Biology subjects

Guerrero, J.

Publications and source records attributed to Guerrero, J..

7 recordsLinked to original sources

bioRxiv2025

End-to-end evaluation of white matter microstructure of the visual pathway in asymmetric glaucoma

Diffusion magnetic resonance imaging is a non-invasive neuroimaging technique that enables in vivo evaluation of white matter microstructure, providing sensitivity to tissue abnormalities caused by disease. Glaucoma, the second leading cause of blindness worldwide, is characterized by progressive loss of retinal ganglion cells and axonal damage in the optic nerve, leading to degeneration along the entire visual pathway. This degeneration includes secondary effects on fiber crossings within the optic chiasm, which are challenging to characterize with conventional diffusion models. In this study, we evaluated 31 patients with asymmetric glaucoma and 31 healthy controls using advanced diffusion magnetic resonance imaging methods, including Diffusion Tensor Imaging, Constrained Spherical Deconvolution, multi-tensor fit via Multi-Resolution Discrete Search method, and Fixel-Based Analysis. We found significant differences of diffusion metrics in white matter tracts of the visual system, including the optic nerve, optic chiasm, optic tracts, and optic radiations. Moreover, diffusion metrics correlated with clinical ophthalmological parameters such as cup-to-disc ratio, visual field mean deviation, and retinal nerve fiber layer thickness. These findings support the use of advanced diffusion magnetic resonance imaging models as sensitive tools for detecting Wallerian degeneration and resolving complex white matter architecture in the human visual pathway, and demonstrate their utility to study other fiber-crossing regions throughout the brain.

neuroscience↗

bioRxiv2025

Exosomes are specialized vehicles to induce fibronectin assembly

Fibronectin is a key stromal matrix molecule whose assembly into fibrils is thought to require cells. In contrast, we find that small exosome-type extracellular vesicles (EVs) are critical initiators of fibronectin assembly. Fibroblasts engineered to be deficient in exosome secretion showed greatly reduced assembly of fibronectin and other stromal matrix molecules in 2D, 3D, and in vivo environments, and led to reduced tumor growth and lung metastasis by breast cancer cells. Furthermore, transgenic mice with defects in exosome secretion had greatly reduced lung fibrosis after treatment with bleomycin. In a direct test of exosome function, we find that the addition of purified small EVs to purified soluble fibronectin in a cell-free system is sufficient to induce fibronectin assembly. The EV-induced fibronectin assembly requires the presence of fibronectin-binding integrins and Syndecan-1 in the EVs. We propose a new model in which secreted exosomes directly drive stromal matrix assembly and tissue fibrosis.

cell biology↗

bioRxiv2025

Inference of VEGFR2 dimerization kinetics on the cell surface by integrating single-molecule imaging and mathematical modeling

Inter-receptor interactions play a key role in receptor signaling, which is the first step in cell signaling in response to external stimuli. In the case of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), dimerization is necessary for activation. VEGFR2 undergoes reversible interactions also in the absence of ligand. For a quantitative understanding of transmembrane signal transduction, it is necessary to quantify the interaction kinetics of VEGFR2 on the cell surface. Live-cell single-molecule imaging (SMI) has the powerful ability to capture receptor interaction events in their native cellular environment with high spatiotemporal resolution. However, it reveals these interactions for only the labeled subset, which is a small fraction of the full population of receptors. We have previously shown that mathematical modeling, combined with SMI data, offers a route to compensate for this lost information and infer the population-level receptor interaction kinetics from SMI data. Here, we applied this approach to VEGFR2, both wildtype full length VEGFR2 (FLR2) and a truncated mutant consisting of its extracellular and transmembrane domains (ECTM), which has been shown to exhibit reduced homotypic interactions in the absence of ligand. We developed a stochastic mathematical model mimicking VEGFR2 diffusion and interactions and determined the unknown model parameters through a combination of direct experimental measurements and stochastic model calibration. We found that a model of dimerization was sufficient to describe VEGFR2 interactions in the absence of ligand. While ECTM was primarily monomeric, FLR2 exhibited a substantial fraction of dimers. Our inference revealed that the difference between FLR2 and ECTM was primarily in the dimer association rate constant, which was about an order of magnitude lower for ECTM than for FLR2. To our knowledge, this is the first time that the interaction kinetics of VEGFR2 have been calculated in live cells. Author summaryInteractions between receptors on the cell surface play a large role in the initiation and modulation of cell signaling. For example, the receptor VEGFR2 in vascular endothelial cells must dimerize as a first step toward signaling. However, many interactions, including VEGFR2 dimerization, also take place in the absence of ligand stimulation. Therefore, elucidating when and where receptor interactions take place, and what their kinetics are, is critical for understanding receptor and cell signaling, especially on a quantitative level. Determining interaction kinetics is particularly challenging, given that membrane proteins do not express well in vitro, and are thus not amenable to traditional biophysical methods that quantify molecular interactions. In addition, the membrane and cellular environment is much more complex than an in vitro setting. To address these challenges, we combine single-molecule imaging, stochastic mathematical modeling, and rigorous model calibration to derive the dimerization kinetics of VEGFR2 (both full-length and a truncation mutant with reduced dimerization) on the surface of live cells. To our knowledge, this is the first time that the interaction kinetics of VEGFR2 have been calculated in live cells. Our approach is generalizable to many other cell surface proteins, opening an avenue for quantifying molecular interaction kinetics in situ.

biophysics↗

bioRxiv2025

Integrins and tetraspanins mediate CD36-actin coupling and regulate CD36 organization and signaling

The actin cortex plays a large role in regulating the dynamic organization of cell surface receptors, which in turn regulates their signaling. However, many receptors have short intracellular domains and no known link to cortical actin. In this work, we identified the {beta}1-integrin subunit and several tetraspanins - CD9, CD81 and CD151 - as part of the hitherto unknown molecular link between the surface receptor CD36 and cortical actin. We found that CD36 in vascular endothelial cells is recruited into complexes/nanodomains containing these proteins, with stronger recruitment near the cell edge. Perturbing this recruitment via the mutation G12V in the N-terminal transmembrane domain of CD36 alters the dynamic organization of CD36 on the vascular endothelial cell surface and weakens its coupling to cortical actin dynamics. Moreover, perturbing this recruitment abolishes thrombospondin-1-induced CD36 signaling through the Src family kinase Fyn. Given their many interactions with other transmembrane proteins, tetraspanins and integrins may provide a ubiquitous mechanism for plasma membrane-cortical actin coupling.

cell biology↗

bioRxiv2025

Two-timepoint assays of neural responses increase the sensitivity and specificity of single-cell whole-brain activity screens

Current approaches for surveying whole brains for neurons activated during a particular state typically rely on immediate early gene (IEG) expression. However, IEG expression is variable across subjects and brain areas, demanding large sample sizes. Further, it cannot determine if the same or different neurons respond to two events. To overcome these issues, we present a whole-brain screening method utilizing transgenic mice to label neurons activated at two timepoints. An imaging and analysis pipeline surveys activity in ~500 brain areas in different conditions. Compared to IEG methods, this approach reduces required sample sizes and enhances sensitivity and specificity. Finally, graph theoretical analyses are utilized to identify key circuit nodes - brain areas whose activity correlates with activity in other areas in a state-dependent manner. We validate this method by surveying whole-brain activity during hunger and satiety, and by investigating neural circuits activated by the GLP1 agonist semaglutide used to treat obesity.

neuroscience↗

bioRxiv2021

Genomic epidemiology of Mycobacterium bovis infection in sympatric badger and cattle populations in Northern Ireland.

BackgroundBovine tuberculosis (bTB) is a costly, epidemiologically complex, multi-host, endemic disease. Pathogen whole genome sequencing can improve the resolution of epidemiological tracing. We genome sequenced an exceptional data set of 619 Mycobacterium bovis isolates from badgers and cattle in a 100km2 bTB hotspot. Historical molecular subtyping data permitted the targeting of an endemic pathogen lineage, whose long-term persistence provided an opportunity to study genome epidemiology in detail. To assess whether badger population genetic structure was associated with the spatial distribution of pathogen genetic diversity, we microsatellite genotyped hair samples from 769 badgers trapped in this area. ResultsEight lineages of M. bovis were circulating in the study area, seven of which were likely non-endemic, and imported by animal movement. The endemic lineage exhibited low genetic diversity with an average inter-isolate genetic distance of 7.6 SNPs (s.d. {+/-} 4.0), consistent with contemporary transmission. Bayesian phylogenetic methods determined an evolutionary rate of 0.30 substitutions per genome per year for this lineage, estimating its emergence 40-50 years before present, while Bayesian Skyline analysis identified significant population expansion of the endemic lineage in the 1990s and again in 2011-2012. The phylogeny revealed distinct sub-lineages, all of which contained isolates from both cattle and badger hosts, indicative of the sharing of closely related strains and inter-species transmission. However, the presence of significant badger population genetic structure was not associated with the spatial distribution of M. bovis genetic diversity. ConclusionsOur data provided unparalleled detail on the evolutionary history of an endemic M. bovis lineage. Findings are consistent with ongoing interspecies transmission in the study area but suggest that badger intra-species transmission may not be a major driver of persistence in this area. In addition, the data collected permitted the tracking of incursions of novel pathogen lineages into the study area and means to determine if they were involved in disease transmission.

evolutionary biology↗

bioRxiv2020

Classical monocytes, the non-inflammatory phenotype, display the most robust pro-inflammatory response to LPS in vitro after immediate cytometric analysis

BACKGROUNDCD14 (Monocyte identifying Toll-Like Receptor) and CD16 (FcyRIII co-receptor, marker of inflammatory monocytes) were used to define 3 subpopulations of circulating monocytes with different attributes in terms of inflammatory and phagocytic capabilities. There are contradictory reports regarding response of circulating monocytes to pro-inflammatory or non-inflammatory stimuli in vitro. Here we aimed to analyze the phenotypic changes in circulating monocytes when stimulated with pro and non-inflammatory stimuli. METHODSWhole blood from 9 healthy donors was extracted and studied. Monocyte subpopulations were directly measured using flow cytometry with PBMC Ficoll extraction method. Pro-inflammatory interleukin IL-1{beta} was measured by intracellular cytometry. Whole blood-extracted monocytes were stimulated using LPS and IL-4 as previously described. Changes against non-stimulated (N-S) populations were statistically analyzed. RESULTSCompared to N-S, LPS-stimulated monocytes display a singular milieu of markers, with higher levels of intracellular IL-1{beta} in parallel raise of CD14+CD163-/CD14+CD163+ ratio. CD163 shows positive correlation with levels of IL-1{beta}. In t-SNE (T-distributed Stochastic Neighbor Embedding) analysis, after LPS stimulation, subpopulation CD14+CD16-CD163-, containing mainly classical monocytes, show a higher number of IL-1{beta}+ cells. CONCLUSIONClassical monocytes, the non-inflammatory subset, show higher levels of IL-1{beta} in response to LPS narrowing down to a new subpopulation of monocytes CD14+CD16-CD163-, which correlates better with this interleukin response than widely used monocytes classification. Using CD163 in addition to CD16, we were able to show that classical monocytes display the most intense response to LPS. Additionally, CD163 appears to be a suitable addition to CD14-CD16 classification to improve its performance.

immunology↗