A rapid electrokinetic chromatography method using short-end injection for the enantioselective separation of tryptophan

A rapid enantioselective methodology for the analysis of tryptophan was developed in this work by electrokinetic chromatography using short-end injection and an anionic cyclodextrin as chiral selector. No previous derivatization of tryptophan was necessary. The influence of different experimental variables on the enantiomeric separation was investigated. The use of a 100 mM formate buffer (pH 2.2) containing 1.25% sulfated-?-cyclodextrin with an uncoated fused-silica capillary of 50 µm inner diameter with a total length of 48.5 cm (effective length of 8.5 cm), and an injection by applying a pressure of -50 mbar (short-end injection) for 4 s, enabled the enantiomeric separation of tryptophan within 2.5 min with a resolution of 7.4. As desirable, the enantiomeric impurity, D-tryptophan, was the first-migrating enantiomer. The analytical characteristics of the developed methodology were evaluated in terms of linearity, precision, accuracy, and limits of detection and quantification, showing its good performance to be applied to the analysis of tryptophan-based dietary supplements. A relative limit of detection of 0.1% was obtained for the enantiomeric impurity, D-tryptophan, in the presence of the L-enantiomer. Results showed that the developed methodology is an interesting alternative for the enantioselective analysis of tryptophan enabling the rapid quality control of dietary supplements

Stereoselective separation of 4-hydroxyproline by electrokinetic chromatography

A chiral methodology was developed in this work enabling for the first time the separation of the four stereoisomers of the amino acid 4-hydroxyproline (4-Hyp) in the format of capillary electrophoresis (CE). After a screening of different neutral cyclodextrins (CDs) in the electrokinetic chromatography (EKC) mode, methyl-?-CD was selected as chiral selector to stereoselectively separate 4-Hyp (previously derivatized with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl)) in a 75 mM phosphate buffer at pH 7.0. The effect of the concentration of the CD, the separation voltage, and the temperature on the chiral separation was investigated. A concentration of 10 mM for methyl-?-CD, a voltage of 30 kV, and a temperature of 15 ºC allowed the separation of the four stereoisomers of 4-Hyp in less than 21 min with resolutions between consecutive peaks of 1.5, 2.7, and 3.6. The injection of individual standard solutions of each stereoisomer enabled peak identification and the methodology was able to detect up to 0.1 % of each stereoisomer. Analytical characteristics of the developed methodology were adequate to be applied to the analysis of nutricosmetic supplements. A good agreement was observed between the content determined for trans-4-L-Hyp and that indicated in the label for the product. No enantiomeric impurities were detected what shows the great potential of this method in the quality control of these product

A Non-Targeted Capillary Electrophoresis-Mass Spectrometry Strategy to Study Metabolic Differences in an In Vitro Model of High-Glucose Induced Changes in Human Proximal Tubular HK-2 Cells

Diabetic nephropathy is characterized by the chronic loss of kidney function due to high glucose renal levels. HK-2 proximal tubular cells are good candidates to study this disease. The aim of this work was to study an in vitro model of high glucose-induced metabolic alterations in HK-2 cells to contribute to the pathogenesis of this diabetic complication. An untargeted metabolomics strategy based on CE-MS was developed to find metabolites affected under high glucose conditions. Intracellular and extracellular fluids from HK-2 cells treated with 25 mM glucose (high glucose group), with 5.5 mM glucose (normal glucose group), and with 5.5 mM glucose and 19.5 mM mannitol (osmotic control group) were analyzed. The main changes induced by high glucose were found in the extracellular medium where increased levels of four amino acids were detected. Three of them (alanine, proline, and glutamic acid) were exported from HK-2 cells to the extracellular medium. Other affected metabolites include Amadori products and cysteine, which are more likely cause and consequence, respectively, of the oxidative stress induced by high glucose in HK-2 cells. The developed CE-MS platform provides valuable insight into high glucose-induced metabolic alterations in proximal tubular cells and allows identifying discriminative molecules of diabetic nephropathy

Exploratory Metabolomic Analysis Based on Reversed-Phase Liquid Chromatography-Mass Spectrometry to Study an In Vitro Model of Hypoxia-Induced Metabolic Alterations in HK-2 Cells

Oxygen deficiency in cells, tissues, and organs can not only prevent the proper development of biological functions but it can also lead to several diseases and disorders. In this sense, the kidney deserves special attention since hypoxia can be considered an important factor in the pathophysiology of both acute kidney injury and chronic kidney disease. To provide better knowledge to unveil the molecular mechanisms involved, new studies are necessary. In this sense, this work aims to study, for the first time, an in vitro model of hypoxia-induced metabolic alterations in human proximal tubular HK-2 cells because renal proximal tubules are particularly susceptible to hypoxia. Different groups of cells, cultivated under control and hypoxia conditions at 0.5, 5, 24, and 48 h, were investigated using untargeted metabolomic approaches based on reversed-phase liquid chromatography-mass spectrometry. Both intracellular and extracellular fluids were studied to obtain a large metabolite coverage. On the other hand, multivariate and univariate analyses were carried out to find the differences among the cell groups and to select the most relevant variables. The molecular features identified as affected metabolites were mainly amino acids and Amadori compounds. Insights about their biological relevance are also provided

Higher constitutive IL15Rα expression and lower IL‐15 response threshold in coeliac disease patients

The IL-15 triggering effect of gliadin is not exclusive to coeliac disease (CD) patients, whereas the secondary response is CD specific. We have studied the expression of the IL-15 receptor, and the IL-15 response upon stimulation, in non-CD and CD patients, and the possible existence of a lower immunological threshold in the latter. Forty-two CD patients (20 on a gluten-containing diet, GCD, and 22 on gluten-free diet, GFD) and 24 non-CD healthy individuals were studied. IL15Rα mRNA expression, and tissue characterization, were assayed in the duodenum. Biopsies from six CD patients on GFD and 10 non-CD individuals were studied in vitro using organ culture in basal conditions, as well as after IL-15 stimulation discarding basal IL-15 production. Secretion of immune mediators was measured in the culture supernatants. IL15Rα mRNA expression was increased in CD patients, as compared with non-CD controls (on GFD P = 0·0334, on GCD P = 0·0062, respectively), and confirmed also by immunofluorescence. No differences were found between CD patients on GFD and on GCD. After in vitro IL-15 stimulation, IL15Rα expression was only triggered in non-CD controls (P = 0·0313), though it remained increased in CD patients. Moreover, IL-15 induced a more intense immunological response in CD patients after triggering the production of both nitrites and IFNγ (P = 0·0313, P = 0·0313, respectively). Gliadin-induced IL15 has a lower response threshold in CD patients, leading to the production of other immune mediators and the development of the intestinal lesion, and thus magnifying its effects within the CD intestine.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun

Effectiveness and Safety of the Switch from Remicade® to CT-P13 in Patients with Inflammatory Bowel Disease

[Background and Aims] To evaluate the clinical outcomes in patients with IBD after switching from Remicade® to CT-P13 in comparison with patients who maintain Remicade®.[Methods] Patients under Remicade® who were in clinical remission with standard dosage at study entry were included. The ‘switch cohort’ [SC] comprised patients who made the switch from Remicade® to CT-P13, and the ‘non-switch’ cohort [NC] patients remained under Remicade®.[Results] A total of 476 patients were included: 199 [42%] in the SC and 277 [58%] in the NC. The median follow-up was 18 months in the SC and 23 months in the NC [p < 0.01]. Twenty-four out of 277 patients relapsed in the NC; the incidence of relapse was 5% per patient-year. The cumulative incidence of relapse was 2% at 6 months and 10% at 24 months in this group. Thirty-eight out of 199 patients relapsed in the SC; the incidence rate of relapse was 14% per patient-year. The cumulative incidence of relapse was 5% at 6 months and 28% at 24 months. In the multivariate analysis, the switch to CT-P13 was associated with a higher risk of relapse (HR = 3.5, 95% confidence interval [CI] = 2–6). Thirteen percent of patients had adverse events in the NC, compared with 6% in the SC [p < 0.05].[Conclusions] Switching from Remicade® to CT-P13 might be associated with a higher risk of clinical relapse, although this fact was not supported in our study by an increase in objective markers of inflammation. The nocebo effect might have influenced this result. Switching from Remicade® to CT-P13 was safe.This research has been funded by grants from the Instituto de Salud Carlos III [PI13/00041 and FI17/00143]

Effectiveness and Safety of the Switch from Remicade® to CT-P13 in Patients with Inflammatory Bowel Disease

BACKGROUND AND AIMS: To evaluate the clinical outcomes in patients with IBD after switching from Remicade® to CT-P13 in comparison with patients who maintain Remicade®. METHODS: Patients under Remicade® who were in clinical remission with standard dosage at study entry were included. The ''switch cohort'' [SC] comprised patients who made the switch from Remicade® to CT-P13, and the ''non-switch'' cohort [NC] patients remained under Remicade®. RESULTS: A total of 476 patients were included: 199 [42%] in the SC and 277 [58%] in the NC. The median follow-up was 18 months in the SC and 23 months in the NC [p < 0.01]. Twenty-four out of 277 patients relapsed in the NC; the incidence of relapse was 5% per patient-year. The cumulative incidence of relapse was 2% at 6 months and 10% at 24 months in this group. Thirty-eight out of 199 patients relapsed in the SC; the incidence rate of relapse was 14% per patient-year. The cumulative incidence of relapse was 5% at 6 months and 28% at 24 months. In the multivariate analysis, the switch to CT-P13 was associated with a higher risk of relapse (HR = 3.5, 95% confidence interval [CI] = 2-6). Thirteen percent of patients had adverse events in the NC, compared with 6% in the SC [p < 0.05]. CONCLUSIONS: Switching from Remicade® to CT-P13 might be associated with a higher risk of clinical relapse, although this fact was not supported in our study by an increase in objective markers of inflammation. The nocebo effect might have influenced this result. Switching from Remicade® to CT-P13 was safe

Molecular and genetic control of plant thermomorphogenesis

Temperature is a major factor governing the distribution and seasonal behaviour of plants. Being sessile, plants are highly responsive to small differences in temperature and adjust their growth and development accordingly. The suite of morphological and architectural changes induced by high ambient temperatures, below the heat-stress range, is collectively called thermomorphogenesis. Understanding the molecular genetic circuitries underlying thermomorphogenesis is particularly relevant in the context of climate change, as this knowledge will be key to rational breeding for thermo-tolerant crop varieties. Until recently, the fundamental mechanisms of temperature perception and signalling remained unknown. Our understanding of temperature signalling is now progressing, mainly by exploiting the model plant Arabidopsis thaliana. The transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) has emerged as a critical player in regulating phytohormone levels and their activity. To control thermomorphogenesis, multiple regulatory circuits are in place to modulate PIF4 levels, activity and downstream mechanisms. Thermomorphogenesis is integrally governed by various light signalling pathways, the circadian clock, epigenetic mechanisms and chromatin-level regulation. In this Review, we summarize recent progress in the field and discuss how the emerging knowledge in Arabidopsis may be transferred to relevant crop systems

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

Nurses' perceptions of aids and obstacles to the provision of optimal end of life care in ICU

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