Identification of a Novel HIF-1α-α_{M}β_{2} Integrin-NET Axis in Fibrotic Interstitial Lung Disease

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMβ2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β2 integrins. Our results identify a novel HIF-1α- αMβ2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD

Adaptations to Endosymbiosis in a Cnidarian-Dinoflagellate Association: Differential Gene Expression and Specific Gene Duplications

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K–dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health

LifeCLEF 2016: Multimedia Life Species Identification Challenges

International audienceUsing multimedia identification tools is considered as one of the most promising solutions to help bridge the taxonomic gap and build accurate knowledge of the identity, the geographic distribution and the evolution of living species. Large and structured communities of nature observers (e.g., iSpot, Xeno-canto, Tela Botanica, etc.) as well as big monitoring equipment have actually started to produce outstanding collections of multimedia records. Unfortunately, the performance of the state-of-the-art analysis techniques on such data is still not well understood and is far from reaching real world requirements. The LifeCLEF lab proposes to evaluate these challenges around 3 tasks related to multimedia information retrieval and fine-grained classification problems in 3 domains. Each task is based on large volumes of real-world data and the measured challenges are defined in collaboration with biologists and environmental stakeholders to reflect realistic usage scenarios. For each task, we report the methodology, the data sets as well as the results and the main outcom

Sleep-wake sensitive mechanisms of adenosine release in the basal forebrain of rodents : an in vitro study

Adenosine acting in the basal forebrain is a key mediator of sleep homeostasis. Extracellular adenosine concentrations increase during wakefulness, especially during prolonged wakefulness and lead to increased sleep pressure and subsequent rebound sleep. The release of endogenous adenosine during the sleep-wake cycle has mainly been studied in vivo with microdialysis techniques. The biochemical changes that accompany sleep-wake status may be preserved in vitro. We have therefore used adenosine-sensitive biosensors in slices of the basal forebrain (BFB) to study both depolarization-evoked adenosine release and the steady state adenosine tone in rats, mice and hamsters. Adenosine release was evoked by high K+, AMPA, NMDA and mGlu receptor agonists, but not by other transmitters associated with wakefulness such as orexin, histamine or neurotensin. Evoked and basal adenosine release in the BFB in vitro exhibited three key features: the magnitude of each varied systematically with the diurnal time at which the animal was sacrificed; sleep deprivation prior to sacrifice greatly increased both evoked adenosine release and the basal tone; and the enhancement of evoked adenosine release and basal tone resulting from sleep deprivation was reversed by the inducible nitric oxide synthase (iNOS) inhibitor, 1400 W. These data indicate that characteristics of adenosine release recorded in the BFB in vitro reflect those that have been linked in vivo to the homeostatic control of sleep. Our results provide methodologically independent support for a key role for induction of iNOS as a trigger for enhanced adenosine release following sleep deprivation and suggest that this induction may constitute a biochemical memory of this state

Identification of a novel HIF-1α-αMβ2 integrin-NETosis axis in fibrotic interstitial lung disease

Neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ILD) particularly in the most severe form, idiopathic pulmonary fibrosis (IPF), although the underlying mechanisms remain unclear. Neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. Hypoxia has an important role in inflammation and may also contribute to pulmonary disease. We aimed to determine how neutrophil activation occurs in ILD and the relative importance of hypoxia. Using lung biopsies and bronchoalveolar lavage (BAL) fluid from ILD patients we investigated the extent of hypoxia and neutrophil activation in ILD lungs. Then we used ex vivo neutrophils isolated from healthy volunteers and BAL from patients with ILD and non-ILD controls to further investigate aberrant neutrophil activation in hypoxia and ILD. We demonstrate for the first time using intracellular staining, HIF-1α stabilization in neutrophils and endothelial cells in ILD lung biopsies. Hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (PMA)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (NET) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). Hypoxia also increased neutrophil expression of the αM (+3.1-fold, p < 0.001) and αX (+1.6-fold, p < 0.01) integrin subunits. Interestingly, NET formation was induced by αMβ2 integrin activation and prevented by cation chelation. Finally, we observed NET-like structures in IPF lung sections and in the BAL from ILD patients, and quantification showed increased cell-free DNA content (+5.5-fold, p < 0.01) and MPO-citrullinated histone H3 complexes (+21.9-fold, p < 0.01) in BAL from ILD patients compared to non-ILD controls. In conclusion, HIF-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β2 integrins. Our results identify a novel HIF-1α- αMβ2 integrin axis in NET formation for future exploration in therapeutic approaches to fibrotic ILD

Monoubiquitination of syntaxin 3 leads to retrieval from the basolateral plasma membrane and facilitates cargo recruitment to exosomes

Syntaxin 3 (Stx3), a SNARE protein located and functioning at the apical plasma membrane of epithelial cells, is required for epithelial polarity. A fraction of Stx3 is localized to late endosomes/lysosomes, although how it traffics there and its function in these organelles is unknown. Here we report that Stx3 undergoes monoubiquitination in a conserved polybasic domain. Stx3 present at the basolateral—but not the apical—plasma membrane is rapidly endocytosed, targeted to endosomes, internalized into intraluminal vesicles (ILVs), and excreted in exosomes. A nonubiquitinatable mutant of Stx3 (Stx3-5R) fails to enter this pathway and leads to the inability of the apical exosomal cargo protein GPRC5B to enter the ILV/exosomal pathway. This suggests that ubiquitination of Stx3 leads to removal from the basolateral membrane to achieve apical polarity, that Stx3 plays a role in the recruitment of cargo to exosomes, and that the Stx3-5R mutant acts as a dominant-negative inhibitor. Human cytomegalovirus (HCMV) acquires its membrane in an intracellular compartment and we show that Stx3-5R strongly reduces the number of excreted infectious viral particles. Altogether these results suggest that Stx3 functions in the transport of specific proteins to apical exosomes and that HCMV exploits this pathway for virion excretion

The Akt inhibitor KP372-1 suppresses Akt activity and cell proliferation and induces apoptosis in thyroid cancer cells

The phosphatidylinositol 3′ kinase (PI3K)/phosphatase and tensin homologue deleted on chromosome ten/Akt pathway, which is a critical regulator of cell proliferation and survival, is mutated or activated in a wide variety of cancers. Akt appears to be a key central node in this pathway and thus is an attractive target for targeted molecular therapy. We demonstrated that Akt is highly phosphorylated in thyroid cancer cell lines and human thyroid cancer specimens, and hypothesised that KP372-1, an Akt inhibitor, would block signalling through the PI3K pathway and inhibit cell proliferation while inducing apoptosis of thyroid cancer cells. KP372-1 blocked signalling downstream of Akt in thyroid tumour cells, leading to inhibition of cell proliferation and increased apoptosis. As thyroid cancer consistently expresses phosphorylated Akt and KP372-1 effectively blocks Akt signalling, further preclinical evaluation of this compound for treatment of thyroid cancer is warranted

Advanced Technologies for Oral Controlled Release: Cyclodextrins for oral controlled release

Cyclodextrins (CDs) are used in oral pharmaceutical formulations, by means of inclusion complexes formation, with the following advantages for the drugs: (1) solubility, dissolution rate, stability and bioavailability enhancement; (2) to modify the drug release site and/or time profile; and (3) to reduce or prevent gastrointestinal side effects and unpleasant smell or taste, to prevent drug-drug or drug-additive interactions, or even to convert oil and liquid drugs into microcrystalline or amorphous powders. A more recent trend focuses on the use of CDs as nanocarriers, a strategy that aims to design versatile delivery systems that can encapsulate drugs with better physicochemical properties for oral delivery. Thus, the aim of this work was to review the applications of the CDs and their hydrophilic derivatives on the solubility enhancement of poorly water soluble drugs in order to increase their dissolution rate and get immediate release, as well as their ability to control (to prolong or to delay) the release of drugs from solid dosage forms, either as complexes with the hydrophilic (e.g. as osmotic pumps) and/ or hydrophobic CDs. New controlled delivery systems based on nanotechonology carriers (nanoparticles and conjugates) have also been reviewed