Designing of sequencing assay assisted by capillary electrophoresis based on DNA folding analysis: an application to the VCAM1 gene

In this work, we describe a fast standardized molecular method for DNA sequencing assisted by capillary electrophoresis with a particular emphasis on bioinformatic approaches to avoid sequencing errors due to complex DNA regions. In this case, the method was applied on the human vascular adhesion molecule 1 (VCAM1) gene. VCAM1 sequence, in fact, shows many thermodynamically critical parameters such as very low GC content (30-40%), many nucleotide stack areas, i.e. hairpins, self-complementary regions. With a traditional primer design approach it was difficult to design correct PCR oligonucleotides, thus sometimes, the chromatogram showed an illegible profile. By a strategy involving various bioinformatic tools (Mfold, Oligo, Highter), we investigated the role of the DNA-folding analysis in the assistance of primer design for the DNA sequencing of fragments with high -ΔG stem-loop regions. This new approach allowed us to sequence nine different VCAM1 regions each containing the respective exon. Our results, based on different DNA samples recruited from oral brushes taken from ten different subjects, identified four different SNPs (c.662-7C/T, c.1793-79A>G, c.2079C/T, c.2208A>G) with high reproducibility.     

A 2DE approach for high-throughput antigen separation applicable to mAb production

A constantly increasing number of mABs are required for the validation of a large proportion of proteomic and protein-protein interaction data. The development of new robotic platforms has greatly enhanced the throughput of monoclonal antibody production; however, the availability of highly purified proteins to use as antigens currently represents the major bottleneck of the process. In this article, we describe a new 2DE approach to purify hundreds of proteins from cellular extracts in a very cost-effective and time-efficient way. The accuracy of the new purification method is shown to be comparable to high-resolution analytical 2DE. The effectiveness and the throughput of the method to purify proteins suitable for the development of mAbs are then assessed. Using this methodology, we were able to separate 447 proteins starting from 50 mg of proteins extracted from HT29 cells. Fractions containing more than 30 μg of protein constantly induced immunization in mice. Using a high-throughput process for monoclonal antibody production, we obtained an average of 3.5 mAbs for each protein. According to pilot experiments, we can predict that starting from an unfractionated cellular extract it is possible to obtain approximately 200 proteins usable for monoclonal antibody development. Our results indicate that the number of antigens available for monoclonal antibody production can be further increased by running parallel separations. The proposed methodology will then facilitate the high-throughput monoclonal antibody process providing a vast array of high quality antigens at very low cost.     

The effectiveness of ‘what if not’ strategy coupled with dynamic geometry software in an inquiry-based geometry classroom

In this paper we present the results of a study which was carried out in an inquiry-based teaching and learning environment with the use of ‘what if not’ methodology coupled with the integration of dynamic geometry software. The vast majority of the students reported that they perceived themselves as participants rather than spectators. Most of the prospective teachers came to the conclusion that the implementation of the findings of this study in their future teachings was a good idea and that it will raise the students’ motivation and enhance and deepen the knowledge pool of the learners.     

Lanchester model for three-way combat

Lanchester (1960) modeled combat situations between two opponents, where mutual attrition occurs continuously in time, by a pair of simple ordinary (linear) differential equations. The aim of the present paper is to extend the model to a conflict consisting of three parties. In particular, Lanchester’s main result, i.e. his Square Law, is adapted to a triple fight. However, here a central factor – besides the initial strengths of the forces – determining the long run outcome is the allocation of each opponent’s efforts between the other two parties. Depending on initial strengths, (the) solution paths are calculated and visualized in appropriate phase portraits. We are able identify regions in the state space where, independent of the force allocation of the opponents, always the same combatant wins, regions, where a combatant can win if its force allocation is wisely chosen, and regions where a combatant cannot win itself but determine the winner by its forces allocation. As such, the present model can be seen as a forerunner of a dynamic game between three opponents.     

A measurement of high-pT correlations in 340 GeV/c pp and 280 GeV/cπ−p reactions

We have measured correlations between single high-p_T(1.5<p_T<3.5 GeV/c) trigger particles on one side of the beam line and groups of particles entering a calorimeter on the opposite side of the beam line. The mean transverse momentum measured in the calorimeter is found to increase with the trigger-particle transverse momentum. The coplanarity of the events increases with trigger-particle transverse momentum. We have compared our data with the predictions of a phenomenological four-jet model. To fit our data we find that we must give large (0.9 GeV/c) mean transverse momenta to the constituents of the initial hadrons.