Generation and analysis of proline mutants in protein G

The pyrrolidine ring of the amino acid proline reduces the conformational freedom of the protein backbone in its unfolded form and thus enhances protein stability. The strategy of inserting proline into regions of the protein where it does not perturb the structure has been utilized to stabilize many different proteins including enzymes. However, most of these efforts have been based on trial and error, rather than rational design. Here, we try to understand proline's effect on protein stability by introducing proline mutations into various regions of the B1 domain of Streptococcal protein G. We also applied the Optimization of Rotamers By Iterative Techniques computational protein design program, using two different solvation models, to determine the extent to which it could predict the stabilizing and destabilizing effects of prolines. Use of a surface area dependent solvation model resulted in a modest correlation between the experimental free energy of folding and computed energies; on the other hand, use of a Gaussian solvent exclusion model led to significant positive correlation. Including a backbone conformational entropy term to the computational energies increases the statistical significance of the correlation between the experimental stabilities and both solvation models.     

A study of the durability of flexible magnetic media in a linear tape system

Travan™ linear tape drives (with the head removed) and custom-made tape cyclers were used for sample generation in a preliminary study of the durability of experimental particulate media. The first tester cycled short (1 m) closed loops of tape, whereas the second cycled cartridge loaded media for an identical number of passes (0, 100, 250, 500, 1000, 5000, 10 000). X-ray photoelectron spectroscopy was used to identify chemical changes to the media surface and in the case of the Travan tester, these changes were compared to dropout growth through the use of a dedicated Travan drive. The results show binder depletion and an increase in surface lubricant with increasing number of cycles, irrespective of the type of tester used. The tapes exhibited good durability since the surface changes did not have a detrimental effect on the dropout characteristics.     

Three-dimensional formulation of rigid-flexible multibody systems with flexible beam elements

Multibody systems generally contain solids with appreciable deformations and which decisively influence the dynamics of the system. These solids have to be modeled by means of special formulations for flexible solids. At the same time, other solids are of such a high stiffness that they may be considered rigid, which simplifies their modeling. For these reasons, for a rigid-flexible multibody system, two types of formulations coexist in the equations of the system. Among the different possibilities provided in the literature on the material, the formulation in natural coordinates and the formulation in absolute nodal coordinates are utilized in this paper to model the rigid and flexible solids, respectively. This paper contains a mixed formulation based on the possibility of sharing coordinates between a rigid solid and a flexible solid. The global mass matrix of the system is shown to be constant and, in addition, many of the constraint equations obtained upon utilizing these formulations are linear and can be eliminated.     

Using a Simple Prioritisation Mechanism to Effectively Interoperate Service and Opportunistic Grids in the EELA-2 e-Infrastructure

Grids currently in production can be broadly classified as either service Grids, composed of dedicated resources, or opportunistic Grids that harvest the computing power of non-dedicated resources when they are idle. While a service Grid provides high and well defined levels of quality of service, an opportunistic Grid provides only a best-effort service. Nevertheless, since opportunistic Grids do not require resources to be fully dedicated to the Grid, they have the potential to assemble a much larger number of resources. Moreover, these Grids cater very well to the execution of the so-called embarrassingly parallel applications, a type of application that is frequently found in practice, and that comprises the largest portion of the typical workload processed in production Grid systems. The EELA-2 e-infrastructure is comprised of a service Grid and an opportunistic Grid that federates computing resources from scientific institutions in both Europe and Latin America. Due to the complementary characteristics of these two types of Grids, a lot of attention has recently been placed in how to interoperate them. In this paper we focus on the less studied problem of assessing the feasibility of such interoperation. We analyse different prioritisation policies that define when the resources of one Grid can be used to run jobs originating from the other. Our results show that in the absence of a suitable prioritisation policy, the benefits that the users of one Grid may have, frequently come with an important negative impact on the users of the other Grid. We also show that a simple reciprocation mechanism is capable of arbitrating the interoperation in such a way that, whenever possible, users profit from the interoperation and, in no case, this benefit leads to a noticeable reduction on the quality of service that the users would experience were the Grids not to interoperate. We conclude discussing how we have implemented, in the context of the EELA-2 project, this prioritisation mechanism, allowing the effective interoperation of a service Grid based on the gLite middleware with an opportunistic Grid that uses the OurGrid middleware.     

Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells

A method was developed for the rapid diagnosis of the infectious hematopoietic necrosis virus (IHNV) based on dot blot hybridization with a non-radioactive probe. When the assay was developed through a color reaction both biotin- and alkaline phosphatase-labeled probes were highly specific for IHNV and the sensitivity reached to 20 pg of viral RNA. When the alkaline phosphatase-labeled probe was developed by a chemiluminiscent reaction, the sensitivity showed a five-fold increase to 4 pg of viral RNA. The procedures successfully detected IHNV directly from infected symptomatic and asymptomatic fishes exhibiting higher sensitivity than the traditional approaches involving viral propagation in cell cultures. Additional advantages of the method are its simplicity and it takes only about 6 h to carry out the procedures from the initial processing of the tissues to diagnosis.     

Analysis of persistent photoconductivity due to potential barriers

Persistent photoconductivity has been seen in thin silicon resistors fabricated with SIMOX material at temperatures between 60 and 220 K. This effect has been attributed to the depletion of carriers near the interface between the top silicon layer and the buried oxide, which is due to the large number of surface traps at this interface. The depletion of carriers is accompanied by a built-in field on the order of 10,000 V/cm, which causes a potential barrier that is nearly a quarter of the energy gap of silicon. The theory of the recombination kinetics of majority carriers with minority carriers trapped at the interface on the other side of a potential barrier is studied. Both the possibilities of tunneling and thermal activation have been considered. The results show that thermal activation dominates at the temperatures of our measurements in SIMOX material, while at lower temperatures tunneling would dominate.     

Practical considerations on the use of predictive models for regulatory purposes

Interest in the use of quantitative structure-activity relationships (QSARs) for regulatory purposes has been growing steadily over the years, and many models have been evaluated under the guidance and acceptability criteria defined at the Setubal workshop held in March 2002. This work explores some of the practical issues related to the use of QSARs for regulatory purposes using results obtained from rat oral lethality and fish acute toxicity estimates generated from computational models (including TOPKAT, MCASE, OASIS, and ECOSAR). Using data submitted under the Environmental Protection Agency's (EPA's) High Production Volume (HPV) Challenge Program, the results on the quality of the estimations are compared using a standard statistical review and an additional classification approach in which the hazard predictions were grouped using well-defined regulatory criteria (those used in EPA's New Chemical Program). Our results indicate that an evaluation of a model's regulatory applicability and predictive power is ultimately dependent on the specific criteria used in the assessment process. This work also discusses the practical difficulties associated with defining the domain of a predictive model using the estimates of four different ready biodegradation models and experimental data submitted under the EPA's New Chemical program. Our results suggest that the method a model employs for its predictions is as important as the training set in determining its domain of applicability. Together, these results highlight the challenges associated with developing reliable and easily applied acceptability criteria for the regulatory use of QSAR models.     

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency.