Multitarget and suspect-screening of antimicrobials in vegetables samples: Uptake experiments and identification of transformation products.

This work provided an accurate analytical method to perform a multitarget analysis of a variety of antimicrobials (AMs) including sulfonamides, tetracyclines, macrolides, fluoroquinolones and quinolones, one imidazole and one nitroimidazole, one triazole, one diaminopyridine and one derivative of Penicillium stoloniferum in vegetables. The analysis is performed using liquid-chromatography coupled to a low-resolution triple quadrupole mass spectrometer (UHPLC-MS/MS) to detect the target analytesor coupled to a high-resolution q-Orbitrap (HRMS) to monitor the formed transformation products (TPs). Both instruments were compared in terms of limits of quantification and matrix effect at the detection. The method was applied to determine the presence of AMs in organic and non-organic vegetables, where sulfadiazine and mycophenolic acid were detected. On the other hand, the transference of four AMs (trimethoprim, sulfamethazine, enrofloxacin, and chlortetracycline) from soils to lettuces was evaluated through controlled uptake experiments. The choice of AMs was based on the classification into different families, and on the fact that those AM families are the most frequently detected in the environment. In this case, each of the AMs with which the soils were contaminated were found in the exposed lettuces. Moreover, in both studies, specific TPs of the AMs were identified, posing the necessity of assessing their effects in relation to food and human safety.

Multitarget and suspect screening of antimicrobials in soil and manure by means of QuEChERS - liquid chromatography tandem mass spectrometry.

The present work aimed to develop an accurate analytical method for the simultaneous analysis of twenty-four antimicrobials in soil:compost and animal manure samples by means of ultra-high performance liquid chromatography coupled to a triple-quadrupole mass spectrometer (UHPLC-QqQ). For this purpose, the effectiveness of two extraction techniques (i.e. focused ultrasound solid-liquid extraction (FUSLE) and QuEChERS (quick, easy, cheap, effective, rugged and safe)) was evaluated, and the clean-up step using solid-phase extraction (SPE) was also thoroughly studied. The method was successfully validated at 10 µg·kg-1, 25 µg·kg-1, and 50 µg·kg-1 showing adequate trueness (70-130%) and repeatability (RSD < 30%), with few exceptions. Procedural limits of quantification (LOQPRO) were determined for soil:compost (0.45 to 7.50 µg·kg-1) and manure (0.31 to 5.53 µg·kg-1) samples. Pefloxacin could not be validated at the lowest level since LOQPRO ≥ 10 µg·kg-1. Sulfamethazine (7.9 ± 0.8 µg·kg-1), danofloxacin (27.1 ± 1.4 µg·kg-1) and trimethoprim (4.9 ± 0.5 µg·kg-1) were detected in soil samples; and tetracycline (56.8 ± 2.8 µg·kg-1), among other antimicrobials, in the plants grown on the surface of the studied soil samples. Similarly, sulfonamides (SAs), tetracyclines (TCs) and fluoroquinolones (FQs) were detected in sheep manure in a range of 1.7 ± 0.3 to 93.3 ± 6.8 µg·kg-1. Soil and manure samples were also analysed through UHPLC coupled to a high-resolution mass-spectrometer (UHPLC-qOrbitrap) in order to extend the multitarget method to suspect screening of more than 22,281 suspects. A specific transformation product (TP) of sulfamethazine (formyl-sulfamethazine) was annotated at 2a level in manure samples, among others. This work contributes to the efforts that have been made in the last decade to develop analytical methods that allow multitarget analysis of a wide variety of antimicrobials, including TPs, which is a complex task due to the diverse physicochemical properties of the antimicrobials.

Physiological response and yield components under greenhouse drought stress conditions in potato.

Due to the effects of climate change, conditions tend to be increasingly extreme, with water availability being one of the main limiting factors in potato production. The objective of this study was to analyze the differential response of physiological and yield components in six potato varieties under water deficit conditions. For this purpose, a greenhouse trial was carried out with the varieties Agata, Agria, Kennebec, Monalisa, Sante and Zorba. The drought stress was applied in stressed plants 36 days after planting (DAP) by withholding water for 25 days. All measurements were taken at four different times: before stress (T0), 17 days (T1) and 24 days (T2) after stress and five days after re-watering. The physiological parameters evaluated were chlorophyll content and fluorescence, relative leaf water content, stomatal conductance, electrolytic leakage and water potential. After the drought period, the aerial part of half of the plants was cut to evaluate the produced biomass. At the end of the cycle yield components were determined. Stomatal conductance and water potential were the parameters that showed the highest differences between the two hydric conditions, and Monalisa was the variety with the best response in tuber production under stress conditions. Indirect selection based on parameters associated with water stress can be a useful tool in potato breeding programs for the identification of more tolerant varieties.

Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine.

Studies of metabolic and physiological bases of plant tolerance and hardening against drought are essential to improve genetic breeding programs, especially in productive species such as Pinus radiata. The exposure to different drought cycles is a highly effective tool that improves plant conditioning, but limited information is available about the mechanisms that modulate this process. To clarify this issue, six P. radiata breeds with well-known differences in drought tolerance were analyzed after two consecutive drought cycles. Survival rate, concentration of several metabolites such as free soluble amino acids and polyamines, and main plant hormones varied between them after drought hardening, while relative growth ratio and water potential at both predawn and dawn did not. Hardening induced a strong increase in total soluble amino acids in all breeds, accumulating mainly those implicated in the glutamate metabolism (GM), especially L-proline, in the most tolerant breeds. Other amino acids from GM such as γ-aminobutyric acid (GABA) and L-arginine (Arg) were also strongly increased. GABA pathway could improve the response against drought, whereas Arg acts as precursor for the synthesis of spermidine. This polyamine showed a positive relationship with the survival capacity, probably due to its role as antioxidant under stress conditions. Finally, drought hardening also induced changes in phytohormone content, showing each breed a different profile. Although all of them accumulated indole-3-acetic acid and jasmonic acid and reduced zeatin content in needles, significant differences were observed regarding abscisic acid, salicylic acid and mainly zeatin riboside. These results confirm that hardening is not only species-dependent but also an intraspecific processes controlled through metabolite changes.

The type of competition modulates the ecophysiological response of grassland species to elevated CO2 and drought.

The effects of elevated CO2 and drought on ecophysiological parameters in grassland species have been examined, but few studies have investigated the effect of competition on those parameters under climate change conditions. The objective of this study was to determine the effect of elevated CO2 and drought on the response of plant water relations, gas exchange, chlorophyll a fluorescence and aboveground biomass in four grassland species, as well as to assess whether the type of competition modulates that response. Elevated CO2 in well-watered conditions increased aboveground biomass by augmenting CO2 assimilation. Drought reduced biomass by reducing CO2 assimilation rate via stomatal limitation and, when drought was more severe, also non-stomatal limitation. When plants were grown under the combined conditions of elevated CO2 and drought, drought limitation observed under ambient CO2 was reduced, permitting higher CO2 assimilation and consequently reducing the observed decrease in aboveground biomass. The response to climate change was species-specific and dependent on the type of competition. Thus, the response to elevated CO2 in well-watered grasses was higher in monoculture than in mixture, while it was higher in mixture compared to monoculture for forbs. On the other hand, forbs were more affected than grasses by drought in monoculture, while in mixture the negative effect of drought was higher in grasses than in forbs, due to a lower capacity to acquire water and mineral nutrients. These differences in species-level growth responses to CO2 and drought may lead to changes in the composition and biodiversity of the grassland plant community in future climate conditions.

Immunolocalization of IAA and ABA in roots and needles of radiata pine (Pinus radiata) during drought and rewatering.

Anatomical, physiological and phytohormonal changes involved in drought tolerance were examined in different Pinus radiata D. Don breeds subjected to soil drying and rewatering. Breeds with the smallest stomatal chamber size had the lowest transpiration rate and the highest intrinsic water-use efficiency. Xylem cell size was positively correlated with leaf hydraulic conductance and needle indole-3-acetic acid (IAA) concentrations, whereas transpiration rate was negatively correlated with needle abscisic acid (ABA) levels. Since these two phytohormones seem important in regulating the P. radiata drought response, they were simultaneously immunolocalized in roots and needles of the most tolerant breed (P. radiata var. radiata × var. cedrosensis) during two sequential drought cycles and after rewatering. During drought, IAA was unequally distributed into the pointed area of the needle cross-section and mainly located in mesophyll and vascular tissue cells of needles, possibly inducing needle epinasty, whereas ABA was principally located in guard cells, presumably to elicit stomata closure. In the roots, at the end of the first drought cycle, while strong IAA accumulation was observed in the cortex, ABA levels decreased probably due to translocation to the leaves. Rewatering modified the distribution of both IAA and ABA in the needles, causing an accumulation principally in vascular tissue, with residual concentrations in mesophyll, likely favouring the acclimatization of the plants for further drought cycles. Contrarily, in the roots IAA and ABA were located in the exodermis, a natural barrier that regulates the phytohormone translocation to other plant tissues and hormone losses to the soil solution after rewatering. These results confirm that immunolocalization is an efficient tool to understand the translocation of IAA and ABA in plants subjected to different water stress situations, and clarify their role in regulating physiological responses such as stomata closure and epinasty in needles and root development.

Solute accumulation and elastic modulus changes in six radiata pine breeds exposed to drought.

Drought is one of the main abiotic factors that determine forest species growth, survival and productivity. For this reason, knowledge of plant drought response and the identification of physiological traits involved in stress tolerance will be of interest to breeding programs. In this work, several Pinus radiata D. Don breeds from different geographical origins were evaluated along a water stress period (4 weeks) and subsequent rewatering (1 week), showing different responses among them. Leaf water potential (Ψ(leaf)) and osmotic potential decreases were accompanied by a variation in the total relative water content (RWC, %). The most tolerant breeds presented the lowest leaf water potential and RWC at turgor loss point, and showed the lowest elastic modulus (ε) values. A high ε value was a characteristic of a less-drought-tolerant plant and was related to membrane alterations (high electrolyte leakage percentages) that could favor cell water loss. Of the group of solutes that contributed to osmotic adjustment, soluble carbohydrates were the most abundant, although stressed plants also increased their content of free amino acids [mainly proline (Pro) and glutamic acid (Glu), and γ-aminobutyric acid (GABA)] and free polyamines. In addition, the most sensitive breeds had a higher GABA/Glu ratio. After rewatering, Pro and GABA were higher in rehydrated plants than in controls.

Physiological response to drought in radiata pine: phytohormone implication at leaf level.

Pinus radiata D. Don is one of the most abundant species in the north of Spain. Knowledge of drought response mechanisms is essential to guarantee plantation survival under reduced water supply as predicted in the future. Tolerance mechanisms are being studied in breeding programs, because information on such mechanisms can be used for genotype selection. In this paper, we analyze the changes of leaf water potential, hydraulic conductance (K(leaf)), stomatal conductance and phytohormones under drought in P. radiata breeds (O1, O2, O3, O4, O5 and O6) from different climatology areas, hypothesizing that they could show variable drought tolerance. As a primary signal, drought decreased cytokinin (zeatin and zeatin riboside-Z + ZR) levels in needles parallel to K(leaf) and gas exchange. When Z + ZR decreased by 65%, indole-3-acetic acid (IAA) and abscisic acid (ABA) accumulation started as a second signal and increments were higher for IAA than for ABA. When plants decreased by 80%, Z + ZR and K(leaf) doubled their ABA and IAA levels, the photosystem II yield decreased and the electrolyte leakage increased. At the end of the drought period, less tolerant breeds increased IAA over 10-fold compared with controls. External damage also induced jasmonic acid accumulation in all breeds except in O5 (P. radiata var. radiata × var. cedrosensis), which accumulated salicylic acid as a defense mechanism. After rewatering, only the most tolerant plants recovered their K(leaf,) perhaps due to an IAA decrease and 1-aminocyclopropane-1-carboxylic acid maintenance. From all phytohormones, IAA was the most representative 'water deficit signal' in P. radiata.

A client-centered approach to family planning: the Davao project.

The reproductive health approach to family planning shifts the focus of service provision from macro-level demographic objectives to meeting clients' needs. Little field experience exists to date, however, to indicate how to implement this approach. This study describes a field project in Davao del Norte and Compostela Valley provinces in the Philippines that implemented the reproductive health approach on a quasi-experimental basis. The intervention was designed to address clients' self-defined reproductive needs by providing them with relevant and accurate information and services of good quality. It consisted of two components: Providers were trained in information exchange at fixed clinics, and supervisors were trained in facilitative supervision. The results presented here indicate that the client-centered intervention was successful in enhancing service providers' knowledge and improving the content of information exchange between providers and clients. One provincial health officer has expanded the intervention throughout his province, while other provinces are interested in duplicating the model.

Glutamine synthetase from mesophyll and bundle sheath maize cells: isoenzyme complements and different sensitivities to phosphinothricin.

Anion-exchange FPLC has been used to resolve the isoforms of glutamine synthetase (GS, EC 6.3.1.2) from Zea mays mesophyll (MC) and bundle sheath cells (BSC). Two different isoforms were detected in both types of photosynthetic cells. The predominantly active isoform was GS1 (61%) in MC and GS2 (67%) in BSC. The relative contribution of GS1 and GS2 to the overall GS activity in BSC in maize here reported resembles the proportion described for most C3 plants. Differences among these isoforms in terms of their susceptibility to phosphinothricin (PPT), an analogue of glutamate and known inhibitor of GS, were found. The GS1 isoenzyme from MC was the most sensitive form, being inhibited by 50% at approximately 2.0 µM DL-PPT, whereas the GS2 from BSC presented the highest tolerance to the inhibitor (I50=30 µM). The transferase-to-semibiosynthetic activity ratio for the MC isoforms, which was higher than the ratio for the BSC isoforms, and the differences shown by the isoforms in susceptibility to PPT predict important differences in the biochemical properties and regulation of GS isoenzymes. In this regard, the cytoplasmic isoenzymes, and especially the one in MC, due to its relatively high contribution to mesophyll cell GS activity, could play a vital role in nitrogen metabolism in maize.

A diagnostic study of the DOH health volunteer workers program.

PIP: A 1993 study in the Philippines sought to 1) examine the characteristics of the Barangay (village) Health Workers (BHWs), 2) describe their recruitment and training, 3) reveal what their work entailed and how they felt about it, 4) identify the factors affecting their ability to deliver FP services, and 5) determine clients' attitudes towards them. Data were collected from structured interviews with 100 active BHWs and 150 clients in each of two regions. Most of the BHWs were women who had at least some high school education. They were longterm residents of their villages, were not gainfully employed, and their family incomes were below the poverty level. They were in favor of FP but considered three or more children ideal. Most had been BHWs for at least six years after being recruited (most often by a midwife). Most BHWs spent an average of 1.7 days assisting in the health centers and 2.0 days in the field. Their FP training was sketchy and largely centered on the use of various methods. The BHWs expressed a desire to learn more about natural FP, the IUD, and newer methods. Most received some monetary or other incentive for their work and were supervised directly by local midwives. FP service delivery was positively associated with education, experience, acquaintance with local residents, and cultural homogeneity with the clients. About half of the clients reported that they had consulted a BHW about FP, but an additional third were not familiar with the BHW program. The image of the BHW in the community was of a friendly, well-intentioned but not terribly knowledgeable person. The BHWs tended to provide follow-up services but often failed to refer clients to the health centers in the first place. Clients also reported that the BHWs gave them no choice about which method to use. This study points out the shortcomings in the BHW program. Improvement in outreach objectives could be reached by improved recruitment and training activities, the provision of more generous incentives, and improved logistics (such as contraceptive supply). The FP record-keeping system should also be improved.^ieng

[Nickel sensitivity in an atopic patient]. / Sensibilización al níquel en una paciente atópica.

A case of atopic dermatitis with it's classics signs in face and extremities, with an eight years evolution is reported. It was treated with corticoids (local and systemic) with no favorable results. In the allergic investigation it was found a sensibilization to nickel. She was cured by eliminating the nickel from her diet and from the objects in contact. Clinic and etiopathogenic considerations between allergic dermatitis by contact to nickel in the atopic and the non-atopic are made.