Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism.

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4 x 10(-8) M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

21st century climate change threatens mountain flora unequally across Europe

Continental-scale assessments of 21st century global impacts of climate change on biodiversity have forecasted range contractions for many species. These coarse resolution studies are, however, of limited relevance for projecting risks to biodiversity in mountain systems, where pronounced microclimatic variation could allow species to persist locally, and are ill-suited for assessment of species-specific threat in particular regions. Here, we assess the impacts of climate change on 2632 plant species across all major European mountain ranges, using high-resolution (ca. 100 m) species samples and data expressing four future climate scenarios. Projected habitat loss is greater for species distributed at higher elevations; depending on the climate scenario, we find 36-55% of alpine species, 31-51% of subalpine species and 19-46% of montane species lose more than 80% of their suitable habitat by 2070-2100. While our high-resolution analyses consistently indicate marked levels of threat to cold-adapted mountain florae across Europe, they also reveal unequal distribution of this threat across the various mountain ranges. Impacts on florae from regions projected to undergo increased warming accompanied by decreased precipitation, such as the Pyrenees and the Eastern Austrian Alps, will likely be greater than on florae in regions where the increase in temperature is less pronounced and rainfall increases concomitantly, such as in the Norwegian Scandes and the Scottish Highlands. This suggests that change in precipitation, not only warming, plays an important role in determining the potential impacts of climate change on vegetation.

Geochemistry of highly acidic mine water following disposal into a natural lake with carbonate bedrock

Acid mine drainage (AMD) from the Zn-Pb(-Ag-Bi-Cu) deposit of Cerro de Pasco (Central Peru) and waste water from a Cu-extraction plant has been discharged since 1981 into Lake Yanamate, a natural lake with carbonate bedrock. The lake has developed a highly acidic pH of similar to 1. Mean lake water chemistry was characterized by 16,775 mg/L acidity as CaCO(3), 4330 mg/L Fe and 29,250 mg/L SO(4). Mean trace element concentrations were 86.8 mg/L Cu, 493 mg/L Zn, 2.9 mg/L Pb and 48 mg/L As, which did not differ greatly from the discharged AMD. Most elements showed increasing concentrations from the surface to the lake bottom at a maximal depth of 41 m (e.g. from 3581 to 5433 mg/L Fe and 25,609 to 35,959 mg/L SO(4)). The variations in the H and 0 isotope compositions and the element concentrations within the upper 10 m of the water column suggest mixing with recently discharged AMD, shallow groundwater and precipitation waters. Below 15 m a stagnant zone had developed. Gypsum (saturation index, SI similar to 0.25) and anglesite (SI similar to 0.1) were in equilibrium with lake water. Jarosite was oversaturated (SI similar to 1.7) in the upper part of the water column, resulting in downward settling and re-dissolution in the lower part of the water column (SI similar to -0.7). Accordingly, jarosite was only found in sediments from less than 7 m water depth. At the lake bottom, a layer of gel-like material (similar to 90 wt.% water) of pH similar to 1 with a total organic C content of up to 4.40 wet wt.% originated from the kerosene discharge of the Cu-extraction plant and had contaminant element concentrations similar to the lake water. Below the organic layer followed a layer of gypsum with pH 1.5, which overlaid the dissolving carbonate sediments of pH 5.3-7. In these two layers the contaminant elements were enriched compared to lake water in the sequence As < Pb approximate to Cu < Cd < Zn = Mn with increasing depth. This sequence of enrichment was explained by the following processes: (i) adsorption of As on Fe-hydroxides coating plant roots at low pH (up to 3326 mg/kg As), (ii) adsorption at increasing pH near the gypsum/calcite boundary (up to 1812 mg/kg Pb, 2531 mg/kg Cu. and 36 mg/kg Cd), and (iii) precipitation of carbonates (up to 5177 mg/kg Zn and 810 mg/kg Mn: all data corrected to a wet base). The infiltration rate was approximately equal to the discharge rate, thus gypsum and hydroxide precipitation had not resulted in complete clogging of the lake bedrocks. (C) 2010 Elsevier Ltd. All rights reserved.

Carbon dioxide in scree slope deposits: A pathway from atmosphere to pedogenic carbonate

A continuum of carbon, from atmospheric CO2 to secondary calcium carbonate, has been studied in a soil associ- ated with scree slope deposits in the Jura Mountains of Switzerland. This approach is based on former studies conducted in other environments. This C continuum includes atmospheric CO2, soil organic matter (SOM), soil CO2, dissolved inorganic carbon (DIC) in soil solutions, and secondary pedogenic carbonate. Soil parameters (pCO2, temperature, pH, Cmin and Corg contents), soil solution chemistry, and isotopic compositions of soil CO2, DIC, carbonate and soil organic matter (δ13CCO2, δ13CDIC, δ13Ccar and δ13CSOM values) have been monitored at different depths (from 20 to 140 cm) over one year. Results demonstrated that the carbon source in secondary carbonate (mainly needle fiber calcite) is related to the dissolved inorganic carbon, which is strongly dependent on soil respiration. The heterotrophic respiration, rather than the limestone parent material, seems to control the pedogenic carbon cycle. The correlation of δ13Corg values with Rock-Eval HI and OI indices demonstrates that, in a soil associated to scree slope deposits, the main process responsible for 13C-enrichment in SOM is related to bac- terial oxidative decarboxylation. Finally, precipitation of secondary calcium carbonate is enhanced by changes in soil pCO2 associated to the convective movement of air masses induced by temperature gradients (heat pump effect) in the highly porous scree slope deposits. The exportation of soil C-leachates from systems such as the one studied in this paper could partially explain the "gap in the European carbon budget" reported by recent studies.

Nuclear Factor I genomic binding associates with chromatin boundaries.

BACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.

Multiplex ultra-performance liquid chromatography-tandem mass spectrometry method for simultaneous quantification in human plasma of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, anidulafungin, and caspofungin.

Therapeutic drug monitoring (TDM) may contribute to optimizing the efficacy and safety of antifungal therapy because of the large variability in drug pharmacokinetics. Rapid, sensitive, and selective laboratory methods are needed for efficient TDM. Quantification of several antifungals in a single analytical run may best fulfill these requirements. We therefore developed a multiplex ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method requiring 100 μl of plasma for simultaneous quantification within 7 min of fluconazole, itraconazole, hydroxyitraconazole, posaconazole, voriconazole, voriconazole-N-oxide, caspofungin, and anidulafungin. Protein precipitation with acetonitrile was used in a single extraction procedure for eight analytes. After reverse-phase chromatographic separation, antifungals were quantified by electrospray ionization-triple-quadrupole mass spectrometry by selected reaction monitoring detection using the positive mode. Deuterated isotopic compounds of azole antifungals were used as internal standards. The method was validated based on FDA recommendations, including assessment of extraction yields, matrix effect variability (<9.2%), and analytical recovery (80.1 to 107%). The method is sensitive (lower limits of azole quantification, 0.01 to 0.1 μg/ml; those of echinocandin quantification, 0.06 to 0.1 μg/ml), accurate (intra- and interassay biases of -9.9 to +5% and -4.0 to +8.8%, respectively), and precise (intra- and interassay coefficients of variation of 1.2 to 11.1% and 1.2 to 8.9%, respectively) over clinical concentration ranges (upper limits of quantification, 5 to 50 μg/ml). Thus, we developed a simple, rapid, and robust multiplex UPLC-MS/MS assay for simultaneous quantification of plasma concentrations of six antifungals and two metabolites. This offers, by optimized and cost-effective lab resource utilization, an efficient tool for daily routine TDM aimed at maximizing the real-time efficacy and safety of different recommended single-drug antifungal regimens and combination salvage therapies, as well as a tool for clinical research.

Threats from climate change to terrestrial vertebrate hotspots in europe.

We identified hotspots of terrestrial vertebrate species diversity in Europe and adjacent islands. Moreover, we assessed the extent to which by the end of the 21(st) century such hotspots will be exposed to average monthly temperature and precipitation patterns which can be regarded as extreme if compared to the climate experienced during 1950-2000. In particular, we considered the entire European sub-continent plus Turkey and a total of 1149 species of terrestrial vertebrates. For each species, we developed species-specific expert-based distribution models (validated against field data) which we used to calculate species richness maps for mammals, breeding birds, amphibians, and reptiles. Considering four global circulation model outputs and three emission scenarios, we generated an index of risk of exposure to extreme climates, and we used a bivariate local Moran's I to identify the areas with a significant association between hotspots of diversity and high risk of exposure to extreme climates. Our results outline that the Mediterranean basin represents both an important hotspot for biodiversity and especially for threatened species for all taxa. In particular, the Iberian and Italian peninsulas host particularly high species richness as measured over all groups, while the eastern Mediterranean basin is particularly rich in amphibians and reptiles; the islands (both Macaronesian and Mediterranean) host the highest richness of threatened species for all taxa occurs. Our results suggest that the main hotspots of biodiversity for terrestrial vertebrates may be extensively influenced by the climate change projected to occur over the coming decades, especially in the Mediterranean bioregion, posing serious concerns for biodiversity conservation.

Superoxide dismutase from Trichuris ovis, inhibiton by benzimidazoles and pyrimidine derivatives

Three superoxide dismutase isoenzymes of different cellular location were detected in an homogenate of Thrichuris ovis. Each of these molecular forms was purified by differential centrifugation and precipitation with ammonium sulphate, followed by chromatography on DEAE-cellulose and Sephadex G-75 columns. The activity levels of the two molecular forms detected in the mitochondrial (one cyanide sensitive Cu-Zn-SOD and the other cyanide intensitive Mn-Sod were higher than that of the superoxide dismutase detected in the cytoplasmic fraction (cyanid sensitive Cu-Zn-SOD). All the mollecular forms present evident differences to the SODs contained in the host liver. Molecular mass and some of the physical and chemical aproperties of the enzyme was determined for all three molecular forms. An inhibitory effect on the SOD of the parasite an the host was detected with a series of compounds, some of wich markedly inhibited parasite ensyme but not host enzyme.

Anells de Liesegang (experimentació amb gels)

Treball de recerca realitzat per un alumne d'ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l'any 2009. Aquest treball de recerca es basa en l'experimentació i, posteriorment, l'obtenció i anàlisi de resultats de l'experiment creador d'anells de Liesegang. Aquest experiment, consistent en la precipitació d'un compost en una base gelificada formant anells distanciats logarítmicament els uns dels altres, ha estat durant més d'un segle objecte d'investigació de moltíssims científics, els quals no han sabut mai treure'n una explicació lògica i raonable d'aquest rar comportament. L'autor ha pretès recrear els curiosos anells intentant formar-los amb diferents inhibidors i compostos als trobats en la bibliografia. Després de realitzar més d'una trentena d'experiments, s'ha realitzat una anàlisi exhaustiva dels resultats. Aquest apartat ha estat un dels més enriquidors, ja que s'han dut a terme en ell comparacions sorprenents i troballes molt curioses, com per exemple la similitud entre els anells de Liesegang i les estructures de Turing, la qual intenta explicar les formes presents en els ocels dels éssers vius; i l'aparició d'anells de Liesegang segons l’òptica visual, efecte inexistent en l’àmplia bibliografia consultada. A més a més, també s'han efectuat una sèrie d'estudis: un en què es confirmen les distàncies logarítmiques entre els anells i on es realitza una comparació entre les dades empíriques i el patró matemàtic; i un altre en què s'estudia el comportament dels anells al variar els factors que regulen la velocitat de reacció.

Effects of increased drought and selective thinning on growth and resprouting of two coexisting Mediterranean oaks with contrasting leaf habit (Quercus ilex and Quercus cerrioides)

According to the IPCC (2007), the Mediterranean basin is expected to suffer important changes in temperature and precipitation in the next few decades, leading the climate warmer and dryer. Therefore, it is necessary to determine the possible effects of increased drought on species with different structural and physiological traits, to be able to predict possible changes in the structure and composition of Mediterranean forests. Moreover, it will be necessary to assess whether traditional management practices can mitigate the effects of climate change on these forests. The main aim of this study has been to analyze the effects of increased drought on the mortality, growth and resprouting patterns of two co-occurring Mediterranean oak species with contrasting leaf habit (the evergreen Quercus ilex and the winter-deciduous Quercus cerrioides), and to assess the effects of selective thinning on their response to increased drought. Our results show a differential effect of increased drought between species: no differences were observed in the growth of Q. ilex while Q. cerrioides reduced its growth under increased drought conditions. Selective thinning reduced the negative effects of increased drought on tree growth, although this beneficial effect tended to decrease during the experiment. Our results show that increasing aridity in Mediterranean areas can be a constraining factor for deciduous oaks, thus potentially causing their decline in mixed forests and favouring their substitution by the evergreen congeneric species. However, as seen in this study, management can strongly encourage growth both for deciduous and evergreen species, thus reversing the effects of increased water stress on Mediterranean coppices.

Turning sunlight into stone: the oxalate-carbonate pathway in a tropical tree ecosystem.

An African oxalogenic tree, the iroko tree (Milicia excelsa), has the property to enhance carbonate precipitation in tropical oxisols, where such accumulations are not expected due to the acidic conditions in these types of soils. This uncommon process is linked to the oxalate-carbonate pathway, which increases soil pH through oxalate oxidation. In order to investigate the oxalate-carbonate pathway in the iroko system, fluxes of matter have been identified, described, and evaluated from field to microscopic scales. In the first centimeters of the soil profile, decaying of the organic matter allows the release of whewellite crystals, mainly due to the action of termites and saprophytic fungi. In addition, a concomitant flux of carbonate formed in wood tissues contributes to the carbonate flux and is identified as a direct consequence of wood feeding by termites. Nevertheless, calcite biomineralization of the tree is not a consequence of in situ oxalate consumption, but rather related to the oxalate oxidation inside the upper part of the soil. The consequence of this oxidation is the presence of carbonate ions in the soil solution pumped through the roots, leading to preferential mineralization of the roots and the trunk base. An ideal scenario for the iroko biomineralization and soil carbonate accumulation starts with oxalatization: as the iroko tree grows, the organic matter flux to the soil constitutes the litter, and an oxalate pool is formed on the forest ground. Then, wood rotting agents (mainly termites, saprophytic fungi, and bacteria) release significant amounts of oxalate crystals from decaying plant tissues. In addition, some of these agents are themselves producers of oxalate (e.g. fungi). Both processes contribute to a soil pool of "available" oxalate crystals. Oxalate consumption by oxalotrophic bacteria can then start. Carbonate and calcium ions present in the soil solution represent the end products of the oxalate-carbonate pathway. The solution is pumped through the roots, leading to carbonate precipitation. The main pools of carbon are clearly identified as the organic matter (the tree and its organic products), the oxalate crystals, and the various carbonate features. A functional model based on field observations and diagenetic investigations with δ13C signatures of the various compartments involved in the local carbon cycle is proposed. It suggests that the iroko ecosystem can act as a long-term carbon sink, as long as the calcium source is related to non-carbonate rocks. Consequently, this carbon sink, driven by the oxalate carbonate pathway around an iroko tree, constitutes a true carbon trapping ecosystem as defined by ecological theory.

A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Anàlisi de la modelització hidrològica com a eina per la predicció d'inundacions i la gestió del territori

Les inundacions són actualment les catàstrofes naturals més recurrents i les que generen un major nombre de danys i víctimes arreu del món. L'ocupació de les zones inundables a les lleres del riu és la causa principal d’aquests desastres naturals. En aquest article es descriu la realització de models hidrològics com a mecanisme per la predicció d’inundacions i la gestió del territori. S’han estudiat les conques de la Riera de Santa Coloma (Catalunya) i del riu San Francisco (Guatemala) mitjançant els programes HEC-HMS i HEC-RAS, dels quals s’avalua la seva capacitat com eina per a la gestió del territori. S’ha analitzat l’efecte de la urbanització en el risc d’inundació en el cas de la Riera de Santa Coloma en base a la previsió del Plà d’Ordenament Urbanístic Municipal. S’han determinat les zones inundables resultants de episodis de precipitació extrems al Riu San Francisco per als episodis de les tempestes Stan(2005) i Agatha(2010).

A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI) medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.