MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child–Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G₀/G₁ phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.     

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.     

The impact of mechanical compression on cortical microtubules in Arabidopsis: a quantitative pipeline

Exogenous mechanical perturbations on living tissues are commonly used to investigate whether cell effectors can respond to mechanical cues. However, in most of these experiments, the applied mechanical stress and/or the biological response are described only qualitatively. We developed a quantitative pipeline based on microindentation and image analysis to investigate the impact of a controlled and prolonged compression on microtubule behaviour in the Arabidopsis shoot apical meristem, using microtubule fluorescent marker lines. We found that a compressive stress, in the order of magnitude of turgor pressure, induced apparent microtubule bundling. Importantly, that response could be reversed several hours after the release of compression. Next, we tested the contribution of microtubule severing to compression‐induced bundling: microtubule bundling seemed less pronounced in the katanin mutant, in which microtubule severing is dramatically reduced. Conversely, some microtubule bundles could still be observed 16 h after the release of compression in the spiral2 mutant, in which severing rate is instead increased. To quantify the impact of mechanical stress on anisotropy and orientation of microtubule arrays, we used the nematic tensor based FibrilTool ImageJ/Fiji plugin. To assess the degree of apparent bundling of the network, we developed several methods, some of which were borrowed from geostatistics. The final microtubule bundling response could notably be related to tissue growth velocity that was recorded by the indenter during compression. Because both input and output are quantified, this pipeline is an initial step towards correlating more precisely the cytoskeleton response to mechanical stress in living tissues.     

Differential Mechanisms of Shedding of the Glycosylphosphatidylinositol (GPI)-anchored NKG2D Ligands

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

The role of nocturnal vision in mate choice: females prefer conspicuous males in the European tree frog (Hyla arborea)

Nocturnal frog species rely extensively on vocalization for reproduction. But recent studies provide evidence for an important, though long overlooked, role of visual communication. In many species, calling males exhibit a conspicuous pulsing vocal sac, a signal bearing visually important dynamic components. Here, we investigate female preference for male vocal sac coloration—a question hitherto unexplored—and male colour pattern in the European tree frog (Hyla arborea). Under nocturnal conditions, we conducted two-choice experiments involving video playbacks of calling males with identical calls and showing various naturally encountered colour signals, differing in their chromatic and brightness components. We adjusted video colours to match the frogs'' visual perception, a crucial aspect not considered in previous experiments. Females prefer males with a colourful sac and a pronounced flank stripe. Both signals probably enhance male conspicuousness and facilitate detection and localization by females. This study provides the first experimental evidence of a preference for specific vocal sac spectral properties in a nocturnal anuran species. Vocal sac coloration is based on carotenoids and may convey information about male quality worthwhile for females to assess. The informative content of the flank stripe remains to be demonstrated.     

Effect of antifungals on itraconazole resistant <Emphasis Type="Italic">Candida glabrata</Emphasis>

In the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC₁₀₀ values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.