A metabolic switch on a yeast arrestin connects glucose signaling to transporter endocytosis

Endocytosis is a critical component of plasma membrane dynamics, by allowing the removal of proteins such as transporters or receptors in response to environmental cues. In yeast, transporter endocytosis requires their ubiquitylation at the plasma membrane by the Nedd4-like E3 ubiquitin ligase, Rsp5. Since the ubiquitylation of a given transporter occurs only in response to specific signals, this raises the question of how substrate specificity is achieved, and how it is regulated dynamically. Various "adaptor" proteins were identified, which may promote the interaction between Rsp5 and its substrates, and may provide a basis for this regulation. However, how they modulate Rsp5 function in response to extracellular stimuli is unknown. We addressed this question by studying a model transporter, Jen1, which is a lactate transporter induced in the presence of lactate and endocytosed in response to glucose (Paiva et al., JBC 2009). We identified an Rsp5 adaptor protein that belongs to the alpha-arrestin family, Art4 (also named Rod1), as essential for Jen1 ubiquitylation and endocytosis of in response to glucose. Interestingly, when cells are grown in lactate medium to induce Jen1 expression, Art4 is strongly phosphorylated by the yeast AMPK homologue, Snf1. Addition of glucose, known to trigger Jen1 endocytosis, leads to a rapid dephosphorylation of Art4, a process that requires the PP1 phosphatase regulatory subunit Reg1. This dephosphorylation allows Art4 ubiquitylation by Rsp5, and we provide details on the molecular mechanism of this regulation. We also show that Art4 ubiquitylation is required for Jen1 endocytosis. Therefore, a switch in Art4 post-translational modifications occurs in response to glucose and is required to modulate its function as an adaptor of Rsp5. This establishes yeast arrestin-like proteins as key regulators of transporter endocytosis in response to extracellular signals

Recent advances on ultrasound contrast agents for blood-brain barrier opening with focused ultrasound

The blood-brain barrier is the primary obstacle to efficient intracerebral drug delivery. Focused ultrasound, in conjunction with microbubbles, is a targeted and non-invasive way to disrupt the blood-brain barrier. Many commercially available ultrasound contrast agents and agents specifically designed for therapeutic purposes have been investigated in ultrasound-mediated blood-brain barrier opening studies. The new generation of sono-sensitive agents, such as liquid-core droplets, can also potentially disrupt the blood-brain barrier after their ultrasound-induced vaporization. In this review, we describe the different compositions of agents used for ultrasound-mediated blood-brain barrier opening in recent studies, and we discuss the challenges of the past five years related to the optimal formulation of agents

Protection against Clostridium difficile infection in a hamster model by oral vaccination using flagellin FliC-loaded pectin beads

International audienceClostridium difficile flagellin FliC is a highly immunogenic pathogen-associated molecular pattern playing a key role in C. difficile pathogenesis and gut colonization. Here, we designed an oral vaccine against C. difficile with FliC encapsulated into pectin beads for colonic release. Bead stability and FliC retention was confirmed in vitro using simulated intestinal media (SIM), while bead degradation and FliC release was observed upon incubation in simulated colonic media (SCM). The importance of FliC encapsulation into pectin beads for protection against C. difficile was assessed in a vaccination assay using a lethal ham-ster model of C. difficile infection. Three groups of hamsters orally received either FliC-loaded beads or unloaded beads in gastro-resistant capsule to limit gastric degradation or free FliC. Two other groups were immunized with free FliC, one intra-rectally and the other intra-peritoneally. Hamsters were then challenged with a lethal dose of C. difficile VPI 10463. Fifty percent of hamsters orally immunized with FliC-loaded beads survived whereas all hamsters orally immunized with free FliC died within 7 days post challenge. No significant protection was observed in the other groups. Only intra-peritoneally immunized hamsters presented anti-FliC IgG antibodies in sera after immunizations. These results suggest that an oral immunization with FliC-loaded beads probably induced a mucosal immune response, therefore providing a protective effect. This study confirms the importance of FliC encapsulation into pectin beads for a protective oral vaccine against C. difficile

Immunotoxicity of poly (lactic-co-glycolic acid) nanoparticles: influence of surface properties on dendritic cell activation

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Co-encapsulation of an antigen and CpG oligonucleotides into PLGA microparticles by TROMS technology.

It seems well established that CpG oligonucleotides Th1 biased adjuvant activity can be improved when closely associated with a variety of antigens in, for example, microparticles. In this context, we prepared 1-μm near non-charged PLGA 502 or PLGA 756 microparticles that loaded with high efficiency an antigen (50% ovalbumin (OVA), approximately) into their matrix and CpG-chitosan complexes (near to 20%) onto their surface maintaining OVA and CpG integrity intact. In the intradermal immunization studies, whereas OVA microencapsulated into PLGA 756 alone induced a strong humoral immune response assisted by a very clear Th1 bias (IgG2a/IgG1=0.875) that was decreased by CpG co-delivery (IgG2a/IgG1=0.55), the co-encapsulation of CpG with OVA in PLGA 502 particles significantly improved the antibody response and isotype shifting (IgG2a/IgG1=0.73) in comparison with mice immunized with OVA loaded PLGA 502 (IgG2a/IgG1=0). This improvement was not correlated with the cellular immune response where the effect of co-encapsulated CpG was rather negative (2030.2 pg/mL and 335.3 pg/mL IFN-g for OVA PLGA 502 for OVA CpG PLGA 502, respectively). These results underscore the critical role of polymer nature and microparticle characteristics to show the benefits of coencapsulating CpG motifs in close proximity with an antigen

Liposomes loaded with everolimus and coated with hyaluronic acid: A promising approach for lung fibrosis

Chronic lung allograft dysfunction (CLAD) and interstitial lung disease associated with collagen tissue diseases (CTD-ILD) are two end-stage lung disorders in which different chronic triggers induce activation of myo-/fibroblasts (LFs). Everolimus, an mTOR inhibitor, can be adopted as a potential strategy for CLAD and CTD-ILD, however it exerts important side effects. This study aims to exploit nanomedicine to reduce everolimus side effects encapsulating it inside liposomes targeted against LFs, expressing a high rate of CD44. PEGylated liposomes were modified with high molecular weight hyaluronic acid and loaded with everolimus (PEG-LIP(ev)-HA400kDa). Liposomes were tested by in vitro experiments using LFs derived from broncholveolar lavage (BAL) of patients affected by CLAD and CTD-ILD, and on alveolar macrophages (AM) and lymphocytes isolated, respectively, from BAL and peripheral blood. PEG-LIP-HA400kDa demonstrated to be specific for LFs, but not for CD44-negative cells, and after loading everolimus, PEG-LIP(ev)-HA400kDa were able to arrest cell cycle arrest and to decrease phospho-mTOR level. PEG-LIP(ev)-HA400kDa showed anti-inflammatory effect on immune cells. This study opens the possibility to use everolimus in lung fibrotic diseases, demonstrating that our lipids-based vehicles can vehicle everolimus inside cells exerting the same drug molecular effect, not only in LFs, but also in immune cells

Anisotropic colloids through non-trivial buckling

We present a study on buckling of colloidal particles, including experimental, theoretical and numerical developments. Oil-filled thin shells prepared by emulsion templating show buckling in mixtures of water and ethanol, due to dissolution of the core in the external medium. This leads to conformations with a single depression, either axisymmetric or polygonal depending on the geometrical features of the shells. These conformations could be theoretically and/or numerically reproduced in a model of homogeneous spherical thin shells with bending and stretching elasticity, submitted to an isotropic external pressure.Comment: submitted to EPJ

Liquid marble-derived solid-liquid hybrid superparticles for CO2 capture.

The design of effective CO2 capture materials is an ongoing challenge. Here we report a concept to overcome current limitations associated with both liquid and solid CO2 capture materials by exploiting a solid-liquid hybrid superparticle (SLHSP). The fabrication of SLHSP involves assembly of hydrophobic silica nanoparticles on the liquid marble surface, and co-assembly of hydrophilic silica nanoparticles and tetraethylenepentamine within the interior of the liquid marble. The strong interfacial adsorption force and the strong interactions between amine and silica are identified to be key elements for high robustness. The developed SLHSPs exhibit excellent CO2 sorption capacity, high sorption rate, long-term stability and reduced amine loss in industrially preferred fixed bed setups. The outstanding performances are attributed to the unique structure which hierarchically organizes the liquid and solid at microscales

Monoolein Lipid Phases as Incorporation and Enrichment Materials for Membrane Protein Crystallization

The crystallization of membrane proteins in amphiphile-rich materials such as lipidic cubic phases is an established methodology in many structural biology laboratories. The standard procedure employed with this methodology requires the generation of a highly viscous lipidic material by mixing lipid, for instance monoolein, with a solution of the detergent solubilized membrane protein. This preparation is often carried out with specialized mixing tools that allow handling of the highly viscous materials while minimizing dead volume to save precious membrane protein sample. The processes that occur during the initial mixing of the lipid with the membrane protein are not well understood. Here we show that the formation of the lipidic phases and the incorporation of the membrane protein into such materials can be separated experimentally. Specifically, we have investigated the effect of different initial monoolein-based lipid phase states on the crystallization behavior of the colored photosynthetic reaction center from Rhodobacter sphaeroides. We find that the detergent solubilized photosynthetic reaction center spontaneously inserts into and concentrates in the lipid matrix without any mixing, and that the initial lipid material phase state is irrelevant for productive crystallization. A substantial in-situ enrichment of the membrane protein to concentration levels that are otherwise unobtainable occurs in a thin layer on the surface of the lipidic material. These results have important practical applications and hence we suggest a simplified protocol for membrane protein crystallization within amphiphile rich materials, eliminating any specialized mixing tools to prepare crystallization experiments within lipidic cubic phases. Furthermore, by virtue of sampling a membrane protein concentration gradient within a single crystallization experiment, this crystallization technique is more robust and increases the efficiency of identifying productive crystallization parameters. Finally, we provide a model that explains the incorporation of the membrane protein from solution into the lipid phase via a portal lamellar phase