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1.
J Proteomics ; 216: 103652, 2020 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-31958637

RESUMO

The composition of endometrial fluid reflects the status of the endometrium; it is a good atraumatic source of information on embryo implantation processes and possible pathological conditions. Although some attempts have been made to characterise its proteome, the catalogue of its proteins remains incomplete and little has been done to analyse the natural peptides it contains. Here, we present a comprehensive analysis of the proteins and natural peptides of the endometrial fluid. The protein content of samples from 11 individuals was analysed using the novel timsTOF Pro mass spectrometer. We identified 4694 proteins with at least one peptide with FDR < 1%, of which 2261 were found in >50% of the samples. A pooled endometrial fluid sample was used for isolation and analysis of the natural peptides. Mass spectrometry analysis identified 3899 naturally occurring peptides from 238 different proteins. Among these, there were some putative natural antibacterial peptides. Antimicrobial activity of peptides derived from elafin and Cu/Zn superoxide dismutase was confirmed using microbiological assays. Our results substantially expand the catalogue of known endometrial fluid proteins and provide extensive new information on the natural peptide content of this fluid. SIGNIFICANCE: The endometrial fluid contains many proteins whose clinical relevance is still unknown. Some might be merely markers of endometrial function, but others might play a role in embryo nutrition and/or implantation. Human endometrial fluid analysis might open the door to new developments in embryo transfer strategies in in-vitro fertilisation programmes and lead to improvements in the composition of embryo culture media. Here, we report, for the first time, antimicrobial activity of endometrial fluid peptides. Such peptides could play an important role in the balance of the recently described uterine microbiota.

2.
Arch Biochem Biophys ; 679: 108223, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31816312

RESUMO

Glucose uptake by mammalian cells is a key mechanism to maintain cell and tissue homeostasis and relies mostly on plasma membrane-localized glucose transporter proteins (GLUTs). Two main cellular mechanisms regulate GLUT proteins in the cell: first, expression of GLUT genes is under dynamic transcriptional control and is used by cancer cells to increase glucose availability. Second, GLUT proteins are regulated by membrane traffic from storage vesicles to the plasma membrane (PM). This latter process is triggered by signaling mechanisms and well-studied in the case of insulin-responsive cells, which activate protein kinase AKT to phosphorylate TBC1D4, a RAB-GTPase activating protein involved in membrane traffic regulation. Previously, we identified protein kinase WNK1 as another kinase able to phosphorylate TBC1D4 and regulate the surface expression of the constitutive glucose transporter GLUT1. Here we describe that downregulation of WNK1 through RNA interference in HEK293 cells led to a 2-fold decrease in PM GLUT1 expression, concomitant with a 60% decrease in glucose uptake. By mass spectrometry, we identified serine (S) 704 in TBC1D4 as a WNK1-regulated phosphorylation site, and also S565 in the paralogue TBC1D1. Transfection of the respective phosphomimetic or unphosphorylatable TBC1D mutants into cells revealed that both affected the cell surface abundance of GLUT1. The results reinforce a regulatory role for WNK1 in cell metabolism and have potential impact for the understanding of cancer cell metabolism and therapeutic options in type 2 diabetes.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31865491

RESUMO

OBJECTIVE: To characterize the most relevant changes in the lipidome of endometrial fluid aspirate (EFA) in non-implantative cycles. DESIGN: Lipidomics in a prospective cohort study. SETTINGS: Reproductive unit of a university hospital. PATIENTS: Twenty-nine women undergoing an IVF cycle. Fifteen achieved pregnancy and 14 did not. INTERVENTION: Endometrial fluid aspiration immediately before performing embryo transfer. MAIN OUTCOME MEASURES: Clinical pregnancy rate and lipidomic profiles obtained on an ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF-MS)-based analytical platform. RESULTS: The comparative analysis of the lipidomic patterns of endometrial fluid in implantative and non-implantative IVF cycles revealed eight altered metabolites: seven glycerophospholipids and an omega-6 polyunsaturated fatty acid. Then, women with a non-implantative cycle were accurately classified with a support vector machine algorithm including these eight lipid metabolites. The diagnostic performances of the algorithm showed an area under the receiver operating characteristic curve, sensitivity, specificity, and accuracy of 0.893 ± 0.07, 85.7%, 80.0%, and 82.8%, respectively. CONCLUSION: A predictive lipidomic signature linked to the implantative status of the endometrial fluid has been found.

4.
Mol Metab ; 29: 40-54, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31668391

RESUMO

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. METHODS: miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. RESULTS: We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid ß-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. CONCLUSION: GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.

5.
J Vis Exp ; (153)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31736480

RESUMO

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest. Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

6.
PLoS Pathog ; 15(11): e1008163, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31738806

RESUMO

Macrophages mediate the elimination of pathogens by phagocytosis resulting in the activation of specific signaling pathways that lead to the production of cytokines, chemokines and other factors. Borrelia burgdorferi, the causative agent of Lyme disease, causes a wide variety of pro-inflammatory symptoms. The proinflammatory capacity of macrophages is intimately related to the internalization of the spirochete. However, most receptors mediating this process are largely unknown. We have applied a multiomic approach, including the proteomic analysis of B. burgdorferi-containing phagosome-enriched fractions, to identify surface receptors that are involved in the phagocytic capacity of macrophages as well as their inflammatory output. Sucrose gradient protein fractions of human monocyte-derived macrophages exposed to B. burgdorferi contained the phagocytic receptor, CR3/CD14 highlighting the major role played by these proteins in spirochetal phagocytosis. Other proteins identified in these fractions include C-type lectins, scavenger receptors or Siglecs, of which some are directly involved in the interaction with the spirochete. We also identified the Fc gamma receptor pathway, including the binding receptor, CD64, as involved both in the phagocytosis of, and TNF induction in response to B. burgdorferi in the absence of antibodies. The common gamma chain, FcγR, mediates the phagocytosis of the spirochete, likely through Fc receptors and C-type lectins, in a process that involves Syk activation. Overall, these findings highlight the complex array of receptors involved in the phagocytic response of macrophages to B. burgdorferi.

7.
Front Immunol ; 10: 2067, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31620119

RESUMO

The ankyrin repeat domain-55 (ANKRD55) gene contains intronic single nucleotide polymorphisms (SNPs) associated with risk to contract multiple sclerosis, rheumatoid arthritis or other autoimmune disorders. Risk alleles of these SNPs are associated with higher levels of ANKRD55 in CD4+ T cells. The biological function of ANKRD55 is unknown, but given that ankyrin repeat domains constitute one of the most common protein-protein interaction platforms in nature, it is likely to function in complex with other proteins. Thus, identification of its protein interactomes may provide clues. We identified ANKRD55 interactomes via recombinant overexpression in HEK293 or HeLa cells and mass spectrometry. One hundred forty-eight specifically interacting proteins were found in total protein extracts and 22 in extracts of sucrose gradient-purified nuclei. Bioinformatic analysis suggested that the ANKRD55-protein partners from total protein extracts were related to nucleotide and ATP binding, enriched in nuclear transport terms and associated with cell cycle and RNA, lipid and amino acid metabolism. The enrichment analysis of the ANKRD55-protein partners from nuclear extracts is related to sumoylation, RNA binding, processes associated with cell cycle, RNA transport, nucleotide and ATP binding. The interaction between overexpressed ANKRD55 isoform 001 and endogenous RPS3, the cohesins SMC1A and SMC3, CLTC, PRKDC, VIM, ß-tubulin isoforms, and 14-3-3 isoforms were validated by western blot, reverse immunoprecipitaton and/or confocal microscopy. We also identified three phosphorylation sites in ANKRD55, with S436 exhibiting the highest score as likely 14-3-3 binding phosphosite. Our study suggests that ANKRD55 may exert function(s) in the formation or architecture of multiple protein complexes, and is regulated by (de)phosphorylation reactions. Based on interactome and subcellular localization analysis, ANKRD55 is likely transported into the nucleus by the classical nuclear import pathway and is involved in mitosis, probably via effects associated with mitotic spindle dynamics.

8.
BMC Vet Res ; 15(1): 188, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174546

RESUMO

BACKGROUND: Both bovine tuberculosis (TB) and paratuberculosis (PTB) are serious and widespread bacterial infections affecting many domestic and wild animal species. However, current vaccines do not confer complete protection and cause interference with other diagnostics tests, including bovine TB. Therefore, the development of "Differentiating Infected from Vaccinated Animals" (DIVA) tests are a pressing need. In this study, we have tested the feasibility of mycobacterial extracellular vesicles (EVs) as potential source of biomarkers to discriminate between Mycobacterium bovis infected, Mycobacterium avium subsp. paratuberculosis (MAP) infected and MAP-vaccinated cows. We have, initially, characterized vesicle production in the two most medically relevant species of mycobacteria for livestock, MAP and M. bovis, for being responsible for tuberculosis (TB) and paratuberculosis (PTB). RESULTS: Our results indicate that these two species produce EVs with different kinetics, morphology and size distribution. Analysis of the immunogenicity of both type of EVs showed some cross reactivity with sera from PTB+ and TB+ cows, suggesting a limited diagnostic capacity for both EVs. Conversely, we noticed that Mycobacterium tuberculosis (Mtb) EVs showed some differential reactivity between sera from MAP-vaccinated or PTB+ cows from TB+ ones. Mass spectrometry analysis (MS) identified a 19-kDa EV-associated lipoprotein as the main source of the differential reactivity. CONCLUSIONS: LpqH could be a good plasma biomarker with capacity to distinguish PTB+ or MAP-vaccinated cows from cows infected with TB.


Assuntos
Doenças dos Bovinos/microbiologia , Vesículas Extracelulares/química , Lipoproteínas/análise , Mycobacterium tuberculosis/química , Paratuberculose/diagnóstico , Tuberculose Bovina/diagnóstico , Animais , Vacinas Bacterianas , Biomarcadores/sangue , Bovinos , Reações Cruzadas , Mycobacterium avium subsp. paratuberculosis/química , Mycobacterium bovis/química , Vacinação/veterinária
9.
Nat Commun ; 10(1): 1184, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862777

RESUMO

The vertical double ß-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 Å 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 ß-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 ß-barrel MCPs.


Assuntos
Vírus de Archaea/fisiologia , Proteínas do Capsídeo/ultraestrutura , Vírus de DNA/fisiologia , Haloarcula/virologia , Montagem de Vírus , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica , Modelos Moleculares , Conformação Proteica em Folha beta , Multimerização Proteica , Vírion/ultraestrutura
10.
Neurobiol Aging ; 73: 123-134, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30342273

RESUMO

Olfactory dysfunction is one of the earliest features in Lewy-type alpha-synucleinopathies (LTSs) such as Parkinson's disease (PD). However, the underlying molecular mechanisms associated to smell impairment are poorly understood. Applying mass spectrometry-based quantitative proteomics in postmortem olfactory bulbs across limbic, early-neocortical, and neocortical LTS stages of parkinsonian patients, a proteostasis impairment, was observed, identifying 268 differentially expressed proteins between controls and PD phenotypes. In addition, network-driven proteomics revealed a modulation in ERK1/2, MKK3/6, and PDK1/PKC signaling axes. Moreover, a cross-disease study of selected olfactory molecules in sporadic Alzheimer's disease (AD) cases revealed different protein derangements in the modulation of secretagogin (SCGN), calcyclin-binding protein (CACYBP), and glucosamine 6 phosphate isomerase 2 (GNPDA2) between PD and AD. An inverse correlation between GNPDA2 and α-synuclein protein levels was also reflected in PD cerebrospinal fluid. Interestingly, PD patients exhibited significantly lower serum GNPDA2 levels than controls (n = 82/group). Our study provides important avenues for understanding the olfactory bulb proteostasis imbalance in PD, deciphering mechanistic clues to the equivalent smell deficits observed in AD and PD pathologies.


Assuntos
Transtornos do Olfato/genética , Bulbo Olfatório/metabolismo , Doença de Parkinson/genética , Proteômica/métodos , Proteostase , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Proteínas de Ligação ao Cálcio/metabolismo , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Proteoma/metabolismo , Secretagoguinas/metabolismo , Biologia de Sistemas/métodos
11.
PLoS One ; 13(10): e0205073, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30312303

RESUMO

PURPOSE: The main objective of this study is to compare the protein expression of human keratocytes treated with Plasma rich in growth factors (PRGF) or autologous serum (AS) and previously induced to myofibroblast by TGF-ß1 treatment. METHODS: Blood from healthy donor was collected and processed to obtain AS and PRGF eye drops. Blood derivates were aliquoted and stored at -80°C until use. Keratocyte cells were pretreated for 60 minutes with 2.5 ng/ml TGF-ß1. After that, cells were treated with PRGF, AS or with TGF-ß1 (control). To characterize the proteins deregulated after PRGF and AS treatment, a proteomic approach that combines 1D-SDS-PAGE approach followed by LC-MS/MS was carried out. RESULTS: Results show a catalogue of key proteins in close contact with a myofibroblastic differentiated phenotype in AS treated-cells, whereas PRGF-treated cells show attenuation on this phenotype. The number of proteins downregulated after PRGF treatment or upregulated in AS-treated cells suggest a close relationship between AS-treated cells and cytoskeletal functions. On the other hand, proteins upregulated after PRGF-treatment or downregulated in AS-treated cells reveal a greater association with processes such as protein synthesis, proliferation and cellular motility. CONCLUSION: This proteomic analysis helps to understand the molecular events underlying AS and PRGF-driven tissue regeneration processes, providing new evidence that comes along with the modulation of TGF-ß1 activity and the reversion of the myofibroblastic phenotype by PRGF.


Assuntos
Técnicas de Cultura de Células , Ceratócitos da Córnea/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Miofibroblastos/metabolismo , Plasma , Soro , Autoenxertos , Diferenciação Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Masculino , Proteoma , Regeneração/fisiologia , Fator de Crescimento Transformador beta1
12.
Hepatol Commun ; 2(9): 1064-1079, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30202821

RESUMO

Drug-induced liver injury (DILI) is a serious worldwide health problem that accounts for more than 50% of acute liver failure. There is a great interest in clinical diagnosis and pharmaceutical industry to elucidate underlying molecular mechanisms and find noninvasive biomarkers for this pathology. Cell-secreted extracellular vesicles (EVs) have provided a new biological source to identify low disease invasive markers. Despite the intense research developed on these vesicles, there is currently a gap on their patho-physiological effects. Here, we study EVs secreted by primary rat hepatocytes challenged with galactatosamine (GalN), acetaminophen, or diclofenac as DILI in vitromodels. Proteomics analysis of these EVs revealed an increase in enzymes already associated with liver damage, such as catecholamine-methyl transferase and arginase 1. An increase in translation-related proteins and a decrease in regulators of apoptosis were also observed. In addition, we show the presence of enzymatic activity of P450 cytochrome 2d1 in EVs. The activity specifically is decreased in EVs secreted by hepatocytes after acetaminophen treatment and increased in EVs derived from GalN-treated hepatocytes. By using in vivo preclinical models, we demonstrate the presence of this cytochrome activity in circulation under normal conditions and an increased activity after GalN-induced injury. Conclusion: Hepatocyte-secreted EVs carry active xenobiotic-metabolizing enzymes that might be relevant in extracellular metabolism of drugs and be associated with DILI. (Hepatology Communications 2018;0:00-00).

13.
Expert Rev Proteomics ; 15(9): 749-756, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30204005

RESUMO

INTRODUCTION: Hepatocellular carcinoma (HCC) is recognized as the fifth most common neoplasm and currently represents the second leading form of cancer-related death worldwide. Despite great progress has been done in the understanding of its pathogenesis, HCC represents a heavy societal and economic burden as most patients are still diagnosed at advanced stages and the 5-year survival rate remain below 20%. Early detection and revolutionary therapies that rely on the discovery of new molecular biomarkers and therapeutic targets are therefore urgently needed to develop precision medicine strategies for a more efficient management of patients. Areas covered: This review intends to comprehensively analyse the proteomics-based research conducted in the last few years to address some of the principal still open riddles in HCC biology, based on the identification of molecular drivers of tumor progression and metastasis. Expert commentary: The technical advances in mass spectrometry experienced in the last decade have significantly improved the analytical capacity of proteome wide studies. Large-scale protein and protein variant (post-translational modifications) identification and quantification have allowed detailed dissections of molecular mechanisms underlying HCC progression and are already paving the way for the identification of clinically relevant proteins and the development of their use on patient care.


Assuntos
Neoplasias Hepáticas/metabolismo , Proteoma/metabolismo , Biomarcadores Tumorais/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Proteômica
14.
Hum Reprod ; 33(10): 1898-1906, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30169642

RESUMO

STUDY QUESTION: Is there any difference in the protein composition of the endometrial fluid aspirate (EFA) obtained the day of embryo transfer in in vitro fertilization (IVF) cycles achieving and not achieving pregnancy? SUMMARY ANSWER: Comparative analysis identified a differential protein expression pattern in 'implantative' and 'non-implantative' IVF cycles. WHAT IS KNOWN ALREADY: EFA allows non-invasive characterization of the endometrium, and may contain important information on its receptivity when performing (IVF) cycles. Endometrial side of implantation has usually been studied with endometrial biopsy in an IVF cycle prior to embryo transfer, focusing on 'receptive/non-receptive' endometria and with low-throughput proteomic techniques. STUDY DESIGN, SIZE, DURATION: We have compared the protein expression patterns in EFA from a total of 110 women undergoing IVF, corresponding to 50 implantative and 60 non-implantative IVF cycles. Discovery (38 patients) and Validation (42 patients) sample cohorts were analyzed using a high-throughput differential proteomic approach. Then, the differential expression of glycogen phosphorylase B (PYGB) was validated by western blotting in an additional cohort (30 patients). The study period was 18 months. PARTICIPANTS/MATERIALS, SETTING, METHODS: The population under study consisted of 110 women aged 18-40 years old, undergoing their first or second IVF/ intracytoplasmic sperm injection cycle, with normal uterus and endometrium, and 1-2 good quality embryos, and embryo transfer being performed on Day 3. Endometrial fluid aspiration was performed immediately before the embryo transfer. Samples (80) were initially distributed in two independent cohorts and analyzed by liquid chromatography-mass spectrometry. The first cohort was used for the discovery and the second for the validation of the results. Filter-aided sample preparation was used for the in-solution tryptic digestion of the proteins present in the samples, followed by label-free mass spectrometry analysis. In order to unravel the molecular features of receptivity, the lists of differential proteins were thoroughly analyzed using different bioinformatic tools, including GSEA, IPA and GO analysis. MAIN RESULTS AND THE ROLE OF CHANCE: A false discovery rate-based correction of the t-test P-values was carried out in order to strengthen the reliability of the results. Functional analyses denoted the deregulation of important processes governing receptivity, such as antimicrobial response, cell-cell interaction, immune response and inflammatory signaling, among others. Overall eight proteins were commonly deregulated in both studied datasets and brain form glycogen phosphorylase (PYGB) was selected for confirmatory analysis. LIMITATIONS, REASONS FOR CAUTION: Our results were obtained from patients with normal uterus and endometrium and with good quality embryos, who had fresh Day-3 embryo transfer, in stimulated cycles. Therefore, our observations may not be applicable to poor prognosis cases or non-stimulated cycles. WIDER IMPLICATIONS OF THE FINDINGS: This work provides insights into the molecular features of implantative IVF cycles using non-invasive methods. It reveals that EFA may reflect an increased inflammatory state in non-implantative endometrium. Additionally, it proposes PYGB as a potential biomarker for endometrial receptivity or implantation success. This knowledge opens a new avenue for developing embryo transfer strategies, through the improvement of embryo culture media or modifying endometrial fluid composition to increase pregnancy rates. STUDY FUNDING/COMPETING INTEREST(S): This study was partially funded by a Grant for Fertility Innovation (GFI, 2011) from Merck (Darmstadt, Germany). Authors declare no competing interests. TRIAL REGISTRATION NUMBER: Not applicable.


Assuntos
Glicemia/metabolismo , Transferência Embrionária/métodos , Endométrio/metabolismo , Glicogênio Fosforilase/metabolismo , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Feminino , Fertilização In Vitro , Glicogênio Fosforilase/análise , Humanos , Gravidez , Proteômica , Reprodutibilidade dos Testes , Adulto Jovem
15.
J Tissue Eng Regen Med ; 12(7): 1769-1779, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29785783

RESUMO

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic Collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin were applied as coatings on sandblasted, acid-etched titanium substrates, and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings.


Assuntos
Substitutos Ósseos , Materiais Revestidos Biocompatíveis , Gelatina , Teste de Materiais , Proteômica , Dióxido de Silício , Animais , Substitutos Ósseos/química , Substitutos Ósseos/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Gelatina/química , Gelatina/farmacologia , Camundongos , Coelhos , Dióxido de Silício/química , Dióxido de Silício/farmacologia
16.
Arch Gynecol Obstet ; 297(6): 1577-1586, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29637268

RESUMO

PURPOSE: To assess whether there are proteins in endometrial fluid aspirate (EFA) that predict implantation. METHODS: The population under study consisted of 285 women undergoing embryo transfer (ET). Endometrial fluid aspiration was performed immediately before ET. Results of proteomic analysis of EFA were compared between 33 cases who achieved pregnancy and 33 who did not. Samples were analysed by 2D electrophoresis and mass spectrometry. Blood samples were studied by ELISA Pregnancy rates and maternal complications were compared to those in women refusing aspiration. RESULTS: We found 23 proteins differentially expressed in the EFA in conception cycles: 4 up-regulated proteins and 19 down-regulated (FC = 0.31 0.78) (among others, arginase-1, actin B, PARK-7, cofilin-1, stathmin, annexin-2 and CAPZB). Among the five studied proteins that were differentially expressed in EFA, none was differentially expressed in serum. The aspiration procedure had no impact on pregnancy rate. No maternal complications were reported. CONCLUSIONS: We found a very different protein profile in implantative cycles, the majority of proteins being down-regulated. This probably reflects a different endometrial functional status, more favourable to implantation. EFA proteomic analysis could be a useful tool in the planning ET strategies.


Assuntos
Implantação do Embrião/fisiologia , Transferência Embrionária/métodos , Endométrio/metabolismo , Fertilização In Vitro/métodos , Proteômica , Adulto , Anexina A2/metabolismo , Proteína de Capeamento de Actina CapZ , Feminino , Humanos , Espectrometria de Massas , Gravidez , Taxa de Gravidez , Estatmina
17.
Emerg Microbes Infect ; 7(1): 19, 2018 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-29511161

RESUMO

Macrophages are cells of the innate immune system with the ability to phagocytose and induce a global pattern of responses that depend on several signaling pathways. We have determined the biosignature of murine bone marrow-derived macrophages and human blood monocytes using transcriptomic and proteomic approaches. We identified a common pattern of genes that are transcriptionally regulated and overall indicate that the response to B. burgdorferi involves the interaction of spirochetal antigens with several inflammatory pathways corresponding to primary (triggered by pattern-recognition receptors) and secondary (induced by proinflammatory cytokines) responses. We also show that the Toll-like receptor family member CD180 is downregulated by the stimulation of macrophages, but not monocytes, with the spirochete. Silencing Cd180 results in increased phagocytosis while tempering the production of the proinflammatory cytokine TNF. Cd180-silenced cells produce increased levels of Itgam and surface CD11b, suggesting that the regulation of CD180 by the spirochete initiates a cascade that increases CR3-mediated phagocytosis of the bacterium while repressing the consequent inflammatory response.


Assuntos
Antígenos CD/imunologia , Borrelia burgdorferi/fisiologia , Doença de Lyme/genética , Macrófagos/imunologia , Animais , Antígenos CD/genética , Borrelia burgdorferi/genética , Citocinas/genética , Citocinas/imunologia , Humanos , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Macrófagos/química , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/química , Monócitos/imunologia , Monócitos/microbiologia , Fagocitose , Proteômica , Receptores Toll-Like/genética , Receptores Toll-Like/imunologia
18.
FASEB J ; 32(7): 3878-3891, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29465313

RESUMO

CD5-like (CD5L) is a soluble scavenger cysteine-rich protein that modulates inflammatory responses. We studied the involvement of CD5L in liver cancer. Immunohistochemistry (IHC) of CD5L in 60 hepatocellular carcinomas and 34 adjacent nontumor livers, showed that CD5L staining was higher in tumor than in nontumor tissue (Mann-Whitney test; P = 0.0039). High CD5L correlated with elevated proliferation (Ki67, linear regression; P < 0.0001) and lower patient event-free survival (log-rank; P = 0.0185). Accordingly, CD5L expression was detected in the liver cancer cell lines Huh7, HepG2, and SNU-398. In vitro technologies using these cell lines, including small interfering RNA (siRNA) and cDNA transfection, showed that CD5L promoted colony formation and cell proliferation and protected against cisplatin-induced apoptosis. To find a molecular explanation for these roles, novel CD5L-interacting protein ligands in liver cancer cells were identified by immunoprecipitation followed by mass spectrometry. Among these, the molecular chaperone of the unfolded protein response (UPR), heat shock protein (HSP)-A5, was selected for validation. The interaction was confirmed by confocal microscopy in the Huh7 and HepG2 cell lines. Furthermore, functional experiments revealed that CD5L activates the UPR and autophagy mechanisms in Huh7 cells, thereby providing a novel molecular link between the UPR and autophagy in liver cancer.-Aran, G., Sanjurjo, L., Bárcena, C., Simon-Coma, M., Téllez, É., Vázquez-Vitali, M., Garrido, M., Guerra, L., Díaz, E., Ojanguren, I., Elortza, F., Planas, R., Sala, M., Armengol, C., Sarrias, M.-R. CD5L is upregulated in hepatocellular carcinoma and promotes liver cancer cell proliferation and antiapoptotic responses by binding to HSPA5 (GRP78).


Assuntos
Apoptose , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Neoplasias Hepáticas/metabolismo , Receptores Depuradores Classe B/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Feminino , Proteínas de Choque Térmico/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Receptores Depuradores Classe B/genética , Resposta a Proteínas não Dobradas , Regulação para Cima
19.
Methods Mol Biol ; 1719: 59-70, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29476504

RESUMO

Peptidomics is an emerging field focused in the analysis of endogenous peptides. Naturally occurring peptides are often endogenously produced protein fragments. Cleavage of precursor proteins by proteases generates peptides that may gain specialized functions not ascribed to their precursors, and which could reflect the state of a cell under certain physiological conditions or pathological processes.Since peptides are found in complex matrices (e.g., serum, tear, urine, cerebrospinal fluid), they need to be isolated from the matrix and concentrated before they can be analyzed on mass spectrometry. This chapter describes methods for sample preparation prior to mass spectrometry analysis. In addition, different peptide fragmentation techniques are described which are complementary when analyzing naturally occurring peptides by liquid chromatography coupled online to tandem mass spectrometry.


Assuntos
Espectrometria de Massas/métodos , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Proteômica/métodos , Lágrimas/metabolismo , Urina/química , Humanos , Lágrimas/química , Urinálise
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