Separation of unsaturated C18 fatty acids using perfluorinated-micellar electrokinetic chromatography: I. Optimization and separation process.

Ammonium perfluorooctanoate (APFOA) was used as a surfactant for the separation of free unsaturated C18 fatty acids by micellar electrokinetic chromatography. A simple background electrolyte of 50 mM APFOA water/methanol (90:10, v/v) at pH = 10 enabled the repeatable separation of oleic acid, elaidic acid, linoleic acid, and alpha-linolenic acid in less than 20 min. Separation conditions were optimized regarding various parameters (organic solvent, counterion, APFOA concentration, and pH). Because the repulsive interactions between fluorocarbon chains and hydrogenated chains are known to lead to segregation and phase separation, the choice of perfluorinated micelles to separate such perhydrogenated long-chain acids could appear astonishing. Therefore, the critical micelle concentration, the charge density, and the mobility of the micelles have been determined, resulting in a first description of the separation process.

Luminescent lanthanide complexes for reactive oxygen species biosensing and possible application in Alzheimer's diseases.

Histopathological hallmarks of Alzheimer's disease (AD) are intracellular neurofibrillary tangles and extracellular formation of senile plaques composed of the aggregated amyloid-beta peptide along with metal ions (copper, iron or zinc). In addition, oxidative stress is considered as an important factor in the etiology of AD and a multitude of metalloproteins and transporters is affected, leading to metal ion misregulation. Redox-active metal ions (e.g., copper) can catalyze the production of reactive oxygen species (ROS) in the presence of molecular oxygen and a reductant such as ascorbate. The ROS thus produced, in particular the hydroxyl radical which is the most reactive one, may contribute to oxidative stress conditions. Thus, detecting ROS in vivo or in biological models of AD is of interest for better understanding AD etiology. The use of biocompatible and highly specific and sensitive probes is needed for such a purpose, since ROS are transient species whose steady-state concentrations are very low. Luminescent lanthanide complexes are sensitive probes that can meet these criteria. The present review focuses on the recent advances in the use of luminescent lanthanide complexes for ROS biosensing. It shows why the use of luminescent lanthanide complexes is of particular interest for selectively detecting ROS ( O2·- , HO• , 1 O2 , H2 O2 , etc.) in biological samples in the µM-nM range. It particularly focuses on the most recent strategies and discusses what could be expected with the use of luminescent lanthanide complexes for better understanding some of the molecular mechanisms underlying the development of Alzheimer's disease.

Electrophoretic Determination of Symmetric and Asymmetric Dimethylarginine in Human Blood Plasma with Whole Capillary Sample Injection.

Asymmetric and symmetric dimethylarginines are toxic non-coded amino acids. They are formed by post-translational modifications and play multifunctional roles in some human diseases. Their determination in human blood plasma is performed using capillary electrophoresis with contactless conductivity detection. The separations are performed in a capillary covered with covalently bonded PAMAPTAC polymer, which generates anionic electroosmotic flow and the separation takes place in the counter-current regime. The background electrolyte is a 750 mM aqueous solution of acetic acid with pH 2.45. The plasma samples for analysis are treated by the addition of acetonitrile and injected into the capillary in a large volume, reaching 94.5% of the total volume of the capillary, and subsequently subjected to electrophoretic stacking. The attained LODs are 16 nm for ADMA and 22 nM for SDMA. The electrophoretic resolution of both isomers has a value of 5.3. The developed method is sufficiently sensitive for the determination of plasmatic levels of ADMA and SDMA. The determination does not require derivatization and the individual steps in the electrophoretic stacking are fully automated. The determined plasmatic levels for healthy individuals vary in the range 0.36-0.62 µM for ADMA and 0.32-0.70 µM for SDMA.

Melanogenesis Promoting Effect, Antioxidant Activity, and UPLC-ESI-HRMS Characterization of Phenolic Compounds of Argan Leaves Extract.

The use of natural products for the regulation of skin pigmentation is gaining popularity. In the present study, we evaluated the effect of argan leaves extract (ALE) on melanogenesis in B16 melanoma cells, determined its antioxidant activity, then quantified and identified its phenolic components. B16 cells were treated with various concentrations of ALE, then the cell viability and proliferation were assessed using MTT assay while the melanin content was determined using spectrophotometric methods. The expression level of tyrosinase (TYR), tyrosinase related protein-1 (TRP-1) and dopachrome tautomerase (DCT) was evaluated by Western blotting. The antioxidant activity of ALE was investigated using four different assays while UPLC-ESI-HRMS analysis was used to characterize the ALE phenolic profile. Fourteen phenolic compounds were identified, of which six are reported for the first time to be present in ALE. ALE treatment increases the melanin content of B16 cells in a dose-dependent manner without cytotoxicity. This was revealed by the observed ALE-increased expression level of TYR, DCT, and TRP-1. These bioactivities may be mainly attributed to its high flavonoids content. Argan leaves have the potential for use as a treatment for hypopigmentation disorders and as a bioactive component of cosmetic products that aim to increase pigmentation.

3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde labeling for direct analysis of amino acids in plasma is not suitable for simultaneous quantification of tryptophan, tyrosine, valine, and isoleucine by CE/fluorescence.

Capillary electrophoresis coupled to LED-induced fluorescence detection is a robust and sensitive technique used for amino acids (AA) analysis in biological media, after labeling with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA). We wanted to quantitate in plasma tryptophan (Trp), tyrosine (Tyr), valine (Val), and isoleucine (Ile). Among the different labeled AA-CBQCA, Trp has the lowest fluorescence yield, which makes its detection and quantification very difficult in biological samples such as plasma. We tried to improve Trp analysis by CE/LED-induced fluorescence detection to its maximal sensitivity by using large volume sample stacking as a preconcentration step in our analytical protocol. At pH 9.5, this step caused a drop in resolution during the separation of the four AAs and it was therefore necessary to work at pH 10. We have found that Tyr, Val, Ile, and Trp are detected and well separated from the other AAs, but Trp cannot be quantified in plasma samples, mainly because of the low fluorescence yield of the Trp-CBQCA derivative. The recorded LOD is 0.18 µM for Trp-CBQCA in standard solution with a resolution between Trp and Tyr of 1.2, while the LOD is 6 µM in plasma with the same resolution. Trp, Tyr, Val, and Ile are, however, efficiently quantified when using a 3 M acetic acid electrolyte and CE associated with capacitively coupled contactless conductivity detection, which also has the advantage of not requiring derivatization or large volume sample stacking. This article demonstrates, for the CE user, that quantitative analysis of these four AA in mouse plasma can be performed by CE-fluorescence after CBQCA labeling, with the exception of Trp. It can be advantageously replaced by CE/capacitively coupled contactless conductivity detection, the only efficient one for Trp, Tyr, Val, and Ile quantification. In this case, the LOD for Trp is 2 µM. The four AAs are separated with resolution with neighbors above 1.5.

Capillary electrophoresis/visible-LED induced fluorescence of tryptophan: What's new?

Tryptophane (Trp) labelled by 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA) is very difficult to identify using CE and fluorescence detection (480 nm). Why in this article some mass spectrometry experiments show that Trp is really labelled by CBQCA as Leucine (Leu)? If the maximum of UV absorption (λmax ) is the same between Leu-CBQCA and Trp-CBQCA, the molar extinction coefficient is around 2 fold higher for Trp-CBQCA. The fluorescence of the Leu-CBQCA derivative is 50 times more important than for Trp-CBQCA. The addition of 7.5 mM of ß-cyclodextrin (ß-CD) was found to be a good mean to improve 2.1 fold the sensitivity of the Trp-CBQCA fluorescence. Using a buffer containing SDS and ß-CD in CE, a LOD of 0.7 µM of L-Trp can be reached and the ratio of the intensities between Leu, Isoleucine, Valine, Trp is 100, 21, 15, 1. Negative ESI/ MS and MS/MS of the labeled amino acids show that a loss of the carboxylate function takes place. In the presence of two enantiomers of Trp-CBQCA, we have shown that this decarboxylation is not due to the derivatization process in the solution but rather occurs in the source of the mass spectrometer.

Sweat chloride quantification using capillary electrophoresis.

BACKGROUND: Cystic fibrosis (CF) is the less rare and severe genetic disease among the European population. Biochemical diagnosis of CF is based on the demonstration of increased chloride concentration in sweat samples, obtained during the sweat test (ST). WynSep developed a capillary electrophoresis with contactless conductivity detection (CE-C4D) able to measure sweat chloride with a low sample volume. We evaluated the clinical feasibility of this device in a cohort of patients suspected of CF, in comparison with the common coulometric method (ChloroChek chloridometer). METHODS: We determined sweat chloride concentration of 65 samples from patients referred to our institution to undergo a sweat test. Each sample was submitted to coulometric method first, then WynSep-CE, with or without internal standard (IS) subject to sufficient volume sample. RESULTS: A total of 53 samples were analysed by both coulometric and WynSep-CE (using IS) methods. The method validation showed comparable analytical performances for both methods; no false positive or false negative was recorded. The two methods showed a high correlation (r = 0.993, p < 0.001) and a close agreement was demonstrated by two different statistical tests (Bland-Altman and Passing-Bablok). CONCLUSIONS: WynSep-CE is an accurate, fast, easy-to-use and an appropriate method for CF diagnosis.

IL13-Mediated Dectin-1 and Mannose Receptor Overexpression Promotes Macrophage Antitumor Activities through Recognition of Sialylated Tumor Cells.

Macrophage-mediated cytotoxicity is controlled by surface receptor expression and activation. Despite the numerous studies documenting the role of macrophage C-type lectin receptors (CLR) in pathogen elimination, little is known about their contribution to antitumor responses. Here, we report that IL13 inhibits T-cell lymphoma and ovarian adenocarcinoma development in tumor-bearing mice through the conversion of tumor-supporting macrophages to cytotoxic effectors, characterized by a CLR signature composed of dectin-1 and mannose receptor (MR). We show that dectin-1 and MR are critical for the recognition of tumor cells through sialic acid-specific glycan structure on their surface and for the subsequent activation of macrophage tumoricidal response. Finally, we validated that IL13 antitumor effect mediated by dectin-1 and MR overexpression on macrophages can extend to various types of human tumors. Therefore, these results identify these CLRs as potential targets to promote macrophage antitumor response and represent an attractive approach to elicit tumor-associated macrophage tumoricidal properties.

A digest of capillary electrophoretic methods applied to lipid analyzes.

Lipids are naturally occurring organic compounds that can be classified into a number of types based on their solubility in nonpolar organic solvents, and are generally insoluble in water. The great structural variety of these various types of lipids has led them to be components of many different biological substances such as oils, waxes, cellular membranes, tissues and biological fluids. The use of capillary electrophoresis (CE) for the study of lipids during the past 30 years has been relatively rare when compared to its use for other classes of biomolecules, primarily due to their insolubility in water. However, a number of interesting studies have been conducted, and as part of this review, we will present the different approaches that were used, which mainly consist of micellar kinetic chromatography and non-aqueous CE. The main advantages of the use of these techniques compared to GC is the very simple sample preparation that is required and, compared to LC, the very robust and quick separations that can be obtained. In this review, we present the various methods that have been reported in the literature that have been used for the study of fatty acids, phospholipids, glycerides, eicosanoids and sterols, with the inclusion of various tables presenting descriptions of the CE methods used as well as the methods of detection, including UV absorbance, fluorescence, mass spectrometry, and conductivity. This review aims to demonstrate that CE can be easily used for the analysis of lipids.

Conserved Composition of Nod Factors and Exopolysaccharides Produced by Different Phylogenetic Lineage Sinorhizobium Strains Nodulating Soybean.

The structural variation of symbiotic signals released by rhizobia determines the specificity of their interaction with legume plants. Previous studies showed that Sinorhizobium strains from different phylogenetic lineages had different symbiotic performance on certain cultivated soybeans. Whether they released similar or different symbiotic signals remained unclear. In this study, we compared their nod and exo gene clusters and made a detailed structural analysis of Nod factors and EPS by ESI-MS/MS and two dimensions NMR. Even if there are some differences among nod or exo gene clusters; they produced much conserved Nod factor and EPS compositions. The Nod factors consist of a cocktail of ß-(1, 4)-linked tri-, tetra-, and pentamers of N-acetyl-D-glucosamine (GlcNAc). The C2 position on the non-reducing terminal end is modified by a lipid chain that contains 16 or 18 atoms of carbon-with or without unsaturations-, and the C6 position on the reducing residue is decorated by a fucose or a 2-O-methylfucose. Their EPS are composed of glucose, galactose, glucuronic acid, pyruvic acid in the ratios 5:1:2:1 or 6:1:2:1. These findings indicate that soybean cultivar compatibility of Sinorhizobium strains does not result from Nod factor or EPS structure variations. The structure comparison of the soybean microbionts with other Sinorhizobium strains showed that Nod factor structures of soybean microbionts are much conserved, although there are no specific genes shared by the soybean microsymbionts. EPS produced by Sinorhizobium strains are different from those of Bradyrhizobium. All above is consistent with the previous deduction that Nod factor structures are related to host range, while those of EPS are connected with phylogeny.

Three-Dimensional Directionality Is a Pivotal Structural Feature for the Bioactivity of Azabisphosphonate-Capped Poly(PhosphorHydrazone) Nanodrug Dendrimers.

Dendrimers are nanosized, nonlinear, hyperbranched polymers whose overall 3D shape is key for their biological activity. Poly(PhosphorHydrazone) (PPH) dendrimers capped with aza-bisphosphonate (ABP) end groups are known to have anti-inflammatory properties enabling the control of inflammatory diseases in different mouse models. Here we screen the anti-inflammatory activity of a series of PPH dendrimers bearing between 2 and 16 ABP end groups in a mouse model of arthritis and confront the biological results with atomistic simulations of the dendrimers. We show that only the PPH dendrimers capped with 10 and 12 ABP end groups can control the flare of the inflammatory disease. All-atom accelerated molecular dynamics simulations show that dendrimers with a low number of ABP end groups are directional but highly flexible/dynamic and have thereby limited efficiency in establishing multivalent interactions. The largest dendrimer appears as nondirectional, having 16 ABP end groups forming patches all over the dendrimer surface. Conversely, intermediate dendrimers having 10 or 12 ABP end groups reach the best compromise between the number of surface groups and their stable directional gathering, a real maximization of multivalency.

G-quadruplex aptamer selection using capillary electrophoresis-LED-induced fluorescence and Illumina sequencing.

One of the major difficulties that arises when selecting aptamers containing a G-quadruplex is the correct amplification of the ssDNA sequence. Can aptamers containing a G-quadruplex be selected from a degenerate library using non-equilibrium capillary electrophoresis (CE) of equilibrium mixtures (NECEEM) along with high-throughput Illumina sequencing? In this article, we present some mismatches of the G-quadruplex T29 aptamer specific to thrombin, which was PCR amplified and sequenced by Illumina sequencing. Then, we show the proportionality between the number of sequenced molecules of T29 added to the library and the number of sequences obtained in Illumina sequencing, and we find that T29 sequences from this aptamer can be detected in a random library of ssDNA after the sample is fractionated by NECEEM, amplified by PCR, and sequenced. Treatment of the data by the counting of double-stranded DNA T29 sequences containing a maximum of two mismatches reveals a good correlation with the enrichment factor (fE). This factor is the ratio of the number of aptamer sequences found in the collected complex sample divided by the total number of sequencing reads (aptamer and non-aptamer) plus the quantity of T29 molecules (spiked into a DNA library) injected into CE.

Recent advances in amino acid analysis by capillary electromigration methods: June 2015-May 2017.

In the tenth edition of this article focused on recent advances in amino acid analysis using capillary electrophoresis, we describe the most important research articles published on this topic during the period from June 2015 to May 2017. This article follows the format of the previous articles published in Electrophoresis. The new developments in amino acid analysis with CE mainly describe improvements in CE associated with mass spectrometry. Focusing on applications, we mostly describe clinical works, although metabolomics studies are also very important. Finally, works focusing on amino acids in food and agricultural applications are also described.

ssDNA degradation along capillary electrophoresis process using a Tris buffer.

Tris-Acetate buffer is currently used in the selection and the characterization of ssDNA by capillary electrophoresis (CE). By applying high voltage, the migration of ionic species into the capillary generates a current that induces water electrolysis. This phenomenon is followed by the modification of the pH and the production of Tris derivatives. By injecting ten times by capillary electrophoresis ssDNA (50 nM), the whole oligonucleotide was degraded. In this paper, we will show that the Tris buffer in the running vials is modified along the electrophoretic process by electrochemical reactions. We also observed that the composition of the metal ions changes in the running buffer vials. This phenomenon, never described in CE, is important for fluorescent ssDNA analysis using Tris buffer. The oligonucleotides are degraded by electrochemically synthesized species (present in the running Tris vials) until it disappears, even if the separation buffer in the capillary is clean. To address these issues, we propose to use a sodium phosphate buffer that we demonstrate to be electrochemically inactive.

Capillary electrophoresis hyphenated with UV-native-laser induced fluorescence detection (CE/UV-native-LIF).

Native laser-induced fluorescence using UV lasers associated to CE offers now a large related literature, for now 30 years. The main works have been performed using very expensive Ar-ion lasers emitting at 257 and 275 nm. They are not affordable for routine analyses, but have numerous applications such as protein, catecholamine, and indolamine analysis. Some other lasers such as HeCd 325 nm have been used but only for few applications. Diode lasers, emitting at 266 nm, cheaper, are extensively used for the same topics, even if the obtained sensitivity is lower than the one observed using the costly UV-Ar-ion lasers. This review presents various CE or microchips applications and different UV lasers used for the excitation of native fluorescence. We showed that CE/Native UV laser induced fluorescence detection is very sensitive for detection as well as small aromatic biomolecules than proteins containing Trp and Tyr amino acids. Moreover, it is a simple way to analyze biomolecules without derivatization.

A revisited structure for nitrosoprodenafil from NMR, mass spectrometry, X-ray and hydrolysis data.

The sildenafil analogue adulterant previously identified as a nitroso derivative (nitrosoprodenafil) in a dietary supplement (DS) marketed to increase sexual performance and sold in Europe in the early 2010s is the same as that found in the same type of DS available in Japan whose structure was established as a nitro derivative (mutaprodenafil or nitroprodenafil). Indeed, the compound isolated from the Man Power DS has identical UV, IR, NMR and MS spectroscopic characteristics and hydrolysis behavior than nitrosoprode-nafil. By revisiting its NMR assignments and MS and MS/MS data interpretation, it is demonstrated that the compound is actually a nitrothioimidazole-methisosildenafil hybrid, i.e. nitroprodenafil, whose structure is unequivocally confirmed by X-ray crystallography and synthesis experiments. Because the product is converted to methisosildenafil by hydrolysis, it is named nitropromethisosildenafil.

Chemical and Instrumental Approaches for Capillary Electrophoresis (CE)-Fluorescence Analysis of Proteins.

Capillary electrophoresis (CE) coupled to fluorescence detection is an invaluable technique for the quantitative analysis of proteins of interest in the field of clinical diagnosis and quality control of novel biotechnology products. The various chemical and instrumental approaches that have been reported to carry out such sensitive analysis are described in this paper. To illustrate the contribution of CE to the analysis of therapeutic proteins, a detailed protocol for impurities profiling of a recombinant antibody sample using CE-LEDIF is given.

Pulsed lasers versus continuous light sources in capillary electrophoresis and fluorescence detection studies: Photodegradation pathways and models.

Pulsed lasers are widely used in capillary electrophoresis (CE) studies to provide laser induced fluorescence (LIF) detection. Unfortunately pulsed lasers do not give linear calibration curves over a wide range of concentrations. While this does not prevent their use in CE/LIF studies, the non-linear behavior must be understood. Using 7-hydroxycoumarin (7-HC) (10-5000 nM), Tamra (10-5000 nM) and tryptophan (1-200 µM) as dyes, we observe that continuous lasers and LEDs result in linear calibration curves, while pulsed lasers give polynomial ones. The effect is seen with both visible light (530 nm) and with UV light (355 nm, 266 nm). In this work we point out the formation of byproducts induced by pulsed laser upon irradiation of 7-HC. Their separation by CE using two Zeta LIF detectors clearly shows that this process is related to the first laser detection. All of these photodegradation products can be identified by an ESI-/MS investigation and correspond to at least two 7HC dimers. By using the photodegradation model proposed by Heywood and Farnsworth (2010) and by taking into account the 7-HC results and the fact that in our system we do not have a constant concentration of fluorophore, it is possible to propose a new photochemical model of fluorescence in LIF detection. The model, like the experiment, shows that it is difficult to obtain linear quantitation curves with pulsed lasers while UV-LEDs used in continuous mode have this advantage. They are a good alternative to UV pulsed lasers. An application involving the separation and linear quantification of oligosaccharides labeled with 2-aminobezoic acid is presented using HILIC and LED (365 nm) induced fluorescence.

Recent advances in amino acid analysis by capillary electromigration methods, 2013-2015.

We describe the most important research articles published on amino acid analysis using CE during the period from June 2013 to May 2015, and follows the format of the previous articles published in electrophoresis the new developments in amino acid analysis with CE are mainly describing improvements in detection means and injection methods. Enantiomeric separation developments are still important. Focusing the applications, we describe the neurochemical and clinical works, but also the metabolomic studies for which the publication number increase greatly. Finally, works focused on amino acids in food and agricultural applications are described.