Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.     

Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome

Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.Prokaryotes are highly diverse (33). One of the explanations of this diversity is the high extinction rate, due to genetic aggression, which leads to the clearance of ecological niches and, as a result, may allow new prokaryotic species to emerge. In the absence of host defense, viral infection of prokaryotic colonies results in colony extinction or the fixation of a fraction of the invader''s genetic material in the host genome, profoundly affecting the life cycle of the host (32). Thus, bacteria and archaea have developed various kinds of defense mechanisms to resist this pressure; the best studied of these mechanisms is restriction-modification systems (4).Along with well-known prokaryotic defense mechanisms, such as rapid evolution of cell receptors or the use of restriction-modification or toxin-antitoxin systems (see, e.g., references 6, 21, and 25), newly discovered clustered regularly interspaced palindromic repeat (CRISPR) systems seem to play an important role in protecting the cell from archaeal virus or bacteriophage assaults (reviewed in reference 36). A typical CRISPR system is a genetic locus comprising CRISPR-associated (cas) genes coding for proteins of several distinct functional classes (8, 19, 29) and a CRISPR cassette. A CRISPR cassette is formed by almost identical direct repeats with an average length of 32 nucleotides (nt), which are separated by similarly sized, unique spacers. A considerable proportion of spacers is similar to known phage or virus sequences, suggesting that the system is involved in antivirus defense (8, 29, 31). This involvement was experimentally demonstrated when a CRISPR system was shown to be essential for cell survival after invasion by foreign DNA (5). The mechanism is thought to be analogous to eukaryotic RNA interference (29), but it has not been characterized in detail yet.CRISPR cassettes retain information that could be used to reveal the evolutionary history of individual systems. First, it has been shown that CRISPR-associated genes could be divided into eight subtypes according to operon organization and gene phylogeny (19). Second, the repeats of different CRISPR cassettes may be similar, which might indicate a common origin of such cassettes. The first attempt to cluster CRISPR cassettes by the similarity of repeat sequences resulted in 12 clusters (27). In that study, the cassettes were obtained by the application of PILER-CR to completely sequenced genomes. Third, pairwise comparison of spacers could also reveal the specific evolutionary history of individual CRISPR cassettes.So far, most large-scale studies of CRISPR systems have been restricted to well-studied organisms with completely sequenced genomes (5, 9, 20, 28, 30). However, the dynamic interaction between viruses or phages and microorganisms in natural environments is of particular interest (2, 10, 15, 23, 35, 38, 40-42). It may be studied using CRISPRs in a metagenome, that is, sequenced DNA fragments collected in one geographical location and therefore representing one ecological niche with all its inhabitants. This approach is interesting for two reasons. First, metagenomic samples provide a common census of coexisting organisms, i.e., in many cases, both the infecting viruses and phages and their victims. Second, most bacteria and archaea from metagenomic samples cannot be cultivated, and hence little is known about their CRISPR systems.To date, three studies have considered host-virus interactions in metagenomes. One study used two thermophilic Synechococcus isolates from microbial mats in hot springs at Yellowstone National Park to demonstrate fast coevolution of the host and phage genomes (22). Two studies described archaeal and bacterial interactions with viruses and phages, respectively, in acidophilic biofilms (2, 39). All environmental communities analyzed so far are extreme and are dominated by few species. Natural samples containing many diverse coexisting organisms may arguably be more interesting.The largest available metagenome, produced by the Sorcerer II Global Ocean Sampling (GOS) expedition, comprises samples of genetic material collected from more than 50 geographical locations of the Pacific and Atlantic oceans (34). This variety provides an opportunity to study the evolution of phage-host interactions reflected in CRISPRs.Three algorithms, PILER-CR (14), the CRISPR recognition tool (CRT) (7), and CRISPRFinder (18), have been developed as tools for the discovery of new CRISPR cassettes. All these algorithms define candidate CRISPR cassette sequences as short direct repeats separated by short unique spacers; they then use a variety of standard repeat-finding techniques. However, the implementation of specific details is different.PILER-CR constructs local alignments of the input sequence to itself; each hit between two close regions is a candidate for an alignment of a repeat with its neighbor copy. In terms of dynamic programming, taking into account the repeat structure of a CRISPR cassette implies looking for hits only within a relatively narrow band around the main diagonal of the dot plot. This process is followed by several refinement steps.CRT does not use alignments to identify candidate repeats; rather, it derives them directly from the analysis of an input sequence. It is based on finding series of short repeats of a specified length (searching for exact k-mer matches) and then extending these repeats (increasing k-mer length) while allowing for a certain level of mismatches.Finally, CRISPRFinder is based on a suffix-tree-based algorithm for repeat discovery, again with additional refinement.All three algorithms were used for the CRISPR cassette search in this study.     

Glucose and mannitol diffusion in human dura mater

An in vitro experimental study of the control of the human dura mater optical properties at administration of aqueous solutions of glucose and mannitol has been presented. The significant increase of the dura mater optical transmittance under action of immersion liquids has been demonstrated. Diffusion coefficients of glucose and mannitol in the human dura mater tissue at 20 degrees C have been estimated as (1.63 +/- 0.29) x 10(-6)cm(2)/s and as (1.31 +/- 0.41) x 10(-6) cm(2)/s, respectively. Experiments show that administration of immersion liquids allows for the effective control of tissue optical characteristics that make dura mater more transparent, thereby increasing the ability of light penetration through the tissue.     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Oncolytic Vesicular Stomatitis Virus in an Immunocompetent Model of MUC1-Positive or MUC1-Null Pancreatic Ductal Adenocarcinoma

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against various malignancies. Here, for the first time, we tested VSV in vitro and in vivo in a clinically relevant, immunocompetent mouse model of pancreatic ductal adenocarcinoma (PDA). Our system allows the study of virotherapy against PDA in the context of overexpression (80% of PDA patients) or no expression of human mucin 1 (MUC1), a major marker for poor prognosis in patients. In vitro, we tested three VSV recombinants, wild-type VSV, VSV-green fluorescent protein (VSV-GFP), and a safe oncolytic VSV-ΔM51-GFP, against five mouse PDA cell lines that either expressed human MUC1 or were MUC1 null. All viruses demonstrated significant oncolytic abilities independent of MUC1 expression, although VSV-ΔM51-GFP was somewhat less effective in two PDA cell lines. In vivo administration of VSV-ΔM51-GFP resulted in significant reduction of tumor growth for tested mouse PDA xenografts (+MUC1 or MUC1 null), and antitumor efficacy was further improved when the virus was combined with the chemotherapeutic drug gemcitabine. The antitumor effect was transient in all tested groups. The developed system can be used to study therapies involving various oncolytic viruses and chemotherapeutics, with the goal of inducing tumor-specific immunity while preventing premature virus clearance.     

Antimicrobial potential of deep surface sediment associated bacteria from the Sea of Japan

The aim of this study was to survey microorganisms from the deep surface sediment samples collected from the Sea of Japan and to screen them for antimicrobial and antagonistic effects. Phylogenetic analysis revealed most isolates sharing 98–100 % sequence similarity to recognized species, including those recovered previously from marine or saline environments. Alteromonas, Halomonas, Marinobacter, Pseudoalteromonas, Salinicola, within the class Gammaproteobacteria, Sulfitobacter (Alphaproteobacteria), Bacillus, Paenibacillus and Paenisporosarcina (Firmicutes), Nocardiopsis and Streptomyces (Actinobacteria) occurred abundantly in all sediment samples. Antimicrobial screening revealed twenty three strains (13 %) capable to inhibit growth of one to eight test cultures and deep sediment isolates. Based on phylogenetic analysis mostly active strains belonged to the genera Bacillus, Brevibacillus, Nocardiopsis, Paenibacillus and Streptomyces. Antimicrobial substances (1–3) were isolated from strain Paenibacillus sp. Sl 79w showing a high inhibitory activity. On the basis of combined spectral analyses (IR, UV, ¹H and ¹³C NMR) the compounds 1, 2 and 3 with [M + H]⁺ at 409.1 and 409.2 m/z, and with [M + Na]⁺ at 822.5 m/z were found to have a carbon skeleton of isocoumarin and peptide antibiotics, respectively. Our findings demonstrated that the deep surface sediments of the Sea of Japan represent an untapped source of diverse microorganisms capable of antimicrobial metabolite production.     

How fast is fast? Eco‐evolutionary dynamics and rates of change in populations and phenotypes

It is increasingly recognized that evolution may occur in ecological time. It is not clear, however, how fast evolution – or phenotypic change more generally – may be in comparison with the associated ecology, or whether systems with fast ecological dynamics generally have relatively fast rates of phenotypic change. We developed a new dataset on standardized rates of change in population size and phenotypic traits for a wide range of species and taxonomic groups. We show that rates of change in phenotypes are generally no more than 2/3, and on average about 1/4, the concurrent rates of change in population size. There was no relationship between rates of population change and rates of phenotypic change across systems. We also found that the variance of both phenotypic and ecological rates increased with the mean across studies following a power law with an exponent of two, while temporal variation in phenotypic rates was lower than in ecological rates. Our results are consistent with the view that ecology and evolution may occur at similar time scales, but clarify that only rarely do populations change as fast in traits as they do in abundance.     

Mathematical frameworks for phenotypical selection and epistasis

A mathematical approach to interactions between genotypes and phenotypes in a multilocus multiallele population is developed. No a priori information on a fitness function is required. In particular, some structural definitions of epistasis and the position effect are given in terms of a decomposition of phenotypical structures. On this base a distance to the additive non-epistasis is introduced and an explicit formula for it is obtained. A class of phenotypical structures including multilocus dominance is described in terms of directed graphs. The evolutionary equations are adjusted to a fitness function compatible with a phenotypical structure. Some results on the finiteness of the equilibria set are presented.     

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.