Living cells are crowded with dynamic distributions of macromolecules and organelles that influence protein diffusion, molecular transport, biochemical reactions, and protein assembly. Here, we test the hypothesis that the diffusion of single molecules deviates from Brownian motion as described by the Stokes-Einstein model in a manner that depends on the viscosity range, the chemical structure of both the diffusing species and the crowding agents, and the spatio-temporal resolution of the employed analytical methods. Our size-dependent fluorescent probes are rhodamine-110, quantum dots, enhanced green fluorescent proteins (EGFP), and mCerulean3-linker-mCitrine FRET probes with various linker length and flexibility. Using fluorescence correlation spectroscopy (FCS), we investigated the translational diffusion of structure-dependent fluorescent probes, at the single-molecule level, in homogeneous (glycerol) and heterogeneous (Ficoll-70) solutions as a function of the bulk viscosity. Complementary rotational diffusion studies using time-resolved anisotropy enable us to assess weak interactions in crowded and viscous environments. Overall, our results show negative deviation from the Stokes-Einstein model in a fluorophore- and environment-dependent manner. In addition, the deviation between the FCS-measured hydrodynamic radius of the FRET probes in a buffer at room temperature and the molecular-weight based estimate (Perrin equation) as the number of the amino acid residues in the linker increases. These studies are essential for quantitative biophysics using fluorescence- and diffusion-based studies of protein-protein interactions and biomolecular transport in living cells.
Most studies using intrinsic NAD(P)H as biomarkers for energy metabolism and mitochondrial anomalies have been conducted in routine two-dimensional (2D) cell culture formats. Cellular metabolism and cell behavior, however, can be significantly different in 2D cultures from that in vivo. As a result, there are emerging interests in integrating noninvasive, quantitative imaging techniques of NAD(P)H with in vivo-like threedimensional (3D) models. The overall features and metabolic responses of the murine breast cancer cells line 4T1 in 2D cultures were compared with those in 3D collagen matrix using integrated optical micro-spectroscopy. The metabolic responses to two novel compounds, MD1 and TPPBr, that target metabolism by disrupting monocarboxylate transporters or oxidative phosphorylation (OXPHOS), respectively, were investigated using two-photon fluorescence lifetime imaging microscopy (2P-FLIM) of intracellular NAD(P)H in 2D and 3D cultures. 4T1 cells exhibit distinct behaviors in a collagenous 3D matrix from those in 2D culture, forming anastomosing multicellular networks and spherical acini in 3D culture, as opposed to simple flattened epithelial plaques in 2D culture. The cellular NAD(P)H in 3D collagen matrix exhibits a longer fluorescence lifetime as compared with 2D culture, which is attributed to an enhanced population of enzyme-bound NAD(P)H in the 3D culture. TPPBr induces mitochondrial hyperpolarization in 2D culture of 4T1 cells along with an enhanced free NAD(P)H population, which suggest an interference with OXPHOS. In contrast, 2P-FLIM of cellular NAD(P)H revealed an enhanced autofluorescence lifetime in 3D 4T1 cultures after MD1 treatment as compared with MD1-treated 2D culture and the control 3D culture. Physical and chemical microenvironmental signaling are critical factors in understanding how therapeutic compounds target cancer cells by disrupting their metabolic pathways. Integrating 2P-FLIM of intrinsic NAD(P)H with refined 3D tumor-matrix in vitro models promises to advance our understanding of the roles of metabolism and metabolic plasticity in tumor growth and metastatic behavior. © 2018International Society for Advancement of Cytometry Key terms NAD(P)H; 4T1; 3D collagen matrix; MD1; TPPBr derivative; two-photon microscopy; FLIM CHANGES in cellular metabolism have long been recognized as a fundamental feature, and are now considered a hallmark, of cancer. A resurgent interest in this area has recently been driven in large measure by significant breakthroughs elucidating relevant mechanisms of cross-talk between not only tumor and stromal cells but also between distinct subpopulations of tumor cells themselves within heterogeneous tumors. In particular, advanced metabolic imaging methods promise to significantly enhance our understanding of how specific microenvironmental factors influence the regulation of cancer cell metabolism on a cell-by-cell basis. Overall, it is becoming clear that a better understanding of the metabolic features of cancer cells-and in particular those that may ...
This study was aimed to evaluate the efficacy of a single administration of long-acting gonadotrophin-releasing hormone agonist (GnRHa) as compared with daily administrations of short-acting GnRHa in controlled ovarian hyperstimulation (COH) for in vitro fertilization and embryo transfer (IVF-ET) cycles. The mean dosage of recombinant follicle-stimulating hormone (rFSH) required for COH (2,354.5±244.2 vs. 2,012.5±626.1 IU) and the rFSH dosage per retrieved oocyte (336.7±230.4 vs. 292.1±540.4 IU) were significantly higher in the long-acting GnRHa group (N=22) than those in the short-acting GnRHa group (N=28) (p<0.05). However, the mean number of visit to the hospital that was required before ovum pick-up (3.3±0.5 vs. 22.2±2.0) and the frequency of injecting GnRHa and rFSH (12.8±1.2 vs. 33.5±3.5) were significantly decreased in the long-acting GnRHa group (p<0.0001). The clinical pregnancy rate, implantation rate, and early pregnancy loss rate were not significantly different between the 2 groups. So, we suggest that a single administration of long-acting GnRHa is a useful alternative for improving patient's convenience with clinical outcomes comparable to daily administrations of short-acting GnRHa in COH for IVF-ET cycles.
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