Patterns of DNA methylation in the normal colon vary by anatomical location,gender, and age

Alterations in DNA methylation have been proposed to create a field cancerization state in the colon, where molecular alterations that predispose cells to transformation occur in histologically normal tissue. However, our understanding of the role of DNA methylation in field cancerization is limited by an incomplete characterization of the methylation state of the normal colon. In order to determine the colon’s normal methylation state, we extracted DNA from normal colon biopsies from the rectum, sigmoid, transverse, and ascending colon and assessed the methylation status of the DNA by pyrosequencing candidate loci as well as with HumanMethylation450 arrays. We found that methylation levels of repetitive elements LINE-1 and SAT-α showed minimal variability throughout the colon in contrast to other loci. Promoter methylation of EVL was highest in the rectum and progressively lower in the proximal segments, whereas ESR1 methylation was higher in older individuals. Genome-wide methylation analysis of normal DNA revealed 8388, 82, and 93 differentially methylated loci that distinguished right from left colon, males from females, and older vs. younger individuals, respectively. Although variability in methylation between biopsies and among different colon segments was minimal for repetitive elements, analyses of specific cancer-related genes as well as a genome-wide methylation analysis demonstrated differential methylation based on colon location, individual age, and gender. These studies advance our knowledge regarding the variation of DNA methylation in the normal colon, a prerequisite for future studies aimed at understanding methylation differences indicative of a colon field effect.     

Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.     

Anisotropic intersubunit and inter-ring interactions revealed in the native bullet-shaped chaperonin complex from Thermus thermophilus

Background

The recent morphological studies on chaperonins have revealed that nearly equivalent amount of symmetric GroEL–(GroES)₂ (football-shaped) and asymmetric GroEL–GroES (bullet-shaped) complexes coexist during the chaperonin reaction cycle, which prompted us to reexamine the equatorial split observed for chaperonin from Thermus thermophilus by implementing semi-empirical molecular orbital (MO) calculations, since it is now believed that the symmetric formation is a precursor to the equatorial split.

Methods

Semi-empirical MO calculations were employed to investigate the intersubunit interactions within the bullet-shaped T. thermophilus chaperonin capturing the substrate of folding intermediates. Interaction energies between each cis-GroEL subunit and closely related remaining subunits in cis-GroEL ring, or in trans-GroEL ring across the equatorial plane, and further, interaction energies between each GroES subunit and adjacent subunits in the same GroES ring and in cis-GroEL ring were simulated.

Results

Anisotropic intensities and energy distribution of the subunits were revealed by the calculations, which are consistent with two conformations of the subunits forming cis-GroEL ring as revealed by X-ray crystal structure, and with an anisotropic critical binding site on cis-GroEL ring for chaperonin functioning.

Conclusions

This is the first application of semi-empirical MO calculations to the macromolecular complex of the native bullet-shaped chaperonin (GroEL–GroES–ADP homolog) from T. thermophilus.

General significance

The results also appear to support the occurrence of the equatorial split for T. thermophilus chaperonin observed via electron microscopy, but has not yet been fully observed for Escherichia coli GroEL–GroES system.     

When size does matter: organelle size influences the properties of transport mediated by molecular motors

Background

Organelle transport is driven by the action of molecular motors. In this work, we studied the dynamics of organelles of different sizes with the aim of understanding the complex relation between organelle motion and microenvironment.

Methods

We used single particle tracking to obtain trajectories of melanosomes (pigmented organelles in Xenopus laevis melanophores). In response to certain hormones, melanosomes disperse in the cytoplasm or aggregate in the perinuclear region by the combined action of microtubule and actin motors.

Results and conclusions

Melanosome trajectories followed an anomalous diffusion model in which the anomalous diffusion exponent (α) provided information regarding the trajectories' topography and thus of the processes causing it. During aggregation, the directionality of big organelles was higher than that of small organelles and did not depend on the presence of either actin or intermediate filaments (IF). Depolymerization of IF significantly reduced α values of small organelles during aggregation but slightly affect their directionality during dispersion.

General significance

Our results could be interpreted considering that the number of copies of active motors increases with organelle size. Transport of big organelles was not influenced by actin or IF during aggregation showing that these organelles are moved processively by the collective action of dynein motors. Also, we found that intermediate filaments enhance the directionality of small organelles suggesting that this network keeps organelles close to the tracks allowing their efficient reattachment. The higher directionality of small organelles during dispersion could be explained considering the better performance of kinesin-2 vs. dynein at the single molecule level.     

Population dynamics of Meloidogyne incognita on black pepper plants in two agro-ecological regions in Vietnam

The objective of our study was to examine the effect of local environmental conditions, especially air temperature and rainfall, in two agro-ecological regions in Vietnam on the population dynamics of Meloidogyne incognita on black pepper plants and on percentage root galling. The two study sites were situated in Cam Lo, Quang Tri province (North Central Coast) and Buon Ma Thuot, Dac Lac province (Central Highlands). At the study sites, 13 plant-parasitic nematode taxa belonging to 12 genera were identified. Ten of these taxa were present in both study sites. Helicotylenchus certus, Hemicriconemoides cocophilus and Pratylenchus coffeae were only found in Buon Ma Thuot. Based on nematode population densities, M. incognita was the most abundant taxon present. Rainfall and air temperature differed significantly between the two study sites. The most important climatic difference was rainfall: in Buon Ma Thuot, the rainy season lasted 2 months longer and the monthly rainfall during the rainy season was much higher compared to Cam Lo. Although this difference resulted in some differences in the population dynamics of M. incognita in the soil and roots of the black pepper variety Vinh Linh, the highest root population densities were observed in both study sites during the first half of the dry season. In Cam Lo, the highest percentage root galling was observed during the first half of the dry season. In Buon Ma Thuot, the highest percentage root galling was observed towards the end of the rainy season. Estimating the population densities if M. incognita to decide on the application of a management strategy should be done during the first half of the dry season.     

THE RELEVANT USE OF THE TRADITIONAL TUNISIAN GAME ‘‘RAQASSA’’ FOR CARDIOVASCULAR STIMULATION IN SCHOOLCHILDREN

The aim of this study was to investigate the heart rate (HR) responses, the rate of perceived exertion (RPE), and the feeling during physical education schooling while performing traditional games activities compared to intermittent exercise. Nineteen pre-pubertal children randomly performed on different days two types of lessons (intermittent running mode vs. traditional Tunisian “Raqassa” game) lasting 12-min each. HR was continuously recorded during both lessons, while ratings of perceived exertion and Feeling values were recorded after the sessions. The mean HR value during the traditional game was significantly higher than during intermittent exercise (p<0.05). Conversely, the perceived exertion score was significantly higher after intermittent exercise than the traditional exercise game (p<0.05), showing that the higher cardiovascular strain of the game was perceived as “lighter” than the run. Simultaneously, the children''s Feeling was significantly higher after the traditional game than intermittent exercise (p<0.001), showing a higher satisfaction from playing with respect to running. Exercise based on the “Raqassa” traditional game could be used in pre-pubertal children as an alternative or as an additional method for suitable cardiovascular stimulation during physical education lessons with lower perceived exertion and better feeling compared to intermittent running.     

Accelerating all-atom protein folding simulations through reduced dihedral barriers

Protein folding requires extensive changes of backbone and sidechain dihedral angles, whose energy barriers constitute obstacles for the folding kinetics. Folding of small proteins is furthermore thought to be path-independent. Here, we propose that time-consuming all-atom protein folding simulations may be accelerated through a reduction of the dihedral barriers of the force field. In order to investigate this hypothesis, we performed various folding simulations of two small proteins. We report an acceleration towards smaller root-mean-square deviations from the native protein structure using our proposed method.     

Controlling the secondary-structure-forming tendencies of proteins by a backbone torsion-energy term

We examined a new backbone torsion-energy term proposed by us in the force field for protein systems. This torsion-energy term is represented by a double Fourier series in two variables, namely the backbone dihedral angles φ and ψ. It gives a natural representation of the torsion energy in the Ramachandran space in the sense that any two-dimensional energy surface periodic in both φ and ψ can be expanded by the double Fourier series. We can then easily control secondary-structure-forming tendencies by modifying the torsion-energy surface. For instance, we can increase or decrease the α-helix-forming-tendencies by lowering or raising the torsion-energy surface in the α-helix region and likewise increase or decrease the β-sheet-forming tendencies by lowering or raising the surface in the β-sheet region in the Ramachandran space. We applied this torsion-energy modification method to six force fields, AMBER parm94, AMBER parm96, AMBER parm99, CHARMM27, OPLS-AA and OPLS-AA/L, and demonstrated that our modifications of the torsion-energy terms resulted in the expected changes of secondary-structure-forming tendencies by performing folding simulations of α-helical and β-hairpin peptides.