Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic pathogenic fungus which causes fatal invasive aspergillosis among immunocompromised patients. To obtain a better understanding of the key elements involved in A. fumigatus virulence and to identify possible drug targets, it is necessary to be able to generate gene-deletion strains. Unfortunately, the molecular techniques available do not include a rapid method to disrupt and identify essential genes. RNA interference, a process in which the presence of double-stranded RNA homologous to a gene of interest results in specific degradation of the corresponding message, has been successfully tested on A. fumigatus. We have shown that expression of double stranded RNA corresponding to portions of the ALB1/PKSP and FKS1 genes results in reduced mRNA levels for those genes, with phenotypic consequences similar to that of gene disruption. The two genes could also be subjected to simultaneous interference through expression of chimeric double-stranded RNA. Use of RNA interference in Aspergillus will allow easier examination of the phenotypic consequences of reducing expression of a gene of interest, especially for essential genes.     

Rapid parasite adaptation drives selection for high recombination rates

The Red Queen hypothesis proposes that sex is maintained through selection pressure imposed by coevolving parasites: susceptible hosts are able to escape parasite pressure by recombining their genome to create resistant offspring. However, previous theoretical studies have shown that the Red Queen typically selects against sex unless selection is strong, arguing that high rates of recombination cannot evolve when parasites are of low virulence. Here we show that under the biologically plausible assumption of a severe fitness cost for parasites that fail to infect, the Red Queen can cause selection for high recombination rates, and that the strength of virulence is largely irrelevant to the direction of selection for increased recombination rates. Strong selection on parasites and short generation times make parasites usually better adapted to their hosts than vice versa and can thus favor higher recombination rates in hosts. By demonstrating the importance of host-imposed selection on parasites, our findings resolve previously reported conflicting results.     

Receptor recognition sites of cytokines are organized as exchangeable modules. Transfer of the leukemia inhibitory factor receptor-binding site from ciliary neurotrophic factor to interleukin-6

Interleukin-6 (IL-6) and ciliary neurotrophic factor (CNTF) are "4-helical bundle" cytokines of the IL-6 type family of neuropoietic and hematopoietic cytokines. IL-6 signals by induction of a gp130 homodimer (e.g. IL-6), whereas CNTF and leukemia inhibitory factor (LIF) signal via a heterodimer of gp130 and LIF receptor (LIFR). Despite binding to the same receptor component (gp130) and a similar protein structure, IL-6 and CNTF share only 6% sequence identity. Using molecular modeling we defined a putative LIFR binding epitope on CNTF that consists of three distinct regions (C-terminal A-helix/N-terminal AB loop, BC loop, C-terminal CD-loop/N-terminal D-helix). A corresponding gp130-binding site on IL-6 was exchanged with this epitope. The resulting IL-6/CNTF chimera lost the capacity to signal via gp130 on cells without LIFR, but acquired the ability to signal via the gp130/LIFR heterodimer and STAT3 on responsive cells. Besides identifying a specific LIFR binding epitope on CNTF, our results suggest that receptor recognition sites of cytokines are organized as modules that are exchangeable even between cytokines with limited sequence homology.     

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.     

A Multilevel Probabilistic Beam Search Algorithm for the Shortest Common Supersequence Problem

The shortest common supersequence problem is a classical problem with many applications in different fields such as planning, Artificial Intelligence and especially in Bioinformatics. Due to its NP-hardness, we can not expect to efficiently solve this problem using conventional exact techniques. This paper presents a heuristic to tackle this problem based on the use at different levels of a probabilistic variant of a classical heuristic known as Beam Search. The proposed algorithm is empirically analysed and compared to current approaches in the literature. Experiments show that it provides better quality solutions in a reasonable time for medium and large instances of the problem. For very large instances, our heuristic also provides better solutions, but required execution times may increase considerably.     

Odor Memory Stability after Reinnervation of the Olfactory Bulb

The olfactory system, particularly the olfactory epithelium, presents a unique opportunity to study the regenerative capabilities of the brain, because of its ability to recover after damage. In this study, we ablated olfactory sensory neurons with methimazole and followed the anatomical and functional recovery of circuits expressing genetic markers for I7 and M72 receptors (M72-IRES-tau-LacZ and I7-IRES-tau-GFP). Our results show that 45 days after methimazole-induced lesion, axonal projections to the bulb of M72 and I7 populations are largely reestablished. Furthermore, regenerated glomeruli are re-formed within the same areas as those of control, unexposed mice. This anatomical regeneration correlates with functional recovery of a previously learned odorant-discrimination task, dependent on the cognate ligands for M72 and I7. Following regeneration, mice also recover innate responsiveness to TMT and urine. Our findings show that regeneration of neuronal circuits in the olfactory system can be achieved with remarkable precision and underscore the importance of glomerular organization to evoke memory traces stored in the brain.     

Faunistic analysis of sharpshooters (Hemiptera: Auchenorrhyncha, Cicadellidae) in a 'Westin' sweet orange orchard

The purpose of this study was to analyze sharpshooter fauna in a five-year-old 'Westin' sweet orange orchard. Yellow sticky traps were placed on the edge of a forest, and on the periphery and inside the citrus stand. The traps were evaluated fortnightly, for three years. The most frequent species were Acrogonia citrina Marucci & Cavichioli, Bucephalogonia xanthophis (Berg), and Oncometopia facialis (Signoret). B. xanthophis occurred more in the forest edge, especially on spring and winter. A. citrina occurred most frequently in the forest edge, especially on spring. The highest incidence of O. facialis was inside the citrus stand, on spring and summer. Other cicadellids occurred more often in the forest edge, especially on summer. A. citrina, B. xanthophis, Dilobopterus costalimai Young, and O. facialis were predominant in all places studied. A. citrina, B. xanthophis and O. facialis were super dominant, super abundant, super frequent, and constant, except inside the stand, where B. xanthophis was dominant, very abundant, very frequent, and constant. D. costalimai and Homalodisca ignorata Melichar were dominant, very abundant, and very frequent in the forest edge and in the periphery of the stand, and D. costalimai was also predominant inside the stand. Scopogonalia subolivacea (St?l) was predominant in the forest edge and inside the stand, while Plesiommata corniculata Young was predominant in the periphery (both were dominant, very abundant, very frequent, and accessory).