Core modified 8-17 DNAzymes with 2'-deoxy-2'-C-methylpyrimidine nucleosides.

The catalytic core of an 8-17 DNAzyme directed against STAT 3 was modified using (2'R) and (2'S) 2'-deoxy-2'-C-methyluridine and cytidine. While 2'-deoxy-2'-C-methyluridine significantly diminished the catalytic activity, 2'-deoxy-2'-C-methylcytidine replacement was better accepted, being the kact of modified DNAzymes at 8- and 11-positions comparable to the non-modified one. When 2'-O-methyl and phosphorothioate nucleotides were tested in the binding arms together with core modified DNAzymes the kcat was affected in a non predictable way, emphasizing the fact that both chemical substitutions should be considered globally. Finally, 2'-deoxy-2'-C-methyl modified DNAzymes stability was assayed finding that the double 2'-C-methyl modification in the catalytic core enhanced 70% the stability against a T47D cell lysate compared to a non-modified control.

siRNA Modified with 2'-Deoxy-2'-C-methylpyrimidine Nucleosides.

(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.

Influence of conformationally restricted pyrimidines on the activity of 10-23 DNAzymes.

The catalytic core of a 10-23 DNAzyme was modified introducing conformationally restricted nucleosides such as (2'R)-, (2'S)-2'-deoxy-2'-C-methyluridine, (2'R)-, (2'S)-2'-deoxy-2'-C-methylcytidine, 2,2'-anhydrouridine and LNA-C, in one, two or three positions. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations using a short RNA substrate. At low Mg(2+) concentrations, triple modified DNAzymes with similar kinetic performance to that displayed by the non-modified control were identified. In the search for a partial explanation of the obtained results, in silico studies were carried out in order to explore the conformational behavior of 2'-deoxy-2'-C-methylpyrimidines in the context of a loop structure, suggesting that at least partial flexibility is needed for the maintenance of activity. Finally, the modified 2'-C-methyl DNAzyme activity was tested assessing the inhibition of Stat3 expression and the decrease in cell proliferation using the human breast cancer cell line T47D.

Thermal paper as a potential source of bisphenol A for humans and the environment: migration and ecotoxicological impact.

The objective of this work was to evaluate thermal paper (TP) tickets used in Argentina as a potential source of bisphenol A (BPA) that could impact humans and the environment. BPA in TP was measured by HPLC ranging from 11.1 to 30.5 mg BPAg-1. In order to estimate the impact on humans, dermal BPA estimated daily intake was calculated as being 79.3 ± 19.5 µgd-1 for workers and 1.6 ± 0.4 µgd-1 for the general population. To evaluate TP's impact on the environment, BPA migration from TP to water and soil was studied. In the case of water, 99.6% of the BPA tickets content migrated in 30 h, while 78.0% moved into the soil in 96 h. BPA degradation kinetics in soil and water were also carried out; while in soil 61.9% of BPA degraded in 120 h, no degradation was observed up to 120 h in tap or river water.Additionally, ecotoxicological effects of BPA on the earthworm Eisenia andrei, a representative terrestrial indicator, were studied performing bioassays on lethality, avoidance, and reproductive and enzymatic activity. BPA showed to be very toxic to E. andrei (LC50 value in contact paper test of 17 µgcm-2, 95% confidence interval 6-46 µgcm-2, 24 h exposure) and also caused an increase of total cocoons for earthworms exposed to 10 and 50 mg BPA kg-1 soil. Evasion response was observed at a concentration of 50 mg BPA kg-1 soil, while no effect was observed on cholinesterases, carboxylesterases, and glutathione S-transferases activities (1, 10, and 50 mg BPA kg-1 soil). Finally, a simple BPA degradation technology using water peroxide and radish (Raphanus sativus) tissue as catalyst was explored as a simple and domestic potential treatment to avoid BPA migration to the environment.

Environmental fate of dibutylphthalate in agricultural plastics: Photodegradation, migration and ecotoxicological impact on soil.

Phthalic acid esters (PAEs) were determined in polyethylene covers used in horticultural production units located at Moreno and La Plata districts (Buenos Aires, Argentina), detecting 0.69-8.75 mg PAEs kg-1 plastic in greenhouse and tunnel films. The PAEs found were diisobutylphthalate (DIBP), dibutylphthalate (DBP) and diethylhexylphthalate (DEHP). DBP was chosen as a model molecule to carry out the photochemical degradation studies that led to the formation of monobutylphthalate (MBP) and phthalic acid (PA). DBP, MBP and PA migration from plastic covers was studied, finding that while DBP and MBP moved to soil and atmosphere in short times (<48 h), PA remained in the agricultural covers. Further experiments with DBP were made to explore the effect on migration of temperature (20 °C, 50 °C), film thickness (25 µm, 100 µm) and plastic ageing by solarization, observing that temperature increase, film thickness reduction and ageing by solarization favored DBP migration to the environment. DBP and MBP impact on soil were evaluated by avoidance and reproduction tests using Eisenia andrei as bioindicator. Both compounds reduced cocoon viability decreasing the number of juveniles at the lowest concentration assayed (0.1 mg kg-1 of soil). At higher DBP and MBP concentrations the reproductive parameters (number of total cocoons, hatchability and number of juveniles) also showed alterations compared with the controls. Carboxylesterases (CaE), cholinesterases (ChE) and glutathion-S-transferases (GST) activities were analyzed in E. andrei exposed to DBP; cholinesterases activities were reduced at 1 and 10 mg DBP kg-1 soil, and glutathione S-transferases activities were increased at 10 mg DBP kg-1 soil while no effect was observed on carboxylesterases activities. These results emphasize the need to continue studying the impact of PAEs and their photodegradation products on the environment.

10-23 DNAzyme modified with (2'R)- and (2'S)-2'-deoxy-2'-C-methyluridine in the catalytic core.

The catalytic core of a 10-23 DNAzyme was modified using (2'R), (2'S)-2'-deoxy-2'-C-methyluridine and LNA-T. Catalytic activities under pseudo first order conditions were compared at different Mg(2+) concentrations, indicating that certain 2'-C-methyl modified DNAzymes have significant activities. Resistance against MCF-7 cell lysate and endonuclease RQ1 was also measured, showing that the introduction of 2'-C-methyl-2'-deoxynucleosides increased the stability.

2-C-methyluridine modified hammerhead ribozyme against the estrogen receptor.

A new synthesis of 2'-C-methyluridine phosphoramidite is presented. Special emphasis is dedicated to the improvement of the protection of the tertiary 2'-hydroxyl group. Comparison to previous protecting strategies and analysis of stability under 5'-DMTr removing conditions are discussed. The synthetic incorporation of this modified nucleoside into the catalytic core of a hammerhead ribozyme against the estrogen receptor alpha protein (ER-alpha), and transfection experiments in MCF-7 cell line are also presented.

Human exposure and mass balance distribution during procymidone application in horticultural greenhouses.

The purpose of this study was to analyze the impact of procymidone application in periurban horticultural greenhouses, especially on workers (applicators and assistants) and soil and plastic mulching, when mechanically pressurized application systems were employed. The mean Potential Dermal Exposure (PDE) was measured using the Whole Body Dosimetry technique. The PDE for the applicators was 188 mL h-1 ± 103 mL h-1, and 14.7 mL h-1 ± 6.3 mL h-1 for the assistants. In the first case, the most exposed body sections were the upper right and left (46.8 mL h-1 ± 23.4 mL h-1; 47.0 mL h-1 ± 23.5 mL h-1) and lower (20.8 mL h-1 ± 10.4 mL h-1; 17.3 mL h-1 ± 8.7 mL h-1) legs, while in the case of assistants, hands and legs were the most impacted limbs. Regarding the Margin of Safety (MOS) during the mix and load stage, two of three pesticide preparations resulted unsafe, while for the applicators, six of six spraying operations were unsafe. For the assistants, five of five operations were safe, but three of them were close to the safety limit. Procymidone distribution between drift (0.03% ± 0.07 %), applicator (0.20% ± 0.15 %), polyethylene mulching (8.5% ± 4.5 %) and soil (3.0% ± 1.1 %) was determined with respect to the total pesticide applied. Procymidone soil impact was also evaluated using Eisenia andrei behavioral tests, finding positive correlations between procymidone application and avoidance and reproduction tests.

Potential dermal exposure to deltamethrin and risk assessment for manual sprayers: influence of crop type.

A comparison of the Potential Dermal Exposure (PDE) of workers to the insecticide deltamethrin was made as a function of crop type, in small agricultural production units in Argentina. Seven experiments were done with two different crops (maize and broccoli, treated area between 600 and 1000 m(2)) with three different operators under typical field conditions using a lever operated knapsack. The methodology is based on the whole body dosimetry technique, presenting separately the data for mixing/loading and application activities. These results indicate a higher concentration of pesticide in lower body sections for broccoli and a wider distribution for maize. The risk inherent in these agricultural procedures is estimated through Margin of Safety (MOS) values and was found to be generally safe. Preliminary results of a mass balance distribution of the pesticide between crop, soil and operator are also presented.

Characterization and electrochemical response of DNA functionalized 2nm gold nanoparticles confined in a nanochannel array.

Polyvalent gold nanoparticle oligonucleotide conjugates are subject of intense research. Even though 2nm diameter AuNPs have been previously modified with DNA, little is known about their structure and electrochemical behavior. In this work, we examine the influence of different surface modification strategies on the interplay between the meso-organization and the molecular recognition properties of a 27-mer DNA strand. This DNA strand is functionalized with different sulfur-containing moieties and immobilized on 2nm gold nanoparticles confined on a nanoporous alumina, working the whole system as an electrode array. Surface coverages were determined by EXAFS and the performance as recognition elements for impedance-based sensors is evaluated. Our results prove that low DNA coverages on the confined nanoparticles prompt to a more sensitive response, showing the relevance in avoiding the DNA strand overcrowding. The system was able to determine a concentration as low as 100pM of the complementary strand, thus introducing the foundations for the construction of label-free genosensors at the nanometer scale.

Human and soil exposure during mechanical chlorpyrifos, myclobutanil and copper oxychloride application in a peach orchard in Argentina.

The objective of this study was to measure the impact of the mechanized chlorpyrifos, copper oxychloride and myclobutanil application in a small peach orchard, on humans (operators, bystanders and residents) and on the productive soil. The mean Potential Dermal Exposure (PDE) of the workers (tractor drivers) was 30.8mL·h-1±16.4mL·h-1, with no specific pesticide distribution on the laborers body. Although the Margin of Safety (MOS) factor for the application stage were above 1 (safe condition) for myclobutanil and cooper oxycloride it was below 1 for chlorpyrifos. The mix and load stage remained as the riskier operation. Pesticide found on the orchard soil ranged from 5.5% to 14.8% of the total chlorpyrifos, copper oxychloride and myclobutanil applied. Pesticide drift was experimentally measured, finding values in the range of 2.4% to 11.2% of the total pesticide applied. Using experimental drift values, bystander (for one application), resident (for 20 applications) and earthworm (for one application) risk indicators (RIs) were calculated for the chlorpyrifos plus copper oxychloride and for myclobutanil treatments for different distances to the orchard border. Earthworm RI was correlated with experimental Eisenia andrei ecotoxicological assays (enzymatic activities: cholinesterases, carboxylesterases and glutathione S-transferases; behavioral: avoidance and bait-lamina tests) with good correlation.

Analytical method for assessing potential dermal exposure to captan, using whole body dosimetry, in small vegetable production units in Argentina.

An analytical method has been developed that can be used to determine the potential dermal exposure (PDE) of workers to the pesticide captan in small-scale horticultural production units. The methodology is based on the whole body dosimetry technique, using a cotton coverall and cotton gloves as sampling media, with protective clothing worn beneath the cotton media to protect the operator. The quantitative determination of captan was done by gas chromatography-electron capture detector (GC-ECD), with the analytical method validated by measuring limits of detection and quantification, linear ranges, sample recovery and precision. Special emphasis is placed on factors that affected the stability of captan during chromatographic determination. The data generated for potential dermal exposure are presented separately for mixing/loading and application activities. These data are compared with values obtained with visible tracers using a similar field technique. Margin of safety (MOS) values are also calculated for the agricultural procedures studied.

Modified Nucleoside Triphosphates for In-vitro Selection Techniques.

The development of SELEX (Selective Enhancement of Ligands by Exponential Enrichment) provides a powerful tool for the search of functional oligonucleotides with the ability to bind ligands with high affinity and selectivity (aptamers) and for the discovery of nucleic acid sequences with diverse enzymatic activities (ribozymes and DNAzymes). This technique has been extensively applied to the selection of natural DNA or RNA molecules but, in order to improve chemical and structural diversity as well as for particular applications where further chemical or biological stability is necessary, the extension of this strategy to modified oligonucleotides is desirable. Taking into account these needs, this review intends to collect the research carried out during the past years, focusing mainly on the use of modified nucleotides in SELEX and the development of mutant enzymes for broadening nucleoside triphosphates acceptance. In addition, comments regarding the synthesis of modified nucleoside triphosphate will be briefly discussed.

Exploring the Use of a Guanine-Rich Catalytic DNA for Sulfoxide Preparation.

A guanine-rich DNA oligonucleotide complexed with hemin was used to catalyze controlled oxygen transfer reactions to different sulfides for sulfoxide preparation in the presence of H2O2. Comparable activities were obtained when using fully modified L-DNA. In addition, oligonucleotide immobilization led to an active catalyst which could be successfully recovered and reused without loss of activity.

Polyethylene film incorporation into the horticultural soil of small periurban production units in Argentina.

Horticulture makes intensive use of soil and extensive use of polyethylene (PE) sheeting and pesticides, producing an environment where the dynamics between soil and plastics can affect pesticide fate. We have determined that the presence of plastic residues in the horticultural soil of small production units equals 10% of the soil area, being meso and macro-sections the predominant fragment sizes. All soil samples were taken from different plots located in Cuartel V, Moreno district, in the suburbs of Buenos Aires city, Argentina. Laboratory experiments were conducted to see the relations among pesticide, soil and PE film. Endosulfan recovery from LDPE films (25µm and 100µm) was studied, observing evidence that indicated migration to the inside of the plastic matrix. To further analyze the dynamics of pesticide migration to soil and atmosphere, experiments using chlorpyrifos, procymidone and trifluralin were performed in soil-plastic-atmosphere microenvironments, showing that up to 24h significant amounts of pesticides moved away from the PE film. To determine whether PE residues could act as potential pesticide collector in soil, column elution experiments were done using chlorpyrifos, procymidone and trifluralin. Results showed an important pesticide accumulation in the mulch film (584µg-2284µg pesticide/g plastic) compared to soil (13µg-32µg pesticide/g soil). Finally, chemical and photochemical degradation of deltamethrin adsorbed in PE film was studied, finding a protective effect on hydrolysis but no protective effect on photodegradation. We believe that a deeper understanding of the dynamics among soil, plastic and pesticides in horticultural productive systems may contribute to alert for the implications of PE use for plastic sheeting.

Confined gold nanoparticles enhance the detection of small molecules in label-free impedance aptasensors.

A controlled architecture of nanoelectrodes, of a similar size to small molecule-binding aptamers, is synthesized inside nanoporous alumina. Gold nanoparticles with a controlled size (about 2 nm) are electrogenerated in the alumina cavities, showing a fast electron transfer process toward ferrocyanide. These uncapped nanoparticles are easily modified with a thiol-containing aptamer for label-free detection of adenosine monophosphate by electrochemical impedance spectroscopy. Our results show that the use of a limited electrical conducting surface inside an insulating environment can be very sensitive to conformational changes, introducing a new approach to the detection of small molecules, exemplified here by the direct and selective detection of adenosine monophosphate at the nanomolar scale.

Synthesis of encoded modified oligonucleotide libraries. Part 1: orthogonal chemistry.

The basis for further development of combinatorial libraries of modified oligonucleotides tagged by a codifying sequence is discussed. The chemistry involved in the orthogonal synthesis of both strands and some representative examples of building blocks are presented.

Activity of core-modified 10-23 DNAzymes against HCV.

The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2'R)-, (2'S)-2'-deoxy-2'-C-methyl- and -2'-O-methylnucleosides at various positions of the catalytic core were assayed against the 5'-internal ribosome entry site element (5'-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes' cores were jointly modified with 2'-C-methyl- and 2'-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10-23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.

Pesticide potential dermal exposure during the manipulation of concentrated mixtures at small horticultural and floricultural production units in Argentina: the formulation effect.

Potential dermal exposure measurements of horticultural and floricultural field operators that handled concentrated pesticides showed a correlation with the types of formulations used (liquid or solid) during the mix and load stage. For liquid formulations, hand exposure was 22-62 times greater than that for solid ones. The dermal exposure mechanism was studied for this formulation under laboratory conditions, finding that the rupture of the aluminum seal of the pesticide container and the color of the liquid formulation are important factors. Additionally, significant external surface contamination of pesticide containers collected at horticultural farms was found. This could partially account for the differences between the exposure levels of field and laboratory experiments for liquid formulations.

Conformational states of 2'-C-methylpyrimidine nucleosides in single and double nucleic acid stranded structures.

The hybridization performance of a set of 12-mer RNA:RNA duplexes containing 2'-C-methyluridine, 5-bromo-2'-C-methyluridine, or (2'S)-2'-deoxy-2'-C-methyluridine was analyzed. Melting point temperatures of the modified duplexes showed an important ΔT(m) decrease (-8.9 to -12.5 °C), while circular dichroism experiments indicated that the helix was still A-type, suggesting a localized disturbance disorder. Molecular dynamics simulations using AMBER were carried out in order to gain structural knowledge about the effect of the 2'-C-methyl modification in double stranded environments. On the other hand, in an attempt to explain the behavior of the 2'-deoxy-2'-C-methyl nucleosides in single stranded environments, like the 10-23 DNAzyme core, molecular dynamic simulations were performed, incorporating the modified analogues into single stranded reported stem-loop structures, studding the sugar conformations along the MD trajectories. It was observed that, despite their preferential conformational states, the 2'-C-methyl analogues are flexible enough to adopt a different puckering in single stranded environments.