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Results for “Optical mapping”

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PubMed2024

Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Humans↗
PubMed2026

A Novel Complete F8 Tandem Duplication Causing Elevated Factor VIII Activity and Associated with Venous Thromboembolism.

Background Coagulation factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. While elevated FVIII levels are an established risk factor for venous thromboembolism (VTE), genetic variants in the F8 gene directly causing such elevations remain scarce. Here, we report a novel complete F8 tandem duplication identified in a female patient with splanchnic venous thrombosis (SVT). Methods We performed genetic testing using a thrombophilia panel targeting 35 genes involved in thrombosis and haemostasis to detect both point variants and copy number variations (CNVs). Family co-segregation analysis and phenotypic assays for FVIII and von Willebrand factor (VWF) were conducted. The structural basis of the identified F8 copy number gain was elucidated using optical genome mapping (OGM). Full-length F8 mRNA amplification, quantitative PCR, plasma FVIII Western blotting, and X-chromosome inactivation analysis were performed to assess the functional consequences of the duplication. Thrombin generation test (TGT) was employed to assess the hypercoagulable state. Results Genetic testing identified three copies of all 26 exons of the F8 gene in the proband, which was also detected in her mother (CNVs = 3) and son (CNVs = 2). One-stage clotting and chromogenic assays confirmed persistently elevated FVIII activity in the proband and her mother, accompanied by increased FVIII antigen levels. The OGM analysis confirmed a 229 kb tandem duplication including the F8 gene on one of the proband's X chromosomes. The junction regions exhibited high sequence homology and were rich in repetitive sequences, which precluded precise breakpoint mapping. Full-length F8 mRNA amplification revealed no aberrant transcripts, whereas quantitative PCR showed increased F8 mRNA expression in all carriers. Plasma FVIII Western blotting indicated FVIII heavy and light chains of expected molecular weights with increased band intensity in carriers. X-chromosome inactivation analysis in female carriers showed no significant skewing. TGT in two available carriers showed increased thrombin generation compared with a normal control at both low (1 pM) and high (5 pM) tissue factor concentrations. Conclusion We identified a novel complete F8 tandem duplication associated with increased FVIII expression and a hypercoagulable phenotype in a female patient with SVT. These findings support F8 gene dosage gain as a rare gain-of-function mechanism contributing to elevated FVIII levels and thrombophilia, while variation in VWF levels and acquired risk factors may modify thrombotic penetrance.

coagulation factor VIII↗
PubMed2026

PULPO: pipeline of understanding large-scale patterns of oncogenomic signatures.

SUMMARY: PULPO v1.0 is a novel; fully automated pipeline designed for the preprocess and extraction of mutational signatures from raw Optical Genome Mapping (OGM) data. Built using Snakemake and executed within an isolated, Conda-managed environment, PULPO transforms complex cytogenetic alterations, captured at ultra-high resolution, into Catalogue of somatic mutations in cancer mutational signatures (COSMIC). This innovative approach not only enables researchers to work directly from raw OGM inputs but also streamlines the traditionally complex process of signature extraction, making advanced oncogenomic analyses accessible to users with varying levels of bioinformatics expertise. By facilitating the integration of comprehensive structural variants (SVs) and copy number variants (CNVs) data with established signature catalogues, PULPO paves the way for improved diagnostic accuracy and personalized therapeutic strategies. AVAILABILITY AND IMPLEMENTATION: The pipeline is open source and freely available under the MIT License at https://github.com/OncologyHNJ/PULPO-v.1.0 and DOI in Zenodo: https://zenodo.org/records/17749097.

Software↗
PubMed2026

Rapid derivation of cloning-competent cells from peripheral blood advances conservation biobanking.

Establishing viable cell lines from endangered species is essential for conservation, yet traditional fibroblast derivation from skin biopsies faces challenges including contamination risk and extended culture timelines. Here, we demonstrate that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood represent robust alternatives to fibroblasts for biobanking. Compared to canid fibroblasts, canid blood-derived cells exhibit 2- to 3-fold faster doubling rates (15 to 20 h vs. ~35 h for fibroblasts) and reduced time to banked cell lines (1.5 to 2 wks vs. 3 to 4 wks for fibroblasts). Proteomic profiling of 32 canonical markers confirmed EPCs and pericytes represent distinct populations with lineage-specific molecular signatures. Optical genome mapping demonstrated equivalent genomic stability across cell types with no detectable structural variants or aneuploidies. Finally, interspecific somatic cell nuclear transfer (iSCNT) experiments confirmed both EPCs and pericytes generate viable canid embryos with efficiency meeting or exceeding fibroblasts. As a proof of concept for conservation cloning, iSCNT embryos made with gray wolf blood-derived cells had a 15% implantation rate following embryo transfer and resulted in six viable fetuses. These findings support integrating blood-derived cell banking into conservation programs, which enables opportunistic genetic preservation during standard management activities and expands options for genetic rescue through assisted reproductive technologies.

Animals↗
PubMed2026

PSIP1::TBL1X: a recurrent gene fusion in pancreatic neuroendocrine tumors.

Effective treatment of metastatic neuroendocrine tumors (NETs) is limited by a lack of targeted therapies and clinically useful predictive biomarkers. We applied complementary genomic profiling technologies, including optical genome mapping (OGM) and whole exome sequencing (WES), to 70 liver metastases of NETs from multiple anatomical primary sites to identify actionable genomic alterations. We detected recurrent fusions involving TBL1X (PSIP1::TBL1X) and BEND2 (CHD7::BEND2 and NEO1::BEND2) by OGM in pancreatic neuroendocrine tumors (pNETs). The expression of the PSIP1::TBL1X fusion was confirmed by PacBio Iso-Seq long-read transcriptome sequencing and nested rtPCR, and fusion protein expression was established by western blotting. Expression of the PSIP1::TBL1X fusion was also assayed in a separate cohort of 31 specimens from 28 pNET cases by rtPCR. Across both cohorts, PSIP1::TBL1X was identified in 11% of pNET patients with available metastatic tissue, but was not detected in primary tumor specimens. All PSIP1::TBL1X fusion isoforms were found to retain early exons of PSIP1 and the complete coding sequence of TBL1X. Consistent with prior reports, BEND2 fusions were associated with high-grade tumors and may represent a clinically useful biomarker for aggressive disease. Notably, TBL1X and BEND2 fusions did not co-occur with ATRX/DAXX mutations, defining a distinct molecular subgroup of pNETs. This study highlights the importance of structural variant profiling in molecular profiling studies and supports a revised view of the role of gene fusions in neuroendocrine malignancies.

Humans↗
PubMed1979

[Topography of the tecto-tectal component of the main ipsilateral visual pathway of the frog (Rana esculenta L.)].

The detailed topography of the tecto-tectal component of the Frog's ipsilateral visual pathway is electrophysiologically obtained by mapping the optic lobes. This linkage transfers the visual information explored along a transversal tectal row on to an homologous line oriented at 130 degrees on the opposite tectum. The ipsilateral projection of the temporo-nasal axis of the retina, but not of the antero-posterior axis of the visual field, is reversed compared to its contralateral projection. Finally, the majority of the homologous tectal points are asymmetrical with respect to the animal's sagittal axis.

Animals↗
PubMed1979

The orientation of the visuotectal map in Xenopus: developmental aspects.

Rotations and translocations of the eye anlage were performed in Xenopus embryos of stages ranging from 21/22 to 30. Some of the operations involved grafting wild-type eye anlagen into albino host orbits. Operations were performed under a variety of operating media and conditions. In later larval life, or after metamorphosis, the visuotectal maps from the operated eyes were recorded electrophysiologically. Results fell into two classes. In the majority, the orientation of the visuotopic map corresponded to the orientation of the eye at the time of recording. In the minority the visuotopic maps were 'compound', consisting of two parts each with its own independent orientation. The organization of the compound maps was such that one component was oriented in correspondence with the orientation of the eye, while the other component was normally oriented. Histological analysis and observations on genetically marked grafts indicated that the component parts of the compound eye were of dual cellular origin. The component giving the rotated (or translocated) map belonged to the originally operated eye tissue; whereas the component giving the normally oriented map was derived from newly grown eye tissue coming from the optic stalk. In no case was a normally oriented map obtained from a rotated or translocated eye. The results are discussed in relation to mechanisms proposed to account for the determination of map-related retinal specificity.

Animals↗
PubMed1979

Anomalous ipsilateral optic fibre projection in Xenopus induced by larval tectal ablation.

Unilateral left tectal ablation was carried out in Xenopus between stage 48 and 1 month after metamorphosis. Six to 12 weeks after metamorphosis the retinal projection from the right eye was examined with the use of [3H]proline autoradiography. The autoradiographs indicated that optic fibres whose tectal target was destroyed recrossed to the ipsilateral tectum and basal optic nucleus via the posterior and pretectal commissures. No anomalous recrossing occurred if the tectal ablation was carried out at stage 58 or later. The aberrant optic fibres were restricted to the rostrolateral and central parts of the ipsilateral tectum and they terminated in a discontinuous manner. It is concluded that available surfaces serving as contact guidance cues are needed to direct aberrant optic fibres to the ipsilateral tectum.

Action Potentials↗
PubMed2026

A STORM-based protocol for nanoscale imaging and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber.

Stochastic Optical Reconstruction Microscopy (STORM) enables nanoscale mapping of molecular components beyond the diffraction limit; however, its reproducible implementation in hydrophobic polymer matrices remains challenging because fluorescence-labeling specificity, fluorophore photoswitching, three-dimensional localization, chromatic registration, and quantitative image analysis must be carefully controlled. This protocol presents a standardized experimental workflow for dual-color labeling, astigmatism-based three-dimensional STORM acquisition, and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber (NR). The workflow covers sample pretreatment, Cy5 NHS ester labeling of protein-associated primary amines, DiI labeling of phospholipid-rich domains, STORM imaging-buffer preparation, three-dimensional single-molecule localization, dual-channel registration, generation of standardized xy projections, aggregate-size analysis, and projected lateral spatial correlation assessment. Reproducibility is supported by defined acquisition and localization criteria, three independent sample preparations with at least five fields of view analyzed per condition, and unlabeled, single-color, dye-only matrix, and processing-associated Cy5 controls. Mean lateral localization precisions of 11.8 ± 2.3 nm for Cy5 and 13.5 ± 2.9 nm for DiI were obtained, while two-dimensional Fourier ring correlation analysis of the xy projections yielded effective lateral image resolutions of approximately 25 and 28 nm, respectively. Image-based particle segmentation and localization-coordinate-based density-based spatial clustering of applications with noise (DBSCAN) were applied to standardized xy projections as complementary quantitative approaches. Application of the protocol to untreated, centrifuged, and protease-treated NR samples demonstrated treatment-associated changes in the detected abundance and projected size distributions of protein- and phospholipid-associated aggregates, together with a non-monotonic change in their projected lateral spatial correlation. These observations describe alterations in nanoscale organization but do not, by themselves, establish stable protein-phospholipid complex formation. Unlike previous studies that primarily demonstrated the feasibility of STORM imaging in rubber materials, the principal contribution of this work is an end-to-end, step-by-step protocol incorporating defined controls, three-dimensional localization, image-quality metrics, chromatic-registration procedures, and complementary quantitative-analysis pipelines for non-expert users. The workflow may be adaptable to other hydrophobic polymers and soft-material systems after appropriate optimization and validation.

Rubber↗
PubMed1977

[Responses of neurons in the nucleus isthmi region of the frog to optic nerve stimulation].

Neuronal responses were studied in the nucleus isthmi area of Rana temporaria. The animals were immobilized by diplacine, a curare-like drug. Single neurons in this area had no background activity. After single-pulse electric stimulation of the contralateral optic nerve these neurons discharged as a rule with one action potential only, its minimal latency being 20-110 msc. The latencies decrease significantly when the stimulus intensities were increased from threshold to maximal, indicating development of summation process in the corresponding afferent pathway. Only 14 per cent (7 of 57) of neurons responded to stimuli exciting myelinated optic fibres and not exciting the ummyelinated ones. All the nucleus isthmi neurons discharged with unstable latency; this means that these neurons were excitated not antidromically. The functional role of nucleus isthmi in Anurans is discussed.

Animals↗
PubMedDate not supplied

[Visual projections in to the brain of the sturgeon Acipenser güldenstädti].

Visual projections have been studied after unilateral lesion of the optic nerve. Degenerated fibers and terminals, as well as fibers labelled by horseradish peroxidase were found contralaterally in the lateral geniculate thalamic nucleus, the second (stratum opticum) and the third (stratum fibrosum et griseum superficiale) layers of the midbrain tectum. In other brain structures (lateral ventrothalamic nucleus, in the pretectal area--cortical nucleus, pretectal nucleus, posterior comissure nucleus) the density of degeneration of optic fibers was significantly lower. Separate fragments of degeneration were found in the fourth layer of the tectum. Ipsilaterally, thalamic, pretectal and tectal visual projections of the same localization were found, although they were less massive than at the opposite side.

Animals↗