An adult Drosophila glioma model to highlight metabolic dysfunctions and evaluate the role of the serotonin 5-HT7 receptor as a potential therapeutic target.

Gliomas account for 50% of brain cancers and are therefore the most common brain tumors. Molecular alterations involved in adult gliomas have been identified and mainly affect tyrosine kinase receptors with amplification and/or mutation of the epidermal growth factor receptor (EGFR) and its associated signaling pathways. Several targeted therapies have been developed, but current treatments remain ineffective for glioblastomas, the most severe forms. Thus, it is a priority to identify new pharmacological targets. Drosophila glioma models established in larvae and adults are useful to identify new genes and signaling pathways involved in glioma progression. Here, we used a Drosophila glioma model in adults, to characterize metabolic disturbances associated with glioma and assess the consequences of 5-HT7 R expression on glioma development. First, by using in vivo magnetic resonance imaging, we have shown that expression of the constitutively active forms of EGFR and PI3K in adult glial cells induces brain enlargement. Then, we explored altered cellular metabolism by using high-resolution magic angle spinning NMR and 1 H-13 C heteronuclear single quantum coherence solution states. Discriminant metabolites identified highlight the rewiring of metabolic pathways in glioma and associated cachexia phenotypes. Finally, the expression of 5-HT7 R in this adult model attenuates phenotypes associated with glioma development. Collectively, this whole-animal approach in Drosophila allowed us to provide several rapid and robust phenotype readouts, such as enlarged brain volume and glioma-associated cachexia, as well as to determine the metabolic pathways involved in glioma genesis and finally to confirm the interest of the 5-HT7 R in the treatment of glioma.

Cefiderocol activity is compromised by acquired extended-spectrum oxacillinases in Pseudomonas aeruginosa.

OBJECTIVES: Cefiderocol has an excellent in vitro activity on clinical strains of Pseudomonas aeruginosa (P. aeruginosa). However, the resistance of some isolates has been associated with the production of some ß-lactamases. Whether some acquired extended-spectrum oxacillinases (ES-OXA) common in this species may compromise the susceptibility of P. aeruginosa to cefiderocol has not been evaluated so far. METHODS: Eighteen genes encoding OXA belonging to the major subgroups identified in P. aeruginosa OXA-1 (n = 3); - 2 (n = 5); - 10 (n = 8), and - 46 (n = 2) were cloned into pUCP24 shuttle vector and transferred into reference strain PAO1. RESULTS: Although production of the OXA-1 subgroup enzymes did not alter cefiderocol MICs, the ß-lactamases of OXA-2, OXA-46, and four variants of the OXA-10 subgroup resulted in an 8-fold to 32-fold decrease in susceptibility in the PAO1 background. Interestingly, point mutations Ala149Pro and Asp150Gly in OXA-2 subgroup, Trp154Cys and Gly157Asp in OXA-10 subgroup (all located in the Ω loop), and the duplication of a Thr206 and a Gly207 in the ß5-ß6 loop of OXA-10 subgroup were related to decreased susceptibility to cefiderocol. We also showed that some ES-OXA, including the most frequent ES-OXA in P. aeruginosa strains, OXA-19 (derived from OXA-10 subgroup), significantly compromised activity of cefiderocol in addition to ceftazidime, ceftolozane/tazobactam, and ceftazidime/avibactam in clinical strains. CONCLUSION: This work shows that several ES-OXA have a significant effect on cefiderocol susceptibility. Of concern are the Trp154Cys and Gly157Asp mutations that occur in some of these ß-lactamases, as they are associated with a decreased activity of the most recent cephalosporins introduced to combat P. aeruginosa infections.

Structure-function relationship of new peptides activating human Nav1.1.

Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.

The diversification of the antimicrobial peptides from marine worms is driven by environmental conditions.

Antimicrobial peptides (AMPs) play a key role in the external immunity of animals, offering an interesting model for studying the influence of the environment on the diversification and evolution of immune effectors. Alvinellacin (ALV), arenicin (ARE) and polaricin (POL, a novel AMP identified here), characterized from three marine worms inhabiting contrasted habitats ('hot' vents, temperate and polar respectively), possess a well conserved BRICHOS domain in their precursor molecule despite a profound amino acid and structural diversification of the C-terminal part containing the core peptide. Data not only showed that ARE, ALV and POL display an optimal bactericidal activity against the bacteria typical of the habitat where each worm species lives but also that this killing efficacy is optimal under the thermochemical conditions encountered by their producers in their environment. Moreover, the correlation between species habitat and the cysteine contents of POL, ARE and ALV led us to investigate the importance of disulfide bridges in their biological efficacy as a function of abiotic pressures (pH and temperature). The construction of variants using non-proteinogenic residues instead of cysteines (α-aminobutyric acid variants) leading to AMPs devoid of disulfide bridges, provided evidence that the disulfide pattern of the three AMPs allows for a better bactericidal activity and suggests an adaptive way to sustain the fluctuations of the worm's environment. This work shows that the external immune effectors exemplified here by BRICHOS AMPs are evolving under strong diversifying environmental pressures to be structurally shaped and more efficient/specific under the ecological niche of their producer.

The Two Domains of the Avian Double-ß-Defensin AvBD11 Have Different Ancestors, Common with Potential Monodomain Crocodile and Turtle Defensins.

Beta-defensins are an essential group of cysteine-rich host-defence peptides involved in vertebrate innate immunity and are generally monodomain. Among bird defensins, the avian ß-defensin 11 (AvBD11) is unique because of its peculiar structure composed of two ß-defensin domains. The reasons for the appearance of such 'polydefensins' during the evolution of several, but not all branches of vertebrates, still remain an open question. In this study, we aimed at exploring the origin and evolution of the bird AvBD11 using a phylogenetic approach. Although they are homologous, the N- and C-terminal domains of AvBD11 share low protein sequence similarity and possess different cysteine spacing patterns. Interestingly, strong variations in charge properties can be observed on the C-terminal domain depending on bird species but, despite this feature, no positive selection was detected on the AvBD11 gene (neither on site nor on branches). The comparison of AvBD11 protein sequences in different bird species, however, suggests that some amino acid residues may have undergone convergent evolution. The phylogenetic tree of avian defensins revealed that each domain of AvBD11 is distant from ovodefensins (OvoDs) and may have arisen from different ancestral defensins. Strikingly, our phylogenetic analysis demonstrated that each domain of AvBD11 has common ancestors with different putative monodomain ß-defensins from crocodiles and turtles and are even more closely related with these reptilian defensins than with their avian paralogs. Our findings support that AvBD11's domains, which differ in their cysteine spacing and charge distribution, do not result from a recent internal duplication but most likely originate from a fusion of two different ancestral genes or from an ancestral double-defensin arisen before the Testudines-Archosauria split.

Real-Time Fluorescence Microscopy on Living E. coli Sheds New Light on the Antibacterial Effects of the King Penguin ß-Defensin AvBD103b.

(1) Antimicrobial peptides (AMPs) are a promising alternative to conventional antibiotics. Among AMPs, the disulfide-rich ß-defensin AvBD103b, whose antibacterial activities are not inhibited by salts contrary to most other ß-defensins, is particularly appealing. Information about the mechanisms of action is mandatory for the development and approval of new drugs. However, data for non-membrane-disruptive AMPs such as ß-defensins are scarce, thus they still remain poorly understood. (2) We used single-cell fluorescence imaging to monitor the effects of a ß-defensin (namely AvBD103b) in real time, on living E. coli, and at the physiological concentration of salts. (3) We obtained key parameters to dissect the mechanism of action. The cascade of events, inferred from our precise timing of membrane permeabilization effects, associated with the timing of bacterial growth arrest, differs significantly from the other antimicrobial compounds that we previously studied in the same physiological conditions. Moreover, the AvBD103b mechanism does not involve significant stereo-selective interaction with any chiral partner, at any step of the process. (4) The results are consistent with the suggestion that after penetrating the outer membrane and the cytoplasmic membrane, AvBD103b interacts non-specifically with a variety of polyanionic targets, leading indirectly to cell death.

In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins.

Photoactivatable drugs targeting ligand-gated ion channels open up new opportunities for light-guided therapeutic interventions. Photoactivable toxins targeting ion channels have the potential to control excitable cell activities with low invasiveness and high spatiotemporal precision. As proof-of-concept, we develop HwTxIV-Nvoc, a UV light-cleavable and photoactivatable peptide that targets voltage-gated sodium (NaV) channels and validate its activity in vitro in HEK293 cells, ex vivo in brain slices and in vivo on mice neuromuscular junctions. We find that HwTxIV-Nvoc enables precise spatiotemporal control of neuronal NaV channel function under all conditions tested. By creating multiple photoactivatable toxins, we demonstrate the broad applicability of this toxin-photoactivation technology.

Complementary Nuclear Magnetic Resonance-Based Metabolomics Approaches for Glioma Biomarker Identification in a Drosophila melanogaster Model.

Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Drosophila melanogaster is a compelling genetic model to better understanding human neurological diseases owing to its high conservation in signaling pathways and cellular content of the brain. Here, glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by 1H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state 1H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism.

Metabolomic Nuclear Magnetic Resonance Studies at Presymptomatic and Symptomatic Stages of Huntington's Disease on a Drosophila Model.

Huntington's disease (HD) is an inherited neurodegenerative disorder, for which diagnostic development and discovery of new therapeutic targets are urgently required. In this study, a model of HD in Drosophila melanogaster has been used to identify metabolic biomarkers at presymptomatic and symptomatic stages of the disease. The pan-neuronal expression of a pathogenic fragment of the human Huntingtin (HTT) protein containing a 93-repeat polyglutamine expansion (Httex1p Q93) in transgenic flies induces a neuropathology with several characteristics of the human disease. The discriminant metabolites between the diseased flies and their controls were identified by 1H nuclear magnetic resonance and orthogonal partial least squares discriminant multivariate analysis. The experiments carried out with 10-day-old flies allowed us to identify a set of 10 biomarkers of the presymptomatic stage: NAD+, AMP, fumarate, asparagine, dimethylamine, ß-alanine, glutamine, succinate, glutamate, and ethanol. Remarkably, the experiments conducted with 16-day-old flies, when the symptoms of the disease were present, highlighted a different set of 6 biomarkers: phosphocholine, ethanolamine, 2-oxoglutarate, succinate, pyruvate, and acetate. Our results provide a better understanding of the metabolic impairments in a widely used HD model and demonstrate that metabolism perturbations change dramatically during the development of the disease.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Impact of an Antifungal Insect Defensin on the Proteome of the Phytopathogenic Fungus Botrytis cinerea.

ETD151, an analogue of the antifungal insect defensin heliomicin, is an antifungal peptide active against yeasts and filamentous fungi. To decipher the mechanisms underlying its molecular action on the phytopathogenic fungus Botrytis cinerea, a necrotrophic pathogen responsible for gray mold disease, we investigated the changes in 3 day-old mycelia upon treatment with different concentrations of ETD151. Optical and fluorescence microscopies were used prior to establishing the peptide/protein profiles through two mass spectrometry approaches: MALDI profiling, to generate molecular mass fingerprints as peptide signatures, and a gel-free bottom-up proteomics approach. Our results show that a concentration of ETD151 above the half-maximal inhibitory concentration can alter the integrity of the mycelial structure of B. cinerea. Furthermore, reproducible modifications of the peptide/protein composition were demonstrated in the presence of ETD151 within a 1500-16,000 mass (m/z) range. After the robustness of LC-ESI-MS/MS analysis on B. cinerea mycelial extracts was confirmed, our analyses highlighted 340 significantly modulated proteins upon treatment with ETD151 within a 4.8-466 kDa mass range. Finally, data mapping on KEGG pathways revealed the molecular impact of ETD151 on at least six pathways, namely, spliceosome, ribosome, protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway, and oxidative phosphorylation.

Structure, function, and evolution of Gga-AvBD11, the archetype of the structural avian-double-ß-defensin family.

Out of the 14 avian ß-defensins identified in the Gallus gallus genome, only 3 are present in the chicken egg, including the egg-specific avian ß-defensin 11 (Gga-AvBD11). Given its specific localization and its established antibacterial activity, Gga-AvBD11 appears to play a protective role in embryonic development. Gga-AvBD11 is an atypical double-sized defensin, predicted to possess 2 motifs related to ß-defensins and 6 disulfide bridges. The 3-dimensional NMR structure of the purified Gga-AvBD11 is a compact fold composed of 2 packed ß-defensin domains. This fold is the archetype of a structural family, dubbed herein as avian-double-ß-defensins (Av-DBD). We speculate that AvBD11 emanated from a monodomain gene ancestor and that similar events might have occurred in arthropods, leading to another structural family of less compact DBDs. We show that Gga-AvBD11 displays antimicrobial activities against gram-positive and gram-negative bacterial pathogens, the avian protozoan Eimeria tenella, and avian influenza virus. Gga-AvBD11 also shows cytotoxic and antiinvasive activities, suggesting that it may not only be involved in innate protection of the chicken embryo, but also in the (re)modeling of embryonic tissues. Finally, the contribution of either of the 2 Gga-AvBD11 domains to these biological activities was assessed, using chemically synthesized peptides. Our results point to a critical importance of the cationic N-terminal domain in mediating antibacterial, antiparasitic, and antiinvasive activities, with the C-terminal domain potentiating the 2 latter activities. Strikingly, antiviral activity in infected chicken cells, accompanied by marked cytotoxicity, requires the full-length protein.

New protein-DNA complexes in archaea: a small monomeric protein induces a sharp V-turn DNA structure.

MC1, a monomeric nucleoid-associated protein (NAP), is structurally unrelated to other DNA-binding proteins. The protein participates in the genome organization of several Euryarchaea species through an atypical compaction mechanism. It is also involved in DNA transcription and cellular division through unknown mechanisms. We determined the 3D solution structure of a new DNA-protein complex formed by MC1 and a strongly distorted 15 base pairs DNA. While the protein just needs to adapt its conformation slightly, the DNA undergoes a dramatic curvature (the first two bend angles of 55° and 70°, respectively) and an impressive torsional stress (dihedral angle of 106°) due to several kinks upon binding of MC1 to its concave side. Thus, it adopts a V-turn structure. For longer DNAs, MC1 stabilizes multiple V-turn conformations in a flexible and dynamic manner. The existence of such V-turn conformations of the MC1-DNA complexes leads us to propose two binding modes of the protein, as a bender (primary binding mode) and as a wrapper (secondary binding mode). Moreover, it opens up new opportunities for studying and understanding the repair, replication and transcription molecular machineries of Archaea.

Synthesis by native chemical ligation and characterization of the scorpion toxin AmmTx3.

The scorpion toxin AmmTx3 is a specific blocker of Kv4 channels. It was shown to have interesting potential for neurological disorders. In this study, we report the first chemical synthesis of AmmTx3 by using the native chemical ligation strategy and validate its biological activity. We determined its 3D structure by nuclear magnetic resonance spectroscopy, and pointed out that AmmTx3 possesses the well-known CSαß structural motif, which is found in a large number of scorpion toxins. Overall, this study establishes an easy synthetic access to biologically active AmmTx3 toxin.

Armadillidin H, a Glycine-Rich Peptide from the Terrestrial Crustacean Armadillidium vulgare, Displays an Unexpected Wide Antimicrobial Spectrum with Membranolytic Activity.

Antimicrobial peptides (AMPs) are key components of innate immunity and are widespread in nature, from bacteria to vertebrate animals. In crustaceans, there are currently 15 distinct AMP families published so far in the literature, mainly isolated from members of the Decapoda order. Up to now, armadillidin is the sole non-decapod AMP isolated from the haemocytes of Armadillidium vulgare, a crustacean isopod. Its first description demonstrated that armadillidin is a linear glycine-rich (47%) cationic peptide with an antimicrobial activity directed toward Bacillus megaterium. In the present work, we report identification of armadillidin Q, a variant of armadillidin H (earlier known as armadillidin), from crude haemocyte extracts of A. vulgare using LC-MS approach. We demonstrated that both armadillidins displayed broad spectrum antimicrobial activity against several Gram-positive and Gram-negative bacteria, fungi, but were totally inactive against yeasts. Membrane permeabilization assays, only performed with armadillidin H, showed that the peptide is membrane active against bacterial and fungal strains leading to deep changes in cell morphology. This damaging activity visualized by electronic microscopy correlates with a rapid decrease of cell viability leading to highly blebbed cells. In contrast, armadillidin H does not reveal cytotoxicity toward human erythrocytes. Furthermore, no secondary structure could be defined in this study [by circular dichroism (CD) and nuclear magnetic resonance (NMR)] even in a membrane mimicking environment. Therefore, armadillidins represent interesting candidates to gain insight into the biology of glycine-rich AMPs.

The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.

Complete 1H, 15N and 13C assignment of trappin-2 and 1H assignment of its two domains, elafin and cementoin.

Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete (1)H, (15)N and (13)C resonance assignment of the recombinant trappin-2 and the (1)H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.

The nucleoid-associated protein HU enhances 8-oxoguanine base excision by the formamidopyrimidine-DNA glycosylase.

The nucleoid-associated protein HU is involved in numerous DNA transactions and thus is essential in DNA maintenance and bacterial survival. The high affinity of HU for SSBs (single-strand breaks) has suggested its involvement in DNA protection, repair and recombination. SSB-containing DNA are major intermediates transiently generated by bifunctional DNA N-glycosylases that initiate the BER (base excision repair) pathway. Enzyme kinetics and DNA-binding experiments demonstrate that HU enhances the 8-oxoguanine-DNA glycosylase activity of Fpg (formamidopyrimidine-DNA glycosylase) by facilitating the release of the enzyme from its final DNA product (one nucleoside gap). We propose that the displacement of Fpg from its end-DNA product by HU is an active mechanism in which HU recognizes the product when it is still bound by Fpg. Through DNA binding, the two proteins interplay to form a transient ternary complex Fpg/DNA/HU which results in the release of Fpg and the molecular entrapment of SSBs by HU. These results support the involvement of HU in BER in vivo.

Macrocyclic Gd(3+) complexes with pendant crown ethers designed for binding zwitterionic neurotransmitters.

A series of Gd(3+) complexes exhibiting a relaxometric response to zwitterionic amino acid neurotransmitters was synthesized. The design concept involves ditopic interactions 1) between a positively charged and coordinatively unsaturated Gd(3+) chelate and the carboxylate group of the neurotransmitters and 2) between an azacrown ether appended to the chelate and the amino group of the neurotransmitters. The chelates differ in the nature and length of the linker connecting the cyclen-type macrocycle that binds the Ln(3+) ion and the crown ether. The complexes are monohydrated, but they exhibit high proton relaxivities (up to 7.7 mM(-1) s(-1) at 60 MHz, 310 K) due to slow molecular tumbling. The formation of ternary complexes with neurotransmitters was monitored by (1) H relaxometric titrations of the Gd(3+) complexes and by luminescence measurements on the Eu(3+) and Tb(3+) analogues at pH 7.4. The remarkable relaxivity decrease (≈80 %) observed on neurotransmitter binding is related to the decrease in the hydration number, as evidenced by luminescence lifetime measurements on the Eu(3+) complexes. These complexes show affinity for amino acid neurotransmitters in the millimolar range, which can be suited to imaging concentrations of synaptically released neurotransmitters. They display good selectivity over non-amino acid neurotransmitters (acetylcholine, serotonin, and noradrenaline) and hydrogenphosphate, but selectivity over hydrogencarbonate was not achieved.