Organic Anion Transporting Polypeptides of the OATP/SLCO Superfamily: Identification of New Members in Nonmammalian Species, Comparative Modeling and a Potential Transport Mode

Organic anion-transporting polypeptides (human, OATPs; other animals, Oatps; gene symbol, SLCO/Slco) form a transport protein superfamily that mediates the translocation of amphipathic substrates across the plasma membrane of animal cells. So far, OATPs/Oatps have been identified in human, rat and mouse tissues. In this study, we used bioinformatic tools to detect new members of the OATP/SLCO superfamily in nonmammalian species and to build models for the three-dimensional structure of OATPs/Oatps. New OATP/SLCO superfamily members, some of which form distinct novel families, were identified in chicken, zebrafish, frog, fruit fly and worm species. The lack of OATP/SLCO superfamily members in plants, yeast and bacteria suggests the emergence of an ancient Oatp protein in an early ancestor of the animal kingdom. Structural models were generated for the representative members OATP1B3 and OATP2B1 based on the known structures of the major facilitator superfamily of transport proteins. A model was also built for the large extracellular region between transmembrane helices 9 and 10, following the identification of a novel homology with the Kazal-type serine protease inhibitors. Along with the electrostatic potential and the conservation of key amino acid residues, we propose a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rocker-switch type of mechanism. Several amino acid residues were identified that may play crucial roles in the proposed transport mechanism. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Adapting test timing to the sleep-wake schedule: effects on diurnal neurobehavioral performance changes in young evening and older morning chronotypes

The synchrony effect refers to the beneficial impact of temporal matching between the timing of cognitive task administration and preferred time-of-day for diurnal activity. Aging is often associated with an advance in sleep-wake timing and concomitant optimal performance levels in the morning. In contrast, young adults often perform better in the evening hours. So far, the synchrony effect has been tested at fixed clock times, neglecting the individual's sleep-wake schedule and thus introducing confounds, such as differences in accumulated sleep pressure or circadian phase, which may exacerbate synchrony effects. To probe this hypothesis, the authors tested older morning and young evening chronotypes with a psychomotor vigilance and a Stroop paradigm once at fixed morning and evening hours and once adapting testing time to their preferred sleep-wake schedule in a within-subject design. The authors observe a persistence of synchrony effects for overall median reaction times during a psychomotor vigilance task, even when testing time is adapted to the specific individual's sleep-wake schedule. However, data analysis also indicates that time-of-day modulations are weakened under those conditions for incongruent trials on Stroop performance and the slowest reaction times on the psychomotor vigilance task. The latter result suggests that the classically observed synchrony effect may be partially mediated by a series of parameters, such as differences in socio-professional timing constraints, the amount of accumulated sleep need, or circadian phase, all leading to differential arousal levels at testing.     

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.     

Coupling between taxonomic and functional diversity in protistan coastal communities

The study of protistan functional diversity is crucial to understand the dynamics of oceanic ecological processes. We combined the metabarcoding data of various coastal ecosystems and a newly developed trait-based approach to study the link between taxonomic and functional diversity across marine protistan communities of different size-classes. Environmental DNA was extracted and the V4 18S rDNA genomic region was amplified and sequenced. In parallel, we tried to annotate the operational taxonomic units (OTUs) from our metabarcoding dataset to 30 biological traits using published and accessible information on protists. We then developed a method to study trait correlations across protists (i.e. trade-offs) in order to build the best functional groups. Based on the annotated OTUs and our functional groups, we demonstrated that the functional diversity of marine protist communities varied in parallel with their taxonomic diversity. The coupling between functional and taxonomic diversity was conserved across different protist size classes. However, the smallest size-fraction was characterized by wider taxonomic and functional groups diversity, corroborating the idea that nanoplankton and picoplankton are part of a more stable ecological background on which larger protists and metazoans might develop.     

Rhamnolipids Elicit Defense Responses and Induce Disease Resistance against Biotrophic, Hemibiotrophic, and Necrotrophic Pathogens That Require Different Signaling Pathways in Arabidopsis and Highlight a Central Role for Salicylic Acid

Plant resistance to phytopathogenic microorganisms mainly relies on the activation of an innate immune response usually launched after recognition by the plant cells of microbe-associated molecular patterns. The plant hormones, salicylic acid (SA), jasmonic acid, and ethylene have emerged as key players in the signaling networks involved in plant immunity. Rhamnolipids (RLs) are glycolipids produced by bacteria and are involved in surface motility and biofilm development. Here we report that RLs trigger an immune response in Arabidopsis (Arabidopsis thaliana) characterized by signaling molecules accumulation and defense gene activation. This immune response participates to resistance against the hemibiotrophic bacterium Pseudomonas syringae pv tomato, the biotrophic oomycete Hyaloperonospora arabidopsidis, and the necrotrophic fungus Botrytis cinerea. We show that RL-mediated resistance involves different signaling pathways that depend on the type of pathogen. Ethylene is involved in RL-induced resistance to H. arabidopsidis and to P. syringae pv tomato whereas jasmonic acid is essential for the resistance to B. cinerea. SA participates to the restriction of all pathogens. We also show evidence that SA-dependent plant defenses are potentiated by RLs following challenge by B. cinerea or P. syringae pv tomato. These results highlight a central role for SA in RL-mediated resistance. In addition to the activation of plant defense responses, antimicrobial properties of RLs are thought to participate in the protection against the fungus and the oomycete. Our data highlight the intricate mechanisms involved in plant protection triggered by a new type of molecule that can be perceived by plant cells and that can also act directly onto pathogens.In their environment, plants are challenged by potentially pathogenic microorganisms. In response, they express a set of defense mechanisms including preformed structural and chemical barriers, as well as an innate immune response quickly activated after microorganism perception (Boller and Felix, 2009). Plant innate immunity is triggered after recognition by pattern recognition receptors of conserved pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs, respectively) or by plant endogenous molecules released by pathogen invasion and called danger-associated molecular patterns (Boller and Felix, 2009; Dodds and Rathjen, 2010). This first step of recognition leads to the activation of MAMP-triggered immunity (MTI). Successful pathogens can secrete effectors that interfere or suppress MTI, resulting in effector-triggered susceptibility. A second level of perception involves the direct or indirect recognition by specific receptors of pathogen effectors leading to effector-triggered immunity (ETI; Boller and Felix, 2009; Dodds and Rathjen, 2010). Whereas MTI and ETI are thought to involve common signaling network, ETI is usually quantitatively stronger than MTI and associated with more sustained and robust immune responses (Katagiri and Tsuda, 2010; Tsuda and Katagiri, 2010).The plant hormones, salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) have emerged as key players in the signaling networks involved in MTI and ETI (Robert-Seilaniantz et al., 2007; Tsuda et al., 2009; Katagiri and Tsuda, 2010; Mersmann et al., 2010; Tsuda and Katagiri, 2010; Robert-Seilaniantz et al., 2011). Interactions between these signal molecules allow the plant to activate and/or modulate an appropriate spectrum of responses, depending on the pathogen lifestyle, necrotroph or biotroph (Glazebrook, 2005; Koornneef and Pieterse, 2008). It is assumed that JA and ET signaling pathways are important for resistance to necrotrophic fungi including Botrytis cinerea and Alternaria brassicicola (Thomma et al., 2001; Ferrari et al., 2003; Glazebrook, 2005). Infection of Arabidopsis (Arabidopsis thaliana) with B. cinerea causes the induction of the JA/ET responsive gene PLANT DEFENSIN1.2 (PDF1.2; Penninckx et al., 1996; Zimmerli et al., 2001). Induction of PDF1.2 by B. cinerea is blocked in ethylene-insensitive2 (ein2) and coronatine-insensitive1 (coi1) mutants that are respectively defective in ET and JA signal transduction pathways. Moreover, ein2 and coi1 plants are highly susceptible to B. cinerea infection (Thomma et al., 1998; Thomma et al., 1999). JA/ET-dependent responses do not seem to be usually induced during resistance to biotrophs, but they can be effective if they are stimulated prior to pathogen challenge (Glazebrook, 2005). Plants impaired in SA signaling are highly susceptible to biotrophic and hemibiotrophic pathogens. Following pathogen infection, SA hydroxylase (NahG), enhanced disease susceptibility5 (eds5), or SA induction-deficient2 (sid2) plants are unable to accumulate high SA levels and they display heightened susceptibility to Pseudomonas syringae pv tomato (Pst), Hyaloperonospora arabidopsidis, or Erysiphe orontii (Delaney et al., 1994; Lawton et al., 1995; Wildermuth et al., 2001; Nawrath et al., 2002; Vlot et al., 2009). Mutants that are insensitive to SA, such as nonexpressor of PATHOGENESIS-RELATED (PR) genes1 (npr1), have enhanced susceptibility to these pathogens (Cao et al., 1994; Glazebrook et al., 1996; Shah et al., 1997; Dong, 2004). According to some reports, plant defense against necrotrophs also involves SA. Arabidopsis plants expressing the nahG gene and infected with B. cinerea show larger lesions compared with wild-type plants (Govrin and Levine, 2002). In tobacco (Nicotiana tabacum), acidic isoforms of PR3 and PR5 gene that are specifically induced by SA (Ménard et al., 2004) are up-regulated after challenge by B. cinerea (El Oirdi et al., 2010). Resistance to some necrotrophs like Fusarium graminearum involves both SA and JA signaling pathways (Makandar et al., 2010). It is assumed that SA and JA signaling can be antagonistic (Bostock, 2005; Koornneef and Pieterse, 2008; Pieterse et al., 2009; Thaler et al., 2012). In Arabidopsis, SA inhibits JA-dependent resistance against A. brassicicola or B. cinerea (Spoel et al., 2007; Koornneef et al., 2008). Recent studies demonstrated that ET modulates the NPR1-mediated antagonism between SA and JA (Leon-Reyes et al., 2009; Leon-Reyes et al., 2010a) and suppression by SA of JA-responsive gene expression is targeted at a position downstream of the JA biosynthesis pathway (Leon-Reyes et al., 2010b). Synergistic effects of SA- and JA-dependent signaling are also well documented (Schenk et al., 2000; van Wees et al., 2000; Mur et al., 2006) and induction of some defense responses after pathogen challenge requires intact JA, ET, and SA signaling pathways (Campbell et al., 2003).Isolated MAMPs trigger defense responses that also require the activation of SA, JA, and ET signaling pathways (Tsuda et al., 2009; Katagiri and Tsuda, 2010). For instance, treatment with the flagellin peptide flg22 induces many SA-related genes including SID2, EDS5, NPR1, and PR1 (Ferrari et al., 2007; Denoux et al., 2008), causes SA accumulation (Tsuda et al., 2008; Wang et al., 2009), and activates ET signaling (Bethke et al., 2009; Mersmann et al., 2010). Local application of lipopolysaccharides elevates the level of SA (Mishina and Zeier, 2007). The oomycete Pep13 peptide induces defense responses in potato (Solanum tuberosum) that require both SA and JA (Halim et al., 2009). Although signaling networks induced by isolated MAMPs are well documented, the contribution of SA, JA, and ET in MAMP- or PAMP-induced resistance to biotrophs and necrotrophs is poorly understood.Rhamnolipids (RLs) are glycolipids produced by various bacteria species including some Pseudomonas and Burkholderia species. They are essential for bacterial surface motility and biofilm development (Vatsa et al., 2010; Chrzanowski et al., 2012). RLs are potent stimulators of animal immunity (Vatsa et al., 2010). They have recently been shown to elicit plant defense responses and to induce resistance against B. cinerea in grapevine (Vitis vinifera; Varnier et al., 2009). They also participate to biocontrol activity of the plant beneficial bacteria Pseudomonas aeruginosa PNA1 against oomycetes (Perneel et al., 2008). However, the signaling pathways used by RLs to stimulate plant innate immunity are not known. To gain more insights into RL-induced MTI, we investigated RL-triggered defense responses and resistance to the necrotrophic fungus B. cinerea, the biotroph oomycete H. arabidopsidis, and the hemibiotroph bacterium Pst in Arabidopsis. Our results show that RLs trigger an innate immune response in Arabidopsis that protects the plant against these different lifestyle pathogens. We demonstrate that RL-mediated resistance involves separated signaling sectors that depend on the type of pathogen. In plants challenged by RLs, SA has a central role and participates to the restriction of the three pathogens. ET is fully involved in RL-induced resistance to the biotrophic oomycete and to the hemibiotrophic bacterium whereas JA is essential for the resistance to the necrotrophic fungus.     

Combined Drug Action of 2-Phenylimidazo[2,1-b]Benzothiazole Derivatives on Cancer Cells According to Their Oncogenic Molecular Signatures

The development of targeted molecular therapies has provided remarkable advances into the treatment of human cancers. However, in most tumors the selective pressure triggered by anticancer agents encourages cancer cells to acquire resistance mechanisms. The generation of new rationally designed targeting agents acting on the oncogenic path(s) at multiple levels is a promising approach for molecular therapies. 2-phenylimidazo[2,1-b]benzothiazole derivatives have been highlighted for their properties of targeting oncogenic Met receptor tyrosine kinase (RTK) signaling. In this study, we evaluated the mechanism of action of one of the most active imidazo[2,1-b]benzothiazol-2-ylphenyl moiety-based agents, Triflorcas, on a panel of cancer cells with distinct features. We show that Triflorcas impairs in vitro and in vivo tumorigenesis of cancer cells carrying Met mutations. Moreover, Triflorcas hampers survival and anchorage-independent growth of cancer cells characterized by “RTK swapping” by interfering with PDGFRβ phosphorylation. A restrained effect of Triflorcas on metabolic genes correlates with the absence of major side effects in vivo. Mechanistically, in addition to targeting Met, Triflorcas alters phosphorylation levels of the PI3K-Akt pathway, mediating oncogenic dependency to Met, in addition to Retinoblastoma and nucleophosmin/B23, resulting in altered cell cycle progression and mitotic failure. Our findings show how the unusual binding plasticity of the Met active site towards structurally different inhibitors can be exploited to generate drugs able to target Met oncogenic dependency at distinct levels. Moreover, the disease-oriented NCI Anticancer Drug Screen revealed that Triflorcas elicits a unique profile of growth inhibitory-responses on cancer cell lines, indicating a novel mechanism of drug action. The anti-tumor activity elicited by 2-phenylimidazo[2,1-b]benzothiazole derivatives through combined inhibition of distinct effectors in cancer cells reveal them to be promising anticancer agents for further investigation.     

B cell-activating factor belonging to the TNF family acts through separate receptors to support B cell survival and T cell-independent antibody formation

The TNF-related ligand, B cell-activating factor belonging to the TNF family (BAFF), is necessary for normal B cell development and survival, and specifically binds the receptors transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), B cell maturation Ag (BCMA), and BAFF-R. Similarities between mice completely lacking BAFF and A/WySnJ strain mice that express a naturally occurring mutant form of BAFF-R suggest that BAFF acts primarily through BAFF-R. However, the nearly full-length BAFF-R protein expressed by A/WySnJ mice makes unambiguous interpretation of receptor function in these animals impossible. Using homologous recombination we created mice completely lacking BAFF-R and compared them directly to A/WySnJ mice and to mice lacking BAFF. BAFF-R-null mice exhibit loss of mature B cells similar to that observed in BAFF(-/-) and A/WySnJ mice. Also, mice lacking both TACI and BCMA simultaneously exhibit no B cell loss, thus confirming that BAFF-R is the primary receptor for transmitting the BAFF-dependent B cell survival signal. However, while BAFF-R-null mice cannot carry out T cell-dependent Ab formation, they differ from BAFF-deficient mice in generating normal levels of Ab to at least some T cell-independent Ags. These studies clearly demonstrate that BAFF regulates Ab responses in vivo through receptors in addition to BAFF-R.     

Sleep-related hippocampo-cortical interplay during emotional memory recollection

Emotional events are usually better remembered than neutral ones. This effect is mediated in part by a modulation of the hippocampus by the amygdala. Sleep plays a role in the consolidation of declarative memory. We examined the impact of sleep and lack of sleep on the consolidation of emotional (negative and positive) memories at the macroscopic systems level. Using functional MRI (fMRI), we compared the neural correlates of successful recollection by humans of emotional and neutral stimuli, 72 h after encoding, with or without total sleep deprivation during the first post-encoding night. In contrast to recollection of neutral and positive stimuli, which was deteriorated by sleep deprivation, similar recollection levels were achieved for negative stimuli in both groups. Successful recollection of emotional stimuli elicited larger responses in the hippocampus and various cortical areas, including the medial prefrontal cortex, in the sleep group than in the sleep deprived group. This effect was consistent across subjects for negative items but depended linearly on individual memory performance for positive items. In addition, the hippocampus and medial prefrontal cortex were functionally more connected during recollection of either negative or positive than neutral items, and more so in sleeping than in sleep-deprived subjects. In the sleep-deprived group, recollection of negative items elicited larger responses in the amygdala and an occipital area than in the sleep group. In contrast, no such difference in brain responses between groups was associated with recollection of positive stimuli. The results suggest that the emotional significance of memories influences their sleep-dependent systems-level consolidation. The recruitment of hippocampo-neocortical networks during recollection is enhanced after sleep and is hindered by sleep deprivation. After sleep deprivation, recollection of negative, potentially dangerous, memories recruits an alternate amygdalo-cortical network, which would keep track of emotional information despite sleep deprivation.     

Emulsified lipids increase endotoxemia: possible role in early postprandial low-grade inflammation

Low-grade inflammation is a risk factor for the onset of atherosclerosis. Little is known about the involvement of endotoxin absorption from the gut during the digestion of lipids. In the present study, we first investigated in humans the impact of a mixed meal containing dispersed lipids on postprandial endotoxemia and inflammation. We then investigated the effect of (i) oil emulsification in vivo in rats and (ii) fatty acid amounts in vitro using Caco-2 cells on postprandial endotoxemia. In humans, postprandial endotoxemia increased early after the meal. Moreover, we evidenced that the endotoxin receptor sCD14 increased during digestion and that chylomicrons could contribute to absorbed endotoxin transport. This could explain the significant peak of inflammatory cytokine IL-6 that we observed 2 h after the mixed meal. Interestingly, in rats, the emulsion led to both higher endotoxemia and hypertriglyceridemia than oil and compared to a control saline load. In vitro, incubation of Caco-2 cells with increasing fatty acid concentrations enhanced epithelial absorption of endotoxin. To our knowledge, this is the first study evidencing in healthy humans that, following a mixed meal containing lipids, increased endotoxemia is associated with raised sCD14 and a peak of IL-6. On a repeated basis, this may thus be a triggering cascade for the onset of atherosclerosis. In this respect, optimizing both dietary fat amount and structure could be a possible strategy to limit such low-grade endotoxemia and inflammation by the control of postprandial lipemia.