Biology subjects

Toledo, D.

Publications and source records attributed to Toledo, D..

4 recordsLinked to original sources

bioRxiv2026

Distinct melanoma EV subpopulations reflect immune- and stress-induced tumor states

Melanoma is the deadliest form of skin cancer, and improved non-invasive approaches to monitor tumor burden and immune dynamics are needed. Although extracellular vesicles (EVs) are increasingly explored as cancer biomarkers, how distinct EV subpopulations reflect dynamic tumor states induced by immune pressure remains insufficiently understood. Using proteomic analyses, we identified the melanoma-associated antigens gp100 (PMEL) and GPNMB in EVs derived from B16F10 melanoma cells and incorporated them into sandwich enzyme-linked immunosorbent assays (ELISAs) that capture total EVs while selectively detecting gp100 and GPNMB EV subpopulations. We subsequently evaluated these EV populations in murine models of anti-tumor vaccination and in plasma samples from melanoma patients. In vivo, melanoma-associated EV subpopulations increased in the serum of tumor-bearing mice and were further augmented following antigen-specific anti-tumor vaccination, whereas total CD81 EVs accumulated more gradually. Notably, gp100 EV levels, but not GPNMB EVs, correlated with tumor-infiltrating lymphocyte densities across multiple time points and treatment conditions. In vitro, TNF/IFN{gamma} stimulation preferentially increased total CD81 EV release, whereas gp100 EVs were promoted by IL-1{beta} stimulation. In contrast, GPNMB EVs accumulated more gradually and broadly across inflammatory, hypoxic, and cytotoxic stress conditions. Together, these findings indicate that immune and stress signals differentially remodel melanoma-associated EV composition. The assay translated to humans, revealing elevated gp100 EV levels in the plasma of melanoma patients compared with healthy donors, whereas GPNMB EVs identified a subset of melanoma patients. We describe a clinically feasible approach that enables direct detection of melanoma-associated EV subpopulations from blood without prior EV isolation. Conceptually, our findings suggest that immune and stress signals dynamically shape circulating EV composition, generating distinct EV signatures that reflect tumor state.

immunology↗

bioRxiv2025

A Grapholita molesta (Lepidoptera: Tortricidae) cadherin in the 99C cadherin clade binds to Bacillus thuringiensis Cry1A proteins

Bt-cadherins, a particular type of midgut cadherins, act as receptors for Bacillus thuringiensis Cry1A pesticidal proteins. The aim of this work was to identify and validate the Cry1A cadherin receptor in Grapholita molesta (GmCad1). The GmCad1 gene was annotated by Blast genome mining using lepidopteran Bt-related cadherins. The phylogenetic analyses grouped GmCad1 with other Torticidae cadherins, in a different clade than the lepidopteran Bt-related cadherins. The in silico analysis of the GmCad1 showed a structure similar to that of the Bt-related cadherins, with 11 cadherin repeats (CRs), a transmembrane region, and an intracellular domain. The full-length mRNA sequencing confirmed GmCad1 expression in vivo in G. molesta guts. To validate the binding ability of Cry1A proteins to GmCad1, a cadherin fragment (CR7-CR11) was expressed and in vitro binding was studied demonstrating that Cry1Aa, Cry1Ab, and Cry1Ac bind to this region in a dose-dependent manner. In silico molecular docking analysis suggested that the interaction may involve mainly the Domains II of Cry1Ab and Cry1Ac, and the Domain III of Cry1Aa. This study provides the first evidence of a 99-C cadherin serving as a receptor for Cry1A in G. molesta.

molecular biology↗

bioRxiv2025

Long-Read Sequencing of the MUC1 VNTR: Genomic Variation, Mutational Landscape, and Its Impact on ADTKD Diagnosis and Progression

BackgroundADTKD-MUC1 is caused by frameshift mutations in MUC1 gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The genes variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations. MethodsWe employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the MUC1 VNTR with the rate of kidney function decline in affected individuals. ResultsWe identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units. ConclusionsSMRT sequencing outperforms current diagnostic methods for ADTKD-MUC1 and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest ([~]6% variance explained), it remains biologically and clinically meaningful. Key Points 3O_LISingle Molecule, Real-Time (SMRT) sequencing of MUC1 outperforms existing genetic and immunohistochemical methods for ADTKD-MUC1 diagnosis. C_LIO_LICLIA-approved mass spectrometry-based genotyping assay, complemented by SMRT sequencing, can detect nearly all ADTKD-MUC1 patients. C_LIO_LIDisease progression correlates with VNTR pattern: fast progressors have fewer WT repeats and more mutant/frameshifted repeats C_LI

genetics↗

bioRxiv2024

Single-cell epigenomic dysregulation of Systemic Sclerosis fibroblasts via CREB1/EGR1 axis in self-assembled human skin equivalents

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin fibrosis, internal organ involvement and vascular dropout. We previously developed and phenotypically characterized an in vitro 3D skin-like tissue model of SSc, and now analyze the transcriptomic (scRNA-seq) and epigenetic (scATAC-seq) characteristics of this model at single-cell resolution. SSc 3D skin-like tissues were fabricated using autologous fibroblasts, macrophages, and plasma from SSc patients or healthy control (HC) donors. SSc tissues displayed increased dermal thickness and contractility, as well as increased -SMA staining. Single-cell transcriptomic and epigenomic analyses identified keratinocytes, macrophages, and five populations of fibroblasts (labeled FB1 - 5). Notably, FB1 APOE-expressing fibroblasts were 12-fold enriched in SSc tissues and were characterized by high EGR1 motif accessibility. Pseudotime analysis suggests that FB1 fibroblasts differentiate from a TGF-{beta}1-responsive fibroblast population and ligand-receptor analysis indicates that the FB1 fibroblasts are active in macrophage crosstalk via soluble ligands including FGF2 and APP. These findings provide characterization of the 3D skin-like model at single cell resolution and establish that it recapitulates subsets of fibroblasts and macrophage phenotypes observed in skin biopsies.

genomics↗