Active barnase variants with completely random hydrophobic cores.

The central structural feature of natural proteins is a tightly packed and highly ordered hydrophobic core. If some measure of exquisite, native-like core packing is necessary for enzymatic function, this would constitute a significant obstacle to the development of novel enzymes, either by design or by natural or experimental evolution. To test the minimum requirements for a core to provide sufficient structural integrity for enzymatic activity, we have produced mutants of the ribonuclease barnase in which 12 of the 13 core residues have together been randomly replaced by hydrophobic alternatives. Using a sensitive biological screen, we find that a strikingly high proportion of these mutants (23%) retain enzymatic activity in vivo. Further substitution at the 13th core position shows that a similar proportion of completely random hydrophobic cores supports enzyme function. Of the active mutants produced, several have no wild-type core residues. These results imply that hydrophobicity is nearly a sufficient criterion for the construction of a functional core and, in conjunction with previous studies, that refinement of a crudely functional core entails more stringent sequence constraints than does the initial attainment of crude core function. Since attainment of crude function is the critical initial step in evolutionary innovation, the relatively scant requirements contributed by the hydrophobic core would greatly reduce the initial hurdle on the evolutionary pathway to novel enzymes. Similarly, experimental development of novel functional proteins might be simplified by limiting core design to mere specification of hydrophobicity and using iterative mutation-selection to optimize core structure.

A synthetic random basic copolymer with promiscuous binding to class II major histocompatibility complex molecules inhibits T-cell proliferative responses to major and minor histocompatibility antigens in vitro and confers the capacity to prevent murine graft-versus-host disease in vivo.

Graft-versus-host disease (GVHD) is a T-cell-mediated disease of transplanted donor T cells recognizing host alloantigens. Data presented in this report show, to our knowledge, for the first time that a synthetic copolymer of the amino acids L-Glu, L-Lys, L-Ala, and L-Tyr (molecular ratio, 1.9:6.0:4.7:1.0; Mr, 6000-8500) [corrected], termed GLAT, with promiscuous binding to multiple major histocompatibility complex class II alleles is capable of preventing lethal GVHD in the B10.D2 --> BALB/c model (both H-2d) across minor histocompatibility barriers. Administration of GLAT over a limited time after transplant significantly reduced the incidence, onset, and severity of disease. GLAT also improved long-term survival from lethal GVHD: 14/25 (56%) of experimental mice survived > 140 days after transplant compared to 2/26 of saline-treated or to 1/10 of hen egg lysozyme-treated control mice (P < 0.01). Long-term survivors were documented to be fully chimeric by PCR analysis of a polymorphic microsatellite region in the interleukin 1beta gene. In vitro, GLAT inhibited the mixed lymphocyte culture in a dose-dependent fashion across a variety of major barriers tested. Furthermore, GLAT inhibited the response of nylon wool-enriched T cells to syngeneic antigen-presenting cells presenting minor histocompatibility antigens. Prepulsing of the antigen-presenting cells with GLAT reduced the proliferative response, suggesting that GLAT inhibits antigen presentation.

Isolation of virus-neutralizing RNAs from a large pool of random sequences.

RNA and ribonuclease-resistant RNA analogs that bound and neutralized Rous sarcoma virus (RSV) were isolated from a large pool of random sequences by multiple cycles of in vitro selection using infectious viral particles. The selected RNA pool of RSV-binding sequences at a concentration of 0.16 microM completely neutralized the virus. Of 19 sequences cloned from the selected pool, 5 inhibited RSV infection. The selected RNA and RNA analogs were shown to neutralize RSV by interacting with the virus, rather than by adversely affecting the host cells. The selection of the anti-RSV RNA and RNA analogs by intact virions immediately suggests the potential application of this approach to develop RNA and RNA analogs as inhibitors of other viruses such as human immunodeficiency virus.

Geographical structuring in the mtDNA of Italians.

Geographical patterns of mtDNA variation were studied in 12 Italian samples (1072 individuals) by two different spatial autocorrelation methods. Separate analyses of the frequencies of 12 restriction morphs show North-South clines, differences between Sardinia and the mainland populations, and the effects of isolation by distance. A recently developed autocorrelation statistic summarizing molecular similarity at all sites (AIDA; autocorrelation index for DNA analysis) confirms the presence of a clinical pattern; differences between random pairs of haplotypes tend to increase with their geographical distance. The partition of gene diversity, however, reveals that most variability occurs within populations, whereas differences between populations are minor (GST = 0.057). When the data from the 12 samples are pooled, two descriptors of genetic variability (number of polymorphic sites and average sequence difference between pairs of individuals) do not behave as expected under neutrality. The presence of clinal patterns, Tajima's tests, and a simulation experiment agree in suggesting that population sizes increased rapidly in Italy and Sicily but not necessarily so in Sardinia. The distribution of pairwise sequence differences in the Italian peninsula (excluding Sardinia) permits a tentative location of the demographic increase between 8000 and 20,500 years ago. These dates are consistent with archaeological estimates of two distinct expansion processes, occurring, respectively, in the Neolithic and after the last glacial maximum in the Paleolithic. Conversely, there is no genetic evidence that such processes have had a major impact on the Sardinian population.

Mapping sites of interaction of p47-phox and flavocytochrome b with random-sequence peptide phage display libraries.

During assembly of the phagocyte NADPH oxidase, cytosolic p47-phox translocates to the plasma membrane and binds to flavocytochrome b, and binding domains for p47-phox have been identified on the C-terminal tails of both flavocytochrome b subunits. In the present report, we further examine the interaction of these two oxidase components by using random-sequence peptide phage display library analysis. Screening p47-phox with the peptide libraries identified five potential sites of interaction with flavocytochrome b, including three previously reported regions of interaction and two additional regions of interaction of p47-phox with gp91-phox and p22-phox. The additional sites were mapped to a domain on the first predicted cytosolic loop of gp91-phox encompassing residues S86TRVRRQL93 and to a domain near the cytosolic C-terminal tail of gp91-phox encompassing residues F450EWFADLL457. The mapping also confirmed a previously reported binding domain on gp91-phox (E554SGPRGVHFIF564) and putative Src homology 3 domain binding sites on p22-phox (P156PRPP160 and G177GPPGGP183). To demonstrate that the additional regions identified were biologically significant, peptides mimicking the gp91-phox sequences F77LRGSSACCSTRVRRQL93 and E451WFADLLQLLESQ463 were synthesized and assayed for their ability to inhibit NADPH oxidase activity. These peptides had EC50 values of 1 microM and 230 microM, respectively, and inhibited activation when added prior to assembly but did not affect activity of the preassembled oxidase. Our data demonstrate the usefulness of phage display library analysis for the identification of biologically relevant sites of protein-protein interaction and show that the binding of p47-phox to flavocytochrome b involves multiple binding sites along the C-terminal tails of both gp91- and p22-phox and other regions of gp91-phox nearer to the N terminus.

Complete congruence between gene diversity estimates derived from genotypic data at enzyme and random amplified polymorphic DNA loci in black spruce.

Controversy still exists over the adaptive nature of variation of enzyme loci. In conifers, random amplified polymorphic DNAs (RAPDs) represent a class of marker loci that is unlikely to fall within or be strongly linked to coding DNA. We have compared the genetic diversity in natural populations of black spruce [Picea mariana (Mill.) B.S.P.] using genotypic data at allozyme loci and RAPD loci as well as phenotypic data from inferred RAPD fingerprints. The genotypic data for both allozymes and RAPDs were obtained from at least six haploid megagametophytes for each of 75 sexually mature individuals distributed in five populations. Heterozygosities and population fixation indices were in complete agreement between allozyme loci and RAPD loci. In black spruce, it is more likely that the similar levels of variation detected at both enzyme and RAPD loci are due to such evolutionary forces as migration and the mating system, rather than to balancing selection and overdominance. Furthermore, we show that biased estimates of expected heterozygosity and among-population differentiation are obtained when using allele frequencies derived from dominant RAPD phenotypes.

Coherent electron dynamics in a two-dimensional random system with mobility edges

We study numerically the dynamics of a one-electron wavepacket in a two-dimensional random lattice with long-range correlated diagonal disorder in the presence of a uniform electric field. The time-dependent Schrodinger equation is used for this purpose. We find that the wavepacket displays Bloch-like oscillations associated with the appearance of a phase of delocalized states in the strong correlation regime. The amplitude of oscillations directly reflects the bandwidth of the phase and allows us to measure it. The oscillations reveal two main frequencies whose values are determined by the structure of the underlying potential in the vicinity of the wavepacket maximum.

Critical properties of the four-state commutative random permutation glassy Potts model in three and four dimensions

We investigate the critical properties of the four-state commutative random permutation glassy Potts model in three and four dimensions by means of Monte Carlo simulations and a finite-size scaling analysis. By using a field programmable gate array, we have been able to thermalize a large number of samples of systems with large volume. This has allowed us to observe a spin-glass ordered phase in d=4 and to study the critical properties of the transition. In d=3, our results are consistent with the presence of a Kosterlitz-Thouless transition, but also with different scenarios: transient effects due to a value of the lower critical dimension slightly below 3 could be very important.

Phase Transitions in Disordered Systems: The Example of the Random-Field Ising Model in Four Dimensions

By performing a high-statistics simulation of the D = 4 random-field Ising model at zero temperature for different shapes of the random-field distribution, we show that the model is ruled by a single universality class. We compute to a high accuracy the complete set of critical exponents for this class, including the correction-to-scaling exponent. Our results indicate that in four dimensions (i) dimensional reduction as predicted by the perturbative renormalization group does not hold and (ii) three independent critical exponents are needed to describe the transition.

Random exams using Sweave

The adaptation of the Spanish University to the European Higher Education Area (EEES in Spanish) demands the integration of new tools and skills that would make the teaching- learning process easier. This adaptation involves a change in the evaluation methods, which goes from a system where the student was evaluated with a final exam, to a new system where we include a continuous evaluation in which the final exam may represent at most 50% in the vast majority of the Universities. Devising a new and fair continuous evaluation system is not an easy task to do. That would mean a student’s’ learning process follow-up by the teachers, and as a consequence an additional workload on existing staff resources. Traditionally, the continuous evaluation is associated with the daily work of the student and a collection of the different marks partly or entirely based on the work they do during the academic year. Now, small groups of students and an attendance control are important aspects to take into account in order to get an adequate assessment of the students. However, most of the university degrees have groups with more than 70 students, and the attendance control is a complicated task to perform, mostly because it consumes significant amounts of staff time. Another problem found is that the attendance control would encourage not-interested students to be present at class, which might cause some troubles to their classmates. After a two year experience in the development of a continuous assessment in Statistics subjects in Social Science degrees, we think that individual and periodical tasks are the best way to assess results. These tasks or examinations must be done in classroom during regular lessons, so we need an efficient system to put together different and personal questions in order to prevent students from cheating. In this paper we provide an efficient and effective way to elaborate random examination papers by using Sweave, a tool that generates data, graphics and statistical calculus from the software R and shows results in PDF documents created by Latex. In this way, we will be able to design an exam template which could be compiled in order to generate as many PDF documents as it is required, and at the same time, solutions are provided to easily correct them.

Handling of Uncertainty in Life Cycle Inventory by Correlated Multivariate Lognormal Distributions: Application to the Design of Supply Chain Networks

Poster presented in the 24th European Symposium on Computer Aided Process Engineering (ESCAPE 24), Budapest, Hungary, June 15-18, 2014.

Modifying enzyme activity and selectivity by immobilization

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.

Handling of Uncertainty in Life Cycle Inventory by Correlated Multivariate Lognormal Distributions: Application to the Design of Supply Chain Networks

In this work, we analyze the effect of incorporating life cycle inventory (LCI) uncertainty on the multi-objective optimization of chemical supply chains (SC) considering simultaneously their economic and environmental performance. To this end, we present a stochastic multi-scenario mixed-integer linear programming (MILP) coupled with a two-step transformation scenario generation algorithm with the unique feature of providing scenarios where the LCI random variables are correlated and each one of them has the desired lognormal marginal distribution. The environmental performance is quantified following life cycle assessment (LCA) principles, which are represented in the model formulation through standard algebraic equations. The capabilities of our approach are illustrated through a case study of a petrochemical supply chain. We show that the stochastic solution improves the economic performance of the SC in comparison with the deterministic one at any level of the environmental impact, and moreover the correlation among environmental burdens provides more realistic scenarios for the decision making process.

A copy of the general map the most part compiled from the actual survey by order of the commisioners appointed to settle the partition line between the provinces of New York and New Jersey : manuscript map, 1769

Map showing the whole of New Jersey and its borders with as well as part of Pennsylvania and New York. Map is drawn in black ink with green, pink, and yellow watercolors used to show features such as waterways, borders, and places of interest. Notes on map concern border disputes between New Jersey and New York.

Copy of the return of the writ of partition relating to the lands of Peter Weare & others of Casco Bay : manuscript, [1730]

Document concerns how certain lands around Casco Bay, York Co., Maine, were partitioned in accordance with an order of a writ.