Biology subjects

Velazquez, D.

Publications and source records attributed to Velazquez, D..

7 recordsLinked to original sources

bioRxiv2026

Bioengineering of Pea (Pisum sativum) for the Expression of Myoglobin, a Heme-containing Animal Protein

Myoglobin, an oxygen-binding animal protein, was engineered in Pisum sativum (pea) to explore its potential as a food ingredient and balance the amino acid profile. In this study, minimal expression cassettes and binary vectors were used to express bovine myoglobin using particle gun and Agrobacterium-mediated transformation, respectively. Successful integration and expression of the myoglobin gene was achieved in P. sativum, with both methods yielding similar transformation efficiencies (~1%). Expression analysis of T2 seeds revealed that Agrobacterium-mediated transformation-derived transgenic lines that expressed myoglobin under the regulation of a Soybean 7S seed-specific promoter and Tobacco Etch Virus (TEV) translation enhancer and a chimeric Rb7MAR Terminator (Ps-BpRG13 events) consistently yielded the highest level of expression (0.32-1.57% of TSP), while transgenic lines with myoglobin expression under the regulation of a Soybean Phaseolin promoter and Rb7MAR Terminator (Ps-BpRG14 events) resulted in moderate levels of heterologous protein expression (0.13-0.83% TSP). Transgenic events with constitutive 2xCaMV35S promoter, TEV translation enhancer and Rb7MAR terminator (Ps-BpRG15 events) exhibited the lowest level of myoglobin expression (0.09-0.14% TSP). Co-bombardment of two minimal expression cassettes - one with myoglobin under the regulation of the Phaseolin promoter and Rb7MAR Terminator and the other with the nptII selectable marker under the regulation of a 2X constitutive CaMV35S promoter, TEV translational enhancer and TNOS Terminator, yielded lines that exhibited variable expression (0.03-0.77% TSP), with some events comparable in expression to Agrobacterium-derived Ps-pRG14 events. To the best of our knowledge, this is the first report of producing a heme-containing animal protein, myoglobin, in peas, with potential implications for sustainable production of food ingredients and nutritionally fortified and value-added plant products using molecular farming.

plant biology↗

bioRxiv2025

Spatial Transcriptomics As Rasterized Image Tensors (STARIT) characterizes cell states with subcellular molecular heterogeneity

Abstract Imaging-based spatially resolved transcriptomics (imSRT) technologies provide high-throughput molecular-resolution spatial characterization of genes within cells. Conventional analysis methods to identify cell-types and states in imSRT data rely on gene count matrices derived from tallying the number of mRNA molecules detected for each gene per segmented cell, thereby overlooking subcellular heterogeneity that can be useful in defining cell states. To take advantage of the molecular-resolution information in imSRT data and potentially identify cell-states based on subcellular heterogeneity, we developed STARIT (Spatial Transcriptomics As Rasterized Image Tensors). STARIT converts transcripts within segmented cells in imSRT data into an image-based tensor representation that can be combined with deep learning computer vision models for downstream analysis. Using simulated and real imSRT data, we demonstrate that STARIT distinguishes transcriptionally distinct cell-types and further separates cell states based on subcellular transcript localization, which conventional gene count analysis fails to capture. By providing a standardized framework to encode subcellular molecular information in imSRT data, STARIT will enable deeper insights into subcellular heterogeneity and enhance the identification and characterization of cell-types and states that are overlooked by gene count representations.

bioinformatics↗

bioRxiv2025

Human Oncogene EWS::FLI1 Functions as a Pioneer Factor in Saccharomyces cerevisiae.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI1s cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1s core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.

cancer biology↗

bioRxiv2025

Spatiotemporal transcriptomic analysis during cold ischemic injury to the murine kidney reveals compartment-specific changes

BackgroundKidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. ResultsTo bridge this knowledge gap, we leveraged the 10x Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We developed a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identified proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This was distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends were validated by qPCR and immunofluorescence in a larger cohort of mice. We provide an interactive online browser at https://jef.works/CellCarto-ColdIschemia/ to facilitate exploration of our results by the broader scientific and clinical community. ConclusionsAltogether, our spatiotemporal transcriptomic analysis identified coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examinations.

bioinformatics↗

bioRxiv2024

scatterbar: an R package for visualizing proportional data across spatially resolved coordinates

SummaryDisplaying proportional data across many spatially resolved coordinates is a challenging but important data visualization task, particularly for spatially resolved transcriptomics data. Scatter pie plots are one type of commonly used data visualization for such data but present perceptual challenges that may lead to difficulties in interpretation. Increasing the visual saliency of such data visualizations can help viewers more accurately identify proportional trends and compare proportional differences across spatial locations. We developed scatterbar, an open-source R package that extends ggplot2, to visualize proportional data across many spatially resolved coordinates using scatter stacked bar plots. We apply scatterbar to visualize deconvolved cell-type proportions from a spatial transcriptomics dataset of the adult mouse brain to demonstrate how scatter stacked bar plots can enhance the distinguishability of proportional distributions compared to scatter pie plots. Availability and implementationScatterbar is available at https://github.com/JEFworks-Lab/scatterbar with additional documentation and tutorials at https://jef.works/scatterbar/.

bioinformatics↗

bioRxiv2022

A needle in a haystack: a new metabarcoding approach to survey diversity at the species level of Arcellinida (Amoebozoa: Tubulinea)

Environmental DNA-based diversity studies have increased in popularity with the development of high throughput sequencing technologies. This permits the potential simultaneous retrieval of vast amounts of molecular data from many different organisms and species, thus contributing to a wide range of biological disciplines. Environmental DNA protocols designed for protists often focused on the highly conserved small subunit of the ribosome gene, that does not permit species-level assignments. On the other hand, eDNA protocols aiming at species-level assignments allow a fine level ecological resolution and reproducible results. These protocols are currently applied to organisms living in marine and shallow lotic freshwater ecosystems, often in a bioindication purpose. Therefore, in this study, we present a species-level eDNA protocol, designed to explore diversity of Arcellinida (Amoebozoa: Tubulinea) testate amoebae taxa, that is based on mitochondrial cytochrome oxidase subunit I (COI). These organisms are widespread in lentic water bodies and soil ecosystems. We applied this protocol to 42 samples from peatlands, estuaries and soil environments, recovering all the infraorders in Glutinoconcha (with COI data), except for Hyalospheniformes. Our results revealed an unsuspected diversity in morphologically homogeneous groups such as Cylindrothecina, Excentrostoma or Sphaerothecina. With this protocol we expect to revolutionize the design of modern distributional Arcellinida surveys. Our approach involve a rapid and cost effective analysis of testate amoeba diversity living in contrasted ecosystems. Therefore, Arcellinida clade have the potential to be established as a model group for an array of theoretical and applied studies.

microbiology↗

bioRxiv2022

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

The human Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity and increased resistance to the HsNHA2-specific inhibitor phloretin that were not only highly active at an acidic pH of 4.0 (like the native antiporter), but also at neutral pH. We also experimentally confirmed the importance of a putative salt bridge between E215 and R432 for antiporter function and structural integrity. Truncations of the first 50 - 70 residues of the N-terminus doubled the transport activity of HsNHA2, whilst changes in the charge at positions E47, E56, K57, or K58 decreased the antiporters transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically autoinhibit its cation transport. Our data also show this in vivo approach to be useful for a rapid screening of SNPs effect on HsNHA2 activity.

molecular biology↗