Effects of cylindrospermopsin, chlorpyrifos and their combination in a SH-SY5Y cell model concerning developmental neurotoxicity.

The cyanotoxin cylindrospermopsin (CYN) has been postulated to cause neurotoxicity, although the studies in this concern are very few. In addition, some studies in vitro indicate its possible effects on development. Furthermore, pesticides can be present in the same environmental samples as cyanotoxins. Therefore, chlorpyrifos (CPF) has been one of the most common pesticides used worldwide. The aim of this report was to study the effects of CYN, isolated and in combination with CPF, in a developmental neurotoxicity in vitro model. The human neuroblastoma SH-SY5Y cell line was exposed during 6 days of differentiation to both toxics to study their effects on cell viability and neurite outgrowth. To further evaluate effects of both toxicants on cholinergic signaling, their agonistic and antagonistic activities on the α7 homomeric nicotinic acetylcholine receptor (nAChR) were studied upon acute exposure. Moreover, a transcriptomic analysis by qPCR was performed after 6 days of CYN-exposure during differentiation. The results showed a concentration-dependent decrease on both cell viability and neurite outgrowth for both toxics isolated, leading to effective concentration 20 (EC20) values of 0.35 µM and 0.097 µM for CYN on cell viability and neurite outgrowth, respectively, and 100 µM and 58 µM for CPF, while the combination demonstrated no significant variations. In addition, 95 µM and 285 µM CPF demonstrated to act as an antagonist to nicotine on the nAChR, although CYN up to 2.4 µM had no effect on the efficacy of these receptors. Additionally, the EC20 for CYN (0.097 µM) on neurite outgrowth downregulated expression of the 5 genes NTNG2 (netrin G2), KCNJ11 (potassium channel), SLC18A3 (vesicular acetylcholine transporter), APOE (apolipoprotein E), and SEMA6B (semaphorin 6B), that are all important for neuronal development. Thus, this study points out the importance of studying the effects of CYN in terms of neurotoxicity and developmental neurotoxicity.

In vitro assessment of the combination of cylindrospermopsin and the organophosphate chlorpyrifos on the human neuroblastoma SH-SY5Y cell line.

Cylindrospermopsin (CYN) is a cyanotoxicant which occurrence is increasing due to climate change. Cylindrospermopsin is able to exert damage in the organism at several levels, among them, in the nervous system. Moreover, it is important to take into account that it is not usually present isolated in nature, but in combination with some other pollutants, being the case of the pesticide chlorpyrifos (CPF). Thus, the aim of the present work was to assess the effects of the interaction of CYN in combination with CPF in the human neuroblastoma cell line SH-SY5Y by evaluating cytotoxicity and mechanistic endpoints. The mixtures 0.25 + 21, 0.5 + 42, 1 + 84 µg/mL of CYN + CPF based on cytotoxicity results, were evaluated, and the isobologram method detected an antagonistic effect after 24 and 48 h of exposure. Moreover, although no alterations of reactive oxygen species were detected, a significant decrease of glutathione levels was observed after exposure to both, CPF alone and the combination, at all the concentrations and times of exposure assayed. In addition, CYN + CPF caused a marked decrease in the acetylcholinesterase activity, providing similar values to CPF alone. However, these effects were less severe than expected. All these findings, together with the morphological study results, point out that it is important to take into account the interaction of CYN with other pollutants. Further research is required to contribute to the risk assessment of CYN and other contaminants considering more realistic exposure scenarios.

Soil biogeochemistry and microbial community dynamics in Pinus pinaster Ait. forests subjected to increased fire frequency.

Fire frequency might increase in many fire-dominated ecosystems of the world due to the combined effects of global warming, land-use change and increased human pressures. Understanding how changes in fire frequency can affect the main soil biogeochemical dynamics, as well as the microbial community, in the long term is utmost important. Here we determined the effect of changes in fire frequency and other fire history characteristics on soil C and N dynamics and the main microbial groups (using soil fatty acid profiles), in Pinus pinaster forests from central Spain. Stands were chosen to differ in the number of fires (1 to 3) occurred between 1976 and 2018, in the time elapsed since the last fire and the interval undergone between the last two consecutive fires. We found that, in general, most of the studied biogeochemical and microbial variables showed clear differences between unburned and burned stands. The time elapsed since the last fire was the most important fire history covariable and governed the main soil nutrient dynamics and microbial groups. Recovery to pre-fire values took 30-40 years. Increased wildfire frequency only modified total C and nitrification rate, but results were not consistent between stands burned twice and thrice. The time interval (years) between the last two fires was not a significant covariable. The fact that some stands burnt up to thrice in a period of 43 years supports the strong capacity of this ecosystem to recover, even under an increased fire frequency.

Evaluation of mRNA markers in differentiating human SH-SY5Y cells for estimation of developmental neurotoxicity.

Current guidelines for developmental neurotoxicity (DNT) evaluation are based on animal models. These have limitations so more relevant, efficient and robust approaches for DNT assessment are needed. We have used the human SH-SY5Y neuroblastoma cell model to evaluate a panel of 93 mRNA markers that are frequent in Neuronal diseases and functional annotations and also differentially expressed during retinoic acid-induced differentiation in the cell model. Rotenone, valproic acid (VPA), acrylamide (ACR) and methylmercury chloride (MeHg) were used as DNT positive compounds. Tolbutamide, D-mannitol and clofibrate were used as DNT negative compounds. To determine concentrations for exposure for gene expression analysis, we developed a pipeline for neurite outgrowth assessment by live-cell imaging. In addition, cell viability was measured by the resazurin assay. Gene expression was analyzed by RT-qPCR after 6 days of exposure during differentiation to concentrations of the DNT positive compounds that affected neurite outgrowth, but with no or minimal effect on cell viability. Methylmercury affected cell viability at lower concentrations than neurite outgrowth, hence the cells were exposed with the highest non-cytotoxic concentration. Rotenone (7.3 nM) induced 32 differentially expressed genes (DEGs), ACR (70 µM) 8 DEGs, and VPA (75 µM) 16 DEGs. No individual genes were significantly dysregulated by all 3 DNT positive compounds (p < 0.05), but 9 genes were differentially expressed by 2 of them. Methylmercury (0.8 nM) was used to validate the 9 DEGs. The expression of SEMA5A (encoding semaphorin 5A) and CHRNA7 (encoding nicotinic acetylcholine receptor subunit α7) was downregulated by all 4 DNT positive compounds. None of the DNT negative compounds dysregulated any of the 9 DEGs in common for the DNT positive compounds. We suggest that SEMA5A or CHRNA7 should be further evaluated as biomarkers for DNT studies in vitro since they also are involved in neurodevelopmental adverse outcomes in humans.

Comparison between pollutants found in breast milk and infant formula in the last decade: A review.

Since ancient times, breastfeeding has been the fundamental way of nurturing the newborn. The benefits of breast milk are widely known, as it is a source of essential nutrients and provides immunological protection, as well as developmental benefits, among others. However, when breastfeeding is not possible, infant formula is the most appropriate alternative. Its composition meets the nutritional requirements of the infant, and its quality is subject to strict control by the authorities. Nonetheless, the presence of different pollutants has been detected in both matrices. Thus, the aim of the present review is to make a comparison between the findings in both breast milk and infant formula in terms of contaminants in the last decade, in order to choose the most convenient option depending on the environmental conditions. For that, the emerging pollutants including metals, chemical compounds derived from heat treatment, pharmaceutical drugs, mycotoxins, pesticides, packaging materials, and other contaminants were described. While in breast milk the most concerning contaminants found were metals and pesticides, in infant formula pollutants such as metals, mycotoxins, and packaging materials were the most outstanding. In conclusion, the convenience of using a feeding diet based on breast milk or either infant formula depends on the maternal environmental circumstances. However, it is important to take into account the immunological benefits of the breast milk compared to the infant formula, and the possibility of using breast milk in combination with infant formula when the nutritional requirements are not fulfilled only with the intake of breast milk. Therefore, more attention should be paid in terms of analyzing these conditions in each case to be able to make a proper decision, as it will vary depending on the maternal and newborn environment.

Previous fire occurrence, but not fire recurrence, modulates the effect of charcoal and ash on soil C and N dynamics in Pinus pinaster Aiton forests.

Understanding the effects of fire history on soil processes is key to characterise their resistance and resilience under future fire events. Wildfires produce pyrogenic carbonaceous material (PCM) that is incorporated into the soil, playing a critical role in the global carbon (C) cycle, but its interactions with soil processes are poorly understood. We evaluated if the previous occurrence of wildfires modulates the dynamic of soil C and nitrogen (N) and microbial community by soil ester linked fatty acids, after a new simulated low-medium intensity fire. Soils with a different fire history (none, one, two or three fires) were heat-shocked and amended with charcoal and/or ash derived from Pinus pinaster. Soil C and N mineralization rates were measured under controlled conditions, with burned soils showing lower values than unburned (without fire for more than sixty years). In general, no effects of fire recurrence were observed for any of the studied variables. Microbial biomass was lower in burned, with a clear dominance of Gram-positive bacteria in these soils. PCM amendments increased cumulative carbon dioxide (CO2) production only in previously burned soils, especially when ash was added. This contrasted response to PCM between burned and unburned soils in CO2 production could be related to the effect of the previous wildfire history on soil microorganisms. In burned soils some microorganisms might have been adapted to the resulting conditions after a new fire event. Burned soils showed a significant positive priming effect after PCM amendment, mainly ash, probably due to an increased pH and phosphorous availability. Our results reveal the role of different PCMs as drivers of C and N mineralization processes in burned soils when a new fire occurs. This is relevant for improving models that evaluate the net impact of fire in C cycling and to reduce uncertainties under future changing fire regimes scenarios.

High fire frequency reduces soil fertility underneath woody plant canopies of Mediterranean ecosystems.

Spatial heterogeneity of soil properties plays a major role in regulating ecosystem structure and functioning. In general, soil resources accumulate beneath woody plant-covered patches more than in the open interspace, making them function as fertility islands. Whilst wildfire is a common disturbance, little information is available on the role of particular plant species in maintaining soil fertility underneath in areas that are subjected to recurrent fires. This is an important issue given that land abandonment, together with a warmer and drier climate, is increasing fire danger in regions such as the Mediterranean. We determined whether increasing fire frequency, producing changes from a Quercus ilex L., woodland to a shrubland, modifies the effect of woody plant canopy on soil fertility. Additionally, the effect of fire history on species-specific leaf and litter nutrient concentration was assessed. Areas affected by none, one, two or three fires were selected. Within each area, soil fertility was measured underneath Cistus ladanifer L., Retama sphaerocarpa L., Phillyrea angustifolia L. and Quercus ilex canopies and in open interspace. Unburned soils located underneath P. angustifolia and Q. ilex canopies were significantly more fertile than in open interspaces. The microsite effect on soil fertility was fire frequency dependent. As fire frequency increased, the plant canopy microsite effect decreased for soil organic matter (SOM), cation exchange capacity (CEC), total C, P, Ca, K and Mg, labile phosphate, arylsulfatase and acid phosphatase activities. Total N, ammonium, nitrate and ß-glucosidase activity decreased with increasing fire frequency, but their spatial variability was maintained along all fire frequency scenarios. Fire frequency decreased foliar N concentration but increased P concentration in some species, leading to a decrease in their N:P ratio. Our findings suggest that soil fertility heterogeneity will be reduced with increasing fire frequency. This could compromise the recovery of soil and ecosystem functioning.

Functional, Clinical, and Sociodemographic Variables Associated with Risk of In-Hospital Mortality by COVID-19 in People over 80 Years Old.

OBJECTIVES: The objective is to assess the role of functional, clinical, and analytic parameters in predicting mortality in older patients hospitalized due to COVID-19. DESIGN: Cohort study with a mean follow-up of 12.8 days. SETTING: Public university hospital (Madrid, Spain). PARTICIPANTS: 499 patients 80 and above consecutively admitted to a Spanish public university hospital between 4 March 2020 and 16 May 2020. MEASUREMENTS: Mortality was the main outcome. Data of sociodemographic variables (age, sex, living), comorbidities, polypharmacy, functional status, date of hospital admission and length of stay was recorded. Clinical symptoms, laboratory and X-ray findings were collected at time of admission. For multivariant analysis, logistic regressions were performed to identify risk factors for death. RESULTS: Mean age was 86.7±4.4 with 37% of death. Mortality was associated with male gender [odds ratio (OR) 1.50; 95% confidence interval (CI) 1.01-2.24], with a 5-points increase on Barthel Index [OR 1.01 (95%CI 1.00-1.02)], higher Charlson Index score [OR 1.13 (95%CI 1.02-1.26)] and comorbidities [OR 1.28 (95%CI 1.06-1.53)], hyperpolipharmacy [OR 2.00 (95%CI 1.04-3.82)], unilateral pneumonia [OR 1.83 (95%CI 1.01-3.30)], higher levels of C-reactive protein [OR 1.09 (95%CI 1.06-1.12)] and creatine [OR 1.48 (95%CI 1.15-1.89)]. Higher oxygen levels were a protective factor [OR 0.92 (95%CI 0.89-0.95)]. CONCLUSIONS: Functional status, being male, a higher burden of comorbidities, hyperpolipharmacy, unilateral pneumonia and some laboratory parameters predict in-hospital mortality in this older population. The knowledge of these mortality risk factors should be used to improve the survival of older hospitalized patients.

Rootstock effects on scion gene expression in maritime pine.

Pines are the dominant conifers in Mediterranean forests. As long-lived sessile organisms that seasonally have to cope with drought periods, they have developed a variety of adaptive responses. However, during last decades, highly intense and long-lasting drought events could have contributed to decay and mortality of the most susceptible trees. Among conifer species, Pinus pinaster Ait. shows remarkable ability to adapt to different environments. Previous molecular analysis of a full-sib family designed to study drought response led us to find active transcriptional activity of stress-responding genes even without water deprivation in tolerant genotypes. To improve our knowledge about communication between above- and below-ground organs of maritime pine, we have analyzed four graft-type constructions using two siblings as rootstocks and their progenitors, Gal 1056 and Oria 6, as scions. Transcriptomic profiles of needles from both scions were modified by the rootstock they were grafted on. However, the most significant differential gene expression was observed in drought-sensitive Gal 1056, while in drought-tolerant Oria 6, differential gene expression was very much lower. Furthermore, both scions grafted onto drought-tolerant rootstocks showed activation of genes involved in tolerance to abiotic stress, and is most remarkable in Oria 6 grafts where higher accumulation of transcripts involved in phytohormone action, transcriptional regulation, photosynthesis and signaling has been found. Additionally, processes, such as those related to secondary metabolism, were mainly associated with the scion genotype. This study provides pioneering information about rootstock effects on scion gene expression in conifers.

Plant treatment, pollutant load, and soil type effects in rhizosphere ecology of trace element polluted soils.

Re-vegetation of trace element contaminated soils can alter the pH and chelating capacity in the rhizosphere, increasing the mobility of pollutants, which, in turn, may impact on rhizosphere ecology. In this study a short-term pot experiment was carried out in order to investigate the multi-factorial effects of: buffering capacity (sandy-loam and loam soils); pollutant load (0%, 1.3%, and 4% of pyrite sludge), and the presence/absence of plant (Lolium perenne L. and Medicago sativa L.) on the mobility of trace elements, soil biochemical functionality (hydrolase activities), and biological diversity (bacterial and nematode communities). The experiment was carried out with representative soils from the Guadiamar basin (SW Spain), an area where the Aznalcóllar mining spill affected over 4000ha. Results indicated that the development of rhizospheres in polluted soils (coarse-textured) increases the mobilization of trace elements. In general the presence of roots has stimulatory effects on soil quality indicators such as hydrolase activities and both bacterial and nematode communities. However, the presence of high amount of metals interferes with these beneficial effects. This study provided evidence about the complexity of the impact of growing plants on trace element polluted soils. Trace element mobilization, hydrolase activities and bacterial and nematode communities in the rhizosphere are dependent on plant species, soil type, and pollution dose.

Shrub encroachment can reverse desertification in semi-arid Mediterranean grasslands.

The worldwide phenomenon of shrub encroachment in grass-dominated dryland ecosystems is commonly associated with desertification. Studies of the purported desertification effects associated with shrub encroachment are often restricted to relatively few study areas, and document a narrow range of possible impacts upon biota and ecosystem processes. We conducted a study in degraded Mediterranean grasslands dominated by Stipa tenacissima to simultaneously evaluate the effects of shrub encroachment on the structure and composition of multiple biotic community components, and on various indicators of ecosystem function. Shrub encroachment enhanced vascular plant richness, biomass of fungi, actinomycetes and other bacteria, and was linked with greater soil fertility and N mineralization rates. While shrub encroachment may be a widespread phenomenon in drylands, an interpretation that this is an expression of desertification is not universal. Our results suggest that shrub establishment may be an important step in the reversal of desertification processes in the Mediterranean region.

Neurotoxicity induced by microcystins and cylindrospermopsin: A review.

Microcystins (MCs) and cylindrospermopsin (CYN) are among the most frequent toxins produced by cyanobacteria. These toxic secondary metabolites are classified as hepatotoxins and cytotoxin, respectively. Furthermore, both may present the ability to induce damage to the nervous system. In this sense, there are many studies manifesting the potential of MCs to cause neurotoxicity both in vitro and in vivo, due to their probable capacity to cross the blood-brain-barrier through organic anion transporting polypeptides. Moreover, the presence of MCs has been detected in brain of several experimental models. Among the neurological effects, histopathological brain changes, deregulation of biochemical parameters in brain (production of oxidative stress and inhibition of protein phosphatases) and behavioral alterations have been described. It is noteworthy that minority variants such as MC-LF and -LW have demonstrated to exert higher neurotoxic effects compared to the most studied congener, MC-LR. By contrast, the available studies concerning CYN-neurotoxic effects are very scarce, mostly showing inflammation and apoptosis in neural murine cell lines, oxidative stress, and alteration of the acetylcholinesterase activity in vivo. However, more studies are required in order to clarify the neurotoxic potential of both toxins, as well as their possible contribution to neurodegenerative diseases.

Neurotoxic assessment of Microcystin-LR, cylindrospermopsin and their combination on the human neuroblastoma SH-SY5Y cell line.

Microcystin-LR (MC-LR) and Cylindrospermopsin (CYN) are produced by cyanobacteria. Although being considered as a hepatotoxin and a cytotoxin, respectively, different studies have revealed neurotoxic properties for both of them. The aim of the present work was to study their cytotoxic effects, alone and in combination, in the SH-SY5Y cell line. In addition, toxicity mechanisms such as oxidative stress and acetylcholinesterase (AChE) activity, and morphological studies were carried out. Results showed a cytotoxic response of the cells after their exposure to 0-100 µg/mL of MC-LR or 0-10 µg/mL CYN in both differentiated and undifferentiated cells. Thus, CYN resulted to be more toxic than MC-LR. Respect to their combination, a higher cytotoxic effect than the toxins alone in the case of undifferentiated cells, and almost a similar response to the presented by MC-LR in differentiated cells were observed. However, after analyzing this data with the isobolograms method, an antagonistic effect was mainly obtained. The oxidative stress study only showed an affectation of glutathione levels at the highest concentrations assayed of MC-LR and the combination in the undifferentiated cells. A significant increase in the AChE activity was observed after exposure to MC-LR in undifferentiated cells, and after exposure to the combination of both cyanotoxins on differentiated cells. However, CYN decreased the AChE activity only on differentiated cultures. Finally, the morphological study revealed different signs of cellular affectation, with apoptotic processes at all the concentrations assayed. Therefore, both cyanotoxins isolated and in combination, have demonstrated to cause neurotoxic effects in the SH-SY5Y cell line.

Effects of pyrite sludge pollution on soil enzyme activities: ecological dose-response model.

A laboratory study was conducted to evaluate the response of soil enzyme activities (acid and alkaline phosphatase, beta-glucosidase, arylsulfatase, urease and dehydrogenase) to different levels of trace elements pollution in soils representative of the area affected by the pyrite sludge mining spill of Aznalcóllar (Guadiamar basin, SW Spain). Three uncontaminated soils from the study area were mixed with different loads of pyrite sludge to resemble field conditions and criteria applied for reclamation practices following the pollution incident: 0% ("reference" or background level), 1.3% ("attention level", further monitoring required), 4% ("intervention level", further cleaning and liming required) and 13% (ten times the "attention level"). Enzyme activities were analysed 4, 7, 14, 21, 34 and 92 days after pollutant addition and those measured after 92 days were used to calculate the ecological dose value (ED50). Soil enzyme activities and pH decreased after the pyrite sludge addition with respect to the "reference level" (0% pyrite sludge), whereas soil bioavailable (DTPA-extractable) trace elements concentration increased. Arylsulfatase, beta-glucosidase and phosphatase activities were reduced by more than 50% at 1.3% pyrite sludge dose. Arylsulfasate was the most sensitive soil enzyme (in average, ED50=0.99), whereas urease activity showed the lowest inhibition (in average, ED50=7.87) after pyrite sludge addition. Our results showed that the ecological dose concept, applied to enzyme activities, was satisfactory to quantify the effect of a multi-metalic pollutant (pyrite sludge) on soil functionality, and would provide manageable data to establish permissible limits of trace elements in polluted soils. Additionally, we evaluate the recovery of enzyme activities after addition of sugar-beet lime (calcium carbonate) to each experimentally polluted soil. The amount of lime added to each soil was enough to raise the pH to the original value (equal to control soil), resembling field remediation practices. After lime amendment, soil recovery was still incomplete in terms of bioavailable trace elements. However, the recovery of soil enzyme activities varied widely, ranging from 0 to 100% depending on soil type and the specific enzyme.

Comparison of two analytical methods for the evaluation of the complexed metal in fertilizers and the complexing capacity of complexing agents.

The aim of this research is to develop an analytical methodology for the determination of complexed element in fertilizers and, then, to obtain an adequate criteria for the inclusion of these products in European Regulations on Fertilizers. This paper compares the CEN method EN 13366:2001, based on the retention of the cations into a sulfonated resin, and an AOAC modified method, based on the precipitation of the inorganic forms at pH 9. A limited interlaboratory trial was carried out to demonstrate the applicability of the AOAC modified method and to study the effect of the removal of organic compounds and the addition of a matrix modifier solution before the element quantification. Then, a global interlaboratory trial was developed to evaluate the validation and quality parameters of the method. As a second objective, the AOAC modified method was applied to the determination of the complexing capacity of complexing agents based on lignosulfonates and amino acids. The AOAC modified method was the choice methodology because it is adequate for the determination of complexing capacity of micronutrients in fertilizer.

Microorganisms causing of clinical pathologies in canines and domestic felines in Valledupar, Colombia / Microorganismos causantes de patologías clínicas en caninos y felinos domésticos en Valledupar, Colombia

ABSTRACT The pathologies caused by microbial groups generate health risks in domestic dogs and cats; showing a zoonotic potential and producing affections in humans. According to that background, the purpose of this study was to establish the main microorganisms causing clinical pathologies in domestic dogs and cats by reviewing medical records in a veterinary clinic in Valledupar, Colombia. In order to do that, a retrospective study was conducted with clinical histories of dogs and cats admitted during 2017 and 2018 to the veterinary clinic "Mis Mejores Amigos", located in the city of Valledupar, Colombia. Based on the information, a descriptive, explanatory and statistical analysis was applied, the latter by means of a Sperman correlation to evaluate the relationship between race, sex, age, microbial group and pathology-pathogen. From a total of 462 reports of canines and felines admitted to the clinic, a total of 273 diagnoses were obtained. 4% of the felines and 24% of the canines were affected by some microbial group. The most prevalent microorganisms in canines were Ancylostoma spp. (9 %) Entamoeba histolytica (5%) Ehrlichia spp. (5%), and Isospora sp. (5%) while in felines were Haemobartonella felis (20%) and Haemobartonella spp. (17%). It was determined that there is only correlation between the age of felines and the microbial group that affects them, in addition to the pathology presented with the pathogen. It was observed a high percentage of Ancylostoma sp. (9%) in canines of mongrel and French Poodle breeds whilst in felines was found the bacterium Haemobartonella felis in 20% of the mongrel breeds.

RESUMEN Las patologías causadas por grupos microbianos generan riesgos en la salud de perros y gatos domésticos, lo que tiene un potencial zoonótico y produce afecciones en seres humanos. De acuerdo con lo anterior, el objetivo de este estudio fue establecer los principales microorganismos causantes de patologías clínicas en perros y gatos domésticos mediante la revisión de historias clínicas en una clínica veterinaria de Valledupar, Colombia. Se realizó un estudio retrospectivo con historias clínicas de perros y gatos ingresados durante 2017 y 2018 a la clínica veterinaria "Mis mejores amigos", ubicada en la ciudad de Valledupar, Colombia. Con base en la información se aplicó un análisis descriptivo, explicativo y estadístico, este último mediante una correlación de Sperman para evaluar relación entre raza, sexo, edad, grupo microbiano y patología-patógeno. De un total 462 reportes de caninos y felinos ingresados a la clínica, se obtuvieron 273 diagnósticos. El 4% de los felinos y el 24 % de los caninos presentaban afecciones por algún grupo microbiano. Los microorganismos más prevalentes en caninos fueron Ancylostoma spp. (9%) Entamoeba histolytica (5%) Ehrlichia spp. (5%) e Isospora sp. (5%), mientras que en felinos fueron Haemobartonella felis (20%) y Haemobartonella spp. (17%). Se determinó que solo existe correlación entre la edad de felinos y el grupo microbiano que los afecta, además de la patología presentada con el patógeno. Se observó un alto porcentaje de Ancylostoma sp. (9%) en caninos de las razas mestiza y French Poodle, y, por otro lado, en felinos se identificó la bacteria Haemobartonella felis en 20% de los sujetos de raza mestiza.

Post-fire soil functionality and microbial community structure in a Mediterranean shrubland subjected to experimental drought.

Fire may cause significant alterations in soil properties. Post-fire soil dynamics can vary depending, among other factors, on rainfall patterns. However, little is known regarding variations in response to post-fire drought. This is relevant in arid and semiarid areas with poor soils, like much of the western Mediterranean. Furthermore, climate change projections in such areas anticipate reduced precipitation and longer annual drought periods, together with an increase in fire severity and frequency. This research evaluates the effects of experimental drought after fire on soil dynamics of a Cistus-Erica shrubland (Central Spain). A replicated (n=4) field experiment was conducted in which the total rainfall and its patterns were manipulated by means of a rain-out shelters and irrigation system. The treatments were: environmental control (natural rainfall), historical control (average rainfall, 2months drought), moderate drought (25% reduction of historical control, 5months drought) and severe drought (45% reduction, 7months drought). After one growing season under these rainfall treatments, the plots were burned. One set of unburned plots under natural rainfall served as an additional control. Soils were collected seasonally. Fire increased soil P and N availability. Post-fire drought treatments reduced available soil P but increased N concentration (mainly nitrate). Fire reduced available K irrespective of drought treatments. Fire reduced enzyme activities and carbon mineralization rate, a reduction that was higher in post-fire drought-treated soils. Fire decreased soil microbial biomass and the proportion of fungi, while that of actinomycetes increased. Post-fire drought decreased soil total microbial biomass and fungi, with bacteria becoming more abundant. Our results support that increasing drought after fire could compromise the resilience of Mediterranean ecosystems to fire.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.

Development of an HPLC method for determination of diphenidol in plasma and its application in an oral multi-dose bioequivalence study in a healthy female Mexican population.

Diphenidol was determined by an HPLC method developed in our laboratory. It was validated and proved to be linear in the 40-400 ng/ml range. Accuracy for quality-control samples for intra and inter day assays ranged from 96.1-98.9% and 98.8-101.4%, respectively. This method was applied to a multi-dose bioequivalence study. No serious side effects were observed in the multi-dose design. Pharmacokinetic parameters (mean+/-standard error [S.E.]) of Cavg (ng/ml) and AUCtau (ngh/ml) for reference and test products were 139.54+/-12.66 versus 148.60+/-16.51 and 551.07+/-53.53 versus 588.78+/-69.02, respectively. Log-transformed values were compared by analysis of variance (ANOVA) followed by the classical 90% confidence interval (CI 90%) test and Schuirmann's test. Confidence limits ranged from 91.48-116.18% for Cmax and from 91.24-117.65% for AUCtau. These results suggest that the analytical method was linear, precise, and accurate for our purpose, and that both assayed formulations were bioequivalent.

Microbial response to heavy metal-polluted soils: community analysis from phospholipid-linked fatty acids and ester-linked fatty acids extracts.

Heavy metal pollution of soil is of concern for human health and ecosystem function. The soil microbial community should be a sensitive indicator of metal contamination effects on bioavailability and biogeochemical processes. Simple methods are needed to determine the degree of in situ pollution and effectiveness of remediating metal-contaminated soils. Currently, phospholipid-linked fatty acids (PLFAs) are preferred for microbial profiling but this method is time consuming, whereas direct soil extraction and transesterification of total ester-linked fatty acids (ELFAs) is attractive because of its simplicity. The 1998 mining acid-metal spill of >4000 ha in the Guadiamar watershed (southwestern Spain) provided a unique opportunity to study these two microbial lipid profiling methods. Replicated treatments were set up as nonpolluted, heavy metal polluted and reclaimed, and polluted soils. Inferences from whole community-diversity analysis and correlations of individual fatty acids with metals suggested Cu, Cd, and Zn were the most important in affecting microbial community structure, along with pH. The microbial stress marker, monounsaturated fatty acids, was significantly lower for reclaimed and polluted soil over nonpolluted soils for both PLFA and ELFA extraction. Another stress marker, the monounsaturated to saturated fatty acids ratio, only showed this for the PLFA. The general fungal marker (18:2omega6c), the arbuscule mycorrhizae marker (16:1omega5c), and iso- and anteiso-branched PLFAs (gram positive bacteria) were suppressed with increasing pollution whereas 17:0cy (gram negative bacteria) increased with metal pollution. For both extraction methods, richness and diversity were greater in nonpolluted soils and lowest in polluted soils. The ELFA method was sensitive for reflecting metal pollution on microbial communities and could be suitable for routine use in ecological monitoring and risk assessment programs because of its simplicity and reproducibility.