Biology subjects

Perez, D.

Publications and source records attributed to Perez, D..

15 recordsLinked to original sources

bioRxiv2026

Fluorescent Biosensors for Cryogenic Correlative Light and Electron Microscopy

Cryogenic electron tomography provides nanometer-scale views of subcellular structures but lacks essential physiological context, such as the metabolic state or ion concentration. To bridge this gap, we incorporated fluorescent biosensors into a cryogenic correlative light and electron microscopy workflow, enabling direct measurement of physiological conditions alongside structural data. Here, we focused on establishing and calibrating biosensors for calcium, pH, and osmotic stress at cryogenic conditions through an in vitro workflow in which biosensors were frozen in defined chemical environments and then imaged ratiometrically. We then applied these biosensors across a range of sample types: an osmotic stress sensor in Saccharomyces cerevisiae, a calcium sensor in human U2OS cells, and a pH sensor in the bacterium Halothiobacillus neapolitanus. This approach links localized physiological changes to the 3D structures of macromolecules and their organization in situ.

biophysics↗

bioRxiv2026

Claudin-dependent clonal expansion couples epicardial activation to regenerative fate acquisition

Epicardial activation is a conserved response to injury across vertebrates, yet only in regenerative species do epicardium-derived cells (EPDCs) acquire the capacity to invade the injury site and contribute to cardiac regeneration. How regenerative competence is established in epicardial cells downstream of activation remains poorly understood. Using multicolour lineage tracing we find that epicardial cells of the salamander species Pleurodeles waltl undergo a transient phase of clonal expansion that precedes EPDC accumulation at the injury site. Depletion of cCpE-sensitive Claudins from otherwise intact tight junctions alters the organisation and the mechanical state of the epicardium, impairs clonal expansion and reduces the capacity of epicardial cells to delaminate. Consequently, EPDCs fail to activate a regenerative transcriptional programme and complete their transition into injury-induced migratory EPDCs that are required to sculpt a Tenascin-C-rich matrix supporting cardiomyocyte protrusion at the injury site. Together, our findings demonstrate that epicardial activation alone is insufficient for regeneration and identify Claudin-dependent clonal expansion as a mechanism that couples epicardial activation to the acquisition of a regenerative EPDC state.

cell biology↗

bioRxiv2026

High Accuracy Fluorescence Guided Focused Ion Beam Milling

Cryo-electron tomography (cryo-ET) is a powerful approach for visualizing macromolecular structures directly within cells, but its broader application is limited by the difficulty of reliably targeting specific structures during sample preparation. In particular, capturing small or rare objects within cryo-focused ion beam (cryo-FIB) milled lamellae remains a major bottleneck. Here, we present two fluorescence-guided cryo-FIB milling workflows that overcome key sources of targeting error and enable routine capture of structures across a broad range of spatial scales. For larger targets (>500 nm), we present an improved registration-based strategy combining FIB-milled fiducials with depth correction for refractive index mismatch. For smaller targets (150-500 nm), we implement real-time fluorescence guided milling on a commercially available cryo-FIB-SEM platform enabling consistent recovery of small, low-copy organelles such as centrosomes and primary cilia. Both workflows are implemented in a new open-source package fibsemOS and together expand the range of cellular structures accessible to cryo-ET and establish a broadly deployable framework for targeted in situ structural biology.

cell biology↗

bioRxiv2025

Prostate-specific EV capture with sufficient RNA yield to enable transcriptomic profiling

Prostate-specific antigen (PSA) screening has reduced prostate cancer (PCa) mortality but suffers from limited specificity, contributing to unnecessary biopsies and overdiagnosis of indolent disease. There is a critical need for biofluid-based biomarkers that improve the precision of PCa detection. Extracellular vesicles (EVs) offer a promising platform for noninvasive diagnostics, as they carry molecular cargo reflective of their tissue of origin. The ExoDx Prostate IntelliScore (EPI) test, a urine-based EV assay, is currently the only commercial EV diagnostic for clinically significant (cs)PCa, but its performance may be constrained by contamination from renal and bladder-derived EVs. To address this, we developed Exosome Diagnostics Depletion and Enrichment (EDDE), a novel immunocapture-based method for isolating prostate-derived EVs with high specificity. By targeting Prostate Specific Membrane Antigen (PSMA), we optimized EDDE to selectively enrich prostate EVs from post-DRE urine and recover sufficient RNA for transcriptomic analysis. Throughout development, we implemented a quantitative framework to track EV stoichiometry and assess depletion efficiency and yield, enabling rigorous optimization of the workflow. Our findings demonstrate that PSMA EDDE enriches prostate-specific EVs and yields RNA quantities compatible with sequencing. This platform enhances the specificity of EV-based biomarker discovery and holds promise for determining if tissue-specific EV biomarkers contribute advantages over bulk EVs.

cancer biology↗

bioRxiv2025

Extracellular Vesicle Gene Expression Enables Sensitive Detection of Colorectal Neoplasia

BACKGROUND & AIMSExtracellular vesicles (EVs), including exosomes, are emerging as promising carriers of disease-specific biomarkers due to their molecular cargo reflective of cellular origin. While cell-free DNA (cfDNA) methylation assays have recently been developed for colorectal cancer (CRC) screening and perform well for cancer detection, they show limited sensitivity for advanced adenomas (AA), a key precursor in the CRC pathway. To address this gap, we sought to determine whether other blood-based analytes, specifically EV-derived long and small RNA transcriptomes and EV proteomes, could improve detection of AA and early-stage CRC. In a prospective cohort, we performed a head-to-head comparison of EV transcriptomics, EV proteomics, and their combinations against cfDNA methylation, all measured from the same patient cohort, enabling a direct performance benchmark and identification of the most promising modality for larger-scale CRC screening studies. METHODSWe prospectively collected pre-colonoscopy plasma samples from 220 participants across three clinical sites. EVs were isolated and profiled using long RNA-seq, small RNA-seq, and Olink-based proteomics. cfDNA was analyzed for methylation patterns. Analyses were conducted according to a statistical analysis plan pre-specified before unblinding. Machine learning models were developed under nested cross-validation to evaluate sensitivity for detecting AA and CRC at a fixed specificity of 91%, consistent with clinical screening benchmarks. RESULTSEV-derived gene expression on long RNA demonstrated the highest sensitivity for detecting AA as well as CRC: 54.8% (95% CI, 26.4%-75.6%) for AA and 94.1% (95% CI, 79.2%-100%) for CRC. This outperformed cfDNA methylation (33.6% [95% CI, 9.7%-60.3%] for AA, 81.3% [95% CI, 64.6%- 93.9%] for CRC) and other EV-based modalities. In addition, for small RNA the sensitivities were 42.6% (95% CI, 35.9%-47.4%) for AA, and 78.9% (95% CI, 69.2%-83.0%) for CRC, while for proteomics the sensitivities were 30.0% (95% CI, 13.9%-40.0%) for AA, and 64.2% (95% CI, 43.6%-87.2%) for CRC. Transcriptomic profiles revealed progressive enrichment of hallmarks of cancer pathways, including apoptosis and epithelial-mesenchymal transition, across disease stages. Multiomic integration did not improve performance beyond EV transcriptomics alone. CONCLUSIONSBy directly comparing multiple EV-based and cfDNA analytes within the same patient cohort, we found that EV transcriptomics delivers the strongest diagnostic performance for both AA and CRC. This rigorous benchmarking approach allows clear prioritization of the most promising modality guiding the design of larger validation studies and accelerating development of next-generation, blood-based CRC screening tools.

genomics↗

bioRxiv2024

Altered B Cell Metabolic Pathways Characterize Type 1 Diabetes Progression

Type 1 diabetes (T1D) results in immune-mediated destruction of insulin-producing beta cells in the pancreas. B cells have been identified as critical, pathogenic antigen presenting cells and their specificity drives disease progression. At the same time, immunosuppressive, IL-10- producing regulatory B cells (Bregs) have been shown to play protective roles in mouse models of several autoimmune diseases, including rheumatoid arthritis and multiple sclerosis. In these models, microenvironmental stimuli induce regulatory B cell differentiation. Specifically, signaling through hypoxia-inducible factor 1 (HIF-1) drives a glycolytic flux that facilitates Breg expansion. While Breg frequencies are decreased in individuals with T1D compared to healthy controls, the role B regs play, how microenvironmental stimuli influence their differentiation, and whether this is altered in T1D progression, which is characterized by progressive, systemic hyperglycemia, are less clear. Here we examine the relationship between B cell differentiation, cellular metabolism, and HIF-1 to reveal that in a mouse model of autoimmune diabetes, B cells have distinct metabolic characteristics that change with disease progression. Further, response to hypoxia in autoimmune B cells is distinct from the response by non-autoimmune, control B cells. Together, these data suggest that dysregulated HIF signaling may skew the B cell repertoire toward inflammatory, rather than regulatory B cell subsets to drive T1D development. Consequently, HIF-1 activation to expand regulatory B cell populations may be a viable option for immune modulation.

immunology↗

bioRxiv2024

Exploring transient states of PAmKate to enable improved cryogenic single-molecule imaging

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nano-scale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) which photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remains a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find a transient state forms with a lifetime of [~]2 ms under cryogenic conditions which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for study of other FPs at cryogenic temperatures.

biophysics↗

bioRxiv2023

Sensory innervation of masseter, temporal and lateral pterygoid muscles in common marmosets

Myogenous temporomandibular disorders (TMDM) is associated with an increased responsiveness of nerves innervating the masseter (MM), temporal (TM), medial pterygoid (MPM) and lateral pterygoid muscles (LPM). This study aimed to examine sensory nerve types innervating MM, TM and LPM of adult non-human primate - common marmosets. Sensory nerves are localized in specific regions of these muscles. Pgp9.5, marker for all nerves, and NFH, a marker for A-fibers, showed that masticatory muscles were predominantly innervated with A-fibers. The proportion of C- to A-fibers was highest in LPM, and minimal (6-8%) in MM. All C-fibers (pgp9.5+/NFH-) observed in masticatory muscles were peptidergic (CGRP+) and lacked mrgprD, trpV1 and CHRNA3, a silent nociceptive marker. All fibers in masticatory muscles were labeled with GFAP+, a myelin sheath marker. There were substantially more peptidergic A-fibers (CGRP+/NFH+) in TM and LPM compared to MM. Almost all A-fibers in MM expressed trkC, with some of them having trkB and parvalbumin. In contrast, a lesser number of TM and LPM nerves expressed trkC, and lacked trkB. Tyrosine hydroxylase antibodies, which did not label TG, highlighted sympathetic fibers around blood vessels of the masticatory muscles. Overall, masticatory muscle types of marmosets have distinct and different innervation patterns.

neuroscience↗

bioRxiv2022

GOAT: Deep learning-enhanced Generalized Organoid Annotation Tool

Organoids have emerged as a powerful technology to investigate human development, model diseases and for drug discovery. However, analysis tools to rapidly and reproducibly quantify organoid parameters from microscopy images are lacking. We developed a deep-learning based generalized organoid annotation tool (GOAT) using instance segmentation with pixel-level identification of organoids to quantify advanced organoid features. Using a multicentric dataset, including multiple organoid systems (e.g. liver, intestine, tumor, lung), we demonstrate generalization of the tool to annotate a diverse range of organoids generated in different laboratories and high performance in comparison to previously published methods. In sum, GOAT provides fast and unbiased quantification of organoid experiments to accelerate organoid research and facilitates novel high-throughput applications.

bioinformatics↗

bioRxiv2022

Exploring masses and internal mass distributions of single carboxysomes in free solution using fluorescence and interferometric scattering in an anti-Brownian trap

Carboxysomes are self-assembled bacterial microcompartments that facilitate carbon assimilation by co-localizing the enzymes of CO2 fixation within a protein shell. These microcompartments can be highly heterogeneous in their composition and filling, so measuring the mass and loading of an individual carboxysome would allow for better characterization of its assembly and function. To enable detailed and extended characterizations of single nanoparticles in solution, we recently demonstrated an improved Interferometric Scattering Anti-Brownian ELectrokinetic (ISABEL) trap, which tracks the position of a single nanoparticle via its scattering of a near-infrared beam and applies feedback to counteract its Brownian motion. Importantly, the scattering signal can be related to the mass of nanoscale proteinaceous objects, whose refractive indices are well-characterized. We calibrate single-particle scattering cross-section measurements in the ISABEL trap and determine individual carboxysome masses in the 50-400 MDa range by analyzing their scattering cross-sections with a core-shell model. We further investigate carboxysome loading by combining mass measurements with simultaneous fluorescence reporting from labeled internal components. This method may be extended to other biological objects, such as viruses or extracellular vesicles, and can be combined with orthogonal fluorescence reporters to achieve precise physical and chemical characterization of individual nanoscale biological objects. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/505029v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@18e9f99org.highwire.dtl.DTLVardef@900912org.highwire.dtl.DTLVardef@4398baorg.highwire.dtl.DTLVardef@1dff103_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Redifferentiated cardiomyocytes retain residual dedifferentiation signatures and are protected against ischaemic injury

Cardiomyocyte renewal by dedifferentiation and proliferation has fueled the field of regenerative cardiology in recent years, while the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterised by the restoration of function that is lost during dedifferentiation and is key to the healing process following injury. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: dedifferentiation, followed by redifferentiation. Here, using temporal RNAseq and proteomics, we survey the landscape of the dedifferentiation-redifferentiation process in the adult mouse heart. We find well characterised dedifferentiation pathways, such as reduced oxphos, increased proliferation and increased EMT-like features, largely return to normal, though elements of residual dedifferentiation remain, even after contractile function is restored. These hearts appeared rejuvenated and showed robust resistance to ischaemic injury. We find that redifferentiation is driven by negative feedback signalling, notably through LATS1/2 Hippo pathway activity. Disabling LATS1/2 in dedifferentiated cardiomyocytes augments dedifferentiation in vitro and prevents redifferentiation in vivo. Taken together, our data reveal the non-trivial nature of redifferentiation, whereby elements of dedifferentiation linger in a surprisingly beneficial manner. This cycle of dedifferentiation-redifferentiation protects against future insult, in what could become a novel prophylactic treatment against ischemic heart disease for at-risk patients.

cell biology↗

bioRxiv2021

Steroid-resistant nephrotic syndrome associated MYO1E mutations have differential effects on myosin 1e localization, dynamics, and activity

Myo1e is a non-muscle motor protein enriched in the podocyte foot processes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Here, we set out to differentiate between the pathogenic and neutral MYO1E variants identified in SRNS patients by exome sequencing. Based on protein sequence conservation and structural predictions, two mutations in the motor domain, T119I and D388H, were selected for this study. EGFP-tagged Myo1e constructs were delivered into the Myo1e-KO podocytes via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculoviral expression system and used to measure Myo1e ATPase and motor activity in vitro. Both mutants were expressed as full-length proteins in the Myo1e-KO podocytes. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs) in podocytes. In contrast, the D388H variant localization was similar to the WT. Surprisingly, the dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting that this mutation also affects Myo1e activity. The ATPase activity and the ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting the findings from cell-based experiments. The experimental pipeline developed in this study allowed us to determine that the T119I and D388H mutations appear to be pathogenic and gain additional knowledge in the Myo1e role in podocytes. This workflow can be applied to the future characterization of novel MYO1E variants associated with SRNS.

cell biology↗

bioRxiv2021

Novel canine high-quality metagenome-assembled genomes, prophages, and host-associated plasmids by long-read metagenomics together with Hi-C proximity ligation

Long-read metagenomics facilitates the assembly of high-quality metagenome-assembled genomes (HQ MAGs) out of complex microbiomes. It provides highly contiguous assemblies by spanning repetitive regions, complete ribosomal genes, and mobile genetic elements. Hi-C proximity ligation data bins the long contigs and their associated extra-chromosomal elements to their bacterial host. Here, we characterized a canine fecal sample combining a long-read metagenomics assembly with Hi-C data, and further correcting frameshift errors. We retrieved 27 HQ MAGs and seven medium-quality (MQ) MAGs considering MIMAG criteria. All the long-read canine MAGs improved previous short-read MAGs from public datasets regarding contiguity of the assembly, presence, and completeness of the ribosomal operons, and presence of canonical tRNAs. This trend was also observed when comparing to representative genomes from a pure culture (short-read assemblies). Moreover, Hi-C data linked six potential plasmids to their bacterial hosts. Finally, we identified 51 bacteriophages integrated into their bacterial host, providing novel host information for eight viral clusters that included Gut Phage Database viral genomes. Even though three viral clusters were species-specific, most of them presented a broader host range. In conclusion, long-read metagenomics retrieved long contigs harboring complete assembled ribosomal operons, prophages, and other mobile genetic elements. Hi-C binned together the long contigs into HQ and MQ MAGs, some of them representing closely related species. Long-read metagenomics and Hi-C proximity ligation are likely to become a comprehensive approach to HQ MAGs discovery and assignment of extra-chromosomal elements to their bacterial host.

microbiology↗

bioRxiv2020

Network variants are similar between task and rest states

Recent work has demonstrated that individual-specific variations in functional networks (that we call "network variants") can be identified in individuals using functional magnetic resonance imaging (fMRI). These network variants exhibit reliability over time with resting-state fMRI data. These properties have suggested that network variants may be trait-like markers of individual differences in brain organization. Another test of this conclusion would be to examine if network variants are stable between task and rest states. Here, we use precision data from the Midnight Scan Club (MSC) to demonstrate that (1) task data can be used to identify network variants reliably, (2) these network variants show substantial spatial overlap with those observed in rest, although state-specific effects are present, (3) network variants assign to similar canonical functional networks in different states, and (4) single tasks or a combination of multiple tasks produce similar network variants to rest. Together, these findings further reinforce the trait-like nature of network variants and demonstrate the utility of using task data to define network variants.

neuroscience↗

bioRxiv2019

Uncovering mechanisms of global ocean change effects on the Dungeness crab (Cancer magister) through metabolomics analysis

The Dungeness crab is an economically and ecologically important species distributed along the North American Pacific coast. To predict how Dungeness crab may physiologically respond to future global ocean change on a molecular level, we performed untargeted metabolomic approaches on individual Dungeness crab juveniles reared in treatments that mimicked current and projected future pH and dissolved oxygen conditions. We found 94 metabolites and 127 lipids responded in a condition-specific manner, with a greater number of known compounds more strongly responding to low oxygen than low pH exposure. Pathway analysis of these compounds revealed that juveniles may respond to low oxygen through evolutionarily conserved processes including downregulating glutathione biosynthesis and upregulating glycogen storage, and may respond to low pH by increasing ATP production. Most interestingly, we found that the response of juveniles to combined low pH and low oxygen exposure was most similar to the low oxygen exposure response, indicating low oxygen may drive the physiology of juvenile crabs more than pH. Our study elucidates metabolic dynamics that expand our overall understanding of how the species might respond to future ocean conditions and provides a comprehensive dataset that could be used in future ocean acidification response studies.

systems biology↗