Javier M. Montserrat

Potential Dermal Exposure in greenhouses for manual sprayers: Analysis of the mix/load, application and re-entry stages

An evaluation of the Potential Dermal Exposure for the mix/load, application and re-entry stages, associated with procymidone and deltamethrin usage, was carried out for tomatoes grown in greenhouses of small production units in Argentina. Eight experiments were done with four different operators, under typical field conditions with a lever operated backpack sprayer. The methodology applied was based on the Whole Body Dosimetry technique, evaluating a set of different data for the mix and load, application and re-entry operations. These results indicated that the Potential Dermal Exposure of the application step was (38+/-17)mLh(-1) with the highest proportion on torso, head and arms. When the three stages were compared, re-entry was found to contribute least towards the total Potential Dermal Exposure; meanwhile in all cases, except one, the mix/load operation was the stage with highest exposure. The Margin of Safety for each different operation was also calculated and the proportion of pesticide drift from the greenhouse to the environment is presented. These results emphasize the importance of improving the personal protection measures in the mix and load stage, an operation that is not usually associated with high-risk in small production units.

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.

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.

Pesticide risk assessment in flower greenhouses in Argentina: the importance of manipulating concentrated products.

An evaluation of the Potential Dermal Exposure of workers to endosulfan and procymidone at the mix/load and application stages was done in small floricultural production units in Argentina. Seven experiments were performed with different operators under typical greenhouse conditions, based on the whole body dosimetry methodology. These results indicate that the mean Potential Dermal Exposure of the application step was 45.0 ± 55.0 mL h(-1) with the highest proportion on torso, head, arms and hands. When the mix/load and application stages were compared, the first was found to contribute the most to the total exposure. Also, the Margin of Safety for the different operations was calculated, and a pesticide surrogate was developed and used to make comparative evaluations of hand exposure for different groups of operators. These results emphasize the importance of the mix/load stage in the exposure process.

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.

Environmental pesticide distribution in horticultural and floricultural periurban production units

The environmental pesticide distribution on non-target systems (soil, drift and agricultural plastics) during the application step at small periurban production units, was studied in open field and greenhouses, for different crops (tomato, lettuce, broccoli, strawberry and flowers) using different pesticides (endosulfan, procymidone, chlorothalonil, chlorpyrifos and deltamethrin). In all cases, soil was the most exposed non-target system. For greenhouses, a general pesticide distribution was found of approximately 2/3 for crop, 1/4 for soil and 1/20 for plastic, of the total amount applied. In horticultural open fields, although the distribution was very dependent on the crop size and type, soil was also the most exposed non-target subsystem. Pesticide drift seems not to be significant in these production units, whilst pesticide accumulation on agricultural plastics reached up to 45% of the total applied, for polyethylene mulching in strawberry fields.

Microbial Hydrolysis of Acetylated Nucleosides

Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5′-selective deprotection or mixtures of mono O-acetylated products were obtained.

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.

Stabilization of Telomeric I-Motif Structures by (2′S)-2′-Deoxy-2′-C-Methylcytidine Residues

G‐quadruplexes and i‐motifs are tetraplex structures present in telomeres and the promoter regions of oncogenes. The possibility of producing nanodevices with pH‐sensitive functions has triggered interest in modified oligonucleotides with improved structural properties. We synthesized C‐rich oligonucleotides carrying conformationally restricted (2′S)‐2′‐deoxy‐2′‐C‐methyl‐cytidine units. The effect of this modified nucleoside on the stability of intramolecular i‐motifs from the vertebrate telomere was investigated by UV, CD, and NMR spectroscopy. The replacement of selected positions of the C‐core with C‐modified residues induced the formation of stable intercalated tetraplexes at near‐neutral pH. This study demonstrates the possibility of enhancing the stability of the i‐motif by chemical modification.

Coupled biocatalysts applied to the synthesis of nucleosides

Biocatalytic procedures offer a good alternative to the chemical synthesis of nucleosides since biocatalyzed reactions are regio- and stereoselective and afford reduced by-products contents. Among them, enzymatic transglycosylation between a pyrimidine nucleoside and a purine base catalyzed by nucleoside phosphorylases or microorganisms that contain them, has attracted considerable attention. In addition, the combination to other enzymatic steps has been explored. In this work we investigate the coupled action of nucleoside phosphorylases with other enzymatic activities: deaminase and phosphopentomutase. Unlike the preparation of other purine nucleosides, transglycosylation from a pyrimidine nucleoside and guanine is difficult because of the low solubility of this base. Therefore, another strategy, based on microbial transglycosylation followed by deamination, is here explored. The direct use of furanose 1-phosphate, the intermediate in the transglycosylation reaction, is an attractive alternative when pyrimidine nucleosides are not available. Its preparation from the more stable furanose 5-phosphate and phosphopentomutase is here applied to different sugars and bases.