The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

PPAR Gamma Coactivator 1 Beta (PGC-1β) Reduces Mammalian Target of Rapamycin (mTOR) Expression via a SIRT1-Dependent Mechanism in Neurons

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism, cell growth, and protein synthesis. Since decreased mTOR activity has been found to slow aging in many species, the aim of this study was to examine the activity of mTOR and its phosphorylated form in in vitro and in vivo models mimicking Alzheimer’s disease (AD), and investigate the potential pathway of PGC-1β in regulating mTOR expression. Primary neurons and N2a cells were treated with Aβ_25–35, while untreated cells served as controls. The expression of mTOR, p-mTOR (Ser2448), and PGC-1β was determined with Western blotting and RT-PCR assay, and the translocation of mTOR was detected using confocal microscopy. Aβ_25–35 treatment stimulated the translocation of mTOR from cytoplasm to nucleus, and resulted in elevated expression of mTOR and p-mTOR (Ser2448) and reduced PGC-1β expression. In addition, overexpression of PGC-1β was found to decrease mTOR expression. The results of this study demonstrate that Aβ increases the expression of mTOR and p-mTOR at the site of Ser2448, and the stimulation of Aβ is likely to depend on sirtuin 1, PPARγ, and PGC-1β pathway in regulating mTOR expression.     

Morphoproteomic Profiling of the Mammalian Target of Rapamycin (mTOR) Signaling Pathway in Desmoplastic Small Round Cell Tumor (EWS/WT1), Ewing’s Sarcoma (EWS/FLI1) and Wilms’ Tumor(WT1)

Background

Desmoplastic small round cell tumor (DSRCT) is a rare sarcoma in adolescents and young adults. The hallmark of this disease is a EWS-WT1 translocation resulting from apposition of the Ewing’s sarcoma (EWS) gene with the Wilms’ tumor (WT1) gene. We performed morphoproteomic profiling of DSRCT (EWS-WT1), Ewing’s sarcoma (EWS-FLI1) and Wilms’ tumor (WT1) to better understand the signaling pathways for selecting future targeted therapies.

Methodology

This pilot study assessed patients with DSRCT, Wilms’ tumor and Ewing’s sarcoma. Morphoproteomics and immunohistochemical probes were applied to detect: p-mTOR (Ser2448); p-Akt (Ser473); p-ERK1/2 (Thr202/Tyr204); p-STAT3 (Tyr 705); and cell cycle-related analytes along with their negative controls.

Principal Findings

In DSRCT the PI3K/Akt/mTOR pathway is constitutively activated by p-Akt (Ser 473) expression in the nuclear compartment of the tumor cells and p-mTOR phosphorylated on Ser 2448, suggesting mTORC2 (rictor+mTOR) as the dominant form. Ewing’s sarcoma had upregulated p-Akt and p-mTOR, predominantly mTORC2. In Wilm’s tumor, the mTOR pathway is also activated with most tumor cells moderately expressing p-mTOR (Ser 2448) in plasmalemmal and cytoplasmic compartments. This coincides with the constitutive activation of one of the downstream effectors of the mTORC1 signaling pathway, namely p-p70S6K (Thr 389). There was constitutive activation of the Ras/Raf/ERK pathway p-ERK 1/2 (Thr202/Tyr204) expression in the Wilms tumor and metastatic Ewing’s sarcoma, but not in the DSRCT.

Conclusion

Morphoproteomic tumor analyses revealed constitutive activation of the mTOR pathway as evidenced by: (a) expression of phosphorylated (p)-mTOR, p-p70S6K; (b) mTORC 2 in EWS and DSRCT; (c) ERK signaling was seen in the advanced setting indicating these as resistance pathways to IGF1R related therapies. This is the first morphoproteomic study of such pathways in these rare malignancies and may have potential therapeutic implications. Further study using morphoproteomic assessments of these tumors are warranted.     

Mammalian Target of Rapamycin (mTOR) Tagging Promotes Dendritic Branch Variability through the Capture of Ca2+/Calmodulin-dependent Protein Kinase II α (CaMKIIα) mRNAs by the RNA-binding Protein HuD

The fate of a memory, whether stored or forgotten, is determined by the ability of an active or tagged synapse to undergo changes in synaptic efficacy requiring protein synthesis of plasticity-related proteins. A synapse can be tagged, but without the “capture” of plasticity-related proteins, it will not undergo long lasting forms of plasticity (synaptic tagging and capture hypothesis). What the “tag” is and how plasticity-related proteins are captured at tagged synapses are unknown. Ca²⁺/calmodulin-dependent protein kinase II α (CaMKIIα) is critical in learning and memory and is synthesized locally in neuronal dendrites. The mechanistic (mammalian) target of rapamycin (mTOR) is a protein kinase that increases CaMKIIα protein expression; however, the mechanism and site of dendritic expression are unknown. Herein, we show that mTOR activity mediates the branch-specific expression of CaMKIIα, favoring one secondary, daughter branch over the other in a single neuron. mTOR inhibition decreased the dendritic levels of CaMKIIα protein and mRNA by shortening its poly(A) tail. Overexpression of the RNA-stabilizing protein HuD increased CaMKIIα protein levels and preserved its selective expression in one daughter branch over the other when mTOR was inhibited. Unexpectedly, deleting the third RNA recognition motif of HuD, the domain that binds the poly(A) tail, eliminated the branch-specific expression of CaMKIIα when mTOR was active. These results provide a model for one molecular mechanism that may underlie the synaptic tagging and capture hypothesis where mTOR is the tag, preventing deadenylation of CaMKIIα mRNA, whereas HuD captures and promotes its expression in a branch-specific manner.     

Interaction between Human Prion Protein and Amyloid-β (Aβ) Oligomers: ROLE OF N-TERMINAL RESIDUES*

Soluble oligomers of Aβ42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP^C, on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128–1132). Here we characterized the interaction between synthetic Aβ42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met¹²⁹ and Val¹²⁹ polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Aβ oligomers. It was also found that in addition to the previously identified ∼95–110 segment, the second region of critical importance for the interaction with Aβ42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23–27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Aβ42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.     

Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase,GCN5

NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.     

Seeded Aggregation and Toxicity of α-Synuclein and Tau: CELLULAR MODELS OF NEURODEGENERATIVE DISEASES*

The deposition of amyloid-like filaments in the brain is the central event in the pathogenesis of neurodegenerative diseases. Here we report cellular models of intracytoplasmic inclusions of α-synuclein, generated by introducing nucleation seeds into SH-SY5Y cells with a transfection reagent. Upon introduction of preformed seeds into cells overexpressing α-synuclein, abundant, highly filamentous α-synuclein-positive inclusions, which are extensively phosphorylated and ubiquitinated and partially thioflavin-positive, were formed within the cells. SH-SY5Y cells that formed such inclusions underwent cell death, which was blocked by small molecular compounds that inhibit β-sheet formation. Similar seed-dependent aggregation was observed in cells expressing four-repeat Tau by introducing four-repeat Tau fibrils but not three-repeat Tau fibrils or α-synuclein fibrils. No aggregate formation was observed in cells overexpressing three-repeat Tau upon treatment with four-repeat Tau fibrils. Our cellular models thus provide evidence of nucleation-dependent and protein-specific polymerization of intracellular amyloid-like proteins in cultured cells.     

Selective Inhibition of Carotenoid Cleavage Dioxygenases: PHENOTYPIC EFFECTS ON SHOOT BRANCHING*S?

Members of the carotenoid cleavage dioxygenase family catalyze the oxidative cleavage of carotenoids at various chain positions, leading to the formation of a wide range of apocarotenoid signaling molecules. To explore the functions of this diverse enzyme family, we have used a chemical genetic approach to design selective inhibitors for different classes of carotenoid cleavage dioxygenase. A set of 18 arylalkyl-hydroxamic acids was synthesized in which the distance between an iron-chelating hydroxamic acid and an aromatic ring was varied; these compounds were screened as inhibitors of four different enzyme classes, either in vitro or in vivo. Potent inhibitors were found that selectively inhibited enzymes that cleave carotenoids at the 9,10 position; 50% inhibition was achieved at submicromolar concentrations. Application of certain inhibitors at 100 μm to Arabidopsis node explants or whole plants led to increased shoot branching, consistent with inhibition of 9,10-cleavage.Carotenoids are synthesized in plants and micro-organisms as photoprotective molecules and are key components in animal diets, an example being β-carotene (pro-vitamin A). The oxidative cleavage of carotenoids occurs in plants, animals, and micro-organisms and leads to the release of a range of apocarotenoids that function as signaling molecules with a diverse range of functions (1). The first gene identified as encoding a carotenoid cleavage dioxygenase (CCD)² was the maize Vp14 gene that is required for the formation of abscisic acid (ABA), an important hormone that mediates responses to drought stress and aspects of plant development such as seed and bud dormancy (2). The VP14 enzyme cleaves at the 11,12 position (Fig. 1) of the epoxycarotenoids 9′-cis-neoxanthin and/or 9-cis-violaxanthin and is now classified as a 9-cis-epoxycarotenoid dioxygenase (NCED) (3), a subclass of the larger CCD family.Open in a separate windowFIGURE 1.Reactions catalyzed by the carotenoid cleavage dioxygenases. a, 11,12-oxidative cleavage of 9′-cis-neoxanthin by NCED; b, oxidative cleavage reactions on β-carotene and zeaxanthin.Since the discovery of Vp14, many other CCDs have been shown to be involved in the production of a variety of apocarotenoids (Fig. 1). In insects, the visual pigment retinal is formed by oxidative cleavage of β-carotene by β-carotene-15,15′-dioxygenase (4). Retinal is produced by an orthologous enzyme in vertebrates, where it is also converted to retinoic acid, a regulator of differentiation during embryogenesis (5). A distinct mammalian CCD is believed to cleave carotenoids asymmetrically at the 9,10 position (6) and, although its function is unclear, recent evidence suggests a role in the metabolism of dietary lycopene (7). The plant volatiles β-ionone and geranylacetone are produced from an enzyme that cleaves at the 9,10 position (8) and the pigment α-crocin found in the spice saffron results from an 7,8-cleavage enzyme (9).Other CCDs have been identified where biological function is unknown, for example, in cyanobacteria where a variety of cleavage specificities have been described (10-12). In other cases, there are apocarotenoids with known functions, but the identity or involvement of CCDs have not yet been described: grasshopper ketone is a defensive secretion of the flightless grasshopper Romalea microptera (13), mycorradicin is produced by plant roots during symbiosis with arbuscular mycorrhyza (14), and strigolactones (15) are plant metabolites that act as germination signals to parasitic weeds such as Striga and Orobanche (16).Recently it was discovered that strigolactones also function as a branching hormone in plants (17, 18). The existence of such a branching hormone has been known for some time, but its identity proved elusive. However, it was known that the hormone was derived from the action of at least two CCDs, max3 and max4 (more axillary growth) (19), because deletion of either of these genes in Arabidopsis thaliana, leads to a bushy phenotype (20, 21). In Escherichia coli assays, AtCCD7 (max3) cleaves β-carotene at the 9,10 position and the apocarotenoid product (10-apo-β-carotene) is reported to be further cleaved at 13,14 by AtCCD8 (max4) to produce 13-apo-β-carotene (22). Also recent evidence suggests that AtCCD8 is highly specific, cleaving only 10-apo-β-carotene (23). How the production of 13-apo-β-carotene leads to the synthesis of the complex strigolactone is unknown. The possibility remains that the enzymes may have different specificities and cleavage activities in planta. In addition, a cytochrome P450 enzyme (24) is believed to be involved in strigolactone synthesis and acts in the pathway downstream of the CCD genes. Strigolactone is thought to effect branching by regulating auxin transport (25). Because of the involvement of CCDs in strigolactone synthesis, the possibility arises that plant architecture and interaction with parasitic weeds and mycorrhyza could be controlled by the manipulation of CCD activity.Although considerable success has been obtained using genetic approaches to probe function and substrate specificity of CCDs in their native biological contexts, particularly in plant species with simple genetic systems or that are amenable to transgenesis, there are many systems where genetic approaches are difficult or impossible. Also, when recombinant CCDs are studied either in vitro or in heterologous in vivo assays, such as in E. coli strains engineered to accumulate carotenoids (26), they are often active against a broad range of substrates (5, 21, 27), and in many cases the true in vivo substrate of a particular CCD remains unknown. Therefore additional experimental tools are needed to investigate both apocarotenoid and CCD functions in their native cellular environments.In the reverse chemical genetics approach, small molecules are identified that are active against known target proteins; they are then applied to a biological system to investigate protein function in vivo (28, 29). This approach is complementary to conventional genetics since the small molecules can be applied easily to a broad range of species, their application can be controlled in dose, time, and space to provide detailed studies of biological functions, and individual proteins or whole protein classes may be targeted by varying the specificity of the small molecules. Notably, functions of the plant hormones gibberellin, brassinosteroid, and abscisic acid have been successfully probed using this approach by adapting triazoles to inhibit specific cytochrome P450 monooxygenases involved in the metabolism of these hormones (30).In the case of the CCD family, the tertiary amines abamine (31) and the more active abamineSG (32) were reported as specific inhibitors of NCED, and abamine was used to show new functions of abscisic acid in legume nodulation (33). However, no selective inhibitors for other types of CCD are known. Here we have designed a novel class of CCD inhibitor based on hydroxamic acids, where variable chain length was used to direct inhibition of CCD enzymes that cleave carotenoids at specific positions. We demonstrate the use of such novel inhibitors to control shoot branching in a model plant.     

Molecular phylogenetics of the moth genus Omiodes Guenée (Crambidae: Spilomelinae), and the origins of the Hawaiian lineage

The moth genus Omiodes (Crambidae) comprises about 80 species and has a circumtropical distribution, with the type species, O. humeralis, occurring in Central America. In Hawaii, there are 23 native species currently placed in Omiodes, but this classification has been disputed, and they were previously placed in various other genera. We used molecular phylogenetic analyses to assess the monophyly of Omiodes as a whole, and specifically of the Hawaiian species, as well as their geographic origins and possible ancestral host plants. Mitochondrial (COI) and nuclear (wingless, EF1α, CAD, and RPS5) DNA was sequenced for Omiodes from Hawaii, South America, and Australasia, along with many other putative outgroup spilomeline genera. Phylogenies were estimated using maximum likelihood and Bayesian inference, and various taxon and character datasets. With the exception of two paleotropical species (O. basalticalis and O. odontosticta, whose placement was unresolved) all Hawaiian, paleotropical and neotropical Omiodes, including the type species, fell within a well-supported, monophyletic clade. Although the center of diversity for Omiodes is in the Neotropics, its center of origin was ambiguous, due to poor resolution of the basal splits between paleotropical and neotropical Omiodes. Very low genetic divergence within the Hawaiian Omiodes suggests a relatively recent colonization of the Hawaiian Islands. Phylogenies constructed using all codon positions were poorly resolved at intergeneric levels, and did not reveal a sister taxon for Omiodes, but phylogenies constructed using only first and second codon positions suggested a close relationship with Cnaphalocrocis. The monophyly of several other spilomeline genera is also discussed.     

Membrane Docking Geometry and Target Lipid Stoichiometry of Membrane-Bound PKCα C2 Domain: A Combined Molecular Dynamics and Experimental Study

Protein kinase Cα (PKCα) possesses a conserved C2 domain (PKCα C2 domain) that acts as a Ca²⁺-regulated membrane targeting element. Upon activation by Ca²⁺, the PKCα C2 domain directs the kinase protein to the plasma membrane, thereby stimulating an array of cellular pathways. At sufficiently high Ca²⁺ concentrations, binding of the C2 domain to the target lipid phosphatidylserine (PS) is sufficient to drive membrane association; however, at typical physiological Ca²⁺ concentrations, binding to both PS and phosphoinositidyl-4,5-bisphosphate (PIP₂) is required for specific plasma membrane targeting. Recent EPR studies have revealed the membrane docking geometries of the PKCα C2 domain docked to (i) PS alone and (ii) both PS and PIP₂ simultaneously. These two EPR docking geometries exhibit significantly different tilt angles relative to the plane of the membrane, presumably induced by the large size of the PIP₂ headgroup. The present study utilizes the two EPR docking geometries as starting points for molecular dynamics simulations that investigate atomic features of the protein-membrane interaction. The simulations yield approximately the same PIP₂-triggered change in tilt angle observed by EPR. Moreover, the simulations predict a PIP₂:C2 stoichiometry approaching 2:1 at a high PIP₂ mole density. Direct binding measurements titrating the C2 domain with PIP₂ in lipid bilayers yield a 1:1 stoichiometry at moderate mole densities and a saturating 2:1 stoichiometry at high PIP₂ mole densities. Thus, the experiment confirms the target lipid stoichiometry predicted by EPR-guided molecular dynamics simulations. Potential biological implications of the observed docking geometries and PIP₂ stoichiometries are discussed.     

The Interplay of Tau Protein and β-Amyloid: While Tauopathy Spreads More Profoundly Than Amyloidopathy,Both Processes Are Almost Equally Pathogenic

Cellular and Molecular Neurobiology - Alzheimer’s disease (AD) is a neurodegenerative disorder, in which amyloid precursor protein (APP) misprocessing and tau protein hyperphosphorylation are...     

Digoxin Derivatives with Enhanced Selectivity for the α2 Isoform of Na,K-ATPase: EFFECTS ON INTRAOCULAR PRESSURE IN RABBITS*

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1β1, α2β1, and α3β1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH₂ substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.     

Lipids as Tumoricidal Components of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET): UNIQUE AND SHARED EFFECTS ON SIGNALING AND DEATH*

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance ¹³C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.     

Discrepancies between subgeneric classification and molecular phylogeny of Ceratitis (Diptera: Tephritidae), can the evolution of host use provide some clues?

Using molecular data from three protein encoding genes and 49 taxa (98 specimens from 20 African countries), we provide an extended phylogeny of Ceratitis and investigate the evolution of stenophagy across clades. Bayesian tree reconstructions support previously proposed monophyletic lineages (Pardalaspis, Pterandrus section A, Pterandrus section B+Ceratitis sensu stricto) and reveal the occurrence of two new monophyletic groups including Ceratalaspis/Hoplolophomyia (viz. Cl(A), and Cl(B)+H). The reconstruction of ancestral character states shows that stenophagy evolved repeatedly and independently in five different clades (Podocarpus, Solanum, Strychnos, Tabernaemontana and Vepris feeders). The evolution of feeding preferences is closely related to the phylogenetic patterns of Ceratalaspis/Hoplolophomyia whose sections include either polyphagous species (Cl(A)) or stenophagous taxa (Cl(B)+H) that are further subdivided in Vepris and Solanum feeders. The evolution of stenophagy in the genus Ceratits appears as the result of a process leading to the exploitation of "unconventional" fruits (viz. toxic and/or not fleshy) and involving either metabolic adaptation to toxic plant compounds and/or the capability of penetrating fruits with thick cuticles.     

Molecular phylogeny and systematics of Australian ‘Iravadiidae’ (Caenogastropoda: Truncatelloidea)

The family Iravadiidae is found to be polyphyletic in a molecular phylogenetic analysis using a subset of Australian taxa. Taxa previously assigned to Iravadia form a monophyletic clade, but Nozeba topaziaca clusters with Auricorona queenslandica n. gen. and n. sp. in an unnamed family related to Tornidae. Aenigmula criscionei n. gen. and n. sp., an iravadiid-like species from the Northern Territory, belongs to another unnamed family related to Caecidae, Calopiidae and Clenchiellidae. A systematic revision of some Australian ‘iravadiids’ raises the subgenera Fluviocingula and Pseudomerelina to full generic rank and reinstates two former synonyms of Iravadia (Fairbankia), Pellamora and Wakauraia, as genera. The species formerly identified in Australia as Iravadia quadrasi is recognised as three allopatric species; Iravadia pilbara n. sp. and the reinstated species Iravadia goliath and Iravadia quadrina. Pellamora splendida n. sp., from Western Australia, is recognised as distinct from Pellamora australis, and Fluviocingula superficialis n. sp. from Fluviocingula resima. Wakauraia fukudai n. sp. is recorded from central Queensland.

http://zoobank.org/urn:lsid:zoobank.org:pub:1B9917F6-48B2-4597-85C1-F90BA9093475