Recent Advances in Capillary Electrochromatography of Proteins and Carbohydrates in the Biopharmaceutical and Biomedical Field.

Capillary electrochromatography (CEC) is a powerful hybrid separation technique that combines capillary electrophoresis and capillary chromatography, capable to address the analytical challenges of proteomics and glycomics. The focus of this paper is to review the recent developments in capillary electrochromatography of proteins and carbohydrates. The different column types applied in capillary electrochromatography such as packed bed, open tubular and monoliths are conferred in detail with respective separation examples. A comprehensive comparison is also given listing the mostly utilized coating methods, stationary phase materials and column preparation methods. The choice of porogenic solvent combinations for monolithic column fabrication is thoroughly discussed, paying close attention to the fine tuning options for the separation driving electroosmotic flow. Application examples of CEC in process analytical technology for the biopharmaceutical and biomarker discovery in the biomedical fields are also given.

Utilization of Analytical Omics Tools in the Molecular Diagnostics of Multiple Myeloma.

Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma B-lymphocytes and even as of today, it is an incurable disease. MM accounts for approximately 10% of all hematologic cancers. Its molecular pathogenesis is poorly understood, but the bone marrow microenvironment of tumor cells and genetic factors have apparent roles in the process. Accurate diagnosis is important to properly identify and stratify the disease, however, MM identification steps are time-consuming and expensive. Thus, development of early molecular diagnostic methods is of high importance in order to start proper therapies as early in the disease progression as possible, given the nature of the poor survival rates/remission periods. Molecular diagnostics via analytical omics represents one of the promising toolsets to speed up the diagnostic process. In this paper, we critically review the utilization of state of the art, high sensitivity analytical omics approaches (genomics, proteomics, metabolomics, lipidomics and glycomics) in MM diagnostics at the molecular level.

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis.

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

Medicinal chemistry meets proteomics: fractionation of the human plasma proteome.

Human plasma and its fractions/derivatives are frequently used materials in biomedicine as it contains thousands and thousands of proteins representing the majority of human proteome. Several important methods were developed in the past for the fractionation of this important biological fluid and its use for medicinal purposes. One of the greatest challenges is the very large dynamic range of plasma proteins ranging up to 10-12 orders of magnitude. Early attempts were mainly based on methods such as salting out or cold ethanol precipitation, as well as chromatography utilizing affinity, size exclusion, ion exchange and hydrophobic interaction techniques. More recently, fractionation applications started with the depletion of the high abundant plasma components, such as serum albumin and immunoglobulins, before isolating lower abundant proteins of interest. Plasma volumes were utilized from the milliliter scale for diagnostic applications to hundreds of liters for industrial scale plasma fractionation (e.g., medicinal product manufacturing). In this paper we review this important part of medicinal chemistry, highlighting the traditional methods along with some of their variations as well as the most significant recent achievements of the field.

Analysis of polar antioxidants in Heartsease (Viola tricolor L.) and Garden pansy (Viola x wittrockiana Gams.).

Heartsease (Viola tricolor L.) is a well-known medicinal plant. Its biological activities are supposed to be related to its antioxidant capacity. Garden pansies (Viola x wittrockiana Gams.) have been crossbred from heartsease and are applied as ornamental plants only. In this study, the mother and the daughter species are compared from a phytochemical point of view. Their flavonoid and anthocyanidin contents are determined by spectroscopic methods recommended by the European Pharmacopoeia 5.0. The compositions of the samples (heartsease and garden pansy varietas of several petal color) are analyzed by high-performance liquid chromatography with UV detection and their antioxidant capacity is determined by trolox equivalent antioxidant capacity assay. Our results suggest that garden pansy, especially its flower, is a promising source of natural antioxidants. In addition, a significant correlation is found between the flavonoid content and antioxidant activity.

Quantitative and qualitative investigation of the main flavonoids in heartsease (Viola tricolor L.).

Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (MSn) is used for the analysis of flavonoids in heartsease (Viola tricolor L.). Our data suggested that the two main flavonoid components were violanthin (6-C-glucosyl-8-C-rhamnosyl apigenin) and rutin (3-O-rutinosyl quercetin). The identification of rutin was confirmed by comparing its retention time, UV spectrum, molecular mass, and fragmentation pattern with the reference standard. In this paper, we also report on the quantitative analysis of rutin by high-performance liquid chromatography. According to our results, heartsease herb contained 420+/-1.17 microg/g rutin.

Raquianestesia isobárica para cesariana: levobupivacaína x bupivacaína

Justificativa e Objetivos: levobupivacaína, enantiomero S(-) da bupivacaína é um anestésico local tipo amida, de longa duração. A potência in vintro, a latência, a duração do bloqueio sensitivo e motor, assim como as propriedades farmacocinéticas da levobupivacaína são muito semelhantes as da bupivacaína. Contradições na literatura sobre sua utilização para cesarianas nos levaram a realizar o presente estudo que tem por objetivo avaliar a efetividade da raquianestesia com levobupivacaína isobárica comparada com a bupivacaína isobárica para cesariana. Método: após aprovação pela Comissão de Ética do hospital, foram selecionadas 60 pacientes com classificação do estado físico da ASA I ou II, submetidas à cesariana, que foram alocadas de forma aleatória em dois grupos e que após punção venosa, hidratação e monitorização dos parâmetros hemodinâmicos, foram submetidos ao bloqueio subaracnóideo para cesariana: Grupo L (n=30) recebeu 12,5mg de levobupivacaína isobárica a 0,5% e o Grupo B (n=30) que recebeu 12,5mg de bupivacaína isobárica a 0,5%. Foram analisados, 5, 10, 15, 30, 45, 60 e 120 minutos após o bloqueio: 1)Tempo de latência 2) Nível do bloqueio sensitivo 3) Intensidade do bloqueio motor: Escala de Bromage. Os dados coletados foram submetidos à análise estatística, sendo considerados estatisticamente significativos os valores de p <0,05. Resultados: os grupos apresentaram semelhança em relação a peso, à altura, à idade e à classificação do estado físico. Foram excluídos quatro casos do Grupo B e um do L devido à falha de bloqueio e uma do B, por bloqueio insuficiente. O Grupo L mostrou tempo de latência de 9,17 ± 3,18 minutos e o Grupo B 15,63 ± 6,40 minutos, sendo esta diferença estatisticamente significativa. Em relação ao bloqueio sensitivo não foram observadas diferenças estatisticamente significativas entre os grupos. O bloqueio motor completo (Bromage I) foi obtido mais rapidamente no Grupo L (25,17 + 9,30) do que no Grupo B (40,20 + 9,41), sendo essa diferença estatisticamente significativa. Conclusão: a levobupivacaína apresentou efetividade no estabelecimento de um bloqueio subaracnóideo adequado para a realização de cesariana

The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region.

BACKGROUND: The human dopamine D4 receptor (DRD4) is a candidate gene of great interest in molecular studies of human personality and psychiatric disorders. This gene is unique in having an exceptionally high amount of polymorphic sites both in the coding and in the promoter region. RESULTS: We report the identification of a new 27 bp deletion starting 524 bp upstream of the initiation codon (27 bp del) of the dopamine D4 receptor (DRD4) gene, in the close vicinity of the -521C>T SNP. The presence of the 27 bp deletion leads to the misgenotyping of the -616C>G SNP by the Sau96 I RFLP method, thus the genotype determination of the mutation is of additional importance. The frequency of this novel sequence variation is considerably low (allele frequency is = 0.16%), as no homozygotes, and only 3 heterozygote carriers were found in a healthy, unrelated Caucasian sample (N = 955). CONCLUSION: Remarkably, the deleted region contains consensus sequences of binding sites for several known transcription factors, suggesting that the different alleles may affect the transcriptional regulation of the gene. A comparison of methods and results for the allelic variations of the DRD4 gene in various ethnic groups is also discussed, which has a high impact in psychiatric genetic studies.

Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene.

DNA-protein interaction in the 5' upstream polymorphic region of the dopamine D4 receptor (DRD4) gene was analyzed by capillary electrophoretic mobility shift assay (CEMSA). The sequence of interest was amplified using a fluorescent primer and applied as a probe in the binding assays with HeLa nuclear extract. Serial dilution of the probe resulted in a concentration dependent DNA-protein complex formation. Sp 1 specific oligonucleotide competitor significantly inhibited the DNA-protein complex formation. A non-specific competitor, differing only in three base pairs, showed weaker effect pointing to the contribution of the Sp 1 recognition sequence in the complex. Polymorphic competitors were also prepared from homozygous individuals possessing either duplicated (2 x 120 bp) or single copy (1 x 120 bp) of the 120 bp repeat sequence and were used against the Sp 1 specific probe in competition assays. Our data provide experimental evidence for the binding of Sp 1 to the 120 bp duplicated sequence of the DRD4 5' upstream region and suggest enhanced binding capacity of the duplicated form.

A novel A/G SNP in the -615th position of the dopamine D4 receptor promoter region as a source of misgenotyping of the -616 C/G SNP.

The polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene containing several single nucleotide polymorphisms (SNPs) has recently become a focus of association studies in psychiatric genetics. Most SNP genotyping methods are based on the two-step procedure of restriction fragment length polymorphism (RFLP). An alternative technique is a single-step method of allele-specific amplification (ASA), previously introduced for genotyping the -521 C/T SNP of the DRD4 promoter region and applied here for the -616 C/G SNP. Parallel genotyping of individuals with the novel ASA method and the conventionally used Ava II RFLP showed a potential underestimation of the -616 GG genotype frequency by the conventional method. Sequencing the dubious samples clearly demonstrated a novel A/G SNP at the -615th position influencing the Ava II digestion and thus resulting in misgenotyping. To avoid this problem, we introduced the Sau96 I RFLP for the -616 C/G genotyping as this restriction enzyme is not sensitive for the -615 A/G sequence variation. Allele (-616 G = 0.48; -616 C = 0.52) and genotype (-616 GG = 0.25; -616 GC = 0.46; -616 CC = 0.29) frequencies were determined by both the novel ASA and the Sau96 I methods. The obtained genotype frequencies corresponded to the Hardy-Weinberg equilibrium in our healthy Caucasian sample (N = 534, P = 0.168). Using these methods, no association was found between the -616 C/G SNP and personality factors of Cloninger's temperament and character inventory (N = 153) in our population.

Noninvasive genotyping of dopamine receptor D4 (DRD4) using nanograms of DNA from substance-dependent patients.

A noninvasive DNA sampling method has been implemented collecting buccal mucosa cells by cotton wool swabs. An amount of 0.2 2 microg DNA per patient was obtained after the phenol-extraction procedure and 0.2 2 ng DNA template was sufficient for PCR amplification of the polymorphic 48 basepair repeat region of dopamine receptor D4 (DRD4) gene. PCR products were visualized during microfabricated electrophoretic separation by laser-induced fluorescent detection and automatic data registration. Initial data of genotyping drug-dependent subjects shows a relatively high ratio of heterozygotes, possessing either longer or shorter variants beside the common 4-repeat DRD4 allele.

High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.

Analysis of dopamine D4 receptor gene polymorphism using microchip electrophoresis.

A microfabricated electrophoresis device was used for rapid polymerase chain reaction product analysis in genotyping the dopamine D4 receptor gene (DRD4) 48 base pairs repeat polymorphism. An allelic ladder, prepared from homozygous individuals, was used as internal standard during the microchip electrophoresis based analysis. Comparison of this novel separation method with the conventional slab gel and previously reported ultra-thin-layer techniques confirmed the reliability of this new method. Genotyping of 332 healthy Hungarian individuals gave the following allele frequencies: two-repeat: 0.089; three-repeat: 0.026; four-repeat: 0.674; five-repeat: 0.011; six-repeat: 0.002; seven-repeat: 0.189; eight-repeat: 0.011. The genotype frequencies obtained showed no deviation from the Hardy-Weinberg equilibrium (p>0.903), further underlying the reliability of this new genotyping technique.

Prenatal diagnosis of steroid 21-hydroxylase deficiency by allele-specific amplification.

OBJECTIVE: Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21-OHD) is the most common cause of ambiguous genitalia in females at birth. Here, we report the first prenatal diagnosis of 21-OHD by DNA analysis in Hungary. METHODS: Allele-specific amplification (ASA) of the DNA obtained by chorionic villus sampling was performed. RESULTS: The fetus had a homozygous nonsense mutation (Gln318Stop), suggesting a salt-wasting phenotype. Dexamethasone treatment of the mother was started on the 8th gestational week and, as the fetus was an affected female, it was continued until term. The newborn had normal external genitalia at birth, and severe salt-wasting crisis and postnatal virilization was prevented by mineralo- and glucocorticoid replacement therapy. CONCLUSION: 21-OHD was genotyped by ASA, and virilization of the fetus was prevented by antenatal dexamethasone therapy.

Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene.

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.

DNA analysis on electrophoretic microchips: effect of operational variables.

Applicability of modern microfabrication technology to electrophoresis microchips initiated a rapidly moving interdisciplinary field in analytical chemistry. Electric field mediated separations in microfabricated devices (electrophoresis microchips) are significantly faster than conventional gel electrophoresis, usually completed in seconds to minutes. Electrophoretic separation of DNA molecules on microfabricated devices proved to have the potential to improve the throughput of analysis by orders of magnitude. The flexibility of electrophoresis microchips allows the use of a plethora of separation matrices and conditions. In this paper, we report on electric field mediated separation of fluorescent intercalator-labeled dsDNA fragments in polyvinylpyrrolidone matrix-filled microchannel structures. The separations were detected in real time by a confocal, single-point laser-induced fluorescence/photomultiplier setup. Effects of the sieving matrix concentration (Ferguson plot), migration characteristics (reptation plot), separation temperature (Arrhenius plot), as well as applied electric field strength and intercalator concentration on the separation of DNA fragments are thoroughly discussed.

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix.

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.

Rapid analysis of covalently and non-covalently fluorophore-labeled proteins using ultra-thin-layer sodium dodecylsulfate gel electrophoresis.

Gel electrophoresis is one of the most frequently used tools for the separation of complex biopolymer mixtures. In recent years, there has been considerable activity in the separation and characterization of protein molecules by sodium dodecylsulfate (SDS) gel electrophoresis with particular interest in using this technique to separate on the basis of size and to estimate molecular mass and protein purity. Although the method is informative, it is cumbersome, time consuming and lacks automation. In this paper we report an automated, high-performance SDS gel electrophoresis system that is based on electric-field-mediated separation of SDS-protein complexes using an ultra-thin-layer platform. The integrated fiber optic bundle-based scanning laser-induced fluorescence detection technology readily provided high sensitivity, real-time detection of the migrating solute molecules. Rapid separations of covalently and non-covalently labeled proteins were demonstrated in the molecular mass range 14,000 to 205,000 in less than 9 and 16 min, respectively. Excellent quantitation and lane-to-lane migration time reproducibility were found for all the solute components using the multilane separation platform. The limit of detection was found to be 1.5-3 ng/band for both labeling methods, with excellent linearity over a six times serial double-dilution range. Molecular mass calibration plots were compared for both covalently and non-covalently labeled proteins. A linear relationship was found between the molecular mass and electrophoretic mobility in the case of covalently labeled samples, while a non-linear relationship was revealed for the non-covalently labeled samples.

Rapid and sensitive genotyping of dopamine D4 receptor tandem repeats by automated ultrathin-layer gel electrophoresis.

Prior studies have revealed possible association between the presence of a seven repeat of the 48 bp variable number tandem repeat polymorphism of the human dopamine D4 receptor gene (DRD4) and some normal and pathological human traits, such as novelty seeking, hyperactivity disorders, and substance abuse. Some reports supported this finding whereas others did not. Incorrect genotyping could be one of the reasons for these controversial results, and might originate from preferential amplification of shorter polymerase chain reaction (PCR) products, resulting in the so-called allele dropout. In this paper we optimized the conditions for simultaneous amplification of shorter and longer amplicons of the 48 bp repeat region of the DRD4 gene in order to avoid the loss of the longer allele and consequent incorrect genotyping, using very low DNA template concentrations and partial replacement of 2'-deoxyguanosine-5'-triphosphate (dGTP) by 2'-deoxyinosine-5'-triphosphate (dITP). The optimized PCR method in combination with high throughput automated ultrathin-layer gel electrophoresis was suitable for rapid genotyping from less than a nanogram DNA using noninvasive sampling (buccal epithelial cells). All detected genotypes are presented, including such rear heterozygotes as the 2 x and 8 x 48 bp repeats in the same sample, showing the reliability of our novel detection method of longer alleles in the presence of shorter alleles.