Square condenser apertures for square cameras in low-dose transmission electron microscopy.

In transmission electron microscopy (TEM), cameras are square or rectangular but beams are round so the circular lobes irradiate adjacent areas, precluding further neighboring acquisition for beam-sensitive samples. We present condenser aperture plates with square and rectangular shapes that improve the efficiency of area usage by 70% and enhance montage imaging for beam-sensitive specimens. We demonstrate the compatibility of these condenser aperture plates with high-resolution cryogenic TEM by reconstructing a 1.8-Å map of equine apo-ferritin.

The Arteria Lymphatica and Lymphatic Microperforators: A Dedicated Blood Supply to Collecting Lymphatics and Their Potential Implications in Lymphedema: Anatomical Description.

Background: Lymphedema is common after lymphatic damage in cancer treatment, with negative impacts on function and quality of life. Evidence suggests that blood vessel microvasculature is sensitive to irradiation and trauma; however, despite knowledge regarding dedicated mural blood supply to arteries and veins (vasa vasorum), equivalent blood vessels supplying lymphatics have not been characterized. We studied collecting lymphatics for dedicated mural blood vessels in our series of 500 lymphaticovenous anastomosis procedures for lymphedema, and equivalent controls. Methods: Microscopic images of lymphatics from lymphedema and control patients were analyzed for lymphatic wall vascular density. Collecting lymphatics from 20 patients with lymphedema and 10 control patients were sampled for more detailed analysis (podoplanin immunostaining, light/confocal microscopy, microcomputed tomography, and transmission electron microscopy) to assess lymphatic wall ultrastructure and blood supply. Results: Analysis revealed elaborate, dense blood microvessel networks associating with lymphatic walls in lymphedema patients and smaller equivalent vessels in controls. These vasa vasora or "arteria lymphatica" were supplied by regular axial blood vessels, parallel to lymphatic microperforators linking dermal and collecting lymphatics. Lymphatic walls were thicker in lymphedema patients than controls, with immunohistochemistry, computed tomography, transmission electron microscopy, and confocal microscopy characterizing abnormal blood vessels (altered appearance, thickened walls, elastin loss, narrow lumina, and fewer red blood cells) on these lymphatic walls. Conclusions: Dedicated blood vessels on lymphatics are significantly altered in lymphedema. A better understanding of the role of these vessels may reveal mechanistic clues into lymphedema pathophysiology and technical aspects of lymphedema microsurgery, and suggest potential novel therapeutic targets.

Genetic Transformation of Plasmid DNA into Escherichia coli Using High Frequency Electromagnetic Energy.

We present a novel technique of genetic transformation of bacterial cells mediated by high frequency electromagnetic energy (HF EME). Plasmid DNA, pGLO (5.4 kb), was successfully transformed into Escherichia coli JM109 cells after exposure to 18 GHz irradiation at a power density between 5.6 and 30 kW m-2 for 180 s at temperatures ranging from 30 to 40 °C. Transformed bacteria were identified by the expression of green fluorescent protein (GFP) using confocal scanning microscopy (CLSM) and flow cytometry (FC). Approximately 90.7% of HF EME treated viable E. coli cells exhibited uptake of the pGLO plasmid. The interaction of plasmid DNA with bacteria leading to transformation was confirmed by using cryogenic transmission electron microscopy (cryo-TEM). HF EME-induced plasmid DNA transformation was shown to be unique, highly efficient, and cost-effective. HF EME-induced genetic transformation is performed under physiologically friendly conditions in contrast to existing techniques that generate higher temperatures, leading to altered cellular integrity. This technique allows safe delivery of genetic material into bacterial cells, thus providing excellent prospects for applications in microbiome therapeutics and synthetic biology.

The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant.

Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family ß-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.

The effect of calcium removal from skim milk by ion exchange on the properties of the ultrafiltration retentate.

New processes are needed to produce concentrated milk feedstocks with tailored calcium content, due to the direct link between calcium concentration and final product texture and functionality. Skim milk treatment with cation exchange resin 1% (w/v) or 2% (w/v) prior to ultrafiltration to a volumetric concentration factor (VCF) of 2.5 or 5 successfully decreased the calcium concentration by 20-30% and produced concentrates with solids content at ∼22-24 g 100 g-1 at a VCF of 5. Calcium reduction partially solubilized the casein micelles, increasing the concentration of soluble protein and individual caseins, leading to decreased turbidity but increased protein hydration and hydrophobicity. Decalcification (2% (w/v) resin treatment) reduced thermal stability, significantly decreasing the denaturation temperature of α-lactalbumin and ß-lactoglobulin in the milk by ∼3 °C and ∼1 °C respectively. Filtration was also altered, reducing permeation flux and the gel concentration and increased filtration time. When combined, calcium reduction and filtration altered functional properties including soluble calcium, soluble protein and sedimentable solids, with increased milk protein hydration also contributing to increased viscosity. This study provides a route to produce calcium-reduced milk concentrates with potential for use in retentate-based dairy products with tailored functionality.

Modeling human skeletal development using human pluripotent stem cells.

Chondrocytes and osteoblasts differentiated from induced pluripotent stem cells (iPSCs) will provide insights into skeletal development and genetic skeletal disorders and will generate cells for regenerative medicine applications. Here, we describe a method that directs iPSC-derived sclerotome to chondroprogenitors in 3D pellet culture then to articular chondrocytes or, alternatively, along the growth plate cartilage pathway to become hypertrophic chondrocytes that can transition to osteoblasts. Osteogenic organoids deposit and mineralize a collagen I extracellular matrix (ECM), mirroring in vivo endochondral bone formation. We have identified gene expression signatures at key developmental stages including chondrocyte maturation, hypertrophy, and transition to osteoblasts and show that this system can be used to model genetic cartilage and bone disorders.

Biochemical Characterization of Caenorhabditis elegans Ferritins.

The nematode Caenorhabditis elegans contains genes for two types of ferritin (ftn-1 and ftn-2) that express FTN-1 and FTN-2. We have expressed and purified both proteins and characterized them by X-ray crystallography, cryo-electron microscopy, transmission electron microscopy, dynamic light scattering, and kinetically by oxygen electrode and UV-vis spectroscopy. Both show ferroxidase activity, but although they have identical ferroxidase active sites, FTN-2 is shown to react approximately 10 times faster than FTN-1, with L-type ferritin character over longer time periods. We hypothesize that the large variation in rate may be due to differences in the three- and four-fold channels into the interior of the protein 24-mer. FTN-2 is shown to have a wider entrance into the three-fold channel than FTN-1. Additionally, the charge gradient through the channel of FTN-2 is more pronounced, with Asn and Gln residues in FTN-1 replaced by Asp and Glu residues in FTN-2. Both FTN-1 and FTN-2 have an Asn residue near the ferroxidase active site that is a Val in most other species, including human H ferritin. This Asn residue has been observed before in ferritin from the marine pennate diatom Pseudo-mitzchia multiseries. By replacing this Asn residue with a Val in FTN-2, we show that the reactivity decreases over long time scales. We therefore propose that Asn106 is involved in iron transport from the ferroxidase active site to the central cavity of the protein.

Small extracellular vesicles promote invadopodia activity in glioblastoma cells in a therapy-dependent manner.

PURPOSE: The therapeutic efficacy of radiotherapy/temozolomide treatment for glioblastoma (GBM) is limited by the augmented invasiveness mediated by invadopodia activity of surviving GBM cells. As yet, however the underlying mechanisms remain poorly understood. Due to their ability to transport oncogenic material between cells, small extracellular vesicles (sEVs) have emerged as key mediators of tumour progression. We hypothesize that the sustained growth and invasion of cancer cells depends on bidirectional sEV-mediated cell-cell communication. METHODS: Invadopodia assays and zymography gels were used to examine the invadopodia activity capacity of GBM cells. Differential ultracentrifugation was utilized to isolate sEVs from conditioned medium and proteomic analyses were conducted on both GBM cell lines and their sEVs to determine the cargo present within the sEVs. In addition, the impact of radiotherapy and temozolomide treatment of GBM cells was studied. RESULTS: We found that GBM cells form active invadopodia and secrete sEVs containing the matrix metalloproteinase MMP-2. Subsequent proteomic studies revealed the presence of an invadopodia-related protein sEV cargo and that sEVs from highly invadopodia active GBM cells (LN229) increase invadopodia activity in sEV recipient GBM cells. We also found that GBM cells displayed increases in invadopodia activity and sEV secretion post radiation/temozolomide treatment. Together, these data reveal a relationship between invadopodia and sEV composition/secretion/uptake in promoting the invasiveness of GBM cells. CONCLUSIONS: Our data indicate that sEVs secreted by GBM cells can facilitate tumour invasion by promoting invadopodia activity in recipient cells, which may be enhanced by treatment with radio-chemotherapy. The transfer of pro-invasive cargos may yield important insights into the functional capacity of sEVs in invadopodia.

CardioVinci: building blocks for virtual cardiac cells using deep learning.

Advances in electron microscopy (EM) such as electron tomography and focused ion-beam scanning electron microscopy provide unprecedented, three-dimensional views of cardiac ultrastructures within sample volumes ranging from hundreds of nanometres to hundreds of micrometres. The datasets from these samples are typically large, with file sizes ranging from gigabytes to terabytes and the number of image slices within the three-dimensional stack in the hundreds. A significant bottleneck with these large datasets is the time taken to extract and statistically analyse three-dimensional changes in cardiac ultrastructures. This is because of the inherently low contrast and the significant amount of structural detail that is present in EM images. These datasets often require manual annotation, which needs substantial person-hours and may result in only partial segmentation that makes quantitative analysis of the three-dimensional volumes infeasible. We present CardioVinci, a deep learning workflow to automatically segment and statistically quantify the morphologies and spatial assembly of mitochondria, myofibrils and Z-discs with minimal manual annotation. The workflow encodes a probabilistic model of the three-dimensional cardiomyocyte using a generative adversarial network. This generative model can be used to create new models of cardiomyocyte architecture that reflect variations in morphologies and cell architecture found in EM datasets. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Effects of altered cellular ultrastructure on energy metabolism in diabetic cardiomyopathy: an in silico study.

Diabetic cardiomyopathy is a leading cause of heart failure in diabetes. At the cellular level, diabetic cardiomyopathy leads to altered mitochondrial energy metabolism and cardiomyocyte ultrastructure. We combined electron microscopy (EM) and computational modelling to understand the impact of diabetes-induced ultrastructural changes on cardiac bioenergetics. We collected transverse micrographs of multiple control and type I diabetic rat cardiomyocytes using EM. Micrographs were converted to finite-element meshes, and bioenergetics was simulated over them using a biophysical model. The simulations also incorporated depressed mitochondrial capacity for oxidative phosphorylation (OXPHOS) and creatine kinase (CK) reactions to simulate diabetes-induced mitochondrial dysfunction. Analysis of micrographs revealed a 14% decline in mitochondrial area fraction in diabetic cardiomyocytes, and an irregular arrangement of mitochondria and myofibrils. Simulations predicted that this irregular arrangement, coupled with the depressed activity of mitochondrial CK enzymes, leads to large spatial variation in adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio profile of diabetic cardiomyocytes. However, when spatially averaged, myofibrillar ADP/ATP ratios of a cardiomyocyte do not change with diabetes. Instead, average concentration of inorganic phosphate rises by 40% owing to lower mitochondrial area fraction and dysfunction in OXPHOS. These simulations indicate that a disorganized cellular ultrastructure negatively impacts metabolite transport in diabetic cardiomyopathy. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Repurposing the mitotic machinery to drive cellular elongation and chromatin reorganisation in Plasmodium falciparum gametocytes.

The sexual stage gametocytes of the malaria parasite, Plasmodium falciparum, adopt a falciform (crescent) shape driven by the assembly of a network of microtubules anchored to a cisternal inner membrane complex (IMC). Using 3D electron microscopy, we show that a non-mitotic microtubule organizing center (MTOC), embedded in the parasite's nuclear membrane, orients the endoplasmic reticulum and the nascent IMC and seeds cytoplasmic microtubules. A bundle of microtubules extends into the nuclear lumen, elongating the nuclear envelope and capturing the chromatin. Classical mitotic machinery components, including centriolar plaque proteins, Pfcentrin-1 and -4, microtubule-associated protein, End-binding protein-1, kinetochore protein, PfNDC80 and centromere-associated protein, PfCENH3, are involved in the nuclear microtubule assembly/disassembly process. Depolymerisation of the microtubules using trifluralin prevents elongation and disrupts the chromatin, centromere and kinetochore organisation. We show that the unusual non-mitotic hemispindle plays a central role in chromatin organisation, IMC positioning and subpellicular microtubule formation in gametocytes.

MeasureIce: accessible on-the-fly measurement of ice thickness in cryo-electron microscopy.

Ice thickness is arguably one of the most important factors limiting the resolution of protein structures determined by cryo-electron microscopy (cryo-EM). The amorphous atomic structure of the ice that stabilizes and protects biological samples in cryo-EM grids also imprints some additional noise in cryo-EM images. Ice that is too thick jeopardizes the success of particle picking and reconstruction of the biomolecule in the worst case and, at best, deteriorates eventual map resolution. Minimizing the thickness of the ice layer and thus the magnitude of its noise contribution is thus imperative in cryo-EM grid preparation. In this paper we introduce MeasureIce, a simple, easy to use ice thickness measurement tool for screening and selecting acquisition areas of cryo-EM grids. We show that it is possible to simulate thickness-image intensity look-up tables, also usable in SerialEM and Leginon, using elementary scattering physics and thereby adapt the tool to any microscope without time consuming experimental calibration. We benchmark our approach using two alternative techniques: the "ice channel" technique and tilt-series tomography. We also demonstrate the utility of ice thickness measurement for selecting holes in gold grids containing an Equine apoferritin sample, achieving a 1.88 Ångstrom resolution in subsequent refinement of the atomic map.

Extracellular Vesicles Secreted by Glioma Stem Cells Are Involved in Radiation Resistance and Glioma Progression.

Glioblastoma is the most aggressive brain tumour with short survival, partly due to resistance to conventional therapy. Glioma stem cells (GSC) are likely to be involved in treatment resistance, by releasing extracellular vesicles (EVs) containing specific molecular cargoes. Here, we studied the EVs secreted by glioma stem cells (GSC-EVs) and their effects on radiation resistance and glioma progression. EVs were isolated from 3 GSCs by serial centrifugation. NanoSight measurement, cryo-electron microscopy and live imaging were used to study the EVs size, morphology and uptake, respectively. The non-GSC glioma cell lines LN229 and U118 were utilised as a recipient cell model. Wound healing assays were performed to detect cell migration. Colony formation, cell viability and invadopodium assays were conducted to detect cell survival of irradiated recipient cells and cell invasion post GSC-EV treatment. NanoString miRNA global profiling was used to select for the GSC-EVs' specific miRNAs. All three GSC cell lines secreted different amounts of EVs, and all expressed consistent levels of CD9 but different level of Alix, TSG101 and CD81. EVs were taken up by both LN229 and U118 recipient cells. In the presence of GSC-EVs, these recipient cells survived radiation exposure and initiated colony formation. After GSC-EVs exposure, LN229 and U118 cells exhibited an invasive phenotype, as indicated by an increase in cell migration. We also identified 25 highly expressed miRNAs in the GSC-EVs examined, and 8 of these miRNAs can target PTEN. It is likely that GSC-EVs and their specific miRNAs induced the phenotypic changes in the recipient cells due to the activation of the PTEN/Akt pathway. This study demonstrated that GSC-EVs have the potential to induce radiation resistance and modulate the tumour microenvironment to promote glioma progression. Future therapeutic studies should be designed to interfere with these GSC-EVs and their specific miRNAs.

Colorimetric histology using plasmonically active microscope slides.

The human eye can distinguish as many as 10,000 different colours but is far less sensitive to variations in intensity1, meaning that colour is highly desirable when interpreting images. However, most biological samples are essentially transparent, and nearly invisible when viewed using a standard optical microscope2. It is therefore highly desirable to be able to produce coloured images without needing to add any stains or dyes, which can alter the sample properties. Here we demonstrate that colorimetric histology images can be generated using full-sized plasmonically active microscope slides. These slides translate subtle changes in the dielectric constant into striking colour contrast when samples are placed upon them. We demonstrate the biomedical potential of this technique, which we term histoplasmonics, by distinguishing neoplastic cells from normal breast epithelium during the earliest stages of tumorigenesis in the mouse MMTV-PyMT mammary tumour model. We then apply this method to human diagnostic tissue and validate its utility in distinguishing normal epithelium, usual ductal hyperplasia, and early-stage breast cancer (ductal carcinoma in situ). The colorimetric output of the image pixels is compared to conventional histopathology. The results we report here support the hypothesis that histoplasmonics can be used as a novel alternative or adjunct to general staining. The widespread availability of this technique and its incorporation into standard laboratory workflows may prove transformative for applications extending well beyond tissue diagnostics. This work also highlights opportunities for improvements to digital pathology that have yet to be explored.

Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome.

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.

Antifungal versus antibacterial defence of insect wings.

HYPOTHESIS: The ability exhibited by insect wings to resist microbial infestation is a unique feature developed over 400 million years of evolution in response to lifestyle and environmental pressures. The self-cleaning and antimicrobial properties of insect wings may be attributed to the unique combination of nanoscale structures found on the wing surface. EXPERIMENTS: In this study, we characterised the wetting characteristics of superhydrophobic damselfly Calopteryx haemorrhoidalis wings. We revealed the details of air entrapment at the micro- and nano scales on damselfly wing surfaces using a combination of spectroscopic and electron microscopic techniques. Cryo-focused-ion-beam scanning electron microscopy was used to directly observe fungal spores and conidia that were unable to cross the air-liquid interface. By contrast, bacterial cells were able to cross the air-water interface to be ruptured upon attachment to the nanopillar surface. The robustness of the air entrapment, and thus the wing antifungal behaviour, was demonstrated after 1-week of water immersion. A newly developed wetting model confirmed the strict Cassie-Baxter wetting regime when damselfly wings are immersed in water. FINDINGS: We provide evidence that the surface nanopillar topography serves to resist both fungal and bacterial attachment via a dual action: repulsion of fungal conidia while simultaneously killing bacterial cells upon direct contact. These findings will play an important role in guiding the fabrication of biomimetic, anti-fouling surfaces that exhibit both bactericidal and anti-fungal properties.

Novel Virus-Like Particle Vaccine Encoding the Circumsporozoite Protein of Plasmodium falciparum Is Immunogenic and Induces Functional Antibody Responses in Mice.

RTS,S is the leading malaria vaccine in development, but has demonstrated only moderate protective efficacy in clinical trials. RTS,S is a virus-like particle (VLP) that uses the human hepatitis B virus as scaffold to display the malaria sporozoite antigen, circumsporozoite protein (CSP). Particle formation requires four-fold excess scaffold antigen, and as a result, CSP represents only a small portion of the final vaccine construct. Alternative VLP or nanoparticle platforms that reduce the amount of scaffold antigen and increase the amount of the target CSP antigen present in particles may enhance vaccine immunogenicity and efficacy. Here, we describe the production and characterization of a novel VLP that uses the small surface antigen (dS) of duck hepatitis B virus to display CSP. The CSP-dS fusion protein successfully formed VLPs without the need for excess scaffold antigen, and thus CSP represented a larger portion of the vaccine construct. CSP-dS formed large particles approximately 31-74 nm in size and were confirmed to display CSP on the surface. CSP-dS VLPs were highly immunogenic in mice and induced antibodies to multiple regions of CSP, even when administered at a lower vaccine dosage. Vaccine-induced antibodies demonstrated relevant functional activities, including Fc-dependent interactions with complement and Fcγ-receptors, previously identified as important in malaria immunity. Further, vaccine-induced antibodies had similar properties (epitope-specificity and avidity) to monoclonal antibodies that are protective in mouse models. Our novel platform to produce VLPs without excess scaffold protein has wide implications for the future development of vaccines for malaria and other infectious diseases.

Structural Characterization of the Type IX Secretion System in Porphyromonas gingivalis.

The type IX secretion system (T9SS) is the most recently discovered secretion system in the gram-negative bacteria and is specific to the Bacteroidetes phylum. It is comprised of at least 19 proteins, which together allows for the secretion and cell surface attachment of a specific group of proteins (T9SS substrates), that harbor a signal sequence at the C-terminus. Here we describe the structural characterization of the PorK, PorN and PorG components of the Porphyromonas gingivalis T9SS using electron microscopy and cross-linking mass spectrometry.

Atypical myelinogenesis and reduced axon caliber in the Scn1a variant model of Dravet syndrome: An electron microscopy pilot study of the developing and mature mouse corpus callosum.

Dravet Syndrome (DS) is a genetic neurodevelopmental disease. Recurrent severe seizures begin in infancy and co-morbidities follow, including developmental delay, cognitive and behavioral dysfunction. A majority of DS patients have an SCN1A heterozygous gene mutation. This mutation causes a loss-of-function in inhibitory neurons, initiating seizure onset. We have investigated whether the sodium channelopathy may result in structural changes in the DS model independent of seizures. Morphometric analyses of axons within the corpus callosum were completed at P16 and P50 in Scn1a heterozygote KO male mice and their age-matched wild-type littermates. Trainable machine learning algorithms were used to examine electron microscopy images of ~400 myelinated axons per animal, per genotype, including myelinated axon cross-section area, frequency distribution and g-ratios. Pilot data for Scn1a heterozygote KO mice demonstrate the average axon caliber was reduced in developing and adult mice. Qualitative analysis also shows micro-features marking altered myelination at P16 in the DS model, with myelin out-folding and myelin debris within phagocytic cells. The data has indicated, in the absence of behavioral seizures, factors that governed a shift toward small calibre axons at P16 have persisted in adult Scn1a heterozygote KO corpus callosum. The pilot study provides a basis for future meta-analysis that will enable robust estimates of the effects of the sodium channelopathy on axon architecture. We propose that early therapeutic strategies in DS could help minimize the effect of sodium channelopathies, beyond the impact of overt seizures, and therefore achieve better long-term treatment outcomes.