Optical micromanipulations inside yeast cells

We present a combination of nonlinear microscopy and optical trapping applied to three-dimensional imaging and manipulation of intracellular structures in living cells. We use Titanium-sapphire laser pulses for nonlinear microscopy of the nuclear envelope and the microtubules marked with green fluorescent protein in fission yeast. The same laser source is also used to trap small lipid granules naturally present in the cell. The trapped granule is used as a handle to exert a pushing force on the cell nucleus. The granule is moved in a raster-scanning fashion to cover the area of the nucleus and hence displace the nucleus away from its normal position in the center of the cell. Such indirect manipulations of an organelle (e.g., nucleus) can be useful when direct trapping of the chosen organelle is disadvantageous or inefficient. We show that nonlinear microscopy and optical manipulation can be performed without substantial damage or heating of the cell. We present this method as an important tool in cell biology for manipulation of specific structures, as an alternative to genetic and biochemical methods. This technique can be applied to several fundamental problems in cell biology, including the mechanism of nuclear positioning and the spatial coordination of nuclear and cell division.     

Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74 degrees C) of native decorsin. The mono-substituted analogs display a Tm of 66-67 degrees C, while the double-substituted analog a Tm of 50 degrees C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro-->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the 'proline rule', previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.     

Caprolactam production: A comparison of different layouts of the liquid-liquid extraction section

Two different layouts for the liquid-liquid extraction section of an industrial caprolactam process are evaluated. Laboratory mass transfer measurements on streams obtained from an industrial and a demonstration plant with different extraction sections are interpreted by a model describing the effect of surface contaminants on mass transfer coefficients. Indications of the impact of these layouts on the complexity of the extraction section, quality of products and efficiency of the extraction processes are given.     

Measurement and numerical simulation of low field magnetization in laminated BSCCO

I present a simulation of the magnetic behaviour of a network of long Josephson junctions, with a statistical distribution of junction lengths and Josephson currents. To support the hypothesis of weak connectivity in laminar materials I measured the magnetization of Bi/Pb (2223) samples, at very low fields and low temperature. Hysteresis cycles were observed.     

World Standards Day Message 14 October 2007 Standards and the Citizen: Contributing to Society

The Global Village brings a broad range of rights and obligations to its citizens. These include rights to safety, security,health and access to information. Obligations include protecting the environment and respecting the safety, property and privacy of others. Standards help citizens to exercise these rights and obligations. They do this, for example, by providing consumers with information and protection, by ensuring the quality and safety of products and services, by defining requirements or giving guidance related to the environment and other issues important to citizens including societal equity, health, security, information and communication, and fair trade.     

Comparative evaluation of the antioxidant capacity of smoke flavouring phenols by crocin bleaching inhibition, DPPH radical scavenging and oxidation potential

The antioxidant capacity of the main phenolic compounds present in wood smoke and smoke flavourings used in the food industry was investigated by three methods, based on a kinetic and thermodynamic approach: the bleaching of the carotenoid crocin, the scavenging of the DPPH radical, and the determination of the oxidation potential. The reaction with the DPPH radical was evaluated calculating the effective concentration (EC₅₀) and the antiradical efficiency (AE). The compounds tested were 2-methoxyphenols (guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-vinylguaiacol, 4-propylguaiacol, eugenol, isoeugenol, vanillin, acetovanillone, 2-propiovanillone), 2,6-dimethoxyphenols (syringol, 4-methylsyringol, 4-allylsyringol, syringaldehyde, acetosyringone) and dihydroxybenzenes (catechol, 3-methylcatechol, 4-methylcatechol, 3-methoxycatechol and hydroquinone). The trend in antioxidant capacity was similar in all the three methods, with dihydroxybenzenes > 2,6-dimethoxyphenols > 2-methoxyphenols, although some discrepancies in the ranking within the groups were present. Considering the overall ranking, isoeugenol was amongst the most active compound, like dihydroxybenzenes, evidencing the role of a conjugated double bond at para position for the stabilization of the phenoxyl radical in the radical scavenging process.     

Notes on the application of the standardized residual sum of squares index for the assessment of intra- and inter-observer variability in color-difference experiments

The standardized residual sum of squares index was proposed to examine the significant merit of a given color-difference formula over another with respect to a given set of visual color-difference data [J. Opt. Soc. Am. A 24, 1823-1829, 2007]. This index can also be employed to determine intra- and inter-observer variability, although the full complexity of this variability cannot be described by just one number. Appropriate utilization of the standardized residual sum of squares index for the assessment of observer variability is described with a view to encourage its use in future color-difference research. The main goal of this paper is to demonstrate that setting the F parameters of the standardized residual sum of squares index to 1 results in a loss of essential properties of the index (for example, symmetry), and is therefore strongly discouraged.     

Identification and measurement of illicit drugs and their metabolites in urban wastewater by liquid chromatography-tandem mass spectrometry

Residues of illicit drugs and their metabolites that are excreted by humans may flow into and through wastewater treatment plants. The aim of this study was to develop a method for the determination of cocaine, amphetamines, morphine, cannabinoids, methadone, and some of their metabolites in wastewater. Composite 24-h samples from urban treatment plants were enriched with deuterated internal standards before solid-phase extraction. High-pressure liquid chromatography tandem mass spectrometry with multiple reaction monitoring was used for quantitation. Recoveries were generally higher than 80%, and limits of quantifications were in the low nanograms-per-liter range for untreated and treated wastewater. The overall variability of the method was lower than 10% for untreated and 5% for treated wastewater. The method was applied to wastewater samples coming from two treatment plants in Italy and Switzerland. Quantification ranges were found to be 0.2-1 microg/L for cocaine and its metabolite benzoylecgonine, 80-200 ng/L for morphine, 10 ng/L for 6-acetylmorphine, 60-90 ng/L for 11-nor-9-carboxy-Delta9-tetrahydrocannabinol, 10-90 ng/L for methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, and lower than 20 ng/L for amphetamines. As previously reported for cocaine, this method could be useful to estimate and monitor drug consumption in the population in real time, helping social scientists and authorities to combat drug abuse.     

A Hypothalamic Mechanism Regulates the Duration of a Migraine Attack: Insights from Microstructural and Temporal Complexity of Cortical Functional Networks Analysis

The role of the hypothalamus and the limbic system at the onset of a migraine attack has recently received significant interest. We analyzed diffusion tensor imaging (DTI) parameters of the entire hypothalamus and its subregions in 15 patients during a spontaneous migraine attack and in 20 control subjects. We also estimated the non-linear measure resting-state functional MRI BOLD signal’s complexity using Higuchi fractal dimension (FD) and correlated DTI/fMRI findings with patients’ clinical characteristics. In comparison with healthy controls, patients had significantly altered diffusivity metrics within the hypothalamus, mainly in posterior ROIs, and higher FD values in the salience network (SN). We observed a positive correlation of the hypothalamic axial diffusivity with migraine severity and FD of SN. DTI metrics of bilateral anterior hypothalamus positively correlated with the mean attack duration. Our results show plastic structural changes in the hypothalamus related to the attacks severity and the functional connectivity of the SN involved in the multidimensional neurocognitive processing of pain. Plastic changes to the hypothalamus may play a role in modulating the duration of the attack.