Immunochemical or fluorescent labeling of vesicular subcellular fractions for microscopy imaging

We describe a procedure for the labeling of membranous vesicular purified subcellular fractions, to image them, typically by confocal laser scanning microscopy. Being intracellular organelles, these fractions, once purified cannot be attached to glass slides as for cells. Fractions are labeled “in batch” without prior embedding or freezing. Each labeling step performed by passages of resuspension/centrifugation is followed by washings. Then samples are dispersed on the glass slides. Mammalian retinal rod outer segment disks, intact brain stem myelin vesicles, and brain synaptosomes were chosen, as these subcellular fractions can be purified by well established procedures. These fractions were immunolabeled with specific antibodies. Moreover, by the earlier procedure, we show that the mitochondrial vital membrane potential probe MitoTracker Deep Red 633 stains myelin vesicles and rod disks before fixation, consistently with our previous reports of a respiring capacity of these membranes. Therefore, the technique seems adequate to become an instrument to study the structure and the function of these and other subcellular fractions. Microsc. Res. Tech. 73:1086–1090, 2010. © 2010 Wiley‐Liss, Inc.     

Non-Coding RNAs in Muscle Dystrophies

ncRNAs are the most recently identified class of regulatory RNAs with vital functions in gene expression regulation and cell development. Among the variety of roles they play, their involvement in human diseases has opened new avenues of research towards the discovery and development of novel therapeutic approaches. Important data come from the field of hereditary muscle dystrophies, like Duchenne muscle dystrophy and Myotonic dystrophies, rare diseases affecting 1 in 7000–15,000 newborns and is characterized by severe to mild muscle weakness associated with cardiac involvement. Novel therapeutic approaches are now ongoing for these diseases, also based on splicing modulation. In this review we provide an overview about ncRNAs and their behavior in muscular dystrophy and explore their links with diagnosis, prognosis and treatments, highlighting the role of regulatory RNAs in these pathologies.     

Instrumental and sensory evaluation of cooked pasta during frozen storage

Food consumption patterns have changed significantly during the last years, showing an increase in the consumption of convenience foods. Frozen foods represent an important segment of this market. The aim of the present paper was to study the influence of frozen storage on the quality of cooked organic tagliatelle. Instrumental measurements of different quality parameters (moisture content, colour, textural and rheological characteristics of frozen cooked pasta) were performed during twelve months of storage. The sensory properties of pasta were also evaluated by means of an acceptability study. In general, both instrumental and sensory analysis found that frozen storage affects negatively the acceptability of cooked pasta. The advantages obtained through fast freezing procedures are not maintained along storage, being hardness (instrumental) and consistency (sensory) the most relevant quality indices. Finally, the shelf life of the product was calculated as 3.6 and 3.8 months in the cryogenic device and air‐blast tunnel, respectively, based on simple kinetic models.     

Integrated mass spectrometry approach to profile proanthocyanidins occurring in food supplements: Analysis of Potentilla erecta L. rhizomes

Potentilla erecta (L.) is known for the high concentration of proanthocyanidin oligomers (PAs) in its underground parts. The use of its preparations as food supplements and the lack of chemical–analytical studies concerning with an efficient chromatographic separation procedure for these compounds led us to develop a strategy to profile PAs occurring in this species.     

New generation of loxP‐mutated deletion cassettes for the genetic manipulation of yeast natural isolates

In this work, we developed molecular tools used in standard laboratory yeast strains, such as the cre–loxP system, so that they can be used with natural and industrial prototrophic yeast species. We constructed a new generation of dominant cassettes, with mutated loxP sites (loxLE and lox2272) and selectable drug markers, to create heterothallic strains and auxotrophic mutants without incurring in the risk of generating chromosomal rearrangements. We have shown that our newly developed loxLE–hphNT1–loxRE and lox2272–natNT2–lox2272 gene‐disruption cassettes can be present in the yeast genome together with the widely used loxP–marker gene–loxP cassettes without any recombination between the lox sequences. Moreover, we also developed a new phleomycin‐resistant Cre‐expressing vector (to excise multiple markers simultaneously) and two new standard loxP deletion cassettes containing hygromicin B and cloNAT as selecatable markers. To validate these cassettes, we created heterothallic auxotrophic S. cerevisiae strains, without the risk of incurring gross chromosomal rearrangements, and we showed an example of a fitness study of intraspecific hybrids deriving from parents with different adaptations to carbon‐limited resources. Copyright © 2010 John Wiley & Sons, Ltd.     

Fiber introduction mass spectrometry: fully direct coupling of solid-phase microextraction with mass spectrometry

This work describes the first fully direct coupling of solid-phase microextraction (SPME) with mass spectrometry. An inlet system using a septum as the only interface between the ambient and the high-vacuum mass spectrometer was constructed to allow the introduction of the SPME needle directly into the ionization region of a mass spectrometer. The PDMS-coated fiber was then placed and exposed exactly between the two ionization filaments. Uniform heating of the fiber, efficient thermal desorption, and electron ionization of the analytes were achieved. Using this new analytical technique, here termed fiber introduction mass spectrometry (FIMS), we have been able to detect and quantitate several volatile (VOC) and semivolatile (SVOC) organic chemicals (carbon tetrachloride, benzene, toluene, xylenes, gamma-terpinene, diisoamyl ether, chlorobenzene, and many PAHs) and two herbicides (Sylvex and its methyl ether) from aqueous solutions at low-ppb to ppt levels using either SPME headspace or solution extraction. FIMS shows high sensitivity (ng/L), good reproducibility, and accuracy, providing therefore a simple and effective approach to rapid analysis of VOC and SVOC in various matrixes.     

Amplitude and phase recovery of rotationally symmetric beams

Two methods are presented for the amplitude and phase recovery of optical beams with rotational symmetry. These are the tomographic method based on the ambiguity function and the one-step wavefront recovery based on the measurement of a phase-space distribution closely related to the Wigner distribution function. The results obtained from these two methods are compared, and the appropriateness of using either one of them for specific situations is discussed.     

Toward overcoming culture, skill and situation hurdles in Human-Computer Interaction

This paper proposes a new effective strategy for designing and implementing interactive systems overcoming culture, skill and situation hurdles in Human-Computer Interaction (HCI). The strategy to identify and reduce these hurdles is developed in the framework of a methodology based on a recently introduced model of HCI, and exploits the technological innovations of XML (Extensible Markup Language). HCI is modelled as a cyclic process in which the user and the interactive system communicate by materializing and interpreting a sequence of messages. The interaction process is formalized by specifying both the physical message appearance and the computational aspect of the interaction. This formalization allows the adoption of notation traditionally adopted by users in their workplaces as the starting point of the interactive system design. In this way, the human–system interaction language takes into account the users’ culture. Moreover, the methodology permits user representatives to build a hierarchy of systems progressively adapted to users’ situations, skills and habits, according to the work organization in the domain considered. The strategy is proved to be effective by describing how to implement it using BANCO (Browsing Adaptive Network for Changing user Operativity), a feasibility prototype based on XML, which allows the hierarchy implementation and system adaptations. Several examples from an environmental case under study are used throughout the paper to illustrate the methodology and the effectiveness of the technology adopted. Published online: 4 June 2002     

Optimal control of advertising and production of a seasonal product with nondifferentiable demand

We consider an application of optimal control theory to a marketing problem, in which a firm seeks the maximum profit by producing and selling a seasonal product. We assume that the firm can advertise its product in order to improve the firm goodwill and affect the product demand positively. In particular the demand is zero when the goodwill is less than a fixed threshold. The original optimal control problem is shown to be equivalent to a nonlinear programming problem, which has an optimal solution, possibly not unique. Editor: G. Leitmann