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Background: Multisystem inflammatory syndrome in children (MIS-C) is a severe post-COVID condition due to a delayed hyperimmune response to SARS-CoV-2. High-density lipoproteins (HDL) are pivotal players in inflammatory and immune modulation through the remodeling of their subfractions. Methods: This study aimed to evaluate serum levels of cholesterol, HDL, and HDL subfractions (HDL-SUB) to define their role in the pathogenesis of MIS-C and their potential use as biomarkers of this condition. We analyzed serum cholesterol, HDL and HDL-SUB (by capillary electrophoresis) in relation to serum values of biomarkers of inflammation and endothelial damage (by microfluidic immunoassays) in 48 patients with MIS-C at hospital admission and in 48 age- and sex-matched healthy controls. Results: Serum cholesterol, as well as HDL, were significantly lower in MIS-C patients than controls. Serum cholesterol was inversely correlated with all biomarkers of inflammation, confirming the impact of cytokines on reverse cholesterol transport, whereas HDL values were inversely correlated with serum biomarkers of endothelial damage, suggesting a role of HDL in endothelial damage in MIS-C patients. Furthermore, we found a remodeling of HDL-SUB with a more pronounced decrease in small HDL that have anti-inflammatory activity. Conclusions: These data confirm the severe impairment of reverse cholesterol transport in MIS-C and indicate serum HDL and HDL-SUB as potential useful diagnostic biomarkers of MIS-C. [ABSTRACT FROM AUTHOR]

AbstractGenotype-phenotype correlation and potential genetic risk in the compound heterozygosity for unstable hemoglobins (UHbs) and α0-thalassemia were discussed. Capillary electrophoresis and gene sequencing helped to establish the diagnosis. Hematological analysis showed the following findings: MCV 80.6 fL, MCH 27 pg, HGB 133 g/L, RBC 4.93 × 1012/L, Hb A: 94%, Hb X: 3.6% (zone 12) and Hb A2: 2.4%. DNA analysis revealed the patient was a Hb Pontoise carrier (HBA1: c.191C > A). Hb Pontoise resulted from an GCC > GAC substitution at codon 63 of the HBA1 genes, but carriers were usually asymptomatic or with only borderline hematological abnormalities. Due to mild instability of Hb Pontoise, its diagnosis relied on genetic diagnosis. Considering the high frequency of thalassemia in South China, accurate genotyping and appropriate genetic counseling should be performed for unstable hemoglobin carriers. [ABSTRACT FROM AUTHOR]

To study the heterogeneity of target membrane proteins in single cells with cellular integrity, we proposed a simple and low-cost method to obtain the copy number of the membrane proteins. HeLa cells were labeled by FITC affinity bodies specifically targeting HER2 membrane proteins. The immunolabeled HeLa cells were quantified by a laboratory-built laser induced fluorescence detector. A series of fluorescent microspheres with known number of FITC molecules on the surface were used to establish the calibration curve, instead of the standard fluorescent solutions, because the morphology of the microspheres was similar to the cells, and the distribution of FITC on the spheres were similar to the distribution of HER2 on the HeLa. The fluorescence intensity of the cells was converted to the molecule number of HER2 by the calibration curve. A capillary electrophoresis system was used to drive the microspheres and cells through the detection window. The copy number of HER2 in HeLa cells ranged from 4,036 to 1,224,920 ± 100 (2.5–97.5%), and the median of copy numbers were 104,438 ± 100 per cell. This method for measuring low-abundance membrane proteins can be utilized for the initial exploration of proteomics in ordinary laboratories. [ABSTRACT FROM AUTHOR]

The cosmetics industry is one of the fastest-growing sectors worldwide. The dynamic evolution of this industry results in an increasingly diverse range of products containing various active ingredients. Ensuring the quality of these products is crucial for consumer safety, necessitating the use of advanced analytical methods and adherence to legal regulations. Electrophoretic techniques, particularly capillary electrophoresis and micellar electrokinetic chromatography, facilitate the rapid and precise separation and identification of cosmetic ingredients. A well-chosen technique and optimized analytical conditions ensure high sensitivity, repeatability, and resolution, achieving detection limits that meet legal requirements. Although electromigration techniques are less common in routine laboratory analyses compared to liquid chromatography, they show potential for broader application in analyzing various substances found in cosmetics. This study reviews the possibilities of applying different electrophoretic techniques to analyze selected cosmetic ingredients serving various functions, including preservatives, dyes, exfoliating agents, UV filters, and also contaminants, while considering sample preparation methods, equipment used, and analysis conditions. The compiled data indicate that capillary electrophoresis, when compared to high-performance liquid chromatography and ion chromatography, shows comparable or superior sensitivity and repeatability, with detection limits adequate to meet regulatory standards. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase CK2 (formerly known as casein kinase II) plays a crucial role in various CNS disorders and is highly expressed in various types of cancer. Therefore, inhibiting this key kinase could be promising for the treatment of these diseases. The CK2 holoenzyme is formed by the recruitment of two catalytically active CK2α and/or CK2α′ subunits by a regulatory CK2β dimer. Starting with the lead furocarbazole W16 (4) inhibiting the CK2α/CK2β interaction, analogous pyrrolocarbazoles were prepared and tested for their protein–protein interaction inhibition (PPII). The key step of the synthesis was a multicomponent Levy reaction of 2-(indolyl)acetate 6, benzaldehydes 7, and N-substituted maleimides 8. Targeted modifications were performed by the saponification of the tetracyclic ester 9a, followed by the coupling of the resulting acid 10 with diverse amines. The replacement of the O-atom of the lead furocarbazole 4 by an N-atom in pyrrolocarbazoles retained or even increased the inhibition of the CK2α/CK2β interaction. The large benzyloxazolidinyl moiety of 4 could be replaced by smaller N-substituents without the loss of the PPII. The introduction of larger substituents at the 2-position and/or at p-position of the phenyl moiety at the 10-position to increase the surface for the inhibition of the PPI did not enhance the inhibition of the CK2α/CK2β association. The strong inhibition of the CK2α/CK2β association by the histidine derivative (+)-20a (Ki = 6.1 µM) translated into a high inhibition of the kinase activity of the CK2 holoenzyme (CK2α2β2, IC50 = 2.5 µM). Thus, 20a represents a novel lead compound inhibiting CK2 via the inhibition of the association of the CK2α and Ck2β subunits. [ABSTRACT FROM AUTHOR]

Escherichia coli β-galactosidase was labelled with 1 mmol/L fluorescein 5-carbamoylmethylthiopropanoic N-hydroxysuccinimidyl ester for 1 and 3 min. The samples were separated by capillary electrophoresis and peak areas compared to that of labelled BSA for the purpose of quantification of the concentration of attached label. Enzyme concentration in the samples was determined by single molecule counting. The average number of labels attached to each molecule of enzyme was found to be 3.1 and 4.5 when labelled for 1 and 3 min, respectively. The distribution of single enzyme molecule electrophoretic mobilities for the unlabelled enzyme and that labelled for 1 and 3 min were measured using capillary electrophoresis. The average mobilities were found determined to be −(1.99 ± 0.13) × 10–8 m2 V−1 s−1 (N = 39), −(2.16 ± 0.19) × 10–8 m2 V−1 s−1 (N = 46), and –(2.18 ± 0.21) × 10–8 m2 V−1 s−1 (N = 39), respectively. A protein electrophoresis model was applied and predicted that the differences in average mobilities could be explained through relatively minor changes in overall charge, Stokes radius, and shape. This difference was similar to the range in mobilities observed in the unlabelled protein. This is consistent with the electrophoretic heterogeneity of the unmodified enzyme being caused by relatively small differences in charge, size, and shape of the individual molecules in the population. [ABSTRACT FROM AUTHOR]

Y-InDels (insertions/deletions) are genetic markers which are extremely understudied. It is unknown whether this type of markers can be utilized for genetic ancestry inference. We have developed an innovative Y chromosome ancestry inference system tailored for forensic applications. This panel amplifies 21 Y chromosome loci, encompassing Y-InDels and Y-SNPs (Single Nucleotide Polymorphism), utilizing the capillary electrophoresis (CE) platform. The system performed well at DNA concentrations greater than 0.125 ng/ul and produced accurate results at a 1:100 mixing ratio of male and female DNA. The Cumulative probability of matching (CPM) was between 0.95 and 0.97 in the experimental population. The system's efficacy in inferring ancestral origins was demonstrated through intercontinental population discrimination, revealing high discrimination power between African and East Asian populations. Population genetic analyses conducted on Han, Qiang and Hui populations in Southwest China, where the smallest FST value was 0.0002 between Han Chinese in Beijing (from 1000 Genomes Project) and Qiang Chinese from Sichuan (CQSC). Phylogenetic tree construction further illuminated distinct haplotypes among populations, with ethnically unique haplotypes observed in 34.6% of Hui and 7.1% of Qiang populations. K-fold cross-validation show the system's inference abilities at the intercontinental level. In addition, our investigations identified potential associations between the Y-InDel locus Y: 15,385,547 (GRCh37) and haplogroup R1a1a1b2a2- Z2124, as well as locus Y: 13,990,180 (GRCh37) and haplogroup F-M89. In conclusion, we have established a Y-chromosome inference system tailored for grassroots-level application, underscoring the value of incorporating Y-InDel markers in forensic analyses. [ABSTRACT FROM AUTHOR]

Rapid advances in messenger RNA (mRNA) technology necessitate effective analytical methods. This study describes the development of a novel in vitro method using ion-pair reversed-phase liquid chromatography coupled to high-resolution mass spectrometry (IP-RP-LC-HRMS) for assessing the poly(A) and complementary poly(T) tail lengths directly into the DNA template used to manufacture mRNA. Briefly, after the validation of poly(A) tail length in the plasmid by Sanger sequencing, double-stranded DNA fragments containing these tails in the plasmid of interest were amplified by the polymerase chain reaction (PCR), purified on silica column, and digested with restriction enzymes ClaI and HindIII. Gel or capillary electrophoresis confirmed sample quality and enzymatic digestion efficiency. Subsequently, poly(A) and complementary poly(T) tails were extracted and analyzed by LC-MS to determine their length and heterogeneity at a single-nucleotide resolution. Three DNA templates containing poly(A) tail lengths of 60A-G, 95A, or 108A were studied. LC-MS results correlated well with Sanger sequencing, identifying major populations of 60A-G, 95A, or 108A. Surprisingly, unlike Sanger sequencing, LC-MS analysis revealed minor poly(A) populations with lengths longer or shorter than the theoretically encoded poly(A) tail length. This finding could be explained by (i) the slippage of bacterial DNA polymerase I during plasmid replication in bacterial culture, which occurs on repeat mononucleotide sequences, or (ii) the slippage of Q5® High-Fidelity DNA Polymerase during PCR amplification. In conclusion, the method is easy, rapid, and accurate and could replace Sanger sequencing to assess the poly(A) and complementary poly(T) tail lengths in plasmid DNA. [ABSTRACT FROM AUTHOR]

This research aimed to investigate the diagnostic value of passive particle agglutination test, Mycoplasma pneumoniae (MP) culture, cold agglutination test (CAT), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction-capillary electrophoresis fragment analysis (PCR-CEFA) for MP infection. Children with respiratory tract infections suspected to be MP infection were subjected to passive particle agglutination test, MP culture, CAT, ELISA, and PCR-CEFA. A total of 146 children (81 males, 65 females, mean age: 5.74 ± 3.32 years, and mean course of disease: 9.07 ± 5.18 days) met the inclusion criteria. The positivity rate of MP detection by MP culture was 69.18% (101/146). Using the MP culture method as the standard, higher sensitivity and positive predictive value were found in the PCR-CEFA compared with the other 3 methods. Appropriate methods are selected following the advantages and disadvantages of pathogen detection, and pediatric MP infection is analyzed by integrating various test results. [ABSTRACT FROM AUTHOR]

This second of the two-part series of a comprehensive survey review provides the diverse applications of two-dimensional correlation spectroscopy (2D-COS) covering different probes, perturbations, and systems in the last two years. Infrared spectroscopy has maintained its top popularity in 2D-COS over the past two years. Fluorescence spectroscopy is the second most frequently used analytical method, which has been heavily applied to the analysis of heavy metal binding, environmental, and solution systems. Various other analytical methods including laser-induced breakdown spectroscopy, dynamic mechanical analysis, differential scanning calorimetry, capillary electrophoresis, seismologic, and so on, have also been reported. In the last two years, concentration, composition, and pH are the main effects of perturbation used in the 2D-COS fields, as well as temperature. Environmental science is especially heavily studied using 2D-COS. This comprehensive survey review shows that 2D-COS undergoes continuous evolution and growth, marked by novel developments and successful applications across diverse scientific fields. [ABSTRACT FROM AUTHOR]

Background: Hemoglobin G-Siriraj is a rare hemoglobin variant caused by a ß-globin gene mutation (HBB: c.22G>A). The focus of this paper is aimed mainly at the chromatographic and electrophoretic properties of hemoglobin G-Siriraj for a presumptive identification. Notably, this is the first documented case of hemoglobin GSiriraj in Morocco. Methods: This hemoglobin variant was discovered in a 44-year-old Moroccan patient while measuring HbA1c with high-performance liquid chromatography. The discrepancies between capillary electrophoresis and the acid agarose electrophoresis led to suspicion of heterozygous hemoglobin G-Siriraj. Only the globin gene analysis can identify definitively this rare hemoglobin variant. Conclusions: Rare hemoglobin variants represent a diagnostic challenge. Contextualizing cytological and biochemical explorations based on clinical data is essential for effectively guiding diagnosis. [ABSTRACT FROM AUTHOR]

Many pharmaceutical active compounds are prepared as hydrochlorides for quick release in the gastrointestinal tract upon oral administration. Their inadvertent escape into the water environment requires efficient analytical separation for accurate quantitation to monitor their environmental fate. The purpose of this study is to demonstrate how best to optimize a capillary electrophoresis method for the separation of four model pharmaceutical hydrochlorides. Concentration of sodium dibasic phosphate in the background electrolyte solution, pH adjustment with HCl or NaOH, and applied voltage across the capillary were the three key factors chosen for optimization. The peak resolutions and total migration time were examined as the response indicators to complete a central composite design in response surface methodology. The examination revealed that CE separation was driven significantly by a linear regression model and minimally by a quadratic regression model, based on the coefficient of determination, the lack of fit, the total sum of squares, and the p values. Under optimal conditions of the background electrolyte concentration of 75 mM, pH 9, and the applied voltage of 10 kV, the model hydrochlorides were separated within five minutes in the migration order of metformin (first) > phenformin > mexiletine > ranitidine (last). The limits of UV detection/quantification attained under optimal CE conditions were 0.015/0.045, 0.020/0.060, 0.142/0.426, and 0.017/0.051, respectively. [ABSTRACT FROM AUTHOR]

New comb-like polyelectrolytes with triethylammonium (pAUTEA-Br), N-methyl-piperidinium (pAUMP-Br), N-methyl-morpholinium (pAUMM-Br) and pyridinium (pAUPy-Br) cation groups were first used to separate steroid hormones, biogenic amines and amino acids under capillary electrophoresis conditions. The polymers acted as modifiers of electrophoretic systems and were used as dynamic coatings of inner surface of fused-silica capillary and additives to background electrolyte to perform capillary electrochromatography and micellar electrokinetic chromatography regimes, leading to increase in separation selectivity and efficiency, and as reagents for indirect spectrophotometric detection of amino acids. New materials also showed activity during on-line preconcentration of biogenic amines and reduced their limits of detections by 10 times. Thus, new comb-like polyelectrolytes were demonstrated to act as multifunctional materials for capillary electrophoresis. This study discusses the features of the influence of the polyelectrolytes on electrophoretic separation capabilities and their possibilities in separation of model biologically active substances. Modifiers of this type significantly expand the analytical capabilities of the capillary electrophoresis method. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Food Safety & Quality is the property of Journal of Food Safety & Quality Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Magyar Állatorvosok Lapja is the property of Herman Otto Intezet Nonprofit Kft. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Metal ions and their interactions with biomolecules play an important role in human health. However, optical detectors commonly used for HPCE cannot directly detect metal ions without UV absorption. To make up for the shortcomings of existing HPCE detectors, a new universal HPCE detection system called an interface-induced current detector (IICRD) was constructed previously, with no need for derivatization procedures or complex instrumental modifications. Meanwhile, most of the reported studies on metal–biomolecule interactions only focused on the detection and analysis of biomolecules, commonly causing inaccurate or false-negative results, which is yet to be resolved. Here, the application of HPCE-IICRD realized the determination of metal–biomolecule interactions by directly measuring the electrophoretic parameters of metal ions for the first time, indicating that the interaction intensity can be measured more directly and accurately. Furthermore, an improved affinity capillary electrophoresis (ACE) method called relative mobility shift partial filling ACE-IICRD (rmsPF-ACE-IICRD) was originally developed to quantitatively analyze the binding strength. Binding behaviors between twelve free metal ions and three types of biomolecules (including two blood proteins, two enzyme proteins and two native DNAs) were investigated, and the values of the equilibrium dissociation constant (KD) of metal–biomolecule complexes were calculated and evaluated by the nonlinear chromatography (NLC) method. The experimental results were basically consistent with the literature values. In particular, heavy metal ions showed stronger interactions with proteins and enzymes, while metal ions tended to show stronger binding with native DNAs than proteins and enzymes, which were in agreement with literature results. The combined use of HPCE-IICRD and rmsPF-ACE showed great advantages such as no need for pretreatment, low operating cost, good repeatability, simple operation and no interference from coexisting substances, which is hopeful to become an efficient metal ion detection method and also to expand the application scope of IICRD in the future. [ABSTRACT FROM AUTHOR]

Objective: This study aims to investigate the prevalence, pathogen spectrum, clinical characteristics, and prognosis-related factors of other respiratory pathogens in COVID-19-infected patients, and to explore the application of molecular detection methods in the epidemiological investigation of multiple pathogen infections. Methods: Respiratory samples and clinical data from 384 patients with outpatient and inpatient respiratory infections were collected and analyzed. Multiplex PCR and capillary electrophoresis were conducted to detect the distribution characteristics of 26 pathogen species, comprising 13 viruses, 13 bacteria. Statistical analysis explored the relationship between pathogen distribution with COVID-19 development. Results: There was no statistical difference in prognosis between the 230 COVID-19-positive patients and the 154 COVID-19-positive patients. COVID-19 cases co-infected with other pathogens do not correlate with patient's age and gender. The main distribution of pathogens was mainly mecA (n = 62, 26.96%), followed by SPN(n = 61; 26.52%) and KP(n = 22; 9.57%). Compared with non-COVID-19 cases, COVID-19 infected patients showed a significantly higher mecA carrying rate (26.96% vs. 15.58%, P < 0.01), while there was no statistical difference for other pathogens. Regression analysis found that mecA and KP were independent risk factors for severe illnesses (mechanical ventilation, endotracheal intubation, ECOMO, etc.) or death in COVID-19 patients had 2.391 times and 3.722 times risk of severe disease or death compared with COVID-19 patients without mecA and KP. Conclusion: COVID-19 patients show a higher mecA carrier rate than non-COVID-19 patients, and mecA and KP may increase the risk of severe disease or death in COVID-19 patients, which requires close attention. [ABSTRACT FROM AUTHOR]

Background Objective Methods Results Conclusions Recent studies have reported that expanded GCA repeats in the GLS gene can cause glutaminase deficiency with ataxia phenotype. However, to data, no studies have investigated the distribution and role of GCA repeats in the GLS gene of Chinese individuals.The aim was to investigate the distribution of GCA repeats in Chinese individuals, including undiagnosed ataxia patients for identifying causal factors, healthy controls for determining the normal range, and ATX−ATXN3 (spinocerebellar ataxia type 3, SCA3) patients for exploring genetic modifiers.We combined whole‐genome sequencing (WGS), repeat‐primed polymerase chain reaction, capillary electrophoresis (RP−PCR/CE), and ExpansionHunter to screen the GCA repeats in the GLS gene of 349 undiagnosed ataxia individuals, 1505 healthy controls, and 1236 ATX−ATXN3 (SCA3) patients from mainland China.No expanded GCA repeats in the GLS gene were detected across any of the samples. The average number of GCA repeats was 11 (range: 8–31), 12 (range: 6–33), and 11 (range: 6–33) for undiagnosed ataxia patients, healthy controls, and SCA3 patients, respectively. The intermediate repeat size (9 < repeat size ≤ 13) of the nonexpanded GCA allele in the GLS gene was associated with later disease onset in ATX−ATXN3 (SCA3) patients.Abnormal expansions of GLS GCA repeats are rare in the Chinese population. However, intermediate‐length normal GCA repeat sizes may influence the age at onset (AAO) in ATX‐ATXN3 (SCA3) patients. © 2024 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Tissue specificity can render mitochondrial uncouplers more promising as leading compounds for creating drugs against serious diseases. In search of tissue‐specific uncouplers, we address anilinothiophenes as possible glutathione‐S‐transferase substrates (GST). Earlier, 'cyclic' uncoupling activity was reported for 5‐bromo‐N‐(4‐chlorophenyl)‐3,4‐dinitro‐2‐thiophenamine (BDCT) in isolated rat liver mitochondria (RLM). The mechanism by which BDCT induced two‐phase changes in mitochondrial respiration (stimulation followed by deceleration) was unknown. To clarify this issue, we synthesized BDCT and its two analogues. Among these, 5‐bromo‐3,4‐dinitro‐N‐(4‐nitrophenyl)‐2‐thiophenamine (BDNT) appeared to be the most effective as a mitochondrial uncoupler, decreasing membrane potential and stimulating respiration at submicromolar concentrations. Importantly, BDNT exerted two‐phase changes in both mitochondrial membrane potential and respiration rate of RLM, which were enhanced by the addition of glutathione (GSH) but inhibited by the compounds capable of GSH depleting, such as 1‐chloro‐2,4‐dinitrobenzene (CDNB). By contrast, the phase of recoupling was not observed in rat heart mitochondria (RHM). Remarkably, BDNT elicited mitochondrial depolarization in primary human fibroblasts but not in cultured human liver (HepG2) cells. By detecting proton‐selective electrical current through planar bilayer lipid membranes, we demonstrated the ability of BDCT and BDNT to transfer protons across membranes. BDNT proved to be an anionic protonophore with a pKa of 7.38. By using LC–MS and capillary electrophoresis, we directly showed the formation of BDNT conjugates with GSH upon incubation with RLM but not RHM. Therefore, we hypothesize that GST is involved in the disappearance of the anilinothiophene uncoupling activity in RLM, ensuring the tissue‐specific behavior of the uncoupler. [ABSTRACT FROM AUTHOR]

This review provides a comprehensive summary of the latest international research on detection methods for glucosinolates in cruciferous plants. This article examines various analytical techniques, including high-performance liquid chromatography (HPLC), liquid chromatography–mass spectrometry (LC-MS), enzyme-linked immunosorbent assay (ELISA), and capillary electrophoresis (CE), while highlighting their respective advantages and limitations. Additionally, this review delves into recent advancements in sample preparation, extraction, and quantification methods, offering valuable insights into the accurate and efficient determination of glucosinolate content across diverse plant materials. Furthermore, it underscores the critical importance of the standardization and validation of these methodologies to ensure reliable glucosinolate analyses in both scientific research and industrial applications. [ABSTRACT FROM AUTHOR]

An in-line sample concentration method for capillary electrophoresis called admicelle to solvent microextraction was proposed. In this technique, analytes were trapped in the cetyltrimethylammonium bromide admicelles formed in situ on the negatively charged capillary surface. A solvent plug was then partially injected hydrodynamically to collapse the admicelles, which liberated and focused the analytes at the solvent front. Voltage was applied across the capillary, completing the stacking process. Various solvents, namely, methanol, ethanol, and acetonitrile, were investigated. The optimal solvent for solvent to admicelle microextraction was 30% acetonitrile in 24 mM sodium tetraborate (pH 9.2). Sample injection time and solvent to sample injection ratio were also optimised. For this demonstration, the optimum sample injection time and solvent to sample injection ratio were 320 s and 1:2, respectively. Using the optimum conditions, UV detection sensitivity was enhanced 132–176-fold for the model anions. The LOQ, %intra-/inter-day (n = 6/n = 12, 2 days) repeatability, and linearity (R2) of admicelle to solvent microextraction were 0.08–2 µg/mL, 1.9–3.9%, 2.8–4.9%, and 0.992, respectively. Admicelle to solvent microextraction was applied to the analysis of various fortified water samples, with good repeatability (%RSD = 0.5–3.6%), and no matrix interferences. [ABSTRACT FROM AUTHOR]

T7 RNA Polymerase (RNAP) is a widely used enzyme with recent applications in the production of RNA vaccines. For over 50 years denaturing sequencing gels have been used as key analysis tools for probing the nucleotide addition mechanisms of T7 RNAP and other polymerases. However, sequencing gels are low-throughput limiting their utility for comprehensive enzyme analysis. Here, we report the development of HiKER; (High-throughput Kinetics using Capillary Electrophoresis and Robotics) a high-throughput pipeline to quantitatively measure enzyme kinetics. We adapted a traditional polymerase misincorporation assay for fluorescent detection at scale allowing rapid estimates of RNAP misincorporation in different experimental conditions. Using this platform with an OT-2 robotics system, ~1500 time points were collected in a single workday. T7 RNAP exhibited dramatic differences in both observed rate constant and amplitude depending on the mismatch examined. An average misincorporation frequency of ~45 misincorporations per million bases was estimated using HiKER and is consistent with previous studies. Misincorporation time courses for T3 RNAP and Sp6 RNAP were also collected and appeared similar to T7 RNAP suggesting conserved kinetic mechanisms. However, differences between the RNAPs were observed in extension from base mismatch experiments. This platform is affordable, open-source, and broadly applicable to many enzymes. [ABSTRACT FROM AUTHOR]

Background/Objectives: Colorectal cancer (CRC) is the third leading cause of cancer death globally, with rising incidence. The immunohistochemistry (IHC) for mismatch repair (MMR) proteins is the first technique used in routine practice to evaluate an MMR status. Microsatellite instability (MSI) may be tested in case of doubt during IHC staining. This study introduces a novel high-resolution melt (HRM) protocol for MSI detection and compares it with traditional fragment length analysis (FLA) via capillary electrophoresis. Methods: A total of 100 formalin-fixed and paraffin-embedded CRC specimens were analyzed using two distinct protocols: one based on FLA (TrueMark MSI Assay kit) and another one based on HRM (AmoyDx® Microsatellite Instability Detection Kit). Results: Overall, 68 (68.0%) of the cases were MSS, and 32 (32.0%) were MSI-H. HRM analysis was first successfully carried out in all the cases. A perfect concordance in MSI evaluation between HRM and FLA was observed. HRM showed slightly shorter hands-on time and turnaround time. Conclusions: We provided evidence of the validity of this new HRM approach in determining the MSI status of colorectal carcinomas. [ABSTRACT FROM AUTHOR]

Understanding the genetic diversity of crops is of fundamental importance for the efficient use and improvement of germplasm resources. Different molecular genotyping systems have been implemented for population structure and phylogenetic relationships analyses, among which, microsatellites (SSRs) and single nucleotide polymorphisms (SNPs) markers have been the most widely used. This study reports the efficacy of SNPs detected via double-digest restriction-site-associated DNA sequencing (ddRADseq) and SSRs analyzed via capillary electrophoresis (CE) and high-resolution melting (HRM) in tomato. In total, 21,020 high-quality SNPs, 20 CE-SSRs, and 17 HRM-SSR markers were assayed in a panel of 72 accessions that included a diversified set of landraces, long-shelf-life cultivars and heirlooms with different origins and fruit typology. The results showed how the population structure analysis was consistent using the three genotyping methods, although SNPs were more efficient in distinguishing cultivar types and in measuring the degree of accessions' similarity. Compared to CE-SSR, the analysis of microsatellites via HRM yielded a slightly higher number of alleles (98 vs. 96). HRM-SSR demonstrated a distinction between European and non-European germplasm, better resolving the collection's diversity and being more consistent with SNP data. Phylogenetic trees drawn with independent marker data, detected specific groups of accessions showing robust clusters, highlighting how heirlooms were less heterogeneous than landraces. In addition, the fixation index (FST) revealed a high genetic differentiation between heirlooms and long-shelf-life cultivars, with SNP and SSR-HRM data emphasizing the distinction between cherry and plum types and CE-SSR data between cherry and oxheart types. In all instances, a greater molecular variance was found within the different considered biological statuses, provenances, and typologies rather than among them. This work presents the first attempt to compare the three tomato genotyping techniques in tomato. Findings highlighted how the markers used are complementary for genetic diversity analysis, with SNPs providing better insight and HRM-SSR as a viable alternative to capillary electrophoresis to dissect the genetic structure. [ABSTRACT FROM AUTHOR]

Background and Objective: Often, stability studies do not cover all facets of ensuring patient safety for biologics, unless the impact of the in-use and out-of-fridge conditions is also assessed. This study investigated the physicochemical and biological stability of Sandoz rituximab biosimilar (SDZ-RTX). Methods: In a worst-case setting, two SDZ-RTX batches in vials were exposed to long-term conditions (5 ± 3 °C) for at least the shelf-life period (36 months). These batches were exposed to out-of-fridge conditions of up to 25 ± 2 °C/60 ± 5% relative humidity in total for 14 days, and subsequently to 30 ± 2 °C/75 ± 5% relative humidity for 7 days. Thereafter, these batches were diluted to 1 mg/mL in 0.9% NaCl in 250-mL polyethylene infusion bags and stored at either 25 ± 2 °C/60 ± 5% relative humidity for 30 days or 30 ± 2 °C/75 ± 5% relative humidity for 14 days, representing in-use conditions. The stability of SDZ-RTX was assessed using a variety of analytical methods, including size-exclusion chromatography, cation exchange chromatography, non-reducing capillary electrophoresis sodium dodecyl sulfate, complement-dependent cytotoxicity-bioactivity, and subvisible particle count by light obscuration. Results: Results for all assessments were within the stringent shelf-life acceptance criteria for SDZ-RTX for both batches under both in-use conditions. Conclusions: These data show that the physicochemical and biological quality of SDZ-RTX diluted in 0.9% NaCl infusion bags is assured, even after prolonged worst-case (out-of-fridge and in-use) storage at elevated temperatures up to 30 °C, if the medication is prepared under aseptic conditions according to the Summary of Product Characteristics. [ABSTRACT FROM AUTHOR]

Background: Gadolinium-based contrast agents (GBCA) are widely used in magnetic resonance imaging (MRI) to enhance image contrast by interacting with water molecules, thus improving diagnostic capabilities. However, understanding the residual accumulation of GBCA in tissues after administration remains an area of active research. This highlights the need for advanced analytical techniques capable of investigating interactions between GBCAs and biopolymers, such as type I collagen, which are abundant in the body. Objective: This study explores the interactions of neutral and charged GBCAs with type I collagen under physiological pH conditions (pH 7.4) using Taylor dispersion analysis (TDA) and frontal analysis continuous capillary electrophoresis (FACCE). Methods: Collagen from bovine achilles tendon was ground using a vibratory ball mill to achieve a more uniform particle size and increased surface area. Laser granulometry was employed to characterize the size distributions of both raw and ground collagen suspensions in water. TDA was used to assess the hydrodynamic radius (Rh) of the soluble collagen fraction present in the supernatant. Results: From the TDA and FACCE results, it was shown that there were no significant interactions between the tested GBCAs and either the ground collagen or its soluble fraction at pH 7.4. Interestingly, we also observed that collagen interacts with filtration membranes, indicating that careful selection of membrane material, or the absence of filtration in the experimental protocol, is essential in interaction studies involving collagen. Conclusion: These findings bring valuable insights into the behavior of GBCAs in biological systems with potential implications for clinical applications. [ABSTRACT FROM AUTHOR]

The new NGS platforms developed allow fast, easy, and most importantly the ability to analyze hundreds of loci in a single run simultaneously, enabling the analysis of mixtures with SNP markers. In this study, DNA samples obtained from 6 different individuals; 2-females and 4-males, were examined. These 6 individuals are formed in double groups artificially prepared in various ratios and a total of 20 samples and 124 SNP loci were analyzed with the HID-Identity panel and Ion-Torrent-PGM platform. To compare the results with the conventional method, which is the capillary electrophoresis platform, the same samples were analyzed by capillary electrophoresis method using the AmpFlSTR®Identifiler®STR Kit. In the SNP analyses performed with the Ion Torrent platform, in all 1 : 1 and 1 : 3 ratios, the average number of readings of minor alleles was obtained at a very successful level. Although the average number of readings decreased as expected from 1 : 10 ratio, the rate of minor allele detection was 21.7%, even at the ratio of 1 : 50. When the results of the SNP analysis were compared with the STR analysis; it was determined that SNP analyzes obtained more successful results compared to STR analysis when the amount of DNA belonging to the minor allele in the mixtures was low. In this study, it has been determined that the HID-Identity panel, which is compatible with the Ion-Torrent-PGM platform and analyzes 124 SNP loci, gives fast, easy, and reliable results and can be successfully used to analyze mixtures. [ABSTRACT FROM AUTHOR]

The present work describes, for the first time, an eco-friendly capillary zone electrophoresis (CZE) methodology for determining apixaban (APX), an oral anticoagulant. Separation was carried out using a fused silica capillary with an applied voltage of 20 kV and hydrodynamic injection for 5 s at 50 mbar. The electrolyte system consisted of 50 mM sodium borate at pH 8.0. The method was validated as recommended by ICH, establishing selectivity, linearity, precision, accuracy, and robustness. The analytical procedure was linear in the range of 10–125 µg mL−1 (r = 0.999). The limits of detection and quantification were determined to be 1.77 µg mL−1 and 5.37 µg mL−1, respectively. Precision was evaluated by relative standard deviation (RSD), with an intra-day RSD (n = 6) of 0.96% and an inter-day RSD (n = 18) of 4.42%. The average recovery rates for the pharmaceutical samples were excellent, ranging from 98.03% to 101.01%, with an RSD of 1.51%. Moreover, the green profile of the method was evaluated using the National Environmental Methods Index (NEMI), Analytical Eco-Scale, Green Analytical Procedure Index (GAPI), and Analytical GREEnness Metric (AGREE) assessment tools. The results suggest that the CZE method aligns with the principles of green chemistry, offering a greener alternative for the routine analysis of APX quality control. Also, the method presents a low-cost analysis, using fewer organic solvents and minimizing waste generation compared to techniques commonly used in routine analysis. [ABSTRACT FROM AUTHOR]

The effect of gravity based on the vector sum of gravity and buoyancy forces working spontaneously for all species was introduced into capillary electrophoresis (CE) as another important force which cooperated with electrophoretic flow and electro-osmotic flow. Their portion was adjusted by simply tilting the whole of the capillary at an angle during CE running. The related formula was proposed and verified through a series of experiments. After investigating the related parameters, results showed that the gravity effect was significantly affected by additives in the buffer, the length, and the inner diameter of the capillary, and the size of the sample molecule. This made the different ions with opposite or significantly different mobilities to be observed at a CE run. It significantly improved separation efficiencies of some small molecules, chiral compounds, macromolecules and cells when the tilt angles of the capillary were adjusted to a special range predicted through the fitting curve. In addition, micrometer level microspheres and cells were firstly separated by the new CE strategy and the resolutions were more than 1.0. After ingeniously designing the gradient of the tilting angle with time, we were able to further enhance the separation efficiency of the targets. For example, the resolution of lysozyme and ribonuclease A could be increased from 3.691 to 7.342. These indicated the huge potential of the new CE strategy and its gradient mode in separation. [ABSTRACT FROM AUTHOR]

Objective: To describe a novel α-thalassemiadeletion identified from a newborn by third-generation sequencing (TGS). Case report: The proband, a newborn subject to neonatal capillary electrophoresis (CE) screening, exhibited suspected α0-thalassemia carrier status (Hb Bart's 3.0%). Notably, both parents had negative results on thalassemia screening during pregnancy. Multiplex ligation-dependent probe amplification (MLPA) presented a deletion between probes 364nt and 472 nt that extended from the HBZ gene to the downstream region of the RGS11 gene. Subsequently, TGS determined the approximated break position of this deletion, indicating a length exceeding 145 kb (chr16:127,815-273,190 del 145376 bp). Sanger sequencing validated the upstream and downstream breakpoints of this deletion. Only maternal data were available for pedigree analysis, with the father's sample lacking. MLPA showed no deletion in the mother, suggesting possible paternal inheritance. The deletion was named Guigang deletion (--Guigang) after the proband's city of origin, Guigang. Conclusions: We reported a novel α-thalassemiadeletion and provided insights into the hematological phenotype and molecular analysis. These findings have implications for genetic counseling and prenatal diagnosis. [ABSTRACT FROM AUTHOR]

The need for consistency in analytical method development reinforces the dependence of pharmaceutical product development and manufacturing on robust analytical data. The Analytical Quality by Design (AQbD), akin to the product Quality by Design (QbD) endows a high degree of confidence to the method quality developed. AQbD involves the definition of the analytical target profile as starting point, followed by the identification of critical method variables and critical analytical attributes, supported on risk assessment and design of experiment tools for the establishment of a method operable design region and control strategy of the method. This systematic approach moves away from reactive troubleshooting to proactive failure reduction. The objective of this review is to highlight the elements of the AQbD framework and provide an overview of their implementation status in various analytical methods used in the pharmaceutical field. These methodologies include but are not limited to, high-performance liquid chromatography, UV-Vis spectrophotometry, capillary electrophoresis, supercritical fluid chromatography, and high-performance thin-layer chromatography. Finally, a critical appraisal is provided to highlight how regulators have encouraged AQbD principles application to boost the prevention of method failures and a better understanding of the method operable design region (MODR) and control strategy, ultimately resulting in cost-effectiveness and regulatory flexibility. [ABSTRACT FROM AUTHOR]

To determine the molecular basis, genotype – phenotype relationship, and genetic origin of Hemoglobin (Hb) Hekinan associated with several forms of α-thalassemia and other hemoglobinopathies for a better understanding of its diverse clinical phenotypes. Seventeen participants with suspected abnormal Hb were studied. Hb analysis was performed using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). Mutational and α-haplotypic and structural analyses were conducted, and the effects of mutations on globin-chain stability were determined. All participants harbored Hb Hekinan II (HBA1:c.84 G>T) co-inherited with another α-globin gene anomaly. Three novel genotypes, (ααHekinan/αCSα), (ααHekinan/αCSα,βA/βE), and (ααHekinan/αCSα,βE/βE), were characterized. Despite being co-inherited with both α- and β-Hb variants Hb Hekinan II led to minimal changes in erythrocyte parameters, suggesting a non-pathological nature. HPLC but not CE revealed a distinct small shoulder-like Hb pattern. Thai Hb Hekinan II was strongly associated with haplotype [+ - S + - - -] and the possibility of four different haplotypes, while two Burmese Hb Hekinan II were associated with haplotypes [± - S + - + -] and [± - S + - - -]. The novel genotypes identified provide a fresh perspective on Hb Hekinan II diversity. HPLC has superior identification capabilities for samples of Hb Hekinan II co-inherited with α-thalassemia. Thai and Burmese Hb Hekinan II have diverse origins. [ABSTRACT FROM AUTHOR]

Dopamine and serotonin are neurotransmitters that are crucial for numerous physiological processes, including mood regulation, reward, and motor function. Dysregulation of these neurotransmitters is associated with various neuropsychiatric disorders. Albumin in plasma modulates the bioavailability of drugs and free concentrations of bioactive constituents. This study aimed to characterize the interactions of dopamine and serotonin with bovine serum albumin. Capillary electrophoresis in the frontal analysis mode was utilized as an effective method to assess dopamine–bovine serum albumin and serotonin–bovine serum albumin affinity. The free neurotransmitter plateaus were distinctly separated from the bovine serum albumin–neurotransmitter complex plateaus. Free dopamine and serotonin concentrations were determined by monitoring the heights of their respective plateaus. In contrast, the bound concentrations were calculated from the difference between the total and free plateau heights. Dopamine and serotonin were found to bind to bovine serum albumin at independent sites with binding constant values of 1.90 × 103 and 2.90 × 103 L/mol, respectively. Additionally, an in silico molecular docking approach revealed the binding sites for dopamine and serotonin near the glutamic acid‐291 and serine‐428 residues of bovine serum albumin, respectively. [ABSTRACT FROM AUTHOR]

Background: Over a thousand types of hemoglobin variants have been reported. It is necessary to understand the impact of variants on the accuracy of various methods. The effect of hemoglobin variants on HbA1c determination depends on different detection methods. Methods: A 53-year-old female presented with abnormally elevated glycated hemoglobin (HbA1c) in a routine medical check-up. Capillary electrophoresis (CE) indicated the presence of hemoglobin variant. In the family screening, we found her mother, sister, and daughter all had the same heterozygous mutation (c.397A > G, K133E) in the HBB gene. Results: This is the first known familial case of Hb Takasago in China. Improving the understanding of hemoglobin variants has important medical significance. In the case of abnormal HPLC chromatograms, it is crucial to apply appropriate detection methods, find accurate causes, and communicate with clinicians in a timely manner. Conclusions: The use of appropriate HbA1c detection methods that are not affected by related variants can ensure the accuracy of the results. In the existence of abnormal HPLC chromatograms, it is necessary to communicate with clinical clinicians. Given its heredity and prevalence, effective screening for abnormal hemoglobinopathies is of great significance for improving the quality of the population. [ABSTRACT FROM AUTHOR]

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Background/Objective: A dysregulated metabolism has been studied as a key aspect of the COVID-19 pathophysiology, but its longitudinal progression in severe cases remains unclear. In this study, we aimed to investigate metabolic dysregulation over time in patients with severe COVID-19 requiring mechanical ventilation (MV). Methods: In this single-center, prospective, observational study, we obtained 236 serum samples from 118 adult patients on MV in an ICU. The metabolite measurements were performed using capillary electrophoresis Fourier transform mass spectrometry, and we categorized the sampling time points into three time zones to align them with the disease progression: time zone 1 (T1) (the hyperacute phase, days 1–3 post-MV initiation), T2 (the acute phase, days 4–14), and T3 (the chronic phase, days 15–30). Using volcano plots and enrichment pathway analyses, we identified the differential metabolites (DMs) and enriched pathways (EPs) between the survivors and non-survivors for each time zone. The DMs and EPs were further grouped into early-stage, late-stage, and consistent groups based on the time zones in which they were detected. Results: With the 566 annotated metabolites, we identified 38 DMs and 17 EPs as the early-stage group, which indicated enhanced energy production in glucose, amino acid, and fatty acid metabolisms in non-survivors. As the late-stage group, 84 DMs and 10 EPs showed upregulated sphingolipid, taurine, and tryptophan–kynurenine metabolisms with downregulated steroid hormone synthesis in non-survivors. Three DMs and 23 EPs in the consistent group showed more pronounced dysregulation in the dopamine and arachidonic acid metabolisms across all three time zones in non-survivors. Conclusions: This study elucidated the temporal differences in metabolic dysregulation between survivors and non-survivors of severe COVID-19, offering insights into its longitudinal progression and disease mechanisms. [ABSTRACT FROM AUTHOR]

BACKGROUND: Phytophthora spp. represent a pivotal genus of plant pathogens with a global distribution, exerting significant deleterious effects on food safety and forestry ecosystems. Numerous pathogenic and invasive Phytophthora species, introduced through imported fruits, have been frequently detected at Chinese ports. With the rise in global trade activities, the plant quarantine of imported fruits is becoming increasingly important but challenging. Fast, simple, and labor‐saving techniques are necessary and anticipated. RESUITS: A polymerase chain reaction restriction fragment length polymorphism capillary electrophoresis (PCR‐RFLP‐CE) technology‐based quarantine approach was developed for 16 Phytophthora species associated with the imported fruits in China. The Ypt1 gene, exhibiting abundant interspecific variations, was selected as the marker gene for PCR. The restriction endonuclease AluI was proven to be capable and compatible in simultaneously separating different Phytophthora species during CE. By combining with a fast and efficient DNA extraction kit, the developed PCR‐RFLP‐CE technique was successfully applied to identify Phytophthora species in artificially infested fruits. CONCLUSION: We provide a quick, practical, and high‐throughput detection approach for hazardous and invasive Phytophthora species associated with imported fruits in China. This strategy can give good convenience and technological support for carrying out massive quarantine activities at Chinese ports. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product. Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal. Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product. Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production. Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process. [ABSTRACT FROM AUTHOR]

This paper introduces a novel contactless single-chip detector that utilizes impedance-to-digital conversion technology to measure impedance in the microfluidic channel or capillary format analytical device. The detector is designed to operate similarly to capacitively coupled contactless conductivity detectors for capillary electrophoresis or chromatography but with the added capability of performing frequency sweeps up to 200 kHz. At each recorded data point, impedance and phase-shift data can be extracted, which can be used to generate impedance versus frequency plots, or phase-shift versus frequency plots. Real and imaginary parts can also be calculated from the data, allowing for the generation of Nyquist diagrams. This detector represents the first of its kind in the contactless conductivity class to provide spectrum-type data, as demonstrated in capillary electrophoresis experiments. [ABSTRACT FROM AUTHOR]

Genetically modified (GM) crops and their products are regulated in many countries. To meet increasing regulatory requirements for a growing number of GM soybean events, high-throughput and cost-effective detection methods are indispensable. In this study, 14 widely commercialized GM soybean events, including GTS40-3-2, MON89788, CV127, A2704-12, A5547-127, 305423, 356043, MON87701, MON87705, MON87708, MON87769, SYHT0H2, FG72, and DAS-444Ø6-6 were utilized as targets for event-specific identification. A high-throughput detection method was established by integrating a single universal primer multiplex PCR (SUP-M-PCR) with capillary electrophoresis technique. This method enables the identification of 14 soybean events in a single PCR reaction, addressing the limitations of conventional multiplex PCR such as self-inhibition and amplification variations between different primers. Furthermore, it significantly enhances the sensitivity and accuracy of previous multiple detection methods, achieving a detection sensitivity of 0.05% (w/w). Overall, this approach greatly improves detection efficiency and holds promising potential for the identification and detection of 14 types of GM soybean events and their derivatives. [ABSTRACT FROM AUTHOR]

To describe a variant hemoglobin that interferes with HbA1c analysis by cation exchange HPLC. A 78 years-old Spanish male patient visited the Internal Medicine Clinic for a routine check-up, with HbA1c included to screen for diabetes. He had suffered hypertension and dyslipidemia, and the patient had no previous symptoms suggestive of diabetes such as hyperglycemia, weight loss, polydipsia, polyuria or tiredness. Diabetes screening by HbA1c measurement was assessed using cation exchange HPLC and an immunoassay point-of-care analyzer. Routine hemoglobinopathy screening was performed including CBC, HbF and HbA2 measurement by cation exchange HPLC and capillary electrophoresis (CE). Further variant characterization was undertaken by DNA sequencing. Discordant HbA1c results were obtained for our subject, with elevated HbA1c of 52 mmol/mol measured by cation exchange HPLC and a normal level of 34 mmol/mol by immunoassay. Abnormal HbA1c peak shape prompted hemoglobinopathy screening to investigate potential variant interference. A globin gene analysis was performed, and the results showed a variant hemoglobin named 'Hb Petie Salpetriere'. This variant arises from a Val → Phe substitution due to a mutation of c.103G>T of the beta-globin gene [BETA34 (B16) Val>Phe; HBB:c.103G>T]. This is the first reported case involving the Hb Petie Salpetriere variant in a Spanish patient. The present results show that the Hb Petie Salpetriere variant can affect the results of HbA1c analysis through ion-exchange HPLC, but not that obtained from the latex agglutination immunoassay. Only ion-exchange HPLC suggested the presence of the Hb variant in this case, suggesting that a careful review of the resulting chromatogram might reveal a potential variant. [ABSTRACT FROM AUTHOR]

Ageing is a complex biological process with variations among individuals, leading to the development of ageing clocks to estimate biological age. Glycans, particularly in immunoglobulin G (IgG), have emerged as potential biomarkers of ageing, with changes in glycosylation patterns correlating with chronological age. For precision analysis, three different plasma pools were analysed over 26 days in tetraplicates, 312 samples in total. In short-term variability analysis, two cohorts were analysed: AstraZeneca MFO cohort of 26 healthy individuals (median age 20) and a cohort of 70 premenopausal Chinese women (median age 22.5) cohort monitored over 3 months. Long-term variability analysis involved two adult men aged 47 and 57, monitored for 5 and 10 years, respectively. Samples were collected every 3 months and 3 weeks, respectively. IgG N-glycan analysis followed a standardized approach by isolating IgG, its subsequent denaturation and deglycosylation followed by glycan cleanup and labelling. Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) and ultra-performance liquid chromatography analyses were employed for glycan profiling. Statistical analysis involved normalization, batch correction, and linear mixed models to assess time effects on derived glycan traits. The intermediate precision results consistently exhibited very low coefficient of variation values across all three test samples. This consistent pattern underscores the high level of precision inherent in the CGE method for analysing the glycan clock of ageing. The AstraZeneca MFO cohort did not show any statistically significant trends, whereas the menstrual cycle cohort exhibited statistically significant trends in digalactosylated (G2), agalactosylated (G0) and fucosylation (F). These trends were attributed to the effects of the menstrual cycle. Long-term stability analysis identified enduring age-related trends in both subjects, showing a positive time effect in G0 and bisected N-acetylglucosamine, as well as a negative time effect in G2 and sialylation, aligning with earlier findings. Time effects measured for monogalactosylation, and F remained substantially lower than ones observed for other traits. The study found that IgG N-glycome analysis using CGE-LIF exhibited remarkably high intermediate precision. Moreover, the study highlights the short- and long-term stability of IgG glycome composition, coupled with a notable capacity to adapt and respond to physiological changes and environmental influences such as hormonal changes, disease, and interventions. The discoveries from this study propel personalized medicine forward by deepening our understanding of how IgG glycome relates to age-related health concerns. This study underscores the reliability of glycans as a biomarker for tracking age-related changes and individual health paths. [ABSTRACT FROM AUTHOR]

Hydrogels like agarose have long been used as sieving media for the electrophoresis-based analysis of biopolymers. During gelation, the individual agarose strands tend to form hydrogen-bond mediated double-helical structures, allowing thermal reversibility and adjustable pore sizes for molecular sieving applications. The addition of tetrahydroxyborate to the agarose matrix results in transitional chemical cross-linking, offering an additional pore size adjusting option. Separation of SDS-proteins during gel electrophoresis is an activated process defined by the interplay between viscosity, gelation/cross-link formation/distortion, and sample conformation. In this paper, the subunits of a therapeutic monoclonal antibody were separated by capillary SDS agarose gel electrophoresis at different temperatures. The viscosity of the separation matrix was also measured at all temperatures. In both instances, Arrhenius plots were used to obtain the activation energy values. It was counterintuitively found that larger SDS–protein complexes required lower activation energies while their low-molecular-weight counterparts needed higher activation energy for their electromigration through the sieving matrix. As a first approximation, we considered this phenomenon the result of the electric force-driven distortion of the millisecond range lifetime reticulations by the larger and consequently more heavily charged electromigrating molecules. In the meantime, the sieving properties of the gel were still maintained, i.e., they allowed for the size-based separation of the sample components, proving the existence of the reticulations. Information about the activation energy sheds light on the possible deformation of the sieving matrix and the solute molecules. In addition, the activation energy requirement study helped in optimizing the separation temperature, e.g., with our sample mixture, the highest resolution was obtained for the high-molecular-weight fragments, i.e., between the non-glycosylated heavy chain and heavy-chain subunits at 25 °C (lower Ea requirement), while 55 °C was optimal for the lower-molecular-weight light chain and non-glycosylated heavy chain pair (lower Ea requirement). Future research directions and possible applications are also proposed. [ABSTRACT FROM AUTHOR]

The DNA within the Human Genome involves loci with high polymorphism such as short tandem repeats (STR). The STR polymorphism analysis is extensively used for numerous purposes such as identification studies in forensic sciences, paternity tests, genome mapping, and population studies. In this study, blood and swab samples were obtained from 200 unrelated healthy individuals living in Elazig province. DNA was amplified via the AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, USA) to analyze the 15 STR loci. Using the ABI PRISM310 Genetic Analyzer capillary electrophoresis, the samples were analyzed and the allelic ladder was used to compare and identify the alleles of the samples, the distribution of the allele frequencies and peak values were examined. For statistical analysis Arlequin v.3.11 and PowerStats v.1.2 were used to assess the data. To determine if the allele frequencies were compatible with the Hardy-Weinberg (HW) equilibrium, the Markov chain and the Fisher's exact test p-value (p<0.05) were examined. It was determined that the D8S1179 (0.00002), D21S11 (0.00593) and vWA (0.04939) loci were not compatible with the HW equilibrium, but the rest of the loci were compatible. For the loci, the combined power of exclusion is 0.9999995, the combined power of discrimination is 0.9999999999998. The probability of matching for the 15 STR loci was calculated to be 1 in 1.364x10ˉ17. The power of discrimination was quite high when all the loci were considered. The results of this study indicate that the populations residing in the Elazığ province may benefit from paternity testing, forensic identification, and genomic mapping using the data as an appropriate marker. [ABSTRACT FROM AUTHOR]

BACKGROUND: The technique of ultrasound processing is widely used for molecular biological analysis. It is of great significance to study the DNA quality of tissue with different storage years under new ultrasonic treatment for further specimen quality control of molecular detection. OBJECTIVE: To explore the effects of different storage durations on DNA quality in specimens with ultrasound processing to investigate the optimal storage time for molecular tests. METHODS: Forty specimens of breast biopsy were collected and paraffin specimens were prepared by ultrasonography. These specimens were divided into four groups based on their storage periods: < 1 year, 1-3 years, > 3-5 years, and > 5 years, which contained 10 cases in each group. Paraffin specimens were sliced; each slice was 3 μm thick; 10-15 slices were taken, and DNA was extracted. The mass concentration of DNA was examined by Nanophotometer N60 ultra-micro spectrophotometer and Qubit 4.0 fluorometer. The purity of the DNA was analyzed by the ratio of A260/A280. DNA fragment integrity was measured by capillary electrophoresis (Qsep 100) to evaluate the quality of the DNA fragments. RESULTS AND CONCLUSION: The mean values of A260/A280 in the four groups were between 1.8 and 2.0, meeting the requirements of tests, without significant differences. The mean values of DNA mass concentration (Qubit concentration) were 30.39, 14.33, 2.52, and 1.95 ng/μL, respectively. The mean values of the N/Q were 6.48, 14.18, 24.56, and 29.86. The mean values of DNA were: 5.64, 1.76, 1.24, and 0.80. The percentage of large DNA fragments averaged 56.08%, 17.72%, 12.68%, and 7.90%. Moreover, the Ct values of the internal control detected by PCR were 15.32, 17.09, 18.39, and 21.24. The three other groups exhibited significantly lower DNA concentration, higher N/Q ratios, decreased DNA quality and percentage of large fragments, and increased values of Ct, compared with the group of within 1 year of storage (P < 0.05). The experimental results suggested that for novel ultrasound processed biopsy specimens, we should prioritize samples stored within 1 year for molecular testing. Samples stored within 3 years can also meet the requirements of second-generation sequencing and other tests. Samples stored within 5 years can only be attempted to carry out PCR. Samples stored for more than 5 years were not recommended to carry out molecular tests. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) represent a cornerstone of modern therapeutic strategies, targeting a wide array of diseases with unprecedented specificity. Developing and validating bioanalytical methods for mAbs are critical to ensuring their efficacy, safety, and regulatory compliance. This review discusses the recent advances in bioanalytical technologies and the persistent challenges faced in the field. Significant progress has been made in chromatographic techniques, mass spectrometry, and capillary electrophoresis, enhancing the analysis of mAbs in terms of resolution, sensitivity, and throughput. Innovations such as high-resolution mass spectrometry and automated capillary electrophoresis systems have transformed the mAb characterization landscape. However, challenges remain, primarily due to the complex nature of mAbs, which necessitates the development of susceptible and specific methods to determine their pharmacokinetic profiles and detect low-abundance impurities accurately. Moreover, the stringent and evolving regulatory requirements demand robust and reproducible analytical methods. This review emphasizes the need for continuous technological innovation and adaptation to address these challenges effectively. [ABSTRACT FROM AUTHOR]

The strict suppression of telomerase activity (TA) in terminally differentiated human cells causes a shortening of the chromosome ends after each cell division. This tumor suppression surveillance mechanism is associated with a limited number of cell divisions known as Hayflick limit. Here we present an optimized protocol for measuring TA that combines a fluorescently labeled bait primer and polymerase chain reaction (PCR) amplification with analytical capillary electrophoresis (CE) to achieve a detection limit of one telomerase-positive cell per ten thousand negative cells. In research laboratories today, a broad panel of TRAP assay protocols enables the assessment of the immortality of newly generated cell lines or the unambiguous evaluation of the reprogramming of induced pluripotent stem cells (iPSCs). The present f-TRAP protocol, optimized for routine use, enables fast ad hoc application for single measurements up to a high throughput of mass samples using a triplicate approach of different lysate concentrations. Final CE analysis facilitates standardized data processing and storage for documentation of results and could make f-TRAP a useful assay in research and clinical oncology.

Background: Application of electromembrane extraction coupled with capillary electrophoresis was studied for ofloxacin extraction from plasma samples. Methods: Ofloxacin migrated from acidic plasma samples through a thin layer of 1-octanol immobilized in the pores of a porous hollow fiber wall into a 10 µL acidic aqueous acceptor solution located inside the lumen of the fiber. Results: Under optimum conditions influencing electromigration (i.e., 20 min of the operation time, stirring speed of 750 rpm, donor phase pH at 4.0, acceptor pH at 3.0, and applied voltage of 30 V across the supported liquid membrane), ofloxacin was extracted from plasma samples with an enrichment factor of 100-fold corresponding to extraction percent of 24%. The calibration curve showed acceptable linearity in the range of 0.2-7.0 µg.mL-1 (R=0.9993). The limits of detection and quantification of 0.05 and 0.2 µg.mL-1 were obtained, respectively. The inter- and intra-day precision and accuracy were obtained below 8.65%. Conclusion: The validated method was successfully processed for the ofloxacin determination in the plasma samples of patients under ofloxacin therapy. There were no interfering peaks which indicates the great selectivity of the developed method.

A fast, simple and cost-effective capillary electrophoresis (CE) method was developed for caffeine determination in different beverages (energy drinks, soft drinks). Because caffeine is neutral from electrophoretic point of view and migrates with the electroosmotic flow (EOF), micellar electrokinetic chromatography (MEKC) was chosen as the separation method. The optimum separation conditions consisted of 25 mM sodium tetraborate, 100 mM sodium dodecyl sulphate, pH 9.30, 20°C temperature, 20 kV voltage, 50 mbar/sec hydrodynamic injection, UV detection at 270 nm. Employing the optimized conditions caffeine was quantified in less than 3 minutes. The analytical performances of the method were verified in terms of accuracy, linearity, limit of detection and quantification, precision and robustness. The method was applied also to detect caffeine in coffee and tea. The advantage of MEKC over other analytical methods, particularly compared with the more frequently used HPLC methods, lies in its lower operating costs and higher environmental friendliness.