Antibody–drug conjugates (ADCs) have revolutionized chemotherapy, allowing it to shift from systemic cytotoxic chemotherapy to specific targeting of cells expressing tumor antigens. ADCs thus offer increased therapeutic efficacy and lower toxicity compared to conventional chemotherapy. Bioconjugation of a highly active therapeutic agent to an antibody is one of the key steps in producing ADCs. The most advanced ADC technologies are based on the production of stoichiometrically homogeneous bioconjugates. Enzymatic modification of antibodies using microbial transglutaminase (mTGase) enables the production of biopharmaceuticals with standardized pharmacokinetic, physicochemical, and therapeutic properties. However, side reactions catalyzed by free mTGase in solution reduce the yield and purity of ADCs. In this study, we evaluated the efficiency of various approaches to mTGase immobilization: streptavidin–biotin interaction, the use of an epoxy-activated polymer, and self-immobilization of mTGase. The highest bioconjugation activity was observed when using streptavidin-modified magnetic particles and biotinylated mTGase. However, mTGase self-immobilization on AH-Sepharose 4B or immobilization using epoxy-activated agarose ensures comparable efficiency in enzymatic antibody modification and is the most rational route from the perspective of process scalability. We believe that the proposed approaches are of high practical significance, opening up avenues for optimizing enzymatic bioconjugation and scaling up the production of stoichiometric ADCs for targeted cancer immunotherapy.
The necropolis of St. Sophia Cathedral in Veliky Novgorod, Russia, as noted in chronicle sources and cathedral inventories, contains the burials of five princes from the Rurikid dynasty and two princesses from their families. To clarify the genetic ancestry of two of the buried individuals, whose remains preserved the skeletal elements required for genetic analysis, DNA extracted from the remains was sequenced. Genetic analysis has revealed that the male individual from the sarcophagus near the southern wall of the Nativity side-chapel carries the Y-chromosomal haplogroup N1a and can be identified with high probability as Mstislav Rostislavich the Brave. Analysis of DNA extracted from female skeletal remains from the reliquary shrine in the Martyrius Porch revealed no close kinship with any of the known genomes of Rurikid dynasty members. A sculptural facial reconstruction based on the skull of Prince Mstislav Rostislavich and a digital facial approximation based on the cranium of the female individual were performed.
Cancer is the second leading cause of death worldwide, after cardiovascular disorders. One of the main constraints of anticancer agents is their limited water solubility, which complicates preclinical in vivo studies. In this work, we propose a versatile approach for stabilizing hydrophobic antitumor agents using liposomes based on natural phospholipids. The efficacy of the method is demonstrated for hydrophobic 1-tridecyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, a promising tetrahydroisoquinoline derivative with proven antitumor activity against lymphoid and epithelial cancer cells. We show that the liposomal formulation ensures drug stability in aqueous media. Modification of the liposome surface with a HER2-specific non-immunoglobulin module strongly improved targeting efficiency. The targeting index differed markedly between cells with low versus high HER2 expression levels (3.02 and 18.98, respectively), demonstrating that the targeted liposomal formulation exerts a substantially enhanced cytotoxicity against HER2-overexpressing cells. The proposed liposomal platform can also be utilized to deliver other hydrophobic anticancer drugs.
Synthetic cell design is one of the daunting challenges of modern biology. While the genome synthesis problem has already been solved technologically, the understanding of the molecular composition of the intracellular environment required for initiating artificial life still remains elusive. One of the mechanisms of self-organization in prebiotic systems is the oscillatory processes (oscillations) arising from feedback regulation. Such dynamics are inherent to glycolysis as the metabolic core of the cell. The objective of this study was to reconstruct the oscillatory glycolytic chain and investigate its behavior upon confinement to microcompartments and upon interaction with nucleic acids. We used the Saccharomyces cerevisiae extract as a model and demonstrated the presence of fluctuations in the concentrations of glycolytic metabolites: ATP, NADH, hexose-6-phosphates, glyceraldehyde-3-phosphate, 3-phosphoglycerate, and phosphoenolpyruvate (PEP). Moreover, the PEP and pyruvate concentrations had a dramatic effect on the stability of oscillations. We also demonstrated that the reduction of the system volume accompanying the encapsulation into a microchip leads to a more rapid onset of oscillations. Furthermore, synthetic mRNA and DNA were found to reduce the amplitude and period of oscillations in a translation-independent manner. Hence, this study demonstrates the feasibility of reconstructing metabolic oscillations in microvolumes and reveals the effect of nucleic acids on the glycolytic dynamics, which is important for the development of protocellular systems.
The nucleolus is a key cellular compartment responsible for the major steps of ribosomal subunit biogenesis. Detailed characterization of the nucleolar proteome is essential for understanding the molecular mechanisms underlying ribosome biogenesis and may help identify novel regulators of this process. However, the nucleolar proteome, and the protein composition of early ribosomal subunit precursors in particular, remain poorly defined, largely because of the methodological inconsistencies and conflicting findings across published studies. Here, we aimed to establish a standardized approach that could be used to identify novel factors involved in the early stages of ribosome biogenesis using an optimized stepwise nucleolar extraction protocol, followed by sucrose density gradient fractionation and mass spectrometry analysis. This optimized approach allowed us to enrich for proteins directly engaged in early biogenesis steps, while depleting non-specific nucleolar “background” components. A total of 529 unique proteins were identified across two biological replicates. Gene Ontology (GO) analysis revealed significant enrichment of proteins annotated as ribosome biogenesis factors, including those directly involved in ribosomal subunit assembly and rRNA processing. A cross-referencing of our dataset against 188 known components of pre-ribosomal complexes represented in the PDB yielded 84 overlapping proteins. Early biogenesis factors prevailed in this group (70 out of 84), supporting the idea of the enrichment of the analyzed fraction with early pre-ribosomal complexes. The resulting protein dataset will facilitate the identification of novel factors governing the poorly understood aspects of human ribosome biogenesis.
