Expanding Horizons in the Validation of GMO Analytical Methods: Fuzzy-based Expert Systems

Validation is the process establishing the suitability of an analytical method for a particular purpose. Various guidelines defining statistical procedures for validation of chemical, bio-chemical, pharmaceutical, and molecular methods have been developed, and ad hoc validation metrics (indices and test statistics) are available and routinely used, for in-house and interlaboratory testing and decision making. However, there is no universally accepted practice for assay validation, and often, subjectivity plays an important role in the interpretation of validation studies’ results. Instead, the key to rational validation studies relies upon the formalization and harmonization of procedures for their design and interpretation of results. Fuzzy-based techniques can be helpful in such respect. Fuzzy logic allows summarizing the information obtained by classic independent validation statistics into one synthetic index of overall method performance. The possibility of having a comprehensive indicator of method performance has the advantage of permitting direct method comparison, facilitating the evaluation of many individual, possibly contradictory metrics. The objectives of this paper are to illustrate the advantages that a fuzzy-based aggregation method could bring into the validation of analytical methods and to propose its application for the evaluation of methods’ performance. Validation metrics are compared for practical examples of assessment of method performance in collaborative studies. Fuzzy logic-based rules are shown to be applicable to improve insights into method quality and interpretation of results.     

Eliminating nuisance parameters: two characterizations

Given a statistical modelP = {P_θ : θ ∈ x} and a surjective functiong: ϑ→Λ the problem of transformingP into a new modelQ= {_λ : λ ∈ Λ} indexed by Λ is investigated. Two characterizations are given for those modelsQ of the form Q_λ = ∫ P_θ π_λ(dθ), where π_λ is some probability such that π_λ(g=λ)=1. The first is related to a geometric property ofQ, while the second rests on the inferential implications of adoptingQ. Also, in the first π_λ is allowed to be finitely additive, while in the second π_λ is σ-additive. Finally, integrated likelihoods are revisited in light of the second characterization.     

Genotype-dependent effects of septal lesions on different types of learning in the mouse.

Tested 80 C57BL/6J, DBA/2J, and SEC/1ReJ mice with septal or control lesions for exploratory behavior, shuttle-box avoidance learning, discriminated avoidance, and maze-learning ability. Control DBA and SEC Ss normally displayed low levels of exploratory behavior and efficient avoidance and maze learning. High exploratory activity and low avoidance and maze learning were characteristically shown by C57 controls. All strains with septal lesions increased levels of exploratory and avoidance behaviors. In contrast, following septal lesions the 3 strains performed more poorly in discriminated-avoidance and maze learning. It is concluded that differences in septal function-e.g., in level of response inhibition-do not substantially account for the learning differences evident between these strains. (19 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Multiple correspondence analysis in S-PLUS

Multiple correspondence analysis (MCA) is a multivariate method for analyzing multidimensional contingency tables. General software procedures to perform MCA are available. Among them SAS Proc CORRESP, SPAD CORMU procedure and the mca function of the MASS library in S-PLUS are probably the most used. However, CORRESP and CORMU output is different from that of mca function. The aim of this short note is showing how to obtain from mca function results compatible with those achieved with SAS or SPAD. A modified code is proposed in order to obtain the same coordinate system computed by SAS and SPAD. Moreover, the computation of the contributions of the levels of the factors to the inertia explained by each axis, the squared cosine of each factor level and the re-evaluation of the inertia explained by each axis have been added in order to improve the interpretations of the results of the decomposition.     

Determination of primary and secondary standards and characterization of appropriate salt bridges for pH measurements in formamide

For the first time, the standardization for pH measurements is here implemented in the domain of superpermittive media. The nonaqueous solvent studied is formamide (epsilon = 109.5 at 298.15 K) for which three primary standards and two secondary standards have been determined, whose excellent internal consistency has also been demonstrated by specific cell measurements. A comparison of the pH scale in formamide with the aqueous scale has been duly tried by accounting for the primary medium effect on the H+ ion. Furthermore, as a result of an ad hoc supplementary systematic investigation, three salt bridges of appropriate level of equitransference in formamide, that is, NH(4)Cl, NH(4)Br, and NH(4)I, have been characterized for abating the liquid junction potentials intervening in the pH-measuring cell to enable the user to carry out regular pH measurements and related controls.     

Chemical synthesis of the RGD-protein decorsin: Pro-->Ala replacement reduces protein thermostability

Decorsin is a 39-residue polypeptide chain, crosslinked by three disulfide bridges, that strongly inhibits platelet aggregation. We report the chemical synthesis and characterization of analogs of decorsin with the aim of investigating the role of proline residues in protein structure, stability and biological activity. Decorsin analogs have been synthesized in which one (P23A and P24A decorsin) or two (P23,24A decorsin) proline residues have been substituted by alanine. The crude synthetic polypeptides were purified by reversed-phase HPLC in their reduced form and allowed to refold oxidatively to their disulfide-crosslinked species. The homogeneity of the synthetic mini-proteins, and also the correct pairing of the three disulfide bridges, were established by a number of analytical criteria, including fingerprinting analysis of the refolded synthetic analogs by using thermolysin and proteinase K as proteolytic enzymes. Replacement of proline by alanine results in a significant and cumulative decrease of the high thermal stability (Tm 74 degrees C) of native decorsin. The mono-substituted analogs display a Tm of 66-67 degrees C, while the double-substituted analog a Tm of 50 degrees C. On the other hand, the overall secondary and tertiary structures were not affected by the Pro-->Ala exchanges, as judged from circular dichroism measurements. Platelet aggregation assays established that the proline substitutions do not impair significantly the biological activity of decorsin. The results of this study clearly indicate that proline residues contribute significantly to the protein thermal stability. Our results are in line with the 'proline rule', previously advanced for explaining the unusual thermal stability of thermophilic enzymes, which usually show an enhanced content of proline residues with respect to their mesophilic counterparts.     

Clinical and Molecular Diagnosis of Beckwith-Wiedemann Syndrome with Single- or Multi-Locus Imprinting Disturbance

Beckwith-Wiedemann syndrome (BWS) is a clinically and genetically heterogeneous overgrowth disease. BWS is caused by (epi)genetic defects at the 11p15 chromosomal region, which harbors two clusters of imprinted genes, IGF2/H19 and CDKN1C/KCNQ1OT1, regulated by differential methylation of imprinting control regions, H19/IGF2:IG DMR and KCNQ1OT1:TSS DMR, respectively. A subset of BWS patients show multi-locus imprinting disturbances (MLID), with methylation defects extended to other imprinted genes in addition to the disease-specific locus. Specific (epi)genotype-phenotype correlations have been defined in order to help clinicians in the classification of patients and referring them to a timely diagnosis and a tailored follow-up. However, specific phenotypic correlations have not been identified among MLID patients, thus causing a debate on the usefulness of multi-locus testing in clinical diagnosis. Finally, the high incidence of BWS monozygotic twins with discordant phenotypes, the high frequency of BWS among babies conceived by assisted reproductive technologies, and the female prevalence among BWS-MLID cases provide new insights into the timing of imprint establishment during embryo development. In this review, we provide an overview on the clinical and molecular diagnosis of single- and multi-locus BWS in pre- and post-natal settings, and a comprehensive analysis of the literature in order to define possible (epi)genotype-phenotype correlations in MLID patients.     

15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells

Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL₃ on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL₃ failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.