BIA Separations (Slovenia)

Melanoma-associated antigens (MAGEs) are cancer-testis antigens (CTAs) aberrantly expressed in multiple cancer types, including hepatocellular carcinoma (HCC), and associated with aggressive phenotypes. Although MAGE proteins are widely studied as cancer immunotherapy targets, their roles in HCC and the regulation of their expression during liver pathogenesis in mouse models, including dietary effects, remain poorly understood. We analyzed Mage gene expression in liver tissues from 78 C3H/HeJ mice with chronic diet-induced obesity. While type I MAGE genes are frequently expressed in human HCC, we found no evidence of their expression in mouse liver tumors, suggesting species-specific regulation. In contrast, type II Maged2, previously reported to be upregulated in human HCC, was significantly increased in mouse liver tumors. Analysis of human HCC samples from The Cancer Genome Atlas (TCGA) database confirmed MAGED2 upregulation and its association with patient prognosis. Together, these findings identify MAGED2 as a conserved marker of liver cancer in both humans and mice and emphasize the importance of cross-species comparative approaches for selecting appropriate models and accurately interpreting results, particularly for CTAs, which often evolved recently and in a species-specific manner.

ABSTRACT In recent years, European countries have intensified efforts to control or limit the spread of invasive freshwater crayfish and the crayfish plague pathogen Aphanomyces astaci , while working to conserve native species such as the noble crayfish ( Astacus astacus ). Although crayfish shed relatively low amounts of DNA into their environment, environmental DNA (eDNA) approaches have proven effective for detecting their presence. A range of protocols and equipment is currently used in eDNA‐based monitoring of freshwater crayfish. To evaluate how methodological variation influences detection accuracy, we conducted the first European interlaboratory ring test using eDNA to detect A. astacus , the invasive signal crayfish Pacifastacus leniusculus , a chronic carrier of A. astaci , and the pathogen itself. The aim is to harmonize monitoring methods for crayfish and disease surveillance across laboratories. Eleven teams from thirteen European countries participated, each using its own equipment and protocols to collect and filter water from indoor tanks and outdoor ponds where the presence of A. astacus and P. leniusculus had been experimentally manipulated, as well as from a natural lake containing a P. leniusculus population. The resulting samples were analyzed in each team's laboratory. Despite methodological differences, all teams successfully detected DNA from both crayfish species in indoor tanks (3–10 crayfish/m 3 ). However, detection accuracy declined in outdoor ponds where crayfish density was an order of magnitude lower (0.32 crayfish/m 3 ). Detection was most variable for A. astaci , likely due to its very low prevalence in the host stock. Our study demonstrates the challenges of achieving consistent eDNA results across laboratories and highlights the importance of interlaboratory comparisons. It also underscores the need to identify sources of variability and error, an essential step toward developing robust and standardized protocols. This multinational intercalibration and exchange of knowledge improved methodology and enhanced reliability in crayfish detection.

This study addresses the residual thrust concept in hybrid thrusters. In this phenomenon, thrust is generated by the gasification of heated fuel post-firing, particularly after long-duration maneuvers. Residual thrust is significantly lower than nominal thrust. However, it poses challenges for attitude control and orbit design in small satellite missions because it can cause postural instability. The residual thrust in hybrid thrusters has not been evaluated to date. This study represents a pioneering study in this field. We conducted a simulation incorporating the rocket equation, unsteady heat conduction equation, fuel pyrolysis characteristics, and radiation from the nozzle and injector to the fuel. Subsequently, we estimated the residual thrust after firing. This estimation technique was applied to a single-port fuel. Simulation results indicate that the residual thrust can exceed 0.3 N for up to 6 s after firing if the fuel regression rate is 0.01 mm/s immediately after firing. The results showed that the residual thrust effect is greater for smaller regression rates. Thus, a thruster for slowly regressing fuel can generate more significant residual thrust than that for regressing fuel quickly, even if the same ΔV is designed.

Lipid nanoparticles (LNPs) are the most established platform for delivery of mRNA payloads. Their tunability and streamlined manufacturing facilitated an unprecedentedly rapid scale-up during the COVID-19 pandemic. However, being multicomponent, complex systems also poses a challenge of controlling their quality and safety. Analytical checkpoints need to be established to characterize LNPs on multiple levels during development and commercialization. This Perspective centres on the chemical reactivity and purity of mRNA-LNP components, which need to be addressed as raw materials, drug substance, excipients, and the fully formed and stored product. Herein, we describe such appropriate orthogonal analytics to design and analyse LNP formulations. For such novel biopharmaceuticals, better controls that go beyond the current analytical workflow and address the nuanced chemical stability, which helps ensure reproducibility, stability and safety, need to be established.

Traditional transcriptomic studies often overlook the complex heterogeneity of skeletal muscle, as they typically isolate RNA from mixed muscle fibre and cell populations, resulting in an averaged transcriptomic profile that obscures fibre type-specific differences. This study assessed the potential of the recently developed Xenium platform for high-resolution spatial transcriptomic analysis of human skeletal muscle histological sections. Human vastus lateralis muscle samples from two individuals were analysed using the Xenium platform and Human Multi-Tissue and Cancer Panel targeting 377 genes complemented by staining of successive sections for Myosin Heavy Chain isoforms to differentiate between type 1 and type 2 muscle fibres. Manual segmentation of muscle fibres allowed accurate comparisons of transcript densities across fibre types and subcellular regions, overcoming limitations in the platform's automated segmentation. The analysis revealed higher transcript density in type 1 fibres, particularly in nuclear and perinuclear areas, and identified 191 out of 377 genes with differential expression between muscle fibres and perimysium. Genes such as PROX1, S100A1, LGR5, ACTA2, and LPL exhibited higher expression in type 1 fibres, whereas PEBP4, CAVIN1, GATM, and PVALB in type 2 fibres. We demonstrated that the Xenium platform is capable of high-resolution spatial in situ transcriptomic analysis of skeletal muscle histological sections. This study demonstrates that, with manual segmentation, the Xenium platform effectively performs fibre type-specific transcriptomic analysis, providing new insights into skeletal muscle biology.

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The Vmax values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min−1), while the Km values (0.310 ± 0.075 g∙L−1) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L−1). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity.

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

Purification of high-quality plasmid DNA in large quantities is a crucial step in its production for therapeutic use and is usually conducted by different chromatographic techniques. Large-scale preparations require the optimization of yield and homogeneity, while maximizing removal of contaminants and preserving molecular integrity. The advantages of Convective Interaction Media® (CIM®) monolith stationary phases, including low backpressure, fast separation of macromolecules, and flow-rate-independent resolution qualified them to be used effectively in separation of plasmid DNA on laboratory as well as on large scale. A development and scale-up of plasmid DNA downstream process based on chromatographic monoliths is described and discussed below. Special emphasis is put on the introduction of process analytical technology principles and tools for optimization and control of a downstream process.

The paper describes how borate-containing BGEs modify ζ-potential and so EOF in bare fused silica capillaries. This surface modification can be used to suppress EOF and improve the separation performance of CZE including capillary sieving electrophoresis (CSE). Boric acid forms complexes with polysaccharides used as sieving matrices in CSE and other compounds containing hydroxyl groups, including polyol bases such as Tris, triethanolamine, and Bis-Tris propane. High concentration of boric acid in BGEs leads to a strong interaction of boric acid with the silica surface of the capillary wall and this suppresses or even completely eliminates ζ-potential and EOF. Using a polyol base with several charge-carrying amino groups, such as Bis-Tris propane, can actually reverse EOF. We demonstrate the use of various borate-containing BGEs in bare fused silica capillaries for size-separation of DNA fragments, size-separation of proteins by SDS CSE, and also by CZE in the absence of any sieving matrix.