Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

Transferència d’electrò i protó als intermediaris de reacció de les hemo-catalases

Projecte de recerca elaborat a partir d’una estada a l’University of Pennsylvania, EUA, entre els mesos d’agosta a desembre del 2006. Les hemo-catalases són enzims que protegeixen les cèl•lules dels efectes tòxics del peròxid d'hidrogen. Aquesta reacció té lloc en dues etapes, via l'intermediari Compost I (Cpd I). Tanmateix, el Compost I pot seguir una reacció secundària, a través de l'intermediari Compost II. Hi ha dos tipus d'hemo-catalases: les hemo-b (com la d'Helicobacter pylori, HPC) i les hemo-d catalases (com la de Penicillium vitale, PVC). Experimentalment s'observa que les hemo-b catalases formen Cpd II més fàcilment que les hemo-d. La formació del Cpd II consta de dos processos: la reducció del catió radical porfirínic i la protonació del grup oxoferril. Durant l'estada, es va estudiar el procès de transferència electrònica a la porfirina utilitzant una metodologia desenvolupada recentment. Els resultats mostren que per PVC la reducció és més fàcil que per HPC. Posteriorment hem realitzat una sèrie de optimitzacions de geometria CPMD QM/MM al llarg del camí per la transferència de protó (PT) de la histidina distal a l'oxoferril. Mentre que per HPC aquesta PT és espontània, per PVC l'isòmer hidroxoferrílic és menys estable que el catió imidazoli.

Combining classification techniques to define topo-climatic landscapes

Landscape classification tackles issues related to the representation and analysis of continuous and variable ecological data. In this study, a methodology is created in order to define topo-climatic landscapes (TCL) in the north-west of Catalonia (north-east of the Iberian Peninsula). TCLs relate the ecological behaviour of a landscape in terms of topography, physiognomy and climate, which compound the main drivers of an ecosystem. Selected variables are derived from different sources such as remote sensing and climatic atlas. The proposed methodology combines unsupervised interative cluster classification with a supervised fuzzy classification. As a result, 28 TCLs have been found for the study area which may be differentiated in terms of vegetation physiognomy and vegetation altitudinal range type. Furthermore a hierarchy among TCLs is set, enabling the merging of clusters and allowing for changes of scale. Through the topo-climatic landscape map, managers may identify patches with similar environmental conditions and asses at the same time the uncertainty involved.

Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family.

NR2E3, a photoreceptor-specific nuclear receptor (PNR), represses cone-specific genes and activates several rod-specific genes. In humans, mutations in NR2E3 have been associated with the recessively-inherited enhanced short-wavelength sensitive S-cone syndrome (ESCS) and, recently, with autosomal dominant (ad) retinitis pigmentosa (RP) (adRP). In the present work, we describe two additional families affected by adRP that carry a heterozygous c.166G>A (p.G56R) mutation in the NR2E3 gene. Functional analysis determined the dominant negative activity of the p.G56R mutant protein as the molecular mechanism of adRP. Interestingly, in one pedigree, the most common causal variant for ESCS (p.R311Q) cosegregated with the adRP-linked p.G56R mutation, and the compound heterozygotes exhibited an ESCS-like phenotype, which in 1 of the 2 cases was strikingly "milder" than the patients carrying the p.G56R mutation alone. Impaired repression of cone-specific genes by the corepressors atrophin-1 (dentatorubral-pallidoluysian atrophy [DRPLA] gene product) and atrophin-2 (arginine-glutamic acid dipeptide repeat [RERE] protein) appeared to be a molecular mechanism mediating the beneficial effect of the p.R311Q mutation. Finally, the functional dominance of the p.R311Q variant to the p.G56R mutation is discussed.

Lack of evidence of the interaction of the Aß peptide with the Wnt signaling cascade in Drosophila models of Alzheimer's disease.

Background Alzheimer's disease (AD) is the leading form of dementia worldwide. The Aß-peptide is believed to be the major pathogenic compound of the disease. Since several years it is hypothesized that Aß impacts the Wnt signaling cascade and therefore activation of this signaling pathway is proposed to rescue the neurotoxic effect of Aß. Findings Expression of the human Aß42 in the Drosophila nervous system leads to a drastically shortened life span. We found that the action of Aß42 specifically in the glutamatergic motoneurons is responsible for the reduced survival. However, we find that the morphology of the glutamatergic larval neuromuscular junctions, which are widely used as the model for mammalian central nervous system synapses, is not affected by Aß42 expression. We furthermore demonstrate that genetic activation of the Wnt signal transduction pathway in the nervous system is not able to rescue the shortened life span or a rough eye phenotype in Drosophila. Conclusions Our data confirm that the life span is a useful readout of Aß42 induced neurotoxicity in Drosophila; the neuromuscular junction seems however not to be an appropriate model to study AD in flies. Additionally, our results challenge the hypothesis that Wnt signaling might be implicated in Aß42 toxicity and might serve as a drug target against AD.

A new chemical tool (C0036E08) supports the role of adenosine A(2B) receptors in mediating human mast cell activation.

Asthma is a chronic inflammatory disease of the airways that involves many cell types, amongst which mast cells are known to be important. Adenosine, a potent bronchoconstricting agent, exerts its ability to modulate adenosine receptors of mast cells thereby potentiating derived mediator release, histamine being one of the first mediators to be released. The heterogeneity of sources of mast cells and the lack of highly potent ligands selective for the different adenosine receptor subtypes have been important hurdles in this area of research. In the present study we describe compound C0036E08, a novel ligand that has high affinity (pK(i) 8.46) for adenosine A(2B) receptors, being 9 times, 1412 times and 3090 times more selective for A(2B) receptors than for A(1), A(2A) and A(3) receptors, respectively. Compound C0036E08 showed antagonist activity at recombinant and native adenosine receptors, and it was able to fully block NECA-induced histamine release in freshly isolated mast cells from human bronchoalveolar fluid. C0036E08 has been shown to be a valuable tool for the identification of adenosine A(2B) receptors as the adenosine receptors responsible for the NECA-induced response in human mast cells. Considering the increasing interest of A(2B) receptors as a therapeutic target in asthma, this chemical tool might provide a base for the development of new anti-asthmatic drugs.

The endodermis--development and differentiation of the plant's inner skin.

Controlling external compound entrance is essential for plant survival. To set up an efficient and selective sorting of nutrients, free diffusion via the apoplast in vascular plants is blocked at the level of the endodermis. Although we have learned a lot about endodermal specification in the last years, information regarding its differentiation is still very limited. A differentiated endodermal cell can be defined by the presence of the "Casparian strip" (CS), a cell wall modification described first by Robert Caspary in 1865. While the anatomical description of CS in many vascular plants has been very detailed, we still lack molecular information about the establishment of the Casparian strips and their actual function in roots. The recent isolation of a novel protein family, the CASPs, that localizes precisely to a domain of the plasma membrane underneath the CS represents an excellent point of entry to explore CS function and formation. In addition, it has been shown that the endodermis contains transporters that are localized to either the central (stele-facing) or peripheral (soil-facing) plasma membranes. These features suggest that the endodermis functions as a polar plant epithelium.

Treatment of essential hypertension with calcium channel blockers: what is the place of lercanidipine?

In all actual clinical guidelines, dihydropyridine calcium channel blockers (CCBs) belong to the recommended first line antihypertensive drugs to treat essential hypertension. Several recent large clinical trials have confirmed their efficacy not only in lowering blood pressure but also in reducing cardiovascular morbidity and mortality in hypertensive patients with a normal or high cardiovascular risk profile. In clinical trials such as ALLHAT, VALUE or ASCOT, an amlodipine-based therapy was at least as effective, when not slightly superior, in lowering blood pressure and sometimes more effective in preventing target organ damages than blood pressure lowering strategies based on the use of diuretics, beta-blockers and blockers of the renin-angiotensin system. One of the main clinical side effects of the first and second generation CCBs including amlodipine is the development of peripheral edema. The incidence of leg edema can be markedly reduced by combining the CCB with a blocker of the renin-angiotensin system. This strategy has now led to the development of several fixed-dose combinations of amlodipine and angiotensin II receptor antagonists. Another alternative to lower the incidence of edema is to use CCBs of the third generation such as lercanidipine. Indeed, although no major clinical trials have been conducted with this compound, clinical studies have shown that lercanidipine and amlodipine have a comparable antihypertensive efficacy but with significantly less peripheral edema in patients receiving lercanidipine. In some countries, lercanidipine is now available in a single-pill association with an ACE inhibitor thereby further improving its efficacy and tolerability profile.

Solid-phase synthesis and NMR studies of modified oligonucleotides forming triplex helix and of oligonucleopeptides mimicking the topoisomerase I-DNA covalent complex

Report for the scientific sojourn carried out at the Institut de Biologia Molecular de Barcelona of the CSIC –state agency – from april until september 2007. Topoisomerase I is an essential nuclear enzyme that modulates the topological status of DNA, facilitating DNA helix unwinding during replication and transcription. We have prepared the oligonucleotide-peptide conjugate Ac-NLeu-Asn-Tyr(p-3’TTCAGAAGC5’)-LeuC-CONH-(CH2)6-OH as model compound for NMR studies of the Topoisomerase I- DNA complex. Special attention was made on the synthetic aspects for the preparation of this challenging compound especially solid supports and protecting groups. The desired peptide was obtained although we did not achieve the amount of the conjugate needed for NMR studies. Most probably the low yield is due to the intrinsic sensitive to hydrolysis of the phosphate bond between oligonucleotide and tyrosine. We have started the synthesis and the structural characterization of oligonucleotides carrying intercalating compounds. At the present state we have obtained model duplex and quadruplex sequences modified with acridine and NMR studies are underway. In addition to this project we have successfully resolved the structure of a fusion peptide derived from hepatitis C virus envelope synthesized by the group of Dr. Haro and we have synthesized and started the characterization of a modified G-quadruplex.

Rhinitis due to acrylates : case report

Background: Acrylates and methacrylates (salts and esters of acrylic and metacrylic acid respectively), are monomers commonly found in polymer plastics, resins and glues, and are widely used in many industry sectors. The first adverse health effects described were skin reactions and asthma. Exposure to acrylates, for instance when using multicomponent glues, is now a well known cause of occupational asthma. Methods: We report the case of a rhinitis - and possible asthma - to acrylates, in a 38-year-old woman, working in a nail beauty salon. She was currently treated for hypertension, and otherwise known for obesity and seasonal rhinoconjunctivitis, but did not have any respiratory problem. Two years after starting this activity, she progressively started to complain of anosmia, rhinitis, and intermittent dyspnea. Her job consisted in decorating nails with a mixture of a polymer powder and a liquid monomer, after removing the previous artificial nail with a small sander. We assessed exposure to acrylates at her working place, both as dust (from sanded nails) and volatile compound (from the mixture described above), and she was asked to measure her peak flow values twice a day for ten days, in order to detect a possible relationship between her occupational activities, the symptoms and the peak flow values. Results: Measures made during the visit of the patient's place of work showed that the existing aspiration system was efficient for eliminating the dust produced by nail sanding, but not for eliminating the volatile components. Thus, occupational exposure to acrylates was demonstrated. Moreover, the peak flow measures showed an average decrease of almost 10 percent when the patient was at work, compared to when she stayed home. We concluded that she actually suffered from professional rhinitis and, possibly, professional asthma (not certain because of the limited number of peak flow measures per day). Conclusion: Although exposure to acrylates is a well known cause of occupational asthma, it should be emphasized that the exact mechanisms of action remain unknown, despite the abundant literature about it. Some professions, which tend to be more frequent nowadays (such as working in a nail beauty salon), can expose the worker to particular risks. This highlights the need of always inquiring not only about the profession, but also the related activities, when facing a case of suspected asthma.

Pharmacokinetic profile of tert-butylaminoethanethiol

A preliminary study of the pharmacokinetic parameters of t-Butylaminoethanethiol (TBAESH) was performed after administration of a single dose (35 mg/kg) either orally or intravenously. Plasma or blood samples were treated with dithiothreitol, perchloric acid and, after filtration, submitted to further purification with anionic resin. In the final step the drug was retained on a cationic resin column, eluted with NaCl lM and detected according to the method of Ellman (1958). The results suggested a pharmacokinetic behavior related to a one open compartment model with the following values for the total drug: area under the intravenous curve (AUC i.v.): 443(+ ou -) 24.0; AUC oral: 85.5(+ ou -) 14.5 ug min.ml(elevado a -1); elimination rate constant: 0.069(+ ou -) 0.0055 min(elevado a -1), biological half-life: 10.0(+ ou -) 0.80 min; distribution volume 1.15(+ ou -) 0.15 ml/g; biodisponibility: 0.19(+ ou -) 0.02. From a pharmacokinetic standpoint, TBAESH seems to have no advantage over the analogous disulfide compound.

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

β-Glucan antigenemia assay for the diagnosis of invasive fungal infections in patients with hematological malignancies: a systematic review and meta-analysis of cohort studies from the Third European Conference on Infections in Leukemia (ECIL-3).

BACKGROUND: Invasive fungal infections (IFIs) are life-threatening complications in patients with hemato-oncological malignancies, and early diagnosis is crucial for outcome. The compound 1,3-β-D-glucan (BG), a cell wall component of most fungal species, can be detected in blood during IFI. Four commercial BG antigenemia assays are available (Fungitell, Fungitec-G, Wako, and Maruha). This meta-analysis from the Third European Conference on Infections in Leukemia (ECIL-3) assessed the performance of BG assays for the diagnosis of IFI in hemato-oncological patients. METHODS: Studies reporting the performance of BG antigenemia assays for the diagnosis of IFI (European Organization for Research and Treatment of Cancer and Mycoses Study Group criteria) in hemato-oncological patients were identified. The analysis was focused on high-quality cohort studies with exclusion of case-control studies. Meta-analysis was performed by conventional meta-analytical pooling and bivariate analysis. RESULTS: Six cohort studies were included (1771 adult patients with 414 IFIs of which 215 were proven or probable). Similar performance was observed among the different BG assays. For the cutoff recommended by the manufacturer, the diagnostic performance of the BG assay in proven or probable IFI was better with 2 consecutive positive test results (diagnostic odds ratio for 2 consecutive vs one single positive results, 111.8 [95% confidence interval {CI}, 38.6-324.1] vs 16.3 [95% CI, 6.5-40.8], respectively; heterogeneity index for 2 consecutive vs one single positive results, 0% vs 72.6%, respectively). For 2 consecutive tests, sensitivity and specificity were 49.6% (95% CI, 34.0%-65.3%) and 98.9% (95% CI, 97.4%-99.5%), respectively. Estimated positive and negative predictive values for an IFI prevalence of 10% were 83.5% and 94.6%, respectively. CONCLUSIONS: Different BG assays have similar accuracy for the diagnosis of IFI in hemato-oncological patients. Two consecutive positive antigenemia assays have very high specificity, positive predictive value, and negative predictive value. Because sensitivity is low, the test needs to be combined with clinical, radiological, and microbiological findings.

Exome sequencing extends the phenotypic spectrum for ABHD12 mutations: from syndromic to nonsyndromic retinal degeneration.

OBJECTIVE: To identify the genetic causes underlying autosomal recessive retinitis pigmentosa (arRP) and to describe the associated phenotype. DESIGN: Case series. PARTICIPANTS: Three hundred forty-seven unrelated families affected by arRP and 33 unrelated families affected by retinitis pigmentosa (RP) plus noncongenital and progressive hearing loss, ataxia, or both, respectively. METHODS: A whole exome sequencing (WES) analysis was performed in 2 families segregating arRP. A mutational screening was performed in 378 additional unrelated families for the exon-intron boundaries of the ABHD12 gene. To establish a genotype-phenotype correlation, individuals who were homozygous or compound heterozygotes of mutations in ABHD12 underwent exhaustive clinical examinations by ophthalmologists, neurologists, and otologists. MAIN OUTCOME MEASURES: DNA sequence variants, best-corrected visual acuity, visual field assessments, electroretinogram responses, magnetic resonance imaging, and audiography. RESULTS: After a WES analysis, we identified 4 new mutations (p.Arg107Glufs*8, p.Trp159*, p.Arg186Pro, and p.Thr202Ile) in ABHD12 in 2 families (RP-1292 and W08-1833) previously diagnosed with nonsyndromic arRP, which cosegregated with the disease among the family members. Another homozygous mutation (p.His372Gln) was detected in 1 affected individual (RP-1487) from a cohort of 378 unrelated arRP and syndromic RP patients. After exhaustive clinical examinations by neurologists and otologists, the 4 affected members of the RP-1292 had no polyneuropathy or ataxia, and the sensorineural hearing loss and cataract were attributed to age or the normal course of the RP, whereas the affected members of the families W08-1833 and RP-1487 showed clearly symptoms associated with polyneuropathy, hearing loss, cerebellar ataxia, RP, and early-onset cataract (PHARC) syndrome. CONCLUSIONS: Null mutations in the ABHD12 gene lead to PHARC syndrome, a neurodegenerative disease including polyneuropathy, hearing loss, cerebellar ataxia, RP, and early-onset cataract. Our study allowed us to report 5 new mutations in ABHD12. This is the first time missense mutations have been described for this gene. Furthermore, these findings are expanding the spectrum of phenotypes associated with ABHD12 mutations ranging from PHARC syndrome to a nonsyndromic form of retinal degeneration.

Bioactivity of neolignans from fructus Schizandrae

Fructus Schisandrae sinensis Baill, a traditional Chinese medicine, used as tonic and sedative, has been shown at the beginning of 70's to lower the elevated serum glutamic-pyruvic transaminase (SGPT) levels of patients suffering from chronic viral hepatitis. Durign past 20 years, a series of neolignans have been isolated and identified as effective principles. Pharmacological studies revealed that they increased liver protein and glicogen synthesis, antagonized liver injuries from CCl4 and thioacetamide. The mechanism of SGPT lowering was considered as a hepato-protective and membrane stabilize action, although inhibition of the activity of liver GPT may also be existed. It was found that some principles of Schisandrae have an inducing effect on hepatic microsomal drug-metabolizing enzynme system P-450, thus explained their anti-toxic, anti-carcinogenic and anti-mutagenic effects. A synthetic derivative compound of Schisandrin called DDB has most of the above mentioned actions now used widely in Chine as a hepato-protective drug with high effectiveness in normalizing liver functions and very low side effects. From Schisandrin to synthesized DDB, pointed out a successful way in the development of new drugs from natural products.