Estudio proteómico de las diferencias entre los tumores benignos y malignos de la glándula tiroides

El cáncer de tiroides es el cáncer endocrino más frecuente. Este tipo de cáncer incluye diferentes categorías la mayoría de comportamiento clínico relativamente indolente. Aunque los carcinomas bien diferenciados papilar y folicular suelen ser poco agresivos, en ocasiones pueden comportarse de una forma inusualmente agresiva y/o transformarse (desdiferenciarse) en carcinomas menos diferenciados. Por otra parte el carcinoma indiferenciado (anaplásico) es una de las formas más letales de cáncer humano. En este proyecto de tesis doctoral se pretende una mejor caracterización de los cánceres de la glándula tiroides humana mediante el empleo de técnicas proteómicas, comparando los perfiles proteicos expresados por los tumores tiroideos primarios

A Novel Nanoproteomic Approach for the Identification of Molecular Targets Associated with Thyroid Tumors

A thyroid nodule is the most common presentation of thyroid cancer; thus, it is extremely important to differentiate benign from malignant nodules. Within malignant lesions, classification of a thyroid tumor is the primary step in the assessment of the prognosis and selection of treatment. Currently, fine-needle aspiration biopsy (FNAB) is the preoperative test most commonly used for the initial thyroid nodule diagnosis. However, due to some limitations of FNAB, different high-throughput “omics” approaches have emerged that could further support diagnosis based on histopathological patterns. In the present work, formalin-fixed paraffin-embedded (FFPE) tissue specimens from normal (non-neoplastic) thyroid (normal controls (NCs)), benign tumors (follicular thyroid adenomas (FTAs)), and some common types of well-differentiated thyroid carcinoma (follicular thyroid carcinomas (FTCs), conventional or classical papillary thyroid carcinomas (CV-PTCs), and the follicular variant of papillary thyroid carcinomas (FV-PTCs)) were analyzed. For the first time, FFPE thyroid samples were deparaffinized using an easy, fast, and non-toxic method. Protein extracts from thyroid tissue samples were analyzed using a nanoparticle-assisted proteomics approach combined with shotgun LC-MS/MS. The differentially regulated proteins found to be specific for the FTA, FTC, CV-PTC, and FV-PTC subtypes were analyzed with the bioinformatic tools STRING and PANTHER showing a profile of proteins implicated in the thyroid cancer metabolic reprogramming, cancer progression, and metastasis. These proteins represent a new source of potential molecular targets related to thyroid tumorsThis work was supported by grants from the Instituto de Salud Carlos III (ISCIII), State Research Agency (AEI), and Ministry of Science and Innovation (Spain), with the participation of European FEDER funds, to C.N. (CP16/00139) and J.M.C.-T. (PI19/01316)S

REVERTIA A Circular Economy Business Case

This report presents the results of the Revertia case study, selected in the framework of the R2PI project, among 17 other cases, because of its focus on an activity linked to priority areas of the Circular Economy, namely Plastics and Critical Raw Materials. The information contained in the report is based on the methodology designed within the framework of the R2PI project to understand the characteristics of the business model, evaluate its outcomes and identify the main barriers and enablers of the CEBM. Revertia is an authorised Waste Electrical and Electronic Equipment (WEEE) manager that combines e-waste management services with a circular value proposal consisting in the preparation of IT equipment for reuse. The company responsibly manages an e-waste flow and, by applying secure reconditioning processes, is able to extend the service life of IT equipment that has been discarded. Refurbished equipment, with a 1-year warranty, are sold in second-hand markets or donated. The activity of preparation for reuse also involves the utilization, to the extent possible, of used and recycled components. In addition, the business model is based on efficient logistics, avoiding e-waste transportation when reconditioning is not an option. The value proposition is based on the provision of high added value services to corporate IT equipment users, who need to manage their WEEE responsibly. The value network is thus formed by the origin-customer that generates e-waste and the EEE manufacturers represented through collective schemes. Downstream there are the destination-customers of second-hand and donated products, as well as the WEEE recycling companies to which Revertia's own waste is destined. Revertia's business model is clearly influenced at the context level by the WEEE Directive 2012/19/EU and the Spanish RD 110/2015, which regulate the industry and set targets for reuse and recycling of WEEE. In addition, the market context given by the rapid technological change in the EEE sector, the collective schemes derived from the implementation of the EPR obligation, the maturity of the WEEE recycling industry, the existence of illegal scrap metal agents, and some socio-cultural aspects such as the lack of awareness of WEEE's environmental costs are factors that strongly affect Revertia's activity. The report also presents an assessment of the circularity of the business model. The model corresponds to the Re-make pattern among the CEBM defined in the R2PI project. Its circularity lies in extending the useful life of a product, also integrating used and recycled components as far as possible. Current circularity is limited, since it is not based on services added to this second-hand product, nor is the organisation further able to act on the products manufacture or end of life. Therefore, Revertia takes opportunity of a gap left by other players in the sector (EEE manufacturers). Therefore the business model is dependent on the generation of an e-waste flow as well as on sales of refurbished computers. With regards to the outcomes assessment, the main advantages of the business model are found in the non-financial aspects: reuse of IT equipment for the same use demonstrates clear environmental benefits compared to recycling. In addition, it also allows for greater local employment generation linked to the management and preparation for reuse activity. In the socio-economic area, second-hand products provide access to quality equipment at very affordable prices. The SWOT analysis shows that the main strengths are the long-lasting and stable relationships with the origin-customers, the know-how and expertise of Revertia. The weaknesses point to its dependence on the linear model, therefore, to being able to capture waste streams, in competition with the mature recycling industry. The opportunities lie in the Spanish regulatory framework, which sets targets for the reuse of WEEE, as well as a state of opinion more favourable to responsible and sustainable consumption; however, there are also clear threats to the model, such as the possible entry of more competitors and the lack of sensitivity of EEE manufacturers towards reuse. Based on the analysis, it is concluded that the Re-make CEBM is replicable and transferable, especially as long as the linear economic paradigm is dominant. However, there are a number of business and policy recommendations that may support greater circularity: first, specific agreements with EEE manufacturers to extend circularity from the conception of EEE products until its final disposal after multiple lifetimes; second, at the policy level, tighter regulation, with inspection and sanction systems, incentives for eco-design and product life extension activities, transparent information and monitoring systems, as well as education and training measures are neededThis project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 73037

Proteomic analysis in morquio a cells treated with immobilized enzymatic replacement therapy on nanostructured lipid systems

[ENG]Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERTS

Inhibition of ATG3 ameliorates liver steatosis by increasing mitochondrial function

Non-alcoholic fatty liver disease (NAFLD) is a major health threat in both developed and developing countries and is a precursor of the more advanced liver diseases, including non-alcoholic steatohepatitis (NASH), cirrhosis, and liver cancer. Currently, understanding the multiple and complex molecular pathways implicated in NAFLD onset and progression is a major priority. The transcription factor p63, which belongs to a family comprising p53, p63, and p73,1 is one of many factors that contributes to the development of liver steatosis. The role of p63 as a tumor suppressor and in cell maintenance and renewal is well studied, but we have recently reported that it is also relevant in the control of lipid metabolism.2 p63 encodes multiple isoforms that can be grouped into 2 categories; isoforms with an acidic transactivation domain (TA) and those without this domain (domain negative). The TAp63α isoform is elevated in the liver of animal models of NAFLD as well as in liver biopsies from obese patients with NAFLD. Furthermore, downregulation of p63α in the liver attenuates liver steatosis in diet-induced obese (DIO) mice, while the activation of TAp63α increases hepatic fat content, mediated by the activation of IKKβ and endoplasmic reticulum stress.2 A specialized form of autophagy that degrades lipid droplets, termed “lipophagy”, is a major pathway of lipid mobilization in hepatocytes. Lipophagy is elevated in hepatoma cells upon exposure to free fatty acids,3 and reduces the fatty acid load in mouse hepatocytes.4 Its impairment has been associated with the development of fatty liver and insulin resistance3,5; in contrast, the autophagic flux is increased during the activation of hepatic stellate cells.6 In the present study, we used an unbiased proteomics approach to gain insight into novel proteins modulating lipid metabolism in the liver of mice with genetic knockdown or overexpression of TAp63α. We found that autophagy-related gene 3 (ATG3) was upregulated by TAp63α activation and downregulated after p63α inhibition. ATG3 is elevated in several animal models of NAFLD and in the liver of patients with NAFLD. Genetic overexpression of ATG3 increased the lipid load in hepatocytes, while its repression alleviated TAp63α- and diet-induced steatosis. ATG3 exerted its role in lipid metabolism by regulating SIRT1 and mitochondrial function. Collectively, these findings identify ATG3 as a novel factor implicated in the development of steatosisThis work has been supported by grants from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación (PA: RTI2018-095134-B-100; DS and LH: SAF2017-83813-C3-1-R; MLMC: RTC2019-007125-1; CD: BFU2017-87721; ML: RTI2018–101840-B-I00; GS; PID2019-104399RB-I00; RN: RTI2018-099413-B-I00 and RED2018-102379-T; MLMC: SAF2017-87301-R; TCD: RTI2018-096759-A-100), FEDER/Instituto de Salud Carlos III (AGR: PI19/00123), Xunta de Galicia (ML: 2016-PG068; RN: 2015-CP080 and 2016-PG057), Fundación BBVA (RN, GS and MLM), Proyectos Investigación en Salud (MLMC: DTS20/00138), Sistema Universitario Vasco (PA: IT971-16); Fundación Atresmedia (ML and RN), Fundación La Caixa (M.L., R.N. and M.C.), Gilead Sciences International Research Scholars Program in Liver Disease (MVR), Marató TV3 Foundation (DS: 201627), Government of Catalonia (DS: 2017SGR278) and European Foundation for the Study of Diabetes (RN and GS). This research also received funding from the European Community’s H2020 Framework Programme (ERC Synergy Grant-2019-WATCH- 810331, to RN, VP and MS). Centro de Investigación Biomédica en Red (CIBER) de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Centro de Investigación Biomédica en Red (CIBER) de Enfermedades Hepáticas y Digestivas (CIBERehd) and CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERdem). CIBERobn, CIBERehd and CIBERdem are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. We thank MINECO for the Severo Ochoa Excellence Accreditation to CIC bioGUNE (SEV-2016-0644)S

INDITEX A Circular Economy Business Model Case

This report presents the case study corresponding to the textile company Inditex, which has been selected by the R2PI project given its importance in the world fashion market. The adoption of a circular model by fashion companies is extremely important, given the relevant environmental impact of this industry. Based on the common methodology of the R2PI project, this report presents the analysis of the “Circular Sourcing" CEBM, although it also highlights other activities of circularity that complement the aforementioned and that the Inditex Group is partly implementing, such as "Co-product recovery" and "Resource-recovery", the latter in collaboration with non-profit organisations in Spain and other markets. The objective of the report is, therefore, to offer a general view of how Inditex is facing the challenge of circularity, taking into account both contextual and internal factors, as well as to assess the level of circularity, the outcomes obtained and the SWOT. Finally, the report tries to offer key insights with regards to the “Circular Sourcing” CEBM pattern. The analysis of the context indicates that there are certain factors in the policy framework that are conditioning the development of circularity in fashion, especially the regulations that affect garments trade, its labelling, and textile waste trade –and will do so even more in the future. The market situation and competition reveal important elements of influence, such as the general race among leading fashion companies towards more sustainable and circular business models. In addition, still uncertain economic factors, such as the evolution of raw materials, water and energy prices and wages, are pointing to the need to adopt measures towards a circular change. As far as the factors of the technological framework are concerned, there is a clear commitment to developing new raw materials and more environmentally friendly processes; likewise, developments in recycling technologies and other advances may change the face of the textile industry in a few years. Finally, while the fast-fashion consumer model continues to be very successful, there is already an increasing demand for healthier and environmentally friendly clothing. The characteristics of Inditex's business model centred on circular sourcing are analysed in detail. This model is based on the group power along the value chain. Thus, a value network is formed in which some suppliers are key partners, because they share Inditex vision and are doing strong efforts towards a more circular sourcing, while also supporting the development of the local textile recycling industry. At the heart of the business model, Inditex continues offering fashion at competitive prices, but the group is increasingly searching for the environmentally friendliness attribute. The analysis of the circularity of Inditex Group indicates that it is currently limited in the sense that it is still focused on the sale of a product, and the revenues and costs are clearly linked to the sale and manufacture of that product, not to added services, for example. Although garments are manufactured with more sustainable raw materials, the industry might become more circular yet if fashion companies decide to innovate the business model in more radical ways. With regard to the financial outcomes, the available data allows to indicate that the manufacturing costs of more sustainable garments are generally higher than those of conventional garments. It is important to remark that Inditex does not pass this higher cost on to end consumers through a price premium. Moreover, sustainable garments are increasing greatly in Inditex's fashion sales. Existing information points to important non-financial outcomes linked to circular sourcing: the reduction of environmental impacts, greater employment generated through social initiatives by means of projects linked to donated garments; and possibilities for the creation of a stronger industry and employment associated with textile recycling. In addition, technological innovation is very important to support this business model, with investments in R&D aimed at the development of new products (fibres) and processes (design, traceability, recycling). Using a SWOT tool, Inditex business model is assessed and put in relation to potential circularity. Weaknesses are identified, such as the dependence of the Inditex circular business model on its ability to influence suppliers and the success of the current dominant business model based on continuous and quick sales. There are also strengths, such as the design skills and the ability to anticipate the market, the power to influence suppliers and to play an intermediary role in the fashion industry. As far as external aspects are concerned, the greatest opportunity lies in taking advantage of the favourable state of opinion and the general movement of the fashion industry towards the Circular Economy. The threats may also be high competition, but they rely mainly in the challenges of regulation for the clothing and waste trade and in the current low cost scenario (raw materials, energy and water, and labour), which make sustainable manufacturing more expensive. Inditex's route towards the Circular Economy will be based on going deeper into the circular sourcing CEBM pattern, especially in the sense of trying to close the loops for some fibres; moreover, in the longer term, it is also part of its plans to explore other business models in which the services added to the product will take on greater relevance. The business model analysed is replicable and transferable. In fact, a large number of leading companies in the sector are already taking action in the same direction. However, there is a need for greater commitment from industry and consumers. Based on this research, some policy measures are highly recommended to further promote the uptake of the circular business model, including: the establishment of clear objectives for garments reuse and recycling; regulation for favouring the implementation of eco-design, durability and quality of garments; and incentives and education for consumers to change towards more responsible and sustainable patterns of fashion consumptionThis project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 73037

Protein Corona Gold Nanoparticles Fingerprinting Reveals a Profile of Blood Coagulation Proteins in the Serum of HER2-Overexpressing Breast Cancer Patients

Breast cancer (BC) is a molecularly heterogeneous disease that encompasses five major molecular subtypes (luminal A (LA), luminal B HER2 negative (LB-), luminal B HER2 positive (LB+), HER2 positive (HER2+) and triple negative breast cancer (TNBC)). BC treatment mainly depends on the identification of the specific subtype. Despite the correct identification, therapies could fail in some patients. Thus, further insights into the genetic and molecular status of the different BC subtypes could be very useful to improve the response of BC patients to the range of available therapies. In this way, we used gold nanoparticles (AuNPs, 12.96 +/- 0.72 nm) as a scavenging tool in combination with Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) to quantitatively analyze the serum proteome alterations in the different breast cancer intrinsic subtypes. The differentially regulated proteins specific of each subtype were further analyzed with the bioinformatic tools STRING and PANTHER to identify the major molecular function, biological processes, cellular origin, protein class and biological pathways altered due to the heterogeneity in proteome of the different BC subtypes. Importantly, a profile of blood coagulation proteins was identified in the serum of HER2-overexpressing BC patients

Protein-Based Salivary Profiles as Novel Biomarkers for Oral Diseases

The Global Burden of Oral Diseases affects 3.5 billion people worldwide, representing the number of people affected by the burden of untreated dental caries, severe periodontal disease, and edentulism. Thus, much more efforts in terms of diagnostics and treatments must be provided in the fight of these outcomes. In this sense, recently, the study of saliva as biological matrix has been identified as a new landmark initiative in the search of novel and useful biomarkers to prevent and diagnose these conditions. Specifically, saliva is a rich reservoir of different proteins and peptides and accessible due to recent advances in molecular biology and specially in targeted and unbiased proteomics technologies. Nonetheless, emerging barriers are an obstacle to the study of the salivary proteome in an effective way. This review aims at giving an overall perspective of salivary biomarkers identified in several oral diseases by means of molecular biology approaches

Proteomic investigation on bio-corona of Au, Ag and Fe nanoparticles for the discovery of triple negative breast cancer serum protein biomarkers

[EN] Nowadays, there are no targeted therapeutic modalities for triple negative breast cancer (TNBC). This disease is associated with poor prognosis and worst clinical outcome because of the aggressive nature of the tumor, delayed diagnosis, and non-specific symptoms in the early stages. Therefore, identification of novel specific TNBC serum biomarkers for screening and therapeutic purposes remains an urgent clinical requirement. New user-friendly and cheap methods for biomarker identification are needed, and nanotechnology offers new opportunities. When dispersed in blood, nanoparticles (NPs) are covered by a protein shell termed ¿protein corona¿ (PC). While alterations in protein patterns are challeging to detect by conventional blood analyses, PC acts as a ¿nano-concentrator¿ of serum proteins with affinity for NPs¿ surface. So, the characterization of PC could allow the detection of otherwise undetectable changes in protein concentration at an early stage of the disease or after chemotherapy or surgery. To explore this research idea, serum samples from 8 triple negative breast cancer (TNBC) patients and 8 patients without malignancy were allowed to interact with gold nanoparticles (AuNPs: 10.02¿±¿0.91¿nm), silver nanoparticles (AgNPs: 9.73¿±¿1.70¿nm) and magnetic nanoparticles (MNPs: (9.30¿±¿0.67¿nm). Here, in order to identify biomarker candidates in serum of TNBC patients, these nanomaterials were combined with electrophoretic separation (SDS-PAGE) to performed qualitative and quantitative comparisons of the serum proteomes of TNBC patients (n = 8) and healthy controls (n = 8) by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis. The results were validated through a sequential window acquisition of all theoretical mass spectra (SWATH) analysis, performed in total serum samples (patients and controls) using this approach as a multiple reaction monitoring (MRM) analysis. Significance It is well known that several proteins presented in human serum are important biomarkers for the diagnosis or prognosis of different diseases, as triple negative breast cancer (TNBC). Determining how nanomaterials as gold nanoparticles (AuNPs: 10.02¿±¿0.91¿nm), silver nanoparticles (AgNPs: 9.73¿±¿1.70¿nm) and magnetic nanoparticles (MNPs: (9.30¿±¿0.67¿nm) interact with human serum will assist not only in understanding their effects on the biological system (biocompability and toxicity), but also to obtain information for developing novel nanomaterials with high specificity and selectivity towards proteins with an important biological function (prognostic and diagnostic protein biomarkers).All authors acknowledge Miguel Servet I Programme (CP16/00139) from the “Instituto de Salud Carlos III” (Plan Estatal de I+D+i 2013-2016 and European Development Regional Fund) of the Spanish Ministry of Science, Innovation and Universities. A. Castro López and M.P. Chantada-Vázquez contributed equally to this work