Disease Models & Mechanisms

Adolescents with Down syndrome face unique menstrual health challenges, yet their experiences remain under-researched. This study aimed to describe the menstruation experiences of adolescents with Down syndrome and their caregivers, in the UK, to inform the development of tailored, evidence-based interventions for this population. Guided by an advisory group of caregivers and young people with Down syndrome, this mixed-methods study (September 2024 -July 2025) involved a national online survey of primary caregivers (N=143) and participatory interviews with adolescents (n=6), mothers (n=11) and healthcare and education professionals (n=8). Quantitative data were analysed descriptively according to support needs (high vs low), and qualitative data were analysed thematically. The median age of menarche (12 years) aligned with the general population. While adolescents generally coped better with menarche than caregivers anticipated, 91% of 120 caregivers of adolescents who had reached menarche had ongoing menstruation concerns. While products like period underwear ("magic pants") improved independence and simplified care, key remaining concerns include: heavy periods (48%); personal care (45%); menstrual pain (45%); and the communication of pain (26%). The impact on adolescent wellbeing was greater for those with greater support needs. Additionally, 33% of caregivers felt "overwhelmed" by menstrual-related care. Decision-making for hormonal intervention was a source of heavy responsibility for caregivers. There is substantial demand for accessible educational and practical resources to support menstruation. Menstrual health is a highly individualised experience for adolescents with Down syndrome. Significant unmet needs persist, particularly for those with higher support needs. Successful outcomes require supporting caregivers through provision of accurate information that dispels pre-menarche anxiety alongside accessible and appropriate guidance to foster young peoples independence, choice and autonomy. Future interventions must be co-developed with the Down syndrome community to ensure safe, dignified menstruation. FundingDowns Syndrome Research Foundation UK

Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme that commonly exhibits altered expression in human diseases such as cancers and neurodegeneration, making it a valuable disease biomarker and putative therapeutic target. However, any treatment targeting LDHA will also disrupt normal metabolism, underscoring the need to investigate physiological consequences of inhibiting this enzyme. We previously established the fruit fly Drosophila melanogaster as a genetic model for studying LDH function in the context of growth, metabolism, and development. Here we expand upon those studies by investigating a serendipitous observation that Ldh mutant larvae exhibit diet-dependent lethality. Using a multiomic approach, we discovered this diet-dependent phenotype is independent of nutritional composition. Instead, Ldh mutant larvae are exercise intolerant and display reduced mobility, rendering mutant larvae sensitive to food consistency. Moreover, tissue-specific analysis reveals that LDH activity within muscle and peripheral glia are essential for larval viability raised on solid food. Intriguingly, these phenotypes mirror the pathophysiology of LDHA deficiency (Glycogen Storage Disease Type XI; GSD Type XI) in humans, where mild symptoms are exacerbated by physical exertion and environmental stress. Together, our findings further highlight the value of using Drosophila to explore the developmental and physiological consequences of Ldh inhibition.

Pathogenic variants in COL3A1 cause Vascular Ehlers-Danlos syndrome (vEDS), a rare connective tissue disorder characterised by vascular fragility, increasing the risk of arterial ruptures/dissection. Advances in genomic sequencing have led to an increasing number of COL3A1 variants where the clinical significance is unclear, with these being termed variants of uncertain significance (VUS). VUS creates challenges for diagnosis and clinical management. Thus major efforts have been made to reclassify these to either pathogenic or benign variants in disease causality. Functional data from model systems can provide significant evidence to clinicians on the pathogenicity of a variant. To address the increasing numbers of VUS in COL3A1, we developed a fast pipeline using F0 crispant zebrafish to provide functional evidence for variant classification despite there being no direct orthologue of COL3A1 in zebrafish. Loss of col5a1 resulted in cardiac defects, dysmorphic blood vessel structures and delayed angiogenic sprouting. Trunk haemorrhage prevalence under physical stress increased in col5a1 knockout zebrafish, recapitulating vEDS patients. Remarkably, co-injection of F0 col5a1 knockout crispants with human wildtype COL3A1 mRNA partially rescued cardiac and vascular phenotypes, indicating a level of functional conservation between zebrafish type V and human type III collagen. These findings establish a tractable in vivo platform for functional assessment of COL3A1 VUS. Phenotypic rescue with wildtype COL3A1 provides a benchmark against which the pathogenicity of variants can be evaluated, generating functional evidence for VUS reclassification. Our model provides both a valuable tool for investigating vEDS disease mechanisms and a clinically relevant platform to improve diagnoses for patients with suspected vEDS.

TBCK Syndrome is a rare Mendelian disorder caused by variants in the TBCK gene. Although symptoms affect multiple organ systems, hallmark features include intellectual and developmental disability, craniofacial differences, hypotonia, and premature death. At the cellular level, TBCK has been implicated in mTOR signaling, autophagy, mitophagy, and mRNA trafficking; however, the mechanisms underlying disease onset and progression remain unclear. To address this gap, we characterized a mouse model of TBCK Syndrome. These mice lack exon 5 of the TBCK gene, resulting in a whole-body knockout of Tbck, modeling the most severe known variant. We performed a comprehensive battery of developmental assays, along with microcomputed tomography and histological analyses, which revealed systemic alterations consistent with those observed in affected individuals. Notably, phenotypic changes arising from Tbck loss emerge early and are detectable in the brain, indicating a primary neurodevelopmental origin of disease pathology. Rigorous characterization of this Tbck-deficient mouse establishes the first in vivo platform to investigate disease mechanisms and provides a foundation for preclinical evaluation of gene and targeted pharmacological therapy strategies. Summary StatementThis study establishes a rigorously validated animal model recapitulating systemic features of TBCK Syndrome, enabling targeted investigation of disease biology and preclinical assessment of candidate therapies.

Subfertility and recurrent pregnancy loss (RPL) affect a significant proportion of couples worldwide. Genetic causes can be seen in up to 30% of these individuals but require multiple genetic tests, which often impede a comprehensive work up. Newer genomic technologies, such as PacBio HiFi long read sequencing (LRS) can detect most subclasses of variations (such as structural rearrangement, monogenic disorders) through one single test. In this multicenter study, we enrolled couples with unexplained subfertility and/or RPL and performed HiFi LRS to determine the underlying genetic etiology. Participants were recruited using a standardized inclusion/ exclusion criteria to rule out other known causes of subfertility and/or RPL. 96 individuals were recruited across the 5 sites. Average age of participants was 36 years (range 30-46 years). Among the 84 individuals who completed sequencing, 4.8% were identified with a likely genetic diagnosis and variants of uncertain significance were identified in another 14.2% of individuals. One individual was identified with an ACMG secondary finding, and while multiple carriers for recessive genetic disorders were identified, none of the couples were identified to be at increased risk. This study highlights the utility of performing genomic sequencing in couples with unexplained subfertility and/or RPL, with 1 in 10 couples harboring a clinically significant variant. In addition, use of HiFi LRS allowed for characterization of different subclasses of genomic variations through a single test. Future studies, including exploring the cost effectiveness and resource utilization of LRS as first line test, will help in optimizing care for such couples. TWEETABLE STATEMENTA single long-read genome sequencing test can consolidate multiple genetic investigations and uncover clinically relevant causes in couples with unexplained subfertility and recurrent pregnancy loss. AT A GLANCEO_LIWhy was this study conducted? O_LIMany couples with subfertility and recurrent pregnancy loss remain undiagnosed after multiple conventional genetic tests C_LIO_LIExisting workflows require sequential testing and may miss complex genomic variants C_LI C_LIO_LIWhat are the key findings? O_LILong-read genome sequencing identified clinically relevant variants in [~]1 in 10 couples with unexplained subfertility or recurrent pregnancy loss C_LIO_LIA single assay enabled detection of multiple variant types, including structural and sequence variants C_LI C_LIO_LIWhat does this study add to what is already known? O_LIDemonstrates feasibility of a unified genomic testing approach in a real-world multicenter cohort C_LIO_LISupports a potential shift from fragmented testing toward a single comprehensive genomic workflow C_LI C_LI

Dominant missense mutations in ATP1A3, encoding a Na+, K+ ATPase -3 subunit, can cause Alternating Hemiplegia of Childhood (AHC), but how these mutations lead to AHC remains unclear. Here, we establish the first C. elegans AHC models by introducing AHC-causing ATP1A3 patient mutations (D801N, E815K, L839P, and G947R) into the orthologous gene, eat-6, using CRISPR/Cas9. Homozygous C. elegans AHC model animals have recessive developmental defects. Heterozygous AHC model animals have dominant defects in neuromuscular junction (NMJ) function that are inconsistent with haploinsufficiency and dominant sleep or arousal defects. Previous work in a Drosophila G755S AHC model found that loss of a K-dependent, Na/Ca{superscript 2} exchanger exacerbated neuronal defects. We introduced a loss-of-function allele of the orthologous C. elegans gene, ncx-4, into C. elegans AHC models; loss of ncx-4 function did not consistently alter C. elegans AHC model defects across alleles. Our results establish novel C. elegans models of AHC with robust phenotypes, demonstrate that AHC mutations disrupt NMJ function, and provide proof-of-concept for discovering cross-species modifiers of AHC-related phenotypes. Summary StatementWe report the first C. elegans models of Alternating Hemiplegia of Childhood. D801N, E815K, L839P, and G947R AHC model animals have recessive development defects and dominant neuromuscular defects.

BackgroundMarfan syndrome (MFS) is a life-threatening heritable connective tissue disorder caused by pathogenic variants in fibrillin-1, characterized by progressive cardiovascular disease. Current medical therapies slow disease progression but do not prevent major complications, underscoring the need for new treatment strategies and unbiased discovery approaches. MethodsWe used a zebrafish model of MFS lacking fibrillin-3 (fbn3-/-), which recapitulates key cardiovascular phenotypes including cardiac stress, valvular defects, arrhythmia, and aortic dilation. To enable sensitive, quantitative assessment of cardiac stress, we generated a novel transgenic zebrafish reporter expressing secreted nanoluciferase under control of the stress-responsive nppb promoter. This reporter was combined with morphological phenotyping and bulbus arteriosus (BA) imaging. We evaluated standard MFS therapies, targeted modulators of TGF-{beta} signaling, and performed an unbiased high-throughput drug screen of over 1 500 clinically approved compounds across multiple developmental treatment windows. Resultsfbn3-/- larvae exhibited markedly elevated nppb activity that correlated with phenotypic severity and peaked during stages of highest mortality. The nanoluciferase reporter provided a [~]1 000-fold dynamic range, substantially outperforming Firefly luciferase-based assays. Pharmacological inhibition of TGF-{beta} signaling produced transient or deleterious effects, while {beta}-blockers, losartan, and allopurinol failed to consistently improve cardiac stress, pericardial edema, or BA dilation. The unbiased high-throughput drug screen identified a small number of primary and secondary hits; however, none demonstrated reproducible phenotypic rescue upon rigorous multi-dose, multi-time window validation. ConclusionsThis study establishes a sensitive zebrafish-based platform for early, quantitative assessment of cardiovascular stress in MFS. Our findings highlight the limited efficacy of current therapies, the context-dependent nature of TGF-{beta} modulation, and the biological complexity underlying MFS pathogenesis. Although no definitive therapeutic candidates were identified, this work lays a robust foundation for expanded unbiased discovery efforts aimed at identifying disease-modifying interventions for MFS.

Tau pathology is central to Alzheimers disease and related tauopathies, yet mechanisms driving neuronal dysfunction and degeneration downstream of pathological changes in tau remain poorly understood. Drosophila melanogaster models provide a genetically tractable system with an intact nervous system and short lifespan that allows investigation of mechanisms of many diseases. However, in Drosophila, developmental expression of human tau frequently causes lethality and developmental phenotypes, limiting the study of neurodegenerative disease processes. Further, sex is rarely considered in Drosophila studies of tau pathology despite clear sex differences being observed in many aspects of human tauopathies. Here, we used an inducible, pan-neuronal GeneSwitch system to express human tau isoforms exclusively in adulthood, enabling the dissection of tau toxicity independent of development. We combined longitudinal behavioural monitoring with lifespan and neurodegeneration analyses, and performed a targeted genetic screen to identify modifiers of tau-induced dysfunction. Adult-onset tau expression produced striking, sexually dimorphic effects on survival and behaviour. Neuronal expression of the human tau isoform with 4 microtubule binding repeats and neither alternatively spliced N-terminal exon (0N4R tau) caused pronounced neurodegeneration and reduced lifespan, which was exacerbated in flies expressing the phospho-mimetic 0N4R-TauE14 variant. Tau expression produced sexually dimorphic effects on survival and behaviour, with females exhibiting a greater reduction in lifespan, while the induction-dependent increase in vacuolar neurodegeneration was broadly comparable between sexes. Behaviourally, tau expression induced elevated daytime inactivity in females, whereas males exhibited hyperactivity, revealing opposing functional outcomes between sexes. A targeted genetic screen further identified modifiers of tau-dependent behavioural impairment. APOE2 expression in glia, syndecan overexpression in neurons, and increased global expression of the chaperone heat shock protein 90 all reduced 0N4R-TauE14-induced behavioural changes. Seventeen candidate perturbations enhanced the TauE14-induced behavioural phenotype, including manipulations of APOE3, CLU, INPP5D/INPP5K, BIN1/Amph, synaptogyrin, LRP1, NPC1, and Hsp90 pathways. Together, these findings establish an adult-onset Drosophila model of tauopathy that uncouples neurotoxicity from development, reveals sex as a major determinant of tau-induced behavioural outcomes in flies, and uncovers genetic modulators of tau-induced dysfunction. This work highlights the importance of incorporating sex as a biological variable and provides a platform for mechanistic and translational studies of tauopathy.

Laminin-2 (gene: LAMA2) is a key protein in the basement membrane of muscle and Schwann cells. A complete lack of this protein results in LAMA2-related congenital muscular dystrophy (LAMA2-RD), a severe muscle disease characterized by progressive muscle weakness, respiratory insufficiency, failure to thrive and shortened life span. One key signature of this disease is early onset of fibrosis coupled with poor muscle growth. We previously showed that TGF-{beta} and its activator, integrin-V, are elevated in dystrophic fibers of DyW mice, a mouse model of LAMA2- RD. Other than activating TGF-{beta}, integrin-V is also known to facilitate the transdifferentiation of various cell types to myofibroblasts. In this study we present evidence for transcriptional dysregulation of genes driving myofibroblast transdifferentiation and extracellular matrix (ECM) remodelling during the early development of DyW mice that is also reflected in muscle biopsies from young LAMA2-RD patients. We hypothesize that the early ECM remodelling, seen in both DyW mice and LAMA2-RD children, may explain the congenital onset of fibrosis with poor muscle growth seen in the disease.

ObjectiveX-linked Hypophosphatemia is associated with dental complications, including spontaneous endodontic infections (abscesses) in non-carious teeth and severe periodontal loss. Previous studies have mainly focused on dentin Hypomineralization; however, the structural basis underlying periodontal tissue failure remains unclear. We aimed to investigate histoanatomical abnormalities in the dentin and periodontium of Hyp mice to clarify structural consequences of Phex deficiency in adult molars. MethodsWe performed detailed histological and scanning electron microscopy analyses on the molar regions of untreated adult Hyp mice and wild-type littermates, with particular attention to the structural integrity of the root and periodontal ligament. Additionally, odontoblast process morphology and periodontal attachment abnormalities were evaluated. ResultsHyp molars exhibited marked root abnormalities, including radicular shunt-like defects and disorganized odontoblast processes, particularly in furcation and radicular dentin. Periodontal attachment showed characteristic asymmetry: detachment from the cementum surface was frequently observed, whereas attachment to the alveolar bone surface was relatively preserved. These changes were accompanied by thinning and discontinuity of Sharpeys fibers and increased vascularity in the periodontal ligament. ConclusionsThese findings provide a histoanatomical framework for understanding refractory dental complications in X-linked hypophosphatemia and support the importance of intervention during root development.

STRUCTURED ABSTRACTO_ST_ABSObjectiveC_ST_ABSTo characterize intestinal transcriptional profiles in gastroschisis, their temporal evolution, and response to fetal intervention. Summary Background DataGastroschisis causes significant intestinal dysfunction, with intra-abdominal bowel dilation clinically shown to correlate with worse outcomes. While inflammation and neurovascular impairment have been implicated, genome-wide transcriptional characterization of disease severity remains lacking. MethodsUsing a fetal ovine model of complex gastroschisis, in which all gastroschisis animals demonstrated significant intra-abdominal bowel dilation at term, bulk RNA sequencing was performed on proximal small intestinal tissue from mid-gestation and term fetuses across three groups: normal, gastroschisis, and prenatally repaired gastroschisis. Differential gene expression (FDR [≤] .05, |log2 fold change| [≥] 1.5) and pathway enrichment analyses were performed, with targeted interrogation of extracellular matrix (ECM), enteric nervous system (ENS), angiogenic, and inflammatory pathways. ResultsAt mid-gestation, gastroschisis intestine showed minimal transcriptional differences (150 differentially expressed genes [DEGs]) and some bowel dilation. By term, dysregulation was substantial (2,423 DEGs) alongside significant dilation. Normal ontogenetic intestinal maturation patterns were altered, with fewer expected developmental gene changes and discordant pathway regulation. ECM pathway aberrations emerged early and persisted, while ENS, angiogenic, and inflammatory pathways were only dysregulated at term. Fetal repair was associated with normalization of gene expression at term (29 DEGs vs controls). ConclusionIntestinal transcriptional changes in experimental gastroschisis parallel progressive bowel dilation, consistent with a mechanical stress contribution to intestinal injury. Prenatal repair normalizes both dilation and gene expression, indicating a dynamic and potentially modifiable transcriptional program that supports the rationale for early fetal intervention. Mini AbstractIn a fetal ovine model, progressive bowel dilation in gastroschisis parallels transcriptomic dysregulation of ECM remodeling, neurovascular impairment, and inflammation which is normalized by prenatal repair.

How cells and their organelles are positioned in three-dimensional, organ level, anatomical context, is rarely investigated. Here we focus on cells, centrioles and primary cilia in the growing limb. Through the ciliums mechanobiological role in skeletal development, we explored the mechanobiology of morphogenesis. A transgenic mouse model (Centrin 2-GFP.ARL13B-mCherry), combined with an image analysis pipeline, can map cellular size, positions and orientations, centriole position and ciliary axoneme orientation, all with respect to the anatomy of the epiphysis or growth plate. The line was crossed with an ift88fl/flCreERT2 line to enable ciliary ift88 deletion. We used limb immobilization, to test for a role of mechanical forces associated with ambulatory loading, in the organization of these elements and transcriptomics to understand the role of forces in regulating growth plate morphogenic programs. The pipeline can accurately quantify expected patterns of cell orientation and size through zones of the growth plate. Analysis across thousands of cells, through regions and zones of multiple murine growth plates, reveals cilia prevalence is increased in the periphery, highest in the resting zone in the outer limb, harboring stem cells. Cilia length is greatest in the hypertrophic cells about to die or transdifferentiate, as part of the formation of bone from cartilage by endochondral ossification. The inducible and cartilage-specific, deletion of ciliary gene ift88, alters cell orientation and sizes and reduces ciliation in the areas where endochondral ossification is most disrupted, the periphery and expanded hypertrophic zones, linking changes in structure to function. Most strikingly, centriole position, including that of the basal body, from which the ciliary axoneme is extended, is not preferentially organised. In contrast, cilia axoneme orientation is preferentially organised. Axonemes are directed posterior or anterior, 45 degrees to the axis of the limb, irrespective of their position, which is defined by basal body position. Immobilization of the limb for 2 weeks markedly alters the transcriptomic profile of the growth plate, with changes to size and orientation of cells and alterations in matrix and cytoskeletal profiles. Within altered genes, primary cilia genes themselves are regulated, including those indicative of altered cilia signaling such as hedgehog signaling. However, despite the role of cilia in mechanobiology of the growing limb, and ciliary signature within changes to loading of the limb, cilia orientation is unaltered by the removal of ambulatory associated forces. Patterns of ciliation in control and IFT88cKO mice help reconcile the previously observed anisotropic effects of cilia perturbation, focusing study on stem cell-resting chondrocytes and hypertrophy, when considering the mechanobiological role of cilia in limb development. Endochondral ossification is apparently highly sensitive to ambulatory loading at transcriptomic level, including effects on ciliary genes and signaling. A highly organized orientation of these putative antennae is governed by centriole position-independent mechanisms and is independent to changes to ambulatory loading, indicating a cell intrinsic mechanism. The resilient position of axonemes in the limb, points to mechano-regulatory mechanisms for how cilia integrate biophysical signals. We propose that predominant ventral or dorsal orientation at 45 degrees to horizonal plane but never parallel to cranial-chordal or medial-lateral axis, ensures multiple signal integration and avoids single signal blindness.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder caused by gain-of-function mutations in MTOR, yet whether these mutations phenocopy TSC2 loss or establish a distinct signaling state remains unclear. Using quantitative proteomics, phosphoproteomics, and transcriptomics in isogenic cell models of SKS (MTOR{Delta}4aa), TSC2 loss (TSC2-/-), and wild-type controls under glucose depletion and refeeding, we find that MTOR{Delta}4aa and TSC2-/- cells occupy fundamentally distinct regulatory states. TSC2-/- cells exhibit broad anabolic remodeling and a transcriptional program dominated by NF-{kappa}B- and STAT-driven inflammatory responses. MTOR{Delta}4aa cells instead display enrichment of nuclear and RNA processing programs, E2F/MYC-driven transcription, and a constrained proteomic dynamic range across nutrient states. Phosphoproteomic analysis of MTOR{Delta}4aa reveals rerouting of nutrient-responsive signaling toward MAPK/ERK- and Ca2+/CaMK-dependent pathways with limited canonical mTORC1/S6K1 engagement. These findings establish SKS as a signaling rewiring disorder distinct from classical mTORC1 hyperactivation, with implications for therapeutic targeting.

Charcot-Marie-Tooth disease type 2D (CMT2D) results from gain-of-function mutations in GARS1, which encodes glycyl-tRNA synthetase (GlyRS), the enzyme responsible for charging transfer RNA (tRNA) with glycine. There are several CMT2D mouse models, but Gars{Delta}ETAQ/+ is the only one that bears a patient-sourced mutation. Created using CRISPR/Cas9 to model a 12-nucleotide de novo GARS1 deletion identified in an unusually severe CMT2D patient, Gars{Delta}ETAQ/+ mice have previously been shown to display several neuromuscular phenotypes; motor axon loss, denervated neuromuscular junctions (NMJs) and reduced muscle function. Here, we extend these analyses to provide a more comprehensive understanding of both motor and sensory nerve deficits across hind- and fore-limbs. At 3 months, Gars{Delta}ETAQ/+ mice possess sex-independent alterations in the levels of neuropathy biomarkers - including decreased NfL and increased periaxin - alongside reduced muscle endurance and strength, and impairments in the sensory modalities of mechanosensation, proprioception and nociception. Underpinning these dysfunctions, we identified site-specific defects comprising altered sensory neuron populations, muscle spindle loss, reduced motor neuron size, disrupted NMJ innervation and maturation, and reduced axonal transport of signalling endosomes in vivo. Together, these experiments show that Gars{Delta}ETAQ/+ mice display robust and selective peripheral nerve pathology that manifests in a general distal-to-proximal fashion, priming this CMT2D allele for testing treatments and evaluating mechanisms underlying peripheral nerve vulnerability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/736541v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@2b25d2org.highwire.dtl.DTLVardef@c00d77org.highwire.dtl.DTLVardef@b41173org.highwire.dtl.DTLVardef@1e5cdcc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Abstract/SummaryFIG4 deficiency is the cause of Charcot Marie Tooth type 4J, a neurological disorder characterized by enlarged lysosomes. Our CRISPR activation genome wide screen found that upregulation of PIKFYVE rescued the enlarged lysosome phenotype in cultured cells. To assess PIKFYVE upregulation treatment in vivo, we generated Fig4 deficient mice with CRISPR activation of Pikfyve in neurons. Pikfyve was increased 2 fold in whole brain of CRISPR activated mice. Pikfyve upregulation did not extend the 3 week survival of Fig4 deficient mice. Vacuolization of brain was not rescued. The data demonstrates that a 2 fold increase of Pikfyve is not sufficient to treat Fig4 deficiency. Further testing will be required to determine if a higher increase of Pikfyve can ameliorate the effects of FIG4 deficiency in vivo.

Aims/hypothesisQuantitative nanoscale analysis of insulin secretory granules (ISGs) in human pancreatic tissue has been limited by the lack of imaging methods that combine high resolution with large-scale sampling. We aimed to establish expansion microscopy (ExM) as a platform for in situ, quantitative analysis of ISG organisation in human {beta}-cells and to assess whether type 2 diabetes (T2D) is associated with alterations in granule size, abundance or spatial organisation. MethodsWe applied Magnify ExM to PFA-fixed, paraffin-embedded pancreatic tissue sections from 6 human donors, 3 non-diabetic (ND) and 3 T2D, enabling super-resolution optical imaging of insulin-labelled granules. Insulin-positive structures were segmented and analysed using a morphometric pipeline to quantitatively assess size, shape and spatial features. Granule clustering was quantified based on combined area and roundness criteria. ResultsThe diameter distribution of highly circular granules was similar between ND and T2D samples and estimates of granule number per cell indicated only a modest reduction in T2D ([~]25%). In contrast, mapping insulin-positive structures in a roundness-area space revealed a marked enrichment of large, irregular objects consistent with granule clustering in T2D. The fraction of clustered granules was significantly increased in T2D and strongly inversely correlated with insulin stimulation index (r = -0.85). Conclusions/interpretationThese results establish expansion microscopy as a powerful platform for quantitative nanoscale analysis of human pancreatic tissue and identify altered spatial organisation of insulin granules, rather than marked granule depletion, as a prominent feature associated with {beta}-cell dysfunction in T2D. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LI{beta}-cell dysfunction in type 2 diabetes is often attributed to reduced insulin content or {beta}-cell loss. C_LIO_LIInsulin secretory granules (ISGs) have been characterised ultrastructurally, but quantitative analysis in human tissue remains limited. C_LIO_LISuper-resolution approaches, including expansion microscopy, are emerging tools for nanoscale imaging in biological tissues. C_LI What is the key question?O_LIIs {beta}-cell dysfunction in type 2 diabetes associated with depletion of insulin granules or with altered spatial organisation? C_LI What are the new findings?O_LIInsulin granule size distribution is largely preserved in type 2 diabetes, with only a modest reduction in granule number per cell. C_LIO_LIA significant increase in insulin granule clustering is observed in diabetic {beta}-cells. C_LIO_LIGranule clustering is strongly inversely correlated with insulin secretion in the same donor tissues. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying altered granule organisation as a feature of {beta}-cell dysfunction may help refine the understanding of disease mechanisms and guide future strategies targeting {beta}-cell function. C_LI

Down syndrome (DS), caused by trisomy of human chromosome 21, is characterized by intellectual disability and cognitive deficits, partly driven by the overexpression of Dual-specificity tyrosine-(Y)-phosphorylation Regulated Kinase 1A (DYRK1A). While postnatal DYRK1A inhibition has shown promise in improving cognition in DS models, its therapeutic potential during embryonic development, a critical window for neurogenesis, remains unexplored. Here, we tested the hypothesis that prenatal inhibition of DYRK1A could mitigate long-term cognitive impairments in DS. We administered Leucettine L41, a potent and selective DYRK1A inhibitor, to pregnant dams carrying two DS mouse models: Ts65Dn and Dp(16)1Yey, both of which recapitulate trisomy of genes homologous to human chromosome 21, including Dyrk1a. Treatment was designed to suppress DYRK1A kinase activity during embryogenesis. In adulthood, we evaluated the progeny for cognitive performance, gene expression profiles linked to DS phenotypes, and neuronal maturation markers. Prenatal L41 treatment produced lasting effects in both models, rescuing specific behavioral deficits and modulating the expression of DS-implicated genes, including the excitatory/inhibitory balance regulator GAD67. However, model-specific responses were observed: hyperactivity, working memory deficits, and GAD67-positive cell counts remained uncorrected in Ts65Dn mice, suggesting divergent molecular pathways underlying shared DS phenotypes. This study demonstrates the therapeutic potential of prenatal DYRK1A inhibition for DS and provides novel insights into its role in neurodevelopmental trajectories and cognitive outcomes. Our findings underscore the importance of timing and genetic context in DS intervention strategies.

Congenital myasthenic syndromes (CMS) are rare inherited diseases of the neuromuscular junction (NMJ). There are 40 identified CMS genes, but many patients go without genetic diagnosis, which suggests new genes have yet to be discovered and characterised. Here, we describe an optogenetic approach to study fatigable muscle weakness and NMJ function in larval zebrafish to facilitate screening approaches for uncovering novel CMS genes. Using blue-light illumination of spinal motoneurons that express channelrhodopsin-2 (ChR2) to induce muscle contraction, we measure motor defects at the behavioural, synaptic, and genetic level through a novel behavioural assay, standard whole-cell electrophysiology of individual muscle fibers and a customized NMJ gene panel. We employ this approach in synaptotagmin-2 (syt2) morphant zebrafish, an identified CMS gene model, to validate its usefulness. Our customized optogenetic behavioural assay successfully demonstrates reduced, fatigable, locomotor response during repeated activation of spinal motoneurons. Whole-cell electrophysiology recordings of optogenetically-elicited endplate currents in muscle fibers reveal similarities to altered properties of NMJ function in syt2 morphants reported in other studies using the standard paired motoneuron-muscle electrophysiology technique. Finally, we develop a genetic panel of CMS and NMJ-related genes to characterize the expression landscape of syt2 morphants to elucidate potential pathomechanisms and novel therapeutic targets. We propose that this three-tiered approach successfully links behaviour, synaptic motor function, and genetic expression and can be used as a tool in the screening of novel genes associated with CMS.

BackgroundPostoperative pancreatic fistula (POPF) is a major cause of morbidity after pancreatoduodenectomy, particularly in patients with high-risk pancreatic remnants. Preventive strategies based solely on surgical technique have yielded inconsistent results, and thus there has been growing interest in strategies aiming to modify the biological behavior of the pancreatic remnant. This preclinical study evaluated the biological and histopathological effects of preoperative endoluminal radiofrequency ablation (ERFA) of the main pancreatic duct (MPD) performed 4 weeks before pancreatic transection in a porcine model. MethodsAnimals underwent laparoscopic MPD occlusion followed by pancreatic transection at 4 weeks and necropsy 15 days thereafter. Feasibility, safety, histological atrophy, and macroscopic findings associated with POPF risk were assessed. As a secondary objective, outcomes were compared with a that underwent MPD occlusion using cyanoacrylate glue. ResultsPreoperative ERFA was technically feasible and safe. At 4 weeks, ERFA induced marked and homogeneous acinar atrophy that was significantly greater than that observed after glue occlusion (p = 0.018), indicating effective biological conditioning of the pancreatic remnant. At necropsy, pseudocyst formation and intra-abdominal adhesions, known surrogate markers of pancreatic fistula in pigs, were significantly more frequent in the glue group and absent in ERFA-treated animals. Serum amylase levels, postoperative weight gain, complication rates, and preservation of endocrine architecture were comparable between groups. ConclusionsDuctal ablation of the MPD via ERFA induced stable, progressive exocrine pancreatic atrophy, effectively preconditioning the gland prior to pancreatic transection. Experimental evidence suggests that its biological effects stabilize approximately 4 weeks after treatment. Compared to cyanoacrylate occlusion, ERFA achieved more homogeneous early biological effects and fewer fistula-related macroscopic complications. These findings support the further investigation of preoperative pancreatic conditioning as a potential adjunct strategy for POPF risk reduction, although clinical studies are needed to clarify its role alongside established reconstructive approaches.

Systemic amyloidosis is typically diagnosed only after irreversible organ damage has occurred, limiting the effectiveness of available therapies. Whether the disease is preceded by detectable molecular changes long before clinical presentation has remained unclear. Here, we leveraged population-scale plasma proteomics and longitudinal follow-up from the UK Biobank to investigate early circulating protein signatures associated with future diagnosis of amyloidosis. Among approximately 53,000 participants with proteomic profiling, we identified 61 individuals who developed amyloidosis up to 14 years after protein assessment. Differential expression and correlation analyses identified a seven-protein panel, including MYL3, MYBPC1, NT-proBNP, NPPB, FCRLB, IGFBP1, and FABP1, consistent with early cardiac stress and immune dysregulation. Time-to-event modelling demonstrated robust stratification of amyloidosis risk and timing. Importantly, a parsimonious subset of these proteins retained strong predictive performance, indicating that a reduced set of biologically informative markers is sufficient for risk stratification. Furthermore, these proteomic signals were not explained by pre-existing cardiac disease, clonal haematopoiesis, or related plasma cell disorders, indicating that they capture disease-specific biological processes preceding clinical diagnosis. Together, these findings show that amyloidosis is preceded by persistent plasma proteomic alterations, providing a framework for early risk stratification and insight into the preclinical biology of this under-recognised disease.