Differential protein expression analysis of activated E2F2-/-T lymphocytes by means of label-free quantification method

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A Three-Protein Panel to Support the Diagnosis of Sepsis in Children

Sepsis is a syndrome without a standard validated diagnostic test. Early recognition is crucial. Serum proteome analysis in children with sepsis may identify new biomarkers. This study aimed to find suitable blood biomarkers for an early diagnosis of sepsis. An analytical observational case-control study was carried out in a single center. Children admitted to a Pediatric Intensive Care Unit with clinical diagnosed sepsis were eligible for study. A proteomic analysis conducted by mass spectrometry was performed. Forty patients with sepsis and 24 healthy donors were recruited. Proteomics results revealed 44 proteins differentially expressed between patients and healthy controls. Six proteins were selected to be validated: lactoferrin, serum amyloid-A1 (SAA-1), complement factor B, leucine-rich alpha-2 glycoprotein (LRG1), soluble interleukin-2 alpha chain receptor (sCD25) and soluble haptoglobin-hemoglobin receptor. Our results showed that sCD25, SAA-1, and LRG1 had high levels of specificity and sensitivity, as well as an excellent area under the ROC curve (>0.9). Our study provides a serum proteomic analysis that identifies new diagnostic biomarkers in sepsis. SAA-1, sCD25 and LRG1 were able to separate septic from healthy donor, so they could be used together with other clinical and analytical features to improve sepsis diagnosis in children.This work was funded by Research Projects from University of Basque Country (US10/02) and from the Basque Government (SAIO10-PE10BF02, SAIO12-PE12BF002, 2012111052, 2019111056). CICbioGUNE is supported by Basque Department of Industry, Tourism and Trade (Etortek and Elkartek programs), the Innovation Technology Department of the Bizkaia County, the ProteoRed-ISCIII (Grant PRB3 IPT17/0019), CIBERehd Network and Severo Ochoa Grant (SEV-2016-0644)

Constitutive Activation of p62/Sequestosome-1-Mediated Proteaphagy Regulates Proteolysis and Impairs Cell Death in Bortezomib-Resistant Mantle Cell Lymphoma

Apoptosis; Autophagy; Proteasome inhibitorApoptosis; Autofagia; Inhibidor del proteasomaApoptosi; Autofàgia; Inhibidor del proteasomaProtein ubiquitylation coordinates crucial cellular events in physiological and pathological conditions. A comparative analysis of the ubiquitin proteome from bortezomib (BTZ)-sensitive and BTZ-resistant mantle cell lymphoma (MCL) revealed an enrichment of the autophagy–lysosome system (ALS) in BTZ-resistant cells. Pharmacological inhibition of autophagy at the level of lysosome-fusion revealed a constitutive activation of proteaphagy and accumulation of proteasome subunits within autophagosomes in different MCL cell lines with acquired or natural resistance to BTZ. Inhibition of the autophagy receptor p62/SQSTM1 upon verteporfin (VTP) treatment disrupted proteaphagosome assembly, reduced co-localization of proteasome subunits with autophagy markers and negatively impacted proteasome activity. Finally, the silencing or pharmacological inhibition of p62 restored the apoptosis threshold at physiological levels in BTZ-resistant cells both in vitro and in vivo. In total, these results demonstrate for the first time a proteolytic switch from the ubiquitin–proteasome system (UPS) to ALS in B-cell lymphoma refractory to proteasome inhibition, pointing out a crucial role for proteaphagy in this phenomenon and paving the way for the design of alternative therapeutic venues in treatment-resistant tumors.This work was supported at early stages by Spanish MINECO, CTQ2011–27874 grant. M.G.-S. is a fellow of the UbiCODE project funded by the EU’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 765445. M.S.R. and L.C. were also funded by the Institut National du Cancer, France (PLBIO16-251), CONACyT-SRE (Mexico) grant 0280365 and the REPERE program of Occitanie. O.C. is funded by “La Ligue contre le cancer du Gard”. ICFO authors were supported by funding from the Spanish MINECO “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), from Fundació Privada Cellex, Fundación Mig-Puig, from Generalitat de Catalunya CERCA program and from LASERLAB Europe (grant agreement No 871124;). G.R. was financially supported by Fondo de Investigación Sanitaria PI15/00102 and PI18/01383, European Regional Development Fund (ERDF) ‘Una manera de hacer Europa’. G.R. and D.R.G. are members of the Spanish Network of Excellence UBIRed funded by the Spanish Ministry Science, Innovation and Universities (SAF2017-90900-REDT)

Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo

The interactions of implanted biomaterials with the host organism determine the success or failure of an implantation. Normally, their biocompatibility is assessed using in vitro tests. Unfortunately, in vitro and in vivo results are not always concordant; new, effective methods of biomaterial characterisation are urgently needed to predict the in vivo outcome. As the first layer of proteins adsorbed onto the biomaterial surfaces might condition the host response, mass spectrometry analysis was performed to characterise these proteins. Four distinct hybrid sol-gel biomaterials were tested. The in vitro results were similar for all the materials examined here. However, in vivo, the materials behaved differently. Six of the 171 adsorbed proteins were significantly more abundant on the materials with weak biocompatibility; these proteins are associated with the complement pathway. Thus, protein analysis might be a suitable tool to predict the in vivo outcomes of implantations using newly formulated biomaterials

Proteomic Analysis of Mesenchymal Stem Cells and Monocyte Co-Cultures Exposed to a Bioactive Silica-Based Sol–Gel Coating

New methodologies capable of extensively analyzing the cell-material interactions are necessary to improve current in vitro characterization methods, and proteomics is a viable alternative. Also, many studies are focused on monocultures, even though co-cultures model better the natural tissue. For instance, human mesenchymal stem cells (MSCs) modulate immune responses and promote bone repair through interaction with other cell types. Here, label-free liquid chromatography tandem mass spectroscopy proteomic methods were applied for the first time to characterize HUCPV (MSC) and CD14+ monocytes co-cultures exposed to a bioactive sol–gel coating (MT). PANTHER, DAVID, and STRING were employed for data integration. Fluorescence microscopy, enzyme-linked immunosorbent assay, and ALP activity were measured for further characterization. Regarding the HUCPV response, MT mainly affected cell adhesion by decreasing integrins, RHOC, and CAD13 expression. In contrast, MT augmented CD14+ cell areas and integrins, Rho family GTPases, actins, myosins, and 14-3-3 expression. Also, anti-inflammatory (APOE, LEG9, LEG3, and LEG1) and antioxidant (peroxiredoxins, GSTO1, GPX1, GSHR, CATA, and SODM) proteins were overexpressed. On co-cultures, collagens (CO5A1, CO3A1, CO6A1, CO6A2, CO1A2, CO1A1, and CO6A3), cell adhesion, and pro-inflammatory proteins were downregulated. Thus, cell adhesion appears to be mainly regulated by the material, while inflammation is impacted by both cellular cross-talk and the material. Altogether, we conclude that applied proteomic approaches show its potential in biomaterial characterization, even in complex systems.This work was supported by MINECO [MAT2017-86043-R; RTC-2017-6147-1], Generalitat Valenciana [GRISOLIAP/2018/091, BEFPI/2021/043, PROMETEO/2020/069], Universitat Jaume I [UJI-B2017-37], and the University of the Basque Country [GIU18/189]. Andreia Cerqueira was supported by the Margarita Salas postdoctoral contract MGS/2022/10 (UP2022-024) financed by the European Union-NextGenerationEU. The University Medical Centre Hamburg-Eppendorf (Hamburg, Germany) and the Clinics for Gynecology AGAPLESION BETHESDA Hospital provided the blood and tissue for cell isolation. The authors would like to thank Raquel Oliver, Jose Ortega, Iraide Escobés, and Anke Borkam-Schuster for their valuable technical assistance and Antonio Coso (GMI-Ilerimplant) for producing the titanium discs

Development and characterisation of strontium-doped sol-gel coatings to optimise the initial bone regeneration processes

Strontium plays an important role in bone regeneration; it promotes the differentiation and maturation of osteoblasts and inhibits the activity of osteoclasts. Our principal objective in this study was to formulate new organic-inorganic hybrid sol-gel coatings applied to titanium discs. These coatings were functionalised with different amounts of SrCl2 and examined using in vitro tests and proteomics. The chemical and morphological characteristics of obtained coatings were scrutinised. The in vitro evaluation using the MC3T3-E1 osteoblasts and RAW264.7 macrophages showed the osteogenic and anti-inflammatory effects of strontium doping. The proteomic assay identified 111 different proteins adhering to the coatings. Six of these proteins reduced their adhesion affinity as a result of Sr-doping, whereas 40 showed increased affinity. Moreover, the proteomic analysis revealed osteogenic and anti-inflammatory properties of these biomaterials. The analysis also showed increased adhesion of proteins related to the coagulation system. We can conclude that proteomic methods are invaluable in developing new biomaterials and represent an important tool for predicting the biocompatibility of dental implants

Structural basis for assembly of vertical single β-barrel viruses

The vertical double beta-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 angstrom resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single beta-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double beta-barrel MCPs.Peer reviewe

Identification of a panel of serum protein markers in early stage of sepsis and its validation in a cohort of patients

Background: Sepsis is a life-threatening illness with a challenging diagnosis. Current serum biomarkers are not sensitive enough for diagnosis. With the aim of finding proteins associated with sepsis, serum protein profile was compared between patients and healthy donors and serum classical inflammatory proteins were analyzed in both groups. Methods: Serum protein profiles were characterized by two-dimensional electrophoresis (2DE). Identification of the proteins was carried out by mass spectrophotometry and their validation was performed by Enzyme-Linked-lmmunoSorbent Assay (ELISA) in a cohort of 85 patients and 67 healthy donors. Seven classical inflammatory proteins were analyzed in the same cohort by ELISA: interleukin-2 receptor alpha-chain (sCD25), scavenger receptor cysteine

Osteointegración de implantes de titanio con superficies activas. Un estudio proteómico

Titanium is a biomaterial largely used on dental implant manufacturing. However, as a consequence of its intrinsically low bioactivity, the development of distinct superficial treatments in order to enhance its osseointegration properties is being studied. In this sense, the use of titanium implants with a higher level of roughness has been broadened, recurring to the application of sand-blasted acid-etched surface treatments. In this article, a study of two distinct titanium surface treatments has been carried out, regarding the physico-chemical properties (roughness, hydrophilicity and chemical composition) of each, as well as the pattern of adhered proteins onto each surface (proteomic study). Hence, mass spectrometry analysis allowed the detection of 2 18 d istinct a dsorbed p roteins, being 37 of those related to bone regenerative processes and dental implant integration. Moreover, using differential quantification between associated proteins, comparing surfaces, it was observable a greater affinity of APOE, ANT3 and PROC proteins to the treated surface, directly linked to the bone regenerative process. On the other hand, the treated surface displays lower affinity of CO3 protein. The variations between the adsorbed protein profiles could be an explanation for distinct in vivo outcomes.El titanio es un biomaterial ampliamente empleado en la fabricación de implantes dentales, sin embargo, como consecuencia de su baja bioactividad se han desarrollado distintos tratamientos superficiales buscando una mejora en su capacidad de osteintegración. De esta forma, se ha extendido el uso de implantes de titanio con un mayor grado de rugosidad gracias a la aplicación de un tratamiento de granallado, al que le sigue un tratamiento de ataque ácido. En este artículo se ha llevado a cabo un estudio de discos de titanio con dos tipos de superficie: sin tratamiento alguno y con tratamiento de granallado más ataque ácido. El estudio reveló diferencias físico-químicas (rugosidad, hidrofilia y composición química) tras la aplicación del tratamiento superficial, pero también en cuanto al perfil de proteínas adheridas a cada superficie (estudio proteómico). Así, la espectrometría de masas permitió la caracterización de las proteínas adsorbidas en ambos tipos de superficies. El análisis permitió la identificación de 218 proteínas distintas, pudiendo relacionar 37 de ellas con el proceso de regeneración ósea y en consecuencia con la osteointegración de un implante dental. Además, tras la cuantificación diferencial entre proteínas asociadas, antes y después de aplicar el tratamiento superficial mencionado, se observó que tras su aplicación se producía un aumento en la afinidad de las proteínas APOE, ANT3 y PROC, directamente relacionadas con el proceso de regeneración ósea. Por el contrario, la proteína CO3 se adhería a esta superficie en menor proporción. Estas variaciones de los perfiles de proteínas podrían explicar la diferencia encontrada en la respuesta de las distintas superficies al ser caracterizadas en cuanto a su comportamiento in vivo