Contribution of Fluorophore Dynamics and Solvation to Resonant Energy Transfer in Protein-DNA Complexes: A Molecular-Dynamics Study

In Förster resonance energy transfer (FRET) experiments, extracting accurate structural information about macromolecules depends on knowing the positions and orientations of donor and acceptor fluorophores. Several approaches have been employed to reduce uncertainties in quantitative FRET distance measurements. Fluorophore-position distributions can be estimated by surface accessibility (SA) calculations, which compute the region of space explored by the fluorophore within a static macromolecular structure. However, SA models generally do not take fluorophore shape, dye transition-moment orientation, or dye-specific chemical interactions into account. We present a detailed molecular-dynamics (MD) treatment of fluorophore dynamics for an ATTO donor/acceptor dye pair and specifically consider as case studies dye-labeled protein-DNA intermediates in Cre site-specific recombination. We carried out MD simulations in both an aqueous solution and glycerol/water mixtures to assess the effects of experimental solvent systems on dye dynamics. Our results unequivocally show that MD simulations capture solvent effects and dye-dye interactions that can dramatically affect energy transfer efficiency. We also show that results from SA models and MD simulations strongly diverge in cases where donor and acceptor fluorophores are in close proximity. Although atomistic simulations are computationally more expensive than SA models, explicit MD studies are likely to give more realistic results in both homogeneous and mixed solvents. Our study underscores the model-dependent nature of FRET analyses, but also provides a starting point to develop more realistic in silico approaches for obtaining experimental ensemble and single-molecule FRET data.     

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.     

The reliability of observational approaches for detecting interspecific parasite interactions: comparison with experimental results

Interactions among coinfecting parasites have the potential to alter host susceptibility to infection, the progression of disease and the efficacy of disease control measures. It is therefore essential to be able to accurately infer the occurrence and direction of such interactions from parasitological data. Due to logistical constraints, perturbation experiments are rarely undertaken to directly detect interactions, therefore a variety of approaches are commonly used to infer them from patterns of parasite association in observational data. However, the reliability of these various approaches is not known. We assess the ability of a range of standard analytical approaches to detect known interactions between infections of nematodes and intestinal coccidia (Eimeria) in natural small-mammal populations, as revealed by experimental perturbations. We show that correlation-based approaches are highly unreliable, often predicting strong and highly significant associations between nematodes and Eimeria in the opposite direction to the underlying interaction. The most reliable methods involved longitudinal analyses, in which the nematode infection status of individuals at one month is related to the infection status by Eimeria the next month. Even then, however, we suggest these approaches are only viable for certain types of infections and datasets. Overall we suggest that, in the absence of experimental approaches, careful consideration be given to the choice of statistical approach when attempting to infer interspecific interactions from observational data.     

Potential pathogenic bacteria in metalworking fluids and aerosols from a machining facility

The metalworking and machining industry utilizes recirculating metalworking fluids for integral aspects of the fabrication process. Despite the use of biocides, these fluids sustain substantial biological growth. Subsequently, the high-shear forces incurred during metalworking processing aerosolize bacterial cells and may cause dermatologic and respiratory effects in exposed workers. We quantified and identified the bacterial load for metalworking fluid and aerosol samples of a machining facility in the US Midwest during two seasons. To investigate the presence of potentially pathogenic bacteria in fluid and air, we performed 16S rRNA gene surveys. The concentration of total bacterial cells (including culturable and nonculturable cells) was relatively constant throughout the study, averaging 5.1 × 10? cells mL?1 in the fluids and 4.8 × 10? cells m?3 in the aerosols. We observed bacteria of potential epidemiologic significance from several different bacterial phyla in both fluids and aerosols. Most notably, Alcaligenes faecalis was identified through both direct sequencing and culturing in every sample collected. Elucidating the bacterial community with gene surveys showed that metalworking fluids were the source of the aerosolized bacteria in this facility.     

Epidermal Growth Factor Receptor Activation Remodels the Plasma Membrane Lipid Environment To Induce Nanocluster Formation

Signal transduction is regulated by the lateral segregation of proteins into nanodomains on the plasma membrane. However, the molecular mechanisms that regulate the lateral segregation of cell surface receptors, such as receptor tyrosine kinases, upon ligand binding are unresolved. Here we used high-resolution spatial mapping to investigate the plasma membrane nanoscale organization of the epidermal growth factor (EGF) receptor (EGFR). Our data demonstrate that in serum-starved cells, the EGFR exists in preformed, cholesterol-dependent, actin-independent nanoclusters. Following stimulation with EGF, the number and size of EGFR nanoclusters increase in a time-dependent manner. Our data show that the formation of EGFR nanoclusters requires receptor tyrosine kinase activity. Critically, we show for the first time that production of phosphatidic acid by phospholipase D2 (PLD2) is essential for ligand-induced EGFR nanocluster formation. In accordance with its crucial role in regulating EGFR nanocluster formation, we demonstrate that modulating PLD2 activity tunes the degree of EGFR nanocluster formation and mitogen-activated protein kinase signal output. Together, these data show that EGFR activation drives the formation of signaling domains by regulating the production of critical second-messenger lipids and modifying the local membrane lipid environment.The epidermal growth factor (EGF) receptor (EGFR) is a single transmembrane domain protein that possesses intrinsic tyrosine kinase (TK) activity. Ligand binding to the extracellular domain induces conformational changes that promote activation of the intracellular TK domain. The kinase domain then autophosphorylates a number of tyrosine residues in the C-terminal region of the protein, creating docking sites for adapter and effector proteins. Thus, the active form of the EGFR could reasonably be expected to be a dimer. However, recent studies using single-molecule imaging, image correlation spectroscopy (ICS), fluorescence correlation spectroscopy (FCS), and immunoelectron microscopy (immuno-EM) show that the EGFR is, in fact, nonrandomly organized into oligomers on the plasma membrane (6, 7, 16, 34, 44). ICS measurements estimate that, in the absence of ligand, there are, on average, 2.2 EGFRs per cluster, which increases to 3.7 receptors per cluster upon stimulation (7). Single-molecule tracking experiments also suggest that unliganded EGFRs continually fluctuate between monomers and dimers that are primed for activation (5). Furthermore, the organization of the EGFR is dynamic and clustering of the EGFR increases over time after EGF stimulation (7, 16). However, neither the precise role of EGFR oligomerization in signal transduction nor the mechanisms driving oligomer formation have been resolved.The organization of the EGFR into oligomers is dependent upon cellular cholesterol. Saffarian et al., using FCS, estimated that 70% of EGFRs exist as monomers, 20% as dimers, and 10% as oligomers (34). However, depletion of cholesterol decreases the percentage of monomeric receptors and increases the proportion of oligomeric receptors. Cholesterol depletion and actin depolymerization also alter the diffusion coefficient of the EGFR and the confinement area size (22). The finding that EGFR membrane organization is dependent upon cholesterol is of particular interest because a number of studies have demonstrated that EGFR activity is negatively regulated by cholesterol (4, 23, 28, 32).Phospholipase D2 (PLD2) hydrolyzes phosphatidylcholine (PC) to produce choline and phosphatidic acid (PA). PLD2 is localized to the plasma membrane (10), associates with the EGFR (39), and is rapidly activated upon EGF stimulation, leading to increased production of PA (15, 38, 39). A number of lines of evidence suggest that PA is an important mediator of EGFR action. First, exogenous PA is mitogenic when incubated with cells (17, 19, 42, 45). Second, direct interaction with membrane PA regulates the activity of a number of components downstream of the EGFR, including Sos (47) and Raf (12, 13, 30, 31).In the current study, we used high-resolution spatial analysis techniques to investigate EGFR plasma membrane organization. Using these approaches, we identified PA as the key molecular component responsible for driving EGFR nanocluster formation in response to EGF binding and demonstrated that the level of PLD2 activity regulates the duration of mitogen-activated protein kinase (MAPK) signal output.     

Green Space Functionality Along an Urban Gradient in Karachi,Pakistan: A Socio-Ecological Study

An important component of urban green space functionality is the way in which such spaces are perceived and used by urban residents. Use is affected by many factors including the nature and dynamics of urban morphology at the city and neighbourhood scale. This paper demonstrates a method for conducting analyses of green space socio-ecological functionality and presents the results of its application to Karachi, Pakistan. The paper explores the use of the results of an urban gradient analysis for site selection and presents the results of an associated survey of functions using three case study green spaces. The results show that, although urban dwellers in the city use green spaces infrequently, when they do it tends to be for long periods of time and for a range of purposes. Levels of overcrowding at the more accessible sites indicate the value that urban dwellers place on these resources, and their importance to the city as a whole. The results of this study provide evidence for the need to protect green spaces from the competing demands associated with increasing urbanisation, especially if the wider goal of urban sustainability is to be achieved.     

Extracellular phospholipases in atherosclerosis

Phospholipases A2 (PLA2) are a family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids liberating lysophospholipids and free fatty acids; important second messengers involved in atherogenesis. Plasma PAF-acetylhydrolase (PAF-AH) or Lp-PLA2 is a Ca²⁺-independent PLA2 which is produced by monocyte-derived macrophages and by activated platelets, and circulates in plasma associated with lipoproteins. PAF-AH catalyzes the removal of the acetyl/short acyl group at the sn-2 position of PAF and oxidized phospholipids produced during inflammation and oxidative stress. In humans, PAF-AH is mainly associated with small dense LDL and to a lesser extent with HDL and with lipoprotein(a). PAF-AH is N-glycosylated prior to secretion which diminishes its association with HDL raising the question of its distribution between the proatherogenic LDL vs the antiatherogenic HDL. Hypercholesterolemic patients have higher plasma PAF-AH activity which is reduced upon hypolipidemic therapy. PAF-AH specific inhibitor darapladib stabilizes human and swine plaques, therefore challenging the antiatherogenic potential of PAF-AH shown in small animal models.