ICM docking score of the compounds selected for biological screening.

<p>ICM docking score of the compounds selected for biological screening.</p

Compound 1 partially reverses MPTP-induced locomotion deficiency in zebrafish larvae.

<p>Zebrafish embryos at 1-incubated with MPTP (250 μM) and <i>L</i>-deprenyl (20 μM) or the indicated concentrations of compound <b>1</b> for 3 d. Each treatment group contained 12 fish larvae, and three independent trials were performed for each fish. The results represented the mean distance travelled by the larvae in 10 min and values are expressed as mean ± SEM. ^###<i>p</i><0.001 <i>vs.</i> negative control and **<i>p</i><0.01 <i>vs.</i> MPTP.</p

Low-energy binding conformations of compound 1 and the acetamide bound to the iNOS molecular model by computational ligand docking.

<p>(a) iNOS is depicted in ribbon form. The heme group and compound <b>1</b> are depicted as ball-and-stick models. (b) A superposition of compound <b>1</b> and the acetamide, shown as ball-and-stick models.</p

Protein expression of iNOS, eNOS and nNOS in HepG2 cells.

<p>HepG2 cells were treated with SMT and compound <b>1</b> (1 or 5 μM) or DMSO (vehicle control) for 12 h analysed by Western blotting. Equal protein loading was confirmed by GAPDH content.</p

Structures of highest-scoring 8 compounds identified in the high-throughput virtual screening chosen for biological validation.

<p>Structures of highest-scoring 8 compounds identified in the high-throughput virtual screening chosen for biological validation.</p

Transcriptome Analysis in Venom Gland of the Predatory Giant Ant <i>Dinoponera quadriceps</i>: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

<div><p>Background</p><p><i>Dinoponera quadriceps</i> is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the <i>D. quadriceps</i> venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation.</p><p>Results</p><p>We conducted a transcriptome analysis of a cDNA library from the <i>D. quadriceps</i> venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences.</p><p>Conclusions</p><p>To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant <i>D. quadriceps</i>. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans.</p></div

Dinoponera quadriceps in the field and its dissected venom apparatus.

<p>Part A - A single specimen of D. quadriceps protecting the nest's entrance. Part B - Dissected <i>D. quadriceps</i> venom apparatus (×40). Abbreviations: <i>Dg</i> - Doffur's gland; <i>cv</i> - convoluted gland (not observable); <i>st</i> - secretory tubule; <i>vs</i>- venom sac; sg – sting.</p

Multiple alignments of the deduced amino acid sequences of venom allergen I (Sol i 1) from <i>D. quadriceps</i> with known sequences from different ant and wasp species.

<p>The identical and conserved amino acid residues of diverse ant and wasp hymenopterans are highlighted in black and gray, respectively. Dots represent gaps.</p

Distribution of the Hymenoptera species as determined by best protein hits.

<p>The percentages of homologs from distinct hymenopterans species with which the 6,429 contigs of the <i>D. quadriceps</i> venom gland shared the high sequence similarities.</p

Multiple alignments of deduced amino acid sequences of different identified dinoponeratoxins from the <i>D. quadriceps</i> venom gland transcriptome and <i>D. australis</i>.

<p>Deduced <i>D. quadriceps</i> dinoponeratoxin cDNA precursor sequences (contig_1 and contig_9). RNA deep sequencing contigs (contig_2 and contig_145) were compared to mature peptide sequences from <i>D. australis</i> (Da-3177 and Da-3105) (part A) and from another species of <i>Dinoponera</i> (Da-2501) (part B). ClustalW was used to multi-align the sequences. Identical amino acid residues are marked with asterisks. Stretches of deduced amino acid sequences supported by EST sequences are boxed. Signal peptides (pre-peptides) are doubled underlined, and pro-regions of pro-peptides are shown with a single line under the sequence. Contigs 1 and 9 were first identified in the EST library, and contigs 2 and 145 came primarily from RNA-Seq.</p