Capillary Gel Electrophoresis Research Articles

Sensitive and rapid analysis of plasmid DNA topology isoforms by capillary gel electrophoresis with laser induced fluorescence in uncoated capillary.

The work describes the use of SYBR Gold to improve the detection sensitivity of plasmid DNA topoisomers by capillary gel electrophoresis with laser induced fluorescence in an uncoated capillary. The impact of different dyes, including ethidium bromide, SYBR Green and SYBR Gold, was compared based on detection and separation of DNA plasmid topoisomers. Use of SYBR Gold enabled improvement of detection sensitivity by 15-fold while maintaining good separation resolution of the different topoisomers. The baseline dropped with the use SYBR Gold but was overcome by the employment of a capillary with longer ineffective length (40 vs. 20cm). Separation resolution and reproducibility were impacted by the concentration of SYBR Gold and hydroxypropyl methylcellulose. With the use of a short capillary (10cm effective length and 50cm total length), fast separations of supercoiled, linear, open circular, and other isoforms were accomplished within 8min. Appropriate capillary cleaning with 0.1M sodium hydroxide/0.1M hydrochloric acid and capillary storage with 0.1M hydrochloric acid ensured good separation reproducibility of 217 runs during an extended period of use.

Urea supplementation improves mRNA in vitro transcription by decreasing both shorter and longer RNA byproducts

ABSTRACT The current letter to the editor describes the presence of RNA byproducts in small-scale in vitro transcription (IVT) reactions as evaluated by capillary gel electrophoresis, asymmetric flow field flow fractionation, immunoblotting, cell-free translation assays, and in IFN reporter cells. We compare standard T7 RNA polymerase (RNAP) based IVT reactions to two recently described protocols employing either urea supplementation or using the VSW3 RNAP. Our results indicate that urea supplementation yields considerably less RNA byproducts and positively affects the overall number of full-length transcripts. In contrast, VSW3 IVT reactions demonstrated a low yield and generated a higher fraction of truncated transcripts. Lastly, both urea mRNA and VSW3 mRNA elicited considerably less IFN responses after transfection in mouse macrophages.

Comprehensive Impurity Profiling of mRNA: Evaluating Current Technologies and Advanced Analytical Techniques.

In vitro transcription (IVT) of mRNA is a versatile platform for a broad range of biotechnological applications. Its rapid, scalable, and cost-effective production makes it a compelling choice for the development of mRNA-based cancer therapies and vaccines against infectious diseases. The impurities generated during mRNA production can potentially impact the safety and efficacy of mRNA therapeutics, but their structural complexity has not been investigated in detail yet. This study pioneers a comprehensive profiling of IVT mRNA impurities, integrating current technologies with innovative analytical tools. We have developed highly reproducible, efficient, and stability-indicating ion-pair reversed-phase liquid chromatography and capillary gel electrophoresis methods to determine the purity of mRNA from different suppliers. Furthermore, we introduced the applicability of microcapillary electrophoresis for high-throughput (<1.5 min analysis time per sample) mRNA impurity profiling. Our findings revealed that impurities are mainly attributed to mRNA variants with different poly(A) tail lengths due to aborted additions or partial hydrolysis and the presence of double-stranded mRNA (dsRNA) byproducts, particularly the dsRNA 3'-loop back form. We also implemented mass photometry and native mass spectrometry for the characterization of mRNA and its related product impurities. Mass photometry enabled the determination of the number of nucleotides of different mRNAs with high accuracy as well as the detection of their size variants [i.e., aggregates and partial and/or total absence of the poly(A) tail], thus providing valuable information on mRNA identity and integrity. In addition, native mass spectrometry provided insights into mRNA intact mass, heterogeneity, and important sequence features such as poly(A) tail length and distribution. This study highlights the existing bottlenecks and opportunities for improvement in the analytical characterization of IVT mRNA, thus contributing to the refinement and streamlining of mRNA production, paving the way for continued advancements in biotechnological applications.

Targeting the glycan epitope type I N-acetyllactosamine enables immunodepletion of human pluripotent stem cells from early differentiated cells.

Cell surface biomarkers are fundamental for specific characterization of human pluripotent stem cells (hPSCs). Importantly, they can be applied for hPSC enrichment and/or purification but also to remove potentially teratoma-forming hPSCs from differentiated populations before clinical application. Several specific markers for hPSCs are glycoconjugates comprising the glycosphingolipid (GSL)-based glycans SSEA-3 and SSEA-4. We applied an analytical approach based on multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection to quantitatively assess the GSL glycome of human embryonic stem cells and human induced pluripotent stem cells as well as during early stages of differentiation into mesoderm, endoderm, and ectoderm. Thereby, we identified the GSL lacto-N-tetraosylceramide (Lc4-Cer, Galβ1-3GlcNAcβ1-3Galβ1-4Glc-Cer), which comprises a terminal type 1 LacNAc (T1LN) structure (Galβ1-3GlcNAc), to be rapidly decreased upon onset of differentiation. Using a specific antibody, we could confirm a decline of T1LN-terminating glycans during the first four days of differentiation by live-cell staining and subsequent flow cytometry. We could further separate T1LN-positive and T1LN-negative cells out of a mixed population of pluripotent and differentiated cells by magnetic activated cell sorting. Notably, not only the T1LN-positive but also the T1LN-negative population was positive for SSEA-3, SSEA-4, and SSEA-5 while expression of nuclear pluripotency markers OCT4 and NANOG was highly reduced in the T1LN-negative population, exclusively. Our findings suggest T1LN as a pluripotent stem cell-specific glycan epitope that is more rapidly down-regulated upon differentiation than SSEA-3, SSEA-4, and SSEA-5.

Genetic characterization of historic Norwegian Erwinia amylovora isolates by SSR-genotyping

AbstractErwinia amylovora, the causative agent of fire blight of pome fruits and other rosaceous plants belongs to the group of regulated quarantine pests. The aim of this work was to characterize the populations of E. amylovora in Norway and their geographical distribution. A total of 238 E. amylovora isolates recovered from symptomatic host plants in Norway between 1986 and 2004 were genotyped by means of a short sequence repeat (SSR) marker (ATTACAGA) on plasmid pEa29. The SSR region was amplified and amplicon size determined using fluorescent labelling and rapid, automated capillary gel electrophoresis. All isolates contained the pEa29 plasmid harbouring the investigated marker. In total, ten genotypes were identified, of which two were detected only once. The number of repeats varied from 3 to 13, with 43% of the isolates containing five repeats. Of 17 isolates collected between 1986 and 1991, all but one contained five repeats, whereas more variation was observed in isolates from the period 2000 to 2004. Most of the isolates (80%) originated from Cotoneaster bullatus, hence no relationship between genotype of the isolate and host species that it was isolated from could be detected. This historic data suggests multiple introductions of E. amylovora to Norway.

Anastrozole and Tamoxifen Impact on IgG Glycome Composition Dynamics in Luminal A and Luminal B Breast Cancers.

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.

Changes in Milk Protein Functionality at Low Temperatures and Rennet Concentrations

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 &lt; 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 &lt; 0.05) effect on decreasing κ-CN, and breakdown increased at higher levels of rennet usage. There was a significant (p &lt; 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 &lt; 0.05) decrease in the foaming ability and foaming stability and a significant (p &lt; 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.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qampmini) with a preprogrammed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces high false positive results. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies) and fast results (less than 120 minutes), which would be suitable for field testing applications. Our high detection sensitivity platform provides fast and accurate results in 120 minutes without doing DNA/RNA extraction.

Synthesis of fluorescent 9-(4-aminoaniline)-acridine for highly specific and rapid detection of human serum albumin by fluorescence-capillary gel electrophoresis

In clinical practice, many diseases can lead to changes in serum albumin concentration (HSA) in patients. Accurate detection of HSA concentration is of great significance for disease diagnosis. Based on this, this study designed and synthesized 9-(4-amino-aniline)-acridine (AAA) as a fluorescent probe. By laser induction and capillary gel electrophoresis (CGE), a new rapid and highly specific HSA detection method based on fluorescence-CGE was established. Various experimental control factors were investigated, and the optimal experimental conditions were determined as follows: the running buffer was H3PO4–KH2PO4 (pH = 2.45, 15.0 mmol L−1), the separation voltage was 30 kV, and the experimental temperature was 25 °C. The sample solution injected 10 s with hydrodynamic mode (3.43×103 Pa), HSA could be directly determined by fluorescence-CGE method. The linear range was 0.10–1.0 μg L−1, the detection limit was 0.012 μg L−1, the relative standard deviation (RSD) was less than 0.30%. This method can be used for the determination of real HSA samples. In addition, in the presence of various biological macromolecules, small molecules, ions and ethanol, the accurate detection of HSA by fluorescence-CGE method will not be affected, suggesting that this method has a high specificity for HSA. The rapid and highly specific fluorescence-CGE method of HSA constructed in this study provides a new way to detect HSA, which is of great significance for the diagnosis of clinical diseases.

Calr Variant Allele Frequency in Essential Thrombocythemia: Molecular Associations and Impact on Disease Phenotype and Outcome

Background. A mutation in calreticulin ( CALRm) is found in 30-35% of patients (pts) with Essential Thrombocythemia (ET). There are 2 main CALRm mutation types, Type 1/Type 1 like (T1) a 52bp deletion, and Type 2/Type 2 like (T2) a 5bp insertion. Other atypical mutations (T3) occur in a minority of pts. Recent study ( Guglielmelli P et al, BCJ 2023) in pts with myelofibrosis (MF) showed that higher CALR variant allele frequency (VAF) was associated with anemia at diagnosis and during FU, and to features of more advanced disease (CD34 + cell counts, ASXL1 mutation (mut), &amp;gt;2 mutated myeloid genes, and shorter leukocytosis-free survival. Aim. To evaluate whether the CALRm VAF in pts with ET was associated with predefined major clinical outcomes: evolution to MF, transformation to acute leukemia (AML), thrombosis, major bleeding, and survival (OS). Patients and Methods. Diagnosis of ET was strictly according to 2023 WHO and ICC to avoid mis-inclusion of prefibrotic MF. CALRm VAF was determined by capillary gel electrophoresis as the ratio (%) of areas under the curve of CALRm/ CALRm+ CALRwt. A panel of 45 myeloid neoplasm-associated genes was sequenced by NGS. Results. A total of 281 CALRm ET pts were identified from CRIMM (Florence, I), Quebec MPN Research Group centers' (Canada) and Mayo Clinic (Rochester, US) databases; 152 (54%), 101 (36%) and 28 (10%) were T1, T2 and T3 CALRm, respectively. Overall FU was 8.6y (0.3-39.4), median survival was not reached. Rate of death at 10y, 20y and 30y was 8%, 15% and 25%. MF transformation occurred in 50 pts (18%), AML in 2 (0.7%), 25 pts (8.9%) died. A major thrombotic event before or at ET diagnosis occurred in 17 (6.0 %) pts (14 arterial and 3 venous), whereas 28 pts (10.0%) had &amp;gt;1 major thrombosis during follow-up (53.6% arterial, 46.4% venous). Major bleeding occurred in 19 pts (6.8%; 16 at or before diagnosis). Ascertainment of correlation of CALRm VAF with predefined outcomes by continuous variable analysis (ROC curve) highlighted a significant correlation of VAF &amp;gt;60% with MF-free survival (MFS), with HR of 2.85 (95%CI, 1.4-5.6; P= .002. Fig. 1A). On the contrary, there was no impact of CALRm VAF as continuous variable on AML, thrombosis, bleeding and OS. We therefore used a threshold of &amp;gt;60% to compare main clinical and hematologic characteristics at diagnosis and outcomes during FU in pts categorized as CALR-high and CALR-low. Of all pts, 21 (7.5%) harbored a VAF &amp;gt;60%; of these, 52%, 19% and 29% were T1, T2, T3, respectively, compared to 54%, 37%, 8% of CALR-low (P= .008). We found no difference in age, gender, constitutional symptoms, splenomegaly, arterial and venous thrombosis at diagnosis and FU, IPSET revised risk stratification, and bleeding events. Hemoglobin was lower (12.8 g/dL (8.4-14.0) versus 13.6 (10.1-16.0); P=.02), while leukocytes and platelets did not differ. The proportion of pts with additional mutations in myeloid genes was significantly greater in CALR-high pts compared to CALR-low (66.7% vs 30.2%; P=0.01), as it was the proportion of pts with &amp;gt;2 mut myeloid genes (25% vs 11.6%, P=0.04). During FU, 52% of CALR-high pts (n=11) transformed to MF compared to 15% (n=39) of CALR-low (P&amp;lt;.0001); AML and death not differ. Anemia (&amp;lt;10g/dL) during the FU developed in 69% of CALR-high pts vs 18%, leukocytosis (&amp;gt;15x10 9/L) in 46% vs 8% and splenomegaly (greater than 5 cm from LCM) in 50% vs 12% (P&amp;lt;.0001 for all). The HR for anemia-free survival was 2.87 (95%CI, 1.3-6.3), for leukocytosis HR 3.71 (95%CI, 1.3-10.5) and splenomegaly HR 3.40 (95%CI, 1.2-9.2), all P&amp;lt;.01. MFS was significantly shorter in T1 (HR 2.0; P=0.04) or T3 (HR 2.7; P=0.03) using T2 as reference. Finally, we compared C ALR-mut, JAK2V617F mut and triple-negative (TN) ET pts. ROC analysis indicated a JAK2VAF &amp;gt;35% ( JAK2-high) as the best cutoff for shorter MFS. As in Fig. 1B, the MFS survival of CALR-high and JAK2-high was superimposable, and significantly shorter that in CALR/JAK2-low and TN pts. Conclusions. A CALR VAF &amp;gt;60% in pts with ET is associated with greater risk and shorter time to MF progression, development of anemia and splenomegaly. Similar findings in JAK2-high pts reinforce that that accumulation of mutated CALR and JAK2 alleles, evident since diagnosis, is detrimental for evolution to post-ET myelofibrosis. Longitudinal studies after diagnosis might provide further information about the kinetics of allele accumulation.

Streamlining the Detection of FLT3 Internal Tandem Duplication (FLT3-ITD) Using the Cepheid Genexpert® Automated System

Objectives: The development of targeted therapies for Acute Myeloid leukemia (AML) demands fast and reliable diagnostic tests. Specifically, FMS-like tyrosine kinase 3 (FLT3-ITD), comprising nearly 25% of all AML cases, is the most common targetable mutation in AML. Current diagnosis of FLT3-ITD by PCR involves numerous laborious steps, with bench-top steps involving time-consuming whole blood processing, DNA isolation, plate-based PCR and subsequent fragment analysis by capillary electrophoresis. Consequently this adds significant overhead, time-consuming personnel training, and numerous steps which could lead to errors, all of which increase the resource burden and time to result. The objective of this study was to create an automated FLT3-ITD diagnostic prototype that is faster, requires less hands-on time that can detect all clinically relevant ITD lengths. Methods: We have developed a simplified FLT3-ITD prototype assay using the Cepheid GeneXpert® automated platform for FLT3-ITD detection. The prototype substantially streamlines the process, using a less than 30 minutes off-board time to prepare a universal blood lysate that is compatible with all Cepheid Hematology Oncology products. Once the sample is loaded into the cartridge and inserted into the instrument, the cartridge performs automated DNA isolation, purification, and qPCR. A Taqman-based probe for ABL is utilized in the qPCR step as a PCR control to ensure that the blood processing and qPCR was successful and avoid wasted downstream steps. Quantitative fragment analysis can easily be performed on the PCR product using capillary electrophoresis (CE) for precise allelic ratio (AR) callout. Additionally, the PCR product is compatible be run on a bioanalyzer or standard Agarose Gel-Electrophoresis if available. Results: The developed automated FLT3-ITD diagnostic prototype demonstrated significant improvements over the existing methodologies with substantially reduced time to result and simplicity in use. To assess the sensitivity of this prototype, we used MV4-11 cancer cells harboring a 30 bp ITD and CRISPR-CAS engineered human cell lines with 3 bp, 21 bp, and 75 bp FLT3 ITD insertions, comprising the most commonly detected ITD lengths spiked into EDTA blood as a test material. For these ITD's, we observed sensitivity and linearity down to 0.01-0.02 by CE for the ITDs tested. Importantly, the prototype detected all lengths of ITD mutations with inserts from 3bp to 300 bp using plasmid test materials. Conclusion: We have successfully developed a novel FLT3-ITD diagnostic prototype that has several major advantages over existing methodologies. The prototype significantly reduces the time and hands-on effort required for analysis, streamlining the diagnostic process for same day results. The larger sample volume used allows improved sensitivity to ensure accurate detection, even at low allelic frequencies. Additionally, the prototype requires fewer reagents in that sample prep and qPCR are performed in a single cartridge and run hands-free. Downstream analysis of amplified products can be easily performed and validated using capillary electrophoresis, bioanalyzer, or gel electrophoresis. Finally, because this prototype uses the same universal blood lysate as other AML tests, it has the potential to improve future diagnostic therapeutic interventions in the clinic.

A duplex droplet digital PCR assay for absolute quantification and characterization of long self-amplifying RNA

Self-amplifying messenger ribonucleic acid (saRNA) provides extended expression of genes of interest by encoding an alphavirus-derived RNA replicase and thus is 2–3 times larger than conventional messenger RNA. However, quality assessment of long RNA transcripts is challenging using standard techniques. Here, we utilized a multiplex droplet digital polymerase chain reaction (ddPCR) assay to assess the quality of saRNA produced from an in vitro transcription reaction and the replication kinetics in human cell lines. Using the one-step reverse transcription ddPCR, we show that an in vitro transcription generates 50–60% full-length saRNA transcripts. However, we note that the two-step reverse transcription ddPCR assay results in a 20% decrease from results obtained using the one-step and confirmed using capillary gel electrophoresis. Additionally, we provided three formulas that differ in the level of stringency and assumptions made to calculate the fraction of intact saRNA. Using ddPCR, we also showed that subgenomic transcripts of saRNA were 19-to-108-fold higher than genomic transcripts at different hours post-transfection of mammalian cells in copies. Therefore, we demonstrate that multiplex ddPCR is well suited for quality assessment of long RNA and replication kinetics of saRNA based on absolute quantification.

The monosialoganglioside GM1a protects against complement attack

The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface, ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, we here generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell death in vitro. Glycolipid-analysis by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF) allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenously administered GM1a integrated into the plasma membrane of trophoblasts, substantially increased binding of complement factor H (FH) and was sufficient to protect the cells from complement attack and cell death. GM1a treatment also rescued human endothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantly reduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). This study demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators.

Enhancement of recombinant adeno-associated virus activity by improved stoichiometry and homogeneity of capsid protein assembly

Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1+ VP2)/VP3= 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1+ VP2)/VP3= 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. Invitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.

Purification free N-glycan analysis by capillary zone electrophoresis: Hunt for the lost glycans

Capillary gel electrophoresis is a widely used method for rapid separation of fluorophore labeled carbohydrates. Even though, many publications conferred about this popular technique, no report yet investigated the possible sample losses during the purification process of the fluorophore labeling reaction mixture. In the present work, normal polarity capillary zone electrophoresis separation mode was applied to take advantage of the opposite migration directions of the electroosmotic flow and the negatively charged sample components using Tris-hexanoic acid running buffer at basic pH. For purification free oligosaccharide analysis, the separation parameters were designed in such a way that the triple charged labeling reagent of aminopyrenetrisulfonate (APTS) could not enter the separation capillary in contrary to the labeled sample components of interest, therefore, the APTS did not have to be removed before analysis. The method was used to show electrophoretic profile differences possibly caused by the cleanup process that was immediately apparent by comparing the electropherograms of the purified and non-purified APTS labeled maltooligosaccharides. Furthermore, qualitative and quantitative N-glycosylation profile alterations were revealed during CZE separation of the fluorophore labeling reaction mixtures before and after purification along with the analysis of the consecutively used washing solutions for the well characterized standard glycoproteins of IgG, ribonuclease B and fetuin.

Characterization and development of 14 microsatellite markers for mountain stewartia (Stewartia ovata): an uncommon, Appalachian-endemic member of the tea family

ABSTRACT Mountain stewartia [Stewartia ovata (Cav.) Weatherby] is a rare, endemic understory tree native to the southeastern U.S.A. While found only in microsites across southeastern Appalachia, the species distribution spans from northern Alabama and Georgia to southern Kentucky and Virginia. However, most occurrences have been recorded in Tennessee. While recent distribution surveys and habitat suitability modeling conducted for this species have improved our understanding of the environmental parameters that constitute its fundamental niche, the ecological profile of S. ovata remains poorly understood. Consequently, its rarity has raised concerns as to the long-term resilience of this species in the wild. Faced with these challenges, the assessment of existing genetic diversity in S. ovata through genetically informative molecular resources is critical to understanding the adaptive potential and ecological resilience of the species. Microsatellites are cost-efficient molecular tools capable of addressing these concerns by elucidating trends in population structure, population demography, and inbreeding. To increase the available genetic resources for S. ovata, we screened 105 microsatellite loci for their suitability to assess these population features using capillary gel electrophoresis. The utility of these markers was evaluated by assessing polymorphism information content (PIC) and population statistics, which yielded 14 highly polymorphic microsatellite markers. These markers displayed an average PIC of 0.695 (PIC range of 0.538–0.851), which supports their ability to capture fine-scale genetic diversity within the species. We also report a low average observed heterozygosity of 0.375 (versus an expected heterozygosity of 0.645) among screened loci, which reinforces the need for broader genetic diversity assessments within this species.

Capillary electrophoresis methods for determining the IVT mRNA critical quality attributes of size and purity.

One result of the Covid-19 pandemic has been an increased awareness of IVT mRNA vaccines and the speed at which they can be produced for disease outbreaks. Currently the only approved IVT mRNA therapeutics are the Covid-19 vaccines, however IVT mRNA is being investigated for other non-Covid prophylactic vaccines, therapeutics, and therapeutic vaccines. IVT mRNAs can range from less than 100 nt in length to longer than 9,000 nt. When producing any IVT mRNA, quality control of the IVT mRNA is essential to ensure that the product is the correct length and does not contain truncated or degraded mRNA. Capillary gel electrophoresis provides high resolution separations of the IVT mRNA of interest from the degraded or truncated impurities allowing for the accurate purity assessment of IVT mRNA. Specialized capillary electrophoresis gels can also be used to provide analysis of purified poly(A) tails enabling characterization of multiple Critical Quality Attributes on a single platform. Here we describe methods for the purity assessment of IVT mRNAs through either 6,000 or 9,000 nt and determination of poly(A) tail length using different capillary gel electrophoresis methods.

A US-based national surveillance study for the susceptibility and epidemiology of Clostridioides difficile isolates with special reference to ridinilazole: 2020-2021.

We have previously reported on the susceptibility and epidemiology of Clostridioides difficile isolates from six geographically dispersed medical centers in the United States. This current survey was conducted with isolates collected in 2020-2021 from six geographically dispersed medical centers in the United States, with specific attention to susceptibility to ridinilazole as well as nine comparators. C. difficile isolates or stools from patients with C. difficile antibiotic-associated diarrhea were collected and referred to a central laboratory. After species confirmation of 300 isolates at the central laboratory, antibiotic susceptibilities were determined by the agar dilution method [M11-A9, Clinical and Laboratory Standards Institute (CLSI)] against the 10 agents. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Ridinilazole had a minimum inhibitory concentration (MIC) 90 of 0.25 mcg/mL, and no isolate had an MIC greater than 0.5 mcg/mL. In comparison, fidaxomicin had an MIC 90 of 0.5 mcg/mL. The vancomycin MIC 90 was 2 mcg/mL with a 0.7% resistance rate [both CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria]. The metronidazole MIC 90 was 1 mcg/mL, with none resistant by CLSI criteria, and a 0.3% resistance rate by EUCAST criteria. Among the 50 different ribotypes isolated in the survey, the most common ribotype was 014-020 (14.0%) followed by 106 (10.3%), 027 (10%), 002 (8%), and 078-126 (4.3%). Ridinilazole maintained activity against all ribotypes and all strains resistant to any other agent tested. Ridinilazole showed excellent in vitro activity against C. difficile isolates collected between 2020 and 2021 in the United States, independent of ribotype.

Determination of Genetic Diversity of Some Upland and Sea Island Cotton Genotypes Using High-Resolution Capillary Electrophoresis Gel

Cotton is a major source of natural fibre for the global textile industry and is also an important oilseed crop. Cotton fibre is the main source of textiles, the seeds are used for oil and the remaining bagasse is used as high-protein animal feed. In addition, cotton’s so-called short fibre is used in more than 50 industries. Cotton breeding is generally based on crossing the best yielding and fibre quality genotypes. However, cotton breeding programmes are negatively affected by the narrow genetic diversity of varieties. It is for this reason that the identification of genetic resources and the disclosure of genetic diversity are so important. Here, the genetic diversity of G. hirsutum and G. barbadense genotypes was determined using high-resolution capillary gel electrophoresis. Using 19 EST-SSR markers, a total of 47 genotypes were screened. The PIC values of the markers used ranged from 0.268 to 0.889. The mean PIC value was calculated to be 0.603. In terms of clustering, PCoA and population structure analyses gave similar results, and the genotypes could be divided into three main groups. Genetic admixture with G. hirsutum was found in some genotypes of the G. barbadense species. We can conclude that (i) the EST-SSR markers used in this study are effective in the determination of genetic diversity, (ii) the genetic diversity should be increased through the collection of genetic resources and (iii) the genetic EST-SSR markers in this study should be considered in breeding programmes by using them in QTL studies.

A single-nucleotide resolution capillary gel electrophoresis workflow for poly(A) tail characterization in the development of mRNA therapeutics and vaccines

The 3′ poly(A) tail is an important component of messenger RNA (mRNA). The length of the poly(A) tail has direct impact on the stability and translation efficiency of the mRNA molecule and is therefore considered to be a critical quality attribute (CQA) of mRNA-based therapeutics and vaccines. Various analytical methods have been developed to monitor this CQA. Methods like ion-pair reversed-phase liquid chromatography (IPRP-LC) can be used to quantify the percentage of mRNA with poly(A) tail but fail to provide further information on the actual length of poly(A). High-resolution methods such as liquid chromatography coupled with mass spectrometry (LC-MS) or next generation sequencing (NGS) can separate poly(A) tail length by one nucleotide (n/n + 1 resolution) but are complicated to implement for release testing of manufactured mRNA. In this study, a workflow utilizing capillary gel electrophoresis (CGE) for characterizing the poly(A) tail length of mRNA was developed. The CGE method demonstrated resolution comparable with the LC-MS method. With UV detection and the addition of poly(A) length markers, this method can provide poly(A) tail length information and can also provide quantitation of each poly(A) length, making it a suitable release method to monitor the CQA of poly(A) tail length.