Carlos Peláez

ERMANIN : AN INSECT DETERRENT FLAVONOID FROM PASSIFLORA FOETIDA RESIN

Abstract Ten flavonoids were isolated from Passiflora foetida L. resin. One of them, ermanin, has a high deterrent activity at 40 ppm against Dione juno larvae.

Development of a solid phase kinetic assay for determination of enzyme activities during composting

Abstract Quantitative determination of enzyme activities can be used to evaluate the dynamics of the composting process. An enzyme activity assay in solid phase was developed in which the enzyme reaction is performed by mixing the compost sample and the corresponding substrate with a water content equivalent to the water retention capacity (WRC) of the compost sample. As opposed to common activity assays that extract and quantify soluble enzymes, this solid phase method evaluates total enzyme activities under conditions closer to those found in the actual composting process. Preliminary results on enzyme activities of a compost of poultry manure and sawdust include amylase, invertase, cellulase, phosphatase, protease, and dehydrogenase activities. With the exception of proteases, all increased from day 9 to 43 of the composting process.

Development of Quantitative Proteomics Using iTRAQ Based on the Immunological Response of Galleria mellonella Larvae Challenged with Fusarium oxysporum Microconidia

Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104 microconidia/mL at 37°C over expressed many more proteins than other treatments.

Alternative oxidase plays an important role in Paracoccidioides brasiliensis cellular homeostasis and morphological transition

Paracoccidioides brasiliensis is the etiologic agent of one of the most common systemic mycoses in Latin America. As a dimorphic fungus, it must adapt to different environments during its life cycle, either in nature or within the host, enduring external stresses such as temperature or host-induced oxidative stress. In this study we addressed the role of alternative oxidase (PbAOX) in cellular homeostasis during batch culture growth and the morphological transition of P. brasiliensis. Using a PbAOX-antisense-RNA (PbAOX-aRNA) strain with a 70% reduction in gene expression, we show that PbAOX is crucial for maintaining cell viability and vitality during batch culture growth of yeast cells, in what appears to be a pH-dependent manner. We also show that silencing of PbAOX drastically reduced expression levels of other detoxifying enzymes (PbY20 and PbMSOD). In addition, our data indicate that PbAOX plays a role during the morphological transition, namely, during the yeast-to-mycelia germination and mycelia/conidia-to-yeast transition, essential events during the establishment of infection by dimorphic fungal pathogens. Altogether, our findings support the hypothesis that PbAOX is important for the maintenance of cellular homeostasis, possibly by assisting redox balancing during cell growth and the morphological switch of P. brasiliensis.

Actividad Fungicida e Insecticida de Emulsiones Agua/Aceite de Mezclas de Extractos de Nicotiana tabacum, Azadiractha indica y Eucalyptus tereticornis

Resumen Se realizo un estudio sobre el potencial insecticida agudo y cronico sobre Drosophila melanogaster y antifungico en Fusarium oxysporum de emulsiones aceite-en-agua de mezclas binarias y ternarias de extractos de Nicotiana tabacum , Azadiractha indica ( neem), y aceite esencial de Eucalyptus tereticornis. Se construyeron curvas dosis/respuesta para el tiempo letal medio, relacion pupa-huevo, adulto-pupa y porcentaje inhibitorio para la actividad antifungica. Se observo alta actividad insecticida aguda del tabaco a 6 g/L (tiempo letal medio=2,3 ± 0,5 minutos ), larvicida en el neem a 0,2 g/L (pupa-huevo= 0,05) y fungicida en el eucalipto a 3 g/L (porcentaje inhibitorio =100%). Las bioactividades se potenciaron en la mayoria de las mezclas binarias, exceptuando la actividad fungicida. La mezcla ternaria presento actividad fungicida antagonica. Se concluye sobre la potencial aplicacion de estos desarrollos para controlar plagas y enfermedades. Palabras clave: actividad fungicida, actividad insecticida. emulsiones agua-en-aceite, accion conjunta, sinergismo

Obtención de Etanol y Biogás a Partir de Banano de Rechazo

In this study was developed a fermentative process to produce ethanol from green banana non optimal for exportation through one induced endogenous hydrolysis. The potential of the stillage to produce biogas was also evaluated. It was compared the induced endogenous hydrolysis, with the exogenous hydrolysis that uses commercial enzymes and the traditional acid hydrolysis done all of them in a final volume of 1 liter, the reference point was the ethanol produced in the fermentation process. The maximum performance was obtained with the endogenous hydrolysis and with these methodology fermentations at 15 liter as done. The average of ethanol produced was 0.065 liters from one kilogram of green banana and the biogas production was 2.24 liter per liter of stillage. The proposed process shows performance comparable with that obtained by sugar cane fermentation. Also the methodology demonstrated several advantages, such as low cost, simple to operate, and because it constitutes a good alternative, which is environmentally compatible in the area of management of crop residues.

Stress responses of tomato protoplasts to copper and paraquat

Plants are often exposed to external biotic or abiotic agents that can damage cells. Biotic agents include molecules produced by pathogens, and abiotic agents include ultraviolet radiation, heavy metals and xenobiotic agents such as herbicides. Because of the importance of abiotic stresses and the limited knowledge of the defense responses of plants to stresses, a study was conducted using flow cytometry to evaluate endocellular events in protoplasts of two tomato species, Lycopersicon hirsutum and Lycopersicon esculentum, following exposure to 10 mM CuCl2 and 1% paraquat. During the first 30 min of exposure to 10 mM CuCl2, mean fluorescence intensity values in both species decreased by more than 50% compared to the untreated control. During the first 30 min of paraquat treatment, the production of reactive oxygen species, evaluated as the incidence of protoplasts in which the superoxide anion was present, was 32.9 % and 25.4%, respectively, for L. hirsutum and L. esculentum. Hyperpolarization of the mitochondria for both tomatoes species was observed during the first 2 h. The absence of early membrane damage and the markedly detrimental effects of the treatments on the mitochondria suggest that additional mechanisms which lead to cell death may be involved in these processes, and that these mechanisms may be associated with apoptosis.

Evaluation of Mutagenic and Genotoxic Activity in Vinasses Subjected to Different Treatments

The mutagenic and genotoxic activity of vinasses collected from a fuel alcohol plant, located in the municipality of Frontino, Northwestern Colombia, were evaluated. Two samples obtained from an 82-L capacity hybrid reactor (UASB-anaerobic filter (AF)-UASB) were studied under laboratory conditions after being treated with biological oxidation, the first, and the second with Fenton reaction consecutively. Mutagenicity was evaluated in vitro by the Ames test using strains TA98 and TA100 with and without S9 metabolic activation. The genotoxic analysis was conducted using the Allium cepa roots assay where chromosomal aberrations were used as clastogenic or aneugenic response markers, and micronuclei as mutagenic response. The Ames test results showed a strain-dependent positive linear association with the vinasse sample concentration before treatment (dose–response effect). Unlike TA100, strain TA98 showed a mutagenic effect in both the presence and absence of metabolic enzymes. After the biological oxidation treatment, vinasse mutagenicity significantly decreased. Finally, after Fenton treatment, the sample did not induce any mutagenic event. Genotoxic activity was observed in all three samples, but there was a higher frequency in the vinasse sample before treatment. Concerning the frequency of micronuclei, no clear association was observed with either the concentration or the type of sample.

iTRAQ, The High Throughput Data Analysis of Proteins to Understand Immunologic Expression in Insect

Isobaric tags for relative and absolute quantification of protein expression (iTRAQ®) is a powerful tool which is combined with the accuracy of Mass Spectrometry for protein identification. This tool was approached to detect proteins associated to the innate immune system of Galleria mellonella in response to pathogenesis caused by Fusarium oxysporum. After experimental approaches, iTRAQ data was used to set up computational analysis based on identification and quantification of peptides and proteins against different protein databases by using ProteinPilotTM and Mascot Distiller search engines. iTRAQ battery was able to identify more than 340 peptides corresponding to 39 putative proteins from G. mellonella and close related species. Despite the low level of genomic and proteomic information available for G. mellonella, iTRAQ demonstrated to be reliable strategy to determine changes in protein expression as a consequence of the infection process induced in G. mellonella. Consequently, it was found differential expression in proteins directly involved in innate immune response such as cecropin-D-like peptide, lysozyme, and hemolin, indicating an active response of G. mellonella in early stages of fungal infection.

Detection of Histoplasma capsulatum in Organic Fertilizers by Hc100 Nested Polymerase Chain Reaction and Its Correlation with the Physicochemical and Microbiological Characteristics of the Samples

Histoplasma capsulatum is the causative agent of histoplasmosis and this fungus inhabits soils rich in phosphorus and nitrogen that are enriched with bird and bat manure. The replacement of organic matter in agroecosystems is necessary in the tropics, and the use of organic fertilizers has increased. Cases and outbreaks due to the presence of the fungus in these components have been reported. The Instituto Colombiano Agropecuario resolution 150 of 2003 contains the parameters set by the Colombian Technical Standard (NTC 5167) on the physicochemical and microbiological features of fertilizers, but it does not regulate the search for H. capsulatum. The aim of this study was to demonstrate H. capsulatum presence in organic fertilizers by nested polymerase chain reaction (PCR). A total of 239 samples were collected: 201 (84.1%) corresponded to organic fertilizers, 30 (12.5%) to bird excrement, and 8 (3.4%) to cave soils. The Hc100 nested PCR had a detection limit of 0.1 pg/µL and a specificity of 100%. A total of 25 (10.5%) samples were positive and validated by sequencing. Seven of the positive samples represented locations where H. capsulatum was previously detected, suggesting the persistence of the fungus. No significant correlations were detected between the physicochemical and microbiological parameters with the presence of H. capsulatum by nested PCR, indicating the fungus existence in organic fertilizers that complied with the NTC 5167. The Hc100 nested PCR targeting H. capsulatum standardized in this work will improve the evaluation of organic fertilizers and ensure the prevention of outbreaks and cases due to manufacturing, marketing, and use of fertilizers contaminated with H. capsulatum.