Rotational and translational diffusion of biomolecules in complex liquids and HeLa cells.

Diffusive motion accompanies many physical and biological processes. The Stokes-Sutherland-Einstein relation for the translational diffusion coefficient, DT, agrees with experiments done in simple fluids but fails for complex fluids. Moreover, the interdependence between DT and rotational diffusion coefficient, DR, also deviates in complex fluids from the classical relation of DT/DR = 4r2/3 known in simple fluids. Makuch et al. Soft Matter, 2020, 16, 114-124 presented a generalization of the classical translational and rotational diffusion theory for complex fluids. In this work, we empirically verify this model based on simultaneous translational and rotational diffusion measurements. We use fluorescently stained cowpea chlorotic mottle virus (CCMV) particles as monodisperse probes and aqueous polyethylene glycol (PEG) solutions as a model complex fluid. The theory and experimental data obtained from fluorescence correlation spectroscopy (FCS) measurements agreed. Finally, we used the same model and analyzed the diffusion of Yo-Pro-1 stained large ribosomal subunits (LSU) in the cytoplasm and nucleus of living HeLa cells.

Epidemiology of Streptococcus pyogenes upper respiratory tract infections in Poland (2003-2017).

Streptococcus pyogenes (group A Streptococcus, GAS) is a major human pathogen and causes every year over 600 millions upper respiratory tract onfections worldwide. Untreated or repeated infections may lead to post-infectional sequelae such as rheumatic heart disease, a major cause of GAS-mediated mortality. There is no comprehensive, longitudinal analysis of the M type distribution of upper respiratory tract strains isolated in Poland. Single reports describe rather their antibiotic resistance patterns or focus on the invasive isolates. Our goal was to analyse the clonal structure of the upper respiratory tract GAS isolated over multiple years in Poland. Our analysis revealed a clonal structure similar to the ones observed in high-income countries, with M1, M12, M89, M28, and M77 serotypes constituting over 80% of GAS strains. The M77 serotype is a major carrier of erythromycin resistance and is more often correlated with upper respiratory tract infections than other serotypes.

Cellular Uptake of Bevacizumab in Cervical and Breast Cancer Cells Revealed by Single-Molecule Spectroscopy.

Bevacizumab is a biological drug that is now extensively studied in clinics against various types of cancer. Although bevacizumab's action is preferably extracellular, there are reports suggesting its internalization into cancer cells, consequently decreasing its therapeutic potential. Here we are solving this issue by applying fluorescence correlation spectroscopy in living cells. We tracked single molecules of fluorescent bevacizumab in MDA-MB-231 and HeLa cells and proved that mobility measurements bring significant added value to standard imaging techniques. We confirmed the presence of the drug in intracellular vesicles. Additionally, we explicitly excluded the presence of a free cytosolic fraction of bevacizumab in both studied cell types. Extracellular and intracellular concentrations of the drug were measured, giving a partition coefficient on the order of 10-5, comparable with the spontaneous uptake of biologically inert nanoparticles. Our work presents how techniques and models developed for physics can answer biological questions.

Electrochemically Synthesized Polyacrylamide Gel and Core-Shell Nanoparticles for 3D Cell Culture Formation.

Biocompatible polyacrylamide gel and core-shell nanoparticles (NPs) were synthesized using a one-step electrochemically initiated gelation. Constant-potential electrochemical decomposing of ammonium persulfate initiated the copolymerization of N-isopropyl acrylamide, methacrylic acid, and N,N'-methylenebisacrylamide monomers. This decomposing potential and monomers' concentrations were optimized to prepare gel NPs and thin gel film-grafted core-shell NPs. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging confirmed the gel NP formation. The lyophilized gel NPs and core-shell NPs were applied to support the three-dimensional (3D) cell culture. In all, core-shell NPs provided superior support for complex 3D tissue structures.

Entanglement of polymer chains in hypertonic medium enhances the delivery of DNA and other biomacromolecules into cells.

HYPOTHESIS: Most experimental procedures applied in modern biology involve cargo delivering into cells. One of the ways to cargo introduction is osmotic-mediated intracellular vesicle swelling. However, its widespread use was hindered due to cargo size (<10 nm) and cell-type-related restrictions. We addressed the issue of the composition of colloidal loading solution to enhance the efficiency of cellular delivery. EXPERIMENTS: We examined the effectiveness of colloidal loading solutions of varied compositions, including various types and sizes of polymers building osmotic pressure. We used confocal imaging coupled with fluorescence correlation spectroscopy to evaluate the introduction of polymers, proteins, nanoparticles, and DNA plasmids (cargos of sizes 1-175 nm) to cells representing eight cell lines: cancer, normal, epithelial, and mesenchymal ones. FINDINGS: We found that cellular delivery effectiveness strongly correlates with the size and concentration of osmotic pressure building polymers and not with the high value of the osmotic pressure itself. We show that polymer solutions at the entangled regime of concentrations enhance the delivery of large biomacromolecules even of size 200 nm (DNA plasmids) into cells, including MDA-MB-231 cells - so far resistant to the osmotic procedure. We show that the colloid loading medium based on entangled polymer chains is a versatile cargo delivery tool for molecular biology.

Quantitative analysis of biochemical processes in living cells at a single-molecule level: a case of olaparib-PARP1 (DNA repair protein) interactions.

Quantitative description of biochemical processes inside living cells and at single-molecule levels remains a challenge at the forefront of modern instrumentation and spectroscopy. This paper demonstrates such single-cell, single-molecule analyses performed to study the mechanism of action of olaparib - an up-to-date, FDA-approved drug for germline-BRCA mutated metastatic breast cancer. We characterized complexes formed with PARPi-FL - fluorescent analog of olaparib in vitro and in cancer cells using the advanced fluorescent-based method: Fluorescence Correlation Spectroscopy (FCS) combined with a length-scale dependent cytoplasmic/nucleoplasmic viscosity model. We determined in vitro olaparib-PARP1 equilibrium constant (6.06 × 108 mol L-1). In the cell nucleus, we distinguished three states of olaparib: freely diffusing drug (24%), olaparib-PARP1 complex (50%), and olaparib-PARP1-RNA complex (26%). We show olaparib accumulation in 3D spheroids, where intracellular concentration is twofold higher than in 2D cells. Moreover, olaparib concentration was tenfold higher (506 nmol L-1vs. 57 nmol L-1) in cervical cancer (BRCA1 high abundance) than in breast cancer cells (BRCA1 low abundance) but with a lower toxic effect. Thus we confirmed that the amount of BRCA1 protein in the cells is a better predictor of the therapeutic effect of olaparib than its penetration into cancer tissue. Our single-molecule and single-cell approach give a new perspective of drug action in living cells. FCS provides a detailed in vivo insight, valuable in drug development and targeting.

Cellular delivery of dinucleotides by conjugation with small molecules: targeting translation initiation for anticancer applications.

Targeting cap-dependent translation initiation is one of the experimental approaches that could lead to the development of novel anti-cancer therapies. Synthetic dinucleoside 5',5'-triphosphates cap analogs are potent antagonists of eukaryotic translation initiation factor 4E (eIF4E) in vitro and could counteract elevated levels of eIF4E in cancer cells; however, transformation of these compounds into therapeutic agents remains challenging - they do not easily penetrate into cells and are susceptible to enzymatic cleavage. Here, we tested the potential of several small molecule ligands - folic acid, biotin, glucose, and cholesterol - to deliver both hydrolyzable and cleavage-resistant cap analogs into cells. A broad structure-activity relationship (SAR) study using model fluorescent probes and cap-ligand conjugates showed that cholesterol greatly facilitates uptake of cap analogs without disturbing the interactions with eIF4E. The most potent cholesterol conjugate identified showed apoptosis-mediated cytotoxicity towards cancer cells.