Evaluation of serum myostatin and sclerostin levels in chronic spinal cord injured patients.

STUDY DESIGN: Case-control study. OBJECTIVES: To assess serum myostatin levels, bone mineral density (BMD), appendicular skeletal muscle mass (ASMM) and serum sclerostin levels in chronic spinal cord injured (SCI) patients and healthy controls. SETTING: SCI centre in Italy. METHODS: Blood samples, whole-body bioelectrical impedance analysis and BMD measurement with the ultrasound technique at the calcaneus level were taken from patients suffering from chronic SCI (both motor complete and incomplete) and healthy control subjects. RESULTS: A total of 28 SCI patients and 15 healthy controls were enrolled. Serum myostatin levels were statistically higher (P<0.01) in SCI patients compared with healthy controls. Similar results were found comparing both the motor complete and the motor incomplete SCI subgroups to healthy controls. Serum sclerostin was significantly higher in patients with SCI compared with healthy controls (P<0.01). BMD, stiffness and mean T-score values in SCI patients were significantly lower than those in healthy controls. Serum myostatin concentrations in the motor complete SCI subgroups correlated only with serum sclerostin levels (r(2)=0.42; P=0.001) and ASMM (r(2)=0.70; P=0.002) but not in healthy controls. DISCUSSION: Serum myostatin and serum sclerostin are significantly higher in chronic SCI patients compared with healthy controls. They are potential biomarkers of muscle and bone modifications after SCI. This is the first study reporting an increase in serum myostatin in patients suffering from chronic SCI and a correlation with ASMM.

Citral induces auxin and ethylene-mediated malformations and arrests cell division in Arabidopsis thaliana roots.

Citral is a linear monoterpene which is present, as a volatile component, in the essential oil of several different aromatic plants. Previous studies have demonstrated the ability of citral to alter the mitotic microtubules of plant cells, especially at low concentrations. The changes to the microtubules may be due to the compound acting directly on the treated root and coleoptile cells or to indirect action through certain phytohormones. This study, performed in Arabidopsis thaliana, analysed the short-term effects of citral on the auxin content and mitotic cells, and the long-term effects of these alterations on root development and ethylene levels. The results of this study show that citral alters auxin content and cell division and has a strong long-term disorganising effect on cell ultra-structure in A. thaliana seedlings. Its effects on cell division, the thickening of the cell wall, the reduction in intercellular communication, and the absence of root hairs confirm that citral is a strong phytotoxic compound, which has persistent effects on root development.

Testing a novel biotechnological passive sampler for monitoring atmospheric PAH pollution.

In this study we evaluated a new type of passive air sampler, the "mossphere" device, filled with a Sphagnum palustre clone. For this purpose, we compared the atmospheric levels of polyaromatic hydrocarbons (PAHs) collected using this device and those collected in conventional bulk deposition and particulate matter (PM10) samplers. All three types of samplers were exposed at 10 sites affected by different levels of pollution and located in two different climate zones. The bulk deposition/ mossphere comparison yielded a greater number of significant regressions with higher coefficients of determination than the PM10/ mossphere comparison. No significant regressions were observed for 3-ring PAHs in either comparison. The mosspheres explain ca. 50% of the variability of the concentrations of 4-, 5- and 6-ring PAHs and total PAHs detected in PM10 and ca. 70% of the corresponding concentrations detected in the bulk deposition. The use of the Sphagnum clone enables standardization of the set-up, thus making the mossphere device a good sampling tool for monitoring 4-, 5- and 6-ring and total PAHs, especially those associated with bulk deposition. The findings indicate the potential usefulness of this innovative technology for mapping PAH levels.

[Assessment of a pharmaceutical interventional programme in patients on medications with renal risk]. / Evaluación de un programa de intervención farmacéutica en pacientes con medicamentos de riesgo renal.

OBJECTIVE: To compare the adaptation of medical prescriptions according to the dosage guides in patients with renal disease, before and after applying a pharmaceutical intervention programme. The secondary objectives were to prepare a guide to dosing in renal disease and to measure the prevalence of prescription of drugs with renal risk. METHOD: Non-randomised, experimental interventional study (before/after) conducted in a general hospital with 800 beds, including hospitalised patients, over the age of 18, with kidney disease and drugs with renal risk prescribed in their pharmacotherapeutic profile. The study was designed to be carried out in two descriptive cross-cutting phases (control group) and a prospective interventional cohort study (intervention group). The primary variable was the percentage non-adaptation according to the stage of renal disease. RESULTS: The study included 185 patients, 88 in the control group and 97 in the intervention group. In the intervention group, the prevalence of non-compliance before and after the intervention was 18.7 % and 2.1 %, representing a statistically significant reduction in non-adaptation of the dose. The costs saved with the pharmaceutical intervention programme were 1,939.63 euro over two months, the average saving per medication intervened amounting to 62.57 euro (CI 95 %, 23.99-101.14 euro; p = 0.02). CONCLUSIONS: The results of the study indicate that the application of a pharmaceutical care model based on the prospective validation of drugs with renal risk, very significantly improved the adaptation of dosing regimens in kidney disease.

Pressurized hot water extraction coupled to solid-phase microextraction-gas chromatography-mass spectrometry for the analysis of polycyclic aromatic hydrocarbons in sediments.

A fully automated, environmentally friendly, simple, and sensitive method was developed for the analysis of polycyclic aromatic hydrocarbons (PAHs) in sediment samples. The procedure is based on pressurized hot water extraction (PHWE) followed by solid-phase microextraction (SPME) and determination by gas chromatography-mass spectrometry. For PHWE, parameters such as organic modifier, percentage of organic modifier, temperature, and static extraction time were studied. For SPME, extraction temperature and time, desorption temperature and time, splitless time, ionic strength adjustments, and effect of an organic modifier were studied. When these parameters were selected, the figures of merit were calculated. The detection and quantification limits were between 0.4-15 microg kg(-1) and 1.2-51 microg kg(-1), respectively. The linearity of the method was assessed spiking sediment samples at seven levels of concentration ranged between 2.5 microg kg(-1) and 500 microg kg(-1) for most of the studied PAHs. The method was validated by two concentration levels reference marine sediment materials (SRM 1944 and SRM 1941b). The obtained results are in very good agreement with the certificate materials. The developed method seems to be suitable for the analysis of PAHs at ultratrace levels in environmental matrices as sediment samples.

Optimisation and validation of a solid-phase microextraction method for simultaneous determination of different types of pesticides in water by gas chromatography-mass spectrometry.

A solid-phase microextraction (SPME) method for the simultaneous determination of a large number of pesticides (46) with a wide range of polarities and chemical structures (organochlorine, organophosphorous, triazines, pyrethroids and others) in water samples by GC-MS has been developed. Three different fibres and parameters that influence the extraction and desorption efficiency were studied. The selected conditions were: a 60 microm polydimethylsiloxane/divinylbenzene (PDMS/DVB) fibre, 45 min of extraction time, sample agitation and temperature control at 60 degrees C; neither pH adjustment nor ionic strength correction were applied. Good detection limits, linearity and repeatability were obtained with this method for the 46 pesticides studied. The method was validated for 29 pesticides following the recommendations of the international norm ISO/IEC 17025 including the calculation of the uncertainties. The detection limits ranged from 4 to 17 ng l(-1). Furthermore, repeatability (6.9-20.5%) and intermediate precision (4.5-19.7%) were shown to be satisfactory. To validate matrix effects for drinking and surface water analytical recoveries were calculated for these matrices. The accuracy of the method was also evaluated by participating in a proficiency inter-laboratory test.

Solid-phase microextraction-gas chromatographic-tandem mass spectrometric analysis of polycyclic aromatic hydrocarbons. Towards the European Union water directive 2006/0129 EC.

This article presents a solid-phase microextraction (SPME) procedure to determine 27 parent and alkylated polycyclic aromatic hydrocarbons (PAHs) with diverging polarities and molecular masses in various types of water samples (tap, well, superficial, and seawater). A 65-microm polydimethylsiloxane-divinylbenzene (PDMS/DVB) fiber was used, and parameters affecting the extraction procedures such as extraction temperature and time, desorption temperature and time, splitless time, effect of an organic modifier or ionic adjustment were studied. The linearity and precision of the proposed method were satisfactory. The use of GC-MS determination in the full scan mode, in the selected ion monitoring (SIM) mode, and in the GC-MS-MS mode provided the unequivocal identification and quantification of the target analytes. Moreover, the proposed SPME-GC-MS-MS method, extracting only 18 ml of sample, reached the very restrictive limits fixed by the 2006/0129 EC proposal for a new water directive to be achieved by 2015. The matrix effects were evaluated through the analysis of tap, well, superficial, and seawater. A discussion on different behaviors, a result of the matrix effects, was included. Analytical recoveries were satisfactory in all cases. The novelty of this work consisted in the wide range of analyzed PAHs, the very low detection limits that were reached, the coupling of SPME-GC-MS-MS, and the study of the matrix effect on water samples. With this developed procedure, detection limits between 0.07 and 0.76 ng l(-1) and quantification limits between 0.10 and 0.98 ng l(-1) were obtained with MS-MS detection. Moreover, the analytical recoveries for different aqueous matrices were near 100% in all cases.

Pressurized liquid extraction of organochlorine pesticides from certified solid materials.

In order to propose a versatile method to use in laboratories devoted to environmental analysis, a scheme for the determination of organochlorine pesticides from different solid matrices is evaluated. Pressurized liquid extraction is chosen as the sample preparation technique, followed by the purification of the extract by means of solid-phase extraction with Envi-carb cartridge (100 m(2)/g). Finally, the extracts are analyzed by programmed temperature vaporization (PTV)-gas chromatography (GC)-electron-capture detection and PTV-GC-mass spectrometry. The suitability of the method for the analysis of different matrices is determined by the extraction and analysis of four certified reference materials of solid matrices: CRM 804-050 (soil), SRM 1941b (organics in marine sediment), SRM 1944 (New York/New Jersey waterway sediment) and SRM 1649a (Urban Dust). Good statistical concordance between the results obtained and the certified or reference concentration are observed for most of the analyzed pesticides.

Plasma membrane depolarization precedes photosynthesis damage and long-term leaf bleaching in (E)-chalcone-treated Arabidopsis shoots.

The plant phenolic compound (E)-chalcone has been previously found to induce noticeable seedling size reduction and progressive de-greening (bleaching) in shoots of Arabidopsis thaliana seedlings. In this work, we demonstrate that this progressive de-greening occurring on Arabidopsis shoots after (E)-chalcone treatment, is directly linked to early plasma membrane depolarization and dramatic effects on chloroplasts structure and function. Later effects in chalcone-treated seedlings included ROS accumulation, pigment degradation, reduced photosynthetic activity, bleaching, and eventually cell death. De-greening and pigment degradation induced by (E)-chalcone were partially reversed when NaCl was added together with chalcone, which could be related to restoration of altered pH gradients. All these results suggest that rapid alteration of plasma membrane potential after chalcone treatment is a major component of the mode of action of (E)-chalcone on Arabidopsis metabolism.

Terpenoid trans-caryophyllene inhibits weed germination and induces plant water status alteration and oxidative damage in adult Arabidopsis.

trans-Caryophyllene (TC) is a sesquiterpene commonly found as volatile component in many different aromatic plants. Although the phytotoxic effects of trans-caryophyllene on seedling growth are relatively explored, not many information is available regarding the phytotoxicity of this sesquiterpenes on weed germination and on adult plants. The phytotoxic potential of TC was assayed in vitro on weed germination and seedling growth to validate its phytotoxic potential on weed species. Moreover, it was assayed on the metabolism of Arabidopsis thaliana adult plants, through two different application ways, spraying and watering, in order to establish the primary affected organ and to deal with the unknown mobility of the compound. The results clearly indicated that TC inhibited both seed germination and root growth, as demonstrated by comparison of the ED50 values. Moreover, although trans-caryophyllene-sprayed adult Arabidopsis plants did not show any effect, trans-caryophyllene-watered plants became strongly affected. The results suggested that root uptake was a key step for the effectiveness of this natural compound and its phytotoxicity on adult plants was mainly due to the alteration of plant water status accompanied by oxidative damage.

Evaluation of HCH isomers and metabolites in soils, leachates, river water and sediments of a highly contaminated area.

In this work we evaluate the contamination caused by HCH residues in the soil, leachates, river water and sediments of an industrial estate of the NW of Spain. We study the distribution of the isomers in the different matrices, analysing 37 soil samples, collected in eight points at several depths, six natural leachates, four river water samples and three river sediments. Soil and leachate samples present very high levels of HCH isomers, higher than the established by legislation, and some pesticides were also detected in the analysed river water whereas no pesticides were detected in the river sediments. The distribution of isomers was different depending on the matrix analysed. Some natural degradation products and also other organochlorine pesticides were detected in the samples analysed.

The plant secondary metabolite citral alters water status and prevents seed formation in Arabidopsis thaliana.

Based on previous results, which showed that the secondary metabolite citral causes disturbances to plant water status, the present study is focused on demonstrating and detailing these effects on the water-related parameters of Arabidopsis thaliana adult plants, and their impact on plant fitness. Clear evidence of effects on water status and fitness were observed: plants treated with 1200 and 2400 µm citral showed decreased RWC, reduced Ψs , increased Ψw and reduced stomatal opening, even 7 days after the beginning of the experiment. Plant protection signals, such as leaf rolling or increased anthocyanin content, were also detected in these plants. In contrast, 14 days after beginning the treatment, treated plants showed signs of citral-related damage. Moreover, the reproductive success of treated plants was critically compromised, with prematurely withered flowers and no silique or seed development. This effect of citral on fitness of adult plants suggests a promising application of this natural compound in weed management by reducing the weed seed bank in the soil.

PAH detection in Quercus robur leaves and Pinus pinaster needles: A fast method for biomonitoring purpose.

Due to the complexity and heterogeneity of plant matrices, new procedure should be standardized for each single biomonitor. Thus, here is described a matrix solid-phase dispersion extraction method, previously used for moss samples, improved and modified for the analyses of PAHs in Quercus robur leaves and Pinus pinaster needles, species widely used in biomonitoring studies across Europe. The improvements compared to the previous procedure are the use of Florisil added with further clean-up sorbents, 10% deactivated silica for pine needles and PSA for oak leaves, being these matrices rich in interfering compounds, as shown by the gas chromatography-mass spectrometry analyses acquired in full scan mode. Good trueness, with values in the range 90-120% for the most of compounds, high precision (intermediate precision between 2% and 12%) and good sensitivity using only 250mg of samples (limits of quantification lower than 3 and 1.5ngg(-1), respectively for pine and oak) were achieved by the selected procedures. These methods proved to be reliable for PAH analyses and, having advantage of fastness, can be used in biomonitoring studies of PAH air contamination.

Peginterferon and ribavirin in patients with HCV cirrhosis after liver transplantation.

The objective of the study was to assess the efficacy of antiviral therapy in patients with hepatitis C virus (HCV) recurrence after liver transplantation (OLT). We included 30 patients of mean age 56 years, who experienced HCV recurrence after OLT. Mean time from OLT to the beginning of therapy was 57 months (median: 43 months). All of them were on monotherapy: tacrolimus (n = 21), cyclosporine (n = 6), and mycophenolate mofetil (n = 3). Fourteen had previously been diagnosed with allograft HCV cirrhosis. Patients were treated with peginterferon alpha 2b (1.5 mug/kg/weekly SC) and ribavirin (10.6 mg/kg/d) for 48 (genotypes 1, 4) or 24 weeks (genotypes 2, 3). After a mean follow-up of 20 months, two patients had died due to biliary sepsis (while on therapy) and acute myocardial infarction (7 months after the end of therapy). End of treatment virological response was achieved in 19 patients (63.3%) and sustained virological response (SUR) in 14 (46.7%). Comparing cirrhotic and noncirrhotic patients, SVR was achieved in seven patients in both groups (50% vs 43.8%; P = .732). Every patient had some adverse event; in 11 patients (36.7%) it was withdrawn (seven cirrhotic and four noncirrhotic; P < .05), and in 12 the starting dose was decreased (40%). There were neither rejection episodes nor cirrhotic complications during therapy, but infections were more common in cirrhotic patients (57% vs 25%; P < .05). In HCV cirrhotic transplanted patients the sustained virological response to combined antiviral therapy was similar to that in noncirrhotic patients, but severe adverse events including infections were much more common.

Therapy of intractable pruritus with MARS.

INTRODUCTION: Pruritus is the most disabling symptom in patients with cholestatic liver diseases. Many drug therapies have been used for the treatment of these diseases, with different outcomes. The molecular adsorbent recirculating system (MARS) has been used in the treatment of intractable pruritus in cholestatic syndromes. We report our experience with MARS in 3 patients with intractable pruritus on the waiting list: 2 liver transplant recipients and a patient with primary biliary cirrhosis. PATIENTS AND RESULTS: Two middle-aged women and 1 middle-aged man, who were recipients of an orthotopic liver transplant for primary biliary cirrhosis, underwent three (n = 2) and two (n = 1) 6-hour sessions of MARS due to medically uncontrollable pruritus. All noted marked improvement of pruritus, with decreased bilirubin levels, but this improvement lasted only a few days in all cases. We observed no changes in transaminase or albumin levels, or prothrombin time. Complications included an episode of angina due to anemia caused by jugular catheter bleeding, and thrombocytopenia in all patients. CONCLUSIONS: MARS is an effective treatment for intractable pruritus in cholestatic liver diseases, although its beneficial effect is short. This extracorporeal liver device is safe, because most related adverse events are mild.

Prevalence of hepatitis C virus genotypes in a Spanish liver transplant unit.

INTRODUCTION: Among the at least six major identified genotypes of HCV, genotype 1b, the one associated with a poorer prognosis, is the most prevalent in Spain. We aimed to compare the distribution of hepatitis C virus genotypes in our liver transplant unit with that of the other HCV patients at our institution (n = 413) in order to assess whether genotype 1b is more prevalent among patients with more severe liver disease. PATIENTS AND METHODS: One hundred eight patients of mean age 56 years included 81 (75%) OLT recipients and 27 (25%) with HCV cirrhosis. Determination of HCV genotypes was made with the Inno-LiPA HCV III. RESULTS: The overall distribution of genotypes was: 1b, 93 patients (86.1%); 1a; eight patients (7.4%); 3, four patients (3.7%); 4; two patients (1.9%), and 2; one patient (0.9%). The distribution was similar among patients with cirrhosis and OLT. Genotype 1b patients were older. Eleven (78.6%) of 14 patients with hepatocellular carcinoma had genotype 1b. In the control group the distribution was: 1b, 287 patients (69.5%); 1a, 54 patients (12.1%); 3, 41 patients (9.9%); 4, 20 patients (4.8%), and genotype 2, 11 patients (2.7%). This differences in the distribution of genotypes between our population and the control group was statistically significant (P < .001). CONCLUSIONS: Genotype 1b, the most prevalent genotype in our liver transplant unit, included older patients in whom hepatocellular carcinoma was common, perhaps due to their higher prevalence of cirrhosis.

Effectiveness of pegylated interferon and ribavirin in patients with liver HCV cirrhosis.

Clearance of HCV before transplantation could avoid recurrence of hepatitis C in the liver allograft, thereby improving graft and patient survival. We report our experience with combined therapy for patients with HCV cirrhosis, including 12 patients with biopsy-proven liver cirrhosis (n = 7) or previous cirrhotic complications (n = 5). The Child-Pugh score was A in eight patients and B in four. Two patients had hepatocellular carcinoma. Genotype distribution was 1a (n = 2), 1b (n = 8) or 3 (n = 1). Patients received peginterferon alpha2b (1.5 microg/kg once weekly) and ribavirin (10.6 g/kg per day) for 48 weeks (genotype 1) or 24 weeks (genotype 3). Twenty-one months after beginning therapy all the patients remained alive; three have undergone liver transplantation. In one patient treatment was discontinued after 2 months due to cachexia. End-of-treatment virologic response was achieved in five patients (41.7%) and sustained virologic response in three patients (25%). Patients who cleared the virus had negative PCR 4 weeks after beginning therapy. All patients had adverse events. The most common clinical events were asthenia, weight loss, fever, and anorexia. Infectious complications resolved in three patients (25%). Hematologic events were common. Seven of 11 patients (63.6%) who completed therapy required dose reduction. We conclude that therapy with peginterferon and ribavirin in patients with HCV cirrhosis has a similar effectiveness to previous treatments. A virologic response 1 month after the beginning of therapy could be a main predictor of a sustained response.

Efficacy of hepatocellular carcinoma locoregional therapies on patients waiting for liver transplantation.

The aim of this study was to evaluate the efficacy of different locoregional therapies in patients with HCC on the waiting list for liver transplantation. From October 2001 to July 2003, 13 patients, all men, with HCC diagnosed by cytology, were transplanted at our center. Locoregional therapies were percutaneous ethanol injection (PEI), transcatheter hepatic arterial chemoembolization (TACE), and radiofrequency microwave ablation (RFA). PEI was employed in seven patients, TACE in five (one of them associated with PEI) and RFA in one. Efficacy was evaluated by determining the percentage of tumoral necrosis in the liver explant. Five tumors were T4, four T3, three T2, and one T1. Ten were well differentiated, two moderately differentiated, and one undifferentiated. One patient died due to primary graft malfunction. After a median posttransplant follow-up of 15 months, 12 patients are alive with no sign of tumor recurrence. Most patients with solitary nodules <4 cm who received PEI had 90% to 100% tumor necrosis. Larger tumors had 25% to 30% necrosis. TACE was employed in six patients with large and/or multiple tumors, obtaining 20% to 50% tumor necrosis. RFA was employed in one case obtaining 85% necrosis (tumor of 4 cm). No serious complications occurred with any technique. According to our experience, PEI and RFA are effective locoregional therapies to treat hepatocellular carcinomas of <4 cm in patients on the waiting list. For larger tumors, their association with other techniques, such as TACE, seems adequate.

Cholinergic agents structurally related to furtrethonium. 2. Synthesis and antimuscarinic activity of a series of N-[5-[(1'-substituted-acetoxy) methyl]-2-furfuryl]dialkylamines.

In the first part of this study, devoted to the discovery of selective antimuscarinic agents, (+/-)- N-[5-[(1'-phenyl-1'-cyclohexylacetoxy)methyl]-2-furfuryl]dimeth yla mine (5a) proved to be at least 20 times more potent in the rat ileum and bladder than in guinea pig atria. Several (+/-)-N- [5-[(1'-substituted-acetoxy)methyl]-2-furfuryl]dialkylamine analogs of 5a were subsequently prepared. This involved exploration of the tertiary nitrogen substituents and modulation of the lipophilic side chain at position 5 of the furan ring, using the Hansch approach. A QSAR study was conducted to correlate activity with physicochemical properties of substituents. The possibility of describing all compounds in a single model indicates that variations of nitrogen and the lipophilic side chain contribute independently to activity. Compounds 5b, c,j, with bulky lipophilic substituents at the tertiary nitrogen, showed unprecedented selectivity between the two smooth muscle tissues, their antimuscarinic potency being from 10 to 90 times higher in the ileum than in the bladder. It is suggested that their interesting tissue selectivity is probably related to nonspecific phenomena involving the receptor environment, rather than real differences between the muscarinic receptors in the two tissues.

Synthesis and pharmacological investigation of the enantiomers of muscarone and allomuscarone.

A strategy based on the use of (R)- and (S)-lactic ester as starting materials allowed the synthesis of the two enantiomers of muscarone [(-)-1 and (+)-1] and allomuscarone [(-)-5 and (+)-5] in greater than 98% enantiomeric excess. The compounds were examined for their ability to bind to membranes from cerebral cortex (M1), heart (M2), and salivary glands (M3) and to recognize affinity agonist states of the muscarinic receptors. The two pairs of enantiomers were also tested in five functional assays, and their muscarinic potency was determined. In both binding and functional tests, (-)-1 (2S,5S) and (-)-5 (2R,5S) were the eutomers of muscarone and allomuscarone, respectively. The eudismic ratio of muscarone, evaluated in the functional tests, spanned a range of 280-440. These values are substantially different from ones (2.4-10.1) reported in the literature. From a stereochemical point of view, muscarone behaves as muscarine and all other major muscarinic agonists; as a consequence, the hypotheses advanced to account for the anomalies of muscarone no longer have reason to exist.