Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.     

Coarse Woody Debris Increases Microbial Community Functional Diversity but not Enzyme Activities in Reclaimed Oil Sands Soils

Forest floor mineral soil mix (FMM) and peat mineral soil mix (PMM) are cover soils commonly used for upland reclamation post open-pit oil sands mining in northern Alberta, Canada. Coarse woody debris (CWD) can be used to regulate soil temperature and water content, to increase organic matter content, and to create microsites for the establishment of microorganisms and vegetation in upland reclamation. We studied the effects of CWD on soil microbial community level physiological profile (CLPP) and soil enzyme activities in FMM and PMM in a reclaimed landscape in the oil sands. This experiment was conducted with a 2 (FMM vs PMM) × 2 (near CWD vs away from CWD) factorial design with 6 replications. The study plots were established with Populus tremuloides (trembling aspen) CWD placed on each plot between November 2007 and February 2008. Soil samples were collected within 5 cm from CWD and more than 100 cm away from CWD in July, August and September 2013 and 2014. Microbial biomass was greater (p<0.05) in FMM than in PMM, in July, and August 2013 and July 2014, and greater (p<0.05) near CWD than away from CWD in FMM in July and August samplings. Soil microbial CLPP differed between FMM and PMM (p<0.01) according to a principal component analysis and CWD changed microbial CLPP in FMM (p<0.05) but not in PMM. Coarse woody debris increased microbial community functional diversity (average well color development in Biolog Ecoplates) in both cover soils (p<0.05) in August and September 2014. Carbon degrading soil enzyme activities were greater in FMM than in PMM (p<0.05) regardless of distance from CWD but were not affected by CWD. Greater microbial biomass and enzyme activities in FMM than in PMM will increase organic matter decomposition and nutrient cycling, improving plant growth. Enhanced microbial community functional diversity by CWD application in upland reclamation has implications for accelerating upland reclamation after oil sands mining.     

Substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs as potent Kv1.3 ion channel blockers. Part 2

We report the synthesis and in vitro activity of a series of novel substituted N-{3-[(1,1-dioxido-1,2-benzothiazol-3-yl)(phenyl)amino]propyl}benzamide analogs. These analogs showed potent inhibitory activity against Kv1.3. Several demonstrated similar potency to the known Kv1.3 inhibitor PAP-1 when tested under the IonWorks patch clamp assay conditions. Two compounds 13i and 13rr were advanced further as potential tool compounds for in vivo validation studies.     

Relationship of aerobic and anaerobic parameters with 400 m front crawl swimming performance

The aims of the present study were to investigate the relationship of aerobic and anaerobic parameters with 400 m performance, and establish which variable better explains long distance performance in swimming. Twenty-two swimmers (19.1±1.5 years, height 173.9±10.0 cm, body mass 71.2±10.2 kg; 76.6±5.3% of 400 m world record) underwent a lactate minimum test to determine lactate minimum speed (LMS) (i.e., aerobic capacity index). Moreover, the swimmers performed a 400 m maximal effort to determine mean speed (S400m), peak oxygen uptake (V.O2PEAK) and total anaerobic contribution (C_ANA). The C_ANA was assumed as the sum of alactic and lactic contributions. Physiological parameters of 400 m were determined using the backward extrapolation technique (V.O2PEAK and alactic contributions of C_ANA) and blood lactate concentration analysis (lactic anaerobic contributions of C_ANA). The Pearson correlation test and backward multiple regression analysis were used to verify the possible correlations between the physiological indices (predictor factors) and S400m (independent variable) (p < 0.05). Values are presented as mean ± standard deviation. Significant correlations were observed between S400m (1.4±0.1 m·s^-1) and LMS (1.3±0.1 m·s^-1; r = 0.80), V.O2PEAK (4.5±3.9 L·min^-1; r = 0.72) and C_ANA (4.7±1.5 L·O₂; r= 0.44). The best model constructed using multiple regression analysis demonstrated that LMS and V.O2PEAK explained 85% of the 400 m performance variance. When backward multiple regression analysis was performed, C_ANA lost significance. Thus, the results demonstrated that both aerobic parameters (capacity and power) can be used to predict 400 m swimming performance.     

Ligand-selective targeting of the glucocorticoid receptor to nuclear subdomains is associated with decreased receptor mobility

The association between nuclear distribution and mobility of the human glucocorticoid receptor was examined in living COS-1 cells using yellow fluorescent protein- and cyan fluorescent protein-tagged receptors. Quantitation of the nuclear distribution induced by an array of glucocorticoid ligands revealed a continuum from a random (cortisone) to a nonrandom (triamcinolone acetonide) receptor distribution. Structure-function analysis revealed that the 9-fluoro and 17-hydroxy groups on the steroid significantly impact nuclear receptor distribution. Using time-lapse microscopy, the triamcinolone acetonide-induced receptor distribution did not change significantly over a period of 15 sec. However, using fluorescence recovery after photobleaching, the individual receptors moved at a much faster rate, indicating rapid exchange of receptors on immobile nuclear subdomains. Receptor mobilities for 13 different steroids, measured by fluorescence recovery after photobleaching, appeared to correlate with receptor distribution. Ligands that induced a nonrandom distribution induced slower receptor mobility and vice versa. Finally, application of 2-photon confocal microscopy revealed differences in receptor mobility between nuclear subdomains. Areas of high receptor concentration showed slower mobility than areas of low receptor concentration. Thus, glucocorticoid receptors can be targeted (depending on the ligand) to relatively immobile nuclear subdomains. The transient association of receptor with these domains decreases the mobility of the receptor.     

Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters

The conserved family of AMT/Rh proteins facilitates ammonium transport across animal, plant, and microbial membranes. A bacterial homologue, AmtB, forms a channel-like structure and appears to function as an NH₃ gas channel. To evaluate the function of eukaryotic homologues, the human RhCG glycoprotein and the tomato plant ammonium transporter LeAMT1;2 were expressed and compared in Xenopus oocytes and yeast. RhCG mediated the electroneutral transport of methylammonium (MeA), which saturated with K_m = 3.8 mM at pH_o 7.5. Uptake was strongly favored by increasing the pH_o and was inhibited by ammonium. Ammonium induced rapid cytosolic alkalinization in RhCG-expressing oocytes. Additionally, RhCG expression was associated with an alkali-cation conductance, which was not significantly permeable to NH₄⁺ and was apparently uncoupled from the ammonium transport. In contrast, expression of the homologous LeAMT1;2 induced pH_o-independent MeA⁺ uptake and specific NH₄⁺ and MeA⁺ currents that were distinct from endogenous currents. The different mechanisms of transport, including the RhCG-associated alkali-cation conductance, were verified by heterologous expression in appropriate yeast strains. Thus, homologous AMT/Rh-type proteins function in a distinct manner; while LeAMT1;2 carries specifically NH₄⁺, or cotransports NH₃/H⁺, RhCG mediates electroneutral NH₃ transport.     

Imaging cell interactions with native and crosslinked polyelectrolyte multilayers

The adhesion of primary chondrocytes to polyelectrolyte multilayer films, made of poly(l-lysine) (PLL) and hyaluronan (HA), was investigated for native and crosslinked films, either ending by PLL or HA. Crosslinking the film was achieved by means of a water-soluble carbodiimide in combination with N-hydroxysulfosuccinimide. The adhesion of macrophages and primary chondrocytes was investigated by microscopical techniques (optical, confocal, and atomic), providing useful information on the cell/film interface. Native films were found to be nonadhesive for the, primary chondrocytes, but could be degraded by macrophages, as could be visualized by confocal laser scanning microscopy after film labeling. Confocal microscopy images show that these films can be deformed by the condrocytes and that PLL diffuses at the chondrocyte membrane. In contrast, the cells adhered and proliferated well on the crosslinked films, which were not degraded by the macrophages. These results were confirmed by a MTT test over a 6-d period and by atomic force microscopy observations. We thus prove that chemical crosslinking can dramatically change cell adhesion properties, the cells being more stably anchored on the crosslinked films. Both authors kcontributed equally.