Biology subjects

Ortiz, J.

Publications and source records attributed to Ortiz, J..

12 recordsLinked to original sources

bioRxiv2026

Discovery of inhibitors of the Pseudomonas aeruginosa NADH:ubiquinone oxidoreductase (NQR) that hinder virulence factors

The growing threat of antimicrobial resistance has created an urgent need to identify novel therapeutic targets in bacteria. The NADH:ubiquinone oxidoreductase (NQR) is a potential target in a number of bacteria that transfers electrons from NADH to ubiquinone while pumping ions from the cytoplasm to the periplasm. In most species, this complex pumps sodium ions, whereas in Pseudomonas aeruginosa it pumps protons, thereby functioning as a member of the electron transport chain. Using a strain of PAO1 with the NQR knocked out, we demonstrate that the NQR complex plays a crucial role in the motility and biofilm development virulence factors in P. aeruginosa. We develop and execute a high-throughput inhibitor screen to identify and confirm compounds that inhibit NADH oxidation by this complex. Using single-particle cryogenic electron microscopy (cryoEM), we determine high-resolution structures of the NQR complex, both inhibitor-free and bound to one of the confirmed hits from our screen, demonstrating that it binds to the ubiquinone binding site. These structures provide insight into conformational dynamics controlled by binding at the ubiquinone site, with potential implications for the coupling between electron transfer and proton pumping in this complex. Biofilm development and motility assays with selected compounds from the screen show that they affect these virulence factors similarly to the NQR knockout.

biochemistry↗

bioRxiv2025

Unraveling novel insights into dual-species cariogenic biofilm formation: a comparative analysis on natural vs artificial bioengineered dentin models

Dental caries is the most prevalent biofilm-associated disease affecting billions of people worldwide, including elderly individuals. Conventional biofilm study methods rely on human or animal-derived samples, posing challenges regarding accessibility, cost, and ethical considerations. While in-vitro systems offer a promising alternative, they often fail to replicate the structural characteristics of dentin, which play a crucial role in bacterial adhesion. To bridge this gap, a bioengineered dentin-like construct has recently been developed as a reproducible and accessible model for studying biofilm formation associated with dental aging. Therefore, this study aimed to assess dual-species Streptococcus mutans and Candida albicans biofilm formation on bioengineered dentin substrates and compare it to biofilm formation on natural human aged dentin. For this, S. mutans UA159 and C. albicans (ATCC 90028) were co-cultured on bioengineered and natural dentin slabs, and polymicrobial biofilm formation and EPS production were characterized via high-resolution confocal laser scanning microscopy. Following biofilm formation, image processing was conducted using COMSTAT software to determine biofilm growth parameters. Additionally, fluorescence intensity was quantified via microplate readings, and cell viability was assessed using a Live/Dead viability kit. Overall, results showed similar biofilm formation patterns between the bioengineered and aged dentin, with no significant differences found in biofilm physical properties or viability. These findings suggest that this bioengineered dentin system provides a reliable platform for studying biofilm formation in the context of dental aging, making it a valuable tool for investigating microbial adhesion and cariogenic biofilm development under controlled conditions, potentially facilitating future research in biofilm-related oral diseases. ImportanceDental caries is one of the most common chronic diseases worldwide, which is driven by complex microbial biofilms formed on the tooths surface. However, existing models for studying these biofilms in the laboratory often rely on human or animal tissues, which are difficult to obtain and standardize, and present ethical challenges. In this study, we validate a bioengineered dentin-like model that accurately mimics the microarchitecture of aged human dentin, a key site for root caries in the elderly. By comparing biofilms formed by the clinically significant Streptococcus mutans and Candida albicans on both artificial and natural substrates, we show that the engineered model supports biofilm development under comparable parameters and enables detection of changes in microbial virulence. Overall, this platform provides a reproducible and scalable alternative for studying oral biofilms with potential applications in understanding disease pathogenesis, novel treatment testing, and integration into next-generation organ-on-a-chip systems.

bioengineering↗

bioRxiv2025

Soil Carbon Sequestration in Nothofagus obliqua Forests with Different Canopy Cover Levels under Silvopastoral Management

Agroforestry contributes to slowing deforestation, favouring ecosystem regeneration and improving land use sustainability. In this study, we evaluated the effect of agroforestry on the recovery of degraded soils in a native forest and its capacity to capture and stabilize carbon (C) and nitrogen (N) in the soil. We conducted the study in a native forest of Nothofagus obliqua (oak) located in Ranchillo Alto (37{degrees}04'52'' S, 71{degrees}39'14'' W), in the Nuble region, Chile. This site includes three silvopastoral systems with different levels of forest degradation: open (Op), semi- open (SOp) and semi-closed (SC), together with a control treatment without silvopastoral treatment (Ctr). The analysis was conducted at four soil depths (0-10, 10-20, 20-30 and 30-60 cm). To evaluate carbon and nitrogen sequestration and fixation in relation to the different forest cover treatments, we performed chemical, physical and biological soil analyses. We also included a physical fractionation by particle size to separate soil organic matter (SOM) into meaningful fractions with different characteristics and dynamics: particulate organic matter (POM) and organic matter associated with the mineral matrix (MAOM). Our results indicated that Op, despite being the most degraded condition, showed the highest values of C, N and non- oxidizable carbon (Cnox). This condition could be associated with the history of potato burning in this sector, which generates stable pyrogenic carbon in the soil. Analyses of C and N, as well as Cnox in the MAOM fraction, and C stock revealed that the three silvopastoral systems showed higher values than the control treatment without silvopastoral treatment. This suggests that the adoption of silvopastoral practices can improve soil quality and contribute to long-term carbon sequestration. These results support the application of sustainable practices to mitigate soil degradation and enhance carbon sequestration in degraded ecosystems.

ecology↗

bioRxiv2024

Ongoing loss of viable neurons for weeks after mild perinatal hypoxia-ischemia

Mild hypoxic-ischemic encephalopathy is common in neonates with no evidence-based therapies, and 30-40% of patients experience adverse outcomes. The nature and progression of mild injury is poorly understood. Thus, we studied the evolution of mild perinatal brain injury using longitudinal two-photon imaging of transgenic fluorescent proteins as a novel readout of neuronal viability and activity at cellular resolution. In vitro, perinatal murine organotypic hippocampal cultures underwent 15-20 minutes of oxygen-glucose deprivation. In vivo, mild hypoxia-ischemia was completed in post-natal day 10 mouse pups of both sexes with carotid ligation and 15 minutes of hypoxia. Consistent with a mild injury, minimal immediate neuronal death was seen and there was no volumetric evidence of injury by ex vivo MRI 2.5 weeks after injury. In both the hippocampus and neocortex, these mild injuries resulted in a significantly delayed and progressive neuronal loss in the second week after injury, measured by fluorophore quenching. Mild hypoxia-ischemia transiently suppressed cortical network activity followed by normal maturation. No post-injury seizures were seen. The participation in network activity of individual neurons destined to die was indistinguishable from those that survived for 4 days post-injury. In conclusion, our results showed that mild perinatal brain injury resulted in a prolonged increase of neuronal death. Neurons that died late were functioning normally for days after injury, suggesting a new pathophysiology of neuronal death. Critically, the neurons destined to die late demonstrated multiple biomarkers of viability long after mild injury, suggesting their later death may be modified with neuroprotective interventions. SIGNIFICANCE STATEMENTNeonatal encephalopathy due to peripartum hypoxia-ischemia (HI) is a major cause of neonatal mortality and morbidity worldwide. Of these infants, most are categorized as having mild HI. Infants with mild HI have significant long-term disabilities. There are currently no evidence-based therapies, largely because the progression and pathophysiology of mild injury is poorly understood. We have identified, for the first time, that mild perinatal HI results in a delayed and prolonged increase in neuronal death. The cortical and hippocampal neurons that die over a week after injury participate normally in neural network activity and exhibit robust viability for many days after injury, indicating a novel pathophysiology of neuronal death. Clinically, these data suggest an extended therapeutic window for mild perinatal HI.

neuroscience↗

bioRxiv2024

Comparative Analysis of AAV Serotypes for Transduction of Olfactory Sensory Neurons

Olfactory sensory neurons within the nasal epithelium detect volatile odorants and relay odor information to the central nervous system. Unlike other sensory inputs, olfactory sensory neurons interface with the external environment and project their axons directly into the central nervous system. The use of adeno-associated viruses to target these neurons has garnered interest for applications in gene therapy, probing olfactory sensory neuron biology, and modeling disease. To date, there is no consensus on the optimal AAV serotype for efficient and selective transduction of olfactory sensory neurons in vivo. Here we utilized serial confocal imaging and single-nucleus RNA sequencing to evaluate the efficacy of 11 different AAV serotypes in transducing murine olfactory sensory neurons via non-invasive nasal inoculation. Our results reveal that AAV1, while highly effective, exhibited broad tropism, whereas AAV-DJ/8 showed the greatest specificity for olfactory sensory neurons.

neuroscience↗

bioRxiv2024

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues

This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers [~]90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types. Graphical abstract created with biorender.com O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/591396v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1cba4c1org.highwire.dtl.DTLVardef@adb231org.highwire.dtl.DTLVardef@18db263org.highwire.dtl.DTLVardef@1babde0_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

CHIR99021 decreases dopamine content in rat brain striatum through inactivation of Tyrosine Hydroxylase

CHIR99021, also known as laduviglusib or CT99021, is a Glycogen-synthase kinase 3{beta} (GSK3{beta}) inhibitor, which has been reported as a promising drug for cardiomyocyte regeneration or treatment of sensorial hearing loss. Since the activation of dopamine (DA) receptors regulates dopamine synthesis and they can signal through the {beta}-arrestin pathway and GSK3{beta}, we decided to check the effect of GSK3{beta} inhibitors (CHIR99021, SB216763 and lithium ion) in the control of DA synthesis. Using ex vivo experiments with minces from rat brain striatum, we observed that CHIR99021, but not SB216763 nor lithium, causes a complete abrogation of DA synthesis and accumulation at low {micro}M concentrations, pointing to off-target effects of CHIR99021. This decrease can be attributed to tyrosine hydroxylase (TH) inhibition since the accumulation of L-DOPA in the presence of a DOPA decarboxylase inhibitor was similarly decreased. On the other hand, CHIR99021 caused a dramatic increase in the DOPAC / DA ratio, an indicator of DA metabolization, and hindered DA incorporation into striatum tissue to the same extent as tetrabenazine, thus pointing to some effect on DA storage that triggers DA feedback inhibition of TH. In addition, CHIR99021 or SB216763, but not lithium, decreased TH phosphorylation in Ser19, but not in Ser31 or Ser40. These results demonstrate that CHIR99021 can lead to TH inactivation and DA depletion in brain striatum, opening the possibility of its use in DA-related disorders, and shows effects to be considered in future clinical trials.

neuroscience↗

bioRxiv2022

Independent innexin radiation shaped signaling in ctenophores.

Innexins facilitate cell-cell communication by forming gap junctions or non-junctional hemichannels, which play important roles in metabolic, chemical, ionic, and electrical coupling. The lack of knowledge regarding the evolution and role of these channels in ctenophores (comb jellies), the likely sister group to the rest of animals, represents a substantial gap in our understanding of the evolution of intercellular communication in animals. Here we identify and phylogenetically characterize the complete set of innexins of four ctenophores: Mnemiopsis leidyi, Hormiphora californensis, Pleurobrachia bachei, and Beroe ovata. Our phylogenetic analyses suggest that ctenophore innexins diversified independently from those of other animals and were established early in the emergence of ctenophores. We identified a four-innexin genomic cluster, which was present in the last common ancestor of these four species and has been largely maintained in these lineages. Evidence from correlated spatial and temporal gene expression of the M. leidyi innexin cluster suggest that this cluster has been maintained due to constraints related to gene regulation. We describe basic electrophysiological properties of putative ctenophore hemichannels from muscle cells using intracellular recording techniques, showing substantial overlap with the properties of bilaterian innexin channels. Together, our results suggest that the last common ancestor of animals had gap junctional channels also capable of forming functional innexin hemichannels, and that innexin genes have independently evolved in major lineages throughout Metazoa.

evolutionary biology↗

bioRxiv2022

Fanca and p53 genes cooperate to suppress oral SCC in transgenic mice

Fanconi anemia (FA) patients frequently develop oral squamous cell carcinoma (OSCC). This cancer in FA patients is diagnosed within the first 3-4 decades of life, very often preceded by lesions that suffer a malignant transformation. In addition, they respond poorly to current treatments due to toxicity or multiple recurrences. Translational research of new chemopreventive agents and therapeutic strategies has been unsuccessful partly due to scarcity of disease models or failure to fully reproduce the disease. Here we report that Fanca gene knockout mice (Fanca-/-) frequently display pre-malignant lesions in the oral cavity. Moreover, when these animals were crossed with animals having conditional deletion of Trp53 gene in oral mucosa (K14cre;Trp53F2-10/F2-10), they spontaneously developed OSCC with a high penetrance and a median latency of less than ten months. Tumors were well differentiated and expressed markers of squamous differentiation, such as keratins K5 and K10. In conclusion, Fanca and Trp53 genes cooperate to suppress oral cancer in mice, and Fanca-/-;K14cre;Trp53F2-10/F2-10 mice constitute the first animal model of spontaneous OSCC in FA.

cancer biology↗

bioRxiv2020

Deep Learning Improves Macromolecules Localization and Identification in 3D Cellular Cryo-Electron Tomograms

Cryo-electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. While this label-free cryogenic imaging technology produces data containing rich structural information, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present a computational procedure that uses artificial neural networks to simultaneously localize with a multi-class strategy several macromolecular species in cellular cryo-electron tomograms. Once trained, the inference stage of DeepFinder is significantly faster than template matching, and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (~3.2 MDa), Rubisco (~540 kDa soluble complex), and photosystem II (~550 kDa membrane complex) with comparable accuracy to expert-supervised ground truth annotations. Furthermore, we show that DeepFinder is flexible and can be combined with template matching to localize the missing macromolecules not found by one or the other method. The DeepFinder algorithm is therefore very promising for the semi-automated analysis of a wide range of molecular targets in cellular tomograms, including macromolecules with weights of 500-600 kDa and membrane proteins.

bioinformatics↗

bioRxiv2019

Cryo-EM structure of the complete E. coli DNA Gyrase nucleoprotein complex

DNA Gyrase is an essential enzyme involved in the homeostatic control of DNA supercoiling and the target of successful antibacterial compounds. Despite extensive studies, the detailed architecture of DNA Gyrase from the model genetic organism E. coli, is still missing, impeding structure-function analysis of E. coli-specific catalytic regulation and limiting the study of conformational intermediates of this highly flexible macromolecule. Herein, we determined the complete molecular structure of the E. coli DNA Gyrase bound to a 180 bp DNA and the antibiotic Gepotidacin, using phase-plate single-particle cryo-electron microscopy. Our data unveil with unprecedented details the structural and spatial organization of the functional domains, their connections and the position of the conserved GyrA-box motif. The deconvolution of closed and pre-opening states of the DNA-binding domain provides a better understanding of the allosteric movements of the enzyme complex. In this region, the local atomic resolution reaching up to 3.0 [A] enables the identification of the antibiotic density in the DNA complex. Altogether, this study paves the way for the cryo-EM determination of gyrase complexes with antibiotics and opens perspectives for targeting conformational intermediates. The type 2A DNA topoisomerases (Top2) are nanomachines that control DNA topology during multiple cellular processes such as replication, transcription and cell division 1-4. These enzymes catalyze the transport of a DNA duplex through a double strand break to perform DNA relaxation, decatenation and unknotting. DNA Gyrase plays a vital role in the compaction of the bacterial genome and is the sole type 2 topoisomerase able to introduce negative supercoils into DNA, a reaction coupled to ATP hydrolysis 5.

biochemistry↗

bioRxiv2017

Facilitation between plants shapes pollination networks

SignificanceAlthough it is known that plant-plant and plant-pollinator interactions can strongly influence biodiversity and its effects on ecosystem functioning, the details of how competition and facili-tation among plants scale up to mutualistic interactions with pollinators and thus affect pollina-tion networks are poorly understood. We introduce a simple experimental system in which we control local plant interactions, measure pollinator responses and characterize plant-pollinator networks. We find that facilitation among plants produces synergistic and antagonistic effects on the pollinator community affecting the architecture and robustness of plant-pollinator net-works. Our results provide evidence for bottom-up non-additive effects of plant interactions on pollination networks and have implications for the way we study and manage ecosystems.

ecology↗