Capillary Electrophoresis Resolution Research Articles

Cytotoxic CD8+ T cells and tissue resident memory cells in colorectal cancer based on microsatellite instability and BRAF status.

BACKGROUNDRecent studies in non-colorectal malignancy have associated T resident memory (TRM) cells with improved patient survival. It is unknown if TRM plays a role in colorectal cancer (CRC).AIMTo examine the potential role of TRM cells in providing immunogenicity in CRC stratified by microsatellite instability (MSI) and BRAF status.METHODSPatients with known MSI and BRAF mutation status were eligible for inclusion in this study. CRC tumour sections stained with haematoxylin and eosin were microscopically reviewed and the images scanned prior to assessment for location of invading edge and core of tumour. Sequential sections were prepared for quantitative multiplex immunohistochemistry (IHC) staining. Opal Multiplex IHC staining was performed with appropriate positive and negative controls and imaged using a standard fluorescent microscope fitted with a spectral scanning camera (Mantra) in conjunction with Mantra snap software. Images were unmixed and annotated in inForm 2.2.0. Statistical analysis was performed using Graphpad Prism Version 7 and Stata Version 15.RESULTSSeventy-two patients with known MSI and BRAF status were included in the study. All patients were assessed for MSI by IHC and high resolution capillary electrophoresis testing and 44 of these patients successfully underwent quantitative multiplex IHC staining. Overall, there was a statistically significant increase in CD8+ TRM cells in the MSI (BRAF mutant and wild type) group over the microsatellite stable (MSS) group. There was a statistically significant difference in CD8+ TRM between high level MSI (MSI-H):BRAF mutant [22.57, 95% confidence interval (CI): 14.31-30.84] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0076 andMSI-H:BRAF wild type [16.18 (95%CI: 10.44-21.93)] vs MSS [8.031 (95%CI: 4.698-11.36)], P = 0.0279. There was no statistically significant difference in CD8 T cells (both CD8+CD103- and CD8+CD103+TRM) between MSI-H: BRAF mutant and wild type CRC.CONCLUSIONThis study has shown that CD8+ TRM are found in greater abundance in MSI-H CRC, both BRAF mutant and MSI-H:BRAF wild type, when compared with their MSS counterpart. CD8+ TRM may play a role in the immunogenicity in MSI-H CRC (BRAF mutant and BRAF wild type). Further studies should focus on the potential immunogenic qualities of TRM cells and investigate potential immunotherapeutic approaches to improve treatment and survival associated with CRC.

Simultaneous determination of twelve biogenic amines in human urine as potential biomarkers of inflammatory bowel diseases by capillary electrophoresis – tandem mass spectrometry

Biogenic amines (BA) are a broad group of biologically active substances, the presence of which in the human body can provide important diagnostic information for many various pathologies, including chronic inflammation. In this work, a capillary electrophoresis (CE) hyphenated with tandem mass spectrometry (MS/MS) method was developed for the simultaneous determination of twelve BA (histamine, serotonin, dopamine, norepinephrine, epinephrine, putrescine, cadaverine, spermine, spermidine, tyramine, tryptamine, phenylethylamine) in human urine as potential biomarkers of inflammatory bowel diseases (IBD). The electrophoretic separations were carried out in an uncoated fused silica capillary (I.D. 50 μm) using 50 mM formic acid (pH 2.0) as a background electrolyte. A reliable identification of the analytes was based on the combination of time resolution in CE and mass resolution in triple quadrupole MS/MS. The total analysis time of the proposed CEMS/MS method was less than 10 min with the limits of detection in the range of 4.47–144 ng/mL. The intra- and inter-day accuracy ranged in the intervals 89.75–109.4% and 89.99–110.2%, respectively, with the RSD values for the intra- and inter-day precision lower than 14 and 13 %, respectively. The recovery values for the samples spiked at three concentration levels ranged from 81.73–105.6% with a precision not exceeding 9.9 %. The favorable performance parameters of the CEMS/MS method highlighted its usefulness for routine clinical applications. In this work, the CEMS/MS method was applied, for the first time, to the analytical profiling of the BA in clinical human samples. The obtained results showed a statistically significant decrease of serotonin and norepinephrine, and an increase of histamine and spermidine, in the studied group of IBD patients when compared with the control group. These findings could be utilized in studying and clarifying the mechanisms of IBD or relevant therapy.

Genetic diversity and genetic relationships among Curcuma accessions based on SRAP and ISSR analysis

Abstract Molecular markers have been used for genetic study and identification of cultivars for a long time. In this study SRAP and ISSR markers were used to evaluate genetic variation and relationships among 25 accessions in China. Both marker systems obtained high polymorphism for the high resolution of capillary electrophoresis. The UPGMA dendrogram based on SRAP and ISSR shared many common clusters: one main cluster (clade 1) included 5 Curcuma alismatifolia accessions (C1, C3, C4, C5, C6), five branches each formed by two most related cultivars (C10 and C17, C24 and C25, C19 and C20, C14 and C22, C15 and C23). Result revealed by two molecular markers highly consistent with each other indicated both of them are credible. The extremely high polymorphism tested made our thoroughly understand the relationships of 25 accessions possible. Our study may benefit in further theoretical research and cross breeding work.

Simultaneous detection of fungal, bacterial, and viral pathogens in insects by multiplex PCR and capillary electrophoresis

Beetles Protaetia brevitarsis seulensis Kolbe (Coleoptera: Cetoniidae) and Allomyrina dichotoma Linn. (Coleoptera: Scarabaeidae) are widely used in traditional medicine, and the number of insect-rearing farms is increasing in South Korea. The purpose of this study was to establish a multiplex PCR-based assay for rapid simultaneous detection of multiple pathogens causing insect diseases. Six insect parasites such as fungi Beauveria bassiana (Bals.-Criv.) Vuill. (Hypocreales: Cordycipitaceae) and Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), bacteria Bacillus thuringiensis Berliner (Bacillales: Bacillaceae), Pseudomonas aeruginosa Migula (Pseudomonadales: Pseudomonadaceae), and Serratia marcescens Bizio (Enterobacteriales: Enterobacteriaceae), and Oryctes rhinoceros nudivirus were chosen based on the severity and incidence rate of insect diseases in South Korea. Pathogen-specific primers were designed and successfully applied for simultaneous detection of multiple infectious agents in farm-bred insects P. b. seulensis and A. dichotoma using multiplex PCR and high resolution capillary electrophoresis. Our results indicate that multiplex PCR is an effective and time-saving method for simultaneous detection of multiple infections in insects, and the QIAxcel capillary electrophoresis system is useful for quantitative evaluation of the individual impact of each infectious agent on the severity of insect disease. The approach designed in this study can be utilized for rapid and accurate diagnostics of infection in insect farms.

Molecular characterization of Glu-B3 locus in wheat cultivars and segregating populations

Bread wheat quality constitutes a key trait for the demands of the baking industry as well as the broad consumer preferences. The role of the low molecular weight glutenin subunits (LMW-GS) with regard to bread quality is so far not well understood owing to their genetic complexity and to the use of different nomenclatures and standards for the LMW-GS assignment by different research groups, which has made difficult the undertaking of association studies between genotypes and bread quality. The development of molecular markers to carry out genetic characterization and allele determination is demanding. Nowadays, the most promising LMW gene marker system is based on PCR and high resolution capillary electrophoresis for the simultaneous analysis of the complete multigene family. The molecular analysis of the bread wheat Glu-B3 locus in F2 and F4:6 populations expressed the expected one-locus Mendelian segregation pattern, thus validating the suitability of this marker system for the characterization of LMW-GS genes in segregating populations, allowing for the successful undertaking of studies related to bread-making quality. Moreover, the Glu-B3 allele characterization of standard cultivars with the molecular marker system has revealed its potential as a complementary tool for the allelic determination of this complex multigene family.

Coupling of Random Amplified Polymorphic DNA Profiles Analysis and High Resolution Capillary Electrophoresis System for the Assessment of Chemical Genotoxicity

Cadmium (Cd) can be toxic to terrestrial snails, but few data are available about its genotoxic effects on early life stages (ELS). The aim of this study was to investigate the genotoxic potential of Cd in embryos of Helix aspersa using a new approach that couples Random Amplified Polymorphic DNA (RAPD) and a high-resolution capillary electrophoresis system (HRS). Clutches of H. aspersa were exposed to Cd solutions (2, 4, and 6 mg/L) from the beginning of their embryonic development. In addition to a dose-dependent effect of Cd on hatching rate, DNA fragmentation was observed in embryos that were exposed to 6 mg Cd/L. The analysis of RAPD products with HRS showed differences between the profiles of exposed and nonexposed embryos, starting at 2 mg Cd/L. In comparison to the profiles of the control samples, all profiles from the exposed snails exhibited an additional 270 bp DNA fragment and lacked a 450 bp DNA fragment. These profile modifications are related to the genotoxic effect of Cd on the ELS of H. aspersa . Our study demonstrates the efficacy of coupling RAPD and HRS for a rapid and efficient screening of the effects of chemicals on DNA.

Unraveling the Glyco-Puzzle: Glycan Structure Identification by Capillary Electrophoresis

State-of-the-art high-resolution separation techniques play an important role in the full structural elucidation of glycans. Capillary electrophoresis (CE) offers a rapid yet simple method for exhaustive carbohydrate profiling. CE is a versatile analytical platform, which can be operated in several separation modes, simply by altering separation conditions during operation. For in-depth glycan structural analysis, CE has also gained significantly from the additional resolution introduced by complementary and orthogonal separation techniques such as ion exchange or hydrophilic interaction chromatography. Commercially available mass spectrometry (MS) interfaces have not only brought this information-rich detection technique within reach, but CE also represents an expedient highly efficient separation inlet for MS, capable of separating isobaric oligosaccharide isomers prior to MS detection and MS/MS fragmentation based identification. This Perspective gives a sophisticated impression of the versatility of capillary electrophoresis for deep structural elucidation of carbohydrates derived from glycoproteins of biomedical interest. Different separation modes for the analysis of both charged and neutral glycans, such as influencing electroosmotic flow, using complexation/interaction based secondary equilibria, and the use of charged and neutral labels are compared. The merits of introducing orthogonal and complementary techniques, such as exoglycosidase digestion arrays, analytical/preparative chromatography and mass spectrometric detection, extending the dynamic range and resolution of CE are all thoroughly discussed.

Recent Advances in Enhancing the Sensitivity and Resolution of Capillary Electrophoresis

An extensive search of published research and review articles indicates that enhancing the sensitivity and resolution of capillary electrophoresis (CE) is a very active area of interest. This review focuses on developments and applications in this field over several years, especially from 2009-present. It first reviews developments in the fields of online sample preconcentration and highly sensitive detection for sensitivity enhancement. The online sample preconcentration techniques cover all methods, from electrophoretic preconcentration, extraction-based preconcentration, micelle-based preconcentration and hybrid preconcentration to nanoparticle-based preconcentration. Attention is also given to multiple dimension separations, additives in buffer solution and capillary inner surface modifications that have been used to enhance the resolution and separation efficiency of complex samples in CE. The additives include nanoparticles, chiral selectors and other buffer additives that have been added to the buffer solution.

Coupling isotachophoresis and capillary electrophoresis: a review and comparison of methods

We present a comprehensive review and comparison of the methodologies for increasing sensitivity and resolution of capillary electrophoresis (CE) using online transient isotachophoresis (tITP). We categorize the diverse set of coupled tITP and CE (tITP-CE) methods based on their fundamental principles for disrupting isotachophoretic preconcentration and triggering electrophoretic separation. Based on this classification, we discuss important features, advantages, limitations, and optimization principles of various tITP-CE methods. We substantiate our discussion with original simulations, instructive examples, and published experimental results.

Equilibrium and structural characterization of ofloxacin–cyclodextrin complexation

The enantiomer-specific characterization of ofloxacin–cyclodextrin complexes was carried out by a set of complementary analytical techniques. The apparent stability constants of the ofloxacin enantiomers with 20 different cyclodextrins at two different pH values were determined to achieve good resolution capillary electrophoresis enantioseparation either to establish enantioselective drug analysis assay, or to interpret and design improved host–guest interactions at the molecular level. The cyclodextrins studied differed in the nature of substituents, degree of substitution (DS), charge and purity, allowing a systematic test of these properties on the complexation. The seven-membered beta-cyclodextrin and its derivatives were found to be the most suitable hosts. Highest stability and best enantioseparation were observed for the carboxymethylated-beta-cyclodextrin (DS ~ 3.5). The effect of substitution pattern (SP) was investigated by molecular modeling, verifying that SP greatly affects the complex stability. Induced circular dichroism was observed and found especially significant on carboxymethylated-beta-cyclodextrin. The complex stoichiometry and the geometry of the inclusion complexes were determined by 1H NMR spectroscopy, including 2D ROESY techniques. Irrespective of the kind of cyclodextrin, the complexation ratio was found to be 1:1. The alfa-cyclodextrin cavity can accommodate the oxazine ring only, whereas the whole tricyclic moiety can enter the beta- and gamma-cyclodextrin cavities. These equilibrium and structural information offer molecular basis for improved drug formulation.

Modification of Resolution in Capillary Electrophoresis for Protein Profiling in Identification of Genetic Modification in Foods

The capillary electrophoresis with UV detection was employed for protein profiling in extracts from maize and soybeans. Modifications of back-ground electrolyte and coating the capillary wall with polybrene was employed in order to decrease the protein adsorption on the capillary walls. The obtained protein profiles were compared for transgenic and non-transgenic variants, showing in some cases signifi- cant changes that might be employed for identification of genetic modifications of plants or foods of plant origin. (doi: 10.5562/cca1777)

Evaluation of common interferences on serum protein high resolution capillary electrophoresis system Capillarys 2

Evaluation of common interferences on serum protein high resolution capillary electrophoresis system Capillarys 2

High resolution capillary electrophoresis (CZE-HR) for serum proteins: Clinical uses and reference values

High resolution capillary electrophoresis (CZE-HR) for serum proteins: Clinical uses and reference values

Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)

BackgroundGenotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported.MethodsThe original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis.ResultsThe CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE.ConclusionThe improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens.

Use of capillary electrophoresis for accurate determination of CAG repeats causing Huntington disease. An oligonucleotide design avoiding shadow bands

Huntington disease (HD) is a neurodegenerative disorder associated with the expansion of a polymorphic trinucleotide CAG repeat in the HD gene. We have developed an assay to accurately determine CAG repeats that combines a novel oligonucleotide design and the resolution of capillary electrophoresis. A mismatch in the second nucleotide from the 3′ end enhanced specificity by avoiding mispriming and diminishing shadow bands and artifactual PCR products. The coupling of capillary electrophoresis analysis with the assay added the advantages of accuracy, high resolution, semi‐automation, rapid analysis and low sample consumption. Analysis of 200 chromosomes in the Spanish population sample studied (control group) gave a peak frequency for 16 CAG repeats and of 7 triplets for CCG repeats. Diagnosis of HD was confirmed in 22 of 34 individuals with a range of CAG repeats from 39 to 52. Predictive testing was also carried out for 19 relatives of the HD families diagnosed at our laboratory. The method proposed in this article provides an accurate sizing of DNA repeats that can be applied to the analysis of DNA size‐related disorders.

Fourier Transform Capillary Electrophoresis with Laminar-Flow Gated Pressure Injection

Fourier transform capillary electrophoresis (FTCE) was developed as a method to improve signal-to-noise ratio (S/N) and resolution in capillary electrophoresis (CE) separation. In FTCE, multiple simultaneous CE separations were performed in the same channel system and interrogated using a single-point detector. To illustrate experimentally the improvement offered by FTCE in S/N ratio and resolution, we carried out a modest number (five) of multiple injections and separations. We show even with this small number of separations, S/N increased by a factor of 2.9, and theoretical plate height improved by a factor of more than 30. We demonstrated this technique with laser-induced fluorescence detection, but a wide variety of detection methods are compatible with FTCE.

High Resolution Capillary Electrophoresis of Oligosaccharide Structural Isomers

In this paper we report on high resolution capillary electrophoresis separation of structural isomers of oligosaccharides of biotechnology interest. Analysis of carbohydrate constituents of various plant and food sources of bioindustrial significance requires high-resolution characterization methods to distinguish between structural isomers of oligosaccharides built of monosaccharides linked together in various anomeric and positional configurations. Development of linkage specific enzymatic biodegradation processes is vital for a variety of applications in agricultural, food, pulp and paper, and other industries. To evaluate and optimize the activity of hundreds of different enzymes and microorganisms, along with their combinations, reliable and automated screening methods are required. In this study, a comprehensive analysis of very similar short oligosaccharide structural isomers has been demonstrated. Excellent resolution was achieved under suppressed electroosmotic flow conditions, employing dynamically coated fused silica capillary columns, in conjunction with a one-step fluorophore labeling procedure and sensitive laser-induced fluorescence detection.

Multiplexed, linkage group-specific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet (Beta vulgaris L.)

A set of single nucleotide polymorphism (SNP) markers has been developed for each of the nine linkage groups of sugar beet. Each set can monitor the polymorphic state at five to six linked marker loci. In each set, the loci selected for marker development are first amplified in a multiplexed reaction. These amplification products are the basis for sequence-specific elongation of primers adjacent to SNP positions. The extension step revealing SNP loci is based on fluorescently labelled nucleotides. In each set, primers developed to reveal SNP alleles differ in length to allow clear peak resolution in capillary electrophoresis. The nine linkage group (LG) - specific sets provide information on the polymorphism at a total of 52 SNP marker loci. Using the SNP-based tool, groups of concerned loci have been anchored to three different linkage maps of sugar beet. In a second experiment, sugar beet breeding lines have been fingerprinted. The use of the nine sets of LG-specific markers in sugar beet genetics and breeding is discussed. The information necessary to specify the 52 marker loci, as well as their map location, and all details concerning SNP assays, including allele type and nature of mutation, are reported.

DNA capillary electrophoresis using poly(vinyl alcohol). I. Inner capillary coating.

Two new methods of inner capillary coating with poly(vinyl alcohol) (PVAL) have been investigated and evaluated by performing DNA capillary electrophoresis (CE) using PVAL as a separation medium and by measuring the electroosmotic flow (EOF) mobility. The treatment of capillaries with a silanol-group modified PVAL (PVAL-Si) has been found to give good coating effects for improving the resolution of DNA CE and for reducing the EOF. This coating must be effectively achieved by combining the adsorptive property of PVAL chains onto silica with the reaction between the silanol groups of PVAL-Si and the silica surface. The adsorption of PVAL onto silica has been observed by using atomic force microscopy (AFM) for PVAL-Si as well as for a nonmodified PVAL as a control. The coating with PVAL that links to the capillary wall surface with more hydrolytically stable bonding, -Si-C-, has been formed by performing the Grignard reaction, followed by in-capillary polymerization of vinyl acetate (VAc) and hydrolysis. This coating has been found to be effective for improving the resolution of DNA CE and for reducing the EOF.

Enantioselective analysis of hydroxymebendazole in plasma by high-performance liquid chromatography and capillary electrophoresis

This paper describes for the first time the enantioselective analysis of hydroxymebendazole (MBZOH), a chiral metabolite of mebendazole (MBZ), in human plasma by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). The plasma samples were prepared by liquid-liquid extraction using diisopropyl ether: ethyl acetate (8 : 2, v/v). The HPLC chiral resolution of MBZOH was obtained using a chiral stationary phase based on tris-3,5-dimethyl-phenylcarbamate amylose derivative (Chiralpak AD column), under normal phase conditions using hexane/2-propanol/ethanol (81 : 14 : 5, v/v/v) as the mobile phase. CE resolution of MBZOH enantiomers was carried out in a fused-silica capillary (30 cm×75 μm ID) using 3.0% w/v sulfated β-cyclodextrin in 20 mmol/L Tris buffer, pH 7.0, as running buffer. Both methods were validated, showing mean recoveries of 70% and linearity in the 25–2500 ng/mL and 62.5–2500 ng/mL range for the HPLC and CE methods, respectively. The coefficient of variations and % relative error for both within-day and between-day assays were lower than 15% for both methods.