The effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA

Most of the G-quadruplex interactive molecules reported to date contain extended aromatic flat ring systems and are believed to bind principally by π–π stacking on the end G-tetrads of the quadruplex structure. One such molecule, TMPyP4, (5,10,15,20-tetra(N-methyl-4-pyridyl)porphyrin), exhibits high affinity and some selectivity for G-quadruplex DNA over duplex DNA. Although not a realistic drug candidate, TMPyP4 is used in many nucleic acid research laboratories as a model ligand for the study of small molecule G-quadruplex interactions. Here we report on the synthesis and G-quadruplex interactions of four new cationic porphyrin ligands having only 1, 2, or 3 (N-methyl-4-pyridyl) substituents. The four new ligands are: P(5) (5-(N-methyl-4-pyridyl)porphyrin), P(5,10) (5,10-di(N-methyl-4-pyridyl)porphyrin), P(5,15) (5,15-di(N-methyl-4-pyridyl)porphyrin), and P(5,10,15) (5,10,15-tri(N-methyl-4-pyridyl)porphyrin). Even though these compounds have been previously synthesized, we report alternative synthetic routes that are more efficient and that result in higher yields. We have used ITC, CD, and ESI-MS to explore the effects of the number of N-methyl-4-pyridyl substituents and the substituent position on the porphyrin on the G-quadruplex binding energetics. The relative affinities for binding these ligands to the WT Bcl-2 promoter sequence G-quadruplex are: K_TMPyP4 ≈ K_P(5,15) > K_P(5,10,15) >>> K_P(5,10), K_P(5). The saturation stoichiometry is 2:1 for both P(5,15) and P(5,10,15), while neither P(5) nor P(5,10) exhibit significant complex formation with the WT Bcl-2 promoter sequence G-quadruplex. Additionally, binding of P(5,15) appears to interact by an ‘intercalation mode’ while P(5,10,15) appears to interact by an ‘end-stacking mode’.     

Inferring Latent States and Refining Force Estimates via Hierarchical Dirichlet Process Modeling in Single Particle Tracking Experiments

Understanding the basis for intracellular motion is critical as the field moves toward a deeper understanding of the relation between Brownian forces, molecular crowding, and anisotropic (or isotropic) energetic forcing. Effective forces and other parameters used to summarize molecular motion change over time in live cells due to latent state changes, e.g., changes induced by dynamic micro-environments, photobleaching, and other heterogeneity inherent in biological processes. This study discusses limitations in currently popular analysis methods (e.g., mean square displacement-based analyses) and how new techniques can be used to systematically analyze Single Particle Tracking (SPT) data experiencing abrupt state changes in time or space. The approach is to track GFP tagged chromatids in metaphase in live yeast cells and quantitatively probe the effective forces resulting from dynamic interactions that reflect the sum of a number of physical phenomena. State changes can be induced by various sources including: microtubule dynamics exerting force through the centromere, thermal polymer fluctuations, and DNA-based molecular machines including polymerases and protein exchange complexes such as chaperones and chromatin remodeling complexes. Simulations aiming to show the relevance of the approach to more general SPT data analyses are also studied. Refined force estimates are obtained by adopting and modifying a nonparametric Bayesian modeling technique, the Hierarchical Dirichlet Process Switching Linear Dynamical System (HDP-SLDS), for SPT applications. The HDP-SLDS method shows promise in systematically identifying dynamical regime changes induced by unobserved state changes when the number of underlying states is unknown in advance (a common problem in SPT applications). We expand on the relevance of the HDP-SLDS approach, review the relevant background of Hierarchical Dirichlet Processes, show how to map discrete time HDP-SLDS models to classic SPT models, and discuss limitations of the approach. In addition, we demonstrate new computational techniques for tuning hyperparameters and for checking the statistical consistency of model assumptions directly against individual experimental trajectories; the techniques circumvent the need for “ground-truth” and/or subjective information.     

A Computational Model for Collective Cellular Motion in Three Dimensions: General Framework and Case Study for Cell Pair Dynamics

Cell migration in healthy and diseased systems is a combination of single and collective cell motion. While single cell motion has received considerable attention, our understanding of collective cell motion remains elusive. A new computational framework for the migration of groups of cells in three dimensions is presented, which focuses on the forces acting at the microscopic scale and the interactions between cells and their extracellular matrix (ECM) environment. Cell-cell adhesion, resistance due to the ECM and the factors regulating the propulsion of each cell through the matrix are considered. In particular, our approach emphasizes the role of receptors that mediate cell-cell and cell-matrix interactions, and examines how variation in their properties induces changes in cellular motion. As an important case study, we analyze two interacting cells. Our results show that the dynamics of cell pairs depends on the magnitude and the stochastic nature of the forces. Stronger intercellular stability is generally promoted by surface receptors that move. We also demonstrate that matrix resistance, cellular stiffness and intensity of adhesion contribute to migration behaviors in different ways, with memory effects present that can alter pair motility. If adhesion weakens with time, our findings show that cell pair break-up depends strongly on the way cells interact with the matrix. Finally, the motility for cells in a larger cluster (size 50 cells) is examined to illustrate the full capabilities of the model and to stress the role of cellular pairs in complex cellular structures. Overall, our framework shows how properties of cells and their environment influence the stability and motility of cellular assemblies. This is an important step in the advancement of the understanding of collective motility, and can contribute to knowledge of complex biological processes involving migration, aggregation and detachment of cells in healthy and diseased systems.     

Response to long-distance relocation in Asian elephants (Elephas maximus): monitoring adrenocortical activity via serum, urine, and feces

Understanding how elephants respond to potentially stressful events, such as relocation, is important for making informed management decisions. This study followed the relocation of eight Asian elephants from the Cocos (Keeling) Islands to mainland Australia. The first goal of this study was to examine patterns of adrenocortical activity as reflected in three different substrates: serum, urine, and feces. We found that the three substrates yielded very different signals of adrenocortical activity. Fecal glucocorticoid metabolites (FGM) increased as predicted post-transport, but urinary glucocorticoid metabolites (UGM) were actually lower following transport. Serum cortisol levels did not change significantly. We suggest that the differences in FGM and UGM may reflect changes in steroid biosynthesis, resulting in different primary glucocorticoids being produced at different stages of the stress response. Additional studies are needed to more thoroughly understand the signals of adrenocortical activity yielded by different substrates. The second goal was to examine individual variation in patterns of adrenal response. There was a positive correlation between baseline FGM value and duration of post-transfer increase in FGM concentration. Furthermore, an individual's adrenocortical response to relocation was correlated with behavioral traits of elephants. Elephants that were described by keepers as being “curious” exhibited a more prolonged increase in FGM post-transfer, and “reclusive” elephants had a greater increase in FGM values. Future research should investigate the importance of these personality types for the management and welfare of elephants.     

Review of the fish parasitic genus Ceratothoa Dana, 1852 (Crustacea,Isopoda, Cymothoidae) from South Africa,including the description of two new species

The genus Ceratothoa Dana, 1852 is revised for South African waters and re-diagnosed. Ceratothoa retusa (Schioedte & Meinert, 1883) is recorded from the eastern coast, and Ceratothoa africanae sp. n. and C. famosa sp. n. are described; C. imbricata (Fabricius, 1775) and C. trigonocephala (Leach, 1818), are redescribed, revised and excluded from the South African fauna. Ceratothoa africanae sp. n. can be distinguished by the stout body shape of the female; triangular cephalon with a pointed rostrum; short uropods which do not extend past the pleotelson; large carinae on the pereopod basis; a broad pleon; and large medial lobes on female pleopods. Ceratothoa famosa sp. n. is characterised by the long rectangular body shape; pereonite 1 with a raised medial protrusion; narrow antenna with antennule article 1 expanded; uropods which reach the posterior margin of the pleotelson; narrow rami on uropods; and no appendix masculina on pleopod 2 of the male specimens.