome was brain maturation assessed by the total maturation score (TMS) on magnetic resonance imaging (MRI). Results:A total of 121 VLBW infants were included and categorized into two cohorts based on the lipid formulation administered: multi-component lipid emulsion (MLE) in 62 and soybean lipid emulsion (SLE) in 49 infants. TMS showed non-statistically significant differences among infants treated with SLE compared with those treated with MLE, as well as in neurodevelopmental outcomes assessed using Griffith's scales. Conclusion:Despite integrating brain imaging and clinical follow-up data, this study could not determine the optimal lipid emulsion for preterm infants.

Abstract Early human development involves dynamic transitions in cell identity, including transient transcriptional modulation and stable lineage commitment. Distinguishing these types of gene expression changes is challenging and can be further exacerbated by genetic and experimental heterogeneity in the context of human pluripotent stem cell (hPSC) research. To address this challenge and help uncover transcriptional changes indicative of true developmental state, we establish a curated, cross-platform marker framework for robust identification of pluripotency and early germ-layer identity. Starting from an unbiased RNA-seq discovery set, we systematically validate candidate markers across qPCR, bulk and single-cell RNA sequencing, and quantitative proteomics platforms, yielding a refined panel of 67 markers (20 for the undifferentiated state, 17 for endoderm, 15 for ectoderm, and 15 for mesoderm). We show that this framework reliably identifies early developmental states across heterogeneous datasets, generalizes to in vivo human embryo cell types, and preserves lineage identity despite substantial transcriptional variability. Furthermore, we demonstrate concordant protein-level expression for a subset of markers, supported by deep proteomic profiling of the reference line KOLF2.1J. To enable broad application, we introduce DeepDiff, a web-based resource integrating the validated markers, allowing automated fate classification in a user-friendly interface. Together, this work provides a standardized framework for defining early human developmental identity and disentangling lineage commitment from context-dependent modulation.

INTRODUCTION:Acquired demyelinating syndromes (ADS) of the central nervous system in children present a diagnostic challenge due to overlapping presentations. Differentiating monophasic from potentially recurrent conditions, such as pediatric-onset multiple sclerosis (POMS), myelin-oligodendrocyte glycoprotein antibody-associated disease (MOGAD), and other seronegative ADS, is essential for treatment and prognosis. This study aimed to characterize the initial presentation of pediatric ADS and evaluate the evolution of diagnosis over time to better guide treatment. METHODS:A retrospective study of 59 children with ADS diagnosed at a tertiary pediatric center in Italy (2012-2024) was conducted. Initial classifications included MS, acute disseminated encephalomyelitis (ADEM), clinically isolated syndrome (CIS), optic neuritis (ON), neuromyelitis optica spectrum disorder (NMOSD), or "Indeterminate". Final diagnoses were categorized as MS, MOGAD, or "Other" demyelinating conditions. Demographic, clinical, MRI, CSF, and outcome were analyzed. Statistical comparisons among groups used Mann-Whitney U, Chi-square, or Fisher's exact tests (p < 0.05). RESULTS:At final diagnosis, older age at onset, absence of preceding infection and characteristic MRI findings (periventricular/callosal lesions, cerebellar involvement) were more frequent in MS group. Younger age, preceding infection, fever, irritability, and cortical involvement were associated with MOGAD. Nearly one-third of final MS cases (29%) were initially CIS or ON, while no ADEM cases converted to MS. "Other" ADS (non-MS/MOG-IgG antibody negative patients) showed more severe initial disability (p = 0.01). Transition to adult neurology was significantly higher in MS (p < 0.001). CONCLUSION:these findings underscore the heterogeneity of pediatric ADS at onset and the value of accurate acute management and longitudinal follow-up to refine diagnosis and guide treatment.

Introduction: Seizures are the most frequent initial symptom of perinatal stroke. Apnea, a less commonly recognized neurological presentation, is a cessation of breathing lasting >15 seconds or shorter if associated with bradycardia. This study explores a monocentric cohort of newborns with perinatal stroke, emphasizing the role of Isolated Central Apnea (ICA) as a potential early indicator of cerebrovascular insult. Methods: A retrospective review was conducted on newborns (>= 36 weeks GA) diagnosed with MRI-confirmed perinatal stroke at our institution between March 2019 and March 2024. Infants with hypoxic-ischemic encephalopathy (HIE), congenital infections, meningoencephalitis, sepsis, or intrauterine stroke were excluded. Clinical data, neuroimaging findings, EEG/aEEG characteristics, and antiepileptic drug (AED) were analyzed. Results: Of 421 newborns who underwent brain MRI, 27 (6%) were diagnosed with perinatal stroke (16 males; median GA 38.5 weeks; mean BW 3137g). Seizures were the presenting symptom in 18 cases (67%), while 8 cases (30%) exhibited ICA as the first sign and an MRI revealing 5 arterial strokes (63%) and 3 venous strokes (37%). EEG abnormalities were detected in 7/8 (88%) cases, while brain ultrasound was abnormal in 5/8 (63%). AED therapy was required in 7/8 (88%) patients. Conclusions: ICA is an early sign of neonatal stroke in approximately one-third of cases. These findings underscore the importance of brain MRI in the diagnostic workup of persistent apnea in term and early-term neonates.

A deep learning algorithm for contrast amplification in brain MRI, trained exclusively on adult data, was tested for cross-population generalization to pediatric patients, including subjects aged 0–2 years. A retrospective monocentric dataset (n = 22 cases) comprising pediatric patients (0–17 years old) diagnosed with various brain tumors was used to evaluate the algorithm, which takes T1-weighted pre- and standard post-contrast images as input and generates an output image with amplified contrast, further post-processed with an HDR algorithm. Quantitative comparisons between standard and amplified images were performed using contrast-to-noise ratio (CNR), contrast enhancement percentage (CEP), and lesion-to-background ratio (LBR). Three neuroradiologists performed qualitative assessment using a 4-point Likert scale, focusing on lesion contrast and delineation. Anatomical similarity was assessed using SSIM and log-Jacobian range. Statistical significance was evaluated using two-tailed paired t-tests. Compared to standard-dose images, contrast-amplified images showed significantly higher values for CNR (+ 186.5

Genome editing often generates complex mixtures of alleles rather than single, predefined outcomes. Resolving these heterogeneous edits across diverse editing modalities, sequencing platforms, and multiplexed designs remains a persistent analytical challenge. To address this, we developed CleanFinder, a browser-native framework for genotyping genome editing outcomes using a constrained semi-global alignment strategy. Context-aware alignment modes support a broad spectrum of editing scenarios, including indels, base substitutions, and complex prime editing modifications across nuclear and mitochondrial targets. Additional modules include an optional turbo mode for high-throughput heuristic alignment in exploratory workflows and an allele-aware module that leverages heterozygous single-nucleotide polymorphisms to detect allelic dropout. To evaluate scalability and practical performance, we applied CleanFinder to a primary small-molecule screen of 1849 compounds in HEK293T cells. The software efficiently processed the dataset, enabling high-throughput comparison of editing outcomes and nomination of candidate compounds for follow-up analysis. Together, CleanFinder provides a flexible and scalable platform for genome editing analysis, enabling detailed genotyping and systematic comparison of editing outcomes across diverse edit types and genomic contexts.

Background: Heterozygosity for pathogenic variants in the ABCC6 gene has been associated with an increased incidence of cerebrovascular diseases. This study aims to characterize the prevalence and clinical and neuroradiological phenotypes associated with monoallelic and biallelic ABCC6 variants in pediatric and adult patients presenting with arterial ischemic stroke or cerebral small vessel disease (CSVD). Methods: We conducted a retrospective observational study on 143 consecutive patients (48 pediatric, 24 juvenile, 71 adult) diagnosed with ischemic stroke or CSVD of unknown etiology. Clinical and neuroradiological data were collected and analyzed in relation to the identified genetic variants through next-generation sequencing. Results: Among the patients, 16 (11.2%) tested positive for causative variants in the ABCC6 gene, with 11 subjects carrying monoallelic variants and 5 carrying biallelic variants. Patients with biallelic variants exhibited severe and complex vasculopathy, with a high incidence of early ischemic events. In contrast, monoallelic carriers predominantly presented with microvascular disease manifestations, including lacunar strokes and signs of CSVD. Conclusions: The results suggest a significant age-dependent phenotypic divergence in patients with ABCC6 variants, highlighting the impact of heterozygosity on cerebrovascular health. Identifying these variants may enhance risk stratification and inform management strategies in patients with traditional vascular risk factors.

Heart failure (HF) represents a growing public health challenge, particularly among older adults. Its prevalence increases with age and is frequently complicated by frailty, multimorbidity, and functional decline, all of which worsen prognosis and complicate management. The relationship between HF and frailty is bidirectional: HF promotes sarcopenia, cachexia, and inflammation, while frailty reduces resilience and tolerance to therapy. The objective of the paper is to summarize current evidence on the management of HF in frail older adults, highlighting recent pharmacological advances, geriatric considerations, and emerging multidisciplinary strategies. A narrative synthesis has been performed, including the most recent European Society of Cardiology and American Heart Association/American College of Cardiology/Heart Failure Society of America guidelines, position papers, and randomized controlled trials focusing on drug therapy, non-pharmacological interventions, and comprehensive geriatric care in older adults with HF. Guideline-directed medical therapy for HF with reduced ejection fraction—comprising sodium-glucose co-transporter 2 (SGLT2) inhibitors, angiotensin receptor-neprilysin inhibitors, β-blockers, and mineralocorticoid receptor antagonists—remains the cornerstone of treatment. Evidence for HF with mildly reduced ejection fraction and HF with preserved ejection fraction supports SGLT2 inhibitors and individualized management of comorbidities. Multidimensional interventions, including nutritional support, tailored physical rehabilitation, cognitive and psychological care, and telemonitoring, significantly improve outcomes. Structured transitional care and early palliative integration reduce readmissions and enhance quality of life. Effective management of HF in frail older adults requires combining evidence-based pharmacological therapy with a holistic, geriatric, and multidisciplinary approach spanning hospital, transitional, and community settings. Strengthening randomized evidence and integrating comprehensive care models are essential to improving prognosis and functional independence in this vulnerable population.

The paper presents two cases of Myelin Oligodendrocyte Glycoprotein Antibody-Associated Disease (MOGAD) and highlights its significant clinical heterogeneity in the paediatric population. Early recognition of neurological red flags is crucial. The availability of specialised diagnostic tools at referral centres enables timely diagnosis and improves clinical outcomes.

Background: Atrial arrhythmias represent a frequent long-term complication in patients with atrial septal defects (ASDs). Interatrial block (IAB), reflecting delayed or impaired conduction across Bachmann's bundle, has been proposed as an electrophysiological substrate predisposing to atrial arrhythmogenesis. However, evidence regarding its prevalence and clinical correlates in pediatric patients with ASD remains limited. The present study aimed to characterize interatrial conduction patterns and assess the occurrence of IAB in children with large secundum ASD undergoing percutaneous closure. Methods: Between January 2020 and March 2024, 37 consecutive pediatric patients (median age 6 years, range 5-11) with large ostium secundum ASD were included in a retrospective analysis of a prospectively maintained institutional database. Standard 12-lead electrocardiograms were recorded before and within 24 h after defect closure. P-wave morphology and duration were systematically analyzed, and IAB was classified according to the Bayés de Luna criteria. Results: The median Qp/Qs ratio was 1.69 (1.32-2.24), with a mean pulmonary artery pressure of 19 mmHg (17-22). IAB was identified in 24.3% of patients before the procedure, predominantly as first-degree IAB. Following device implantation, IAB prevalence (29.7%) and P-wave parameters remained unchanged, with no significant differences compared with baseline. No associations were observed between IAB and defect size, hemodynamic burden, or device characteristics, whereas anthropometric variables, including weight, height, and body surface area, showed a significant correlation with IAB occurrence. During a median follow-up of 199 days, no atrial arrhythmias were documented. Conclusions: In this pediatric cohort with large ASD, IAB was present in approximately one quarter of patients and appeared unrelated to anatomical or procedural factors, supporting the hypothesis of an underlying congenital conduction abnormality. Early recognition of IAB may therefore have implications for long-term arrhythmic risk stratification in this population.

Human induced pluripotent stem cell (hiPSC)-derived neural models combined with microelectrode array (MEA)-based readouts are increasingly used in next-generation neurotoxicity assessment. However, neural induction of hiPSCs into multipotent neural progenitor cells (hiNPCs) remains highly variable, and current quality control (QC) efforts focus largely on the pluripotent starting material. As a result, failed neural inductions are often recognized only after weeks of differentiation during functional network analysis, causing substantial resource loss. Here, we present a tiered QC framework spanning the entire workflow from hiPSC banking, neural induction into proliferative hiNPCs, the differentiation of hiNPCs into 3D neuron-glia BrainSpheres, and their subsequent organization into functional 2D networks on MEAs, with emphasis on early detection of induction failures. Using eleven independent dual-SMAD inductions derived from a single hiPSC line, we show that hiPSCore, a machine-learning-based classifier for early cell-fate decisions, reliably distinguishes successful from unsuccessful neuroectodermal inductions at early stages (days 6-12). Successful induction depends on timely neuroectodermal commitment, reflected in hiPSCore trajectories and PAX6 expression, as well as preserved hiNPC proliferation. Inductions passing these early QC checkpoints generated BrainSphere-derived networks with reproducible MEA maturation trajectories, balanced neurotransmitter-responsive unit distributions, and conserved subtype-specific pharmacological responses across GABAergic, glutamatergic, dopaminergic, and serotonergic modalities. In contrast, signaling pathway-related markers associated with dual SMAD inhibition (BMP, TGFβ, and MAPK) were not predictive of downstream QC performance. Together, these findings demonstrate that early fate-level QC is predictive of the functional performance of hiPSC-derived neural networks. Our adaptable QC framework allows early termination of failed inductions, reduces resource burden, and strengthens confidence in hiPSC-based neural network assays for neurotoxicity testing.

Arginine vasopressin deficiency (AVP-D) is an uncommon but clinically important cause of the polyuria-polydipsia syndrome. Establishing the diagnosis extends beyond confirming hypotonic polyuria and requires differentiation from primary polydipsia and arginine vasopressin resistance, together with identification of the underlying etiology. Unlike adults, children and adolescents with AVP-D frequently present with the earliest manifestation of an evolving neoplastic, infiltrative, inflammatory, congenital, autoimmune, or genetic disorder, making longitudinal clinical, endocrine, and magnetic resonance imaging (MRI) surveillance integral to the diagnostic process. Using three illustrative clinical cases, this review presents a practical approach to the evaluation of AVP-D, integrating clinical assessment, biochemical investigation, dedicated hypothalamic-pituitary MRI, and risk-adapted longitudinal surveillance. We discuss the strengths and limitations of the water deprivation test, the emerging role of copeptin-based diagnostics, and current evidence supporting arginine-, urea-, and glucagon-stimulated copeptin testing. Emphasis is placed on the longitudinal interpretation of MRI, consensus recommendations for PST, and the concept that idiopathic AVP-D should be regarded as a provisional diagnosis requiring continued etiological reassessment. Emerging biomarkers, including neurophysin I and oxytocin, may further refine the assessment of hypothalamic-neurohypophyseal dysfunction but remain investigational in children. Ultimately, AVP-D should be viewed not as the end of the diagnostic process but as its beginning, with clinical assessment, neuroimaging, endocrine evaluation, and structured surveillance integrated to achieve the earliest possible etiological diagnosis.

Cockayne Syndrome type B (CSB) is caused by mutations in the ERCC6 gene, which encodes a key protein involved in transcription-coupled nucleotide excision repair (TC-NER) and chromatin remodeling. Deficiency in CSB leads to defective transcriptional recovery after DNA damage, oxidative stress accumulation, and progressive neurodegeneration. In this work, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, IUFi004-A-12, carrying a homozygous mutation in ERCC6 causing a premature stop codon in its 10th exon. The modified iPSCs displayed normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This model enables mechanistic studies of CSB dysfunction and facilitates therapeutic development for Cockayne Syndrome.

Preterm birth is a significant risk factor for atypical neurodevelopment, yet early electrophysiological markers of brain maturation are still lacking. Non-invasive electroencephalographic (EEG) monitoring of cortical maturation in these patients holds promise as a tool for neurodevelopmental prediction. However, its clinical application is limited by technical challenges in maintaining stable, long-term electrode placement on very small neonate scalps and by the highly specialised, multi-level expertise required to care for these fragile patients. Using video-polysomnographic EEG recordings in very low birth weight (VLBW, < 1500 g) preterm infants, we characterised large-scale neuronal dynamics during distinct vigilance states and tested whether they could serve as indicators of early cortical maturation. We analysed EEG recordings obtained at 33.9 ± 1.4 weeks postmenstrual age (PMA), during active sleep (AS), sleep onset active sleep (SOAS), quiet sleep (QS), and quiet wakefulness (QW). For each vigilance state, we assessed large-scale neuronal dynamics in terms of phase synchronisation, neuronal bistability, and local phase-amplitude coupling (PAC), both globally and separately for anterior and posterior regions, and correlated them with PMA. We found that phase synchronisation peaked in the δ band during QS and in the θ band during more active states (QW, SOAS, AS). δ-band bistability was lower in posterior regions across all states, while δ-PAC was lower posteriorly during sleep but reversed during wakefulness. Also, bistability and PAC decreased with advancing PMA. These findings suggest that vigilance-state-dependent neuronal dynamics capture aspects of early cortical maturation-even with low-density EEG cap-offering novel candidate biomarkers to monitor neurodevelopment in infants born preterm.

Patients with heart failure with reduced ejection fraction (HFrEF) and atrial fibrillation (AF) may show positive remodeling after catheter ablation, yet rhythm control is not consistently prioritized alongside guideline-directed medical therapy (GDMT) before implantable cardioverter-defibrillator (ICD) implantation. This meta-analysis evaluated the effect of AF ablation on primary prevention ICD eligibility in patients with left ventricular ejection fraction (LVEF) ≤ 35

Arterial Spin Labelling MRI is a neuroimaging technique able to evaluate brain perfusion, an indirect measure of brain metabolism and function. Arterial Spin Labelling MRI showed to have performances comparable to [18F]fluorodeoxyglucose-PET in in epilepsy, yet literature data is still lacking about its use in children and the value of voxel-based asymmetry index analysis. Purpose of the project is to compare the Arterial Spin Labelling MRI and [18F]fluorodeoxyglucose-PET ability to identify the epileptogenic zone before and after asymmetry index analysis in children. In this observational study, paediatric patients with focal onset drug-resistant epilepsy that underwent presurgical evaluation, including Arterial Spin Labelling MRI and [18F]fluorodeoxyglucose-PET, were enrolled. The epileptogenic zone was defined by anatomo-electroclinical correlation and post-surgical outcome, when feasible. The rates of concordance with the epileptogenic zone of Arterial Spin Labelling MRI and [18F]fluorodeoxyglucose-PET before (visual analysis) and after asymmetry index analysis, were calculated. Statistically significant differences between were determined using Mc Nemar’s test (p < 0.05). 28 paediatric patients (mean age 10.07 years, 15 females) with focal epilepsy were enrolled; 22 underwent epilepsy surgery (mean age 9.86 years, 12 females). When comparing the techniques, visual analysis of Arterial Spin Labelling MRI had a significantly lower rate of concordance with the epileptogenic zone (p < 0.05). Voxel-based asymmetry index analysis increased significantly the rate of concordance of Arterial Spin Labelling MRI with the epileptogenic zone, achieving results comparable with [18F]fluorodeoxyglucose-PET in a cohort of paediatric patients.

Anaplastic pleomorphic xanthoastrocytoma (APXA) is a rare pediatric high-grade glioma (pHGG). We report a 10-year-old boy with a left temporal APXA harboring a BRAF V600E mutation, who presented with generalized tonic-clonic seizures 3-4 times weekly, treated with combined BRAF and MEK inhibition. This case illustrates the utility of [18F]FDOPA PET in monitoring targeted therapy, providing metabolic information that preceded and complemented conventional imaging.