Assessment of DNA damage in Down Syndrome patients by means of a new, optimised single cell gel electrophoresis technique

Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.     

Selective interactions of perylene derivatives having different side chains with inter- and intramolecular G-quadruplex DNA structures. A correlation with telomerase inhibition

While the importance of the aromatic core in small organic molecules, studied as G-quadruplex mediated telomerase inhibitors, appears well studied by a number of researches, the role of side chains has been less well characterized. In this paper, we have studied the ability of six perylene derivatives with different side chains to induce both inter- and intramolecular G-quadruplex structures. The distance between the aromatic core and the positive charges in the side chains emerges as a significant molecular feature in G-quadruplex formation. Furthermore, the G-quadruplex formation appears also related to drugs 'self-association', influenced by the side chains basicity. The different efficiencies of the six perylene derivatives in interacting both with inter- and intramolecular G-quadruplex structures satisfactorily correlate with telomerase inhibition in cell-free systems.     

Catalytic enantioselective addition of indoles to arylnitroalkenes: an effective route to enantiomerically enriched tryptamine precursors

A new valuable catalytic protocol for the preparation of synthetically useful beta-indolyl nitro compounds bearing benzhydryl stereocenters is presented. The combined use of catalytic amounts of a commercially available chiral [SalenAlCl] complex and pyridine allowed, for the Friedel-Crafts alkylation reaction of indoles with aromatic nitro-olefins to be carried out in good yields and enantioselectivity (up to 63% ee).     

Evaluation of dog semen quality after slow (biological freezer) or rapid (nitrogen vapours) freezing

Three ejaculates were collected from each of five dogs. After initial evaluation, the sperm-rich fractions were diluted to 100 x 10(6) spermatozoa x mL(-1) in two steps with an egg yolk-TRIS extender containing a final concentration of 5% glycerol and 0.5% Equex STM paste. Half of the 0.5 mL straws obtained from each ejaculate were frozen on nitrogen vapours (4 cm above the liquid surface) ("rapid freezing"), while the other half was frozen in a biological freezer at a rate of 0.5 degrees C x min(-1) between 5 degrees C and -10 degrees C and of 8 degrees C x min(-1) between -10 degrees C and -60 degrees C, followed by immersion in liquid nitrogen ("slow freezing"). After an average storage of 30 days, the straws were thawed in a water-bath at 37 degrees C for 1 min. Progressive motility was subjectively estimated hourly for 8 h on semen incubated at 38 degrees C. Immediately after thawing and after 2 h of incubation, motility parameters were also measured by a motility analyser. Sperm membrane function and chromatin stability were assessed immediately post-thaw, using the hypo-osmotic swelling test and acridine orange staining, respectively. Slow freezing significantly improved total post-thaw motility, which showed a slower decline over time, although spermatozoal average path and straight line velocity were lower compared to the fast rate. Also the number of intact membrane spermatozoa was significantly higher in slow-frozen samples while the proportion of spermatozoa with single-stranded DNA was minimal after both freezing procedures.     

Polymorphism of pyridinium amphiphiles for gene delivery: influence of ionic strength, helper lipid content, and plasmid DNA complexation

Two double-tailed pyridinium cationic amphiphiles, differing only in the degree of unsaturation of the alkyl chains, have been selected for a detailed study of their aggregation behavior, under conditions employed for transfection experiments. The transfection efficiencies of the two molecules are remarkably different, especially when combined with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as helper lipid. The phase behavior of the cationic amphiphile/DOPE mixtures have been studied using (31)P- and (2)H-NMR (on deuterated cationic amphiphiles) as main techniques, to monitor independently the behavior of the two components. In water, the lamellar organization is dominant for both the surfactants in their mixtures with the helper lipid. In HEPES saline buffer (HBS), the mixtures of the unsaturated surfactant form inverted phases and, in particular, stable H(II) phases for DOPE contents > or =30 mol %. By contrast, the saturated surfactant does not form homogeneously mixed inverted phases in mixtures with DOPE at room temperature. However, mixed inverted phases are observed for this system at higher temperatures and, after mixing has been achieved by heating, the metastable mixed phases remain present for several hours at 5 degrees C. At 35 degrees C the dominant phase is the cubic phase. The lipoplex composed of equimolar mixtures of the unsaturated surfactant with DOPE and plasmid DNA was found to be organized in highly curved bilayers.     

The high-resolution architecture and structural dynamics of Bacillus spores

The capability to image single microbial cell surfaces at nanometer scale under native conditions would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, environmental resistance, and biotransformation. Here, using in vitro atomic force microscopy, we have directly visualized high-resolution native structures of bacterial endospores, including the exosporium and spore coats of four Bacillus species in air and water environments. Our results demonstrate that the mechanisms of spore coat self-assembly are similar to those described for inorganic and macromolecular crystallization. The dimensions of individual Bacillus atrophaeus spores decrease reversibly by 12% in response to a change in the environment from fully hydrated to air-dried state, establishing that the dormant spore is a dynamic physical structure. The interspecies distributions of spore length and width were determined for four species of Bacillus spores in water and air environments. The dimensions of individual spores differ significantly depending upon species, growth regimes, and environmental conditions. These findings may be useful in the reconstruction of environmental and physiological conditions during spore formation and for modeling the inhalation and dispersal of spores. This study provides a direct insight into molecular architecture and structural variability of bacterial endospores as a function of spatial and developmental organizational scales.     

Optimal Transmission Radius for Flooding in Large Scale Sensor Networks

One of the principal characteristics of large scale wireless sensor networks is their distributed, multi-hop nature. Due to this characteristic, applications such as query propagation rely regularly on network-wide flooding for information dissemination. If the transmission radius is not set optimally, the flooded packet may be holding the transmission medium for longer periods than are necessary, reducing overall network throughput. We analyze the impact of the transmission radius on the average settling time—the time at which all nodes in the network finish transmitting the flooded packet. Our analytical model takes into account the behavior of the underlying contention-based MAC protocol, as well as edge effects and the size of the network. We show that for large wireless networks there exists an intermediate transmission radius which minimizes the settling time, corresponding to an optimal tradeoff between reception and contention times. We also explain how physical propagation models affect small wireless networks and why there is no intermediate optimal transmission radius observed in these cases. The mathematical analysis is supported and validated through extensive simulations.Marco Zuniga is currently a PhD student in the Department of Electrical Engineering at the University of Southern California. He received his Bachelors degree in Electrical Engineering from the Pontificia Universidad Catolica del Peru in 1998, and his Masters degree in Electrical Engineering from the University of Southern California in 2002. His interests are in the area of Wireless Sensor Networks in general, and more specifically in studying the interaction amongst different layers to improve the performance of these networks. He is a member of IEEE and the Phi Kappa Phi Honor society.Bhaskar Krishnamachari is an Assistant Professor in the Department of Electrical Engineering at the University of Southern California (USC), where he also holds a joint appointment in the Department of Computer Science. He received his Bachelors degree in Electrical Engineering with a four-year full-tuition scholarship from The Cooper Union for the Advancement of Science and Art in 1998. He received his Masters degree and his Ph.D. in Electrical Engineering from Cornell University in 1999 and 2002, under a four-year university graduate fellowship. Dr. Krishnamacharis previous research has included work on critical density thresholds in wireless networks, data centric routing in sensor networks, mobility management in cellular telephone systems, multicast flow control, heuristic global optimization, and constraint satisfaction. His current research is focused on the discovery of fundamental principles and the analysis and design of protocols for next generation wireless sensor networks. He is a member of IEEE, ACM and the Tau Beta Pi and Eta Kappa Nu Engineering Honor Societies     

JACOP: A simple and robust method for the automated classification of protein sequences with modular architecture

Background  

Whole-genome sequencing projects are rapidly producing an enormous number of new sequences. Consequently almost every family of proteins now contains hundreds of members. It has thus become necessary to develop tools, which classify protein sequences automatically and also quickly and reliably. The difficulty of this task is intimately linked to the mechanism by which protein sequences diverge, i.e. by simultaneous residue substitutions, insertions and/or deletions and whole domain reorganisations (duplications/swapping/fusion).     

Proteome analysis of a recombinant <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain during heterologous production of a glucosyltransferase

A recombinant B. megaterium strain was used for the heterologous production of a glucosyltransferase (dextransucrase). To better understand the physiological and metabolic responses of the host cell to cultivation and induction conditions, proteomic analysis was carried out by combined use of two-dimensional gel electrophoresis and mass spectrometry (2-DE/MS) for protein separation and identification.