Optimal Superpositioning of Flexible Molecule Ensembles

Analysis of the internal dynamics of a biological molecule requires the successful removal of overall translation and rotation. Particularly for flexible or intrinsically disordered peptides, this is a challenging task due to the absence of a well-defined reference structure that could be used for superpositioning. In this work, we started the analysis with a widely known formulation of an objective for the problem of superimposing a set of multiple molecules as variance minimization over an ensemble. A negative effect of this superpositioning method is the introduction of ambiguous rotations, where different rotation matrices may be applied to structurally similar molecules. We developed two algorithms to resolve the suboptimal rotations. The first approach minimizes the variance together with the distance of a structure to a preceding molecule in the ensemble. The second algorithm seeks for minimal variance together with the distance to the nearest neighbors of each structure. The newly developed methods were applied to molecular-dynamics trajectories and normal-mode ensembles of the Aβ peptide, RS peptide, and lysozyme. These new (to our knowledge) superpositioning methods combine the benefits of variance and distance between nearest-neighbor(s) minimization, providing a solution for the analysis of intrinsic motions of flexible molecules and resolving ambiguous rotations.     

Disease-causing Mutations in the Cystic Fibrosis Transmembrane Conductance Regulator Determine the Functional Responses of Alveolar Macrophages

Alveolar macrophages (AMs) play a major role in host defense against microbial infections in the lung. To perform this function, these cells must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Recently, we demonstrated that murine alveolar macrophages employ the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel as a determinant in lysosomal acidification (Di, A., Brown, M. E., Deriy, L. V., Li, C., Szeto, F. L., Chen, Y., Huang, P., Tong, J., Naren, A. P., Bindokas, V., Palfrey, H. C., and Nelson, D. J. (2006) Nat. Cell Biol. 8, 933–944). Lysosomes and phagosomes in murine cftr^−/− AMs failed to acidify, and the cells were deficient in bacterial killing compared with wild type controls. Cystic fibrosis is caused by mutations in CFTR and is characterized by chronic lung infections. The information about relationships between the CFTR genotype and the disease phenotype is scarce both on the organismal and cellular level. The most common disease-causing mutation, ΔF508, is found in 70% of patients with cystic fibrosis. The mutant protein fails to fold properly and is targeted for proteosomal degradation. G551D, the second most common mutation, causes loss of function of the protein at the plasma membrane. In this study, we have investigated the impact of CFTR ΔF508 and G551D on a set of core intracellular functions, including organellar acidification, granule secretion, and microbicidal activity in the AM. Utilizing primary AMs from wild type, cftr^−/−, as well as mutant mice, we show a tight correlation between CFTR genotype and levels of lysosomal acidification, bacterial killing, and agonist-induced secretory responses, all of which would be expected to contribute to a significant impact on microbial clearance in the lung.     

CLC-3 channels modulate excitatory synaptic transmission in hippocampal neurons

It is well established that ligand-gated chloride flux across the plasma membrane modulates neuronal excitability. We find that a voltage-dependent Cl(-) conductance increases neuronal excitability in immature rodents as well, enhancing the time course of NMDA receptor-mediated miniature excitatory postsynaptic potentials (mEPSPs). This Cl(-) conductance is activated by CaMKII, is electrophysiologically identical to the CaMKII-activated CLC-3 conductance in nonneuronal cells, and is absent in clc-3(-/-) mice. Systematically decreasing [Cl(-)](i) to mimic postnatal [Cl(-)](i) regulation progressively decreases the amplitude and decay time constant of spontaneous mEPSPs. This Cl(-)-dependent change in synaptic strength is absent in clc-3(-/-) mice. Using surface biotinylation, immunohistochemistry, electron microscopy, and coimmunoprecipitation studies, we find that CLC-3 channels are localized on the plasma membrane, at postsynaptic sites, and in association with NMDA receptors. This is the first demonstration that a voltage-dependent chloride conductance modulates neuronal excitability. By increasing postsynaptic potentials in a Cl(-) dependent fashion, CLC-3 channels regulate neuronal excitability postsynaptically in immature neurons.     

Investigation of readiness and perceived workload in junior female basketball players during a congested match schedule

This study aimed to: a) investigate the differences in workload and readiness between two junior female national basketball teams competing at different European Championships (EC); b) compare workload, readiness and match performance for players with longer and shorter playing times, and; c) examine the relationship between workload, readiness and match performance variables. Under-18 (U18) (n = 10, height = 179.9 ± 6.6 cm, body mass = 70.2 ± 5.1 kg) and under-20 (U20) female national basketball teams (n = 11, height = 178.4 ± 8.8 cm, body mass = 73.0 ± 9.7 kg) were monitored during congested match schedules encompassing 7 matches within 9 days. Daily workload was determined via the session rating of perceived exertion (sRPE workload); readiness was measured by heart-rate variability (HRV) and well-being (WB); and match performance was assessed using the efficiency statistic and playing time. Analysis of workload and readiness during the EC showed no statistically significant between-team differences in any variables except WB for the U18 team, which was lower on Day 8 compared to the U20 team (p = 0.03; effect size [ES] = large). Players accumulating longer playing time showed a higher sRPE workload (p = 0.01, ES = moderate) and efficiency statistic (p = 0.04, ES = moderate) while no readiness variable differed significantly (p > 0.05) compared to players with shorter playing time. Trivial-to-small correlations were observed between workload, readiness and match performance variables. The study shows that junior female basketball players were able to cope with a congested schedule of 7 matches in 9 days irrespective of the competition context or individual differences in workload. Finally, combining objective and subjective methods to assess workload and readiness is recommended due to the weak relationships observed between these methods.     

Genome of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2

Despite the fact that multidrug-resistant Klebsiella sp. strains emerge rapidly (Xu J, et al., Adv. Mater. Res. 268-270:1954-1956, 2011) and bacteriophages have been reported to be useful in controlling these bacteria (Kumari S, Harjai K, Chhibber S, J. Med. Microbiol. 60:205-210, 2011), the complete genome sequences of only five Klebsiella phages (four siphoviruses and one myovirus) can be found in databases. In this paper, we report on the complete genome sequence of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2. With a genome size of 345,809 bp, this is the second largest myovirus and the largest Klebsiella phage sequenced to date. This phage differs substantially from other myoviruses since 411 out of 534 vB_KleM_RaK2 open reading frames have no known functions and lack any reliable database matches. Comparative analysis of the genome sequence of vB_KleM_RaK2 suggests that this phage forms a distinct phylogenetic branch within the family Myoviridae of tailed bacteriophages.     

Pyrrolidinone-bearing methylated and halogenated benzenesulfonamides as inhibitors of carbonic anhydrases

Two series of benzenesulfonamides bearing methyl groups at ortho/ortho or meta/ortho positions and a pyrrolidinone moiety at para position were synthesized and tested as inhibitors of the twelve catalytically active human carbonic anhydrase (CA) isoforms. Observed binding affinities were determined by fluorescent thermal shift assay and intrinsic binding affinities representing the binding of benzenesulfonamide anion to the Zn(II)-bound water form of CA were calculated. Introduction of dimethyl groups into benzenesulfonamide ring decreased the binding affinity to almost all CA isoforms, but gained in selectivity towards one CA isoform. A chloro group at the meta position of 2,6-dimethylbenzenesulfonamide derivatives did not influence the binding to CA I, but it increased the affinity to all other CAs, especially, CA VII and CA XIII (up to 500 fold). The compounds may be used for further development of CA inhibitors with higher selectivity to particular CA isoforms.     

Strongmen sport is associated with larger absolute heart size and impaired cardiac relaxation

This study was carried out to compare cardiac structure and function and blood lipids among Strongmen, sedentary controls, and marathoners. Echocardiography was performed, and endothelial function, blood lipids and maximal oxygen uptake were measured in 27 Caucasian adult men (8 Strongmen, 10 marathoners, 9 controls). Absolute cardiac size parameters such as left ventricular (LV) diameter and wall thickness of Strongmen were higher (p < 0.05), but relative (body surface area indexed) parameters were not different between controls and Strongmen. In Strongmen, the relative LV diameter (p < 0.05), wall thickness (p < 0.001), and LV mass index (p < 0.01) were lower than in marathoners. The absolute but not relative right ventricular diameter was larger in Strongmen as compared with controls, whereas all of the measured relative cardiac size parameters were higher in marathoners as opposed to in controls. The endothelial function and the ratio of wall thickness to chamber diameter were similar among the groups (p > 0.05). Maximal oxygen uptake of Strongmen was lower than in controls (p < 0.05) and marathoners (p < 0.001). Global diastolic LV function of Strongmen was impaired in comparison to controls (p < 0.05) and marathoners (p < 0.05). Plasma lipids were not different between Strongmen and sedentary controls, but in comparison to runners, Strongmen had higher low-density lipoprotein-cholesterol (p < 0.05) and lower high-density lipoprotein cholesterol (p < 0.01). Participation in Strongmen sport is associated with higher absolute but not relative cardiac size parameters, impaired myocardial relaxation, and low cardiorespiratory fitness. Therefore, Strongmen may demand greater attention as an extreme group of athletes with regard to cardiovascular risk.