Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS-7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose-dependent and time-dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent-resistant cellular membranes showed that sticholysin II colocalizes with caveolin-1 in fractions corresponding to raft-like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane-associated protein. Toxin binding to raft-like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS-7 cells through interaction with membrane domains which behave like cellular rafts.     

Interleukin-6 is a potential biomarker for severe pandemic H1N1 influenza A infection

Pandemic H1N1 influenza A (H1N1pdm) is currently a dominant circulating influenza strain worldwide. Severe cases of H1N1pdm infection are characterized by prolonged activation of the immune response, yet the specific role of inflammatory mediators in disease is poorly understood. The inflammatory cytokine IL-6 has been implicated in both seasonal and severe pandemic H1N1 influenza A (H1N1pdm) infection. Here, we investigated the role of IL-6 in severe H1N1pdm infection. We found IL-6 to be an important feature of the host response in both humans and mice infected with H1N1pdm. Elevated levels of IL-6 were associated with severe disease in patients hospitalized with H1N1pdm infection. Notably, serum IL-6 levels associated strongly with the requirement of critical care admission and were predictive of fatal outcome. In C57BL/6J, BALB/cJ, and B6129SF2/J mice, infection with A/Mexico/4108/2009 (H1N1pdm) consistently triggered severe disease and increased IL-6 levels in both lung and serum. Furthermore, in our lethal C57BL/6J mouse model of H1N1pdm infection, global gene expression analysis indicated a pronounced IL-6 associated inflammatory response. Subsequently, we examined disease and outcome in IL-6 deficient mice infected with H1N1pdm. No significant differences in survival, weight loss, viral load, or pathology were observed between IL-6 deficient and wild-type mice following infection. Taken together, our findings suggest IL-6 may be a potential disease severity biomarker, but may not be a suitable therapeutic target in cases of severe H1N1pdm infection due to our mouse data.     

Thermal degradation of allicin in garlic extracts and its implication on the inhibition of the in-vitro growth of Helicobacter pylori

Allicin, the main active principle related to Allium sativum chemistry, is considered to be responsible for the bacteriostatic properties of garlic. The work described here has demonstrated the direct implication of the allicin present in solvent-free garlic extracts obtained with ethanol (ethanolic garlic extract, EGE) and acetone (acetonic garlic extract, AGE) in the inhibition of the in-vitro growth of Helicobacter pylori (Hp), the bacterium responsible for serious gastric diseases such as ulcers and even gastric cancer. The evolution of allicin concentration as a function of time and temperature has been the subject of a kinetic study. The reaction order, activation energy, and preexponential factor (in accordance with Arrhenius theory) have been determined for the decomposition process of allicin in these organic media. First-order decomposition, an activation energy of 97.4 kJ/mol, and an Arrhenius preexponential factor of 8.9 x 10(10) s(-1) have been determined for allicin in EGE. For allicin in AGE the kinetic order determined was 1.5, the activation energy 184.5 kJ/mol, and the preexponential factor 3.1 x 10(24) s(-1) (mg/L)(-0.5). The presence or absence of allicin in these garlic products was found to be crucial for the inhibition of the in-vitro growth of Hp, as demonstrated by microbiological analysis for AGE. A relationship has been identified between the effectiveness and durability of the anti-Hp properties shown by AGE and the allicin content of these products. The bacteriostatic properties were active for up to 10 months if the samples were maintained at 6 degrees C.     

MACROALGAE SURVIVE DIGESTION BY FISHES1

Small individuals (<15 cm long) of the clingfish Sicyases sanguineus consume as many as 18 seaweed species in Central Chile. Enteromorpha sp. was the only species able to survive digestion by fish recently collected from the field. Enteromorpha compressa and Gelidium chilense also survived by tissue regeneration when offered as food in the laboratory, but only E. compressa showed stimulation of swarmer production. In general, our results on a vertebrate species reproduce previous findings on diverse invertebrate species. Since fish are highly motile, their capacity to disperse algal species by defecation might be of great ecological importance.     

First phylogenetic analysis of the family Neriidae (Diptera), with a study on the issue of scaling continuous characters

Neriidae are a small family of acalyptratae flies, mostly distributed in the tropics. Very little is known about their biology, and the evolutionary relationships among species have never been evaluated. We perform the first comprehensive phylogenetic analysis of the family, including 48 species from all biogeographic regions inhabited, as well as five species of Micropezidae and one Cypselosomatidae as outgroups. We build a morphological data matrix of 194 characters, including 72 continuous characters. We first explore ways to deal with the issue of scaling continuous characters, including rescaling ranges to unity and using implied weighting. We find that both strategies result in very different phylogenetic hypotheses, and that implied weighting reduces the problem of scaling, but only partially. Furthermore, using implied weighting after rescaling characters improves the congruence between partitions and results in higher values of group support. With respect to the Neriidae, we confirm the monophyly of the family and of most its genera, although we do not obtain any of the currently accepted suprageneric groups. We propose to restrict the Eoneria and Nerius groups exclusively to the Neotropical fauna, and synonymize Glyphidops subgenus Oncopsia Enderlein with Glyphidops subgenus Glyphidops Enderlein, eliminating the subgeneric divisions. This revised phylogeny presents a striking biogeographic consistency, and shows that previous main divisions of the family were based on events of convergence.     

Late toxicity for prostate cancer patients treated with hypofractionated helical tomotherapy

AimThe purpose of this study is to evaluate the long term tolerability of hypofractionated helical tomotherapy (HT) in localized prostate cancer patients.BackgroundPrevious hypofractionated schedules with conventional RT were associated with excessive toxicity, likely due to inadequate sophistication of treatment delivery. There are few data about late toxicity after HT.Materials and methodsWe evaluated 38 patients with primary adenocarcinoma of the prostate. There were 9 (24%), 15 (39%), and 14 (37%) patients with high, intermediate, and low risk, respectively. Patients were treated with hypofractionated HT from May 2008 to February 2011. Hypofractionation regimens included: 68.04 Gy at 2.52 Gy/fraction (N = 25; 66%), 70 Gy at 2.5 Gy/fraction (N = 4; 11%) and 70.2 Gy at 2.6 Gy/fraction (N = 9; 23%). Late genitourinary (GU) and gastrointestinal (GI) toxicity was scored using the Radiation Therapy Oncology Group scoring system.ResultsMedian age at diagnosis was 70 years (range 49–80) and median follow-up, 5.8 years. Late grade 1, 2 and 3 GI toxicity were 13%, 24%, and 2.6%, respectively. Late grade 1, 2, 3 GU toxicity were 29%, 21%, and 8%, respectively. Sexual toxicity was evaluated in 19 patients to be grade 1, 2 in 11% and grade 3 in 16%. Multivariate analysis showed that patients with higher values of rectum V50 associated with late GI toxicity (P = 0.025). Patients with PSA ≤8 (P = 0.048) or comorbidities (P = 0.013) at diagnosis were associated with higher late GU toxicity. Additionally, PSA ≤8 also associated with moderate (grade ≥2) late GU toxicity in the multivariate analysis (P = 0.028).ConclusionsHypofractionated HT can be delivered safely with limited rates of moderate and severe late toxicity. The proportion of the rectum that receives a moderate and high dose, having comorbidities, and PSA at diagnosis seem to associate with long term toxicity.     

Effect of emulsified feeding of oily substrate via submerged ceramic membranes on surfactant production in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> fermentation

Aqueous two-phase systems often face mass transfer limitations due to very poor miscibility of the fluids, and to enhance the homogeneity (or emulsification) in the reaction volume, high energy inputs are required which result in high shear forces in the culture medium. For the purposes of emulsification, microporous systems have advantages over other conventional methods due to mild operating conditions and narrow droplet-size distribution. In this study, emulsification within the culture volume was achieved by feeding the oily substrate (dispersed phase) into the aqueous medium (dispersion phase) via ceramic membranes integrated in the bioreactor. The method was investigated for bioprocesses aimed at producing rhamnolipids and polyhydroxyalkanoates (PHA). Better homogenization of the mixed volume and hence improved consumption of oily substrate was successful. Surfactants are produced by various bacterial cultures, especially Pseudomonas aeruginosa species, when oil is present as the only carbon source. Are surfactants produced only as a result of bacteria feeding on the oily substrate, or as a requirement to feed on the oily substrate, owing to their surface-active characteristics? This paper also intends to draw some conclusions in this respect.     

Pulmonary embolism and 3-month outcomes in 4036 patients with venous thromboembolism and chronic obstructive pulmonary disease: data from the RIETE registry

Background

Patients with chronic obstructive pulmonary disease (COPD) have a modified clinical presentation of venous thromboembolism (VTE) but also a worse prognosis than non-COPD patients with VTE. As it may induce therapeutic modifications, we evaluated the influence of the initial VTE presentation on the 3-month outcomes in COPD patients.

Methods

COPD patients included in the on-going world-wide RIETE Registry were studied. The rate of pulmonary embolism (PE), major bleeding and death during the first 3 months in COPD patients were compared according to their initial clinical presentation (acute PE or deep vein thrombosis (DVT)).

Results

Of the 4036 COPD patients included, 2452 (61%; 95% CI: 59.2-62.3) initially presented with PE. PE as the first VTE recurrence occurred in 116 patients, major bleeding in 101 patients and mortality in 443 patients (Fatal PE: first cause of death). Multivariate analysis confirmed that presenting with PE was associated with higher risk of VTE recurrence as PE (OR, 2.04; 95% CI: 1.11-3.72) and higher risk of fatal PE (OR, 7.77; 95% CI: 2.92-15.7).

Conclusions

COPD patients presenting with PE have an increased risk for PE recurrences and fatal PE compared with those presenting with DVT alone. More efficient therapy is needed in this subtype of patients.     

Retinoic acid binds to the C2-domain of protein kinase C(alpha)

Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.     

Effects of sex, body size, temperature, and location on the antipredator tactics of free-ranging gartersnakes (Thamnophis sirtalis, Colubridae)

Gartersnakes (Thamnophis sirtalis parietalis) in southern Manitoba are subject to intense predation (primarily by crows) duringtheir spring breeding season. The huge numbers of snakes providea unique opportunity to quantify behavioral traits. We simulatedpredator attacks by "pecking" more than 500 free-ranging snakes,to explore the determinants of snake response. Snakes respondedto a human finger in the same way as they did to a more realisticstimulus (a model crow). A snake's response to attack dependedon several factors, which interacted in complex ways. The primaryinfluences on response were body temperature (warmer snakes tended to flee, whereas colder snakes remained cryptic or flattenedand/or gaped and struck) and sex (males were more likely toflee). Responses also depended on microhabitat (i.e., insidethe winter den versus in adjacent grassland) and on the snake'sprior activity (e.g., courting snakes often ignored our closeapproach). These factors interacted in significant ways; for example, snakes outside the den were smaller and warmer thanthose inside, male snakes were smaller and warmer than females,and mean body temperatures were higher in larger snakes withineach sex. Thus, a snake's body size and its location affectedits defensive response indirectly (via their influence on bodytemperature). Our results differ from those of previous studiesand suggest that antipredator responses in these animals dependin a flexible and complex way upon biotic and abiotic variables.Interactions among these variables also must be consideredbefore we can identify underlying causal processes.