Validation of a nutrition knowledge questionnaire in Italian students attending the University of Parma.

OBJECTIVE: The aim of this study was to assess the validity and reliability of a self-administered nutrition knowledge (NK) questionnaire for Italian university students. DESIGN: The NK questionnaire included ninety questions on experts' nutritional recommendations, nutritional content of food, health aspects of food and diets, relationship between diet and diseases, and proper food choices. It was administered to the same population under the same conditions on two different occasions with a time interval of 3 weeks between the two administrations. SETTING: The survey was carried out at the University of Parma (Italy) during the 2018-2019 academic year. PARTICIPANTS: Data were collected for 132 bachelor and master degree students attending the University of Parma, either attending or not nutrition classes during their studies (19-30 years, 29·5 % males, 57·6 % with an academic nutrition background). RESULTS: The questionnaire revealed high overall internal consistency reliability (Cronbach's α > 0·8) and a good temporal stability with high correlation of the total score (r = 0·835, P < 0·001). Moreover, it showed a good ability to discriminate between subjects with potentially different NK. CONCLUSIONS: This NK questionnaire proved to be a reliable, valid and easy-to-use tool for assessing the NK of Italian university students, either with or without nutrition background.

Local conformational changes in the 8-17 deoxyribozyme core induced by activating and inactivating divalent metal ions.

The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational change was generally hyperbolic (suggesting it mirrored the binding by a single metal ion) and yielded apparent dissociation constants close to those required for activation. In contrast, Cu2+, a divalent metal ion which does not support catalysis, caused in the deoxyribozyme a slow, reversible inactivation, which correlated with a very distinct conformational change at position 15.

1H[19F] NOE NMR structural signatures of the insulin R6 hexamer: evidence of a capped HisB10 site in aryl- and arylacryloyl-carboxylate complexes.

NEW AND IMPROVED INSULIN: 1H[19F] NOE NMR difference spectra for CF(3)-substituted aromatic carboxylates bound at the HisB10 sites of the R(6) human insulin (HI) hexamer show strong NOEs between the CF(3) groups and the LeuB6, AsnB3, and PheB1 sidechains. The NOEs and structural modeling establish that these carboxylates form closed complexes with the HisB10 site capped by the PheB1 rings.

A mutational analysis of the 8-17 deoxyribozyme core.

The 8-17 deoxyribozyme is a small RNA-cleaving DNA enzyme of significant applicative interest. We measured the kinetics of over 60 variants of 8-17, mutated within the "core" region. The data were analyzed according to a conceptual framework in which deleterious substitutions can either decrease the stability of the reaction's transition state, or favor unreactive ground-state conformations. In agreement with earlier in vitro evolution studies, the most severe functional effects were observed upon mutating four conserved residues, whose role was further explored by replacing them with non-standard nucleotides. Removal or modification of individual functional groups on the A6 and G7 bases suggested that these residues are involved in a close-contact interaction and form a network of functionally important hydrogen bonds. Mutagenesis of residues C13 and G14 was less revealing, but argued strongly against a role of C13 as a general acid/base catalyst. The use of non-standard nucleotides also led to the identification of one deoxyribozyme variant that, under some ionic conditions, is substantially more active than the wild-type construct. Finally, the effects of mutations in the intramolecular "core stem" correlated only in part with changes in helical stability, suggesting that a stable stem is required but not sufficient for optimal activity.

Insulin allosteric behavior: detection, identification, and quantification of allosteric states via 19F NMR.

The insulin hexamer is an allosteric protein widely used in formulations for the treatment of diabetes. The hexamer exhibits positive and negative cooperativity and apparent half-site binding activity, reflecting the interconversion of three allosteric states, designated as T6, T3R3, and R6. The hexamer contains two symmetry-related Zn2+ located 16 A apart on the 3-fold symmetry axis. In the transition of T3 units to R3 units, Zn2+ switches from an octahedral Zn2+ N3O3 complex (N is HisB10, O is H2O) to a distorted tetrahedral Zn2+ N3L complex (L is a monovalent anion). Hence, monovalent anions are allosteric ligands that stabilize R3 units of T3R3 and R6. Herein, we exploit the high sensitivity of 19F NMR chemical shifts and fluorinated carboxylates to reveal subtle differences in the anion-binding sites of T3R3 and R6. We show that the chemical shifts of 4- and 3-trifluoromethylbenzoate and 4- and 2-trifluoromethylcinnamate give bound resonances that distinguish between T3R3 and R6. 3-Trifluoromethylbenzoate and 2-trifluoromethylcinnamate also were shown to bind to the R3 units of T3R3 and R6 in two alternative, slowly interconverting modes with different microenvironments for the CF3 groups. Line width analysis shows that ligand off rates are slower by 1/10(3) than the diffusion limit, indicating a rate-limiting protein conformational transition. These studies confirm that the Seydoux, Malhotra, and Bernhard allosteric model (Bloom, C. R., Choi, W. E., Brzovic, P. S., Ha, J. J., Huang, S. T., Kaarsholm, N. C., and Dunn, M. F. (1995). J. Mol. Biol. 245, 324-330), provides a robust description of the insulin hexamer.

Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme.

The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechanism. The 8-17 bound its substrate with a rate constant approximately 10-fold lower than those typical for the annealing of short complementary oligonucleotides. The observed free energy of substrate binding indicates that an energetic penalty near to +7 kcal/mol is attributable to the deoxyribozyme core. Substrate cleavage required divalent metal ion cofactors, and the dependence of activity on the concentration of Mg2+, Ca2+ or Mn2+ suggests the occurrence of a single, low-specificity binding site for activating ions. The efficiency of activation correlated with the Lewis acidity of the ion cofactor, compatible with a metal-assisted deprotonation of the reactive 2'-hydroxyl group. However, alternative roles of the metal ions cannot be excluded, because those ions that are stronger Lewis acids are also capable of forming stronger interactions with ligands such as the phosphate oxygens. The apparent enthalpy of activation for the 8-17 reaction was close to the values observed for hydroxide-catalyzed and hammerhead ribozyme-catalyzed RNA cleavage.