Bovine Serum Albumin as a Platform for Designing Biologically Active Nanocarriers-Experimental and Computational Studies.

Due to the specificity of their structure, protein systems are adapted to carry various ligands. The structure of many proteins potentially allows for two types of immobilization of a therapeutic agent, either on the outer surface of the protein or within the protein structure. The existence of two active sites in BSA's structure, the so-called Sudlow I and II, was confirmed. The conducted research involved determining the effectiveness of BSA as a potential carrier of 5-fluorouracil (5FU). 5-fluorouracil is a broad-spectrum anticancer drug targeting solid tumors. The research was carried out to estimate the physicochemical properties of the system using complementary measurement techniques. The optimization of the complex formation conditions made it possible to obtain significant correlations between the form of the drug and the effective localization of the active substance in the structure of the protein molecule. The presence of two amino groups in the 5FU structure contributes to the deprotonation of the molecule at high pH values (pH > 8) and the transition to the anionic form (AN1 and AN3). To investigate the binding affinity of the tautomeric form with BSA, UV-vis absorption, fluorescence quenching, zeta potential, QCM-D, and CD spectroscopic studies were performed. The experimental research was supported by molecular dynamics (MD) simulations and molecular docking. The simulations confirm the potential location of 5FU tautomers inside the BSA structure and on its surface.

Raman Research on Bleomycin-Induced DNA Strand Breaks and Repair Processes in Living Cells.

Even several thousands of DNA lesions are induced in one cell within one day. DNA damage may lead to mutations, formation of chromosomal aberrations, or cellular death. A particularly cytotoxic type of DNA damage is single- and double-strand breaks (SSBs and DSBs, respectively). In this work, we followed DNA conformational transitions induced by the disruption of DNA backbone. Conformational changes of chromatin in living cells were induced by a bleomycin (BLM), an anticancer drug, which generates SSBs and DSBs. Raman micro-spectroscopy enabled to observe chemical changes at the level of single cell and to collect hyperspectral images of molecular structure and composition with sub-micrometer resolution. We applied multivariate data analysis methods to extract key information from registered data, particularly to probe DNA conformational changes. Applied methodology enabled to track conformational transition from B-DNA to A-DNA upon cellular response to BLM treatment. Additionally, increased expression of proteins within the cell nucleus resulting from the activation of repair processes was demonstrated. The ongoing DNA repair process under the BLM action was also confirmed with confocal laser scanning fluorescent microscopy.

Raman Microspectroscopic Analysis of Selenium Bioaccumulation by Green Alga Chlorella vulgaris.

Selenium (Se) is an element with many commercial applications as well as an essential micronutrient. Dietary Se has antioxidant properties and it is known to play a role in cancer prevention. However, the general population often suffers from Se deficiency. Green algae, such as Chlorella vulgaris, cultivated in Se-enriched environment may be used as a food supplement to provide adequate levels of Se. We used Raman microspectroscopy (RS) for fast, reliable, and non-destructive measurement of Se concentration in living algal cells. We employed inductively coupled plasma-mass spectrometry as a reference method to RS and we found a substantial correlation between the Raman signal intensity at 252 cm-1 and total Se concentration in the studied cells. We used RS to assess the uptake of Se by living and inactivated algae and demonstrated the necessity of active cellular transport for Se accumulation. Additionally, we observed the intracellular Se being transformed into an insoluble elemental form, which we further supported by the energy-dispersive X-ray spectroscopy imaging.

Substrate Stiffness Mediates Formation of Novel Cytoskeletal Structures in Fibroblasts during Cell-Microspheres Interaction.

It is well known that living cells interact mechanically with their microenvironment. Many basic cell functions, like migration, proliferation, gene expression, and differentiation, are influenced by external forces exerted on the cell. That is why it is extremely important to study how mechanical properties of the culture substrate influence the cellular molecular regulatory pathways. Optical microscopy is one of the most common experimental method used to visualize and study cellular processes. Confocal microscopy allows to observe changes in the 3D organization of the cytoskeleton in response to a precise mechanical stimulus applied with, for example, a bead trapped with optical tweezers. Optical tweezers-based method (OT) is a microrheological technique which employs a focused laser beam and polystyrene or latex beads to study mechanical properties of biological systems. Latex beads, functionalized with a specific protein, can interact with proteins located on the surface of the cellular membrane. Such interaction can significantly affect the cell's behavior. In this work, we demonstrate that beads alone, placed on the cell surface, significantly change the architecture of actin, microtubule, and intermediate filaments. We also show that the observed molecular response to such stimulus depends on the duration of the cell-bead interaction. Application of cytoskeletal drugs: cytochalasin D, jasplakinolide, and docetaxel, abrogates remodeling effects of the cytoskeleton. More important, when cells are plated on elastic substrates, which mimic the mechanical properties of physiological cellular environment, we observe formation of novel, "cup-like" structures formed by the microtubule cytoskeleton upon interaction with latex beads. These results provide new insights into the function of the microtubule cytoskeleton. Based on these results, we conclude that rigidity of the substrate significantly affects the cellular processes related to every component of the cytoskeleton, especially their architecture.