Publications

Background: Malaria during pregnancy implies a high risk for the mother and the developing child. However, the therapeutic options for pregnant women have historically been very limited, especially during the first trimester of pregnancy due to potential adverse effects on embryo-fetal development. Recently, there has been great controversy regarding these potential embryo-fetal adverse effects because the results of rodent studies were not in accordance with the clinical data available, and finally the WHO has changed the recommendations for pregnant women with uncomplicated P. falciparum malaria to treatment with artemether-lumefantrine during the first trimester. The discrepancy between pre-clinical and clinical studies has been attributed to speciesdifferences in the duration of the window of susceptibility of circulating primitive erythroblasts. Methods: Here we provide a tool based on an alternative method to animal experimentation that accelerates the research of novel drugs for pregnant women. We have adapted the zebrafish embryo developmental toxicity assay to include hemoglobin staining in the embryos and two time-points of lethality and dysmorphogenesis evaluation. These two time-points were selected to include one when the development is independent of and one when the development is dependent of erythrocytes function. The method was used to test four marketed antimalarial drugs and three new antimalarial drug candidates. Results: Our combination of tests can correctly predict the teratogenic and non-teratogenic effects of several antimalarial marketed drugs (artemisinin, quinine, chloroquine, and dihydroartemisinin + desbutyl-lumefantrine). Furthermore, we have tested three new drug candidates (GS-GUAN, DONE3TCl, and YAT2150) with novel mechanisms of action, and different from those of the marketed antimalarial drugs. Conclusions: We propose a decision tree combining the results of the two time-points of evaluation together with the information on significant erythrocyte depletion. The aim of this decision tree is to identify compounds with no or lower hazard on teratogenicity or erythrocyte depletion at an early phase of the drug development process.

JTD Keywords: Alternative methods to animal experimentation, Artesunate, Culture, Dihydroartemisinin, Drug discovery, Embryotoxicity test, In-vitro, Inhibitors, Mode, Nams, Paludism, Rat, Resistance, Safety, Teratogenesis, Toxicity testin

The inclusion of microexons by alternative splicing occurs frequently in neuronal proteins. The roles of these sequences are largely unknown, and changes in their degree of inclusion are associated with neurodevelopmental disorders1. We have previously shown that decreased inclusion of a 24-nucleotide neuron-specific microexon in CPEB4, a RNA-binding protein that regulates translation through cytoplasmic changes in poly(A) tail length, is linked to idiopathic autism spectrum disorder (ASD)2. Why this microexon is required and how small changes in its degree of inclusion have a dominant-negative effect on the expression of ASD-linked genes is unclear. Here we show that neuronal CPEB4 forms condensates that dissolve after depolarization, a transition associated with a switch from translational repression to activation. Heterotypic interactions between the microexon and a cluster of histidine residues prevent the irreversible aggregation of CPEB4 by competing with homotypic interactions between histidine clusters. We conclude that the microexon is required in neuronal CPEB4 to preserve the reversible regulation of CPEB4-mediated gene expression in response to neuronal stimulation.

JTD Keywords: Alternative splicing, Animals, Autism spectrum disorder, Cpeb4 protein, human, Cpeb4 protein, mouse, Exons, Gene expression regulation, Humans, Mice, Neurons, Protein aggregates, Protein biosynthesis, Rna-binding proteins

Cholesterol, a crucial component of cell membranes, influences various biological processes, including membrane trafficking, signal transduction, and host-pathogen interactions. Disruptions in cholesterol homeostasis have been linked to congenital and acquired conditions, including neurodegenerative disorders such as Alzheimer's disease (AD). Previous research from our group has demonstrated that herpes simplex virus type I (HSV-1) induces an AD-like phenotype in several cell models of infection. This study explores the interplay between cholesterol and HSV-1-induced neurodegeneration. The impact of cholesterol was determined by modulating its levels with methyl-beta-cyclodextrin (M beta CD) using the neuroblastoma cell lines SK-N-MC and N2a. We have found that HSV-1 infection triggers the intracellular accumulation of cholesterol in structures resembling endolysosomal/autophagic compartments, a process reversible upon M beta CD treatment. Moreover, M beta CD exhibits inhibitory effects at various stages of HSV-1 infection, underscoring the importance of cellular cholesterol levels, not only in the viral entry process but also in subsequent post-entry stages. M beta CD also alleviated several features of AD-like neurodegeneration induced by viral infection, including lysosomal impairment and intracellular accumulation of amyloid-beta peptide (A beta) and phosphorylated tau. In conclusion, these findings highlight the connection between cholesterol, neurodegeneration, and HSV-1 infection, providing valuable insights into the underlying mechanisms of AD.

JTD Keywords: Alzheimer's disease, Arterial cells, Beta-amyloid, Cholesterol, Hsv-1, Hyperphosphorylated ta, Infection, Lysosomal alterations, Methyl-beta-cyclodextrin, Neuroblastoma cells, Neurodegeneration, Syste

Simple Summary Neuroblastoma (NB), a prevalent childhood cancer, presents challenges in treatment due to its cellular diversity and the presence of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs, hindering effective therapies. We developed a stiffness-based in vitro platform simulating arterial and venous conditions to address this gap. Notably, adrenergic NB cells transdifferentiated into TECs where there was an arterial-like stiffness, while mesenchymal cells did not. This platform facilitated the identification of Globotriaosylceramide (GB3) as a novel TEC biomarker. Moreover, we harnessed Shiga toxin-functionalized nanoparticles for the specific targeting of GB3-positive cells, showing promise for future therapeutic strategies. Our study provides insights into NB heterogeneity, offers a predictive tool for assessing aggressiveness, and introduces potential targets for precision therapies.Abstract Neuroblastoma (NB) is a childhood cancer in sympathetic nervous system cells. NB exhibits cellular heterogeneity, with adrenergic and mesenchymal states displaying distinct tumorigenic potentials. NB is highly vascularized, and blood vessels can form through various mechanisms, including endothelial transdifferentiation, leading to the development of tumor-derived endothelial cells (TECs) associated with chemoresistance. We lack specific biomarkers for TECs. Therefore, identifying new TEC biomarkers is vital for effective NB therapies. A stiffness-based platform simulating human arterial and venous stiffness was developed to study NB TECs in vitro. Adrenergic cells cultured on arterial-like stiffness transdifferentiated into TECs, while mesenchymal state cells did not. The TECs derived from adrenergic cells served as a model to explore new biomarkers, with a particular focus on GB3, a glycosphingolipid receptor implicated in angiogenesis, metastasis, and drug resistance. Notably, the TECs unequivocally expressed GB3, validating its novelty as a marker. To explore targeted therapeutic interventions, nanoparticles functionalized with the non-toxic subunit B of the Shiga toxin were generated, because they demonstrated a robust affinity for GB3-positive cells. Our results demonstrate the value of the stiffness-based platform as a predictive tool for assessing NB aggressiveness, the discovery of new biomarkers, and the evaluation of the effectiveness of targeted therapeutic strategies.

JTD Keywords: Alternative vasculature, Angiogenesis, Cells, Differentiation, Gb3, Neuroblastoma, Origin, Tumor-derived endothelial cells

Exposure of experimental rodents to controlled cycles of light, food, and temperature is important when investigating alterations in circadian cycles that profoundly influence health and disease. However, applying such stimuli simultaneously is difficult in practice. We aimed to design, build, test, and open-source describe a simple device that subjects a conventional mouse cage to independent cycles of physiologically relevant environmental variables. The device is based on a box enclosing the rodent cage to modify the light, feeding, and temperature environments. The device provides temperature-controlled air conditioning (heating or cooling) by a Peltier module and includes programmable feeding and illumination. All functions are set by a user-friendly front panel for independent cycle programming. Bench testing with a model simulating the CO2 production of mice in the cage showed: a) suitable air renewal (by measuring actual ambient CO2), b) controlled realistic illumination at the mouse enclosure (measured by a photometer), c) stable temperature control, and d) correct cycling of light, feeding, and temperature. The cost of all the supplies (retail purchased by e-commerce) was <300 US$. Detailed technical information is open-source provided, allowing for any user to reliably reproduce or modify the device. This approach can considerably facilitate circadian research since using one of the described low-cost devices for any mouse group with a given light-food-temperature paradigm allows for all the experiments to be performed simultaneously, thereby requiring no changes in the light/temperature of a general-use laboratory. 1 Introduction

JTD Keywords: Animal experiment, Animal model, Animal research, Article, Circadian alteration, Circadian rhythm, Commercial phenomena, Controlled study, Cycling, Energy consumption, Energy-expenditure, Experimental model, Feeding, Food, Food availability, Illumination, Intermittent fasting, Light, Light cycle, Light dark cycle, Mouse, Nonhuman, Open source technology, Open-source hardware, Performance, Photography, Research, Rhythms, Rodent, Temperature, Temperature cycle

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

JTD Keywords: 3 & prime, alternative splicing, gene expression, meiosis, oocyte competence, program, rna, splicing, untranslated region processing, untranslated regions, 3′ untranslated region processing, 3′ untranslated regions, Alternative splicing, Expression, Gene expression, Human oocytes, Meiosis, Oocyte competence, Splicing

Peri-implantitis, caused by an inflammatory response to pathogens, is the leading cause of dental implant failure. Poor soft tissue healing surrounding implants - caused by inadequate surface properties - leads to infection, inflammation, and dysregulated keratinocyte and macrophage function. One activated inflammatory response, active around peri-implantitis compared to healthy sites, is the IL-23/IL-17A cytokine axis. Implant surfaces can be synthesized with peptide nanocoatings to present immunomodulatory motifs to target peri-implant keratinocytes to control macrophage polarization and regulate inflammatory axises toward enhancing soft tissue healing.We synthesized an IL-23 receptor (IL-23R) noncompetitive antagonist peptide nanocoating using silanization and evaluated keratinocyte secretome changes and macrophage polarization (M1-like "pro-inflammatory" vs. M2-like "pro-regenerative").IL-23R antagonist peptide nanocoatings were successfully synthesized on titanium, to model dental implant surfaces, and compared to nonfunctional nanocoatings and non-coated titanium. IL-23R antagonist nanocoatings significantly decreased keratinocyte IL-23, and downstream IL-17A, expression compared to controls. This peptide noncompetitive antagonistic function was demonstrated under lipopolysaccharide stimulation. Large scale changes in keratinocyte secretome content, toward a pro-regenerative milieu, were observed from keratinocytes cultured on the IL-23R antagonist nanocoatings compared to controls. Conditioned medium collected from keratinocytes cultured on the IL-23R antagonist nanocoatings polarized macrophages toward a M2-like phenotype, based on increased CD163 and CD206 expression and reduced iNOS expression, compared to controls.Our results support development of IL-23R noncompetitive antagonist nanocoatings to reduce the pro-inflammatory IL-23/17A pathway and augment macrophage polarization toward a pro-regenerative phenotype. Immunomodulatory implant surface engineering may promote soft tissue healing and thereby reduce rates of peri-implantitis.Copyright © 2023 Elsevier Inc. All rights reserved.

JTD Keywords: agents, alter, bioactivity, cells, dental implant, growth, keratinocyte, macrophage, peptide, peri -implant infection, peri-implant infection, Surface chemistry, Titanium

Different copolymers incorporating terpene oxide units (e.g., limonene oxide) have been evaluated considering thermal properties, degradability, and biocompatibility. Thus, polycarbonates and polyesters derived from aromatic, monocyclic and bicyclic anhydrides have been considered. Furthermore, ring substitution with myrcene terpene has been evaluated. All polymers were amorphous when evaluated directly from synthesis. However, spherulites could be observed after the slow evaporation of diluted chloroform solutions of polylimonene carbonate, with all isopropene units possessing an R configuration. This feature was surprising considering the reported information that suggested only the racemic polymer was able to crystallize. All polymers were thermally stable and showed a dependence of the maximum degradation rate temperature (from 242 °C to 342 °C) with the type of terpene oxide. The graduation of glass transition temperatures (from 44 °C to 172 °C) was also observed, being higher than those corresponding to the unsubstituted polymers. The chain stiffness of the studied polymers hindered both hydrolytic and enzymatic degradation while a higher rate was detected when an oxidative medium was assayed (e.g., weight losses around 12% after 21 days of exposure). All samples were biocompatible according to the adhesion and proliferation tests performed with fibroblast cells. Hydrophobic and mechanically consistent films (i.e., contact angles between 90° and 110°) were obtained after the evaporation of chloroform from the solutions, having different ratios of the studied biobased polyterpenes and poly(butylene succinate) (PBS). The blend films were comparable in tensile modulus and tensile strength with the pure PBS (e.g., values of 330 MPa and 7 MPa were determined for samples incorporating 30 wt.% of poly(PA-LO), the copolyester derived from limonene oxide and phthalic anhydride. Blends were degradable, biocompatible and appropriate to produce oriented-pore and random-pore scaffolds via a thermally-induced phase separation (TIPS) method and using 1,4-dioxane as solvent. The best results were attained with the blend composed of 70 wt.% PBS and 30 wt.% poly(PA-LO). In summary, the studied biobased terpene derivatives showed promising properties to be used in a blended form for biomedical applications such as scaffolds for tissue engineering.

JTD Keywords: alternating copolymerization, biobased materials, biodegradability, composites, crystallization, cyclohexene oxide, induced phase-separation, limonene oxide, mechanical-properties, polyesters, scaffolds, spherulites, terpene derivatives, thermal properties, thermally-induced phase separation, Acetone, Bio-based, Bio-based materials, Biobased materials, Biocompatibility, Biodegradability, Butenes, Cell culture, Chlorine compounds, Degradation, Evaporation, Glass transition, Limonene oxide, Monoterpenes, Phase separation, Poly (butylenes succinate), Polybutylene succinate, Property, Ring-opening copolymerization, Scaffolds, Spheru-lites, Tensile strength, Terpene derivatives, Thermal properties, Thermally induced phase separation, Thermally-induced phase separation, Thermally?induced phase separation, Thermodynamic properties, Thermogravimetric analysis

The ability to control neural activity is essential for research not only in basic neuroscience, as spatiotemporal control of activity is a fundamental experimental tool, but also in clinical neurology for therapeutic brain interventions. Transcranial-magnetic, ultrasound, and alternating/direct current (AC/DC) stimulation are some available means of spatiotemporal controlled neuromodulation. There is also light-mediated control, such as optogenetics, which has revolutionized neuroscience research, yet its clinical translation is hampered by the need for gene manipulation. As a drug-based light-mediated control, the effect of a photoswitchable muscarinic agonist (Phthalimide-Azo-Iper (PAI)) on a brain network is evaluated in this study. First, the conditions to manipulate M2 muscarinic receptors with light in the experimental setup are determined. Next, physiological synchronous emergent cortical activity consisting of slow oscillations-as in slow wave sleep-is transformed into a higher frequency pattern in the cerebral cortex, both in vitro and in vivo, as a consequence of PAI activation with light. These results open the way to study cholinergic neuromodulation and to control spatiotemporal patterns of activity in different brain states, their transitions, and their links to cognition and behavior. The approach can be applied to different organisms and does not require genetic manipulation, which would make it translational to humans.

JTD Keywords: brain states, light-mediated control, muscarinic acetylcholine receptors, neuromodulation, Activation, Alternating/direct currents, Animals, Basal forebrain, Brain, Brain states, Clinical research, Clinical translation, Controlled drug delivery, Cortex, Ferrets, Forebrain cholinergic system, Genetic manipulations, Higher frequencies, Hz oscillation, Light‐, Light-mediated control, Mediated control, Mice, Mice, inbred c57bl, Models, animal, Muscarinic acetylcholine receptors, Muscarinic agonists, Muscarinic receptor, Neurology, Neuromodulation, Neurons, Noradrenergic modulation, Parvalbumin-positive interneurons, Photopharmacology, Receptor-binding, Slow, Spatiotemporal control, Spatiotemporal patterns

Tau protein is largely responsible for tauopathies, including Alzheimer’s disease (AD), where it accumulates in the brain as insoluble aggregates. Tau mRNA is regulated by alternative splicing, and inclusion or exclusion of exon 10 gives rise to the 3R and 4R isoforms respectively, whose balance is physiologically regulated. In this sense, one of the several factors that regulate alternative splicing of tau is GSK3?, whose activity is inhibited by the cellular prion protein (PrPC), which has different physiological functions in neuroprotection and neuronal differentiation. Moreover, a relationship between PrPC and tau expression levels has been reported during AD evolution. For this reason, in this study we aimed to analyze the role of PrPC and the implication of GSK3? in the regulation of tau exon 10 alternative splicing. We used AD human samples and mouse models of PrPC ablation and tau overexpression. In addition, we used primary neuronal cultures to develop functional studies. Our results revealed a paralleled association between PrPC expression and tau 4R isoforms in all models analyzed. In this sense, reduction or ablation of PrPC levels induces an increase in tau 3R/4R balance. More relevantly, our data points to GSK3? activity downstream from PrPC in this phenomenon. Our results indicate that PrPC plays a role in tau exon 10 inclusion through the inhibitory capacity of GSK3?. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: alternative splicing, alzheimer's disease, alzheimers-disease, alzheimer’s disease, amyloid-beta, cellular prion protein, frontotemporal dementia, glycogen-synthase kinase-3, gsk3 beta, gsk3?, gsk3β, messenger-rna, microtubule-associated protein tau, neurofibrillary tangles, progressive supranuclear palsy, promotes neuronal differentiation, stem-cells, tauopathies, Alternative splicing, Alzheimer’s disease, Cellular prion protein, Gsk3?, Microtubule-associated protein tau, Tauopathies

During alternating ventilation (AV) one lung is inflating while the other is deflating. Considering the possible respiratory and hemodynamic advantages of AV, we investigated its effects during increased intra-abdominal pressure (IAP = 10 mmHg). In Sprague-Dawley rats (n = 6, 270–375 g) the main bronchi were independently cannulated, and respiratory mechanics determined while animals underwent different ventilatory patterns: synchronic ventilation without increased IAP (SV-0), elevated IAP during SV (SV-10), and AV with elevated IAP (AV-10). Thirty-three other animals (SV-0, n = 10; SV-10, n = 11 and AV-10, n = 12) were ventilated during 3 h. Mean arterial pressure (MAP), and lung histology were assessed. Increased IAP resulted in significantly higher elastances (p < 0.001), being AV-10 lower than SV-10 (p < 0.020). SV-10 showed higher central venous pressure (p < 0.003) than S-0; no change was observed in AV-10. Wet/dry lung weight ratio was lower in AV-10 than SV-10 (p = 0.009). Application of AV reduced hemodynamic and lung impairments induced by increased IAP during SV.

JTD Keywords: Alternating ventilation, Respiratory mechanics, Intra-abdominal pressure, Hemodynamic, Mechanical ventilation, Animal model