Bacterial polysaccharides suppress induced innate immunity by calcium chelation

Bacterial pathogens and symbionts must suppress or negate host innate immunity. However, pathogens release conserved oligomeric and polymeric molecules or MAMPs (Microbial Associated Molecular Patterns), which elicit host defenses [1], [2] and [3]. Extracellular polysaccharides (EPSs) are key virulence factors in plant and animal pathogenesis, but their precise function in establishing basic compatibility remains unclear [4], [5], [6] and [7]. Here, we show that EPSs suppress MAMP-induced signaling in plants through their polyanionic nature [4] and consequent ability to chelate divalent calcium ions [8]. In plants, Ca2+ ion influx to the cytosol from the apoplast (where bacteria multiply [4], [5] and [9]) is a prerequisite for activation of myriad defenses by MAMPs [10]. We show that EPSs from diverse plant and animal pathogens and symbionts bind calcium. EPS-defective mutants or pure MAMPs, such as the flagellin peptide flg22, elicit calcium influx, expression of host defense genes, and downstream resistance. Furthermore, EPSs, produced by wild-type strains or purified, suppress induced responses but do not block flg22-receptor binding in Arabidopsis cells. EPS production was confirmed in planta, and the amounts in bacterial biofilms greatly exceed those required for binding of apoplastic calcium. These data reveal a novel, fundamental role for bacterial EPS in disease establishment, encouraging novel control strategies.     

Randomised Controlled Trials May Underestimate Drug Effects: Balanced Placebo Trial Design

Background

It is an inherent assumption in randomised controlled trials that the drug effect can be estimated by subtracting the response during placebo from the response during active drug treatment.

Objective

To test the assumption of additivity. The primary hypothesis was that the total treatment effect is smaller than the sum of the drug effect and the placebo effect. The secondary hypothesis was that non-additivity was most pronounced in participants with large placebo effects.

Methods

We used a within-subject randomised blinded balanced placebo design and included 48 healthy volunteers (50% males), mean (SD) age 23.4 (6.2) years. Experimental pain was induced by injections of hypertonic saline into the masseter muscle. Participants received four injections with hypertonic saline along with lidocaine or matching placebo in randomised order: A: received hypertonic saline/told hypertonic saline; B: received hypertonic saline+lidocaine/told hypertonic saline; C: received hypertonic saline+placebo/told hypertonic saline+pain killer; D: received hypertonic saline+lidocaine/told hypertonic saline+pain killer. The primary outcome measure was the area under the curve (AUC, mm²) of pain intensity during injections.

Results

There was a significant difference between the sum of the drug effect and the placebo effect (mean AUC 6279 mm² (95% CI, 4936–7622)) and the total treatment effect (mean AUC 5455 mm² (95% CI, 4585–6324)) (P = 0.049). This difference was larger for participants with large versus small placebo effects (P = 0.015), and the difference correlated significantly with the size of the placebo effect (r = 0.65, P = 0.006).

Conclusion

Although this study examined placebo effects and not the whole placebo response as in randomised controlled trials, it does suggest that the additivity assumption may be incorrect, and that the estimated drug effects in randomised controlled trials may be underestimated, particularly in studies reporting large placebo responses. The implications for randomised controlled trials and systematic reviews need to be discussed.     

Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors

We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.     

Effects of exposure to 17alpha-ethinylestradiol during early development on sexual differentiation and induction of vitellogenin in zebrafish (Danio rerio)

The effect of acute stress on plasma beta-corticosterone (B), testosterone (T) and estradiol-17beta (E2) concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observed in alligators from all of the sites after 2 h of capture although females from the intermediate contaminant site exhibited a significantly lower percentage increase in B than females from the other two sites. Males from the site with the highest contaminant levels exhibited elevated initial B concentrations relative to the other sites. This pattern was not observed after 2 h of restraint. Females from the highest contaminant site exhibited depressed initial T when compared to the other sites although this pattern was not observed after 2 h of restraint. Neither E2 nor T decreased after 2 h in females, whereas T concentrations decreased in all males over the same time period. The variance associated with these endpoints was also examined to determine whether it could serve as a more sensitive marker for perturbations of the endocrine system and stress response. Females from the higher and intermediate contaminant sites exhibited the lowest and highest standard errors (respectively) associated with 2 h plasma B concentrations with no differences among mean concentrations suggesting a perturbation of the stress response in these animals that was not detected by examining the means. We concluded that the environmental contaminants could be acting as stressors, leading to the observed differences.     

Chemotaxis ofRhizobium sp. Towards root exudate ofCicer Arietinum L.

Summary Root exudate from seedlings ofCicer arietinum L. was collected in a chamber under aseptic conditions. The exudate was fractionated into anion, cation and neutral fractions. The anionic fraction was made up of galacturonic acid, gluconic acid, mannuronic acid and two unidentified compounds withR _f values 0.56 and 0.62. The cationic fraction contained alanine, arginine, aspartic acid, cystine, glycine, histidine, isoleucine, leucine, lysine and serine. The neutral fraction was made up of arabinose, galactose, glucose, ribose and xylose. The amino acids contributed to the bulk of the root exudate. The ratio of anionic, cationic and neutral fraction was 1∶7∶2. The crude root exudate was tested for its chemotactic ability using the capillary tube method. It was highly chemotactic for theRhizobium sp. The individual fractions and their various combinations were tested for chemotaxis. The chemotactic response of the Cicer strain of Rhizobium was least with anionic fraction most with cationic fraction and intermediate with neutral fraction. Maximum chemotactic response among the fractional combinations was obtained with all the three fractions and least with cationic plus neutral factions. Individual compounds constituting the various fractions were also tried for their ability to elicit chemotactic response. The organism exhibited maximum positive chemotactic response to histidine and negative response to alanine among the amino acids and to glucose and gluconic acid among the sugars and sugar acids.     

Ketogenic diet is antiepileptogenic in pentylenetetrazole kindled mice and decrease levels of N-acylethanolamines in hippocampus

The ketogenic diet (KD) is used for the treatment of refractory epilepsy in children, however, the mechanism(s) remains largely unknown. Also, the antiepileptogenic potential in animal models of epilepsy has been poorly addressed. Activation of cannabinoid type-1 receptor (CB₁-R) upon seizure activity may mediate neuroprotection as may several N-acylethanolamines. It is unknown how the KD interfere with the endocannabinoid system.We investigated the antiepileptogenic potential of the KD in the pentylenetetrazole kindling model in young mice and measured the hippocampal levels of CB₁-R by Western blot and of endocannabinoids and N-acylethanolamines by mass spectrometry.The KD significantly decreased incidence and severity of seizures, and significantly increased the latency to clonic convulsions. There were no changes in levels of endocannabinoids or CB₁-R expression by either seizure activity or type of diet. The level of oleoylethanolamide as well as the sum of N-acylethanolamines were significantly decreased by the KD, but were unaffected by seizure activity.The study shows that the KD had clear antiepileptogenic properties in the pentylenetetrazole kindling model and does not support a role of endocannabinoids in this model. The significance of the decreased hippocampal level of oleoylethanolamide awaits further studies.     

Description of Ulvella elegans sp. nov. and U. islandica sp. nov. (Ulvellaceae,Ulvophyceae) from Iceland – a study based on morphology of species in culture and tufA gene sequences

Two new Ulvella species, U. elegans R. Nielsen & K. Gunnarsson and U. islandica R. Nielsen & K. Gunnarsson are described. These microfilamentous marine green algae were found in the sublittoral zone in northern Iceland, epiphytic on Euthora cristata and associated with a calcareous polychaete tube, respectively. Unialgal cultures were established from field-collected material for morphological observations. In culture, Ulvella elegans was characterized by rosettes of monostromatic pseudoparenchyma consisting of radiating filaments with a margin of mutually free filaments. Each cell had one pyrenoid. Hairs were not observed. Ulvella islandica had a heterotrichous morphology, consisting of dense tufts of upright broad branches and much narrower, rhizoid-like branches. Acrochaete-type hairs occurred; these are hyaline non-septate merocytic extensions from a more or less bulbous base, which may be separated from the vegetative cell below. Most cells had one pyrenoid except for a few broad cells which had two or three. In a phylogenetic reconstruction based on the chloroplast-encoded tufA gene, the sequences for the two species were clearly distinct from any other Ulvella sequence available for this gene. Ulvella islandica was placed in a clade together with U. lens, U. wittrockii, U. reticulata and U. pseudorepens. Ulvella elegans occupied a branch deep in the phylogeny but the position was poorly supported.     

A taxonomic revision of Secale (Triticeae, Poaceae)

Several taxa have previously been recognized within Secale , but most of them are difficult or even impossible to distinguish morphologically. We recognize only three species: S. sylvestre, S. strictum , and S. cereale. Secale strictum has priority over S. montanum and has two subspecies, ssp. strictum and ssp. africanum , and two varieties within ssp. strictum , van strictum and var. ciliatoglume comb. nov. Secale cereale is also treated as having two subspecies. The cultivated taxa, marked by their tough rachises, are placed in ssp. cereale and the wild or weedy taxa that have more or less fragile rachis, in ssp. ancestrale. A complete synonymy is given for S. cereale , but typification has been omitted because, in many instances, type material does not exist or has been impossible to trace.     

A versatile and tunable coating strategy allows control of nanocrystal delivery to cell types in the liver

There are many liver diseases that could be treated with delivery of therapeutics such as DNA, proteins, or small molecules. Nanoparticles are often proposed as delivery vectors for such therapeutics; however, achieving nanoparticle accumulations in the therapeutically relevant hepatocytes is challenging. In order to address this issue, we have synthesized polymer coated, fluorescent iron oxide nanoparticles that bind and deliver DNA, as well as produce contrast for magnetic resonance imaging (MRI), fluorescence imaging, and transmission electron microscopy (TEM). The composition of the coating can be varied in a facile manner to increase the quantity of poly(ethylene glycol) (PEG) from 0% to 5%, 10%, or 25%, with the aim of reducing opsonization but maintaining DNA binding. We investigated the effect of the nanoparticle coating on DNA binding, cell uptake, cell transfection, and opsonization in vitro. Furthermore, we exploited MRI, fluorescence imaging, and TEM to investigate the distribution of the different formulations in the liver of mice. While MRI and fluorescence imaging showed that each formulation was heavily taken up in the liver at 24 h, the 10% PEG formulation was taken up by the therapeutically relevant hepatocytes more extensively than either the 0% PEG or the 5% PEG, indicating its potential for delivery of therapeutics to the liver.