Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway

Aluminum (Al³⁺) has been recognized as a main toxic factor in crop production in acid lands. Phosphatidic acid (PA) is emerging as an important lipid signaling molecule and has been implicated in various stress-signaling pathways in plants. In this paper, we focus on how PA generation is affected by Al³⁺ using Coffea arabica suspension cells. We pre-labeled cells with [³²P]orthophosphate (³²Pi) and assayed for ³²P-PA formation in response to Al³⁺. Treating cells for 15 min with either AlCl₃ or Al(NO₃)₃ inhibited the formation of PA. In order to test how Al³⁺ affected PA signaling, we used the peptide mastoparan-7 (mas-7), which is known as a very potent stimulator of PA formation. The Al³⁺ inhibited mas-7 induction of PA response, both before and after Al³⁺ incubation. The PA involved in signaling is generated by two distinct phospholipid signaling pathways, via phospholipase D (PLD; EC: 3.1.4.4) or via Phospholipase C (PLC; EC: 3.1.4.3), and diacylglycerol kinase (DGK; EC 2.7.1.107). By labeling with ³²Pi for short periods of time, we found that PA formation was inhibited almost 30% when the cells were incubated with AlCl₃ suggesting the involvement of the PLC/DGK pathway. Incubation of cells with PLC inhibitor, U73122, affected PA formation, like AlCl₃ did. PLD in vivo activation by mas-7 was reduced by Al³⁺. These results suggest that PA formation was prevented through the inhibition of the PLC activity, and it provides the first evidence for the role of Al toxicity on PA production.     

Phosphatidylinositol 3-Phosphate 5-Kinase,FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P₂, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P₂, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.Phosphoinositides play an important role in various cellular processes, including determination of organelle identity and mediating signal transduction by recruiting effector molecules to various organelles (Balla, 2013). Among those, D3-phosphorylated phosphoinositides, phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂], play essential roles in the endosomal trafficking and the vacuolar sorting. PtdIns3P is produced from phosphatidylinositol by class III PI3-kinase, vacuolar protein sorting34 (VPS34). In animal cells, PtdIns3P predominantly localizes to the early endosomes and controls endosome maturation, recycling, and degradation of cargo proteins coordinated with Rab5 GTPases (Jean and Kiger, 2012). In Arabidopsis (Arabidopsis thaliana), PtdIns3P mainly resides on the late endosomes and the prevacuolar membrane (Vermeer et al., 2006; Simon et al., 2014). Dysfunction of AtVPS34 resulted in a defect in growth (Welters et al., 1994), root hair elongation (Lee et al., 2008a), and pollen development (Lee et al., 2008b), indicating an important role for AtVPS34 and its product PtdIns3P in plant development. VPS34-mediated PtdIns3P synthesis at the endosomes recruits phosphatidylinositol 3-phosphate 5-kinase formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), then FAB1/PIKfyve produces PtdIns(3,5)P₂ from PtdIns3P to mediate late endosome maturation in yeast (Saccharomyces cerevisiae) and animals (Ho et al., 2012; Jean and Kiger, 2012). PtdIns(3,5)P₂ has crucial roles in the maintenance of lysosome/vacuole morphology and acidification, membrane trafficking of proteins, autophagy, and signaling mediation in response to various stresses (Shisheva, 2008).FAB1 was discovered in yeast, where mutations were found to result in the formation of aploid and binucleate cells (hence its name FAB). In addition, a loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth (Yamamoto et al., 1995). In mammalian cells, this kinase is called PIKfyve (FYVE is a PI3P-binding domain). FAB1/PIKfyve forms a protein complex with an adaptor-like protein, Vacuole14 (Bonangelino et al., 1997) and PtdIns(3,5)P₂ 5-phosphatase (Fig. 4; Gary et al., 2002), indicating that the FAB1 complex catalyzes both PtdIns(3,5)P₂ synthesis and turnover simultaneously. In mammalian cells, interference of FAB1/PIKfyve function causes severe defects during embryogenesis, resulting in embryonic lethality in Drosophila spp., Caenorhabditis elegans, and mice (Nicot et al., 2006; Rusten et al., 2006; Ikonomov et al., 2011; Takasuga et al., 2013). Whereas most genomes from human to yeast contain a single-copy gene, the Arabidopsis genome codes for four FAB1 genes (FAB1A–D), of which only FAB1A and FAB1B contain a FYVE domain (Mueller-Roeber and Pical, 2002), and fab1a/fab1b double mutant reveals male gametophyte lethality phenotype in Arabidopsis (Whitley et al., 2009). The mutant pollen shows severe defects in vacuolar reorganization following the first mitotic division of development, suggesting an important role of FAB1 and PtdIns(3,5)P₂ in vacuolar rearrangement for pollen development (Whitley et al., 2009).Open in a separate windowFigure 4.Localization of endosomal markers upon down-regulation of FAB1A/B or inhibition of PtdIns(3,5)P₂ synthesis in young root cortical cells. Localization of mRFP-SYP43, mRFP-vesicle-associated membrane protein (VAMP727), mRFP-ARA7, and SNX1-mRFP without estradiol (A, E, I, and M) or with estradiol (B, F, J, and N) in the FAB1A/B-amiRNA line, or wild-type (WT) plants without YM201636 (C, G, K, and O) or with YM201636 (D, H, L, and P). Bar = 10 μm. Measurement of fluorescent dot structures (Q). Data represent fluorescent dots per cell (mean ± sd). *, P < 0.001 (Student’s t test).We previously developed a transgenic Arabidopsis line that is able to conditionally down-regulate FAB1A and FAB1B expression simultaneously, and demonstrated that a loss of FAB1 function causes various abnormal phenotypes, including growth inhibition, hypersensitivity to exogenous auxin, disturbance of root gravitropism, and floral organ abnormalities (Hirano et al., 2011). In addition, we found that down-regulation of FAB1A/B expression impaired endomembrane homeostasis, including endocytosis, vacuole formation, and vacuolar acidification, likely causing pleiotropic developmental phenotypes that mostly related to the auxin signaling in Arabidopsis (Hirano et al., 2011; Hirano and Sato, 2011). In plants, auxin is a crucial phytohormone that has a wide variety of physiological roles associated with growth, development, and tropic responses (Zhao, 2010). The polar cell-to-cell transport of auxin is mediated by auxin transporters localized on the plasma membrane (PM), such as PIN-FORMED (PIN) proteins (Vieten et al., 2007; Feraru and Friml, 2008). PINs are used as model molecules for polarity establishment on the PM in Arabidopsis. The establishment of PIN polarity is accomplished by the recycling of PINs between the PM and endosomal compartments comprising the trans-Golgi network/early endosomes (TGN/EEs) and the late endosomes (LEs)/prevacuolar compartments. The PIN-recycling pathway is mediated by multiple endosomal regulatory proteins, such as Rab family GTPases and Sorting Nexin (SNX; Jaillais et al., 2006; Park and Jürgens, 2011).Rab proteins function as molecular switches to regulate the tethering and fusion step of transport vesicles to target membranes. Rab5 members of the Rab GTPases have various functions in the endocytic pathway in eukaryotes. The maturation of the early-to-late endosomes is regulated by Rab5-to-Rab7 conversion, which is regulated by the Mon1/Sand-1-Ccz1 complex (Nordmann et al., 2010; Poteryaev et al., 2010). In plants, Rab5-family proteins, Ara6 and Ara7, and Rha1 play important roles in Rab5-mediated endosomal trafficking including the vacuolar trafficking pathway, thereby regulating of the polar transport of auxin and responses to environmental conditions (Ebine et al., 2011; Inoue et al., 2013).SNXs are composed of two conserved domains: the PHOX domain, involved in the interaction with the phosphoinositides, PtdIns3P and PtdIns (3,5)P₂, in the endosomal membrane in animals (Cozier et al., 2002), and the BAR domain, mediating dimerization and binding to curved membranes (Peter et al., 2004). Loss of SNX function disrupts the stable association of the retromer subcomplex, VPS26-Vps29-Vps35, with endosomal membranes, and thus results in retromer dysfunction, indicating that SNXs have a crucial role in the assembly and maintenance of the core retromer function (Teasdale et al., 2001; Cullen and Korswagen, 2012). The first plant SNX was identified as a protein that interacts with various receptor kinases in Brassica oleracea (Vanoosthuyse et al., 2003), and then three SNX genes (SNX1, SNX2a, and SNX2b) were identified in Arabidopsis. The snx1 null mutant exhibits a semidwarf phenotype with other subtle developmental defects (Pourcher et al., 2010). SNX1 is localized to the late endosome and is involved in PIN2 recycling between endosomes and the PM (Jaillais et al., 2006). SNX1 has been reported to interact with cortical microtubules via the microtubule-associated protein Cytoplasmic Linker Associated Protein (CLASP), and the clasp1 null mutant displays aberrant SNX1 endosomes and enhanced PIN2 degradation in the lytic vacuoles, suggesting that an association of SNX1 endosomes and CLASP is important for recycling of PIN transporters (Ambrose et al., 2013).Although many analyses of FAB1/PIKfyve, Rab5 family GTPases, SNXs, and microtubles have been reported, and there are significant similarities in endosomal trafficking, a functional relationship between them is still largely obscure.In this study, we demonstrate that FAB1 produced PtdIns(3,5)P₂ in Arabidopsis, and knockdown of FAB1 expression or inhibition of FAB1 activity with a FAB1/PIKfyve inhibitor, YM201636, decreased PtdIns(3,5)P₂ content. We also found that FAB1 and its product PtdIns(3,5)P₂ mediate the late endosome maturation by recruiting endosomal effector molecules, Ara7 and SNX1, onto endosomes to establish endosome-cortical microtubule interaction. Subsequently, the basal polarity of PIN2 in young cortical cells and PIN1 in stele cells is achieved.     

Modeling the Cost Effectiveness of Malaria Control Interventions in the Highlands of Western Kenya

Introduction

Tools that allow for in silico optimization of available malaria control strategies can assist the decision-making process for prioritizing interventions. The OpenMalaria stochastic simulation modeling platform can be applied to simulate the impact of interventions singly and in combination as implemented in Rachuonyo South District, western Kenya, to support this goal.

Methods

Combinations of malaria interventions were simulated using a previously-published, validated model of malaria epidemiology and control in the study area. An economic model of the costs of case management and malaria control interventions in Kenya was applied to simulation results and cost-effectiveness of each intervention combination compared to the corresponding simulated outputs of a scenario without interventions. Uncertainty was evaluated by varying health system and intervention delivery parameters.

Results

The intervention strategy with the greatest simulated health impact employed long lasting insecticide treated net (LLIN) use by 80% of the population, 90% of households covered by indoor residual spraying (IRS) with deployment starting in April, and intermittent screen and treat (IST) of school children using Artemether lumefantrine (AL) with 80% coverage twice per term. However, the current malaria control strategy in the study area including LLIN use of 56% and IRS coverage of 70% was the most cost effective at reducing disability-adjusted life years (DALYs) over a five year period.

Conclusions

All the simulated intervention combinations can be considered cost effective in the context of available resources for health in Kenya. Increasing coverage of vector control interventions has a larger simulated impact compared to adding IST to the current implementation strategy, suggesting that transmission in the study area is not at a level to warrant replacing vector control to a school-based screen and treat program. These results have the potential to assist malaria control program managers in the study area in adding new or changing implementation of current interventions.     

Learning the lipid language of plant signalling

Plant cells respond to different biotic and abiotic stresses by producing various uncommon phospholipids that are believed to play key roles in cell signalling. We can predict how they work because animal and yeast proteins have been shown to have specific lipid-binding domains, which act as docking sites. When such proteins are recruited to the membrane locations where these phospholipids are synthesized, the phospholipids activate them directly, by inducing a conformational change, or indirectly, by juxtaposing them with an activator protein. The same lipid-binding domains are present in Arabidopsis proteins. We believe that they represent an untapped well of information about plant lipid signalling.     

Bandoniozyma gen. nov., a Genus of Fermentative and Non-Fermentative Tremellaceous Yeast Species

Background

Independent surveys across the globe led to the proposal of a new basidiomycetous yeast genus within the Bulleromyces clade of the Tremellales, Bandoniozyma gen. nov., with seven new species.

Methodology/Principal Findings

The species were characterized by multiple methods, including the analysis of D1/D2 and ITS nucleotide sequences, and morphological and physiological/biochemical traits. Most species can ferment glucose, which is an unusual trait among basidiomycetous yeasts.

Conclusions/Significance

In this study we propose the new yeast genus Bandoniozyma, with seven species Bandoniozyma noutii sp. nov. (type species of genus; CBS 8364^T  =  DBVPG 4489^T), Bandoniozyma aquatica sp. nov. (UFMG-DH4.20^T  =  CBS 12527^T  =  ATCC MYA-4876^T), Bandoniozyma complexa sp. nov. (CBS 11570^T  =  ATCC MYA-4603^T  =  MA28a^T), Bandoniozyma fermentans sp. nov. (CBS 12399^T  =  NU7M71^T  =  BCRC 23267^T), Bandoniozyma glucofermentans sp. nov. (CBS 10381^T  =  NRRL Y-48076^T  =  ATCC MYA-4760^T  =  BG 02-7-15-015A-1-1^T), Bandoniozyma tunnelae sp. nov. (CBS 8024^T  =  DBVPG 7000^T), and Bandoniozyma visegradensis sp. nov. (CBS 12505^T  =  NRRL Y-48783^T  =  NCAIM Y.01952^T).     

Mosquito Feeding Assays to Determine the Infectiousness of Naturally Infected Plasmodium falciparum Gametocyte Carriers

Introduction

In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes.

Methods

We compared two commonly used mosquito feeding assay procedures: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum.

Results

930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94–2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p<0.0001). Replacing the plasma of the gametocyte donor with malaria naïve control serum resulted in higher mosquito infection rates compared to own plasma (OR 1.92, 95% CI 1.68–2.19) while the infectiousness of gametocytes may be reduced during the replacement procedure (OR 0.60, 95% CI 0.52–0.70).

Conclusions

Despite a higher efficiency of direct skin feeding assays, membrane feeding assays appear suitable tools to compare the infectiousness between individuals and to evaluate transmission-reducing interventions. Several aspects of membrane feeding procedures currently lack standardization; this variability makes comparisons between laboratories challenging and should be addressed to facilitate future testing of transmission-reducing interventions.     

IgG responses to Anopheles gambiae salivary antigen gSG6 detect variation in exposure to malaria vectors and disease risk

Assessment of exposure to malaria vectors is important to our understanding of spatial and temporal variations in disease transmission and facilitates the targeting and evaluation of control efforts. Recently, an immunogenic Anopheles gambiae salivary protein (gSG6) was identified and proposed as the basis of an immuno-assay determining exposure to Afrotropical malaria vectors. In the present study, IgG responses to gSG6 and 6 malaria antigens (CSP, AMA-1, MSP-1, MSP-3, GLURP R1, and GLURP R2) were compared to Anopheles exposure and malaria incidence in a cohort of children from Korogwe district, Tanzania, an area of moderate and heterogeneous malaria transmission. Anti-gSG6 responses above the threshold for seropositivity were detected in 15% (96/636) of the children, and were positively associated with geographical variations in Anopheles exposure (OR 1.25, CI 1.01-1.54, p?=?0.04). Additionally, IgG responses to gSG6 in individual children showed a strong positive association with household level mosquito exposure. IgG levels for all antigens except AMA-1 were associated with the frequency of malaria episodes following sampling. gSG6 seropositivity was strongly positively associated with subsequent malaria incidence (test for trend p?=?0.004), comparable to malaria antigens MSP-1 and GLURP R2. Our results show that the gSG6 assay is sensitive to micro-epidemiological variations in exposure to Anopheles mosquitoes, and provides a correlate of malaria risk that is unrelated to immune protection. While the technique requires further evaluation in a range of malaria endemic settings, our findings suggest that the gSG6 assay may have a role in the evaluation and planning of targeted and preventative anti-malaria interventions.     

Removing celiac disease-related gluten proteins from bread wheat while retaining technological properties: a study with Chinese Spring deletion lines

Background  

Gluten proteins can induce celiac disease (CD) in genetically susceptible individuals. In CD patients gluten-derived peptides are presented to the immune system, which leads to a CD4⁺ T-cell mediated immune response and inflammation of the small intestine. However, not all gluten proteins contain T-cell stimulatory epitopes. Gluten proteins are encoded by multigene loci present on chromosomes 1 and 6 of the three different genomes of hexaploid bread wheat (Triticum aestivum) (AABBDD).     

PA,a stress-induced short cut to switch-on ethylene signalling by switching-off CTR1?

Constitutive triple response 1 (CTR1) is a protein kinase that represses plant responses to ethylene. Recently, we have shown that CTR1 function is negatively regulated by the lipid second messenger phosphatidic acid (PA) in vitro.¹ PA was shown to inhibit (1) CTR1''s protein kinase activity, (2) the intramolecular interaction between N-terminus and kinase domain, and (3) the interaction of CTR1 with the ethylene receptor ETR1. PA typically accumulates within minutes in response to biotic or abiotic stresses, which are known to induce ethylene formation. Although long-term treatment with ethephon does stimulate PA accumulation, our results show no fast increase in PA in response to ethylene. A speculative model is presented which explains how stress-induced PA formation could switch on downstream ethylene responses via interaction of the lipid with CTR1.Key words: lipid signaling, phosphatidic acid, ethylene, constitutive triple response 1, plant stress signaling, protein kinase, phospholipase D