New statistical software for intralaboratory and interlaboratory quality control in clinical cytology. Validation in a simulation study on clinical samples

OBJECTIVE: To design a statistical software package to provide automated calculations of normal and weighted and 3 indices. STUDY DESIGN: Prompted by the lack of commonly available software to compute weighted kappa and the nonproportionate workload needed to calculate our 3 variability indices manually, the new statistical software package was designed. To demonstrate the performance of the new CONQUISTADOR software, a simulation study (both intralaboratory and interlaboratory) was designed using 5,000 clinical samples randomly selected from a data file of > or = 200,000 conventional Pap smears and programmed to become "analyzed" by 12 cytologists in 5 imaginary laboratories. RESULTS: A representative set of both complete and partial outputs provided by the software, in Excel format (Microsoft, Redmond, Washington, U.S.A.) are shown to illustrate the different functions of the program. In the interlaboratory mode, the software calculates accuracy indicators (sensitivity, specificity, positive and negative predictive value, and their 95% CI), which are not common features of regular statistical packages; kappa and weighted kappa; and their 95% CI (comparison of single laboratories to all laboratories and pairwise comparisons between single laboratories). The 3 diagnostic variability indices can be computed separately for all samples or for only the positive samples. In the intralaboratory mode, the software calculates the same indices for individual cytologists. CONCLUSION: The CONQUISTADOR statistical package has properties that are useful in monitoring cytologic laboratory quality in both intralaboratory and interlaboratory settings. The software will be distributed by the National Institute of Health, Rome, for the delivery costs only.     

Lipase-catalysed synthesis of new acetylcholinesterase inhibitors: N-benzylpiperidine aminoacid derivatives

New acetylcholinesterase inhibitors were synthetized via a lipase-mediated regioselective amidation using Candida antarctica lipase B as a biocatalyst in the key step. The new compounds have two different structural fragments: a N-benzylpiperidine moiety to anchor the enzyme active site and a dicarboxylic aminoacid to act as a biological carrier. Some analogues of N-benzylpiperazine were also synthesised and studied but they did not display AChE inhibitor activity. A preliminary structure activity relationship study was performed employing some computational techniques as similarity indices and electrostatic potential maps.     

A two-stage probabilistic approach to multiple-community similarity indices

SUMMARY: A traditional approach for assessing similarity among N (N > 2) communities is to use multiple pairwise comparisons. However, pairwise similarity indices do not completely characterize multiple-community similarity because the information shared by at least three communities is ignored. We propose a new and intuitive two-stage probabilistic approach, which leads to a general framework to simultaneously compare multiple communities based on abundance data. The approach is specifically used to extend the commonly used Morisita index and NESS (normalized expected species shared) index to the case of N communities. For comparing N communities, a profile of N- 1 indices is proposed to characterize similarity of species composition across communities. Based on sample abundance data, nearly unbiased estimators of the proposed indices and their variances are obtained. These generalized NESS and Morisita indices are applied to comparison of three size classes of plant data (seedling, saplings, and trees) within old-growth and secondary rain forest plots in Costa Rica.     

An extension of presence/absence coefficients to abundance data: a new look at absence

Abstract. Alternative community analyses, based on quantitative and presence/absence data, are comparable logically if the data type is the only factor responsible for differences among results. For presence/absence indices that consider mutual absences, no quantitative alternatives are known. To facilitate such comparisons, a new family of similarity coefficients is proposed for abundance data. Formally, this extension is achieved by generalizing the four cells of the usual 2 × 2 contingency table to the quantitative case. This implies an expanded meaning of absence: for a given species at a given site it is understood as the difference between the actual value and the maximum detected in the entire study. The correspondence between 10 presence/absence coefficients and their quantitative counterparts is evaluated by graphical comparisons based on artificial data. The behaviour of the new functions is also examined using field data representing post‐fire regeneration processes in grasslands and a chronosequence pertaining to forest regeneration after clear‐cut. The examples suggest that the new coefficients are most informative for data sets with low beta‐diversity and temporal background changes.     

Electrostatic Similarities between Protein and Small Molecule Ligands Facilitate the Design of Protein-Protein Interaction Inhibitors

One of the underlying principles in drug discovery is that a biologically active compound is complimentary in shape and molecular recognition features to its receptor. This principle infers that molecules binding to the same receptor may share some common features. Here, we have investigated whether the electrostatic similarity can be used for the discovery of small molecule protein-protein interaction inhibitors (SMPPIIs). We have developed a method that can be used to evaluate the similarity of electrostatic potentials between small molecules and known protein ligands. This method was implemented in a software called EleKit. Analyses of all available (at the time of research) SMPPII structures indicate that SMPPIIs bear some similarities of electrostatic potential with the ligand proteins of the same receptor. This is especially true for the more polar SMPPIIs. Retrospective analysis of several successful SMPPIIs has shown the applicability of EleKit in the design of new SMPPIIs.     

Discriminating D1 and D2 agonists with a hydrophobic similarity index

Currently, methods for calculating molecular similarity indices have been developed for comparing steric, charge density, and molecular electrostatic potential (MEP) properties. Much of the existing technology may, however, be applied to the quantitative comparison of molecular hydrophobicities. In this article we present an empirical hydrophobic similarity index. We utilize atomic hydrophobic parameters derived from a quantum mechanical semiempirical wavefunction. Hydrophobicity at points on a grid is computed with a recently introduced “molecular lipophilicity potential”. The overlap of pairs of molecules is calculated with the metric introduced by Carbó. This approach is applied to a case in which steric and electrostatic criteria have already been shown to be inadequate in rationalizing selectivity, namely, requirements for recognition at the dopamine D1 and D2 receptors. We demonstrate that, for a set of dopamine agonists, D1 ligands show higher similarity in this property than D2 analogs. This indicator of similarity is more successful at accounting for D1 selectivity than previous methods.     

Identification of protein biochemical functions by similarity search using the molecular surface database eF-site

The identification of protein biochemical functions based on their three-dimensional structures is strongly required in the post-genome-sequencing era. We have developed a new method to identify and predict protein biochemical functions using the similarity information of molecular surface geometries and electrostatic potentials on the surfaces. Our prediction system consists of a similarity search method based on a clique search algorithm and the molecular surface database eF-site (electrostatic surface of functional-site in proteins). Using this system, functional sites similar to those of phosphoenoylpyruvate carboxy kinase were detected in several mononucleotide-binding proteins, which have different folds. We also applied our method to a hypothetical protein, MJ0226 from Methanococcus jannaschii, and detected the mononucleotide binding site from the similarity to other proteins having different folds.     

The Two Sides of Complement C3d: Evolution of Electrostatics in a Link between Innate and Adaptive Immunity

The interaction between complement fragment C3d and complement receptor 2 (CR2) is a key aspect of complement immune system activation, and is a component in a link between innate and adaptive immunities. The complement immune system is an ancient mechanism for defense, and can be found in species that have been on Earth for the last 600 million years. However, the link between the complement system and adaptive immunity, which is formed through the association of the B-cell co-receptor complex, including the C3d-CR2 interaction, is a much more recent adaptation. Human C3d and CR2 have net charges of −1 and +7 respectively, and are believed to have evolved favoring the role of electrostatics in their functions. To investigate the role of electrostatics in the function and evolution of human C3d and CR2, we have applied electrostatic similarity methods to identify regions of evolutionarily conserved electrostatic potential based on 24 homologues of complement C3d and 4 homologues of CR2. We also examine the effects of structural perturbation, as introduced through molecular dynamics and mutations, on spatial distributions of electrostatic potential to identify perturbation resistant regions, generated by so-called electrostatic “hot-spots”. Distributions of electrostatic similarity based on families of perturbed structures illustrate the presence of electrostatic “hot-spots” at the two functional sites of C3d, while the surface of CR2 lacks electrostatic “hot-spots” despite its excessively positive nature. We propose that the electrostatic “hot-spots” of C3d have evolved to optimize its dual-functionality (covalently attaching to pathogen surfaces and interaction with CR2), which are both necessary for the formation B-cell co-receptor complexes. Comparison of the perturbation resistance of the electrostatic character of the homologues of C3d suggests that there was an emergence of a new role of electrostatics, and a transition in the function of C3d, after the divergence of jawless fish.     

Immunophysical properties and prediction of activities for vaccinia virus complement control protein and smallpox inhibitor of complement enzymes using molecular dynamics and electrostatics

We present immunophysical modeling for VCP, SPICE, and three mutants using MD simulations and Poisson-Boltzmann-type electrostatic calculations. VCP and SPICE are homologous viral proteins that control the complement system by imitating, structurally and functionally, natural regulators of complement activation. VCP and SPICE consist of four CCP modules connected with short flexible loops. MD simulations demonstrate that the rather complex modules of VCP/SPICE and their mutants exhibit a high degree of intermodular spatial mobility, which is affected by surface mutations. Electrostatic calculations using snapshots from the MD trajectories demonstrate variable spatial distribution of the electrostatic potentials, which suggests dynamic binding properties. We use covariance analysis to identify correlated modular oscillations. We also use electrostatic similarity indices to cluster proteins with common electrostatic properties. Our results are compared with experimental data to form correlations between the overall positive electrostatic potential of VCP/SPICE with binding and activity. We show how these correlations can be used to predict binding and activity properties. This work is expected to be useful for understanding the function of native CCP-containing regulators of complement activation and receptors and for the design of antiviral therapeutics and complement inhibitors.     

The beauty of molecular surfaces as revealed by self-organizing neural networks

The Kohonen neural network is a self-organizing network that can be used for the projection of the surface properties of molecules. This allows one to view properties on a molecular surface, like the electrostatic potential in a single picture. These maps are useful for the comparison of molecules and provide a new definition of molecular similarity.     

Exhaustive comparison and classification of ligand‐binding surfaces in proteins

Many proteins function by interacting with other small molecules (ligands). Identification of ligand‐binding sites (LBS) in proteins can therefore help to infer their molecular functions. A comprehensive comparison among local structures of LBSs was previously performed, in order to understand their relationships and to classify their structural motifs. However, similar exhaustive comparison among local surfaces of LBSs (patches) has never been performed, due to computational complexity. To enhance our understanding of LBSs, it is worth performing such comparisons among patches and classifying them based on similarities of their surface configurations and electrostatic potentials. In this study, we first developed a rapid method to compare two patches. We then clustered patches corresponding to the same PDB chemical component identifier for a ligand, and selected a representative patch from each cluster. We subsequently exhaustively as compared the representative patches and clustered them using similarity score, PatSim. Finally, the resultant PatSim scores were compared with similarities of atomic structures of the LBSs and those of the ligand‐binding protein sequences and functions. Consequently, we classified the patches into ～2000 well‐characterized clusters. We found that about 63% of these clusters are used in identical protein folds, although about 25% of the clusters are conserved in distantly related proteins and even in proteins with cross‐fold similarity. Furthermore, we showed that patches with higher PatSim score have potential to be involved in similar biological processes.     

Species dependence of enzyme-substrate encounter rates for triose phosphate isomerases

Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate–protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate. Proteins 31:406–416, 1998. © 1998 Wiley-Liss, Inc.     

A new protein folding recognition potential function

On the study of protein inverse folding problem, one goal is to find simple and efficient potential to evaluate the compatibility between structure and a given sequence. We present here a novo empirical mean force potential to address the importance of electrostatic interactions in protein inverse folding study. It is based on protein main chain polar fraction and constructed in a way similar with Sippl's from a database of 64 known independent three-dimensional protein structures. This potential was applied to recognize the protein native conformations among a conformation pool. Calculated results show that this potential is powerful in picking out native conformations, in addition it can also find structure similarity between proteins with low sequence similarity. The success of this new potential clearly shows the importance of electrostatic factors in protein inverse folding studies. © 1995 Wiley-Liss, Inc.     

Individual typological features reflected in different stages of learning and related changes in dopamine transport in the mesolimbic brain

A hypothesis is proposed according to which there is a strong similarity between the individual typological features of animals (humans) and different stages of learning processes. The hypothesis is supported by the results of comparisons of psychophysiological and neurochemical indices of different stages of conditioning and various typologies of higher nervous activity. Such comparisons reveal a high similarity between the generalization phase and impulsivity, the phase a learned conditioned reflex and self-controlled behaviour, and overlearned CR and a state of "psychosis". Functional states close to pathology and possibilities of development and manifestation of several psychotic states depending on psychophysiological and neurochemical organization of learning processes are also considered.     

Identification of free fatty acid receptor 2 agonists using virtual screening

Structure- and ligand-based virtual-screening methods (docking, 2D- and 3D-similarity searching) were analyzed for their effectiveness in virtual screening against FFAR2. To evaluate the performance of these methods, retrospective virtual screening was performed. Statistical quality of the methods was evaluated by BEDROC and RIE. The results revealed that electrostatic similarity search protocol using EON (ET combo) outperformed all other protocols with outstanding enrichment of >95% in top 1% and 2% of the dataset with an AUC of 0.958. Interestingly, the hit lists that are obtained from different virtual-screening methods are generally highly complementary to hits found from electrostatic similarity searching. These results suggest that considering electrostatic similarity searching first increases the chance of identifying more (and more diverse) active compounds from a virtual-screening campaign. Accordingly, prospective virtual screening using electrostatic similarity searching was used to identify novel FFAR2 ligands. The discovered compounds provide new chemical matter starting points for the initiation of a medicinal chemistry campaign.     

Electrostatic Potential of B-DNA: Effect of Interionic Correlations

Modified Poisson-Boltzmann (MPB) equations have been numerically solved to study ionic distributions and mean electrostatic potentials around a macromolecule of arbitrarily complex shape and charge distribution. Results for DNA are compared with those obtained by classical Poisson-Boltzmann (PB) calculations. The comparisons were made for 1:1 and 2:1 electrolytes at ionic strengths up to 1 M. It is found that ion-image charge interactions and interionic correlations, which are neglected by the PB equation, have relatively weak effects on the electrostatic potential at charged groups of the DNA. The PB equation predicts errors in the long-range electrostatic part of the free energy that are only ∼1.5 kJ/mol per nucleotide even in the case of an asymmetrical electrolyte. In contrast, the spatial correlations between ions drastically affect the electrostatic potential at significant separations from the macromolecule leading to a clearly predicted effect of charge overneutralization.     

Genetic relationships among Neisseria species assessed by comparative enzyme electrophoresis

The electrophoretic mobilities of 12 enzymes from 19 Neisseria species (including 6 strains of N. perflava), Gemella haemolysans, Escherichia coli and Branhamella catarrhalis were characterized by polyacrylamide slab gel electrophoresis. All strains and species tested exhibited qualitatively different zymogram patterns. Species and strain relationships were quantified by pairwise comparisons of all 12 enzyme systems to obtain similarity indices; these data were subjected to numerical clustering methods to obtain groups and a phenogram. The electrophoretic classification compared favourably with those obtained by other criteria. In addition, the quantitative clustering data indicated that N. ovis and N. caviae are sufficiently different from the other Neisseria species to warrant their separation into a distinct group. These two species also lacked the characteristic NADPH-diaphorase zymogram pattern found in all the other Neisseria species. Intra-species similarity indices were generally greater than the inter-species index values. However, certain species such as N. meningitidis and N. gonorrhoeae had similarity index values in the range of inter-strain index values.