Biology subjects

Vazquez, S.

Publications and source records attributed to Vazquez, S..

16 recordsLinked to original sources

bioRxiv2026

Complex-trait dissection with Evolve-and-Resequence: Selection for improved flight performance in laboratory fruit fly populations

Evolve-and-Resequence studies combine experimental evolution with molecular sequencing, enabling associations between complex trait phenotypes and evolved genomic changes. For this, a targeted complex trait is subjected to artificial laboratory selection in genetically variable populations. Using populations of fruit flies Drosophila melanogaster from the DSPR background, we have conducted a short-term selection study on a highly complex trait : flight performance. We selected for improved flight performance using a custom-built flight tunnel system, with light as a motivator and airflow as a resistive factor. As a result, flight performance significantly improved in selected populations compared to controls. We also observed signatures of evolution in flight related traits, such as activity level and wing shape, and in life-history related traits such as adult dry mass and fecundity. We used whole genome pooled sequencing on the selected and control populations, revealing significantly differentiated genomic sites. Gene ontology and enrichment analyses revealed the evolution of pathways involved in response to stimuli, growth, and development. We identified several genes of interest (e.g., Sdc, dally). The range of evolved phenotypes and associated genomic changes show that flight performance is an ideal complex trait for future Evolve-and-Resequence studies.

evolutionary biology↗

bioRxiv2026

A brain-penetrant P2X7R antagonist mitigates Alzheimer's disease pathology

The ATP-gated P2X7 receptor (P2X7R) activates inflammatory signaling pathways in the central nervous system. In particular, P2X7Rs drive chronic glia-mediated neuroinflammation, which is increasingly recognized as a key contributor to Alzheimers disease, a neurodegenerative disorder that lacks effective disease-modifying therapies. Here we identify a potent and selective negative allosteric modulator of P2X7Rs with therapeutic potential. We synthesize a series of small molecules based on a polycyclic scaffold and confirm blood-brain barrier penetration by testing a radiolabeled analogue using positron emission tomography imaging. Through a structure-guided medicinal chemistry campaign centered on our scaffold, we identify four promising P2X7R antagonists. Of these, UB-ALT-P2 exhibits the most favorable safety profile, high oral bioavailability and robust brain penetration. High-resolution cryo-EM structures of UB-ALT-P2 bound to human, mouse, and rat P2X7Rs reveal a conserved antagonist binding mode with steric features that favor prolonged binding to human receptors. In the 5xFAD mouse model of AD, oral UB-ALT-P2 blunts weight loss, improves short- and long-term memory, reduces amyloid-{beta} plaque burden, lowers hyperphosphorylated tau, and diminishes oxidative and inflammatory markers. These results establish UB-ALT-P2 as a potent and safe P2X7R antagonist that can mitigate core AD pathologies, providing a compelling foundation for further development.

neuroscience↗

bioRxiv2025

Exploring polycyclic scaffolds as adamantane replacements in M2 channel inhibitors of Influenza A virus

The increasing resistance of influenza A viruses to adamantane-based antivirals underscores the need for new inhibitors targeting both wild-type (WT) and mutant M2 ion channels. Here, we report the synthesis and biological evaluation of polycyclic cage amines designed to replace the adamantane scaffold as M2 inhibitors. These include ring-contracted and ring-expanded analogues, evaluated both as primary amines and as aryl-/heteroaryl-substituted derivatives. Most of the polycyclic amines inhibited the WT M2 channel as demonstrated by electrophysiological assays. Among them, compound 10, a 3,4,8,9-tetramethyltetracyclo[4.4.0.03..0.]decan-1-amine, emerged as a triple blocker active against M2 WT, M2 L27F, and M2 V27A channels. In contrast, compound 6c, a noradamantane-isoxazole derivative, showed selective inhibition of the S31N mutant. Although no antiviral activity was observed against influenza A virus in infected cell assays, both compounds 6c and 10 displayed significant antiviral activity against human coronavirus 229E. Furthermore, compound 10 demonstrated favourable pharmacokinetic properties. MD simulations show that noradamantane 6c binds inside the M2 S31N pore, with its ammonium forming H-bonds to Asn31 and the isoxazole positioned near Val27, restricting water entry. In contrast, larger polycyclic amines likely cannot access the pore due to steric hindrance.

pharmacology and toxicology↗

bioRxiv2025

N-benzamides as Influenza Virus Fusion Inhibitors Acting on H1 and H5 Hemagglutinins

Novel antiviral drugs are needed to prepare against infections from influenza A virus (IAV). Here a series of N-[(thiophen-3-yl)methylbenzamides which target the hemagglutinin (HA)-mediated fusion process is reported. The most active compound, VF-57a, displays a 50% effective concentration (EC50) of [~]0.8 M and antiviral selectivity index >130, in Madin-Darby canine kidney (MDCK) cells infected with A/H1N1 virus. VF-57a proved to be a strong inhibitor of A/H1N1- and A/H5N1-pseudovirus entry (EC50 values of 0.3 and 0.8 {micro}M, respectively). Cell-cell fusion assays in HA-expressing cells, surface plasmon resonance-based assessment of HA protein refolding, and resistance studies suggested that VF-57a prevents the conformational change of HA at acidic pH. Molecular modelling highlighted the role of the dimethylthiophene moiety and the amide-based tether in the anchoring to the binding cavity of HA. Our findings support further development of this class of IAV fusion inhibitors against A/H1N1 and A/H5N1 viruses.

microbiology↗

bioRxiv2025

UB-MBX-46 is a potent and selective antagonist of the human P2X7 receptor developed by structure-based drug design

The P2X7 receptor is an ATP-gated ion channel that activates inflammatory pathways involved in diseases such as cancer, atherosclerosis, and neurodegeneration. However, despite the potential benefits of blocking overactive signaling, no P2X7 receptor antagonists have been approved for clinical use. Interspecies variation among existing antagonists has proven challenging, in part due to the dearth of molecular information on different receptor orthologs. Here, to identify distinct molecular features in the human receptor, we determine high-resolution cryo-EM structures of the full-length wild-type human P2X7 receptor in apo closed and ATP-bound open state conformations and draw comparisons with new and existing structures of other orthologs. We also report a cryo-EM structure of the human receptor in complex with an adamantane-based inhibitor, which we leverage, in conjunction with functional data and molecular dynamics simulations, to design a potent and selective antagonist with a unique polycyclic scaffold. Functional and structural analysis reveal how this optimized ligand, UB-MBX-46, interacts with the classical allosteric pocket of the human P2X7 receptor with picomolar potency and high selectivity, revealing its significant therapeutic potential.

biochemistry↗

bioRxiv2024

A guanidine-based coronavirus replication inhibitor which targets the nsp15 endoribonuclease and combines a direct antiviral effect with subtle boosting of interferon

The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target. Author summaryDespite major achievements in ending the global COVID-19 crisis through vaccines and antiviral drugs, SARS-CoV-2 infection remains a serious health threat for vulnerable individuals, such as elderly and immunocompromised patients. These populations could benefit from innovative treatments that target the virus from multiple angles. In this study, we focus on the nsp15 endoribonuclease (EndoU), a poorly understood coronavirus protein that aids in viral evasion from the host innate immune response and is also assumed to regulate viral RNA synthesis. Our research centers around a newly identified, first-in-class inhibitor of coronavirus nsp15, designated EPB-113. Although nsp15 is best-known for its function in interferon evasion, EPB-113 mainly acts via an interferon-independent mechanism that is situated during viral RNA synthesis. When incubated with nsp15 protein, EPB-113 induces a structural change and enhances its EndoU enzyme activity. EPB-113 selects for escape mutants that carry substitutions in the core of EndoU, leading to a severe replication defect. Hence, our study introduces a novel drug concept to disturb the multifunctional nsp15 protein and suppress coronavirus replication on multiple fronts.

microbiology↗

bioRxiv2024

TORC1-dependent control of fission yeast cohesin

Cohesin is a DNA tethering complex essential for chromosome structure and function. In fission yeast, defects in the cohesin loader Mis4 result in chromosome segregation defects and dysregulated expression of genes near chromosome ends. A genetic screen for suppressors of the thermosensitive growth defect of mis4-G1487D identified several hypomorphic mutants of the Target of Rapamycin Complex 1 (TORC1), a conserved kinase that integrates cellular signals to regulate growth and metabolism through substrate-specific phosphorylation. Here, we demonstrate that the TORC1 pathway modulates cohesin functions in chromosome segregation and gene expression. In the context of compromised cohesin loading, the incidence of chromosome segregation defects was modulated by the growth medium in a TORC1-dependent manner. Pharmacological or genetic down-regulation of TORC1 activity restored cohesin binding to its chromosomal sites and improved mitotic chromosome segregation. Notably, reduced TORC1 activity also increased cohesin binding and chromosome transmission fidelity in wild-type cells. These results suggest that environmental cues influence chromosome stability via TORC1. Biochemically, TORC1 co-purified with cohesin and reduced TORC1 activity correlated with decreased phosphorylation of specific residues on Mis4 and cohesin. Mutations in cohesin that mimic the non-phosphorylated state mirrored the effects of TORC1 downregulation, showing that TORC1 is part of the network that controls cohesin phosphorylation to modulate its functions. Finally, we show that the functional interaction between TORC1 and Mis4 extends to the regulation of stress-responsive genes. Our findings reveal a TORC1-cohesin link that may facilitate cellular adaptation to environmental changes. Given that TORC1 inhibitors and calorie restriction extend lifespan in diverse species, this connection raises the intriguing possibility that cohesin-mediated changes in chromosome structure contribute to these effects.

molecular biology↗

bioRxiv2024

Memantine Inhibits Calcium-Permeable AMPA Receptors

Memantine is an US Food and Drug Administration (FDA) approved drug that selectively inhibits NMDA-subtype ionotropic glutamate receptors (NMDARs) for treatment of dementia and Alzheimers. NMDARs enable calcium influx into neurons and are critical for normal brain function. However, increasing evidence shows that calcium influx in neurological diseases is augmented by calcium-permeable AMPA-subtype ionotropic glutamate receptors (AMPARs). Here, we demonstrate that these calcium-permeable AMPARs (CP-AMPARs) are inhibited by memantine. Electrophysiology unveils that memantine inhibition of CP-AMPARs is dependent on their calcium permeability and the presence of their neuronal auxiliary subunit transmembrane AMPAR regulatory proteins (TARPs). Through cryo-electron microscopy we elucidate that memantine blocks CP-AMPAR ion channels in a unique mechanism of action from NMDARs. Furthermore, we demonstrate that memantine reverses a gain of function AMPAR mutation found in a patient with a neurodevelopmental disorder and inhibits CP-AMPARs in nerve injury. Our findings alter the paradigm for the memantine mechanism of action and provide a blueprint for therapeutic approaches targeting CP-AMPARs.

biophysics↗

bioRxiv2024

Novel Pentafluorosulfanyl-containing Triclocarban Analogs selectively kill Gram-positive bacteria.

The antibacterial and antibiofilm efficacy of our novel pentafluorosulfanyl-containing triclocarban analogs was explored against seven different Gram-positive and Gram-negative indicator strains. After initial screening, they had bactericidal and bacteriostatic activity against Gram-positive bacteria, especially Staphylococcus aureus and MRSA (methicillin-resistant staphylococcus aureus) in a very low concentration. Our results were compared with the most common antibiotic being used (Ciprofloxacin and Gentamycin); the novel components had significantly better antibacterial and antibiofilm activity in lower concentrations in comparison to the antibiotics. For instance, EBP-59 minimum inhibitory concentration was < 0.0003 mM, while ciprofloxacin 0.08 mM. Further antibacterial activity of novel components was surveyed against 10 clinical antibiotic resistance MRSA isolates. Again, novel components had significantly better antibacterial and antibiofilm activity in comparison with antibiotics. Mechanistic studies have revealed that none of these novel compounds exhibit any effect on the reduced thiol, disrupting iron sulfur clusters, or hydrogen peroxide pathways. Instead, their impact is attributed to the disruption of the Gram-positive bacterial cell membrane. Toxicity and safety testing on tissue cell culture showed promising results for the safety of components to the host.

microbiology↗

bioRxiv2023

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 hr post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labeled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

immunology↗

bioRxiv2022

Looking for the mechanism of arsenate respiration in an arsenate-dependent growing culture of Fusibacter sp. strain 3D3, independent of ArrAB.

The literature has reported the isolation of arsenate-dependent growing (ADG) microorganisms which lack a canonical homolog for respiratory arsenate reductase, ArrAB. We recently isolated an ADG bacterium from arsenic-bearing environments in Northern Chile, Fusibacter sp. strain 3D3 (Fas) and studied the arsenic metabolism in this Gram-positive isolate. Features of Fas deduced from genome analysis and comparative analysis with other arsenic-reducing microorganisms revealed the lack of ArrAB coding genes and the occurrence of two arsC genes encoding for putative cytoplasmic arsenate reductases named ArsC-1 and ArsC-2. Interestingly, ArsC-1 and ArsC-2 belong to the thioredoxin-coupled family (because of the redox-active disulfide protein used as reductant), but they conferred differential AsV resistance to the E. coli WC3110 {Delta}arsC strain. PCR experiments confirmed the absence of arrAB genes and results obtained using uncouplers revealed that Fas growth is linked to the proton gradient. In addition, Fas harbors ferredoxin-NAD+ oxidoreductase (Rnf) coding genes. These are key molecular markers of a recently discovered flavin-based electron bifurcation mechanism involved in energy conservation, mainly in anaerobic metabolisms regulated by the cellular redox state and mostly associated with cytoplasmic enzyme complexes. At least three electron-bifurcating flavoenzyme complexes were evidenced in Fas, some of them shared in conserved genomic regions by other members of the Fusibacter genus. These physiological and genomic findings permit us to hypothesize the existence of an uncharacterized arsenate-dependent growth metabolism regulated by the cellular redox state in Fusibacter genus.

microbiology↗

bioRxiv2020

RL-118 and 11β-HSD1 target engagement through TAPS assay: behaviour and molecular analysis

1.Taking into consideration the convergence of ageing, stress and neurodegenerative diseases, such as AD, there is impaired GC signalling. Therefore, the study of GC-mediated stress response to chronic moderate stressful situations, as account in daily life, becomes of huge interest to design pharmacological strategies to prevent neurodegeneration. To address this issue, SAMP8 were exposed for 4 weeks to the CMS paradigm and treated with RL-118, an 11{beta}-HSD1 inhibitor. In fact, several pieces of evidence link the inhibition of this enzyme with reduction of GC levels and cognitive improvement, while CMS exposure has been associated with reduced cognitive performance. The aim of this project was to assess whether RL-118 treatment could restore the deleterious effects of CMS on cognition and behavioural abilities, but also on molecular mechanisms that compromise healthy ageing in SAMP8 mice. On the one hand, we determined the target engagement between RL-118 and 11{beta}-HSD1. Therefore all the beneficial effects previously described in SAMP8 treated with the drug can undoubtedly be attributed to the inhibition of this enzyme. Besides, herein we observed decreased DNA methylation, hydroxymethylation and histone phosphorylation induced by CMS but, on the contrary, increased after RL-118 treatment. In addition, CMS exposure produced ROS damage accumulation, and increments of pro-oxidant enzymes as well as pro-inflammatory mediators through NF-{kappa}B pathway and astrogliosis markers, like Gfap. Of note, those modifications were recovered by 11{beta}-HSD1 inhibition. Remarkably, although CMS altered mTORC1 signalling, autophagy was increased in SAMP8 treated with RL-118 mice. Also, we found amyloidogenic APP processing pathway favoured and decreased synaptic plasticity and neuronal remodelling markers in mice under CMS, but changed after RL-118 treatment. In consequence, detrimental effects on behaviour and cognitive performance were detected in CMS exposed mice, but restored after concomitant 11{beta}-HSD1 inhibition by RL-118. Overall, CMS is a feasible intervention to understand the influence of stress on epigenetic mechanisms underlying cognition and accelerating senescence. However and most important, 11{beta}-HSD1 inhibition through RL-118 turned up to restore the majority of these detrimental effects caused by CMS, indicating that GC excess attenuation may become a potential therapeutic strategy for age-related cognitive decline and AD.

molecular biology↗

bioRxiv2020

GMO LABELING IN FOOD PRODUCTS IN MONTEVIDEO, URUGUAY

Montevideo establishes the mandatory labeling of foods containing genetically modified material through the Departmental Decree No. 36.554, positioning Uruguay within the 65 countries that have incorporated this type of regulation. The Food Regulation Service, in its role of sanitary police, and through its Laboratory of Bromatology, in agreement with the Food Molecular Traceability Laboratory (Faculty of Sciences, University of the Republic), carried out the analysis of 206 products made with ingredients derived from corn and/or soybean, during the 2015-2017 period, within the framework of compliance with the aforementioned Decree. The strategy used consisted of the application of molecular techniques (Real Time PCR), for the detection of common sequences present in the transgenic events of soybean and corn, and the subsequent quantification of the content of GM material, in relation to an established labeling threshold of 1%. As a result of this study, it was found that 36.9% of the analyzed foods presented sequences derived from genetically modified plant organisms (GMOs); and in 95% of the cases, its content exceeded the threshold established for its labeling. This study, constitutes the first approach to the knowledge of different transgenic elements distribution in food commercialized in Montevideo. These results provide valuable information to both the consumer, for decision making about the food to be consumed, and also for the official control organizations, which must enforce the regulations. This type of work has demonstrated, once again, the importance of the interrelation between academy and state agencies, in the generation of knowledge and in the implementation of new analytical methodologies, as well as in the training of qualified human resources and in the compliance with current regulations.

molecular biology↗

bioRxiv2020

The Biogeography of Dromiciops in Southern South America: middle Miocene transgressions, speciation and associations with Nothofagus

AimSeveral geological events affecting Southern South America during the middle Miocene climatic optimum acted as important drivers of diversification to the biota. This is the case of Microbiotheria, for which Dromiciops is considered the sole surviving lineage, the sister group of Eomarsupialia (Australian marsupials). Three main Dromiciops genetic lineages are known, whose divergence was initially attributed to recent Pleistocene glaciations. Using fossil-calibrated dating on nuclear and mitochondrial genes, here we reevaluate this hypothesis and report an older (Miocenic) biogeographic history for the genus. LocationSouthern South America. MethodsPhylogenetic reconstruction using sequences from two mitochondrial DNA and four nuclear DNA genes in 159 specimens, from 31 sites across Chile and Argentina. Divergence time estimation using fossil calibration. ResultsOur phylogenetic analysis resolved four well supported clades with discrete geographic distributions. The oldest and most differentiated clade corresponds to that of the northern distribution (35.2{degrees}S to 39.3{degrees}S), which would be a different species (D. bozinovici, sensu Delia et al. 2016). According to our estimations, this species shared a common ancestor with D. gliroides (southern clades) about 13 million years ago (95% CI: 6.4-25.3). The southern clades (39.6{degrees}S to 42.0{degrees}S), showed a divergence time ranging from 9.57 to 6.5 Mya. Strong genetic structure was detected from north to south but not across the Andes, or between Chiloe island/ mainland. Demographic equilibrium is inferred to the northern clade, and recent demographic expansions was detected in the central and southern clades. Main conclusionsThe whole diversification of Dromiciops occurred within the Miocene, being the Middle Miocene transgression (MMT), the massive marine flooding that covered several lowlands of the western face of los Andes between 38-48{degrees} S, the most likely diversifying force. This was the result of an increase in global sea levels due to the Miocene climatic optimum, which shaped the biogeographic origin of several species, including Nothofagus forests, the habitat main of Dromiciops.

evolutionary biology↗

bioRxiv2019

The CDK Pef1 and Protein Phosphatase 4 oppose each other for regulating cohesin binding to fission yeast chromosomes

Cohesin has essential roles in chromosome structure, segregation and repair. Cohesin binding to chromosomes is catalyzed by the cohesin loader, Mis4 in fission yeast. How cells fine tune cohesin deposition is largely unknown. Here we provide evidence that Mis4 activity is regulated by phosphorylation of its cohesin substrate. A genetic screen for negative regulators of Mis4 yielded a CDK called Pef1, whose closest human homologue is CDK5. Inhibition of Pef1 kinase activity rescued cohesin loader deficiencies. In an otherwise wild-type background, Pef1 ablation stimulated cohesin binding to its regular sites along chromosomes while ablating Protein Phosphatase 4 had the opposite effect. Pef1 and PP4 control the phosphorylation state of the cohesin kleisin Rad21. The CDK phosphorylates Rad21 on Threonine 262. Pef1 ablation, non phosphorylatable Rad21-T262 or mutations within a Rad21 binding domain of Mis4 alleviated the effect of PP4 deficiency. Such a CDK/PP4 based regulation of cohesin loader activity could provide an efficient mechanism for translating cellular cues into a fast and accurate cohesin response.

molecular biology↗

bioRxiv2019

Pharmacological inhibition of soluble epoxide hydrolase as a new therapy for Alzheimer’s Disease

The inhibition of the enzyme soluble epoxide hydrolase (sEH) has demonstrated clinical therapeutic effects in several peripheral inflammatory-related diseases, with two compounds that have entered clinical trials. However, the role of this enzyme in the neuroinflammation process has been largely neglected. Herein, we disclose the pharmacological validation of sEH as a novel target for the treatment of Alzheimers Disease (AD). Of interest, we have found that sEH is upregulated in brains from AD patients. We have evaluated the cognitive impairment and the pathological hallmarks in two models of age-related cognitive decline and AD using three structurally different and potent sEH inhibitors as chemical probes. Our findings supported our expectations on the beneficial effects of central sEH inhibition, regarding of reducing cognitive impairment, tau hyperphosphorylation pathology and the number of amyloid plaques. Interestingly, our results suggest that reduction of inflammation in the brain is a relevant therapeutic strategy for all stages of AD.

pharmacology and toxicology↗