Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non-toxic can, in principle, be rationalized. Armed with supercomputers and accurate force fields for biomolecular interactions, we can now investigate phenomena that span hundreds of nanoseconds. Although the phenomena involved in antimicrobial activity, (i.e., diffusion of peptides, interaction with lipid layers, secondary structure attainment, possible surface aggregation, possible formation of pores, and destruction of the lipid layer integrity) collectively span time scales still prohibitively long for classical mechanics simulations, it is now feasible to investigate the initial approach of single peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential.     

Endosomal lipid accumulation in NPC1 leads to inhibition of PKC, hypophosphorylation of vimentin and Rab9 entrapment

Background information. Within the group of lysosomal storage diseases, NPC1 [NPC (Niemann‐Pick type C) 1] disease is a lipidosis characterized by excessive accumulation of free cholesterol as well as gangliosides, glycosphingolipids and fatty acids in the late E/L (endosomal/lysosomal) system (Chen et al., 2005 ) due to a defect in late endosome lipid egress. We have previously demonstrated that expression of the small GTPase Rab9 in NPC1 cells can rescue the lipid transport block phenotype (Walter et al., 2003 ), albeit by an undefined mechanism. Results. To investigate further the mechanism by which Rab9 facilitates lipid movement from late endosomes we sought to identify novel Rab9 binding/interacting proteins. In the present study, we report that Rab9 interacts with the intermediate filament phosphoprotein vimentin and this interaction is altered by lipid accumulation in late endosomes, which results in inhibition of PKC (protein kinase C) and hypophosphorylation of vimentin, leading to late endosome dysfunction. Intermediate filament hypophosphorylation, aggregation and entrapment of Rab9 ultimately leads to transport defects and inhibition of lipid egress from late endosomes. Conclusions. These results reveal a previously unappreciated interaction between Rab proteins and intermediate filaments in regulating intracellular lipid transport.     

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

Background

DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell.

Results

We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods.

Conclusion

Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.     

Probing corticular photosynthesis through in vivo chlorophyll fluorescence measurements: evidence that high internal CO levels suppress electron flow and increase the risk of photoinhibition

Twigs of many woody plants possess chlorenchyma under a well-developed periderm which lacks stomata and impedes both gas diffusion and light penetration. The so-called corticular photosynthesis, occurring in the shade and under extremely high CO(2) concentrations, was probed in this study through in vivo chlorophyll fluorescence measurements. Field comparisons between twigs and corresponding leaves in five species indicated that both the dark- and light-adapted PSII photochemical efficiencies are considerably lower in twigs at all incident photon fluence rates, in spite of the significant attenuation of solar radiation by the periderm. Light saturation curves for linear electron transport rates (corrected according to the actual light intensities reaching twig chlorenchyma) were compatible with a shade-acclimated photosynthetic machinery, showing very low maximum electron transport rates (at approximately 5% of the corresponding leaf values) and threshold irradiances for light saturation. However, removing periderms from twig segments (i.e. relieving the twig interior form the high CO(2) partial pressures) considerably improved the light-adapted (but not the dark-adapted) PSII photochemical efficiency, allowing the assumption that the high internal CO(2) levels may interfere with the smooth functioning of photosynthesis. Indeed, laboratory experiments with twig segments equilibrated under various CO(2) levels (0.036-20%), resulted in a progressive decrease of light-adapted PSII photochemical yield, with the values obtained at 20% CO(2) being similar to those obtained with intact twigs in the field. Further experiments indicated that high CO(2) combined with high light suppressed the development of a photoprotective non-photochemical quenching through a reduction of its fast relaxing component, accompanied by a higher risk of photoinhibition. It is suggested that high internal CO(2) concentrations in twigs impede photosynthesis possibly through acidification of protoplasm and impairment of the pH-dependent high energy state quenching followed by reduction in the efficiency of heat dissipation.     

Thigh-muscles strength training, dance exercise, dynamometry, and anthropometry in professional ballerinas

The purpose of the present study was to assess the effects of 12 weeks of quadriceps and hamstring strength training on torque levels after a dance exercise and on selected anthropometric parameters. The sample consisted of 22 (ages, 25 +/- 1.3 years) full-time professional ballerinas who were randomly assigned into experimental (n = 12) and control (n = 10) groups. A dance routine designed to cause fatigue within 5 minutes, isokinetic dynamometry, and anthropometric assessments were conducted before and after strength training in both groups. Before strength training, the dance routine resulted in significant reductions of hamstring (p < 0.001) and quadriceps (p < 0.001) peak torques in both subject groups. However, after strength training, only control subjects demonstrated such torque decrements (p < 0.001) after the dance routine. Furthermore, the experimental group revealed greater knee extension (119 vs. 138 N.m; p < 0.001) and flexion (60 vs. 69 N.m; p < 0.001) torques, smaller sum of skinfolds (33.6 vs. 27.8 mm; p < 0.01), more fat-free mass (37.7 vs. 39.4 kg; p < 0.05), but unchanged body mass (p > 0.05) and thigh circumferences (p > 0.05). A negative relationship (p < 0.001) was found between initial strength levels and improvements measured at the end of the 12-week program. These results suggest that supplementary strength training for hamstring and quadriceps muscles is beneficial to professional ballerinas and their dancing; weaker individuals are more likely to benefit from such regimens than their stronger counterparts, whereas increases in thigh-muscle strength do not alter selected aesthetic components.     

Escherichia coli SecA truncated at its termini is functional and dimeric

Terminal residues in SecA, the dimeric ATPase motor of bacterial preprotein translocase, were proposed to be required for function and dimerization. To test this, we generated truncation mutants of the 901aa long SecA of Escherichia coli. We now show that deletions of carboxy-terminal domain (CTD), the extreme CTD of 70 residues, or of the N-terminal nonapeptide or of both, do not compromise protein translocation or viability. Deletion of additional C-terminal residues upstream of CTD compromised function. Functional truncation mutants like SecA9-861 are dimeric, conformationally similar to SecA, fully competent for nucleotide and SecYEG binding and for ATP catalysis. Our data demonstrate that extreme terminal SecA residues are not essential for SecA catalysis and dimerization.     

Guilty until proven innocent: the case of NPC1 and cholesterol

Cholesterol accumulation in the endosomes and lysosomes of Niemann-Pick C (NPC) cells is considered to be the hallmark of this disorder, so the main focus of NPC research has revolved around cholesterol and its role in disease pathogenesis. However, recent data indicate that cholesterol is not the primary culprit in this human lipidosis. I propose a new hypothesis for the potential action or function of the NPC1 protein in the endosome. In this context, the relationship of NPC2 and NPC1 is also discussed.     

Equal volumes of high and low intensity of eccentric exercise in relation to muscle damage and performance

We examined differences in muscle damage and muscle performance perturbations in relation to the same volumes of high (HI) and low intensity (LI) of eccentric exercise. Untrained young healthy men (n = 12) underwent 2 isokinetic quadriceps eccentric exercise sessions, 1 on each randomly selected leg, separated by a 2-week interval. In the first session subjects performed HI exercise (i.e., 12 sets of 10 maximal voluntary efforts). In the second session, volunteers were subjected to continuous exercise of LI (50% of peak torque) until the total work done was approximately equal to that generated during HI. Muscle damage (serum creatine kinase concentration [CK], delayed onset of muscle soreness, and range of motion) and muscle performance (eccentric [EPT] and isometric peak torque [IPT]) indicators were assessed pre-exercise and 24, 48, 72, and 96 hours postexercise. Compared to baseline data, changes in muscle damage indicators were significantly different (p < 0.05) at almost all postexercise time points in both conditions. However, apart from the significant elevation of CK at 24 hours after HI (p < 0.05), no other significant differences were observed between the 2 exercise conditions (p > 0.05). The main finding in relation to muscle performance was that decrements following HI exercise were significantly greater (p < 0.05) compared to LI. Compared with baseline data, the EPT values following HI and LI exercise were as follows: 24 hours, 72.1% vs. 92%; 48 hours, 81.9% vs. 94.8%; 72 hours, 77.7% vs. 100.6%; 96 hours, 86.8% vs. 107.9%. The corresponding data for IPT were as follows: 24 hours, 86.4% vs. 102.8%; 48 hours, 84.2% vs. 107%; 72 hours, 84.8% vs. 109.2%; 96 hours, 86.8% vs. 114.4%. These results indicate that matching volumes of HI and LI eccentric exercise have similar effects on muscle damage, but HI has a more prominent effect on muscle performance.