APO1 promotes the splicing of chloroplast group II introns and harbors a plant-specific zinc-dependent RNA binding domain

Arabidopsis thaliana APO1 is required for the accumulation of the chloroplast photosystem I and NADH dehydrogenase complexes and had been proposed to facilitate the incorporation of [4Fe-4S] clusters into these complexes. The identification of maize (Zea mays) APO1 in coimmunoprecipitates with a protein involved in chloroplast RNA splicing prompted us to investigate a role for APO1 in splicing. We show here that APO1 promotes the splicing of several chloroplast group II introns: in Arabidopsis apo1 mutants, ycf3-intron 2 remains completely unspliced, petD intron splicing is strongly reduced, and the splicing of several other introns is compromised. These splicing defects can account for the loss of photosynthetic complexes in apo1 mutants. Recombinant APO1 from both maize and Arabidopsis binds RNA with high affinity in vitro, demonstrating that DUF794, the domain of unknown function that makes up almost the entirety of APO1, is an RNA binding domain. We provide evidence that DUF794 harbors two motifs that resemble zinc fingers, that these bind zinc, and that they are essential for APO1 function. DUF794 is found in a plant-specific protein family whose members are all predicted to localize to mitochondria or chloroplasts. Thus, DUF794 adds a new example to the repertoire of plant-specific RNA binding domains that emerged as a product of nuclear-organellar coevolution.     

Immunolocalization of endocan during the endothelial-mesenchymal transition process

Endocan is a dermatan sulfate proteoglycan (DSPG) that has been observed in the cytoplasm of endothelial cells of small and large vessels in lung, kidney, liver, colon, ovary and brain tumors. This DSPG has been implicated in the regulation of cellular activities such as adhesion, migration, and proliferation. Given the important roles played by endocan in such processes, we sought to determine whether this DSPG is present in the chicken embryo aortic wall in embryonic days 12 and 14, when intimal thickening and endothelial transformation are notorious. Immunolabeling of serial paraffin cross-sections revealed endocan immunoreactivity at the endothelium and some mesenchymal cells constituting the intimal thickening but not in the cells arranged in lamellar layers. We also investigated whether endocan was present in monolayers of primary embryonic aortic endothelial cells attached to fibronectin when they were deprived of serum and stimulated with epidermal growth factor. Immunofluorescence determined that in the epidermal growth factor (EGF) condition where separating, detaching, and migrating cells were observed, endocan appeared organized in arrays typical of focal complexes in the leading edge of these cells. In serum-free medium condition in which the endothelial cells displayed a cobblestone appearance, endocan appeared mainly delineating the margin of many cells. This study demonstrates for the first time the presence of endocan during the aortic wall remodeling, and provides evidence that suggests a possible contribution of this DSPG in the endothelial-mesenchymal transition (EndoMT) process.     

Generating balanced learning and test sets for function approximation problems

In function approximation problems, one of the most common ways to evaluate a learning algorithm consists in partitioning the original data set (input/output data) into two sets: learning, used for building models, and test, applied for genuine out-of-sample evaluation. When the partition into learning and test sets does not take into account the variability and geometry of the original data, it might lead to non-balanced and unrepresentative learning and test sets and, thus, to wrong conclusions in the accuracy of the learning algorithm. How the partitioning is made is therefore a key issue and becomes more important when the data set is small due to the need of reducing the pessimistic effects caused by the removal of instances from the original data set. Thus, in this work, we propose a deterministic data mining approach for a distribution of a data set (input/output data) into two representative and balanced sets of roughly equal size taking the variability of the data set into consideration with the purpose of allowing both a fair evaluation of learning's accuracy and to make reproducible machine learning experiments usually based on random distributions. The sets are generated using a combination of a clustering procedure, especially suited for function approximation problems, and a distribution algorithm which distributes the data set into two sets within each cluster based on a nearest-neighbor approach. In the experiments section, the performance of the proposed methodology is reported in a variety of situations through an ANOVA-based statistical study of the results.     

Comparative evaluation of pyrethroid insecticide formulations against Triatoma infestans (Klug): residual efficacy on four substrates

We investigated the residual efficacy of four insecticide formulations used in Chagas disease vector control campaigns: cyfluthrin 12.5% suspension concentrace (SC), lambda-cyhalothrin 10% wettable powder (WP), deltamethrin 2.5% SC, and 2.5% WP on four types of circular blocks of wood, straw with mud, straw with mud painted with lime, and mud containing 5% of cement. Three concentrations of these insecticides were tested: the LC90 (previously determined on filter paper), the double of the LC90, and the recommended operational dose. For each bioassay test, 15 third-stage nymphs of Triatoma infestans (Klug) (Hemiptera: Reduviidae) were exposed for 120 h to each treatment at 24 h, 30, 60, 90, and 180 days post-spraying. Mortality rates, moulting history and behaviour were recorded at 24, 48, 72, and 120 h of exposure. Mortality rates were highest during the first 30 days post-spraying. Highest mortality rates (above 50%) were observed for deltamethrin 2.5% SC and lambda-cyhalothrin 10% WP on wood blocks up to three months post-spraying. Mud was the substrate on which treatments showed lowest persistence, with the other two substrates showing intermediate residual efficacy of all treatments. During the first 30 days WP formulations were not as effective as SC flowable formulations but, overall in the longer term, WP gave grater mortality rates of T. infestans nymphs exposed at up to six months post-spraying. Porous surfaces, especially mud, showed most variability presumably due to absorption of the insecticide. In contrast the less porous surfaces (i.e. wood and lime-coated mud) kept mortality rates high for longer post-treatment, irrespective of the insecticide concentration used.     

Bait matrix for delivery of chitin synthesis inhibitors to the formosan subterranean termite (Isoptera: Rhinotermitidae)

The efficacy of three chitin synthesis inhibitors, diflubenzuron, hexaflumuron, and chlorfluazuron, incorporated into a novel bait matrix to kill the Formosan subterranean termite, Coptotermes formosanus Shiraki, was evaluated in the laboratory. The bait matrix was significantly preferred by C. formosanus over southern yellow pine wood in a two-choice feeding test. Bait formulations containing 250 ppm of the three chitin synthesis inhibitors were presented to termite nests with 2,500 individuals (80% workers and 20% soldiers) in the presence of alternative food sources consisting of cardboard and southern yellow pine, Pinus taeda L., wood. None of the bait formulations were significantly repellent or feeding deterrent to the termite workers evidenced by the lack of full consumption of alternative food sources. All nests presented with the bait formulations died within 9 wk, whereas the control nests (bait with no chitin synthesis inhibitors) remained alive 6 mo after the end of the study. No significant differences in consumption were observed among the chitin synthesis inhibitor treatments. Importance of this study for the improvement of current bait technology is discussed.     

Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.     

Regulation of miR319 during cold stress in sugarcane

MicroRNAs (miRNAs) are part of a novel mechanism of gene regulation that is active in plants under abiotic stress conditions. In the present study, 12 miRNAs were analysed to identify miRNAs differentially expressed in sugarcane subjected to cold stress (4 °C). The expression of miRNAs assayed by stem–loop RT‐PCR showed that miR319 is up‐regulated in sugarcane plantlets exposed to 4 °C for 24 h. The induction of miR319 expression during cold stress was observed in both roots and shoots. Sugarcane miR319 was also regulated by treatment with abscisic acid. Putative targets of this miRNA were identified and their expression levels were decreased in sugarcane plantlets exposed to cold. The cleavage sites of two targets were mapped using a 5′ RACE PCR assay confirming the regulation of these genes by miR319. When sugarcane cultivars contrasting in cold tolerance were subjected to 4 °C, we observed up‐regulation of miR319 and down‐regulation of the targets in both varieties; however, the changes in expression were delayed in the cold‐tolerant cultivar. These results suggest that differences in timing and levels of the expression of miR319 and its targets could be tested as markers for selection of cold‐tolerant sugarcane cultivars.     

Pyrethroid insecticide evaluation on different house structures in a Chagas disease endemic area of the Paraguayan Chaco

Insecticide effects of deltamethrin 2.5% SC (flowable solution) on different substrates and triatomine infestation rates in two indigenous villages (Estancia Salzar and Nueva Promesa) of the Paraguayan Chaco are reported. This field study was carried out to determine the extent to which variability in spray penetration may affect residual action of the insecticide. A total of 117 houses in the two villages were sprayed. Filter papers discs were placed on aluminium foil pinned to walls and roofs in selected houses and the applied insecticide concentration was determined by high pressure liquid chromatography (HPLC). The target dose rate was 25 mg a.i./m2. The mean actual applied dose in Estancia Salazar was 11.2 +/- 3.1 mg a.i./m2 in walls and 11.9 +/- 5.6 mg a.i./m2 in roofs while in Nueva Promesa, where duplicates were carried out, the mean values were 19.9 +/- 6.9 mg a.i./m2 and 34.7 +/- 10.4 mg a.i./m2 in walls and 28.8 +/- 19.2 mg a.i./m2 and 24.9 +/- 21.8 mg a.i./m2 in roofs. This shows the unevenness and variability of applied doses during spraying campaigns, and also the reduced coverage over roof surfaces. However, wall bioassays with Triatoma infestans nymphs in a 72 h exposure test showed that deposits of deltamethrin persisted in quantities sufficient to kill triatomines until three months post spraying. Knockdown by deltamethrin on both types of surfaces resulted in 100% final mortality. A lower insecticidal effect was observed on mud walls. However, three months after treatment, sprayed lime-coated mud surfaces displayed a twofold greater capacity (57.5%) to kill triatomines than mud sprayed surfaces (25%). Re-infestation was detected by manual capture only in one locality, six months after spraying.