Mycolactone-dependent depletion of endothelial cell thrombomodulin is strongly associated with fibrin deposition in Buruli ulcer lesions

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tisischemia could contribute to the development of the tissue necrosis seen in BU lesions

Solulin reduces infarct volume and regulates gene-expression in transient middle cerebral artery occlusion in rats

<p>Abstract</p> <p>Background</p> <p>Thrombolysis after acute ischemic stroke has only proven to be beneficial in a subset of patients. The soluble recombinant analogue of human thrombomodulin, Solulin, was studied in an <it>in vivo </it>rat model of acute ischemic stroke.</p> <p>Methods</p> <p>Male SD rats were subjected to 2 hrs of transient middle cerebral artery occlusion (tMCAO). Rats treated with Solulin intravenously shortly before reperfusion were compared to rats receiving normal saline i.v. with respect to infarct volumes, neurological deficits and mortality. Gene expression of IL-6, IL-1β, TNF-α, MMP-9, CD11B and GFAP were semiquantitatively analyzed by rtPCR of the penumbra.</p> <p>Results</p> <p>24 hrs after reperfusion, rats were neurologically tested, euthanized and infarct volumes determined. Solulin significantly reduced mean total (p = 0.001), cortical (p = 0.002), and basal ganglia (p = 0.036) infarct volumes. Hippocampal infarct volumes (p = 0.191) were not significantly affected. Solulin significantly downregulated the expression of IL-1β (79%; p < 0.001), TNF-α (59%; p = 0.001), IL-6 (47%; p = 0.04), and CD11B (49%; p = 0.001) in the infarcted cortex compared to controls.</p> <p>Conclusions</p> <p>Solulin reduced mean total, cortical and basal ganglia infarct volumes and regulated a subset of cytokines and proteases after tMCAO suggesting the potency of this compound for therapeutic interventions.</p

The relationship of air pollution and surrogate markers of endothelial dysfunction in a population-based sample of children

<p>Abstract</p> <p>Background</p> <p>This study aimed to assess the relationship of air pollution and plasma surrogate markers of endothelial dysfunction in the pediatric age group.</p> <p>Methods</p> <p>This cross-sectional study was conducted in 2009-2010 among 125 participants aged 10-18 years. They were randomly selected from different areas of Isfahan city, the second large and air-polluted city in Iran. The association of air pollutants' levels with serum thrombomodulin (TM) and tissue factor (TF) was determined after adjustment for age, gender, anthropometric measures, dietary and physical activity habits.</p> <p>Results</p> <p>Data of 118 participants was complete and was analyzed. The mean age was 12.79 (2.35) years. The mean pollution standards index (PSI) value was at moderate level, the mean particular matter measuring up to 10 μm (PM₁₀) was more than twice the normal level. Multiple linear regression analysis showed that TF had significant relationship with all air pollutants except than carbon monoxide, and TM had significant inverse relationship with ozone. The odds ratio of elevated TF was significantly higher in the upper vs. the lowest quartiles of PM₁₀, ozone and PSI. The corresponding figures were in opposite direction for TM.</p> <p>Conclusions</p> <p>The relationship of air pollutants with endothelial dysfunction and pro-coagulant state can be an important factor in the development of atherosclerosis from early life. This finding should be confirmed in future longitudinal studies. Concerns about the harmful effects of air pollution on children's health should be considered a top priority for public health policy; it should be underscored in primordial and primary prevention of chronic diseases.</p

First RNA-seq approach to study fruit set and parthenocarpy in zucchini (Cucurbita pepo L.)

[EN] Background: Zucchini fruit set can be limited due to unfavourable environmental conditions in off-seasons crops that caused ineffective pollination/fertilization. Parthenocarpy, the natural or artificial fruit development without fertilization, has been recognized as an important trait to avoid this problem, and is related to auxin signalling. Nevertheless, differences found in transcriptome analysis during early fruit development of zucchini suggest that other complementary pathways could regulate fruit formation in parthenocarpic cultivars of this species. The development of next-generation sequencing technologies (NGS) as RNA-sequencing (RNA-seq) opens a new horizon for mapping and quantifying transcriptome to understand the molecular basis of pathways that could regulate parthenocarpy in this species. The aim of the current study was to analyze fruit transcriptome of two cultivars of zucchini, a non-parthenocarpic cultivar and a parthenocarpic cultivar, in an attempt to identify key genes involved in parthenocarpy. Results: RNA-seq analysis of six libraries (unpollinated, pollinated and auxin treated fruit in a non-parthenocarpic and parthenocarpic cultivar) was performed mapping to a new version of C. pepo transcriptome, with a mean of 92% success rate of mapping. In the non-parthenocarpic cultivar, 6479 and 2186 genes were differentially expressed (DEGs) in pollinated fruit and auxin treated fruit, respectively. In the parthenocarpic cultivar, 10,497 in pollinated fruit and 5718 in auxin treated fruit. A comparison between transcriptome of the unpollinated fruit for each cultivar has been performed determining that 6120 genes were differentially expressed. Annotation analysis of these DEGs revealed that cell cycle, regulation of transcription, carbohydrate metabolism and coordination between auxin, ethylene and gibberellin were enriched biological processes during pollinated and parthenocarpic fruit set. Conclusion: This analysis revealed the important role of hormones during fruit set, establishing the activating role of auxins and gibberellins against the inhibitory role of ethylene and different candidate genes that could be useful as markers for parthenocarpic selection in the current breeding programs of zucchini.Research worked is supported by the project RTA2014-00078 from the Spanish Institute of Agronomy Research INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria) and also PP.AVA.AVA201601.7, FEDER y FSE (Programa Operativo FSE de Andalucia 2007-2013 "Andalucia se mueve con Europa"). TPV is supported by a FPI scholarship from RTA2011-00044-C02-01/02 project of INIA. 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Structure-Based Predictive Models for Allosteric Hot Spots

In allostery, a binding event at one site in a protein modulates the behavior of a distant site. Identifying residues that relay the signal between sites remains a challenge. We have developed predictive models using support-vector machines, a widely used machine-learning method. The training data set consisted of residues classified as either hotspots or non-hotspots based on experimental characterization of point mutations from a diverse set of allosteric proteins. Each residue had an associated set of calculated features. Two sets of features were used, one consisting of dynamical, structural, network, and informatic measures, and another of structural measures defined by Daily and Gray [1]. The resulting models performed well on an independent data set consisting of hotspots and non-hotspots from five allosteric proteins. For the independent data set, our top 10 models using Feature Set 1 recalled 68–81% of known hotspots, and among total hotspot predictions, 58–67% were actual hotspots. Hence, these models have precision P = 58–67% and recall R = 68–81%. The corresponding models for Feature Set 2 had P = 55–59% and R = 81–92%. We combined the features from each set that produced models with optimal predictive performance. The top 10 models using this hybrid feature set had R = 73–81% and P = 64–71%, the best overall performance of any of the sets of models. Our methods identified hotspots in structural regions of known allosteric significance. Moreover, our predicted hotspots form a network of contiguous residues in the interior of the structures, in agreement with previous work. In conclusion, we have developed models that discriminate between known allosteric hotspots and non-hotspots with high accuracy and sensitivity. Moreover, the pattern of predicted hotspots corresponds to known functional motifs implicated in allostery, and is consistent with previous work describing sparse networks of allosterically important residues

Human malarial disease: a consequence of inflammatory cytokine release

Malaria causes an acute systemic human disease that bears many similarities, both clinically and mechanistically, to those caused by bacteria, rickettsia, and viruses. Over the past few decades, a literature has emerged that argues for most of the pathology seen in all of these infectious diseases being explained by activation of the inflammatory system, with the balance between the pro and anti-inflammatory cytokines being tipped towards the onset of systemic inflammation. Although not often expressed in energy terms, there is, when reduced to biochemical essentials, wide agreement that infection with falciparum malaria is often fatal because mitochondria are unable to generate enough ATP to maintain normal cellular function. Most, however, would contend that this largely occurs because sequestered parasitized red cells prevent sufficient oxygen getting to where it is needed. This review considers the evidence that an equally or more important way ATP deficency arises in malaria, as well as these other infectious diseases, is an inability of mitochondria, through the effects of inflammatory cytokines on their function, to utilise available oxygen. This activity of these cytokines, plus their capacity to control the pathways through which oxygen supply to mitochondria are restricted (particularly through directing sequestration and driving anaemia), combine to make falciparum malaria primarily an inflammatory cytokine-driven disease