Mathematical models and fractal analysis for the investigation of the photodynamic inactivation in phytopathogenic microorganisms.

The increasing and indiscriminate use of pesticides may lead to the intoxication and contamination of the environment and foods. In addition, pesticides can cause fungal resistance promoting the selection of resistant phytopathogenic fungi. This is a problem in the agricultural and human health areas, which leads to a need for developing new methodologies to address this problem. Photodynamic inactivation is a promising strategy involving the association of a photosensitizer (PS), light, and molecular oxygen to inhibit the growth of microorganisms. In this work, the PS acridine orange (AO) was deposited using the spray layer-by-layer technique. The effectiveness of the method was evaluated by the analysis of the growth evolution of the colonies as a function of the amount of PS layers applied in field in the presence of sunlight. Image processing and analysis of the fractal theory were used to evaluate the growth of the colonies. The results revealed that AO is a candidate PS for use in field. It was possible to observe the reduction of the growth dynamics of the colonies with the increase of the number of PS layers. The parameters related to the fractality of the system were described by mathematical models of the fractal growth of interfaces. The knowledge of these parameters can help to identify new strategies for the control of phytopathogenic microorganisms that directly affect agricultural production.

Incorporation of triclosan and acridine orange into liposomes for evaluating the susceptibility of Candida albicans.

Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. Experiments were carried out to examine the susceptibility of C. albicans to PDI. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15J/cm2) than in non-irradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by photodynamic inactivation. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains.

Acridine orange interaction with DNA: Effect of ionic strength.

BACKGROUND: The study of acridine orange (AO) spectral characteristics and the quenching of its singlet and triplet excited states by TEMPO radical at its binding to DNA in the function of the DNA concentration and in the absence and presence of NaCl is reported. METHODS: The study was performed using steady-state and time resolved optical absorption and florescence, fluorescence correlation spectroscopy and resonant light scattering techniques. RESULTS: The presence of different species in equilibrium: AO monomers and aggregates bound to DNA, has been demonstrated, their relative content depending on the DNA and the AO concentrations. At high DNA concentration the AO monomers are protected against the contact with other molecules, thus reducing the AO excited state quenching. The addition of NaCl reduces the AO binding constant to DNA, thus reducing the AO and DNA aggregation. CONCLUSIONS: The interaction of AO with DNA is a complex process, including aggregation and disaggregation of both components. This modifies the AO excited state characteristics and AO accessibility to other molecules. The salt reduces the DNA effects on the AO excited state characteristics thus attenuating its effects on the AO efficacy in applications. GENERAL SIGNIFICANCE: This study demonstrates that the interaction of photosensitizers with DNA, depending on their relative concentrations, can both decrease and increase the photosensitizer efficacy in applications. The salt is able to attenuate these effects.

Spray layer-by-layer films for photodynamic inactivation.

BACKGROUND: A novel approach for photodynamic inactivation of Candida albicans is proposed. This method consists of realizing inactivation using ultraviolet light (254nm) combined with spraying layer-by-layer films of acridine orange. METHODS: To evaluate the effectiveness of the approach, the C. albicans were immobilized on quartz slices and covered with the spray layer-by-layer films. The fungi were analyzed using experiments to determine cell viability, as well as by fluorescence and atomic force microscopy. RESULTS: Viability analysis of C. albicans after photodynamic inactivation assisted by the films indicates cell death. The extent of cell death increases as the number of film layers increases. Fluorescence and atomic force microscopy analyses corroborated the cell death of C. albicans, which is posited to be due to damages to the fungi cell wall. CONCLUSIONS: Our approach has the potential to be used as an alternative for photodynamic inactivation of C. albicans. In addition, this method could be used in clinical procedures, such as for the decontamination of medical devices.

Reliable Screening of Dye Phototoxicity by Using a Caenorhabditis elegans Fast Bioassay.

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.

Time-resolved fluorescence anisotropy as a tool to study guest-cucurbit[n]uril-protein ternary supramolecular interactions.

Ternary supramolecular complexes involving cucurbit[n]urils and proteins are of potential interest for improving drug transport and delivery. We report here time-resolved fluorescence studies for acridine orange complexes with cucurbit[7]uril and cucurbit[8]uril in the presence of human serum albumin as a model system. A detailed characterization of the fluorescence lifetime and anisotropy properties of the different acridine orange complexes with cucurbit[n]urils and human serum albumin was performed. Of particular importance is the analysis of the stepwise binding for acridine orange-cucurbit[8]uril complexes and the assignment of the fluorescence and anisotropy properties to the 2 : 1 complex. Anisotropy decay measurements were essential to detect protein-bound species and to discriminate between different complexes. Based on the fluorescence evidence, ternary interactions with the protein are suggested for the acridine orange-cucurbit[7]uril complex but not for the cucurbit[8]uril complex. We highlight here the usability and sensitivity of the combined fluorescence analysis.

In situ analysis of apoptosis in Aspergillus nidulans with ethidium bromide and acridine orange.

Apoptosis and necrosis are among several types of cell death. We stained the nuclei of Aspergillus nidulans grown in micro-colonies with ethidium bromide and acridine orange to detect in situ apoptosis. Suspensions of conidia from 5-day-old colonies of the A. nidulans strains biA1methG1, G422, CLC100, and CLB3 were each put into two tubes. The suspension of one tube was irradiated with ultraviolet light for 20 s, whereas the other tube was not exposed to irradiation. The two suspensions were inoculated in complete liquid medium and 50-µL samples were placed on sterilized cover slips, spread on the surface of solid culture media on Petri dishes. After the micro-colonies were formed, the material on the cover slips was stained with ethidium bromide and acridine orange, placed on the lamina and observed under a fluorescence microscope. This staining method was efficient in discriminating normal nuclei from those going apoptosis and necrosis. Results have shown that irradiation provokes apoptosis but does not induce necrosis. There were no differences between the three strains and all data were considered to be statistically significant.

Photocatalytic degradation of acridine dyes using anatase and rutile TiO2.

The adsorption and photodegradation of acridine orange (AO) and acriflavine (AF) dyes on two mesoporous titania crystalline phases, anatase and rutile, were experimentally studied. Anatase and rutile were characterized by nitrogen adsorption, electron scanning and transmission microscopy, and X-ray diffraction. The adsorption capacity of rutile was higher than that of anatase, while the reverse is observed for photodegradation of both dyes. The adsorption of AF on both adsorbents was higher than that of AO, which was related with the smaller size of AF molecules compared with those of AO, therefore the access of AF to the adsorption sites is favored.

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri.

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.

Evaluación de la coloración diferencial de fluorescencia modificada en Pseudomonas spp. aisladas de suelo / Evaluation of the modified flourescence staining differential on Pseudomonas spp. isolated from soil

Se evaluó la coloración diferencial de fluorescencia modificada en Pseudomonas spp. aisladas de suelos de cultivos agrícolas del estado Sucre, a fin de observar eventos microscópicos relacionados con el ciclo celular. Cada especie de Pseudomonas identificada bioquímicamente se sembró en caldos incubados a temperatura ambiente, aerobiosis, durante 15, 20, 30 y 45 minutos, y 1, 24, 48 y 72 horas; luego, se elaboraron y colorearon los extendidos. En las 24 cepas de Pseudomonas identificadas, P. mendocina (41,67 por ciento), P. aeruginosa (37,50 por ciento) y P. putida (20,83 por ciento), se observaron variaciones de tinción en los diferentes tiempos de incubación como verde, amarilla y anaranjada, fluorescentes y de baja fluorescencia. La coloración emplea naranja de acridina que se intercala al ADN, provocando fluorescencia verde, e interactúa con el ARN provocando fluorescencia anaranjada; el decolorante remueve el naranja de acridina no unido al material genético y la fluoresceína de sodio produce color amarillo en bacterias que retienen suficiente cantidad de naranja de acridina. Las variaciones de tinción citoplasmática en Pseudomonas spp., están asociadas a la cantidad de ARN y ADN presente en la célula de acuerdo a la fase de su ciclo celular.

The modified fluorescence staining differential was evaluated using Pseudomonas spp. isolated from cultivated agricultural soils in the State of Sucre, in order to observe microscopic events related to the cellular cycle. Each species of biochemically identified Pseudomonas was inoculated into a broth and incubated at room temperature, aerobiosis, for 15, 20, 30 and 45 minutes and 1, 24, 48 and 72 hours; then, slides were made and stained. For the 24 identified strains of Pseudomonas, P. mendocina (41.67 percent), P. aeruginosa (37.50 percent) and P. putida (20.83 percent), staining variations such as green, yellow and orange, fluorescent and low fluorescence were observed for the different incubation times. The stain uses acridine orange that interacts with DNA by intercalation, causing green fluorescence; it interacts with RNA by electrostatic attraction causing orange fluorescence; the alcohol-acetone decolorant removes the acridine orange not united with the genetic material and sodium fluorescein produces a yellow color in bacteria that retain a sufficient amount of acridine orange. Cytoplasmatic staining variations in Pseudomonas spp., are associated with the amount of RNA and DNA present in the cells according to the phase of their cellular cycle.

Titration of cardiolipin by either 10-N-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition.

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.

Programmed cell death in salivary glands of Drosophila arizonae and Drosophila mulleri

Programmed cell death (PCD) in insect metamorphosis assumes a great diversity of morphology and controlling processes that are still not well understood. With the objective of obtaining information about the PCD process, salivary glands of Drosophila arizonae and D. mulleri were studied during larval-pupal development. From the results, it can be concluded that the type of the PCD that occurs in these organs is morphologically typical of apoptosis (formation of apoptotic nuclei, followed by fragmentation into apoptotic bodies). Histolysis happens in both species, between 22 and 23 h after pupation. There were no significant differences between the species studied. Apoptosis does not occur simultaneously in all cells. Cytoplasmic acid phosphatase activity gradually increases during development, suggesting the existence of acid phosphatases that are only expressed during the apoptotic stage. Twenty hours after pupation, salivary glands already show biochemical alterations relative to nuclear permeability such as acidification, possibly due to the fusion of lysosomes with the nucleus a few hours before apoptosis. Autophagy seems to act together with apoptosis and has a secondary role in cell death.