Biology subjects

Ruiz, R.

Publications and source records attributed to Ruiz, R..

12 recordsLinked to original sources

bioRxiv2025

Metabolic and transcriptional adaptations to phagocytosis in microglia sustain their functionality and regenerative properties

Phagocytosis of apoptotic cells, or efferocytosis, is a tightly regulated process that ensures tissue homeostasis and prevents mounting inflammatory responses. In the brain parenchyma, it is executed by microglia, which are encumbered by large numbers of apoptotic debris generated during development, in adult neurogenic niches, aging, and brain diseases. Emerging evidence suggest that phagocytosis is not limited to garbage disposal, but triggers adaptations in the phagocytes that may have a functional impact. To test it we developed an in vivo model of superphagocytosis induced by low cranial irradiation (LCI, 2Gy) that specifically induced apoptosis in the neurogenic niche of the adult hippocampus, synchronizing microglia in a phagocytic state within 6h and leading to full clearance by 24h. Single cell RNA sequencing and metabolomics revealed an unexpected oxidative stress in post-phagocytic microglia, accompanied by catabolic shutdown, mitochondrial remodeling, increased expression of galectin 3, and production of polyamines that led to cell death and compensatory proliferation. To test whether these changes impaired subsequent microglial phagocytosis, we used a glioblastoma model treated with sequential irradiation to induce tumor cell apoptosis. The phagocytosis efficiency of tumor-associated microglia/macrophages was comparable in the first and second apoptotic challenge, suggesting that the metabolic remodeling induced by phagocytosis was adaptive and destined to sustain their functionality. Finally, we assessed the functional impact of post-phagocytosis adaptations using galectin 3 deficient mice under LCI. We found that the recovery of the neurogenic niche after LCI strongly depended on galectin 3, demonstrating the regenerative capacity of post-phagocytic microglia. Overall, our data unveils the complexity of post-phagocytosis adaptations in microglia, underscoring their unexplored therapeutic potential in brain disorders.

neuroscience↗

bioRxiv2025

Remodeling-mediated changes in left ventricular mechanics under settings of chronic pressure overload and exercise

Left ventricular (LV) remodeling, whether occurring as a part of somatic growth or as a chronic response to a sustained stimulus, is a primary factor underlying cardiac mechanical function. Although LV remodeling is a complex process that can be described at several levels (i.e. biochemical, cellular, tissue, organ, system), response variables that govern cardiac mechanics include changes in LV wall and chamber geometry, the mechanical properties of the LV myocardium, and functionally-deterministic LV structural mechanical properties such as LV chamber stiffness. In this study, we leverage two-dimensional speckle-tracking echocardiography (STE) to serially monitor key LV remodeling response variables in porcine models of LV pressure overload (LVPO), chronic exercise (CE), and the superposition of both settings (CE+LVPO), and compare changes to those occurring in age-matched referent control (RC) animals. Our findings show that over a 28-day study period, LVPO and CE both induce hypertrophy in comparison to RC, but passive LV myocardial stiffness increases with the former and decreases with the latter. As a net effect of geometrical and mechanical property changes, these settings induce divergent changes in LV chamber stiffness, namely an elevation with LVPO and reduction with CE. In the CE+LVPO cohort, exercise was found to attenuate the LVPO-induced increase in LV myocardial and LV chamber stiffnesses and insomuch supports its continued integration and refinement as a cardiac rehabilitation therapy. Data obtained across study cohorts were used to identify a phenomenological model of LV chamber stiffness, providing the first explicit relation of LV geometry and myocardial mechanical properties to a key LV structural property. Additional data processing was performed to develop a predictive mathematical model of late changes in LV chamber stiffness based on early remodeling response variables irrespective of stimulus, suggesting that STE can be extended to predict cardiac disease risk/progression in certain patient populations.

bioengineering↗

bioRxiv2025

Widespread and Abundant CRISPR-Cas Systems in the Deep Ocean

CRISPR-Cas systems have revolutionized modern biology. Most CRISPR-Cas systems in use for biotechnological applications derive from cultivated bacteria, but evidence suggests that environmental microbiomes harbor a large untapped diversity of these systems. Yet, our understanding of which environmental and biological factors drive the prevalence of CRISPR-Cas systems in the oceans remains limited. A search for CRISPR-Cas systems was conducted among 176 globally-distributed marine microbial metagenomes from the Malaspina expedition, which sampled both free-living and particle-attached microbiomes with emphasis on the deep ocean. We show that CRISPR-Cas systems are proportionally more abundant among microbiomes from the deep ocean than in the photic layers and among free-living microbes compared to those attached to particles, reflecting the higher concentrations of archaea and their viruses in these habitats. We identified 1,146 CRISPR-cas loci, some of which displayed unique loci architectures. From these loci, a total of 48 Cas9 proteins were identified, many of which are potentially novel. These discoveries expand the scope of CRISPR-Cas diversity and point at the deep-sea as a rich reservoir of these resources, which helps guide future bioprospecting efforts.

microbiology↗

bioRxiv2025

CRISPR-Cas9 trans-cleavage is hindered by a closed R-loop, an elongated spacer, and an inactive HNH domain

Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key structural determinants underlying this function. First, we show that open R-loops in the PAM-distal region favor trans-cleavage activity, which occur when targeting short double-stranded or single-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impairs this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Structural analyses of domain rearrangements provide mechanistic insight. Together, these findings illustrate a fine modulation of Cas9 function.

biochemistry↗

bioRxiv2025

Aetokthonotoxin, the causative agent of vacuolar myelinopathy, uncouples oxidative phosphorylation due to protonophore activity

Aetokthonotoxin (AETX) is an emerging environmental toxin produced by the freshwater cyanobacterium Aetokthonos hydrillicola. Accumulating in the food chain, it causes vacuolar myelinopathy, a neurological disease affecting a wide range of wildlife characterized by the development of large intra-myelinic vacuoles in the white matter of the brain. So far, the mode of action of AETX is unknown. After discovering that AETX is cytostatic and arrests cancer cell lines in G1-phase, metabolomic profiling of AETX-treated cells as well as an assessment of the physico-chemical properties of the compound suggested that AETX is a weakly acidic uncoupler of mitochondrial respiration. We confirmed this hypothesis by in vitro assays on mammalian cells, finding that AETX has the expected effects on the mitochondrial network morphology, mitochondrial membrane potential, and oxygen consumption rates, resulting in affected ATP generation. We confirmed that AETX is capable of transporting protons across lipid bilayers. In summary, we demonstrate that AETX is a protonophore that uncouples oxidative phosphorylation in mitochondria, the primary event of AETX intoxication. Significance statementAetokthonotoxin (AETX) is an emerging cyanotoxin. Produced by the cyanobacterium Aetokthonos hydrillicola, it is transferred through the food chain, affects the nervous system, and eventually causes mortality in animals of various taxa. Our finding that AETX is an unspecific uncoupler of mitochondrial respiration implies that it might also be harmful for human health upon ingestion and trophic accumulation. First steps towards a full risk assessment are needed. An important aspect in this regard is the elucidation of the toxins mode of action. We anticipate our findings to be a starting point for the development of an adverse outcome pathway addressing the formation of vacuolar myelinopathy, expanding the significance of our results to the future risk assessment of other environmental neurotoxins.

pharmacology and toxicology↗

bioRxiv2024

Drug screening targeting TREM2-TYROBP transmembrane binding

TREM2 encodes a microglial membrane receptor involved in the disease-associated microglia (DAM) phenotype whose activation requires the transmembrane interaction with TYROBP. Mutations in TREM2 represent a high-impact risk factor for Alzheimers disease (AD) which turned TREM2 into a significant drug target. We present a bacterial two-hybrid (B2H) system designed for high-throughput screening of modulators for the TREM2-TYROBP transmembrane interaction. In a pilot study, 315 FDA-approved drugs were analyzed to identify potential binding modifiers. Our pipeline includes multiple filtering steps to ensure candidate specificity. The screening suggested two potential candidates that were finally assayed in the human microglial cell line HMC3. Upon stimulation with anti-TREM2 mAb, pSYK/SYK ratios were calculated in the presence of the candidates. As a result, we found that varenicline, a smoking cessation medication, can be considered as a transmembrane agonist of the TREM2-TYROBP interaction.

molecular biology↗

bioRxiv2022

Repurposing the mammalian RNA-binding protein Musashi-1 as an allosteric translation repressor in bacteria

The RNA recognition motif (RRM) is the most common RNA-binding protein domain identified in nature. However, RRM-containing proteins are only prevalent in eukaryotic phyla, in which they play central regulatory roles. Here, we engineered an orthogonal post-transcriptional control system of gene expression in the bacterium Escherichia coli with the mammalian RNA-binding protein Musashi-1, which is a stem cell marker with neurodevelopmental role that contains two canonical RRMs. In the circuit, Musashi-1 is regulated transcriptionally and works as an allosteric translation repressor thanks to a specific interaction with the N-terminal coding region of a messenger RNA and its structural plasticity to respond to fatty acids. We fully characterized the genetic system at the population and single-cell levels showing a significant fold change in reporter expression, and the underlying molecular mechanism by assessing the in vitro binding kinetics and in vivo functionality of a series of RNA mutants. The dynamic response of the system was well recapitulated by a bottom-up mathematical model. Moreover, we applied the post-transcriptional mechanism engineered with Musashi-1 to specifically regulate a gene within an operon, to implement combinatorial regulation, and to reduce protein expression noise. This work illustrates how RRM-based regulation can be adapted to simple organisms, thereby adding a new regulatory layer in prokaryotes for translation control.

synthetic biology↗

bioRxiv2022

Identification of the EH CRISPR-Cas9 system on a metagenome and its application to genome engineering

Non-coding RNAs (crRNAs) produced from clustered regularly interspaced short palindromic repeats (CRISPR) loci, and CRISPR associated (Cas) proteins of the prokaryotic CRISPR-Cas systems, form complexes that interfere with the spread of transmissible genetic elements through Cas-catalysed cleavage of foreign genetic material matching the guide crRNA sequences. The easily programmable targeting of nucleic acids enabled by these ribonucleoproteins has facilitated the implementation of CRISPR-based molecular biology tools for in vivo and in vitro modification of DNA and RNA targets. Despite the diversity of DNA-targeting Cas nucleases so far identified, native and engineered derivatives of the Streptococcus pyogenes SpCas9 are the most used for genome engineering, at least in part due to its catalytic robustness and the requirement of an exceptionally short motif (5-NGG-3 PAM) flanking the target sequence. However, the large size of the SpCas9 variants impairs the delivery of the tool to eukaryotic cells and smaller alternatives are demanded. Here we identify in a metagenome a new CRISPR-Cas9 system associated with a smaller Cas9 protein (EHCas9) that targets DNA sequences flanked by 5-NGG-3 PAMs. We develop a simplified EHCas9 tool that specifically cleaves DNA targets and is functional for genome editing applications in prokaryotes and eukaryotic cells.

molecular biology↗

bioRxiv2021

Arg1+ microglia are critical for shaping cognition in female mice

Diversity within microglia, the resident brain immune cells, is reported. Whether microglial subsets constitute different subtypes with intrinsic properties and unique functions has not been fully elucidated. Here, we describe a microglial subtype characterized by the expression of the enzyme Arginase-1, i.e. Arg1 +microglia, which is found predominantly in the cholinergic neuron-rich forebrain region during early postnatal development. Arg1+ microglia are frequently observed in close apposition to neurons and exhibit a distinctive molecular signature reflecting a reactive profile. Arg1 deficiency in microglia results in impaired dendritic spine maturation in the hippocampus where cholinergic neurons project, and cognitive behavioural deficiencies in female mice. Our results expand on microglia diversity and provide insights into distinctive spatiotemporal functions exerted by microglial subtypes.

neuroscience↗

bioRxiv2019

Structure of the cell-binding component of the Clostridium difficile binary toxin reveals a novel macromolecular assembly

Targeting Clostridium difficile infection (CDI) is challenging because treatment options are limited, and high recurrence rates are common. One reason for this is that hypervirulent CDI often has a binary toxin termed the C. difficile toxin (CDT), in addition to the enterotoxins TsdA and TsdB. CDT has an enzymatic component, termed CDTa, and a pore-forming or delivery subunit termed CDTb. CDTb was characterized here using a combination of single particle cryoEM, X-ray crystallography, NMR, and other biophysical methods. In the absence of CDTa, two novel di-heptamer structures for activated CDTb (aCDTb; 1.0 MDa) were solved at atomic resolution including a symmetric (SymCDTb; 3.14 [A]) and an asymmetric form (AsymCDTb; 2.84 [A]). Roles played by two receptor-binding domains of aCDTb were of particular interest since RBD1 lacks sequence homology to any other known toxin, and the RBD2 domain is completely absent in other well-studied heptameric toxins (i.e. anthrax). For AsymCDTb, a novel Ca2+ binding site was discovered in RBD1 that is important for its stability, and RBD2 was found to be critical for host cell toxicity and the novel di-heptamer fold for both forms of aCDTb. Together, these studies represent a starting point for structure-based drug-discovery strategies to targeting CDT in the most severe strains of CDI. SIGNIFICANCE STATEMENTThere is a high burden from C. difficile infection (CDI) throughout the world, and the Center for Disease Control (CDC) reports more than 500,000 cases annually in the United States, resulting in an estimated 15,000 deaths. In addition to the large clostridial toxins, TcdA/TcdB, a third C. difficile binary toxin (CDT) is associated with the most serious outbreaks of drug resistant CDI in the 21st century. Here, structural biology and biophysical approaches were used to characterize the cell binding component of CDT, termed CDTb, at atomic resolution. Surprisingly, two novel structures were solved from a single sample that help to explain the molecular underpinnings of C. difficile toxicity. These structures will also be important for targeting this human pathogen via structure-based therapeutic design methods.

biophysics↗

bioRxiv2019

Overt and covert paths for sound in the auditory system of mammals

Current scientific consensus holds that sound is transmitted, solely mechanically, from the tympanum to the cochlea via ossicles.\n\nBut this theory does not explain the hearing extreme quality regarding high frequencies in mammals. So, we propose a bioelectronic pathway (the covert path) that is complementary to the overt path.\n\nWe demonstrate experimentally that the tympanum produces piezoelectric potentials isochronous to acoustic vibrations thanks to its collagen fibers and that their amplitude increases along with the frequency and level of the vibrations. This finding supports the existence of an electrical pathway, specialized in transmitting high-frequency sounds, that works in unison with the mechanical pathway. A bio-organic triode, similar to a field effect transistor, is the key mechanism of our hypothesized pathway. We present evidence that any deficiency along this pathway produces hearing impairment. By augmenting the classical theory of sound transmission, our discovery offers new perspectives for research into both normal and pathological audition and may contribute to an understanding of genetic and physiological problems of hearing.

physiology↗

bioRxiv2018

Galectin-3, A Novel Endogenous Trem2 Ligand, Regulates Inflammatory Response and Aβ Fibrilation in Alzheimer’s Disease

Alzheimers disease (AD) is a progressive neurodegenerative disease in which the formation of extracellular aggregates of amyloid beta (A{beta}) peptide, intraneuronal tau neurofibrillary tangles and microglial activation are major pathological hallmarks. One of the key molecules involved in microglial activation is galectin-3 (gal3), and we demonstrate here for the first time a key role of gal3 in AD pathology. Gal3 was highly upregulated in the brains of AD patients and 5xFAD (familial Alzheimers disease) mice, and found specifically expressed in microglia associated with A{beta} plaques. Single nucleotide polymorphisms in the LGALS3 gene, which encodes gal3, were associated to an increased risk of AD. Gal3 deletion in 5xFAD mice attenuated microglia-associated immune responses, particularly those associated with TLR and TREM2/DAP12 signaling. In vitro data demonstrated the requirement of gal3 to fully activate microglia in response to fibrillar A{beta}. Gal3 deletion decreased the A{beta} burden in 5xFAD mice and improved cognitive behavior. Electron microscopy of gal3 in AD mice demonstrated i) a preferential expression of gal3 by plaque-associated microglia, ii) its presence in the extracellular space and iii) its association to A{beta} plaques. Low concentrations (1 nM) of pure gal3 promoted cross-seeding fibrilization of pure A{beta}. Importantly, a single intrahippocampal injection of gal3 along with A{beta} monomers in WT mice was sufficient to induce the formation of insoluble A{beta} aggregates that were absent when gal3 was lacking. High-resolution microscopy (STORM) demonstrated close co-localization of gal3 and TREM2 in microglial processes, and a direct interaction was shown by a fluorescence anisotropy assay involving the gal3 CRD domain. Furthermore, gal3 stimulated the TREM2-DAP12 signaling pathway. In conclusion, we provide evidence that gal3 is a central regulator of microglial immune response in AD. It drives proinflammatory activation and A{beta} aggregation, as well as acting as an endogenous ligand to TREM2, a key receptor driving microglial response under disease conditions. Gal3 inhibition may, hence, be a potential pharmacological approach to counteract AD.

neuroscience↗