1. Investigación

Although atherosclerosis preferentially develops at arterial curvatures and bifurcations where disturbed flow (DF) activates endothelium, therapies targeting flow-dependent mechanosensing pathways in the vasculature are unavailable. Here, we provided experimental evidence demonstrating a previously unidentified causal role of DF-induced endothelial TXNDC5 (thioredoxin domain containing 5) in atherosclerosis. TXNDC5 was increased in human and mouse atherosclerotic lesions and induced in endothelium subjected to DF. Endothelium-specific Txndc5 deletion markedly reduced atherosclerosis in ApoE−/− mice. Mechanistically, DF-induced TXNDC5 increases proteasome-mediated degradation of heat shock factor 1, leading to reduced heat shock protein 90 and accelerated eNOS (endothelial nitric oxide synthase) protein degradation. Moreover, nanoparticles formulated to deliver Txndc5-targeting CRISPR-Cas9 plasmids driven by an endothelium-specific promoter (CDH5) significantly increase eNOS protein and reduce atherosclerosis in ApoE−/− mice. These results delineate a new molecular paradigm that DF-induced endothelial TXNDC5 promotes atherosclerosis and establish a proof of concept of targeting endothelial mechanosensitive pathways in vivo against atherosclerosis.

Precision nutrition in broilers requires tools capable of identifying amino acid imbalances individually or in groups, as well as knowledge on how more digestible proteins can be designed for innovative feeding programs adjusted to animals’ dynamic requirements. This work proposes two potential tools, combining traditional nutrition with biotechnological, metabolomic, computational and protein engineering knowledge, which can contribute to improving the precise amino acid nutrition of broilers in the future: (i) the use of serum uric nitrogen content as a rapid biomarker of amino acid imbalances, and (ii) the design and modeling of de novo proteins that are fully digestible and fit exactly to the animal’s requirements. Each application is illustrated with a case study. Case study 1 demonstrates that serum uric nitrogen can be a useful rapid indicator of individual or group amino acid deficiencies or imbalances when reducing dietary protein and adjusting the valine and arginine to lysine ratios in broilers. Case study 2 describes a stepwise approach to design an ideal protein, resulting in a potential amino acid sequence and structure prototype that is ideally adjusted to the requirements of the targeted animal, and is theoretically completely digestible. Both tools can open up new opportunities to form an integrated framework for precise amino acid nutrition in broilers, helping us to achieve more efficient, resilient, and sustainable production. This information can help to determine the exact ratio of amino acids that will improve the efficiency of the use of nitrogen by poultry.

The ideal protein concept refers to dietary protein with an amino acid profile that exactly meets an animal’s requirement. Low-quality protein levels in the diet have negative implications for productive and reproductive traits, and a protein oversupply is energetically costly and leads to an excessive N excretion, with potentially negative environmental impact. Urea Nitrogen (UN), which corresponds to the amount of nitrogen in the form of urea circulating in the bloodstream, is a metabolite that has been widely used to detect amino acid imbalances and deficiencies and protein requirements. This review aims to critically analyse how UN can contribute to accurately implementing the ideal protein concept in monogastric animals, particularly in pig, poultry, and rabbit nutrition (14,000 animals from 76 published trials). About 59, 37, and 4% of trials have been conducted in pigs, poultry, and rabbits, respectively. UN level was negatively correlated to main performance traits (Pearson Correlation Coefficient [PCC] of 􀀀0.98 and 􀀀0.76, for average daily gain and feed conversion ratio, respectively), and lower UN level was related to higher milk yield and concentration. High level of UN was positively correlated to N excretion (PCC = 0.99) and negatively correlated to protein retention (PCC = 􀀀0.99). Therefore, UN in blood seems to be a proper indicator of amino acid imbalance in monogastric animals. Great variability in the use of UN was observed in the literature, including uses as determination medium (blood, plasma, or serum), units, and feeding system used (ad libitum or restricted), among others. A standardization of the methods in each of the species, with the aim to harmonize comparison among works, is suggested. After review, UN measurement in plasma and, whenever possible, the utilization of the same nutritional methodology (ad libitum conditions or restriction with blood sampling after refeeding at standardised time) are recommended. More studies are necessary to know the potential of UN and other bioindicators for amino acid deficiencies evaluation to get closer to the ideal protein concept.

El mieloma múltiple (MM) es una neoplasia hematológica muy común, caracterizada por la proliferación excesiva de células plasmáticas diferenciadas a partir de linfocitos B en el interior de la médula ósea. Esta enfermedad presenta diferentes etapas en las que evoluciona desde periodos tempranos asintomáticos hasta la manifestación de síntomas clínicos típicos del MM. A pesar de que actualmente existen diversos tratamientos para el MM, es frecuente la aparición de recaídas, siendo la tasa relativa de supervivencia a 5 años es del 50%. Esto implica que, a pesar de los avances científicos de los últimos años, la enfermedad sigue considerándose incurable, por lo que es necesario conocer con mayor profundidad los mecanismos celulares que intervienen tanto en el desarrollo de la enfermedad como en la aparición de resistencias al tratamiento. El objetivo de este trabajo es estudiar el papel de Rnd3 en el MM. Rnd3 es una proteína Rho atípica incluida dentro de la subfamilia Rnd. Las proteínas Rho están clásicamente implicadas en la reorganización del citoesqueleto de actina y han sido ampliamente relacionadas con procesos de transformación celular, destacando su implicación en la proliferación y la migración de las células tumorales. En el caso concreto de Rnd3 también existen evidencias que la relacionan con la progresión tumoral, aunque su función oncogénica u oncosupresora es dependiente del tipo celular y tumoral. Datos previos indican un incremento de la expresión de RND3 en muestras de MM procedentes de modelos murinos que mimetizan la enfermedad humana, así como también en células plasmáticas procedentes de pacientes con MM respecto a células plasmáticas sanas. Para profundizar en el papel de Rnd3 en el MM hemos generado dos líneas celulares de MM (RPMI 8226 y JJN3) deficientes en Rnd3, y hemos analizado su efecto en los procesos de proliferación, adhesión celular y sensibilidad a fármacos. Por otro lado, hemos realizado un estudio transcriptómico para determinar el papel de Rnd3 en la regulación de la expresión de genes implicados en MM. Como resultado destaca la expresión diferencial del gen que codifica para una quimiocina, CXCL10, que se encuentra implicada en la migración de las células del sistema inmuntario y que recientemente se ha relacionado con fenómenos de inmunoterapia e inmunovigilancia en diferentes tumores. Finalmente, hemos validado estos resultados en células deficientes en Rnd3 observando un incremento en la expresión de esta quimiocina, tanto a nivel de mRNA como de expresión proteica. Así pues, estos resultados indican un papel inhibidor de Rnd3 sobre la expresión de CXCL10. Sin embargo, esto no tiene un efecto sobre las propias células tumorales, sugiriendo un papel paracrino de CXCL10 sobre el microambiente tumoral y sobre el sistema inmunitario. En conjunto, este trabajo sugiriere un nuevo papel de Rnd3 en la regulación de la expresión de CXCL10 y su posible papel en la patología del MM.

In some species, female steroid hormones modify the profile of acute phase proteins (APPs) during the estrous cycle and pregnancy, according to the ovulation, embryonic implantation and placental development; however, nowadays there’s no experimental evidence for equine species. Objectives of this study were: to compare the serum amyloid A (SAA), haptoglobin (Hp) and C-reactive protein (CRP) concentrations between cyclic and pregnant mares, and to analyze the influence of estradiol-17β (E2) during estrous cycle or estrone sulfate (E1) during pregnancy, and progesterone (P4) on these proteins to assess their potential role to identify the cyclicity or pregnancy in Spanish mares. Blood samples were taken from 20 Purebred Spanish mares on the day of ovulation (day 0), on days +5 and +16 post-ovulation, and then, monthly during the whole pregnancy. SAA, Hp and CRP did not change between day 0, +5 and +16 post-ovulation days. P4 concentrations were significantly higher on day +16 than on days +5 and 0; and E2 concentrations were significantly higher on day 0 than day +5. On the other hand, pregnancy was characterized by a progressive increase in the Hp, variable modifications of E1 and P4 concentrations, without changes in SAA and CRP. The absence of significant differences in the APPs between days 0, +5 and +16, suggested that these proteins cannot be used as biomarkers of diagnosis of heat or preg- nancy in Spanish mares, at least early, since the Hp later increases during the gestation. Nevertheless, it is possible to use them for comparative purposes with other equine breeds, as supervisor instrument of health status in breeding females as diagnostic tools to monitor pregnancy’s development and/or subclinical reproductive inflammations, that could lead to the early embryonic death.

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.

Autofagia es el proceso por el cual se media el direccionamiento de material citoplasmático (proteínas y orgánulos) hacia los lisosomas para su posterior degradación. Su correcto funcionamiento es clave para mantener la homeostasis celular y controlar los niveles y la calidad de los constituyentes intracelulares, contribuyendo a la obtención de energía y permitiendo la adaptación de la célula a condiciones de estrés. Se ha descrito que un mal funcionamiento en el proceso de autofagia está relacionado con numerosas enfermedades neurodegenerativas (Parkinson, Alzhéimer o esclerosis lateral amiotrófica), siendo una característica común la presencia de agregados proteicos u orgánulos disfuncionales, que, en parte, acaban explicando la aparición de los distintos síntomas clínicos. Las proteínas Rho, implicadas en la regulación de la dinámica del citoesqueleto de actina, juegan un papel clave en el desarrollo y función del sistema nervioso central. Rnd3/RhoE es un miembro de la subfamilia Rnd y se caracteriza por estar constitutivamente activada. Su función en migración, proliferación y supervivencia celular ha sido ampliamente estudiada. Rnd3 es necesario para el correcto desarrollo y funcionamiento del SNC. En nuestro grupo, la utilización de un modelo de ratón genéticamente modificado (Rnd3gt/gt) ha demostrado que la ausencia de esta proteína produce graves alteraciones motoras y de desarrollo neural, destacando la presencia de agregados proteicos y mitocondrias morfológicamente aberrantes en neuronas, un fenotipo compatible con problemas en el proceso de autofagia, tal y como se ha descrito en algunas patologías neurodegenerativas. En este estudio nos planteamos analizar el posible papel de Rnd3 en autofagia, con el objetivo de poder entender alguno de los fenotipos que aparecen en el modelo murino deficiente en esta proteína. Nuestros resultados demuestran que la ausencia de Rnd3 aumenta la tasa de degradación proteica y provoca un aumento en autofagia en condiciones basales, sugiriendo que Rnd3 es un regulador negativo de este proceso, actuando a nivel de la maduración del autofagosoma. Además, hemos demostrado un nuevo papel de Rnd3 en la homeostasis mitocondrial. La presencia de mitocondrias no funcionales en células deficientes para Rnd3 provoca una alteración del metabolismo oxidativo y una activación de la glucólisis como mecanismo alternativo para la obtención de energía. Todos estos resultados muestran un nuevo papel de Rnd3 en la regulación de autofagia, así como en la homeostasis mitocondrial, lo que podría explicar algunos de los fenotipos observados en el modelo de ratón deficiente para Rnd3. / Autophagy is the cellular process by which lysosomes contribute to the degradation of intracellular components, including proteins and organelles. The correct function of this catabolic process is necessary for cell homeostasis and controls the levels and quality of intracellular constituents, not only contributing to obtaining energy but also allowing cells to adapt to stress conditions. It has been described that an alteration in the autophagy process is related to numerous neurodegenerative diseases (Parkinson's, Alzheimer's or amyotrophic lateral sclerosis). Moreover, autophagy malfunction leads to protein aggregates and dysfunctional organelles accumulation that can explain some of the clinical symptoms. Rho proteins are involved in the regulation of the actin cytoskeleton dynamics and also have been postulated to play a key role in the development and function of the Central Nervous System. Rnd3/RhoE is a member of the Rnd subfamily, being constitutively activated. Its role in cell migration, proliferation and survival has been extensively studied. Rnd3 is necessary for the correct development and function of the CNS. In our group, the use of a genetically modified mouse model (Rnd3gt/gt) revealed that the lack of this protein produces serious motor and neural development alterations. Moreover, Rnd3 deficient neurons show protein aggregates and aberrant mitochondria, a phenotype compatible with problems in autophagy, as also described in some neurodegenerative pathologies. In this study we sought to analyze the possible role of Rnd3 in autophagy, in order to understand some of the phenotypes that appear in the Rnd3 deficient murine model. Our results demonstrate that the absence of Rnd3 promotes higher protein degradation rates and increased levels of autophagy even in basal conditions, suggesting that Rnd3 negatively regulates this process, at the level of the autophagosome maturation. In addition, we have demonstrated a new role of Rnd3 in mitochondrial homeostasis. The presence of nonfunctional mitochondria in Rnd3-deficient cells causes an alteration in the oxidative metabolism and an activation of glycolysis as an alternative mechanism to obtain energy. Altogether, these results highlight a new role of Rnd3 in the regulation of autophagy and mitochondrial homeostasis, which could explain some of the phenotypes observed in the Rnd3gt/gt mouse model.

Acute phase proteins (APP) are biomarkers of systemic inflammation, which allow monitoring the evolution of diseases, the response to treatments, and post-operative complications. Ovariectomy (OVE) is frequently performed in veterinary medicine and can be a useful model to evaluate surgical trauma and inflammation in the bitch. The objective was to investigate and compare the acute phase response (APR) after applying three di erent OVE techniques by measuring serum levels of C-reactive protein (CRP), haptoglobin (Hp), albumin (Alb), and paraoxonase-1 (PON-1). Forty-five intact bitches were included in the study, being randomly distributed into three groups: laparoscopic OVE (L), midline OVE (M), and flank OVE (F). Serum CRP, Hp, Alb, and PON-1 were measured before surgery, 1, 24, 72, and 168 h post-intervention. CRP levels increased significantly 24 h post-surgery in theMand F groups, but no significant variation was observed in the L group at any time of the study period. Hp was significantly higher in group L than in group F 72 h post-surgery. Alb and PON-1 showed no statistical di erence among groups or among sampling periods. CRP response suggests that the use of laparoscopic procedures produce lower inflammation compared to open conventional approaches when performing OVE in the bitch.

Objectives: To investigate the contribution to virulence of the surface protein internalin B (InlB) in the Listeria monocytogenes lineage I strain F2365, which caused a deadly listeriosis outbreak in California in 1985. Methods: The F2365 strain displays a point mutation that hampers expression of InlB. We rescued the expression of InlB in the L. monocytogenes lineage I strain F2365 by introducing a point mutation in the codon 34 (TAA to CAA). We investigated its importance for bacterial virulence using in vitro cell infection systems and a murine intravenous infection model. Results: In HeLa and JEG-3 cells, the F2365 InlBþ strain expressing InlB was z9-fold and z1.5-fold more invasive than F2365, respectively. In livers and spleens of infected mice at 72 hours after infection, bacterial counts for F2365 InlBþ were significantly higher compared to the F2365 strain (z1 log more), and histopathologic assessment showed that the F2365 strain displayed a reduced number of necrotic foci compared to the F2365 InlBþ strain (Mann-Whitney test). Conclusions: InlB plays a critical role during infection of nonpregnant animals by a L. monocytogenes strain from lineage I. A spontaneous mutation in InlB could have prevented more severe human morbidity and mortality during the 1985 California listeriosis outbreak.