Search Results (48)

Background: FLT3 mutation testing is a key ancillary molecular assay for diagnosing and managing patients with acute myeloid leukemia (AML), including assessing the utility of FLT3 inhibitors during induction chemotherapy. FLT3 PCR utilizing fluorescently labeled primers and capillary electrophoresis readout is the most used technique for the rapid detection of FLT3 internal tandem duplications (ITDs) (including very small ITDs) and tyrosine kinase domain (TKD) mutations. However, capillary electrophoresis (CE) is a relatively lengthy and technically demanding result readout mode that could potentially be replaced by faster alternatives. Methods: Here, we describe the validation of a modified FLT3 PCR assay that uses the Agilent 4200 TapeStation platform for result readouts. This platform generates quantifiable electropherograms and gel images in under two minutes and at a low cost. We validated its ability to detect FLT3-ITD and -TKD mutations using 22 and 18 previously tested patient samples, respectively. Results: The TapeStation 4200 instrument is 100% sensitive, specific, and highly reproducible for post-PCR fragment analysis in detecting FLT3-ITD (greater than 15 bp in size) and TKD mutations in AML patients. Its results are nearly 100% concordant with those obtained from our previously validated NGS and PAGE methods. However, the limitation of this readout mode is its inability to reliably detect FLT3-ITDs smaller than 15 bp in size. Conclusions: Given the widespread use of TapeStation instruments in molecular diagnostics laboratories as part of next-generation sequencing (NGS) workflows, this modified assay is well-suited as a companion test for rapid NGS platforms to detect larger FLT3-ITDs, which are NGS often miscalledor under-called by the NGS bioinformatics algorithms. However, it is not suitable for use as a standalone assay, as it is unable to reliably detect very short FLT3-ITDs. Full article

Background/Objective: Oligodeoxynucleotides (ODNs) containing base-labile modifications such as N4-acetyldeoxycytidine (4acC), N6-acetyladenosine (6acA), N2-acetylguanosine (2acG), and N4-methyoxycarbonyldeoxycytidine (4mcC) are highly challenging to synthesize because standard ODN synthesis methods require deprotection and cleavage under strongly basic and nucleophilic conditions, and there is a lack of ideal alternative methods to solve the problem. The objective of this work is to explore the capability of the recently developed 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) method for the incorporation of multiple 4acC modifications into a single ODN molecule and the feasibility of using the method for the incorporation of the 6acA, 2acG and 4mcC modifications into ODNs. Methods: The sensitive ODNs were synthesized on an automated solid phase synthesizer using the Dmoc group as the linker and the methyl Dmoc (meDmoc) group for the protection of the exo-amino groups of nucleobases. Deprotection and cleavage were achieved under non-nucleophilic and weakly basic conditions. Results: The 4acC, 6acA, 2acG, and 4mcC were all found to be stable under the mild ODN deprotection and cleavage conditions. Up to four 4acC modifications were able to be incorporated into a single 19-mer ODN molecule. ODNs containing the 6acA, 2acG, and 4mcC modifications were also successfully synthesized. The ODNs were characterized using RP HPLC, capillary electrophoresis, gel electrophoresis and MALDI MS. Conclusions: Among the modified nucleotides, 4acC has been found in nature and proven beneficial to DNA duplex stability. A method for the synthesis of ODNs containing multiple 4acC modifications is expected to find applications in biological studies involving 4acC. Although 6acA, 2acG, and 4mcC have not been found in nature, a synthetic route to ODNs containing them is expected to facilitate projects aimed at studying their biophysical properties as well as their potential for antisense, RNAi, CRISPR, and mRNA therapeutic applications. Full article

Affinity gel electrophoresis was introduced about 50 years ago. Proteins interact with a ligand immobilized in the support. Specific interactions cause a decrease in electrophoretic mobility. The presence of a free ligand, competing with an immobilized ligand, restores electrophoretic mobility. In affinity capillary electrophoresis, the ligand is mobile, and its interaction with a specific protein changes the mobility of the protein–ligand complex. This review mostly focuses on gel affinity electrophoresis. The theoretical basis of this technique, ligand immobilization strategies, and principles for determination of ligand affinity are addressed. Factors affecting specificity and strength of interactions are discussed, in particular, the structure of the affinity matrix, pH, temperature, hydrostatic pressure, solvent, co-solvents, electric field, and other physico-chemical conditions. Capillary affinity electrophoresis principles and uses are also briefly introduced. Affinity gel electrophoresis can be used for qualitative and quantitative purposes. This includes detection of specific proteins in complex media, investigation of specific interactions, protein heterogeneity, molecular and genetic polymorphism, estimation of dissociation constants of protein–ligand complexes, and conformational stability of binding sites. Future prospects, in particular for screening of engineered mutants and potential new drugs, coupling to other analytical methods, and ultra-microtechnological developments, are addressed in light of trends and renewal of this old technique. Full article

Capillary electrophoresis based on laser-induced fluorescence (CE-LIF) plays an important role in the analysis of nucleic acids. However, the commercial CE-LIF is not only quite expensive but also inflexible, thus hindering its widespread use in the lab. Herein, we proposed a compact, low-cost, and flexible CE-LIF system. We also investigated its stability by separating the DNA ladders. Experiments demonstrated that the relative standard error of the relative fluorescence intensity and migration time was lower than 6.2% and 1.1%, respectively. The aperture size of the light source illuminating the capillary can affect the separation performance. Smaller apertures offer higher resolution length for the adjacent DNA fragments but may reduce the number of theoretical plates. Various fluorescent dyes (e.g., SYBR Green I, Gel Green, EvaGreen) can be employed in the self-built system. The limit of detection of dsDNA was as low as 0.05 ng/μL. The working range for DNA was 0.05 ng/μL~10 ng/μL. Finally, we have successfully separated the PCR products of the target gene of Porphyromonas gingivalis and Candida albicans in the home-built CE system. Such a robust CE-LIF system is easy to assemble in the lab. The total cost of the assembled CE system did not exceed 1100 USD. We believe this work can advance the application of CE and hope it will facilitate the easy assembly of flexible CE instruments in labs. Full article

Background/Objectives: The purpose of the current study was to compare the methylation of five regions of the CpG island of MLH1 with the presence of microsatellite instability (MSI) in colorectal cancer (CRC) patients. Methods: The study analyzed 138 CRC tumor samples. DNA extraction was performed, followed by bisulfite conversion. MLH1 gene methylation was assessed by methylation-specific PCR (MS-PCR), and the resulting fragments were analyzed using polyacrylamide gels. MSI was evaluated using multiplex PCR, and the fragments were run through capillary electrophoresis. R studio (v4.4.1) and SPSS (v29.0) software were used for the statistical analysis, and values of p < 0.05 were considered statistically significant. Results: The study showed 75.4% unmethylated, 21% partially methylated, and 3.6% fully methylated samples, with region A frequently methylated. MSI was observed in 7.2% of cases (MSI-H: 5.8%, MSI-L: 1.4%). BAT-26 was the most unstable marker. A significant difference between MLH1 methylation and MSI-H (p < 0.01) was identified, but there was no relationship with specific MLH1 regions. Conclusions: No differences were identified when analyzing specific methylation regions in relation to MSI. This study is the first to describe MSI frequency in Mexican patients regardless of age. Full article

The diagnostic utility and reference intervals for blood studies in Aldabra giant tortoises (Aldabrachelys gigantea) are not well described. Capillary zone electrophoresis (CZE) has been evaluated in non-mammalian vertebrates and shows a higher fraction resolution and less overall variation in results than agarose gel electrophoresis. To date, the investigation of novel biomarkers has been limited in reptiles. MRP-126, a calgranulin homologue in reptiles, has not been evaluated for its diagnostic potential in tortoises. The goals of this study were to establish preliminary reference intervals for CZE protein electrophoresis and to examine MRP-126 as a potential biomarker of inflammation in Aldabra giant tortoises. In 27 clinically healthy tortoises, CZE resolved seven protein fractions. In tortoises with an inflammatory or infectious disease process (n = 4), MRP-126 concentrations and CZE fractions did not consistently increase or were abnormal. To strengthen the understanding of the diagnostic value of CZE and MRP-126 concentration in this species, future studies should evaluate a larger sample set inclusive of repeated measures of clinically abnormal tortoises as well as CZE and MRP-126 variations in regard to additional health conditions, age, sex, season, and geographic location. Full article

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 E_a requirement), while 55 °C was optimal for the lower-molecular-weight light chain and non-glycosylated heavy chain pair (lower E_a requirement). Future research directions and possible applications are also proposed. Full article

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. Full article

Quantifying M-proteins is an important part of diagnosing and monitoring patients with monoclonal gammopathies. Historically, laboratories use one of two methods to accomplish this. The splice method utilizes a perpendicular drop on each side of the M-protein on the electrophoretogram. In contrast, the skim method applies a tangent skimming line connecting the points above the polyclonal background. In this study, we compared the bias between these two methods across two different instruments (Helena SPIFE 3000 and Sebia Capillarys 3) in 118 patients. First, we compared the splice technique on both instruments and observed a significant average bias of 58.3% (slope = 1.437, y-intercept = 0.76, and r = 0.9682). We next compared the splice technique on the SPIFE 3000 to the skim technique on the Capillarys 3 and observed an average bias of only −2.10% (slope = 1.363, y-intercept = −1.98, and r = 0.9716), although there was significant scatter along the line of best fit. Lastly, we compared splice vs. skim on the Capillarys 3 and observed an average bias of −38.2% (slope = 0.947, y-intercept = −2.65, and r = 0.9686). Based on these results, care should be taken when switching instruments or integration techniques to ensure consistent monitoring of patients. Full article

The present study aimed to conduct a comparative investigation of the biological properties of phenolic and polysaccharide extracts obtained using an ultrasound-assisted technique from Aloe vera gel and their effects on each stage of the wound healing process in in vitro experimental models. HPLC analysis showed that the phenolic extract contained aloin, ferulic, and caffeic acid, as well as quercetin dihydrate, as major compounds. Capillary zone electrophoresis indicated the prevalence of mannose and glucose in the polysaccharide extract. Cell culture testing revealed the anti-inflammatory properties of the phenolic extract at a concentration of 0.25 mg/mL through significant inhibition of pro-inflammatory cytokines—up to 28% TNF-α and 11% IL-8 secretion—in inflamed THP-1-derived macrophages, while a pro-inflammatory effect was observed at 0.5 mg/mL. The phenolic extract induced 18% stimulation of L929 fibroblast proliferation at a concentration of 0.5 mg/mL, enhanced the cell migration rate by 20%, and increased collagen type I synthesis by 18%. Moreover, the phenolic extract exhibited superior antioxidant properties by scavenging free DPPH (IC₅₀ of 2.50 mg/mL) and ABTS (16.47 mM TE/g) radicals, and 46% inhibition of intracellular reactive oxygen species (ROS) production was achieved. The polysaccharide extract demonstrated a greater increase in collagen synthesis up to 25%, as well as antibacterial activity against Staphylococcus aureus with a bacteriostatic effect at 25 mg/mL and a bactericidal one at 50 mg/mL. All these findings indicate that the phenolic extract might be more beneficial in formulations intended for the initial phases of wound healing, such as inflammation and proliferation, while the polysaccharide extract could be more suitable for use during the remodeling stage. Moreover, they might be combined with other biomaterials, acting as efficient dressings with anti-inflammatory, antioxidant, and antibacterial properties for rapid recovery of chronic wounds. Full article

Clostridioides difficile is a complex of anaerobic bacteria responsible for the epidemics of post-antibiotic diarrhea as one of the examples of CDI (Clostridioides difficile infection). As many as 70% of cases concern hospitalized patients, particularly those in intensive care units. Ribotyping is one of the most common methods for differentiating bacterial strains. The purpose of this work was to show the effectiveness of the gel electrophoresis-based PCR ribotyping method and the Webribo database for typing C. difficile isolates, including the hypervirulent 027 ribotype. DNA samples extracted from 69 C. difficile strains with previously marked genotypes were included in this study. PCR was performed using 16S–23S primers, and capillary gel electrophoresis was performed on the Applied Biosystem 3130xl Genetic Analyzer. The Webribo database was applied for ribotype assignment. Out of 69 samples, 48 belonged to already known ribotypes, 13 represented new ribotypes and 8 was indicated as similar to the existing ones, having some differences. Capillary gel electrophoresis-based PCR is an effective method for the differentiation of C. difficile ribotypes and can be recognized as a very useful tool in epidemiological studies, while the Webribo database is a useful and an accessible database for a quick analysis of C. difficile ribotypes. Full article

Botrytis cinerea is a well-known pathogen that can be challenging to control in crops, such as wine grapes. To adapt to the increasing problems of climate change and strain resistance, it is important to find new methods to detect Botrytis cinerea and differentiate strains. These methods include strain differentiation and classification by simple sequence repeats (SSRs) and early detection of the fungus by qPCR. Various strains were analysed using SSR markers and either agarose gel electrophoresis or capillary sequencing via PCR. A sensitive qPCR method was refined to achieve an early detection method for the pathogen. The results demonstrate promising ways to distinguish between strains using both agarose gel electrophoresis and capillary sequencing as well as to detect infection before it becomes visible on grapes. This can be used to further understand and analyse different Botrytis cinerea strain characteristics such as laccase activity, regional or annual effects. The early detection method can be used to better prepare growers for an impending infection so that targeted efforts can be made. Full article

This study examines the intricate relationship between protein glycosylation dynamics and therapeutic responses in Luminal A and Luminal B breast cancer subtypes, focusing on anastrozole and tamoxifen impacts. The present methods inadequately monitor and forecast patient reactions to these treatments, leaving individuals vulnerable to the potential adverse effects of these medications. This research investigated glycan structural changes by following patients for up to 9 months. The protocol involved a series of automated steps including IgG isolation, protein denaturation, glycan labelling, purification, and final analysis using capillary gel electrophoresis with laser-induced fluorescence. The results suggested the significant role of glycan modifications in breast cancer progression, revealing distinctive trends in how anastrozole and tamoxifen elicit varied responses. The findings indicate anastrozole’s association with reduced sialylation and increased core fucosylation, while tamoxifen correlated with increased sialylation and decreased core fucosylation. These observations suggest potential immunomodulatory effects: anastrozole possibly reducing inflammation and tamoxifen impacting immune-mediated cytotoxicity. This study strongly emphasizes the importance of considering specific glycan traits to comprehend the dynamic mechanisms driving breast cancer progression and the effects of targeted therapies. The nuanced differences observed in glycan modifications between these two treatments underscore the necessity for further comprehensive research aimed at thoroughly evaluating the long-term implications and therapeutic efficacy for breast cancer patients. Full article

This study aimed to evaluate the influence of low-concentration rennet on the chemical, rheological characteristics, and protein fractions of skim milk (SM) at 4 ± 1 °C. Skimmed milk (SM) was divided into four lots of 500 mL, and diluted rennet (1:10,000) was added at different levels at 4 ± 1 °C. The treatments included control (no rennet), T1 (0.001 mL/rennet), T2 (0.01 mL rennet), and T3 (0.1 mL rennet) treatments, which were incubated for 24 h. The sampling was performed at 0, 1, 2, 6, 12, and 24 h, and the SM after incubation time was heated to 73 °C/16 s to denature the rennet enzyme. Skim milk samples (SMS) (control and rennet-added samples) were evaluated for proximate composition, capillary gel electrophoresis (CGE), hydrodynamic diameter, zeta potential, and rheology at 0, 1, 2, 6, 12, and 24 h. Foaming ability, foaming stability, water-holding capacity (WHC), oil emulsifying activity (OEA), and emulsion stability (ES) were performed at 0, 12, and 24 h of incubation time. There was a significant (p < 0.05) increase in non-proteins by 0.50% and in non-casein nitrogen by 0.81% as incubation progressed. The results showed that aggregation or curd was not formed during storage time. The CGE data indicated that increasing the rennet concentration had a significant (p < 0.05) effect on decreasing κ-CN, and breakdown increased at higher levels of rennet usage. There was a significant (p < 0.05) increase in the hydrodynamic diameter and a decrease in the zeta potential values in rennet-added samples at the end of the incubation time (24 h). The rheological results showed no changes in the storage modulus (G′), loss modulus (G″), or viscosity values. Increasing the rennet amount and storage time led to a significant (p < 0.05) decrease in the foaming ability and foaming stability and a significant (p < 0.05) increase in the oil emulsifying activity and emulsion stability of rennet-added SMS. This study concluded that milk protein functionality can be changed without aggregating or curd formation, and rennet milk can be processed. Full article

Due to complex polyploid, sugarcane whole genome sequencing and characterization lag far behind other crops. PCR-based DNA markers are a viable low-cost option to evaluate genetic diversity and verify genotypes. In this study, the 5S ribosomal RNA-intergenic spacer (ITS) of 171 accessions of Saccharum spp. and Tripidium spp. was dissected, including 30 accessions of S. officinarum, 71 of S. spontaneum, 17 of S. robustum, 25 of S. barberi, 13 of S. sinense, 2 of S. edule, 5 sugarcane cultivars (Saccharum spp. hybrids), 6 of Tripidium spp. (formally Erianthus spp.), and 2 of unknown species. The ITS spacers were amplified from 10 ng of the leaf DNA of each accession with the universal PCR primers PI and PII. The PCR-amplified spacers (amplicons) were analyzed by both agarose gel and capillary electrophoresis (CE). While agarose gel electrophoresis revealed five banding patterns, a total of 42 polymorphic amplicons, ranging from 60 to 506 bp, were detected by CE. Three amplicons, 234-, 235-, and 236-bp in size, were amplified from all accessions of six Saccharum species, except for three S. robustum accessions (Molokai 5573, NG 57-054, and NG 77-235) that lacked the 236-bp amplicon. The 234-, 235-, 236-bp banding pattern found in S. spontaneum was less consistent than other Saccharum species, sometimes missing a few but not all the bands in this region. An amplicon of 61-bp was amplified only from the sugarcane hybrid varieties. The PI/PII patterns indicated diversity and subpopulations within Saccharum, which could potentially be used in Breeding. Moreover, all Saccharum-specific amplicons were mostly absent in Tripidium spp. accessions, which produced 405-bp and 406-bp amplicons, and any pattern of the exceptions indicated misidentification. The T. bengalense accession Kalimpong had a unique CE-banding pattern that was different from all other accessions. Although the clustering pattern of the 42 amplicons only discriminated at the genus level, these amplicons helped identify nine misclassified accessions. This study further demonstrates that these PI/PII amplicons could be particularly useful markers for breeders at sugarcane field stations to quickly confirm and discriminate among the accessions of germplasm collections. Full article