Biology subjects

Ayala, J.

Publications and source records attributed to Ayala, J..

5 recordsLinked to original sources

bioRxiv2025

Toll-like Receptor 4 Contributes to PCOS-like Metabolic and Reproductive Pathogenesis

Polycystic ovary syndrome (PCOS) is a reproductive disorder with heterogeneous symptoms and severity. Despite extensive research documenting chronic immune dysfunction as a hallmark of PCOS, the specific molecular mechanisms driving immune activation and its connection to the syndromes diverse symptoms remain poorly understood. Emerging evidence suggests that gut-derived bacterial endotoxins, particularly lipopolysaccharide (LPS), may breach the intestinal barriers in PCOS patients and trigger systemic inflammation through Toll-like receptor 4 (TLR4), a pattern recognition receptor of the innate immune system. This study investigated whether TLR4 serves as a critical mechanistic driver of PCOS pathogenesis by examining the effect of genetic TLR4 knockout (TLR4-/-) in a letrozole (LET)-induced mouse model of PCOS. Our results demonstrate that TLR4 deficiency reduces many PCOS-like symptoms, including elevated luteinizing hormone, anovulation, and metabolic dysfunction. TLR4 knockout also preserved estrous cycling and fertility, improved glucose tolerance, maintained gut barrier integrity, and reduced inflammatory markers in LET-treated females. These findings establish TLR4 as a key mediator orchestrating PCOSs multi-system pathology, positioning TLR4 as a critical convergence point rather than affecting individual symptoms in isolation. This novel work reveals that TLR4-mediated inflammation drives multiple PCOS pathologies, opening avenues for targeted anti-inflammatory treatments in women with this disorder. Significance StatementPolycystic ovary syndrome (PCOS) affects up to 15% of reproductive-age women worldwide. This study reveals that TLR4, an innate immune receptor, is key to the pathophysiology of PCOS-like symptoms in female mice. When TLR4 was genetically deleted, mice treated with letrozole to induce PCOS-like symptoms maintained normal weight, glucose regulation, estrous cycling, and fertility. The improvements coincided with preserved gut barrier breakdown and reduced inflammation. These findings identify TLR4 as a key mediator between gut health, immune activation, and PCOS pathophysiology, suggesting that targeting TLR4 could offer new therapeutic approaches for this common but poorly understood syndrome affecting millions of women.

immunology↗

bioRxiv2024

Termination sequence between an inducible promoter and ubiquitous chromatin opening element (UCOE) reduces gene expression leakage and silencing

AbstractInducible gene expression circuits offer precise control over target gene activation, making them essential tools for direct reprogramming, where cells are guided to differentiate into specific cell types. However, stable circuit function and consistent expression of key inducible proteins are crucial for effective reprogramming, and DNA methylation-induced silencing hinders this stability. To address this issue, A2-ubiquitous chromatin opening elements (A2UCOE) have gained attention for their ability to maintain open chromatin and prevent methylation, thereby stabilizing gene expression. In this study, we aimed to forward program iPSCs into thymic epithelial cells (TECs) using a compact, all-in-one gene circuit composed of a doxycycline-inducible Tet-On system, 863-bp A2UCOE (0.9 UCOE), and FOXN1, a master transcription factor for TEC differentiation. This compact construct enables site-specific genome integration and stable inducible expression within iPSCs for renewably generating TECs. While the 0.9 UCOE promoted stable expression of constitutively expressed genes, it also caused unintended FOXN1 gene leakage, leading to unprogrammed gradual differentiation of the iPSCs. We generated A2UCOE fragments of varying lengths and found that gene leakage persisted regardless of fragment size. We tested spacer sequences between the A2UCOE and the Tet-On promoter consisting of varying AT-nucleotide content (35, 50, and 65%) and the 65% AT-rich SV40 poly-A terminator sequence and found that only the SV40 poly-A mitigated this leakage, and surprisingly, enhanced desired anti-silencing effects. These findings highlight the benefits and potential risks of using A2UCOE in iPSCs, and provides insights into a regulatory DNA architecture optimal for compact forward programming circuits for controlled iPSC differentiation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/629244v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@8398d7org.highwire.dtl.DTLVardef@1a75beeorg.highwire.dtl.DTLVardef@84767eorg.highwire.dtl.DTLVardef@7f5cec_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

The Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy

Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

pathology↗

bioRxiv2023

Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor

Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae, the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, {beta}-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.

biochemistry↗

bioRxiv2021

Open-Access STRS Database Of Populations From The 1000 Genomes Project Using High Coverage Phase 3 Data

Accurate STR genotyping from next-generation sequencing (NGS) data has been challenging. Haplotype inference and phasing for STRs (HipSTR) was specifically developed to deal with genotyping errors and obtain reliable STR genotypes from whole-genome sequencing datasets. The objective of this investigation was to perform a comprehensive genotyping analysis of a set of STRs of broad forensic interest from the 1000 Genomes populations and release a reliable open-access STR database to the forensic genetics community. A set of 22 STR markers were analyzed using the CRAM files of the 1000 Genomes Project Phase 3 high-coverage (30x) dataset generated by the New York Genome Center (NYGC). HipSTR was used to call genotypes from 2,504 samples from 26 populations organized into five groups: African, East Asian, European, South Asian, and admixed American. The D21S11 marker could not be detected in the present study. Moreover, the Hardy-Weinberg equilibrium analysis, coupled with a comprehensive analysis of allele frequencies, revealed that HipSTR could not identify longer Penta E (and Penta D at a lesser extent) alleles. This issue is probably due to the limited length of sequencing reads available for genotype calling, resulting in heterozygote deficiency. Notwithstanding that, AMOVA, a clustering analysis using STRUCTURE, and a Principal Coordinates Analysis revealed a clear-cut separation between the four major ancestries sampled by the 1000 Genomes Consortium (AFR, EUR, EAS, SAS). Meanwhile, the AMOVA results corroborated previous reports that most of the variance is (97.12%) observed within populations. This set of analyses revealed that except for larger Penta D and Penta E alleles, allele frequencies and genotypes defined by HipSTR from the 1000 Genomes Project phase 3 data and offered as an open-access database are consistent and highly reliable.

genetics↗