Presence of pharmaceuticals and heavy metals in the waters of a Mediterranean coastal wetland: Potential interactions and the influence of the environment.

The occurrence of 17 relevant pharmaceuticals and 7 heavy metals in the waters of the Pego-Oliva Marsh Natural Park (Valencia Community, Spain) were monitored. Thirty four zones (including the lagoon and the most important irrigation channels), covering the main land uses and water sources, were selected for sampling. Thirty three of them were contaminated with at least one pharmaceutical. Ibuprofen and codeine were the pharmaceuticals more frequently detected, in concentrations between 4.8 and 1.2 ng/L and a maximum of 59 ng/L and 63 ng/L, respectively. Regarding metals, Zn showed values under the detection limit in all the samples, while Cd, Co, Cr, Cu, Ni and Pb were detected at concentrations lower than the WHO and EU maximum levels for drinking waters. Ni showed significant direct correlations with diazepam, norfloxacin, ofloxacin and fenofibrate, and inverse relationships with ibuprofen, at 99 and 95% of significance. Cu, Co and Cr also showed significant correlations with some of the pharmaceuticals. These interactions could favor the synergistic/antagonistic interactions among pharmaceuticals and metals in the marsh, which can affect its aquatic fauna and flora or even human health. The influences of the water sources, land uses and spatial distribution of both types of contaminants were also studied.

Archaeal community dynamics and abiotic characteristics in a mesophilic anaerobic co-digestion process treating fruit and vegetable processing waste sludge with chopped fresh artichoke waste.

This study evaluated the feasibility of obtaining methane in anaerobic digestion (AD) from the waste products generated by the processing of fruit and vegetables. During the first phase (0-55 d) of the AD using sludge from fruit and vegetable processing, an average value of 244±88 L kg(-1) dry matter d(-1)of biogas production was obtained, and methane content reached 65% of the biogas. Co-digestion with chopped fresh artichoke wastes in a second phase (55-71 d) enhanced biogas production, and resulted in an average value of 354±68 L kg(-1) dry matter d(-1), with higher methane content (more than 70%). The archaeal community involved in methane production was studied using the ANAEROCHIP microarray and real-time PCR. Results indicated that species of Methanosaeta and Methanosarcina were important during the AD process. Methanosarcina numbers increased after the addition of chopped fresh artichoke, while Methanosaeta numbers decreased.

Purification of erythropoietin from human plasma samples using an immunoaffinity well plate.

A method is described to isolate human erythropoietin (hEPO) from plasma using an EPO-specific immunoaffinity micro well plate (IAP). The operating conditions of the method (binding, blocking and elution) were optimised to avoid isoform discrimination and cross-contamination with other glycoproteins. The overall hEPO recovery was ca. 56% and significant clean-up for plasmatic hEPO was achieved. Polyvinylpyrrolidone (PVP) was used as a blocking reagent and elution took place at pH 11.0. Under these conditions all isoforms from recombinant human EPOs (rhEPOs) and analogues were uniformly recovered guaranteeing lack of discrimination. The resulting procedure allowed isolating erythropoietin from plasma in conditions amenable to hEPO analysis by other techniques such as SDS-PAGE or IEF. Moreover, avoiding contamination with other glycosylated material allowed the identification in human plasma samples of the non-human N-glycolyl-neuraminic acid (Neu5Gc) using HPLC-FLD. Neu5Gc is present as 1-2% of the sialic acid content in rhEPO so this approach could be used to unequivocally detect abuse of rhEPOs or analogues as part of the doping control.

Utilisation of citrus compost-based growing media amended with Trichodermaharzianum T-78 in Cucumismelo L. seedling production.

Two citrus composts (C1: composed of 40% citrus wastes, 20% sludge obtained from a citrus industry waste-water treatment facility and 40% green residues; C2: composed of 60% citrus wastes and 40% green residues, and no sludge) and their water extracts amended with Trichodermaharzianum T-78 (T. harzianum T-78) were assayed in order to verify if these composts could act as a partial substitute for peat-based growing media as well as enhance suppressiveness against Fusarium wilt in the production of melon (Cucumismelo L.) seedlings at greenhouse nurseries. Over a 43-day growth cycle of melon seedlings, measurements were taken of the nutriactive effect (the capability of a substrate to express additional and/or synergistic nutritional and biostimulating effects), the pathogen incidence (percentage of fresh weight loss of melon plants grown on treatments infected with Fusariumoxysporum with respect to the same treatment without inoculation of the phytopathogen) and the trend of the T.harzianum T-78 population. A nutriactive effect was observed in the tested citrus compost-based growing media (96% and 112% plant weight increase with respect to peat for C1Th and C2Th, respectively). Pathogen incidence was significantly lower in C2Th than peat (12% compared to 33%), while no difference was observed in C1Th. The T.harzianum T-78 population showed a significant decrease at the first sampling time compared to the initial quantity (from 10(6) to 10(5)CFUg(-1)), but later recovered over time. These results demonstrate that the combination of citrus compost and T.harzianum T-78 can be a viable alternative to peat and can minimise the application of chemicals necessary to control Fusarium wilt in greenhouse nurseries for melon seedling production.

New screening protocol for recombinant human erythropoietins based on differential elution after immunoaffinity purification.

A screening method able to differentiate recombinant human erythropoietins (rhEPOs) and analogues like CERA from human urinary erythropoietin (uhEPO) is described. The method is based on the discrimination between isoforms observed when the protein is eluted under acidic followed by basic conditions from immunoaffinity microtiter wells. From a comparison with the complex IEF protocol currently applied in anti-doping analysis, the newly developed assay procedure is amenable to wide screening application and presents good resolving power between rhEPOs and uhEPO.

[Scrotal mass presenting as giant urethral diverticulum]. / Masa escrotal como presentación de divertículo uretral gigante.

Urethral diverticulum in the male is a rare entity that may be congenital or acquired. They are common in paraplegic patients, who are prone to developing this disorder on an acquired basis because of prolonged catheterization. The most common diseases in patients with spinal cord injury are stricture, fistula and diverticula. Patients with diverticula typically present with symptoms of urinary incontinence, dysuria, perineal pain, or a mass on the ventral aspect of the genitalia or perineum. Treatment of choice is always surgical and a complete extirpation should be performed.

Citrus compost and its water extract for cultivation of melon plants in greenhouse nurseries. Evaluation of nutriactive and biocontrol effects.

Two different types of citrus composts, and their water extracts, were tested with regard to their utilisations as partial substitutes for peat in growing media for melon seedlings in greenhouse nurseries. Both compost showed higher plant growth than peat. Compost composed by citrus waste and green residue (C2) showed greater plant growth than compost obtained from the same organic matrices mentioned above further the addition of sludge obtained from citrus industry (C1). Compost C2 showed a greater auxinic effect than C1 and it was the only one that showed cytokinic effect. Both composts also demonstrated a biocontrol effect against Fusarium oxysporum for melon plants: the effects were also higher in C2 than in C1. Higher number of isolated fungi was active against F. oxysporum in compost C2, than compost C1. No different bacterial biocontrol efficacy was observed between both composts. The water extracts of both composts gave lower plant yields than their solid matrices, their relative effects being similar to those of the solid composts (C2 extract gave higher plant yields than the extract from C1). The biocontrol effects of compost water extracts followed the same trend.

Monitoring gene therapy by external imaging of mRNA: pilot study on murine erythropoietin.

Gene therapy is anticipated as being an important medical development. Essential to its effectiveness is the appropriate activity (protein expression) in the expected target cells. A noninvasive diagnostic procedure of successful gene expression will be of paramount importance to validate its use or its misuse (eg, sports gene doping). Externally detectable labeled oligonucleotide hybridizing with the messenger RNA generated by the transferred gene has been proposed as a possibility to monitor successful gene therapy. The authors selected the erythropoietin gene (Epo) for a pilot study on erythropoietin protein expression in mouse muscle. Oligonucleotides of peptide nucleic acid (PNA) type capable of antisense binding to unique murine Epo-mRNA sequences were synthesized by solid phase methods, and elongated at the N-terminus with the HIV Tat (48-60) cell penetrating peptide. They were labeled with fluorescence and radioactive tags to verify penetration and longer half-life properties in Epo gene transfected C2C12 mouse muscle cells as compared with corresponding wild-type cells. Downregulation of newly expressed erythropoietin protein in such cells additionally confirmed the penetration and hybridizing properties of the selected labeled oligonucleotide. I-labeled Tat-PNAs were intravenously injected into mice that had previously received the Epo gene into the right tibialis muscle by DNA electrotransfer. Preferential accumulation of radioactivity in the transferred limb as compared with the contralateral limb was ascertained, especially for I-Tat-CTA CGT AGA CCA CT (labeled Tat-PNA 1). This study provides experimental data to support the potential use of external noninvasive image detection to monitor gene therapy. The extension of the approach to more sensitive methods for whole-body external detection such as positron emission tomography appears feasible.

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP.

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.

Effect of physical fitness and endurance exercise on indirect biomarkers of recombinant erythropoietin misuse.

Erythropoietin (EPO) and soluble transferrin receptor (sTfR) in serum have been proposed as indirect biomarkers for the detection of recombinant human EPO (rhEPO) misuse in sport. The purpose of the present study was to investigate the influence of different levels of physical fitness, sport, different training workload during the sport season, and endurance exercise in the concentrations of these serum biomarkers for their application into mathematical models to indirectly detect rhEPO misuse. Serum EPO and sTfR concentrations were measured in 96 elite athletes of various sports along the sport season, in 21 recreational athletes at baseline (non exercising) conditions and in 129 other recreational athletes before and after long-distance races (10 and 21 km). In elite athletes, hemoglobin concentrations and percentage of reticulocytes were also measured, and indirect detection models applied. In recreational athletes, for EPO and sTfR, significant differences were only observed after the 21-km race. In baseline conditions, no differences were observed between recreational and elite athletes for EPO and sTfR. In elite athletes, individual EPO and sTfR concentrations slightly changed over the sport season, with coefficients of variation (CV) of 26.1 % and 9.0 %, respectively. Hemoglobin and reticulocytes were influenced by sport, but their individual variation over the sport season was not physiologically relevant (CV of 3.7 % and 21.3 %, respectively). When applying mathematical models for detection of rhEPO administration, only one elite athlete obtained an individual model score above the established thresholds. Physical fitness, sport and different training workload during the sport season had no substantial effect on serum EPO and sTfR concentrations, except in recreational athletes after a 21-km race. Variations observed in mathematical models to detect EPO administration were mainly due to fluctuation in hemoglobin concentrations, commonly observed in elite athletes.

Effect of physical fitness and endurance exercise on indirect biomarkers of recombinant growth hormone misuse: insulin-like growth factor I and procollagen type III peptide.

Serum insulin-like growth factor-I (IGF-I) and procollagen type III peptide (P-III-P) have been proposed as indirect biomarkers of rhGH misuse in sports. The purpose of the present study was to investigate concentrations of these biomarkers in athletes at different levels of physical fitness and endurance exercise. Serum total IGF-I and P-III-P were measured in 96 elite athletes of various sports along the training season; in 21 recreational athletes at baseline non-exercising conditions and in another 129 recreational athletes before and after long-distance races (10 and 21 km). No differences were evidenced for IGF-I concentrations, but statistically higher values of serum P-III-P were found in elite athletes compared to recreational ones. Among elite athletes, the specific sport did not affect serum IGF-I. However, P-III-P was statistically higher in the sport performed by the youngest athletes (rhythmic gymnastics), even after correction of the logarithm of the concentration by the reciprocal of age. Over the training season, the within-athlete variabilities of IGF-I and P-III-P in elite athletes were low (22.8 % and 21.7 %, respectively). Recreational athletes taking part in a 21 km competition race showed a significant increase in serum values of IGF-I and P-III-P immediately after the event. Exercise workload and age had a significant effect on serum concentration of P-III-P, while age alone affected IGF-I serum concentrations. Therefore, athlete's reference concentration ranges for doping detection should include subjects from as many different ages and sports as possible.

Microbial community structure at different depths in disturbed and undisturbed semiarid Mediterranean forest soils.

Metabolic abilities and micrfiobial community structure were investigated through three semiarid Mediterranean soils of SE Spain. The soils were (1) a Typic Calcixerept under an adult pine plantation (PP), growing on abandoned agricultural terraces; (2) a Typic Calcixeroll under a native pinewood (NP); and (3) a Typic Haploxerept covered with a grass steppe (GS). PP and NP were similar as regards their genesis, but the former used to be tilled. NP and GS were undisturbed and supported natural and seminatural vegetation, respectively. Seven samples in 10-cm depth increments were taken in triplicate along each soil profile. Community-level physiological profiles based on sole-C-source use were determined to characterize the metabolic abilities. A 16S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis analysis was performed to investigate the microbial genetic structure. Plant cover and land-use history were major determinants of microbial community structure. Microbial communities residing in soils under a native pinewood, the most diverse and stable plant cover, were the most complex both metabolically and genetically. The microbial community structure distinctly changed with depth, related to the quantity and quality of total organic carbon. Both undisturbed soils showed falling gradients of metabolic and genetic complexity, which were invariably of a greater magnitude in the mature woodland than in the grass steppe. In the planted pinewood, however, the substrate-use diversity increased with depth, apparently a response to the depleted metabolic abilities within its upper layer (0-30 cm). Tilling and plant cover removal might be responsible for such a perturbation. In the same profile, molecular fingerprint patterns of the topsoil layer (0-10 cm) indicated a disturbed genetic structure that might underlie the loss of metabolic abilities. However, the genetic structure of the deeper layers of the planted and native pinewoods was not dissimilar, revealing that equivalent genetic resources perform different environmental functions under changing soil scenarios.

Biopesticide effect of green compost against fusarium wilt on melon plants.

AIMS: The biopesticide effect of four green composts against fusarium wilt in melon plants and the effect of soil quality in soils amended with composts were assayed. METHODS AND RESULTS: The composts consisted of pruning wastes, with or without addition of coffee wastes (3/1 and 4/1, dry wt/dry wt) or urea (1000/1, dry wt/dry wt). In vitro experiments suggested the biopesticide effect of the composts against Fusarium oxysporum, while only the compost of pine bark and urea (1000/1dry wt/dry wt) had an abiotic effect. Melon plant growth with composts and F. oxysporum was one to four times greater than in the non-amended soil, although there was no significant decrease in the level of the F. oxysporum in the soil. The addition of composts to the soil also improved its biological quality, as assessed by microbiological and biochemical parameters: ATP and hydrolases involved in the P (phosphatase), C (beta-glucosidase) and N (urease) cycles. CONCLUSIONS: Green composts had greater beneficial characteristics, improved plant growth and controlled fusarium wilt in melon plants. These composts improve the soil quality of semi-arid agricultural soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Biotic and abiotic factors from composts have been tested as responsible of their biopesticide activity against fusarium wilt.

[Study of the extent and rate of bioavailability of ibuprofen tablets]. / Estudio de la biodisponibilidad en magnitud y en velocidad de comprimidos de ibuprofeno.

The extent and rate of bioavailability of ibuprofen tablets were determined in a crossover clinical trial in 18 healthy subjects of both sexes. The study was approved by the local ethical committee and was authorized by the Spanish Medicines Agency. Volunteers signed an informed consent form and were included in accordance with the standard procedures for this type of study. In two distinct sessions participants received a single 600 mg ibuprofen dose as Gelofeno(®) 600 mg tablets (Laboratorios Gelos S.L.), or as the reference formulation, Neobrufen(®) 600 mg tablets. Ibuprofen concentrations in plasma were determined immediately before (0 h) and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12 and 24 h after drug administration. The pharmacokinetic parameters were then calculated. In all subjects, Gelofeno(®) 600 mg tablets produced plasma concentrations above the quantification limit between 15 and 30 minutes after administration, and in 9 (50%) of these subjects maximal plasma concentrations were reached at 1 h. The median t(max) was 1.25 h, and the average maximal plasma concentration was 40.7 mg/l. Gelofeno(®) 600 mg tablets were bioequivalent both in extent and in rate of bioavailability compared with the reference drug. The formulation showed good tolerability and no medication-related adverse effects were observed.

[Intraocular metastatic renal carcinoma presenting as progressive unilateral blindness]. / Carcinoma renal metastásico intraocular. Ceguera unilateral progresiva como forma de presentación.

Clear cell renal carcinoma is the most common histological type, representing 70-80% of all renal carcinomas. Metastases are already present in about 25-30% of patients at the time of diagnosis of renal cell carcinoma. Ocular metastasis is extremely rare.

Influence of the stabilisation of organic materials on their biopesticide effect in soils.

Some organic materials have shown a suppressive effect on several diseases induced by soilborne plant pathogens. We have carried out a laboratory experiment (microcosm) to ascertain the influence of the stabilisation process of sewage sludge on it biopesticide effect when Pythium ultimum or Phytophthora sp. were introduced to soil as pathogens for pea or pepper. When P. ultimum was introduced there was a 63.8% reduction in the weight of the stems grown in the control, 47.6% in the presence of sewage sludge, but only 24.7% with compost. The same biopesticide effect was evident from the weight of the roots. The biopesticide effect was also strong when compost was used as organic amendment in the presence of Phytophthora, as could be seen from stem and shoot weight and length. The data showed that the degree of stabilisation of the organic material (compost) had a positive influence on it biopesticide effect. The changes undergone by a soil after the addition of organic materials helped to explain the biopesticide effect of the amendment. The organic treatments reduced P. ultimum and Phytophthora sp. populations.

Recombinant erythropoietin and analogues: a challenge for doping control.

Erythropoietin (EPO) increases the number of circulating erythrocytes and thus muscle oxygenation. The availability of the recombinant protein (rEPO) has increased the risk of its illegal use in sports, its detection being a difficult challenge. Five different hematopoietic parameters were initially chosen as indirect markers of rEPO abuse: concentration of serum EPO, concentration of serum-soluble transferrin receptors (sTFr), hematocrit, percentage of reticulocytes, and percentage of macrocytes. New models considering only hemoglobin, serum EPO concentration, and percentage of reticulocytes are simpler and seem to be more sensitive when low doses of rEPO are used. A more direct method of urine analysis (isoelectrofocusing, double blotting, and chemiluminescent detection) based on the charge differences between rEPO and endogenous EPO, related to their carbohydrate composition, provides proof of rEPO use. Furthermore, this approach permits the detection of darbepoetin, a direct analogue of EPO also known as NESP ("new erythropoiesis stimulating protein"). Recently a protein conjugate, "synthetic erythropoiesis protein" (SEP), containing precision-length, monodisperse, negatively charged polymers instead of oligosaccharides has been synthesized. Finally, EPO-mimetics are molecules capable of acting as EPO in dimerizing the EPO receptor. Two kinds of EPO-mimetics have been described: peptides and nonpeptides. The enhancement of oxygen availability to muscles by rEPO, analogues, and mimetics constitutes one of the main challenges to doping control. Major steps have already been developed for detection ofrEPO and some analogues. In the near future, the transfection to an athlete's body of genes that code for erythropoietin might be an emerging doping issue, and sports authorities have incorporated "gene doping" among the prohibited practices.

Evaluation of immunoassays for the measurement of soluble transferrin receptor as an indirect biomarker of recombinant human erythropoietin misuse in sport.

Soluble transferrin receptor (sTfR) has been proposed as an indirect biomarker of the misuse of recombinant human erythropoietin in sport. An extended validation of four commercially available immunoassays for its measurement in serum is presented. Two ELISA techniques (ELISA1: Orion Diagnostica; ELISA2: R&D Systems), an immunoturbidimetric technique (Turbid: Roche Diagnostics), and a nephelometric technique (Nephel: Dade Behring) were investigated. Intra-laboratory precision better than 3% and correct accuracies were obtained for the Turbid and Nephel techniques using autoanalysers. Slightly worse precision (but always better than 11%) and correct accuracies were also obtained in almost all cases for the two ELISA techniques. Inter-laboratory results showed higher concordances for the ELISA procedures (intraclass correlation coefficients of 0.848 for ELISA1 and 0.973 for ELISA2 which was clearly better). Inter-technique correlations were good for the four techniques with lower dispersions found for the techniques using autoanalysers, i.e. Turbid and Nephel. While Turbid and ELISA1 results (expressed in mg/l) were comparable, results obtained with Nephel were approximately 2.7 times lower. The relationship between those three techniques was maintained when compared with ELISA2, which uses different units (nmol/l). We conclude that ELISA2 and Nephel in our hands were the most suitable techniques in terms of sensitivity, precision and accuracy, and adequacy of the calibration curve for the measurement of sTfR in real serum samples. Discrepancies observed in the results obtained with the different sTfR techniques showed that different reference standards were used and harmonization is recommended in order to obtain comparable results.

[Giant stone in a Bricker urinary diversion. Obstruction uropathy as the presenting form]. / Litiasis gigante en derivación urinaria tipo Bricker. Uropatía obstructiva como forma de presentación.

Bowel is used in urological surgery to replace the bladder, either as a conduit to drain urine to the abdominal wall as a urinary stoma or refashioned to form a substitute bladder. Many factors contribute to stone formation, being urinary stasis, mucus production and bacteriuria the most important. Metabolic changes induced by exposure of segments of the alimentary tract to urine promote struvite, calcium oxalate and calcium phosphate stone formation. Generally, the majority of patients with stones in a urinary diversion can be treated with minimally invasive techniques. Open surgical removal is considered when other modality of treatments cannot be accomplished safely and expeditiously.