Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins

The analysis of the complexes between metal-based chemotherapeutic drugs and proteins in biological samples, such as cisplatin or oxaliplatin, can be a challenge due to metal strong reactivity towards S-donor molecules such as dithiothreitol (DTT) or b-mercaptoethanol (BME), usually employed as reducing agents in electrophoretic separations and proteolytic digestions for LC–MS/MS analysis. This protocol describes the use of the thiol-free reducing trialkylphosphines, such as tributylphosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP) as suitable reagents for the preservation of the metal-protein complexes during OFFGEL-IEF and SDS-PAGE separations, respectively. Moreover, the filter-aided sample preparation (FASP) method is presented as an advantageous option to perform tryptic in-solution digestions of metal–protein complexes in combination with OFFGEL-IEF separations

Bridging the gap between molecular and elemental mass spectrometry: Higher energy collisional dissociation (HCD) revealing elemental information

Molecular mass spectrometry has been applied to simultaneously obtain molecular and elemental information from metal-containing species. Energy tuning of the higher-energy collision dissociation (HCD) fragmentation cell allows the controlled production of typical peptide fragments or elemental reporter ions informing about the metallic content of the analyzed species. Different instrumental configurations and fragmentation techniques have been tested, and the efficiency extracting the elemental information has been compared. HCD fragmentation operating at very high energy led to the best results. Platinum, lanthanides, and iodine reporter ions from peptides interacting with cisplatin, peptides labeled with lanthanides-MeCAT-IA, and iodinated peptides, respectively, were obtained. The possibility to produce abundant molecular and elemental ions in the same analysis simplifies the correlation between both signals and open pathways in metallomics studies enabling the specific tracking of metal-containing species. The proposed approach has been successfully applied to in solution standards and complex samples. Moreover, interesting preliminary MALDI-imaging experiments have been performed showing similar metal distribution compared to laser ablation (LA)-ICPMS

Evolution after Anti-TNF Discontinuation in Patients with Inflammatory Bowel Disease: A Multicenter Long-Term Follow-Up Study

OBJECTIVES:The aims of this study were to assess the risk of relapse after discontinuation of anti-tumor necrosis factor (anti-TNF) drugs in patients with inflammatory bowel disease (IBD), to identify the factors associated with relapse, and to evaluate the overcome after retreatment with the same anti-TNF in those who relapsed.METHODS:This was a retrospective, observational, multicenter study. IBD patients who had been treated with anti-TNFs and in whom these drugs were discontinued after clinical remission was achieved were included.RESULTS:A total of 1, 055 patients were included. The incidence rate of relapse was 19% and 17% per patient-year in Crohn''s disease and ulcerative colitis patients, respectively. In both Crohn''s disease and ulcerative colitis patients in deep remission, the incidence rate of relapse was 19% per patient-year. The treatment with adalimumab vs. infliximab (hazard ratio (HR)=1.29; 95% confidence interval (CI)=1.01-1.66), elective discontinuation of anti-TNFs (HR=1.90; 95% CI=1.07-3.37) or discontinuation because of adverse events (HR=2.33; 95% CI=1.27-2.02) vs. a top-down strategy, colonic localization (HR=1.51; 95% CI=1.13-2.02) vs. ileal, and stricturing behavior (HR=1.5; 95% CI=1.09-2.05) vs. inflammatory were associated with a higher risk of relapse in Crohn''s disease patients, whereas treatment with immunomodulators after discontinuation (HR=0.67; 95% CI=0.51-0.87) and age (HR=0.98; 95% CI=0.97-0.99) were protective factors. None of the factors were predictive in ulcerative colitis patients. Retreatment of relapse with the same anti-TNF was effective (80% responded) and safe.CONCLUSIONS:The incidence rate of inflammatory bowel disease relapse after anti-TNF discontinuation is relevant. Some predictive factors of relapse after anti-TNF withdrawal have been identified. Retreatment with the same anti-TNF drug was effective and safe

Anti-tumour necrosis factor discontinuation in inflammatory bowel disease patients in remission: study protocol of a prospective, multicentre, randomized clinical trial

Background: Patients with inflammatory bowel disease who achieve remission with anti-tumour necrosis factor (anti-TNF) drugs may have treatment withdrawn due to safety concerns and cost considerations, but there is a lack of prospective, controlled data investigating this strategy. The primary study aim is to compare the rates of clinical remission at 1?year in patients who discontinue anti-TNF treatment versus those who continue treatment. Methods: This is an ongoing, prospective, double-blind, multicentre, randomized, placebo-controlled study in patients with Crohn?s disease or ulcerative colitis who have achieved clinical remission for ?6?months with an anti-TNF treatment and an immunosuppressant. Patients are being randomized 1:1 to discontinue anti-TNF therapy or continue therapy. Randomization stratifies patients by the type of inflammatory bowel disease and drug (infliximab versus adalimumab) at study inclusion. The primary endpoint of the study is sustained clinical remission at 1?year. Other endpoints include endoscopic and radiological activity, patient-reported outcomes (quality of life, work productivity), safety and predictive factors for relapse. The required sample size is 194 patients. In addition to the main analysis (discontinuation versus continuation), subanalyses will include stratification by type of inflammatory bowel disease, phenotype and previous treatment. Biological samples will be obtained to identify factors predictive of relapse after treatment withdrawal. Results: Enrolment began in 2016, and the study is expected to end in 2020. Conclusions: This study will contribute prospective, controlled data on outcomes and predictors of relapse in patients with inflammatory bowel disease after withdrawal of anti-TNF agents following achievement of clinical remission. Clinical trial reference number: EudraCT 2015-001410-1

Thiol-free reducing agents in electrophoretic separations and FASP proteolytic digestions for the analysis of metal-binding proteins

The analysis of the complexes between metal-based chemotherapeutic drugs and proteins in biological samples, such as cisplatin or oxaliplatin, can be a challenge due to metal strong reactivity towards S-donor molecules such as dithiothreitol (DTT) or β-mercaptoethanol (BME), usually employed as reducing agents in electrophoretic separations and proteolytic digestions for LC–MS/MS analysis. • This protocol describes the use of the thiol-free reducing trialkylphosphines, such as tributylphosphine (TBP) and tris(2-carboxyethyl)phosphine (TCEP) as suitable reagents for the preservation of the metal-protein complexes during OFFGEL-IEF and SDS-PAGE separations, respectively. • Moreover, the filter-aided sample preparation (FASP) method is presented as an advantageous option to perform tryptic in-solution digestions of metal–protein complexes in combination with OFFGEL-IEF separations. • The FASP procedure allows including previous reduction and alkylation steps in addition to proteolysis, ensuring the preservation of the metal–protein complexes. The limited time that proteins remain in contact with the reducing agent, either TBP or even DTT, during FASP could be a key factor for its extraordinary performance on the digestion of metal–protein complexes

Compuestos y sus usos como sondas fluorescentes

Compuestos y sus usos como sondas fluorescentes. Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o marcaje fluorescente especifico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic

Compuestos y sus usos como sondas fluorescentes

Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o marcaje fluorescente especifico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)B2 Patente con examen previ

Compounds and their uses as fluorescent probes

Organic dyes based on F-BODIPY derivatives and carnitine, method of producing same and their use for specific fluorescent labeling or tagging of mitochondria in live cellsPeer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic

Compuestos y sus usos como sondas fluorescentes

[EN] Organic dyes based on F -BODIPY derivatives and camitine, method for producing same and their use for the specific fluorescent labelling or tagging of mitochondria in living cells.[ES] Colorantes orgánicos basados en derivados de F-BODIPY y carnitina, su procedimiento de obtención y su aplicación para el etiquetado o mareaje fluorescente específico de mitocondrias en células vivas.Peer reviewedConsejo Superior de Investigaciones Científicas (España),Fundación para el fomento en Asturias de la investigacion aplicada y la tecnología (FICYT), Fundación para la investigación e innovación biosanitaria en el principado de Asturias (FINBA)A1 Solicitud de patente con informe sobre el estado de la técnic