Modulación de la proteostasis celular por el inhibidor Ala-Ala-Phe- clorometilcetona

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 04-04-201

p73 as a Tissue Architect

Sec. Molecular and Cellular Pathology[EN] The TP73 gene belongs to the p53 family comprised by p53, p63, and p73. In response to physiological and pathological signals these transcription factors regulate multiple molecular pathways which merge in an ensemble of interconnected networks, in which the control of cell proliferation and cell death occupies a prominent position. However, the complex phenotype of the Trp73 deficient mice has revealed that the biological relevance of this gene does not exclusively rely on its growth suppression effects, but it is also intertwined with other fundamental roles governing different aspects of tissue physiology. p73 function is essential for the organization and homeostasis of different complex microenvironments, like the neurogenic niche, which supports the neural progenitor cells and the ependyma, the male and female reproductive organs, the respiratory epithelium or the vascular network. We propose that all these, apparently unrelated, developmental roles, have a common denominator: p73 function as a tissue architect. Tissue architecture is defined by the nature and the integrity of its cellular and extracellular compartments, and it is based on proper adhesive cell-cell and cell-extracellular matrix interactions as well as the establishment of cellular polarity. In this work, we will review the current understanding of p73 role as a neurogenic niche architect through the regulation of cell adhesion, cytoskeleton dynamics and Planar Cell Polarity, and give a general overview of TAp73 as a hub modulator of these functions, whose alteration could impinge in many of the Trp73–/– phenotypesSIThis work was supported by Grant PID2019-105169RB-I00 from Spanish Ministerio de Ciencia e Innovación cofinanced by FEDER funds (to MCM). LM-A was a holder of a predoctoral scholarship from the Asociación Española contra el Cáncer (AECC) and was funded by a postdoctoral contract from Junta de Castilla y León

Conventional Feed-Grade or Slow-Release Coated Urea as Sources of Dietary Nitrogen for Fattening Lambs

[EN]Twenty-two Assaf male lambs (29.2 ± 0.9 kg live weight and 89 ± 0.2 days of age), distributed in two experimental groups, were used to evaluate the use of either feed-grade conventional urea (Control diet; n = 11) or slow-release urea (SRU diet; n = 11) as sources of dietary nitrogen on animal performance, ruminal fermentation, blood acid-base status, plasmatic metabolic profile, and carcass and meat quality. Animals were housed individually and fed ad libitum. At the end of the fattening period (day 70), the animals were slaughtered to compare the fermentation patterns in ruminal digesta and to evaluate the carcass and meat characteristics. No statistically significant differences (p > 0.05) were observed between treatments in the dry matter intake, final live weight, average daily gain, and feed conversion rate. Regarding the ruminal fermentation parameters, the molar proportion of propionic acid was higher (p 0.05) between experimental treatments in the blood acid-base status and biochemical profile, except for the concentration of urea in plasma, which was significantly (p 0.05) in the carcass and meat characteristics. In conclusion, the use of slow-release urea as a replacement for feed-grade conventional urea in the diet of Assaf fattening lambs, under the experimental conditions of this study, did not improve animal performance and increased the feeding costs.SIThis research was funded by JUNTA DE CASTILLA Y LEÓN, project CSI042 P17. Cristina Saro acknowledges the receipt of a postdoctoral contract funded by the EUROPEAN REGIONAL DEVELOPMENT FUND

Deciphering the Nature of Trp73 Isoforms in Mouse Embryonic Stem Cell Models: Generation of Isoform-Specific Deficient Cell Lines Using the CRISPR/Cas9 Gene Editing System

This article belongs to the Special Issue The Isoforms of the p53 Gene Family and Their Role in Cancer and Aging：Selection Papers from International p53/p63/p73 Isoforms Workshop[EN] The p53 family has been widely studied for its role in various physiological and pathological processes. Imbalance of p53 family proteins may contribute to developmental abnormalities and pathologies in humans. This family exerts its functions through a profusion of isoforms that are generated by different promoter usage and alternative splicing in a cell type dependent manner. In particular, the Trp73 gene gives rise to TA and DN-p73 isoforms that confer p73 a dual nature. The biological relevance of p73 does not only rely on its tumor suppression effects, but on its pivotal role in several developmental processes. Therefore, the generation of cellular models that allow the study of the individual isoforms in a physiological context is of great biomedical relevance. We generated specific TA and DN-p73-deficient mouse embryonic stem cell lines using the CRISPR/Cas9 gene editing system and validated them as physiological bona fide p73-isoform knockout models. Global gene expression analysis revealed isoform-specific alterations of distinctive transcriptional networks. Elimination of TA or DN-p73 is compatible with pluripotency but prompts naïve pluripotent stem cell transition into the primed state, compromising adequate lineage differentiation, thus suggesting that differential expression of p73 isoforms acts as a rheostat during early cell fate determinationSIThis work was supported by Grants PID2019-105169RB-I00 from Spanish Ministerio de Ciencia e Innovación cofinanced by FEDER funds (to M.C.M.) and LE021P17 from Junta de Castilla y Leon. L.L.-F. was a holder of a postdoctoral contract “Juan de de la Cierva-Incorporacion” from Ministerio de Ciencia e Innovación. N.M.-G. and H.A.-O. are supported by a predoctoral scholarship from the Asociación Española contra el Cáncer (AECC). M.M.-L. was a recipient of a Torres Quevedo contract from Ministerio de Ciencia e Innovación at Biomar Microbial Technologies. Á.D.-M., J.V.-F. and L.M.-A. are funded by Junta de Castilla y Leó

Key role for hypothalamic interleukin-6 in food-motivated behavior and body weight regulation

The pro-inflammatory role of interleukin-6 (IL-6) is well-characterized. Blockade of IL-6, by Tocilizumab, is used in patients with rheumatoid arthritis and those diagnosed with cytokine storm. However, brain-produced IL-6 has recently emerged as  a critical mediator of gut/adipose communication with the brain. Central nervous system (CNS) IL-6 is engaged by peripheral and central signals regulating energy homeostasis. IL-6 is critical for mediating hypophagia and weight loss effects of a GLP-1 analog, exendin-4, a clinically utilized drug. However, neuroanatomical substrates and behavioral mechanisms of brain IL-6 energy balance control remain poorly understood. We propose that the lateral hypothalamus (LH) is an IL-6-harboring brain region, key to food intake and food reward control. Microinjections of IL-6 into the LH reduced chow and palatable food intake in male rats. In contrast, female rats responded with reduced motivated behavior for sucrose, measured by the progressive ratio operant conditioning test, a behavioral mechanism previously not linked to IL-6. To test whether IL-6, produced in the LH, is necessary for ingestive and motivated behaviors, and body weight homeostasis, viroge-netic knockdown by infusion of AAV-siRNA-IL6 into the LH was utilized. Attenuation of LH IL-6 resulted in a potent increase in sucrose-motivated behavior, without any effect on ingestive behavior or body weight in female rats. In  contrast, the  treatment did  not  affect any  parameters measured (chow intake, sucrose-motivated behavior, locomotion, and body weight) in chow-fed males. However, when challenged with a high-fat/high- sugar diet, the male LH IL-6 knockdown rats displayed rapid weight gain and hyperphagia. Together, our data suggest that LH-produced IL-6 is necessary and sufficient for ingestive behavior and weight homeostasis in male rats. In females, IL-6 in the LH plays a critical role in food-motivated, but not ingestive behavior control or weight regulation. Thus, collectively these data support the idea that brain-produced IL-6 engages the hypothalamus to control feeding behavior.</p

Parabrachial Interleukin-6 reduces body weight and food intake and increases thermogenesis to regulate energy metabolism

Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6This research was funded by the Swedish Research Council ( 2014-2945 to K.P.S.; 2017-00792 to I.W.A.; and 2013-7107 to Patrik Rorsman), the Novo Nordisk Foundation Excellence project grant (to K.P.S. and I.W.A.), the Ragnar Söderberg Foundation (to K.P.S.), Harald Jeanssons Stiftelse and Greta Jeanssons Stiftelse (to K.P.S.), Magnus Bergvalls Stiftelse (to K.P.S.), the Wallenberg Foundation and the Center for Molecular and Translational Medicine (to K.P.S.), postdoctoral stipendium from The Swedish Brain Foundation (to D.M.), the ERC ( BFU2015-70664-R and StG-281408 ) (to R.N.), and the NIH ( DK-21397 ) (to H.J.G.)S

Effect of Sunflower and Marine Oils on Ruminal Microbiota, In vitro Fermentation and Digesta Fatty Acid Profile

This study using the rumen simulation technique (RUSITEC) investigated the changes in the ruminal microbiota and anaerobic fermentation in response to the addition of different lipid supplements to a ruminant diet. A basal diet with no oil added was the control, and the treatment diets were supplemented with sunflower oil (2%) only, or sunflower oil (2%) in combination with fish oil (1%) or algae oil (1%). Four fermentation units were used per treatment. RUSITEC fermenters were inoculated with rumen digesta. Substrate degradation, fermentation end-products (volatile fatty acids, lactate, gas, methane, and ammonia), and microbial protein synthesis were determined. Fatty acid profiles and microbial community composition were evaluated in digesta samples. Numbers of representative bacterial species and microbial groups were determined using qPCR. Microbial composition and diversity were based on T-RFLP spectra. The addition of oils had no effect on substrate degradation or microbial protein synthesis. Differences among diets in neutral detergent fiber degradation were not significant (P = 0.132), but the contrast comparing oil–supplemented diets with the control was significant (P = 0.039). Methane production was reduced (P &lt; 0.05) with all oil supplements. Propionate production was increased when diets containing oil were fermented. Compared with the control, the addition of algae oil decreased the percentage C18:3 c9c12c15 in rumen digesta, and that of C18:2 c9t11 was increased when the control diet was supplemented with any oil. Marine oils decreased the hydrogenation of C18 unsaturated fatty acids. Microbial diversity was not affected by oil supplementation. Cluster analysis showed that diets with additional fish or algae oils formed a group separated from the sunflower oil diet. Supplementation with marine oils decreased the numbers of Butyrivibrio producers of stearic acid, and affected the numbers of protozoa, methanogens, Selenomonas ruminantium and Streptococcus bovis, but not total bacteria. In conclusion, there is a potential to manipulate the rumen fermentation and microbiota with the addition of sunflower, fish or algae oils to ruminant diets at appropriate concentrations. Specifically, supplementation of ruminant mixed rations with marine oils will reduce methane production, the acetate to propionate ratio and the fatty acid hydrogenation in the rumen

Differential Diet and pH Effects on Ruminal Microbiota, Fermentation Pattern and Fatty Acid Hydrogenation in RUSITEC Continuous Cultures

The aim of this study was to distinguish effects due to diet composition from those triggered by ruminal pH on fermentation patterns and microbial profiles in a continuous culture system (RUSITEC). The study followed a 2 × 2 factorial design, with two diets varying in the proportions of forage and concentrate and two pH levels in the culture medium. RUSITEC fermenters were used to simulate rumen fermentation and feed digestibility, fermentation end-products, microbial protein synthesis, microbial community, and long-chain fatty acid profiles in the digesta were determined. Multivariate analyses were applied to summarize the overall results. High concentrate (34% cereal grain, 32% hay) diets were more digestible (p p p > 0.05) differences between diets in methane production. Ciliate protozoa, anaerobic fungi, some fibrolytic bacteria, hydrogenation of oleic acid, and relative proportion of conjugated linoleic acid were increased (p p p > 0.05) on the synthesis of microbial protein, and on the output of methane, propionate, butyrate or lactate. Minor changes in microbial community profile or the fatty acid relative proportions were observed within this pH range. The overall multivariate analysis revealed a clear discrimination between high-concentrate and high-forage diets, with subtler and less-defined pH effects on ruminal fermentation and microbial communities

Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis

Extraction of polyphenols and synthesis of new activated carbon from spent coffee grounds

A valorization process of spent coffee grounds (SCG) was studied. Thus, a two-stage process, the first stage of polyphenols extraction and synthesis of a carbonaceous precursor and a subsequent stage of obtaining activated carbon (AC) by means of a carbonization process from the precursor of the previous stage, was performed. The extraction was carried out with a hydro-alcoholic solution in a pressure reactor, modifying time, temperature and different mixtures EtOH:HO. To optimize the polyphenols extraction, a two-level factorial experimental design with three replicates at the central point was used. The best results were obtained by using a temperature of 80 °C during 30 min with a mixture of EtOH:HO 50:50 (v/v). Caffeine and chlorogenic acid were the most abundant compounds in the analysed extracts, ranging from 0.09 to 4.8 mg∙g and 0.06 to 9.7 mg∙g, respectively. Similarly, an experimental design was realized in order to analyze the influence of different variables in the AC obtained process (reaction time, temperature and KOH:precursor ratio). The best results were 1 h, 850 °C, and a mixture of 2.5:1. The obtained activated carbons exhibit a great specific surface (between 1600 m∙g and 2330 m∙g) with a microporous surface. Finally, the adsorption capacity of the activated carbons was evaluated by methylene blue adsorption.To the Technical Research Support Unit of the Institute of Catalysis and Petroleum Chemistry (CSIC) for support in the textural characterization of activated carbon. Mª Eugenia Léon-González and Noelia Rosales-Conrado thank the Spanish Commission of Science and Technology (CTQ2017-83569-C2-1-R) and the Comunidad of Madrid and European funding from FSE and FEDER programs for financial support (project S2018/BAA-4393, AVANSECAL-II-CM)