[Comparison between pre- and postnatal echographic screening of malformative uropathies]. / Confronto tra lo screening ecografico pre- e postnatale delle uropatie malformative.

AIM: The aim of this study has been to compare the validity of postnatal echographic screening in respect of prenatal echography in early diagnosis of malformative uropathies (MU). METHODS: In 6578 infants, who have been submitted to fetal echography, and to a postnatal screening of MU in our Neonatal Service of Echography (University of Messina), we have compared the diagnostic agreement of prenatal with postnatal echography. RESULTS: Our comparison demonstrates that, in respect of postnatal screening, only 35.71% of pyelectasies and 73.17% of hydronephrosis have been diagnosed by fetal echography, and, in particular, only 18.75% of no-dilated MU. CONCLUSIONS: These data confirm that, in our country, the postnatal screening of MU has still significance and suggest that, before excluding this screening, it is necessary to verify everywhere the validity of fetal echography.

Hierarchy of simulation models in predicting molecular recognition mechanisms from the binding energy landscapes: structural analysis of the peptide complexes with SH2 domains.

Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.

Incidence of dehydration and hypernatremia in exclusively breast-fed infants.

OBJECTIVES: To verify in exclusively breast-fed, term infants the incidence of hypernatremic dehydration and identify possible maternal and/or infant factors that interfere with successful breast-feeding. STUDY DESIGN: We prospectively included all healthy breast-fed neonates referred to our Neonatology Unit between October 1999 and March 2000. All neonates with a weight loss > or = 10% of birth weight had a breast-feeding test and a determination of serum sodium, urea, and base excess. Student t test and chi-square test were used for statistical analysis of the data. RESULTS: Of 686 neonates, 53 (7.7%) had a weight loss > or = 10% of the birth weight, and 19 also had hypernatremia. These 53 neonates had a significantly higher incidence of caesarean delivery and lower maternal education than neonates with a weight loss < 10%. CONCLUSION: Our prospective study demonstrates that a weight loss > or = 10% during the first days of life is frequent. Daily weight evaluation, careful breast-feeding assessment, and early routine postpartum follow-up are effective methods to prevent hypernatremic dehydration and promote breast-feeding.

Deciphering common failures in molecular docking of ligand-protein complexes.

Common failures in predicting crystal structures of ligand-protein complexes are investigated for three ligand-protein systems by a combined thermodynamic and kinetic analysis of the binding energy landscapes. Misdocked predictions in ligand-protein docking are classified as 'soft' and 'hard' failures. While a soft failure arises when the search algorithm is unable to find the global energy minimum corresponding to the crystal structure, a hard failure results from a flaw of the energy function to qualify the crystal structure as the predicted lowest energy conformation in docking simulations. We find that neither the determination of a single structure with the lowest energy nor finding the most common binding mode is sufficient to predict crystal structures of the complexes, which belong to the category of hard failures. In a proposed hierarchical approach, structural similarity clustering of the conformations, generated from equilibrium simulations with the simplified energy function, is followed by energy refinement with the AMBER force field. This protocol, that involves a hierarchy of energy functions, resolves some common failures in ligand-protein docking and detects crystallographic binding modes that were not found during docking simulations.

Towards understanding the mechanisms of molecular recognition by computer simulations of ligand-protein interactions.

The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.

Solvation studies of DMP323 and A76928 bound to HIV protease: analysis of water sites using grand canonical Monte Carlo simulations.

We examine the water solvation of the complex of the inhibitors DMP323 and A76928 bound to HIV-1 protease through grand canonical Monte Carlo simulations, and demonstrate the ability of this method to reproduce crystal waters and effectively predict water positions not seen in the DMP323 or A76928 structures. The simulation method is useful for identifying structurally important waters that may not be resolved in the crystal structures. It can also be used to identify water positions around a putative drug candidate docked into a binding pocket. Knowledge of these water positions may be useful in designing drugs to utilize them as bridging groups or displace them in the binding pocket. In addition, the method should be useful in finding water sites in homology models of enzymes for which crystal structures are unavailable.

Enzyme-inhibitor association thermodynamics: explicit and continuum solvent studies.

Studying the thermodynamics of biochemical association reactions at the microscopic level requires efficient sampling of the configurations of the reactants and solvent as a function of the reaction pathways. In most cases, the associating ligand and receptor have complementary interlocking shapes. Upon association, loosely connected or disconnected solvent cavities at and around the binding site are formed. Disconnected solvent regions lead to severe statistical sampling problems when simulations are performed with explicit solvent. It was recently proposed that, when such limitations are encountered, they might be overcome by the use of the grand canonical ensemble. Here we investigate one such case and report the association free energy profile (potential of mean force) between trypsin and benzamidine along a chosen reaction coordinate as calculated using the grand canonical Monte Carlo method. The free energy profile is also calculated for a continuum solvent model using the Poisson equation, and the results are compared to the explicit water simulations. The comparison shows that the continuum solvent approach is surprisingly successful in reproducing the explicit solvent simulation results. The Monte Carlo results are analyzed in detail with respect to solvation structure. In the binding site channel there are waters bridging the carbonyl oxygen groups of Asp189 with the NH2 groups of benzamidine, which are displaced upon inhibitor binding. A similar solvent-bridging configuration has been seen in the crystal structure of trypsin complexed with bovine pancreatic trypsin inhibitor. The predicted locations of other internal waters are in very good agreement with the positions found in the crystal structures, which supports the accuracy of the simulations.

Structure-based drug design: computational advances.

Structure-based computational methods continue to enhance progress in the discovery and refinement of therapeutic agents. Several such methods and their applications are described. These include molecular visualization and molecular modeling, docking, fragment methods, 3-D database techniques, and free-energy perturbation. Related issues that are discussed include the use of simplified potential energy functions and the determination of the positions of tightly bound waters. Strengths and weaknesses of the various methods are described.

Theoretical study of inhibition of adenosine deaminase by (8R)-coformycin and (8R)-deoxycoformycin.

Molecular dynamics and free energy simulations were performed to examine the binding of (8R)-deoxycoformycin and (8R)-coformycin to adenosine deaminase. The two inhibitors differ only at the 2' position of the sugar ring; the sugar moiety of conformycin is ribose, while it is deoxyribose for deoxycoformycin. The 100 ps molecular dynamics trajectories reveal that Asp 19 and His 17 interact strongly with the 5' hydroxyl group of the sugar moiety of both inhibitors and appear to play an important role in binding the sugar. The 2' and 3' groups of the sugars are near the protein-water interface and can be stabilized by either protein residues or water. The flexibility of the residues at the opening of the active site helps to explain the modest difference in binding of the two inhibitors and how substrates/inhibitors can enter an otherwise inaccessible binding site.

Computational science new horizons and relevance to pharmaceutical design.

Computer methods are used extensively in the design and refinement of drug leads. A short summary is given for several computational methods followed by a description of how some of these methods have been applied to design drugs targeted to the renin-angiotensin system and to cholinergic synapses. These methods include quantitative structure-activity relationship (QSAR) methods, comparative molecular field analyses (CoMFA), 3D database searching, de novo design of ligands, docking, and computational alchemy [free energy perturbation (FEP) and thermodynamic integration (MCTI)]. Most of these methods can be used whether or not detailed structural information about the binding site is available, although without an x-ray structure, the analyses are more qualitative. All of these methods are used extensively in the commercial design of pharmaceuticals. The main problem with most of these methods is in the scoring (ranking) of interactions or matches. Advances in this area and others (methods development and increases in capabilities of computers) will increase the predictive power of these methods and help to speed the time to market of new pharmaceuticals. (Trends Cardiovasc Med 1996;6:198-203).

Infection potential of nondisposable pressure transducers prepared prior to use.

This study was designed to examine whether pressure transducer systems prepared hours or days before actual use represent an infection potential. Twenty-one nondisposable transducers assembled with sterile plastic disposable domes and extension tubing were utilized. The systems were prefilled with heparinized flush solution (2 U sodium heparin per ml normal saline). Flush solution samples were cultured from the distal end of the equipment immediately after set-up (time 0) and 24, 48, and 72 h after set-up in all 21 transducers. In 11 of 21 transducers, cultures were also taken at 168 h (1 wk) and 336 h (2 wk) after set-up. Out of the total 106 cultures, only two showed growth, both of which proved to be external skin or mouth contaminants. Binomial distribution testing indicated that if a .05 probability level was used, the chance was 1.3% that one or more transducers in 100 could become contaminated by 72 h. Using the same statistical analyses for the 66 cultures involving the 11 transducers cultured for 2 wk, the chance of contamination in 100 cultures is 2.7% at 2 wk. We conclude from these data that nondisposable transducer systems, when preassembled using standard aseptic precautions and sterile disposable plastics, show no evidence of significant contamination when allowed to stand in readiness after assembly but not connected to a patient. We feel it is safe to preassemble transducers for at least a 72-h period before use. Longer intervals from set-up to use may also be justified.