Biology subjects

Contreras, O.

Publications and source records attributed to Contreras, O..

9 recordsLinked to original sources

bioRxiv2026

Nanoparticle Albumin-Bound Paclitaxel Targets Pulmonary Neutrophils

Nanoparticle albumin-bound paclitaxel (Abraxane) was among the first clinically approved nanomedicines and is a first-line chemotherapeutic. It enhances therapeutic efficacy and reduces side effects of paclitaxel. Despite its extensive clinical use, mechanisms of Abraxane's therapeutic effects are the subject of ongoing study. This work posits a new mechanism for Abraxane's effects in lung cancers and metastasis. Tracing the albumin component of Abraxane with radioisotopes or fluorophores, we find that Abraxane homes to the lungs. Abraxane's lung tropism is elevated by inflammation and is eliminated in complement protein C3 knockout mice, showing that complement pathway drives the uptake. Flow cytometry and histology show that neutrophils are the dominant cell type taking up Abraxane in the lungs. We demonstrate Abraxane's tropism to pulmonary neutrophils in mice and in human donor lungs. We note previously undocumented side effects associated with Abraxane's lung tropism, including pro-thrombotic and pro-inflammatory responses in acute lung inflammation. We show that Abraxane's efficacy against lung metastases is affected when lung uptake is eliminated by C3 knockout. Our results provide insight into why Abraxane is effective in lung cancer and metastases. These findings may be used to improve outcomes in patients receiving Abraxane and help design next-generation cancer nanomedicines.

bioengineering↗

bioRxiv2026

A scalable MNase-seq framework for reproducible nucleosome profiling across pluripotent stem cell and cardiomyocyte models

Micrococcal nuclease (MNase) digestion is widely used to profile chromatin accessibility and nucleosome footprinting. However, its application is often limited by sensitivity to reaction conditions, high cell input requirements, and the lack of standardized protocols across cell types. Here we developed a robust MNase workflow encompassing buffer composition, DNA purification chemistry, fixation and decrosslinking parameters, cell input scalability, and an in-house yeast spike-in for quantitative normalization. We validated this unified framework across human induced pluripotent stem cells (hiPSCs), hiPSC-derived cardiomyocytes at multiple differentiation stages, primary murine embryonic cardiac cells, and adult mouse cardiomyocytes, and demonstrated comparable digestion efficiencies and kinetics despite marked differences in cellular architecture and chromatin organization. Genome-wide MNase-seq in hiPSCs, combined with the nucMACC bioinformatic pipeline, resolved concentration-dependent nucleosomal occupancy and precise nucleosome positioning at pluripotency-related regulatory elements. This modular, end-to-end, and scalable workflow provides a standardized platform for reproducible MNase-based chromatin profiling across diverse in vitro and in vivo models. TEASERA unified, rapid, and scalable MNase-seq workflow for reproducible mononucleosomal and subnucleosomal profiling from stem cells to adult cardiomyocytes. HIGHLIGHTSO_LISystematic MNase optimization across buffer, DNA purification, and cell input variables C_LIO_LIUnified workflow validated in hiPSCs, hiPSC-CMs, embryonic, and adult cardiomyocytes C_LIO_LIFixed-cell protocol enables weeks of storage without loss of DNA quality C_LIO_LICost-effective yeast spike-in ensures quantitative normalization for MNase-seq C_LIO_LIGenome-wide analyses confirm robust and precise nucleosome positioning at regulatory elements C_LI

genomics↗

bioRxiv2026

Proteo-transcriptomics and morphometrics of teleost cardiac cells define regulatory networks and exercise-induced cardiomyocyte hypertrophy and hyperplasia

Zebrafish and medaka are powerful cardiovascular models, yet cellular and molecular investigations of adult heart cells have been constrained by suboptimal dissociation and characterization methods. To overcome these barriers, we developed a physiological-temperature workflow that generates high-yield, viable single-cell suspensions for FACS, imaging, and low-input molecular profiling. Using transgenic fluorescent reporters, we consistently isolate [~]6,000 cardiomyocytes per adult zebrafish ventricle and [~]12,000 from medaka, preserving cellular, structural, and molecular integrity. Single-cell morphometrics revealed cardiomyocyte heterogeneity and demonstrated that swimming exercise induces both hypertrophy and hyperplasia, while ventricular injury triggers expansion of regenerative gata4 cardiomyocytes. We integrated proteomics and RNA-seq from FACS-purified cells to construct cell-type-specific proteo-transcriptomic atlases. Functional enrichment, transcription factor, and network analyses identified protein hubs and regulatory circuits defining cardiomyocyte and endothelial cell identity. Our platform delivers cell-type-resolved molecular datasets of adult teleost cardiac cells, establishing a systems-level resource for heart regeneration research, cardiovascular disease modeling, and drug discovery. TeaserA robust workflow for isolation, FACS, imaging, and multi-omics profiling of cardiomyocytes and cardiac endothelial cells in fish. HIGHLIGHTSO_LITemperature-optimized dissociation and standardized FACS enable high-yield isolation of viable cardiac cells. C_LIO_LISingle-cell imaging uncovers morphological heterogeneity in adult ventricular cardiomyocytes. C_LIO_LISustained exercise induces both hypertrophy and hyperplasia of zebrafish cardiomyocytes. C_LIO_LIProteo-transcriptomics defines core molecular programs and interaction networks in cardiomyocytes and endothelial cells. C_LI

systems biology↗

bioRxiv2025

OpenEMMU: a versatile, open-source EdU multiplexing methodology for studying DNA replication and cell cycle dynamics

5-Ethynyl-2-deoxyuridine (EdU) has transformed DNA replication and cell cycle analyses through fast and efficient click chemistry detection. However, commercial EdU kits are expensive, contain proprietary reagents, are suboptimal for antibody multiplexing, and are challenging to use with larger biological specimens, highlighting the need for innovation. Here, we report Open-source EdU Multiplexing Methodology for Understanding DNA replication dynamics (OpenEMMU), an optimized, affordable, and user-friendly click chemistry resource using off-the-shelf reagents. OpenEMMU improves the efficiency, brightness, and multiplexing capabilities of EdU thymidine analog staining with both non-conjugated and conjugated antibodies across various cell types. We validated its effectiveness for fluorescent imaging of nascent DNA in developing embryos and organs, including the embryonic heart and forelimbs, and in 3D hiPSC-derived cardiac organoids. OpenEMMU also enabled 3D imaging of DNA synthesis in zebrafish larvae at fine resolution. This opens new avenues for insights into organismal development and growth, cell proliferation, and DNA replication, with precision and flexibility that surpass traditional methods. TeaserAn open-source and cost-effective resource for DNA replication profiling and cell cycle analysis using click chemistry.

developmental biology↗

bioRxiv2024

Targeting de novo lipogenesis improves gemcitabine efficacy in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to discover new therapeutic targets for improving treatment efficacy. We show that GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 cells through increased activity and expression of acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). We also discovered alternate fatty acid desaturase 2 (FADS2) activity in Panc1 cells, which led to the production of sapienic acid (FA 16:1n-10, cis) from palmitic acid (FA 16:0). Knockdown of key lipid synthesis enzymes sensitised cells to GEMC treatment, with FAS (both cell lines), SCD1 (MiaPaCa2 only) and SCD1+FADS2 (Panc1) knockdown showing the greatest reduction in cell growth when combined with GEMC treatment. In Panc1 cells, both desaturases upregulated their activity when the alternate was knocked down, necessitating the need for dual desaturase knockdown in this cell line. PDAC cells attenuated to grow in combination GEMC/paclitaxel (CombAT) also displayed enhanced de novo lipogenesis; however, combination chemotherapy significantly downregulated FADS2 expression and activity in Panc1 CombAT cells rendering them more sensitive to SCD1 knockdown. We conclude that co-targeting lipid synthesis in PDAC could be a viable strategy for improving the efficacy of both GEMC monotherapy and combination GEMC/PTX therapy.

cancer biology↗

bioRxiv2024

Fragmentation of genomic DNA using Covaris g-Tubes prior to library preparation for sequencing

Covaris g-TUBEs can be used to fragment DNA to pre-determined sizes based on the relative centrifugal force that they are run at. They are recommended for use while preparing Oxford Nanopore Technology libraries by the manufacturer. However, the volumes and DNA concentration typically used for ONT libraries are outside the range of the example data provided by Covaris. Here, we ran g-TUBEs at three different relative centrifugal forces and determined the effect on DNA fragmentation in the range 0.5 - 4 {micro}g. This dataset can be used to inform the effective fragmentation of DNA for creating Oxford Nanopore libraries of an optimal size.

molecular biology↗

bioRxiv2022

Temporal transcriptomic dynamics of the ATX-LPAR-PLPP axis during skeletal muscle regeneration at single cell resolution

Lysophosphatidic acid (LPA) is a growth factor-like bioactive phospholipid recognising LPA receptors (LPARs) and mediating signalling pathways that regulate embryonic development, wound healing, carcinogenesis, and fibrosis, via effects on cell migration, proliferation and differentiation. Extracellular LPA is generated from lysophospholipids by the secreted hydrolase - ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2; also, AUTOTAXIN / ATX) and metabolised by different membrane-bound phospholipid phosphatases (PLPPs). Here, we use public bulk and singlecell RNA sequencing datasets to explore the gene expression of Lpar1-6, Enpp2, and Plpp genes under skeletal muscle homeostasis and regeneration conditions. We show that the skeletal muscle system dynamically expresses the Enpp2-Lpar-Plpp gene axis, with Lpar1 being the highest expressed member among LPARs. Lpar1 was expressed by mesenchymal fibro-adipogenic progenitors (FAPs) and tenocytes, whereas FAPs mainly expressed Enpp2. Clustering of FAPs identified populations representing distinct cell states with robust Lpar1 and Enpp2 transcriptome signatures in homeostatic cells expressing higher levels of markers Dpp4 and Hsd11b1. However, tissue injury induced transient repression of Lpar genes and Enpp2. The role of LPA in modulating the fate and differentiation of tissue-resident FAPs has not yet been explored. Ex vivo, LPAR1/3 and ENPP2 inhibition significantly decreased the cell-cycle activity of FAPs and impaired fibro-adipogenic differentiation, implicating LPA signalling in the modulation of the proliferative and differentiative fate of FAPs. Together, our results demonstrate the importance of the ENPP2-LPAR-PLPP axis in different muscle cell types and FAP lineage populations in homeostasis and injury, paving the way for further research on the role of this signalling pathway in skeletal muscle homeostasis and regeneration, and that of other organs and tissues, in vivo. SummaryOur reanalysis of single-cell transcriptomics revealed the involvement and temporally dynamic expression of the ENPP2-LPAR-PLPP axis in response to skeletal muscle regeneration.

cell biology↗

bioRxiv2021

Targeting fibrosis in the Duchenne Muscular Dystrophy mice model: an uphill battle

AimFibrosis is the most common complication from chronic diseases, and yet no therapy capable of mitigating its effects is available. Our goal is to unveil specific signallings regulating the fibrogenic process and to identify potential small molecule candidates that block fibrogenic differentiation of fibro/adipogenic progenitors. MethodWe performed a large-scale drug screen using muscle-resident fibro/adipogenic progenitors from a mouse model expressing EGFP under the Collagen1a1 promotor. We first confirmed that the EGFP was expressed in response to TGF{beta}1 stimulation in vitro. Then we treated cells with TGF{beta}1 alone or with drugs from two libraries of known compounds. The drugs ability to block the fibrogenic differentiation was quantified by imaging and flow cytometry. From a two-rounds screening, positive hits were tested in vivo in the mice model for the Duchenne muscular dystrophy (mdx mice). The histopathology of the muscles was assessed with picrosirius red (fibrosis) and laminin staining (myofiber size). Key findingsFrom the in vitro drug screening, we identified 21 drugs and tested 3 in vivo on the mdx mice. None of the three drugs significantly improved muscle histopathology. SignificanceThe in vitro drug screen identified various efficient compounds, none of them strongly inhibited fibrosis in skeletal muscle of mdx mice. To explain these observations, we hypothesize that in Duchenne Muscular Dystrophy, in which fibrosis is a secondary event due to chronic degeneration and inflammation, the drugs tested could have adverse effect on regeneration or inflammation, balancing off any positive effects and leading to the absence of significant results.

cell biology↗

bioRxiv2020

Extracellular TGF-β downregulates the expression of Wnt transcription factor TCF7L2/TCF4 in mesenchymal stromal cells and fibroblasts

Mesenchymal stromal/stem cells (MSCs) are multipotent progenitors essential ororganogenesis, tissue homeostasis, regeneration, and scar formation. Tissue injury upregulates TGF-{beta} signaling, which modulates myofibroblast fate, extracellular matrix remodeling, and fibrosis. However, the molecular determinants of MSCs differentiation and survival remain poorly understood. The canonical Wnt Tcf/Lef transcription factors regulate development and stemness, but the mechanisms by which injury-induced cues modulate their expression remain underexplored. Here, we studied the cell-specific gene expression of Tcf/Lef and, more specifically, we investigated whether damage-induced TGF-{beta} impairs the expression and function of TCF7L2, using several models of MSCs, including skeletal muscle fibro-adipogenic progenitors. We show that Tcf/Lefs are differentially expressed and that TGF-{beta} reduces the expression of TCF7L2 in MSCs but not in myoblasts. We also found that the ubiquitin-proteasome system regulates TCF7L2 proteostasis and participates in TGF-{beta}-mediated TCF7L2 protein downregulation. Finally, we show that TGF-{beta} requires HDACs activity to repress the expression of TCF7L2. Thus, our work found a novel interplay between TGF-{beta} and Wnt canonical signaling cascades in PDGFR+ fibroblasts and suggests that this mechanism could be targeted in tissue repa ir and regeneration. Summary statementTGF-{beta} signaling suppresses the expression of the Wnt transcription factor TCF7L2 and compromises TCF7L2-dependent functions in tissue-resident PDGFR+ fibroblasts.

cell biology↗