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Tuberous sclerosis complex (TSC) is a rare multisystemic disorder caused by a pathogenic variant in the TSC1 or TSC2 gene. A great phenotypic variability characterises TSC. The condition predisposes to the formation of hamartomas in various tissues, neurologic and neurodevelopmental disorders such as epilepsy, psychiatric disorders, as well as intellectual disability in 50%. TSC may be responsible for cardiac rhabdomyomas (CRs), cortical tubers, or subependymal nodules during foetal life. Detecting multiple CRs is associated with a very high risk of TSC, but the CR could be single and isolated. Few data exist to estimate the risk of TSC in these cases. We report the largest series of prenatal genetic tests for TSC with a retrospective study of 240 foetuses presenting with suggestive antenatal signs. We also provide a review of the literature to specify the probability of clinical or genetic diagnosis of TSC in case of detection of single or multiple CRs. Indeed, an early diagnosis is crucial for the counselling of the couple and their families. In this series, a definite diagnosis was assessed in 50% (41/82) of foetuses who initially presented with a single CR and 80.3% (127/158) in cases of multiple CRs. The prevalence of parental germinal mosaicism was 2.6% (3/115)., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: This study was approved by the Ethics Committee of the University Hospital of Angers, France (Approval ID: 2022-127), and has been performed in compliance with the Declaration of Helsinki. Informed consent was obtained from all participants. Individual-level data were de-identified., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

The ultimate aim of fluorescence microscopy is to achieve high-resolution imaging of increasingly larger biological samples. Extended depth of field presents a potential solution to accelerate imaging of large samples when compression of information along the optical axis is not detrimental to the interpretation of images. We have implemented an extended depth of field (EDF) approach in a random illumination microscope (RIM). RIM uses multiple speckled illuminations and variance data processing to double the resolution. It is particularly adapted to the imaging of thick samples as it does not require the knowledge of illumination patterns. We demonstrate highly-resolved projective images of biological tissues and cells. Compared to a sequential scan of the imaged volume with conventional 2D-RIM, EDF-RIM allows an order of magnitude improvement in speed and light dose reduction, with comparable resolution. As the axial information is lost in an EDF modality, we propose a method to retrieve the sample topography for samples that are organized in cell sheets., (© 2024. The Author(s).)

The polygonal shape of cells in proliferating epithelia is a result of the tensile forces of the cytoskeletal cortex and packing geometry set by the cell cycle. In the larval Drosophila epidermis, two cell populations, histoblasts and larval epithelial cells, compete for space as they grow on a limited body surface. They do so in the absence of cell divisions. We report a striking morphological transition of histoblasts during larval development, where they change from a tensed network configuration with straight cell outlines at the level of adherens junctions to a highly folded morphology. The apical surface of histoblasts shrinks while their growing adherens junctions fold, forming deep lobules. Volume increase of growing histoblasts is accommodated basally, compensating for the shrinking apical area. The folded geometry of apical junctions resembles elastic buckling, and we show that the imbalance between the shrinkage of the apical domain of histoblasts and the continuous growth of junctions triggers buckling. Our model is supported by laser dissections and optical tweezer experiments together with computer simulations. Our analysis pinpoints the ability of histoblasts to store mechanical energy to a much greater extent than most other epithelial cell types investigated so far, while retaining the ability to dissipate stress on the hours time scale. Finally, we propose a possible mechanism for size regulation of histoblast apical size through the lateral pressure of the epidermis, driven by the growth of cells on a limited surface. Buckling effectively compacts histoblasts at their apical plane and may serve to avoid physical harm to these adult epidermis precursors during larval life. Our work indicates that in growing nondividing cells, compressive forces, instead of tension, may drive cell morphology., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Rigato et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Anoctamins are a family of Ca 2+ -activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca 2+ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patch-clamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca 2+ -independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca 2+ -dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease., Competing Interests: Declaration of interests K.H. is currently employed by Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutseweg 30, Beerse B-2340, Belgium., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Spinal muscular atrophy (SMA) is a rare genetic neuromuscular disorder due to an autosomal recessive mutation in the survival motor neuron 1 gene (SMN1), causing degeneration of the anterior horn cells of the spinal cord and resulting in muscle atrophy. This study aimed to report on the 36-month follow-up of children with SMA treated with nusinersen before the age of 3 years. Changes in motor function, nutritional and ventilatory support, and orthopedic outcomes were evaluated at baseline and 36 months after intrathecal administration of nusinersen and correlated with SMA type and SMN2 copy number., Results: We found that 93% of the patients gained new motor skills during the 3 years-standing without help for 12 of 37 and walking with help for 11 of 37 patients harboring three SMN2 copies. No patients with two copies of SMN2 can stand alone or walk. Patients bearing three copies of SMN2 are more likely to be spared from respiratory, nutritional, and orthopedic complications than patients with two SMN2 copies., Conclusion: Children with SMA treated with nusinersen continue to make motor acquisitions at 3 years after initiation of treatment. Children with two SMN2 copies had worse motor, respiratory, and orthopedic outcomes after 3 years of treatment than children with three copies., Competing Interests: Declaration of Competing Interest JD, CV, MGGB, and UWL received funding as scientific advisory boards member from Biogen. VL, FA, JD, AI, MCN, JBD, CET, MGGB, and UWL received funding as scientific advisory boards member from Novartis. JD, CC, MGGB, and ID received funding as scientific advisory boards member from Roche. ID received funding as scientific advisory boards member from PTC therapeutics. CC, CET, and UWL received funding as scientific advisory boards member from Pfizer. VL, FA, CB, AI, JBD, CS, and ID received compensations for presentation from Novartis. FA, CB, JBD, CV, and CET received compensations for presentation from Biogen. CC received compensations for presentation from Roche. CS received compensations for presentation from PTC therapeutics and Sanofi Adventis. CC and ID received compensations for presentation from Pfizer. JBD is investigator for ongoing Roche clinical trials. MGGB is sub-investigator in SMA studies for Biogen, Novartis, and Roche. SMD, MC, and MB, declare that they have no competing interests., (Copyright © 2023. Published by Elsevier Masson SAS.)

Neonicotinoids are a widely used class of insecticides that are being applied in agricultural fields. We examined the capacity of a neonicotinoid, thiacloprid ( thia ), to induce transgenerational effects in male mice. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5-E15.5 using different doses. Testis sections were used for morphology analysis, ELISAs for testosterone level analysis, RT-qPCR and RNA-seq for gene expression analysis, MEDIP-seq and MEDIP-qPCR techniques for DNA methylation analysis, and Western blot for a protein analysis. The number of meiotic double-strand breaks and the number of incomplete synapsed chromosomes were higher in the thia 6-treated group of F3 males. Genome-wide analysis of DNA methylation in spermatozoa revealed that differentially methylated regions were found in all three generations at the promoters of germ cell reprogramming responsive genes and many superenhancers that are normally active in embryonic stem cells, testis, and brain. DNA methylation changes induced by thia exposure during embryonic period are preserved through several generations at important master regulator regions., (© 2023 Dali et al.)

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω 1 , ω 2 ) resulting in excitation energies at 2ω 1 , 2ω 2 , and the mixing energy ω 1 +ω 2 . Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

Objective: To assess the genetic and epigenetic effects promoted by Bisphenol A (BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, and in human cells., Methods: DNA methylation was analysed using MEDIP. Repeat number variation in genomic DNA was evaluated, along with the analysis of H3K4me3 by using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Analyses were performed with material extracted from whole blood of the adolescents, complemented by in vitro assessments of human (HeLa) cells exposed to 10 nM BPA, specifically, immunofluorescence evaluation of protein levels, gene expression analysis and ChIP‒qPCR analysis., Results: Adolescents in the high urinary BPA levels group presented a higher level of Satellite A (SATA) repetitive region copy numbers compared to those in the low BPA group and a tendency towards increase in telomere length. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2; at LINE1 retroelements; and at the ARID2, EGFR and ESRRA and TERT genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. Results were supported in HeLa cells exposed to 10 nM BPA in vitro. In accordance with the data obtained in blood samples, we observed higher H3K4me3 occupancy and lower DNA methylation at some specific targets in HeLa cells. In exposed cells, changes in the expression of genes encoding DNA repair factors (ATM, ARID2, TRP53) were observed, and increased expression of several genes encoding telomeric DNA binding factors (SMG7, TERT, TEN1, UPF1, ZBTB48) were also found. Furthermore, an increase in ESR1/ERa was observed in the nuclei of HeLa cells along with increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter. The DNA damage marker p53/TP53 was also increased., Conclusion: In this pilot study, genome-wide analysis of histone trimethylation in adolescent males exposed to BPA revealed a global impact on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors with similar results in HeLa cells. Nevertheless, larger studies should confirm our findings., (© 2022. The Author(s).)

Background: Brain-derived neurotrophic factor (BDNF) plays an important role in brain development by regulating multiple pathways within the central nervous system. In the Human Biomonitoring for Europe Project (HBM4EU), this neurotrophin is being implemented as a novel effect biomarker to evaluate the potential threats of environmental chemicals on neurodevelopment., Objectives: To explore the relationships among exposure to environmental metals, BDNF biomarkers at two levels of biological complexity, and behavioral function in adolescent males., Methods: Data were gathered from 125 adolescents on: spot urine sample total concentrations of the neurotoxic metal(oid)s arsenic (As), cadmium (Cd), mercury (Hg), and lead (Pb); serum BDNF protein concentrations; and concurrent behavioral functioning according to the Child Behavior Check List (CBCL/6-18). In 113 of the participants, information was also collected on blood BDNF DNA methylation at six CpGs. Associations were evaluated by multivariate linear regression analysis adjusted for confounders., Results: As, Cd, Hg, and Pb were detected in 100%, 98.5%, 97.0%, and 89.5% of urine samples, respectively. Median serum BDNF concentration was 32.6 ng/mL, and total percentage of BDNF gene methylation was 3.8%. In the adjusted models, urinary As was non-linearly associated with more internalizing problems and Cd with more externalizing behaviors. The percentage BDNF DNA methylation at CPGs #5 and the mean percentage CpG methylation increased across As tertiles (p-trend = 0.04 and 0.03, respectively), while 2nd tertile and 3rd tertile of Cd concentrations were associated with lower serum BDNF and higher CpG3 methylation percentage. Additionally, when BDNF was categorized in tertiles, serum BDNF at the 3rd tertile was associated with fewer behavioral problems, particularly withdrawn (p-trend = 0.04), social problems (p-trend = 0.12), and thought problems (p-trend = 0.04)., Conclusion: Exposure to As and Cd was associated with BDNF gene DNA methylation BDNF gene and serum BDNF, respectively. Associations with DNA methylation may be attributable to a higher variability over time in circulating BDNF concentrations than in the methylation status of this gene. Caution should be taken when interpreting the results relating postnatal Pb and Hg to behavioral functioning. Further studies are needed to verify these findings., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)