2. Universidad Cardenal Herrera-CEU

The ability to communicate scientific information effectively is crucial for veterinary professionals. Consequently, veterinary students require consistent and proactive training in communication. In parallel, digital transformation has undoubtedly impacted educational institutions. To address these priorities, the TranspaVET project aimed to design an immersive educational experience through advanced and innovative technology. This article aims to share our experience involving first-year veterinary students in producing scientific posters and how augmented reality (AR) and virtual reality (VR) impact their scientific outreach. The project, developed in the academic year 2022-23, involved 35 students and eight mentor professors and resulted in nine scientific posters. The posters were digitized allowing their access through links or Quick Response (QR) codes. Firstly, they could be visualised in 3D Web preview and in AR, as images overlayed into reality through mobile devices. Secondly, they could be viewed in an immersive VR educational metaverse. Visitors could access the metaverse via their personal electronic devices and via VR headsets. Users can view, download, and share the posters and talk together inside the virtual environment. By January 7th, 2024, the posters were digitally viewed 1,795 times, and 207 unique users entered the TranspaVET metaverse from different Spanish regions (Valencian Community, Castile and Leon, Andalusia, Madrid, Catalonia, Asturias, and Galicia), as well as from Argentina and Costa Rica. The TranspaVET project represents a creative connection between educational innovation and scientific research dissemination. It sets an example for the future of immersive, technology-driven learning through a platform that combines AR and VR.

Background: Multiple myeloma (MM) is the second most common hematologic neoplasm which is characterized by proliferation and infiltration of plasmatic cells in the bone marrow. Currently, MM is considered incurable due to resistance to treatment. The CRISPR/Cas9 system has emerged as a powerful tool for understanding the role of different genetic alterations in the pathogenesis of hematologic malignancies in both cell lines and mouse models. Despite current advances of gene editing tools, the use of CRISPR/Cas9 technology for gene editing of MM have not so far been extended. In this work, we want to repress Rnd3 expression, an atypical Rho GTPase involved in several cellular processes, in MM cell lines using a CRISPR interference strategy. Results: We have designed different guide RNAs and cloning them into a lentiviral plasmid, which contains all the machinery necessary for developing the CRISPR interference strategy. We co-transfected the HEK 293T cells with this lentiviral plasmid and 3rd generation lentiviral envelope and packaging plasmids to produce lentiviral particles. The lentiviral particles were used to transduce two different multiple myeloma cell lines, RPMI 8226 and JJN3, and downregulate Rnd3 expression. Additionally, the impact of Rnd3 expression absence was analyzed by a transcriptomic analysis consisting of 3' UTR RNA sequencing. The Rnd3 knock-down cells showed a different transcriptomic profile in comparison to control cells. Conclusions: We have developed a CRISPR interference strategy to generate stable Rnd3 knockdown MM cell lines by lentiviral transduction. We have evaluated this strategy in two MM cell lines, and we have demonstrated that Rnd3 silencing works both at transcriptional and protein level. Therefore, we propose CRISPR interference strategy as an alternative tool to silence gene expression in MM cell lines. Furthermore, Rnd3 silencing produces changes in the cellular transcriptomic profile.

Macrophage activation is a complex process with multiple control elements that ensures an adequate response to the aggressor pathogens and, on the other hand, avoids an excess of inflammatory activity that could cause tissue damage. In this study, we have identified RND3, a small GTP-binding protein, as a new element in the complex signaling process that leads to macrophage activation. We show that RND3 expression is transiently induced in macrophages activated through Toll receptors and potentiated by IFN-γ. We also demonstrate that RND3 increases NOTCH signaling in macrophages by favoring NOTCH1 expression and its nuclear activity; however, Rnd3 expression seems to be inhibited by NOTCH signaling, setting up a negative regulatory feedback loop. Moreover, increased RND3 protein levels seem to potentiate NFκB and STAT1 transcriptional activity resulting in increased expression of proinflammatory genes, such as Tnf-α, Irf-1, or Cxcl-10. Altogether, our results indicate that RND3 seems to be a new regulatory element which could control the activation of macrophages, able to fine tune the inflammatory response through NOTCH.

Glucose uptake into the mammalian nervous system is mediated by the family of facilitative glucose transporter proteins (GLUT). In this work we investigate how the expression of the main neuronal glucose transporters (GLUT3, GLUT4 and GLUT8) is modified during cerebellar cortex maturation. Our results reveal that the levels of the three transporters increase during the postnatal development of the cerebellum. GLUT3 localizes in the growing molecular layer and in the internal granule cell layer. However, the external granule cell layer, Purkinje cell cytoplasm and cytoplasm of the other cerebellar cells lack GLUT3 expression. GLUT4 and GLUT8 have partially overlapping patterns, which are detected in the cytoplasm and dendrites of Purkinje cells, and also in the internal granule cell layer where GLUT8 displays a more diffuse pattern. The differential localization of the transporters suggests that they play different roles in the cerebellum, although GLUT4 and GLUT8 could also perform some compensatory or redundant functions. In addition, the increase in the levels and the area expressing the three transporters suggests that these roles become more important as development advances. Interestingly, the external granule cells, which have been shown to express the monocarboxylate transporter MCT2, express none of the three main neuronal GLUTs. However, when these cells migrate inwardly to differentiate in the internal granule cells, they begin to produce GLUT3, GLUT4 and GLUT8, suggesting that the maturation of the cerebellar granule cells involves a switch in their metabolism in such a way that they start using glucose as they mature.

Peripheral nerve axotomy in adult mice elicits a complex response that includes increased glucose uptake in regenerating nerve cells. This work analyses the expression of the neuronal glucose transporters GLUT3, GLUT4 and GLUT8 in the facial nucleus of adult mice during the first days after facial nerve axotomy. Our results show that whereas GLUT3 levels do not vary, GLUT4 and GLUT8 immunoreactivity increases in the cell body of the injured motoneurons after the lesion. A sharp increase in GLUT4 immunoreactivity was detected 3 days after the nerve injury and levels remained high on Day 8, but to a lesser extent. GLUT8 also increased the levels but later than GLUT4, as they only rose on Day 8 post-lesion. These results indicate that glucose transport is activated in regenerating motoneurons and that GLUT4 plays a main role in this function. These results also suggest that metabolic defects involving impairment of glucose transporters may be principal components of the neurotoxic mechanisms leading to motoneuron death.

Autophagy is a highly conserved process that mediates the targeting and degradation of intracellular components to lysosomes, contributing to the maintenance of cellular homeostasis and to obtaining energy, which ensures viability under stress conditions. Therefore, autophagy defects are common to different neurodegenerative disorders. Rnd3 belongs to the family of Rho GTPases, involved in the regulation of actin cytoskeleton dynamics and important in the modulation of cellular processes such as migration and proliferation. Murine models have shown that Rnd3 is relevant for the correct development and function of the Central Nervous System and lack of its expression produces several motor alterations and neural development impairment. However, little is known about the molecular events through which Rnd3 produces these phenotypes. Interestingly we have observed that Rnd3 deficiency correlates with the appearance of autophagy impairment profiles and irregular mitochondria. In this work, we have explored the impact of Rnd3 loss of expression in mitochondrial function and autophagy, using a Rnd3 KO CRISPR cell model. Rnd3 deficient cells show no alterations in autophagy and mitochondria turnover is not impaired. However, Rnd3 KO cells have an altered mitochondria oxidative metabolism, resembling the effect caused by oxidative stress. In fact, lack of Rnd3 expression makes these cells strictly dependent on glycolysis to obtain energy. Altogether, our results demonstrate that Rnd3 is relevant to maintain mitochondria function, suggesting a possible relationship with neurodegenerative diseases.

Las GTPasas de la familia Rho están adquiriendo un gran interés en los últimos años por su papel en el desarrollo y función del sistema nervioso central (SNC). RhoE es un miembro de esta gran familia cuyas funciones in vivo no están aún bien definidas. En este trabajo estudiamos la expresión de RhoE en el desarrollo embrionario y postnatal y su papel el SNC analizando las características de ratones modificados genéticamente, que no expresan el gen RhoE. Nuestros resultados muestran que RhoE se expresa en diferentes órganos embrionarios, entre los que se encuentra el SNC, desde edades tempranas. En el SNC postnatal se expresa con altos niveles en las primeras etapas y se localiza en numerosos sitios, destacando su presencia en las células de la corriente migratoria rostral, la capa granular externa del cerebelo o en las motoneuronas La eliminación de la expresión de RhoE se traduce en la aparición de graves alteraciones en el sistema nervioso. Los ratones que no expresan RhoE presentan un acúmulo anormal en la zona subventricular de células que hemos caracterizado como neuroblastos postmitóticos, que migran en una tasa mucho menor a través de la corriente migratoria rostral. En consecuencia, a los bulbos olfatorios llegan menos células, que, además, se disponen de forma alterada, de manera que las células calbindina positivas están en menor proporción y muy desorganizadas. Además, en los ratones que carecen de RhoE la rama anterior de la comisura anterior, que conecta ambos bulbos olfatorios, está ausente, y hay en todo su cerebro una reducción en la mielinización. Por otra parte, estos ratones tienen alterado el sistema motor, muestran un retraso en la maduración de las sinapsis neuromusculares y, sorprendentemente, no forman nervio peroneo común, que está ausente en todos los ratones analizados. Estos resultados muestran que RhoE es una proteína esencial en el desarrollo del sistema nervioso, que controla eventos tan diferentes como la migración neuronal y la formación de nervios periféricos. / Rnd proteins are a subfamily of Rho GTPases involved in the control of actin cytoskeleton dynamics, which differently to the other Rho members are constitutively active. In this work we describe Rnd3/RhoE expression and we show the in vivo effect of RhoE gene ablation in the central nervous system (CNS). RhoE is widely expressed during the embryonic development, including the CNS. RhoE is found at high levels in the postnatal CNS and is extensively localized, including the cells of the rostral migratory stream, cerebral cortex and olfactory bulb and those of the external granule cell layer of the cerebellum and the motoneurons. Mice lacking RhoE expression (RhoE gt/gt) accumulate cells in the subventricular zone, which show a delayed migration through the rostral migratory stream to the olfactory bulb. As a consequence, the calbindin positive cells of this region are disorganized. Moreover, RhoE null mice have reduced central myelination, lack of the anterior branch of the anterior commissure and are occasionally hydrocephalic. Finally, these mice have an altered motor system, with a delayed maturation of the neuromuscular junctions and, strikingly, they do not form the common peroneal nerve. These results show that RhoE is an essential protein for the nervous system development controlling important event such as neuronal migration and peripheral nerve formation.

Rho small GTPases are proteins with key roles in the development of the central nervous system. Rnd proteins are a subfamily of Rho GTPases characterized by their constitutive activity. Rnd3/RhoE is a member of this subfamily ubiquitously expressed in the CNS, whose specific functions during brain development are still not well defined. Since other Rho proteins have been linked to the myelination process, we study here the expression and function of Rnd3 in oligodendrocyte development. We have found that Rnd3 is expressed in a subset of oligodendrocyte precursor cells and of mature oligodendrocytes both in vivo and in vitro. We have analyzed the role of Rnd3 in myelination using mice lacking Rnd3 expression (Rnd3gt/gt mice), showing that these mice exhibit hypomyelination in the brain and a reduction in the number of mature and total oligodendrocytes in the corpus callosum and striatum. The mutants display a decreased expression of several myelin proteins and a reduction in the number of myelinated axons. In addition, myelinated axons exhibit thinner myelin sheaths. In vitro experiments using Rnd3gt/gt mutant mice showed that the differentiation of the precursor cells is altered in the absence of Rnd3 expression, suggesting that Rnd3 is directly required for the differentiation of oligodendrocytes and, in consequence, for the correct myelination of the CNS. This work shows Rnd3 as a new protein involved in oligodendrocyte maturation, opening new avenues to further study the function of Rnd3 in the development of the central nervous system and its possible involvement in demyelinating diseases.