Biology subjects

Maldonado, J.

Publications and source records attributed to Maldonado, J..

11 recordsLinked to original sources

bioRxiv2026

Ex vivo human tumor slices more accurately predict patient responses to an oncolytic virus than in vivo mouse models

Immunotherapies, including oncolytic viruses (OV), are promising therapies that can enhance anti-tumor immune responses. However, preclinical success of immunotherapies in mouse models has not always translated to clinical benefit in cancer patients. This study compared preclinical efficacy and mechanism of action for ASP9801, a vaccinia virus expressing IL-7 and IL-12, using mouse models of colorectal cancer (CRC) in vivo and in human organotypic tumor slice models ex vivo. The murine surrogate for ASP9801 significantly reduced tumor volumes in treated and abscopal tumors in two different CRC models in vivo (MC38 and RO100). Treatment efficacy was accentuated when combined with anti-PD1 treatment, and single-cell RNA sequencing analysis revealed depletion of tumor cells and increased T cell infiltration and activation in both treated and abscopal tumors. However, human tissue analysis ex vivo (E-slices) using PDX models and patient samples showed that ASP9801 is not effective in CRC, consistent with clinical trial results. On the other hand, ASP9801 was highly effective in GBM, indicating indication-specific efficacy of ASP9801, and how E-slice assays can be used to identify treatment-sensitive indications. This study demonstrates the superiority of E-slices over mouse models for predicting clinical response and its utility in planning clinical trials.

cancer biology↗

bioRxiv2026

Microvascular architecture and dynamics of the choroid plexus brain barrier

The choroid plexus is a specialized blood-cerebrospinal fluid barrier that supports cerebrospinal fluid production, immune surveillance, and molecular exchange between the circulation and the central nervous system, yet its vascular bed remains poorly understood. Here, we combine whole-tissue clearing and light-sheet imaging, single-nucleus transcriptomic reanalysis, and live calcium imaging of intact murine explants to define the structural and functional organization of choroid plexus endothelial networks across development. Three-dimensional imaging and reconstruction highlights a dense, epithelial-ensheathed vascular plexus that is continuous with the broader cerebrovasculature and organized into anatomically distinct inflow and ventricular margin regions. Transcriptomic analysis identified developmentally stratified endothelial subtypes, with embryonic populations enriched for proliferative, motor, and membrane remodeling programs and adult and aged populations enriched for adhesion, extracellular matrix, transport, and mechanosensory pathways. Endothelial subsets across stages expressed genes linked to flow sensing and calcium-dependent mechanotransduction, including Piezo1, Piezo2, and Trpv4. Consistent with these signatures, intact explants exhibited spontaneous, spatially graded calcium oscillations, and pharmacologic activation by Piezo1, triggered robust network-wide calcium responses in embryonic and adult tissue with distinct temporal dynamics. Piezo1 activation also promoted stabilization of PECAM1-associated endothelial adhesion under ex vivo flow conditions. Together, these findings establish the choroid plexus endothelium as a structurally specialized, developmentally dynamic, and mechanosensitive vascular network and provide a framework for investigating endothelial contributions to blood-cerebrospinal fluid barrier function in health and disease.

neuroscience↗

bioRxiv2026

Preferential vulnerability of cortical GABAergic interneurons to Nalcn deficiency

Neuronal resting membrane potential (RMP) reflects the balance of leak conductances and varies systematically across cortical cell types. Many GABAergic interneurons exhibit more depolarized RMPs than neighbouring excitatory neurons, but the molecular mechanisms underlying these systematic differences in intrinsic excitability remain incompletely defined. The sodium leak channel non-selective (NALCN) mediates a major fraction of basal sodium conductance and cause neurodevelopmental syndromes characterized by developmental delay and cognitive impairment. Here we define NALCN contribution to cortical circuit development and function with cell-type precision. Using Nalcn-GFP reporter line, we map Nalcn expression across cortical types revealing enrichment in GABAergic hippocampal interneurons relative to hippocampal pyramidal neurons. Using conditional mouse models, we selectively deleted Nalcn in cortical glutamatergic lineages or forebrain GABAergic cells from early embryogenesis. Electrophysiological analysis show that developmental loss of Nalcn preferentially reduces intrinsic excitability across GABAergic interneuron subtypes while sparing pyramidal neurons. To understand the impact of a depolarized GABAergic RMP on brain function, we assessed the behavioral performance of Nalcn-deficient mice that revealed persistent deficits in contextual adaptation and spatial short-term memory. These findings reveal a neuron type-specific function of NALCN in the cerebral cortex and position NALCN as a crucial ion channel regulating basal excitability of GABAergic inhibitory circuits and cortical circuit function.

neuroscience↗

bioRxiv2026

Biomimetic model for the identification of distinctive microenvironmental factors in glioblastoma radiosensitivity

Radiation therapy (RT) has long been included in the treatment of glioblastoma (GBM). However, radioresistance in cancer cells as well as toxicity in normal tissues are major obstacles to clinical efficacy. Improved understanding of the mechanisms of tumor microenvironment-induced radioresistance during and after radiation therapy can provide fundamental insights to improve clinical outcomes in GBM. Here, using three-dimensional engineered hydrogel models in vitro, we report the influence of extracellular matrix, hypoxia, and adjacent neuronal cells in radiotherapeutic sensitivity. We find that mechanical cues and oxygen availability regulate cellular response to radiation, with softer matrices allowing for more DNA damage. Hyaluronan fragments from the extracellular matrix also modulate rapid metabolic response to radiation, especially in hypoxic environments. We show that neuronal networks influence tumor metabolic activity and the inflammatory response. Overall, we demonstrate here that alternative radiation strategies, such as low dose rate radiation therapy and microenvironmental regulation, have the potential to be more effective in a specific subset of radiosensitive GBM tumors.

bioengineering↗

bioRxiv2025

Mapping CD4+ T Cell Landscapes in Glioblastoma Reveals Effectors and Bystanders

The tumor immune microenvironment (TME) has been demonstrated to significantly shape glioblastoma (GBM) progression and therapeutic response, yet the role of CD4+ T cells remains incompletely defined. Here, by integrating single-cell RNA, surface-protein, and TCR profiling of 39,788 T cells from 16 high-grade gliomas with five matched blood samples, we mapped 23,550 CD4+ T cells across 11 states and revealed predominance of CD4+ T cells in primary, but not recurrent, tumors, alongside pronounced tumor-blood discordance. Clonal analyses revealed expansion of cytotoxic effector CD4+ T cells within tumors and TEMRA CD4+ T cells in blood. A small set of dominant clonotypes (0.2% of unique TCR{beta} sequences) accounted for >6% of all CD4+ T cells across both compartments and were shared across different transcriptional subsets, suggesting diverse transcriptional development stemming from a shared progenitor. In contrast, virally annotated clonotypes were broadly dispersed and largely unexpanded, consistent with bystander populations. Collectively, we investigated the cellular and clonal architecture of human CD4+ T cells in GBM and highlight the contrast between PBMC and TIL compartments.

immunology↗

bioRxiv2024

Arteriolar degeneration and stiffness in cerebral amyloid angiopathy are linked to β-amyloid deposition and lysyl oxidase.

Cerebral amyloid angiopathy (CAA) is a vasculopathy characterized by vascular {beta}-amyloid (A{beta}) deposition on cerebral blood vessels. CAA is closely linked to Alzheimers disease (AD) and intracerebral hemorrhage. CAA is associated with the loss of autoregulation in the brain, vascular rupture, and cognitive decline. To assess morphological and molecular changes associated with the degeneration of penetrating arterioles in CAA, we analyzed post-mortem human brain tissue from 26 patients with mild, moderate, and severe CAA end neurological controls. The tissue was optically cleared for three-dimensional light sheet microscopy, and morphological features were quantified using surface volume rendering. We stained A{beta}, vascular smooth muscle (VSM), lysyl oxidase (LOX), and vascular markers to visualize the relationship between degenerative morphological features, including vascular dilation, dolichoectasia (variability in lumenal diameter) and tortuosity, and the volumes of VSM, A{beta}, and LOX in arterioles. Atomic force microscopy (AFM) was used to assess arteriolar wall stiffness, and we identified a pattern of morphological features associated with degenerating arterioles in the cortex. The volume of VSM associated with the arteriole was reduced by around 80% in arterioles with severe CAA and around 60% in cases with mild/moderate CAA. This loss of VSM correlated with increased arteriolar diameter and variability of diameter, suggesting VSM loss contributes to arteriolar laxity. These vascular morphological features correlated strongly with A{beta} deposits. At sites of microhemorrhage, A{beta} was consistently present, although the morphology of the deposits changed from the typical organized ring shape to sharply contoured shards with marked dilation of the vessel. AFM showed that arteriolar walls with CAA were more than 400% stiffer than those without CAA. Finally, we characterized the association of vascular degeneration with LOX, finding strong associations with VSM loss and vascular degeneration. These results show an association between vascular A{beta} deposition, microvascular degeneration, and increased vascular stiffness, likely due to the combined effects of replacement of VSM by {beta}-amyloid, cross-linking of extracellular matrices (ECM) by LOX, and possibly fibrosis. This advanced microscopic imaging study clarifies the association between A{beta} deposition and vascular fragility. Restoration of physiologic ECM properties in penetrating arteries may yield a novel therapeutic strategy for CAA.

neuroscience↗

bioRxiv2023

Comparative analysis of myoglobin in Cetaceans and humans reveals novel regulatory elements and evolutionary flexibility

Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles, especially those responsible for swimming. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke exercise, hypoxia, and other physiological pathways that impact gene regulation, and increase myoglobin gene expression. Here we explore an alternative hypothesis: that Cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, has a low level of expression, less than 10% that of humans. This low expression level is broadly shared among Cetaceans, and Artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5 of the AT element termed the "CCAC-box". Comparative analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3 of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5 of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. They also support the hypothesis that the induction of transcription by physiological signaling pathways, and evolved protein stability are important in leading to the high myoglobin protein levels found in Cetaceans.

molecular biology↗

bioRxiv2022

Sex-biased Yap1 oncogene function

The incidence of many human cancers differs according to sex, but little is known about the interplay between oncogenic events and sex as a variable in tumorigenesis. Here we report that the oncogene Yap1 is sexually dimorphic in medulloblastoma progression and immune suppression. We show that Yap1 promotes stemness and blocks differentiation in sonic hedgehog (SHH)-subtype medulloblastoma by at least two distinct but complementary molecular mechanisms to regulate the RNA expression and protein functions of Sox2, Atoh1, NeuroD1, and Zic1/2. Yap1 also promotes an immune suppressive tumor microenvironment by directly regulating Csf1, Igf1, and Igfbp3 transcription and modulating IL6-JAK-STAT3, TNFR1, TGF-{beta}, and CCL5 immune pathways. Notably, Yap1 function is more critical in males and this is evolutionarily conserved: genes downstream of YAP1 identified in mouse models stratify male but not female medulloblastoma patient survival. In summary, we demonstrate a sex-based function for an oncogene, underscoring the critical need to incorporate sex as a variable in cancer mechanism and clinical response studies, particularly those involving YAP1.

cancer biology↗

bioRxiv2021

RNA-Seq analysis and transcriptome assembly of Salicornia neei reveals powerful system for ammonium detoxification

BackgroundSalicornia neei is a halophyte plant that has been proposed for use in the phytoremediation of the saline wastewater generated by land-based aquaculture, which usually contains elevated concentrations of ammonium resulting from protein metabolism. To identify the molecular mechanisms related to ammonium response, we analyzed the transcriptome of S. neei in response to growth in saline water containing 3 mM ammonium and the Michaelis-Menten ammonium removal biokinetics. ResultsThe RNA sequencing generated a total of 14,680,108 paired-end reads from the control and stressed conditions. De novo assembly using the CLC Genomic Workbench produced 86,020 transcripts and a reference transcriptome with an N50 of 683 bp. A total of 45,327 genes were annotated, representing 51.2% of the contig predicted from de novo assembly. As regards DEGs, a total of 9,140 genes were differentially expressed in response to ammonium in saline water; of these, 7,396 could be annotated against functional databases. The upregulated genes were mainly involved in cell wall biosynthesis, transmembrane transport and antiporter activities, including biological KEGG pathways linked to the biosynthesis of secondary metabolites, plant hormone signal transduction, autophagy, and nitrogen metabolism. In addiction, a set of 72 genes was directly involved in ammonium metabolism, including glutamine synthetase 1 (GLN1), glutamate synthase 1 (GLT1), and ferredoxin-dependent glutamate synthase chloroplastic (Fd-GOGAT). Finally, we observed that the ammonium uptake rate increased with increasing ammonium concentrations, and tended toward saturation in the range of 3 to 4 mM. ConclusionOur results support the hypothesis that an ammonium detoxification system mediated by glutamine and glutamate synthase was activated in S. neei when exposed to ammonium and saline water. The present transcriptome profiling method could be useful when investigating the response of halophyte plants to saline wastewater from land-based aquaculture.

genomics↗

bioRxiv2020

Phylogenomic systematics of the spotted skunks (Carnivora, Mephitidae, Spilogale): Additional species diversity and Pleistocene climate change as a major driver of diversification

Four species of spotted skunks (Carnivora, Mephitidae, Spilogale) are currently recognized: Spilogale angustifrons, S. gracilis, S. putorius, and S. pygmaea. Understanding species boundaries within this group is critical for effective conservation given that regional populations or subspecies (e.g., S. p. interrupta) have experienced significant population declines. Further, there may be currently unrecognized diversity within this genus as some taxa (e.g., S. angustifrons) and geographic regions (e.g., Central America) never have been assessed using DNA sequence data. We analyzed species limits and diversification patterns in spotted skunks using multilocus nuclear (ultraconserved elements) and mitochondrial (whole mitogenomes and single gene analysis) data sets from broad geographic sampling representing all currently recognized species and subspecies. We found a high degree of genetic divergence among Spilogale that reflects seven distinct species and eight unique mitochondrial lineages. Initial divergence between S. pygmaea and all other Spilogale occurred 29 in the Early Pliocene (~ 5.0 million years ago) which was followed by subsequent diversification of the remaining Spilogale into an "eastern" and "western" lineage during the Early Pleistocene (~1.5 million years ago). These two lineages experienced temporally coincident patterns of diversification at ~0.66 and ~0.35 million years ago into two and ultimately three distinct evolutionary units, respectively. Diversification was confined almost entirely within the Pleistocene during a timeframe characterized by alternating glacial-interglacial cycles, with the origin of this diversity occurring in northeastern Mexico and the southwestern United States of America. Mitochondrial-nuclear discordance was recovered across three lineages in geographic regions consistent with secondary contact, including a distinct mitochondrial lineage confined to the Sonoran Desert. Our results have direct consequences for conservation of threatened populations, or species, as well as for our understanding of the evolution of delayed implantation in this enigmatic group of small carnivores.

evolutionary biology↗

bioRxiv2019

Rapidly processed stool swabs approximate stool microbiota profiles

Studies of the intestinal microbiome commonly utilize stool samples to measure microbial composition in the distal gut. However, collection of stool can be difficult from some subjects under certain experimental conditions. In this study we validate the use of swabs of fecal matter to approximate measurements of microbiota in stool using 16S rRNA gene Illumina amplicon sequencing, and evaluate the effects of shipping time at ambient temperatures on accuracy. Results indicate that swab samples reliably replicate stool microbiota bacterial composition, alpha diversity, and beta diversity when swabs are processed quickly (< 2 days), but sample quality quickly degrades after this period, accompanied by increased abundances of Enterobacteriaceae. Fresh swabs appear to be a viable alternative to stool sampling when standard collection methods are challenging, but extended exposure to ambient temperatures prior to processing threatens sample integrity.

microbiology↗