Biology subjects

Romero, P.

Publications and source records attributed to Romero, P..

12 recordsLinked to original sources

bioRxiv2026

Dual profiling of DNA modifications with enhancer features during the exit of naive pluripotency

Cis-regulatory elements, such as enhancers, play an essential role in coordinating gene expression programs during cellular transitions. As such, substantial efforts have been made to characterize enhancer elements, e.g., chromatin accessibility, transcription factor (TF) binding sites, histone post-translational modifications (PTMs), and 5-cytosine DNA methylation and hydroxymethylation (5mC and 5hmC). Elevated 5mC levels are typically correlated with the inactive enhancer state. However, whether 5mC precludes TF binding or is simply a downstream consequence of enhancer decommissioning is difficult to determine. Bulk genomics assays from cell populations fail to fully capture cellular heterogeneity, and single-cell assays often suffer from limited read counts per cell. Here, we leveraged tagmentation-based technologies to assess chromatin features with 5mC simultaneously: Methyl-ATAC and Methyl-CUT&Tag. In addition, we modified the technique to interrogate 5hmC dynamics (hM-ATAC). We employed these techniques during the exit of naive pluripotency in mouse embryonic stem cells (mESCs), which recapitulates the embryonic DNA methylation establishment program. This system withstands the complete absence of DNA methylation and demethylation machinery, allowing us to further dissect the temporal contributions of 5mC and 5hmC. Given the affordability of these techniques, we were able to obtain robust, dynamic single-molecule 5mC/5hmC information during this cellular transition for chromatin accessibility and histone marks in wild-type and mutant conditions. The sum of these data allowed for unprecedented insight into the role that 5mC turnover plays at enhancers during a key window of mammalian embryonic development.

Molecular Biology↗

bioRxiv2026

KLRG1 identifies circulating cytotoxic CD4 T cells with selective anti-tumor function in human cancer

Given the critical role of CD4 T cells in anti-tumor immunity, strategies to harness these cells for cancer immunotherapy are gaining increasing interest. Historically overshadowed by CD8 T cells, cytotoxic CD4 T cells can directly kill MHC class II-expressing tumor cells. However, the defining molecular signature and the mechanisms underlying their cytolytic activity remain poorly understood, particularly in cancer patients. Here, using ex vivo single-cell transcriptomic and spatial analyses of CD4 T cells from paired blood and tumor samples of melanoma patients, we identified Killer Cell Lectin-Like Receptor G1 (KLRG1) as a defining surface marker of cytotoxic CD4 T cells. The CD4+ KLRG1+ T cell subset was notably enriched among circulating cells compared with tumor-infiltrating populations, which were instead enriched in T follicular helper (Tfh) states. Functionally, KLRG1+ CD4 T cells expressed elevated levels of cytotoxic genes and exhibited superior tumor-killing capacity compared with their KLRG1- counterparts. We demonstrated that their cytotoxicity is granulysin-dependent, as confirmed by CRISPR/Cas9-mediated gene deletion. Mechanistically, CD4 T cells spared MHC class II+ cells lacking the KLRG1 ligands CD324 and CD325, such as professional antigen-presenting cells (APCs), indicating that cytotoxicity was selectively directed towards tumor cells while preserving immune cells. Finally, by investigating how the tumor microenvironment may impair CD4 T cell cytotoxicity, we showed that tumor-derived factors, including interleukin-6 (IL-6), are key drivers promoting the transition of cytotoxic CD4 T cells toward a Tfh phenotype. In summary, our findings define KLRG1 as a defining cell surface marker of cytotoxic CD4 T cells in cancer patients, as well as a key regulator that protects MHC class II+ APCs. Moreover, targeting the IL-6 signalling pathway may enhance CD4 T cell anti-tumor cytotoxicity, offering new avenues for cancer immunotherapy.

immunology↗

bioRxiv2026

Hypophosphite is a naturally-occurring selective inhibitor of syntrophic methanogenesis

Microbial methanogenesis is a major contributor to global warming and methane fluxes represent a loss of energy and electrons from industrial ecosystems. The chemical space of methane control strategies is still under-explored. Most known methanogenesis inhibitors target methanogenic archaeal enzymes. However, interference with syntrophic electron exchange in methanogenic systems presents an additional target for methane control. Here we show that hypophosphite (H2PO2-), an inorganic formate analog, is a potent and selective inhibitor of syntrophic methanogenesis versus primary fermentation in rice field sediments and cattle rumens. Hypophosphite is also generally recognized as safe and relatively non-toxic to plants and animals. Genetic screens and physiological assays in the model methanogen Methanococcus maripaludis S2 implicate formate metabolism as the target of hypophosphite inhibition. Currently, there is no known biological pathway for anaerobic hypophosphite oxidation and hypophosphite is stable in anoxic sediments for weeks to months. Given its widespread natural occurrence, we propose that hypophosphite may modulate carbon cycling in natural environments. Taken together, our results suggest that hypophosphite could be used as a safe, inexpensive, strategy for methane control in syntrophic methanogenic ecosystems.

microbiology↗

bioRxiv2025

Purinergic receptor activation rectifies autism-associated endothelial dysfunction

Early cerebrovascular alterations affect brain maturation by impacting trophic support and energy supply. Recent evidence in a 16p11.2 deletion mouse model of autism spectrum disorder (ASD) revealed brain endothelial abnormalities postnatally. Yet, the endothelial alterations eliciting these changes remain unknown. Isolation of brain endothelial cells (ECs) from 14-day old male 16p11.2-deficient and wild-type mice revealed that 16p11.2 deletion-induced endothelial dysfunction is linked to a bioenergetic failure, with reduced intracellular ATP. Intra- or extra-cellular ATP supplementation rescued the function of 16p11.2-deficient ECs in vitro via P2 purinergic receptor activation, specifically P2Y2 receptors. Activating P2Y2 receptors restored cerebrovascular reactivity in 16p11.2-deficient parenchymal arterioles ex vivo and rescued 16p11.2 deletion-associated mouse behaviors. Taken together, this study demonstrates that metabolic reprogramming of brain ECs via purinergic receptor engagement represents a possible therapeutic avenue for ASD.

cell biology↗

bioRxiv2025

Multiobjective learning and design of bacteriophage specificity

To better understand and design proteins, it is crucial to consider the multifunctional landscapes on which all proteins exist. Proteins are often optimized for single functions during design and engineering, without considering the countless other functionalities that may contribute to or interfere with the intended outcome. In this work, we apply deep learning to understand and design the multifunctional host-targeting landscape of the T7 bacteriophage receptor binding protein for enhanced infectivity, pre-defined specificity, and high generality in virulence toward unseen strains. We compare several different model architectures and design approaches and experimentally characterize designed phages optimized for 26 diverse tasks. We demonstrate that with multiobjective machine learning, it is possible to design complex specificities at success rates that can enable low-throughput validation of predicted hits. Our results show that the targeting capabilities of T7 are highly plastic, with opposite specificities often separated by only a few mutations. This level of tunability underscores how models trained on multifunctional data can uncover key principles of phage biology and specificity. The same modeling framework can be applied to guide the multiobjective design of other proteins or mutable biological systems, offering a general strategy for navigating multifunctional landscapes.

synthetic biology↗

bioRxiv2024

Red seaweed supplementation suppresses methanogenesis in the rumen, revealing potentially advantageous traits among hydrogenotrophic bacteria

Macroalgae belonging to the genus Asparagopsis have been shown to reduce the production of methane (CH4) during rumen fermentation, while increasing feed efficiency when added to the feed of cattle. However, little is known about how the microbial community in the rumen responds to Asparagopsis supplementation, and how changes in the rumen microbiome may contribute to shifts in rumen function and ultimately the hosts phenotype. In this study, we generated and analyzed metagenomic and metatranscriptomic data from the microbiome associated with rumen fluid collected from two cohorts of lactating dairy cows, one fed a diet supplemented with Asparagopsis armata (treatment) and another fed the same diet without A. armata supplementation (control). The reduction of CH4 emission from animals that received A. armata was coupled to a qualitative decrease in relative archaeal abundance and a significant reduction in the transcription of methanogenesis pathways. Additionally, a significant decrease in the transcription of genes for complex carbon catabolism and a re-organization of the expression profile of carbon catabolic genes at the species level was observed in treated animals. Increased H2 production, a consequence of methanogenesis suppression, was coupled to a significant increase in the transcription of hydrogenases that mediate hydrogenotrophic metabolism in the treatment group. Analysis of metatranscriptome data identified a single uncultured hydrogenotrophic bacterial species (a Duodenibacillus sp.) as the dominant driver of this transcriptional change. Comparative genomic analysis between the Duodenibacillus sp. and other hydrogenotrophic rumen organisms revealed metabolic traits in the Duodenibacillus that may provide a competitive advantage in H2 scavenging. These findings provide an initial understanding of how rumen microbiota respond to a promising CH4 reducing feed additive, and may serve as a model to understand alternative stable rumen microbiome states that produce less methane and increase animal productivity.

microbiology↗

bioRxiv2024

Biosynthesis of oxyresveratrol in mulberry (Morus alba L.) is mediated by a group of p-coumaroyl-CoA 2'-hydroxylases acting upstream of stilbene synthases

Mulberry (Morus alba L.) is considered a millenary medicinal plant and a food source for silkworms. Different M. alba extracts offer a variety of biological and pharmacological properties that are in part attributed to stilbenoids, a small group of phenylpropanoids that include resveratrol and oxyresveratrol. These are naturally present in non-renewable parts of mulberry trees, impeding their efficient extraction. As a way to bypass this spatiotemporal restriction, we generated cell suspensions from mulberry twigs and demonstrated that the combined use of methyl jasmonate and methyl- or hydroxypropyl-{beta}-cyclodextrins elicited a high production of resveratrol and oxyresveratrol, both intra and extracellularly. To identify oxyresveratrol-producing enzymes (unknown to date), we first improved the structural and functional annotation of the mulberry genome by integrating short and long-read sequencing data. We further combined this data with transcriptome, metabolite and proteome time-series evidence to identify a complete set of elicited phenylpropanoid- and stilbenoid-related genes. These included 22 stilbene synthase (STS) genes and a group of six p-coumaroyl-CoA 2-hydroxylases (C2Hs) that were highly co-expressed with resveratrol and oxyresveratrol accumulation. We transiently transformed Nicotiana benthamiana plants and grapevine (Vitis vinifera L.) cell suspensions to functionally validate the role of C2Hs as the first committed step of oxyresveratrol synthesis, providing an alternative substrate for STSs by hydroxylating p-coumaroyl-coA into 24-dihydroxycinnamoyl-CoA. We offer tools for genomic and transcriptomic exploration in the context of jasmonate elicitation aiding in the characterization of novel stilbenoid-modifying and regulatory genes in the Morus genus.

molecular biology↗

bioRxiv2023

Estrogen regulates myogenic tone in hippocampal arterioles by enhanced basal release of nitric oxide and endothelial SKCa channel activity

Arteries and arterioles exhibit myogenic tone, a partially constricted state that allows further constriction or dilation in response to moment-to-moment fluctuations in blood pressure. The vascular endothelium that lines the internal surface of all blood vessels controls a wide variety of essential functions, including the contractility of the adjacent smooth muscle cells by providing a tonic vasodilatory influence. Studies conducted on large (pial) arteries on the surface of the brain have shown that estrogen lowers myogenic tone in female mice by enhancing nitric oxide (NO) release from the endothelium, however, whether this difference extends to the intracerebral microcirculation remains ambiguous. The existing incomplete picture of sex differences in cerebrovascular physiology combined with a deficiency in treatments that fully restore cognitive function after cerebrovascular accidents places heavy emphasis on the necessity to investigate myogenic tone regulation in the microcirculation from both male and female mice. We hypothesized that sex-linked hormone regulation of myogenic tone extends its influence on the microcirculation level, and sought to characterize it in isolated arterioles from the hippocampus, a major cognitive brain area. Using diameter measurements both in vivo (acute cranial window vascular diameter) and ex vivo (pressure myography experiments), we measured lower myogenic tone responses in hippocampal arterioles from female than male mice. By using a combined surgical and pharmacological approach, we found myogenic tone in ovariectomized (OVX) female mice matches that of males, as well as in endothelium-denuded arterioles. Interestingly, eNOS inhibition induced a larger constriction in female arterioles but only partially abolished the difference in tone. We identified that the remnant difference was mediated by a higher activity and expression of the small-conductance Ca2+-sensitive K+ (SK) channels. Collectively, these data indicate that eNOS and SK channels exert greater vasodilatory influence over myogenic tone in female mice at physiological pressures.

neuroscience↗

bioRxiv2023

EZH2 mutations in follicular lymphoma distort H3K27me3 profiles and alter transcriptional responses to PRC2 inhibition

Mutations in chromatin regulators or their histone substrates are widespread in cancer and often play decisive roles in tumorigenesis. These include Polycomb Repressive Complex 2 (PRC2), a histone H3 lysine 27 methyltransferase that shows distinct alterations in each of a range of tumor types. Mechanistically, this tumor-type specificity is poorly understood. Here, we model several of these alterations in a single isogenic system in order to reveal their comparative impacts on chromatin and transcription. Focusing then on gain-of-function substitutions in catalytic subunit EZH2, which occur in [~]25% of follicular lymphomas, we show that Ezh2Y641F induces aberrant H3K27 methylation patterns even without wild-type Ezh2, and that these are alleviated by partial PRC2 inhibition. Ezh2Y641F also causes gains in existing H3K27 acetylation peaks and extensive gene expression changes. Remarkably, Ezh2Y641F transforms the transcriptomic response to PRC2 inhibition, leading notably to the induction of antigen presentation genes in mutant cells. Using a unique longitudinal cohort of FL patient samples we further strengthen the link between EZH2 mutation status and abnormal H3K27 methylation. This analysis also uncovered unexpected variability in the mutational landscape of successive biopsies from the same patient that points to the frequent co-existence of different clones. On a clinical level, this urges caution when stratifying patients based on single tumor sampling. Altogether, our results provide a mechanistic foundation for understanding how oncogenic PRC2 mutations disrupt chromatin and transcription, and the therapeutic vulnerabilities this creates.

cancer biology↗

bioRxiv2022

NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer

PD-1/PD-L1-blockade immunotherapies have limited efficacy in the treatment of muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Here, we show that KLRC1 (NKG2A) expression associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in CD8Ahigh bladder tumors. The loss of antigen presentation is a common mechanism for tumor escape in bladder cancer. NKG2A+ CD8 T cells are able to circumvent HLA-ABC loss through TCR-independent cytotoxicity, which is partly mediated by DNAM-1. In bladder tumors, NKG2A is acquired on a subset of PD-1+ CD8 T cells, alongside stronger tissue-residency memory features, TCR-independent cytotoxicity and evidence of recent proliferation. HLA-E is low but variably expressed on bladder tumors. When expressed, NKG2A+ CD8 T cell anti-tumor responses to HLA-ABC-deficient tumors are inhibited and partly restored upon NKG2A blockade. Overall, our study identifies an alternative path for CD8 T cell exhaustion, that is mediated by NKG2A upregulation and TCR-independent cytotoxicity.

immunology↗

bioRxiv2022

Orthogonal Gene Engineering Enables CD8+ T Cells to Control Tumors through a Novel PD-1+ TOX-indifferent Synthetic Effector State

Adoptive immunotherapy offers opportunities to reprogram T cells and the tumor microenvironment. Orthogonal engineering of adoptively transferred T cells with an IL-2R{beta}{gamma}-binding IL-2 variant, PD1-decoy and IL-33 led to cell-autonomous T-cell expansion, T-cell engraftment and tumor control in immunocompetent hosts through reprogramming of both transferred and endogenous CD8+ cells. Tumor-infiltrating lymphocytes adopted a novel effector state characterized by TOX suppression and specific expression of multiple effector molecules, most prominently granzyme C. While the IL-2 variant promoted CD8+ T-cell stemness and persistence, and was associated with downregulation of TOX, the combination with IL-33 was necessary to trigger the novel polyfunctional effector state. Rational T-cell engineering without host lymphodepletion enables optimal reprogramming of adoptively transferred T cells as well as mobilization of endogenous immunity into new functional CD8+ states mediating tumor control.

immunology↗

bioRxiv2020

Lack of mucosal cholinergic innervation is associated with increased IL-23-induced T-helper-17 response in Hirschsprung disease

Background & AimsHirschsprung’s disease (HSCR) is a congenital intestinal motility disorder defined by the absence of enteric nervous cells (ganglia). The development of HSCR-associated enterocolitis remains a life-threatening complication. Absence of enteric ganglia implicates extramural innervation of acetylcholine-secreting (cholinergic) nerve fibers. Cholinergic signals have been reported to control excessive inflammation, but the impact on HSCR-associated enterocolitis is unknown.Methods We enrolled 44 HSCR patients in a prospective multicenter study and grouped them according to their degree of colonic mucosal cholinergic innervation using immunohistochemistry. The fiber phenotype was correlated with the tissue cytokine profile as well as immune cell frequencies using quantitative reverse-transcribed real-time polymerase chain reaction (qRT-PCR) of whole colonic tissue and fluorescence-activated cell sorting (FACS) analysis of isolated colonic immune cells. Fiber-associated immune cells were identified using confocal immunofluorescence microscopy and characterized by RNA-seq analysis. Microbial dysbiosis was analyzed in colonic patient tissue using 16S rDNA gene sequencing. Finally, the fiber phenotype was correlated with postoperative enterocolitis manifestation.Results We provided evidence that extrinsic mucosal innervation correlated with reduced interleukin (IL)-17 cytokine levels and T-helper-17 (Th17) cell frequencies. Bipolar CD14high macrophages colocalized with neurons and expressed significantly less interleukin-23, a Th17-promoting cytokine. HSCR patients lacking mucosal cholinergic nerve fibers showed microbial dysbiosis and had a higher incidence of postoperative enterocolitis.Conclusion The mucosal fiber phenotype might serve as a new prognostic marker for enterocolitis development in HSCR patients and may offer an approach to personalized patient care and new future therapeutic options. (www.clinicaltrials.gov accessing number NCT03617640)Competing Interest StatementThe authors have declared no competing interest.Abbrevations used in this paperHSCRHirschsprung’s diseaseFACSFluorescence-activated cell sortingqRT-PCRQuantitative reverse-transcribed real-time polymerase chain reactionILInterleukinTh17T-helper-17ENSEnteric nervous systemAChAcetylcholineα7nAChRα7-nicotinic ACh receptorCAIPCholinergic-anti-inflammatory-pathwayVNVagus nerveMΦMacrophagesMMΦMuscle macrophagesTNFtumor necrosis factorPOIPostoperative ileusCDColon descendensRECRectumSCSigmoid colonVIPVasoactive intestinal peptideTHTyrosine hydroxylaseNOSNitric oxide synthaseNKNatural killerNCRNK cell receptorsAPCAntigen-presenting cellPCAPrincipal component analysisHLAHuman leucocyte antigenGDFGrowth/differentiation factorNRP1Neuropilin-1UNC5BUNC-5 homology BEPHB2Ephrin type B-receptor 2SEMASemaphorinLPLamina propriaFISHFluorescence in situ hybridizationOTUsOperational taxonomic unitsBMPBone morphogenetic proteinTLRToll-like receptorView Full Text

immunology↗