Modulación de la actividad de la fosfoenolpiruvato carboxiquinasa citosólica (PEPCK-C) porcina por efectores alostéricos, por mutagénesis y por las regiones reguladoras de la expresión

La fosfoenolpiruvato carboxiquinasa citosólica (PEPCK-C) porcina es una enzima relevante en la determinación de los caracteres de calidad de la carne y de la canal, como el espesor de la grasa dorsal, la grasa intramuscular o la exudación. En un trabajo previo de nuestro grupo demostramos la importancia de la mutación p.M139L de la PEPCK-C porcina y el probable papel in vivo de los dos sentidos de la reacción. En esta tesis se exploran nuevas formas de regulación de la actividad de la PEPCK-C.La primera forma de regulación estudiada fue el uso de efectores enzimáticos. Mediante el uso del inhibidor ácido 3-mercaptopicolínico se ha determinado que la sustitución M139L produce modificaciones en el sitio de unión de GTP/GDP al enzima causantes de las diferencias en la actividad enzimática. A su vez se han descrito dos nuevos inhibidores no competitivos de la PEPCK-C, los cuales podrían modificarse químicamente para hacerlos más solubles y ser utilizados in vivo para modular la actividad de la PEPCK-C.La segunda forma de regulación estudiada fue la creación de mutantes artificiales de la PEPCK-C con una de sus dos actividades inhibida y la otra sin modificar. Estos mutantes son estables en cultivo celular, por lo que podrían utilizarse para estudiar in vivo como ambos sentidos de la reacción catalizada por la PEPCK-C modifican las variaciones en la infiltración grasa del cerdo.La tercera forma de regulación estudiada fue la búsqueda de nuevos SNPs en las regiones reguladoras del gen de la PEPCK-C. Se han descrito 62 SNPs, de los cuales uno situado en la región 3’UTR era interesante por aparecer en cerdos Duroc y por estar en el sitio de unión de un microARN que regula el metabolismo de la glucosa. Al analizar el SNP se observó que aparece en poblaciones Duroc x Landrace/Large White actuales pero no está asociado a ningún carácter fenotípico de infiltración grasa.<br /

Role of mTOR-regulated autophagy in spine pruning defects and memory impairments induced by binge-like ethanol treatment in adolescent mice

Adolescence is a brain maturation developmental period during which remodeling and changes in synaptic plasticity and neural connectivity take place in some brain regions. Different mechanism participates in adolescent brain maturation, including autophagy that plays a role in synaptic development and plasticity. Alcohol is a neurotoxic compound and its abuse in adolescence induces neuroinflammation, synaptic and myelin alterations, neural damage and behavioral impairments. Changes in synaptic plasticity and its regulation by mTOR have also been suggested to play a role in the behavioral dysfunction of binge ethanol drinking in adolescence. Therefore, by considering the critical role of mTOR in both autophagy and synaptic plasticity in the developing brain, the present study aims to evaluate whether binge ethanol treatment in adolescence would induce dysfunctions in synaptic plasticity and cognitive functions and if mTOR inhibition with rapamycin is capable of restoring both effects. Using C57BL/6 adolescent female and male mice (PND30) treated with ethanol (3 g/kg) on two consecutive days at 48-hour intervals over 2 weeks, we show that binge ethanol treatment alters the density and morphology of dendritic spines, effects that are associated with learning and memory impairments and changes in the levels of both transcription factor CREB phosphorylation and miRNAs. Rapamycin administration (3 mg/kg) prior to ethanol administration restores ethanol-induced changes in both plasticity and behavior dysfunctions in adolescent mice. These results support the critical role of mTOR/autophagy dysfunctions in the dendritic spines alterations and cognitive alterations induced by binge alcohol in adolescence

Age-Mediated Transcriptomic Changes in Adult Mouse Substantia Nigra

Substantia nigra pars compacta (SNpc) is highly sensitive to normal aging and selectively degenerates in Parkinson's disease (PD). Until now, molecular mechanisms behind SNpc aging have not been fully investigated using high throughput techniques. Here, we show early signs of aging in SNpc, which are more evident than in ventral tegmental area (VTA), a region adjacent to SNpc but less affected in PD. Aging-associated early changes in transcriptome were investigated comparing late middle-aged (18 months old) to young (2 months old) mice in both SNpc and VTA. A meta-analysis of published microarray studies allowed us to generate a common >transcriptional signature> of the aged (≥ 24 months old) mouse brain. SNpc of late-middle aged mice shared characteristics with the transcriptional signature, suggesting an accelerated aging in SNpc. Age-dependent changes in gene expression specific to SNpc were also observed, which were related to neuronal functions and inflammation. Future studies could greatly help determine the contribution of these changes to SNpc aging. These data help understand the processes underlying SNpc aging and their potential contribution to age-related disorders like PD. © 2013 Gao et al.This work was funded by Spanish Ministry of Science and Education, Andalusian Government, and “Marcelino Botín” Foundation. “CIBERNED” (Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas) was funded by the Spanish “Carlos III” Institute of Health. LME was supported by the Spanish “Carlos III” Institute of Health. Support from the Spanish Ministry of Science and Education for MHF (“FPI” predoctoral fellowship) is also acknowledged.Peer Reviewe

Survival Time after Surgical Debulking and Temozolomide Adjuvant Chemotherapy in Canine Intracranial Gliomas

Intracranial gliomas are associated with a poor prognosis, and the most appropriate treatment is yet to be defined. The objectives of this retrospective study are to report the time to progression and survival times of a group of dogs with histologically confirmed intracranial gliomas treated with surgical debulking and adjuvant temozolomide chemotherapy. All cases treated in a single referral veterinary hospital from 2014 to 2021 were reviewed. Inclusion criteria comprised a histopathological diagnosis of intracranial glioma, adjunctive chemotherapy, and follow-up until death. Cases were excluded if the owner declined chemotherapy or there was insufficient follow-up information in the clinical records. Fourteen client-owned dogs were included with a median time to progression (MTP) of 156 days (95% CI 133-320 days) and median survival time (MST) of 240 days (95% CI 149-465 days). Temozolomide was the first-line adjuvant chemotherapy but changed to another chemotherapy agent (lomustine, toceranib phosphate, or melphalan) when tumour relapse was either suspected by clinical signs or confirmed by advanced imaging. Of the fourteen dogs, three underwent two surgical resections and one, three surgeries. Survival times (ST) were 241, 428, and 468 days for three dogs treated twice surgically and 780 days for the dog treated surgically three times. Survival times for dogs operated once was 181 days. One case was euthanized after developing aspiration pneumonia, and all other cases after progression of clinical signs due to suspected or confirmed tumour relapse. In conclusion, the results of this study suggest that debulking surgery and adjuvant chemotherapy are well-tolerated options in dogs with intracranial gliomas in which surgery is a possibility and should be considered a potential treatment option. Repeated surgery may be considered for selected cases

Developing Ubiquitous Sensor Network Platform Using Internet of Things: Application in Precision Agriculture

The application of Information Technologies into Precision Agriculture methods has clear benefits. Precision Agriculture optimises production efficiency, increases quality, minimises environmental impact and reduces the use of resources (energy, water); however, there are different barriers that have delayed its wide development. Some of these main barriers are expensive equipment, the difficulty to operate and maintain and the standard for sensor networks are still under development. Nowadays, new technological development in embedded devices (hardware and communication protocols), the evolution of Internet technologies (Internet of Things) and ubiquitous computing (Ubiquitous Sensor Networks) allow developing less expensive systems, easier to control, install and maintain, using standard protocols with low-power consumption. This work develops and test a low-cost sensor/actuator network platform, based in Internet of Things, integrating machine-to-machine and human-machine-interface protocols. Edge computing uses this multi-protocol approach to develop control processes on Precision Agriculture scenarios. A greenhouse with hydroponic crop production was developed and tested using Ubiquitous Sensor Network monitoring and edge control on Internet of Things paradigm. The experimental results showed that the Internet technologies and Smart Object Communication Patterns can be combined to encourage development of Precision Agriculture. They demonstrated added benefits (cost, energy, smart developing, acceptance by agricultural specialists) when a project is launched.This research was supported by Industrial Computers and Computer Networks program (I2RC) (2015/2016) funded by the University of Alicante

Exploring the interplay between metabolic pathways and taxane production in elicited Taxus baccata cell suspensions

Taxus cell cultures are a reliable biotechnological source of the anticancer drug paclitaxel. However, the interplay between taxane production and other metabolic pathways during elicitation remains poorly understood. In this study, we combined untargeted metabolomics and elicited Taxus baccata cell cultures to investigate variations in taxane-associated metabolism under the influence of 1 µM coronatine (COR) and 150 µM salicylic acid (SA). Our results demonstrated pleiotropic effects induced by both COR and SA elicitors, leading to differential changes in cell growth, taxane content, and secondary metabolism. Metabolite annotation revealed significant effects on N-containing compounds, phenylpropanoids, and terpenoids. Multivariate analysis showed that the metabolomic profiles of control and COR-treated samples are closer to each other than to SA-elicited samples at different time points (8, 16, and 24 days). The highest level of paclitaxel content was detected on day 8 under SA elicitation, exhibiting a negative correlation with the biomarkers kauralexin A2 and taxusin. Our study provides valuable insights into the intricate metabolic changes associated with paclitaxel production, aiding its potential optimization through untargeted metabolomics and an evaluation of COR/SA elicitor effects.Agencia Estatal de Investigación | Ref. PID2020-113438RB-I00Agència de Gestió d’Ajuts Universitaris i de Recerca | Ref. 2021 SGR00693Ministerio de Ciencia, Innovación y Universidades | Ref. FPU18/00850Ministerio de Universidades | Ref. 33.50.460A.75

Impact of Elicitation on Plant Antioxidants Production in Taxus Cell Cultures

Elicited cell cultures of Taxus spp. are successfully used as sustainable biotechnological production systems of the anticancer drug paclitaxel, but the effect of the induced metabolomic changes on the synthesis of other bioactive compounds by elicitation has been scarcely studied. In this work, a powerful combinatorial approach based on elicitation and untargeted metabolomics was applied to unravel and characterize the effects of the elicitors 1 M of coronatine (COR) or 150 M of salicylic acid (SA) on phenolic biosynthesis in Taxus baccata cell suspensions. Differential effects on cell growth and the phenylpropanoid biosynthetic pathway were observed. Untargeted metabolomics analysis revealed a total of 83 phenolic compounds, mainly flavonoids, phenolic acids, lignans, and stilbenes. The application of multivariate statistics identified the metabolite markers attributed to elicitation over time: up to 34 compounds at 8 days, 41 for 16 days, and 36 after 24 days of culture. The most notable metabolic changes in phenolic metabolism occurred after 8 days of COR and 16 days of SA elicitation. Besides demonstrating the significant and differential impact of elicitation treatments on the metabolic fingerprint of T. baccata cell suspensions, the results indicate that Taxus ssp. biofactories may potentially supply not only taxanes but also valuable phenolic antioxidants, in an efficient optimization of resources