Search Results (205)

Oral squamous cell carcinoma (OSCC) is an aggressive disease with a glycoproteomically unmapped progression and a low five-year survival rate. Thus, the aim of this pilot study was to explore the N-glycosylation pattern differences in malignant, adjacent mucosal and healthy tissues in the context of OSCC. Oral mucosal soft tissue samples was obtained by incisional biopsy from five patients with OSCC, both from the malignant and the opposite healthy gingival sides, and from seven age-sex-matched healthy controls. The collected tissues were homogenized, followed by N-glycan profiling of the endoglycosidase-released and fluorophore-labeled carbohydrates using capillary electrophoresis with ultra-sensitive laser-induced fluorescent detection (CE-LIF). Six out of the twenty-two identified N-glycan structures, including glycogens, showed significant (p < 0.05) differences between the malignant tissue samples of the OSCC patients and the healthy controls. Comparing the healthy and the positive control oral mucosal samples, differences in four N-glycan structures were revealed, while only one alteration was observed between the N-glycan profiles of the malignant tumor and positive control samples. However, the results are presented descriptively, reflecting the limited sample size of the pilot study, it shows the potential of high-resolution CE-LIF-based glyocoanalytical protocol to be highly efficient and sensitive for glycobiomarker-based molecular diagnostics of oral malignant lesions. Full article

Metabolomics, which is typically referred to as the post-genomic methodology addressing low-molecular-weight metabolites, became a powerful tool in post-genomic research over the last two decades. Indeed, the state-of-the-art metabolomics relies on several well-established complementary platforms—nuclear magnetic resonance (NMR) spectroscopy, liquid and gas chromatography coupled on-line with mass spectrometry (LC- and GC-MS, respectively), and capillary electrophoresis–mass spectrometry (CE-MS). Among them, GC-MS represents one of the oldest and most well-established techniques currently employed in the metabolomics of volatile compounds and non-volatiles—polar low-molecular-weight metabolites, which can be efficiently converted in volatile form by comprehensive derivatization of polar functional groups. Currently, GC-MS is established as the principal analytical method for characterizing primary plant metabolism, although other methods also contribute significantly to determining the complete metabolite profile. Therefore, here, we address the role of GC-MS in plant metabolomics and its potential for the profiling of low-molecular-weight metabolites. Further, we comprehensively review the methods of sample preparation with special emphasis on extraction and derivatization approaches, which are currently employed to improve the method performance and its metabolome coverage. Full article

In this research, 59 samples from 31 animals (19 European rabbits, 11 Iberian hares, and 1 cat) and an axenic culture of the Leishmania infantum isolate (MCAN/ES/97/10445, zymodeme ZM/MON-1) used as a reference were studied based on the analysis of kinetoplast minicircle (kDNA) restriction fragments by combining polymerase chain reaction and length polymorphisms (PCR-RFLP). This analysis was performed in parallel with polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE), as well as in silico digestion of the abovementioned reference. These analyses did not reveal differences between the L. infantum isolates detected in the different samples of wild lagomorphs (rabbits and hares) from various areas of the Community of Madrid or with the axenically cultured promastigotes of the L. infantum isolate (MCAN/ES/97/10445, zymodeme ZM/MON-1) used as a reference. Consequently, it was proven that with the implemented approaches, only one isolate of L. infantum was responsible for infection in wild leporids and that these animals sustained the pathogen’s life cycle, both in the area of the human leishmaniasis outbreak that has been occurring in the Community of Madrid since 2009 and outside of it. Additionally, this isolate has been circulating since at least the 1990s. Full article

Capillary electrophoresis (CE) is an effective tool for the analysis of many biocomponents, such as dsDNA, RNA, amino acids and bacteria, which are extremely important not only in research work but also in numerous practical applications. However, there are many factors that affect the separation performance, including the polymers inside the capillary, the electric field strength, the capillary coating and the effective length of the capillary. So far, various CE techniques have been developed to increase the resolution, sample volume consumption and limit of detection. To better understand the development of techniques for the separation of these biomolecules by CE, this review provides a comprehensive summary of polymers (e.g., polyvinylpyrrolidone, hydroxyethyl cellulose and polyethylene glycol), optimization methods, capillary coating methods, technological advancement of microchips for CE and the limitation of detection proposed by different groups worldwide. We also discuss the challenges and future directions associated with CE technology. Full article

The bioanalysis of body fluids plays a crucial role in clinical diagnostics, pharmaceutical research, forensic science, and biomarker discovery. Traditional chromatographic techniques are widely used in clinical laboratories but are often costly and time-consuming. Capillary electrophoresis (CE) and its modifications, such as capillary zone electrophoresis, isotachophoresis, and micellar electrokinetic chromatography, have emerged as efficient, cost-effective, and miniaturized alternatives for analyzing small organic and inorganic molecules in biological fluids. This paper deals with the applications of CE-based electromigration techniques in the determination of various analytes in urine, blood, saliva, and cerebrospinal fluid. The study further discusses the advantages and limitations of different detection methods, including ultraviolet-visible spectroscopy, laser-induced fluorescence, mass spectrometry, conductivity, and amperometric detection. A focus is given to the identification and quantification of amino acids and their metabolites as potential biomarkers for metabolic and degenerative disorders. The work highlights recent advancements in CE methodologies and their potential to enhance sensitivity and selectivity in bioanalytical applications. Full article

This study explored IgG N-glycosylation pattern differences in maternal and infant serum in the context of gestational diabetes mellitus (GDM). Serum samples from 15 mother–infant pairs were collected at 12 weeks postpartum and categorized according to maternal body mass index (BMI) and GDM status. The N-glycosylation patterns of the isolated IgG pools were analyzed by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Descriptive comparison of the relative area percentage of IgG N-glycan structures revealed differences between the groups. Comparison of the maternal and infant sialo-form/neutral-form ratio (SF/NF) of the N-glycans suggested differences between control mothers and their children, as well as between obese mothers and their children. The maternal SF/NF ratio of IgG varied between the obese and normal-weight GDM mothers. The SF/NF ratios of IgG from the infants showed variation between infants of control mothers and infants of obese mothers, between infants of obese and infants of obese GDM mothers, and between infants of GDM with normal-weight and GDM with obese mothers. The observed differences in maternal and infant IgG N-glycosylation profiles suggest potentially selective placental transfer mechanisms. Full article

Compared with capillary electrophoresis (CE), gel electrophoresis (GE) is a traditional method for the analysis of nucleic acids because of its low cost, although the operation process is complicated. The electropherogram from CE can offer more information (e.g., DNA size and its concentration) for researchers. Based on the self-built integrated biochip GE system, we proposed a computational method that converts conventional agarose GE images into CE-like fluorescence profiles for enhanced DNA analysis. The gel images were processed using an image-based algorithm involving median filtering to remove background noise and pixel-wise intensity summation along the migration axis to generate one-dimensional records of electrophoretic separations. Each DNA band in the gel was thereby transformed into a distinct fluorescence peak, reflecting its migration distance and relative intensity. To further enhance resolution and peak separation, Gaussian modeling was applied to fit the fluorescence intensity distribution, providing smoother and more distinguishable spectral peaks. To validate the method, three periodontal pathogens—Porphyromonas gingivalis (P.g), Treponema denticola (T.d), and Tannerella forsythia (T.f)—were amplified using PCR and analyzed by gel electrophoresis. The method successfully identified distinct electrophoretic patterns for the three pathogens by using a 50 bp DNA ladder as an internal calibration reference. The results demonstrate that image-based reconstruction of electrophoretic data provides a reliable, quantitative, and visually interpretable representation of DNA migration, comparable to CE output. This approach bridges a gap between traditional GE and modern capillary systems, allowing for the semi-quantitative analysis of DNA fragments without specialized CE instrument. The proposed method offers a valuable analysis method for the separation of DNA, RNA, protein and polypeptides. Full article

High temperatures during grain filling degrade rice quality, yet the metabolite-level basis of varietal tolerance—particularly root contributions—remains unclear. We compared the heat-tolerant ‘Fusaotome’ and the widely grown ‘Akitakomachi’ under control and high-temperature conditions. Panicles and roots were sampled at heading and profiled by capillary electrophoresis–mass spectrometry (CE–MS), followed by PCA, univariate testing, and KEGG pathway analysis. PCA resolved treatment and cultivar differences in an organ-specific manner. In panicles, ‘Fusaotome’ showed 8 increased metabolites (≥1.5-fold) and 11 decreased (≤1/1.5), whereas ‘Akitakomachi’ showed 19 increases and 6 decreases (p < 0.05). In roots, 12 metabolites increased in ‘Fusaotome’ and 9 in ‘Akitakomachi’; no significant decreases were detected. Pathway analysis indicated activation in ‘Fusaotome’ panicles of tryptophan, nicotinate/nicotinamide, arginine/proline, glycolysis/TCA, pyruvate, and vitamin B6 pathways, while ‘Akitakomachi’ emphasized phenylpropanoid, isoquinoline alkaloid, caffeine, and ubiquinone/terpenoid–quinone biosynthesis. In roots, ‘Fusaotome’ prioritized phenylalanine/phenylpropanoid, aromatic amino acids, lysine degradation, branched-chain amino acids, glycerophospholipids, and alkaloids, whereas ‘Akitakomachi’ favored nitrogen- and antioxidant-related routes. Collectively, the tolerant cultivar maintained antioxidant capacity and energy supply while coordinating root–panicle metabolism, whereas the susceptible cultivar shifted toward secondary defenses. These signatures nominate candidate metabolic markers and targets for breeding and management to stabilize rice production under warming climates. Full article

Intermittent fasting (IF) has emerged as a promising strategy for managing obesity and related metabolic disorders. Although metabolic adaptations in adipose tissue during IF are well documented, the specific reprogramming of white adipose tissue (WAT) under prolonged cycles of fasting and refeeding remains incompletely understood. Using mass spectrometry-based approaches, including liquid chromatography (LC) and capillary electrophoresis (CE), we identified a marked increase in inositol monophosphates (InsP1s) in obese adipose tissue following extended IF. Specifically, myo-inositol-1-phosphate and myo-inositol-3-phosphate, which are typically present at low levels in gonadal WAT (gWAT) of diet-induced obese mice, were significantly elevated after 15 cycles of IF. Additionally, extended IF upregulated the expression levels of inositol tetrakisphosphate 1-kinase (ITPK1) and inositol monophosphatase 1 (IMPA1), two key enzymes involved in InsP1 metabolism. These increases coincide with reductions in body weight and fat mass, as well as improved insulin sensitivity. This reprogramming was further supported by enhanced tricarboxylic acid (TCA) cycle activity. Collectively, these findings suggest the inositol monophosphate pathway as a novel mechanism underlying fasting-induced metabolic adaptation in adipose tissue and highlight the potential of these metabolites as biomarkers for obesity and related metabolic conditions. Full article

We developed and validated a capillary electrophoresis (CE) method for the separation of two opioid–neurotensin hybrid peptides, recently presented as potent analgesics being decapeptides with a hybridic nature (i.e., H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH; PK20 and its structural analogue H-Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Ile-Leu-OH; [Ile9]PK20). As these two chimeras differ by only one amino acid, Tle→Ile, and are characterized by possessing the same molecular weight while having different spatial conformations, the aim of the study was to determine their potential separation in terms of the presence of any differences resulting from this structural modification. The separation process was performed using an eCAP fused silica capillary at a detection wavelength of 200 nm in 25 mM phosphate buffer at pH 2.5. The analysis was performed at 25 °C and 10 kV. The developed method was validated by assessing linearity in the concentration range from 50 to 5000 ng/mL. Very good linearity was obtained, with the coefficient of determination (R²) ranging from 0.9991 to 0.9999 for both analyzed derivatives. The method demonstrated baseline resolution (Rs = 1.4). The limit of quantification ranged from 34.72 ng/mL to 34.98 ng/mL. The recoveries of all derivatives ranged from 94.8% to 100%. The total analysis time was only 6 min. The developed method enables the determination of PK20 and [Ile9]PK20 derivatives both in aqueous solutions and in serum. Full article

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is widely used to promote bone regeneration. However, conventional surface-attached delivery on absorbable collagen sponges causes a rapid burst release, excessive inflammation, and suboptimal healing. To overcome these limitations, we developed a thermally controlled Poly(DL-lactide-co-glycolide) (P_DLLGA) encapsulation system, designed to stabilize rhBMP-2 and enable controlled release. rhBMP-2 was incorporated in P_DLLGA pellets using the hot-melt extrusion of a lyophilized mixture containing poloxamer 407 and hydroxypropyl-β-cyclodextrin, and terminal sterilization (X-ray irradiation). The released rhBMP-2 maintained its molecular integrity after sterilization and remained stable for up to 732 days in storage, as confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis (CE). Further, high-affinity binding between released rhBMP-2 and BMPR-IA was confirmed by bio-layer interferometry (BLI), and the released protein induced a robust in vitro ALP response, confirming preserved osteogenic activity. Our encapsulation approach for rhBMP-2 using P_DLLGA, including the combination product with β-TCP (LDGraft; Locate Bio, Nottingham, UK), provides a stable and bioactive rhBMP-2 delivery strategy with inherent dose-shielding properties, supporting safe, controlled, and effective bone regeneration therapies. Full article

Additives such as surfactants (Tween-20) and cryoprotectants (glycerol and trehalose) are often used in enzymatic assays to improve the quality and long-term stabilization of proteins. However, these additives can affect the enzymatic activity and the enzyme’s affinity for active compounds, such as inhibitors, and must be considered during assay design since a slight shift in enzyme behavior may compromise the reliability of the results. In this study, the effects of Tween-20, glycerol, and trehalose on hyaluronidase (Hyal) were systematically evaluated by assessing their influence both directly—through microscale thermophoresis (MST) signals of the labeled enzyme (Hyal*)—and indirectly, by monitoring the formation of the final product of the degradation of hyaluronic acid, tetrasaccharide (Tet), using capillary electrophoresis (CE/UV). Hyal was labeled for the first time with ATTO-647 NHS ester, a commercial dye compatible with MST. Efficient labeling was achieved in a phosphate-based buffer without loss of catalytic activity. Tween-20 showed no impact on MST signals nor on enzymatic performance when used between 0.005 and 0.05% (v/v). Glycerol also did not interfere with MST measurements; however, it significantly reduced catalytic activity at concentrations above 2% (v/v). Trehalose affected Hyal* fluorescence in a concentration-dependent manner and enhanced catalytic activity even at 0.02% (v/v). Full article

Background: Carrimycin is a mixture of spiramycin derivatives with antibacterial functions. However, recent studies have shown that it possesses certain anticancer properties. The specific mechanism of the anticancer activity is unknown. Methods: To study the anticancer mechanism of carrimycin, we synthesized a derivative of spiramycin, n-hexyl spiramycin (h-SPM), and used a combination of metabolomics and transcriptomics methods. Capillary electrophoresis–mass spectrometry (CE-MS) was used to detect polar small molecule metabolites, and liquid chromatography–mass spectrometry (LC-MS) was used to detect lipid metabolites in cells. Transcriptomics was used to measure mRNA content in cells. Finally, by processing these data using specific bioinformatics methods, the mechanism underlying anticancer effect of carrimycin was determined. Results: Metabolomics and transcriptomic results showed that lipid metabolism and mitochondrial biogenesis pathways in the cells changed after hSPM treatment, NR1D1 genes and ceramide were enriched from these pathways, implicating the involvement of ROS and pro-inflammatory response. Western blotting verified that the protein levels of NR1D1 decreased after h-SPM treatment, and ROS stating and qPCR demonstrated that ROS levels and the mRNA levels of pro-inflammatory genes were greatly induced by h-SPM. Conclusions: h-SPM reduced the protein level of NR1D1, disrupted metabolic regulation, accumulating ceramide, and the subsequent increased ROS generation promoted apoptosis and pro-inflammatory-like response of cells. Our findings unveiled the anticancer mechanism of a potent anticancer derivative of spiramycin and unveiled its mechanism of action. Full article

Deep eutectic solvents (DESs) represent a versatile and sustainable class of solvents, characterized by their low volatility, favorable biodegradability, and the ability to tailor their viscosity, polarity, and hydrogen-bonding capacity through the choice of their individual components. These characteristics have established them as powerful media in various analytical extraction and separation processes. This review presents a critical evaluation of the expanding role of DESs within the field of capillary electromigration techniques, summarizing key advancements from 2019 to mid-2025. We synthesize the current literature to delineate the benefits, persistent challenges, and future prospects of integrating DESs into capillary electrophoresis (CE)-based analytical workflows. Specifically, it systematically documents the following: (i) the diverse types of DESs employed in electrophoretic separations, (ii) proposed mechanisms underlying their influence on chiral compound resolution, and (iii) their utilization as separation media and pseudostationary phases (PSP) in capillary electromigration systems. By critically assessing their advantages and drawbacks, this review aims to provide a comprehensive perspective on the application of DESs in modern capillary electromigration techniques. Full article

Background/Objectives: Forced degradation studies are critical for identifying potential degradation pathways of monoclonal antibodies (mAbs), particularly under thermal stress. Due to their structural complexity and sensitivity to elevated temperatures, mAbs are prone to fragmentation, aggregation, and post-translational modifications. This study aimed to evaluate and compare the degradation profiles of biosimilar anti-VEGF mAb and its originator counterparts sourced from both the United States (U.S.) and the European Union (EU) under thermal stress conditions. To our knowledge, this represents one of the few studies conducting a direct head-to-head comparability assessment across biosimilar and two geographically sourced originators, integrating orthogonal analytical approaches. Methods: Biosimilar candidate and originator products (U.S. and EU) were incubated at 37 °C and 50 °C for 3, 7, and 14 days. Fragmentation profiles were assessed using validated non-reduced and reduced capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) methods. Additionally, size-exclusion ultra-performance liquid chromatography (SE-UPLC) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) assays were performed on samples stressed for 14 days to provide deeper insights into degradation pathways. Results: Non-reduced CE-SDS analysis indicated a time- and temperature-dependent increase in low-molecular-weight fragments and a corresponding decrease in the intact form, with more pronounced effects observed at 50 °C. Reduced CE-SDS revealed a more rapid increase in total impurity levels at 50 °C, accompanied by a decrease in total light and heavy chain content. SE-UPLC showed enhanced aggregation under thermal stress, more pronounced at 50 °C. LC-MS/MS analysis identified increased asparagine deamidation in the PENNY peptide and pyroglutamic acid formation (pE) at the N-terminus of the heavy chain. Conclusions: The degradation profiles of the biosimilar and originator mAbs were highly comparable under thermal stress, with no significant qualitative differences detected. By applying a multi-tiered analytical characterization technique, this study provides a comprehensive comparability assessment that underscores the robustness of biosimilarity even under forced degradation conditions. Full article