Handbook of instrumental techniques for materials, chemical and biosciences research

This Handbook contains a collection of articles describing instrumental techniques used for Materials, Chemical and Biosciences research that are available at the Scientific and Technological Centers of theUniversity of Barcelona (CCiTUB). The CCiTUB are a group of facilities of the UB that provide both the research community and industry with ready access to a wide range of major instrumentation.Together with the latest equipment and technology, the CCiTUB provide expertise in addressing the methodological research needs of the user community and they also collaborate in R+D+i Projectswith industry. CCiTUB specialists include technical and Ph.D.-level professional staff members who are actively engaged in methodological research. Detailed information on the centers’ resources andactivities can be found at the CCiTUB website www.ccit.ub.edu ...

Physical and chemical characteristics of sapota fruit at different stages of maturation

In the present work, the physical and chemical characteristics in three stages of maturation of sapota (Manilkara zapota L.P. Royen) fruit were studied as well as its post-harvest behavior during storage at ambient and refrigerated conditions. With the advance of maturation, the concentration of the reducing sugars increased while the total acidity and tannin contents decreased. The fruits which did not have their pedicel removed during the post-harvest presented the storage time superior when compared with the fruits having their pedicels removed. The fruits stored under refrigeration had higher weight retention as compared to the fruits stored under ambient conditions.

Space Competition and Time Delays in Human Range Expansions. Application to the Neolithic Transition

Space competition effects are well-known in many microbiological and ecological systems. Here we analyze such an effectin human populations. The Neolithic transition (change from foraging to farming) was mainly the outcome of a demographic process that spread gradually throughout Europe from the Near East. In Northern Europe, archaeological data show a slowdown on the Neolithic rate of spread that can be related to a high indigenous (Mesolithic) population density hindering the advance as a result of the space competition between the two populations. We measure this slowdown from a database of 902 Early Neolithic sites and develop a time-delayed reaction-diffusion model with space competition between Neolithic and Mesolithic populations, to predict the observed speeds. The comparison of the predicted speed with the observations and with a previous non-delayed model show that both effects, the time delay effect due to the generation lag and the space competition between populations, are crucial in order to understand the observations

Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).

Biological monitoring of chemical exposure : toxicokinetic differences due to age and sex

Human biomonitoring is a widely used method in the assessment of occupational exposure to chemical substances and recommended biological limits are published periodically for interpretation and decision-making. However, it is increasingly recognized that a large variability is associated with biological monitoring, making interpretation less efficient than assumed. In order to improve the applicability of biological monitoring, specific factors responsible for this variability should be identified and their contribution quantified. Among these factors, age and sex are easily identifiable, and present knowledge about pharmaceutical chemicals suggests that they play an important role on the toxicokinetics of occupational chemical agents, and therefore on the biological monitoring results.The aim of the present research project was to assess the influence of age and sex on biological indicators corresponding to organic solvents. This has been done experimentally and by toxicokinetic computer simulation. Another purpose was to explore the effect of selected CYP2E1 polymorphisms on the toxicokinetic profile.Age differences were identified by numerical simulations using a general toxicokinetic model from a previous study which was applied to 14 chemicals, representing 21 specific biological entities, with, among others, toluene, phenol, lead and mercury. These models were runn with the modified parameters, indicating in some cases important differences due to age. The expected changes are mostly of the order of 10-20 %, but differences up to 50 % were observed in some cases. These differences appear to depend on the chemical and on the biological entity considered.Sex differences were quantified by controlled human exposures, which were carried out in a 12 m3 exposure chamber for three organic solvents separately: methyl ethyl ketone, 1-methoxy-2-propanol and 1,1,1-trichloroethane. The human volunteer groups were composed 12 of ten young men and fifteen young women, the latter subdivided into those with and without hormonal contraceptive. They were exposed during six hours at rest and at half of the threshold limit value. The kinetics of the parent compounds (organic volatiles) and their metabolite(s) were followed in blood, urine and expired air over time. Analyses of the solvent and their metabolites were performed by using headspace gas chromatography, CYP2E1 genotypes by using PCR-based RFLP methods. Experimental data were used to calibrate the toxicokinetic models developed for the three solvents. The results obtained for the different biomarkers of exposure mainly showed an effect on the urinary levels of several biomarkers among women due to the use of hormonal contraceptive, with an increase of about 50 % in the metabolism rate. The results also showed a difference due to the genotype CYP2E1*6, when exposed to methyl ethyl ketone, with a tendency to increase CYP2E1 activity when volunteers were carriers of the mutant allele. Simulations showed that it is possible to use simple toxicokinetic tools in order to predict internal exposure when exposed to organic solvents. Our study suggests that not only physiological differences but also exogenous sex hormones could influence CYP2E1 enzyme activity. The variability among the urinary biological indicators levels gives evidence of an interindividual susceptibility, an aspect that should have its place in the approaches for setting limits of occupational exposure.

Impact of controlled changes in grain size and pore space characteristics on the hydraulic conductivity and spectral induced polarization response of "proxies" of saturated alluvial sediments

Understanding the influence of pore space characteristics on the hydraulic conductivity and spectral induced polarization (SIP) response is critical for establishing relationships between the electrical and hydrological properties of surficial unconsolidated sedimentary deposits, which host the bulk of the world's readily accessible groundwater resources. Here, we present the results of laboratory SIP measurements on industrial-grade, saturated quartz samples with granulometric characteristics ranging from fine sand to fine gravel, which can be regarded as proxies for widespread alluvial deposits. We altered the pore space characteristics by changing (i) the grain size spectra, (ii) the degree of compaction, and (iii) the level of sorting. We then examined how these changes affect the SIP response, the hydraulic conductivity, and the specific surface area of the considered samples. In general, the results indicate a clear connection between the SIP response and the granulometric as well as pore space characteristics. In particular, we observe a systematic correlation between the hydraulic conductivity and the relaxation time of the Cole-Cole model describing the observed SIP effect for the entire range of considered grain sizes. The results do, however, also indicate that the detailed nature of these relations depends strongly on variations in the pore space characteristics, such as, for example, the degree of compaction. The results of this study underline the complexity of the origin of the SIP signal as well as the difficulty to relate it to a single structural factor of a studied sample, and hence raise some fundamental questions with regard to the practical use of SIP measurements as site- and/or sample-independent predictors of the hydraulic conductivity.

Chemical nature of soil humified fractions and their bioactivity

The aim of this work was to evaluate the humus composition from an Ultisol from Campos dos Goytacazes, RJ, Brazil. Soil samples of four depths (0-0.05, 0.05-0.10, 0.10-0.20 and 0.20-0.40 m) and its chemical nature were analysed by elemental composition, E4/E6 ratios and Fourier transformed infrared spectroscopy. The bioactivity of these humified substances was evaluated through their action on maize root growth and H+-ATPase activity of roots microsomes. In topsoil, the content of high condensed alkaline soluble humic substances is greater than that found in the subsuperficial layers. The chemical nature of humic and fulvic acids also varied with the soil depth. The humic acids isolated from the soil samples exhibited higher bioactivity compared with the fulvic acids. Moreover, the results suggest that more condensed humic substances can promote highest stimulation of the microsomal H+-ATPases from maize roots. These data reinforce the concept that the activity of the H+ pumps can be used as a biochemical marker for evaluation of humic substances bioactivity.

Meta-structure correlation in protein space unveils different selection rules for folded and intrinsically disordered proteins

The number of existing protein sequences spans a very small fraction of sequence space. Natural proteins have overcome a strong negative selective pressure to avoid the formation of insoluble aggregates. Stably folded globular proteins and intrinsically disordered proteins (IDP) use alternative solutions to the aggregation problem. While in globular proteins folding minimizes the access to aggregation prone regions IDPs on average display large exposed contact areas. Here, we introduce the concept of average meta-structure correlation map to analyze sequence space. Using this novel conceptual view we show that representative ensembles of folded and ID proteins show distinct characteristics and responds differently to sequence randomization. By studying the way evolutionary constraints act on IDPs to disable a negative function (aggregation) we might gain insight into the mechanisms by which function - enabling information is encoded in IDPs.

Chemical probes that differentially modulate peroxisome proliferator-activated receptor alpha and BLTR, nuclear and cell surface receptors for leukotriene B(4).

Peroxisome proliferator-activated receptor alpha (PPARalpha)is a nuclear receptor for various fatty acids, eicosanoids, and hypolipidemic drugs. In the presence of ligand, this transcription factor increases expression of target genes that are primarily associated with lipid homeostasis. We have previously reported PPARalpha as a nuclear receptor of the inflammatory mediator leukotriene B(4) (LTB(4)) and demonstrated an anti-inflammatory function for PPARalpha in vivo (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43). LTB(4) also has a cell surface receptor (BLTR) that mediates proinflammatory events, such as chemotaxis and chemokinesis (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624). In this study, we report on chemical probes that differentially modulate activity of these two LTB(4) receptors. The compounds selected were originally characterized as synthetic BLTR effectors, both agonists and antagonists. Here, we evaluate the compounds as effectors of the three PPAR isotypes (alpha, beta, and gamma) by transient transfection assays and also determine whether the compounds are ligands for these nuclear receptors by coactivator-dependent receptor ligand interaction assay, a semifunctional in vitro assay. Because the compounds are PPARalpha selective, we further analyze their potency in a biological assay for the PPARalpha-mediated activity of lipid accumulation. These chemical probes will prove invaluable in dissecting processes that involve nuclear and cell surface LTB(4) receptors and also aid in drug discovery programs.

Development and validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method for the determination of ethyl glucuronide in hair and its application to forensic toxicology.

Ethyl glucuronide (EtG) is a minor and direct metabolite of ethanol. EtG is incorporated into the growing hair allowing retrospective investigation of chronic alcohol abuse. In this study, we report the development and the validation of a method using gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS) for the quantification of EtG in hair. EtG was extracted from about 30 mg of hair by aqueous incubation and purified by solid-phase extraction (SPE) using mixed mode extraction cartridges followed by derivation with perfluoropentanoic anhydride (PFPA). The analysis was performed in the selected reaction monitoring (SRM) mode using the transitions m/z 347-->163 (for the quantification) and m/z 347-->119 (for the identification) for EtG, and m/z 352-->163 for EtG-d(5) used as internal standard. For validation, we prepared quality controls (QC) using hair samples taken post mortem from 2 subjects with a known history of alcoholism. These samples were confirmed by a proficiency test with 7 participating laboratories. The assay linearity of EtG was confirmed over the range from 8.4 to 259.4 pg/mg hair, with a coefficient of determination (r(2)) above 0.999. The limit of detection (LOD) was estimated with 3.0 pg/mg. The lower limit of quantification (LLOQ) of the method was fixed at 8.4 pg/mg. Repeatability and intermediate precision (relative standard deviation, RSD%), tested at 4 QC levels, were less than 13.2%. The analytical method was applied to several hair samples obtained from autopsy cases with a history of alcoholism and/or lesions caused by alcohol. EtG concentrations in hair ranged from 60 to 820 pg/mg hair.

Are niche-based species distribution models transferable in space?

Aim To assess the geographical transferability of niche-based species distribution models fitted with two modelling techniques. Location Two distinct geographical study areas in Switzerland and Austria, in the subalpine and alpine belts. Methods Generalized linear and generalized additive models (GLM and GAM) with a binomial probability distribution and a logit link were fitted for 54 plant species, based on topoclimatic predictor variables. These models were then evaluated quantitatively and used for spatially explicit predictions within (internal evaluation and prediction) and between (external evaluation and prediction) the two regions. Comparisons of evaluations and spatial predictions between regions and models were conducted in order to test if species and methods meet the criteria of full transferability. By full transferability, we mean that: (1) the internal evaluation of models fitted in region A and B must be similar; (2) a model fitted in region A must at least retain a comparable external evaluation when projected into region B, and vice-versa; and (3) internal and external spatial predictions have to match within both regions. Results The measures of model fit are, on average, 24% higher for GAMs than for GLMs in both regions. However, the differences between internal and external evaluations (AUC coefficient) are also higher for GAMs than for GLMs (a difference of 30% for models fitted in Switzerland and 54% for models fitted in Austria). Transferability, as measured with the AUC evaluation, fails for 68% of the species in Switzerland and 55% in Austria for GLMs (respectively for 67% and 53% of the species for GAMs). For both GAMs and GLMs, the agreement between internal and external predictions is rather weak on average (Kulczynski's coefficient in the range 0.3-0.4), but varies widely among individual species. The dominant pattern is an asymmetrical transferability between the two study regions (a mean decrease of 20% for the AUC coefficient when the models are transferred from Switzerland and 13% when they are transferred from Austria). Main conclusions The large inter-specific variability observed among the 54 study species underlines the need to consider more than a few species to test properly the transferability of species distribution models. The pronounced asymmetry in transferability between the two study regions may be due to peculiarities of these regions, such as differences in the ranges of environmental predictors or the varied impact of land-use history, or to species-specific reasons like differential phenotypic plasticity, existence of ecotypes or varied dependence on biotic interactions that are not properly incorporated into niche-based models. The lower variation between internal and external evaluation of GLMs compared to GAMs further suggests that overfitting may reduce transferability. Overall, a limited geographical transferability calls for caution when projecting niche-based models for assessing the fate of species in future environments.