Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

The Cu(II)-fluconazole complex revisited. Part I: Structural characteristics of the system

Protonation equilibria and Cu(II) binding processes by an antifungal agent fluconazole, α-(2,4-difluorophenyl)-α-(1H-1,2,4-triazol-1-yl-methyl)-1H-1,2,4-triazole-1-ethanol, were studied using the UV-Vis, EPR and NMR spectroscopic techniques. The protonation constant of fluconazole was determined from NMR titration and attributed to N4′ nitrogen atoms using the DFT methods. The spectroscopic data suggest that at pH as low as 0.4 the first complex is formed, in which one or two Cu(II) ions are bound to one of the nitrogen atoms (N4′) from triazole rings. Above pH 1.5 each Cu(II) ion is surrounded by two nitrogen atoms (also N4′) from two different ligand molecules, forming primary monomeric complexes and above pH = 5, both dimeric or oligomeric species occur, which is well registered by the EPR technique.The mixture of Cu(NO₃)₂ with fluconazole in a 1:1 molar ratio in a water (pH = 4.5)/ethanol solution gave crystals of [Cu₂(H₂O){(C₆H₃-2,4-F₂)(CH₂N₃C₂H₂)₂C-OH}{(C₆H₃-2,4-F₂)(CH₂N₃C₂H₂)₂C-O}(NO₃)](NO₃)₂·9(H₂O). This complex is the first example of a cupric 3D polymeric structure with a fluconazole ligand coordinated via both N2′ and N4′ atoms from the same triazole rings. At higher pH values, we obtained a binuclear complex [Cu₂(L)₂(H₂O)₂(NO₃)₂], in which the copper(II) atoms were bridged by the oxygen atoms of the deprotonated OH group of fluconazole.The hypothetical oxidative properties of this system were also examined, however it failed to generate either reactive oxygen species or DNA scission products.     

Depth-sensing analysis of cytoskeleton organization based on AFM data

Atomic force microscopy is a common technique used to determine the elastic properties of living cells. It furnishes the relative Young’s modulus, which is typically determined for indentation depths within the range 300–500 nm. Here, we present the results of depth-sensing analysis of the mechanical properties of living fibroblasts measured under physiological conditions. Distributions of the Young’s moduli were obtained for all studied cells and for every cell. The results show that for small indentation depths, histograms of the relative values of the Young’s modulus described the regions rich in the network of actin filaments. For large indentation depths, the overall stiffness of a whole cell was obtained, which was accompanied by a decrease of the modulus value. In conclusion, the results enable us to describe the non-homogeneity of the cell cytoskeleton, particularly, its contribution linked to actin filaments located beneath the cell membrane. Preliminary results showing a potential application to improve the detection of cancerous cells, have been presented for melanoma cell lines.     

Fine-scale genetic analysis of Daphnia host populations infected by two virulent parasites - strong fluctuations in clonal structure at small temporal and spatial scales

Numerous theoretical studies suggest that parasites impose a strong selection pressure on their host, driving genetic changes within host populations. Yet evidence of this process in the wild is scarce. In the present study we surveyed, using high resolution microsatellite markers, the genetic structure of cyclically parthenogenetic Daphnia hosts within two different Daphnia communities belonging to the Daphnia longispina hybrid complex. One community, consisting of a single host species, was infected with the protozoan parasite Caullerya mesnili. The second community consisted of two parental Daphnia spp. and their hybrids, and was infected with the yeast parasite Metschnikowia. Significant differences in the clonal composition between random and infected sub-samples of Daphnia were detected on several occasions within both communities, indicating that host genotypes differ in resistance to both parasites. In addition, one parental species in the multi-taxon community was consistently under-infected, compared with the other taxa. Overall, our field data confirm that infection patterns are strongly affected by host genetic composition in various Daphnia-microparasite systems. Thus, parasite-driven selection operates in natural Daphnia populations and microparasites influence the clonal structure of host populations.     

Effects of diet and temperature on condition, proximate composition and three major macro elements, Ca, P and Mg, in barbel Barbus barbus juveniles

Six feeding groups of 60 early juveniles (0.6?g) of a rheophilic cyprinid barbel Barbus barbus were reared in triplicate in 18?glass aquaria. Fish fed a commercial formulated dry diet Aller Futura were compared with those on natural food??commercially available frozen Chironomidae larvae at 17, 21 and 25°C. Daily food rations were adjusted according to fish biomass, differences in hydration between the two diets, and rearing temperature. No mortality occurred during experiment. Temperature, and especially diet, both affected whole body proximate and mineral composition of B. barbus juveniles. Under conditions of this experiment the formulated diet Aller Futura seems to satisfy the demands for calcium and magnesium, while an elevated lipid content in this diet was found and marginally deficient phosphorus content can be concluded. These resulted in a depressed content of phosphorus and total minerals in the body, and in elevated lipid content and condition coefficient in the Futura-fed fish, but no visible body deformities occurred. Advantages and limitations of the condition coefficient K were discussed. This is a non-destructive, express indicator that can be used as a supplementary tool for estimation of changes in body proximate composition in fish juveniles of similar size within a population.     

Enterococcus in wound infections: virulence and antimicrobial resistance

Enterococci, a complex group of facultative pathogens have become increasingly isolated in various hospital settings. They are considerable frequently cultured from traumatic and surgical wounds. We investigated 57 strains of the species E. faecalis, E. faecium and E. casseliflavus isolated from infected wounds. Their ability to produce virulence factors and their sensitivity to antibiotics were evaluated using phenotypic and genotyping methods. In the phenotype studies, significant portion of the isolates produced biofilm (66.7%) and gelatinase (36.8%). Nearly 30% of the strains expressed hemolytic properties. Only a few produced DNAse (15.8%) and lipase (7.0%). The genes esp, gelE, cylA, cylB, cylM and agg were detected in most of the isolates (38.6-87.7%). All the isolated enterococci were susceptible to vancomycin and were characterized by their low resistance to antibiotics, except aminoglycosides (HLR).     

Regional Differences in AIDS and Non-AIDS Related Mortality in HIV-Positive Individuals across Europe and Argentina: The EuroSIDA Study

Background

Differences in access to care and treatment have been reported in Eastern Europe, a region with one of the fastest growing HIV epidemics, compared to the rest of Europe. This analysis aimed to establish whether there are regional differences in the mortality rate of HIV-positive individuals across Europe, and Argentina.

Methods

13,310 individuals under follow-up were included in the analysis. Poisson regression investigated factors associated with the risk of death.

Findings

During 82,212 person years of follow-up (PYFU) 1,147 individuals died (mortality rate 14.0 per 1,000 PYFU (95% confidence interval [CI] 13.1–14.8). Significant differences between regions were seen in the rate of all-cause, AIDS and non-AIDS related mortality (global p<0.0001 for all three endpoints). Compared to South Europe, after adjusting for baseline demographics, laboratory measurements and treatment, a higher rate of AIDS related mortality was observed in East Europe (IRR 2.90, 95%CI 1.97–4.28, p<.0001), and a higher rate of non-AIDS related mortality in North Europe (IRR 1.51, 95%CI 1.24–1.82, p<.0001). The differences observed in North Europe decreased over calendar-time, in 2009–2011, the higher rate of non-AIDS related mortality was no longer significantly different to South Europe (IRR 1.07, 95%CI 0.66–1.75, p = 0.77). However, in 2009–2011, there remained a higher rate of AIDS-related mortality (IRR 2.41, 95%CI 1.11–5.25, p = 0.02) in East Europe compared to South Europe in adjusted analysis.

Interpretations

There are significant differences in the rate of all-cause mortality among HIV-positive individuals across different regions of Europe and Argentina. Individuals in Eastern Europe had an increased risk of mortality from AIDS related causes and individuals in North Europe had the highest rate of non-AIDS related mortality. These findings are important for understanding and reviewing HIV treatment strategies and policies across the European region.     

Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes

Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.     

A quantum-chemical study of the binding ability of βXaaHisGlyHis towards copper(II) ion

The present study analyzed binding of Cu²⁺ to tetrapeptides in water solution at several levels of theoretical approximation. The methods used to study the energetic and structural properties of the complexes in question include semiempirical hamiltonians, density functional theory as well as ab initio approaches including electron correlation effects. In order to shed light on the character of interactions between Cu²⁺ and peptides, which are expected to be mainly electrostatic in nature, decomposition of interaction energy into physically meaningful components was applied.     

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.