Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus

We present the genome sequences of a new clinical isolate of the important human pathogen, Aspergillus fumigatus, A1163, and two closely related but rarely pathogenic species, Neosartorya fischeri NRRL181 and Aspergillus clavatus NRRL1. Comparative genomic analysis of A1163 with the recently sequenced A. fumigatus isolate Af293 has identified core, variable and up to 2% unique genes in each genome. While the core genes are 99.8% identical at the nucleotide level, identity for variable genes can be as low 40%. The most divergent loci appear to contain heterokaryon incompatibility (het) genes associated with fungal programmed cell death such as developmental regulator rosA. Cross-species comparison has revealed that 8.5%, 13.5% and 12.6%, respectively, of A. fumigatus, N. fischeri and A. clavatus genes are species-specific. These genes are significantly smaller in size than core genes, contain fewer exons and exhibit a subtelomeric bias. Most of them cluster together in 13 chromosomal islands, which are enriched for pseudogenes, transposons and other repetitive elements. At least 20% of A. fumigatus-specific genes appear to be functional and involved in carbohydrate and chitin catabolism, transport, detoxification, secondary metabolism and other functions that may facilitate the adaptation to heterogeneous environments such as soil or a mammalian host. Contrary to what was suggested previously, their origin cannot be attributed to horizontal gene transfer (HGT), but instead is likely to involve duplication, diversification and differential gene loss (DDL). The role of duplication in the origin of lineage-specific genes is further underlined by the discovery of genomic islands that seem to function as designated "gene dumps" and, perhaps, simultaneously, as "gene factories".     

Evaluation of a fast implicit solvent model for molecular dynamics simulations.

A solvation term based on the solvent accessible surface area (SASA) is combined with the CHARMM polar hydrogen force field for the efficient simulation of peptides and small proteins in aqueous solution. Only two atomic solvation parameters are used: one is negative for favoring the direct solvation of polar groups and the other positive for taking into account the hydrophobic effect on apolar groups. To approximate the water screening effects on the intrasolute electrostatic interactions, a distance-dependent dielectric function is used and ionic side chains are neutralized. The use of an analytical approximation of the SASA renders the model extremely efficient (i.e., only about 50% slower than in vacuo simulations). The limitations and range of applicability of the SASA model are assessed by simulations of proteins and structured peptides. For the latter, the present study and results reported elsewhere show that with the SASA model it is possible to sample a significant amount of folding/unfolding transitions, which permit the study of the thermodynamics and kinetics of folding at an atomic level of detail.     

Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.     

Effects of the Diet on the Microbiota of the Red Palm Weevil (Coleoptera: Dryophthoridae)

Rhynchophorus ferrugineus, also known as the red palm weevil, is regarded as the major pest of palm trees. Although studies of the microbiota associated with this species have been performed in recent years, little attention has been dedicated to the influence of the diet in shaping the host bacterial community. Here, we investigated the influence of food sources (i.e. palm tissues vs apple based substrate) on the microbial diversity associated with RPW, which was compared with the microbiota associated with wild individuals of the sister species Rhynchophorus vulneratus. The bacterial characterization was performed using a culture independent approach, i.e. the 16S rRNA pyrotag, and a culture dependent approach for a subset of the samples, in order to obtain bacterial isolates from RPW tissues. The bacterial community appeared significantly influenced by diet. Proteobacteria resulted to be the most abundant clade and was present in all the specimens of the three examined weevil groups. Within Proteobacteria, Enterobacteriaceae were identified in all the organs analysed, including hemolymph and reproductive organs. The apple-fed RPWs and the wild R. vulneratus showed a second dominant taxon within Firmicutes that was scarcely present in the microbiota associated with palm-fed RPWs. A comparative analysis on the bacteria associated with the palm tissues highlighted that 12 bacterial genera out of the 13 identified in the plant tissues were also present in weevils, thus indicating that palm tissues may present a source for bacterial acquisition.     

Evidence of serotonin-dopamine involvement in the inhibition of d-amphetamine stereotypy and appearance of stereotyped movements found respectively after acute and long-term treatment with morphine and methadone in rats

P-chlorophenylalanine, an inhibitor of serotonin synthesis, was found to completely prevent the inhibitory effect of morphine and methadone on the stereotypy caused by d-amphetamine and methyl-phenidate in rats. d-Fenfluramine and m-chlorophenylpiperazine, two drugs supposed to increase serotonin transmission, and halo-peridol, an antagonist of dopamine at central receptors, blocked the stereotyped movements induced by repeated treatment with morphine and methadone. The results suggest that a) brain serotonin mediates the effect of morphine and methadone on amphetamine and methylphenidate stereotypy b) serotonin and dopamine are involved in the stereotyped movements caused by long-term treatment with these narcotics in the rat.     

Cloning and characterization of barley long chain acyl-CoA oxidase and its possible regulation by glucose

A barley ( Hordeum vulgare L.) full-length clone coding for long chain acyl-CoA oxidase (ACX), key enzyme of β -oxidation, was isolated by cDNA library screening and 5'-rapid amplification of cDNA ends. The cDNA encodes for a polypeptide of 667 amino acids, with a molecular mass of 74.5 kDa. The amino acid sequence, beside an extensive similarity with other plant and mammalian ACXs, showed a PTS1 peroxisomal targeting signal at the C terminus and a conserved FAD-binding domain. The gene was over-expressed in E. coli and the fusion protein was shown to possess long chain acyl-CoA oxidase activity. Polyclonal antibodies were raised against a large fragment of the protein encoded by the barley putative ACX gene. Northern and Western analysis demonstrated that a basal level of long chain ACX is always present along the barley life cycle, while a higher level of expression is typical of actively growing tissues such as germinating embryos, ovary before anthesis, developing embryos, shoots and roots apexes. In vitro germination experiments with glucose and glucose analogues provided evidence about the involvement of a glucose-deriving signal in the positive modulation of ACX expression. This result highlights the role of ACX, not only during oil reserve mobilization, but also in plant growth and metabolism.     

Effect of preincubation UV irradiation of abscisic acid and gibberellin A3 on {alpha}-amylase induction in barley half-seeds

A 24 hr preincubation UV (254 nm) irradiation of RS-ABA andGA₃ solutions completely negated the inhibitory effect of ABAon GA₃ induced -amylase production with barley half-seeds. Thisirradiation, however, caused no significant inhibition of GA₃-induced-amylase production. UV treatment of an extract from Tulipaindicated that this technique can be used to photooxidize ABA-likesubstances present in plant extracts. ¹ Michigan Agricultural Experiment Station Journal Article No.5774. This research was supported in part by a grant from theNetherlands Flower-Bulb Institute, New York and the OrnamentalMarketing Board, The Hague. ² Present address: Istituto di Orticoltura e Floricoltura, Universitàdegli Studi di Pisa, 56100, Italy. (Received February 26, 1972; )     

The 3A6-TCR/superagonist/HLA-DR2a complex shows similar interface and reduced flexibility compared to the complex with self-peptide

T-cell receptor (TCR) recognition of the myelin basic protein (MBP) peptide presented by major histocompatibility complex (MHC) protein HLA-DR2a, one of the MHC class II alleles associated with multiple sclerosis, is highly variable. Interactions in the trimolecular complex between the TCR of the MBP83-99-specific T cell clone 3A6 with the MBP-peptide/HLA-DR2a (abbreviated TCR/pMHC) lead to substantially different proliferative responses when comparing the wild-type decapeptide MBP90-99 and a superagonist peptide, which differs mainly in the residues that point toward the TCR. Here, we investigate the influence of the peptide sequence on the interface and intrinsic plasticity of the TCR/pMHC trimolecular and pMHC bimolecular complexes by molecular dynamics simulations. The intermolecular contacts at the TCR/pMHC interface are similar for the complexes with the superagonist and the MBP self-peptide. The orientation angle between TCR and pMHC fluctuates less in the complex with the superagonist peptide. Thus, the higher structural stability of the TCR/pMHC tripartite complex with the superagonist peptide, rather than a major difference in binding mode with respect to the self-peptide, seems to be responsible for the stronger proliferative response.     

Discovery of a Non-Peptidic Inhibitor of West Nile Virus NS3 Protease by High-Throughput Docking

Background

The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections.

Methodology

In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by ¹⁵N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay.

Conclusion

The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.