Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Electrochemical chlorination of azulene derivatives: Insight into the mechanism of anodic oxidative chlorination

The cyclic voltammetry behavior of a series of five 1-substituted azulenes has been investigated as well as the electrochemical chlorination of these compounds. In the case of azulene compounds containing electron withdrawing groups which have higher oxidation potentials than that of the chloride ion, the electrochemical chlorination led usually to 3-chloro derivatives. The electrochemical chlorination fails for azulenes with lower oxidation potentials. Additionally some polyhalogenated compounds were obtained by controlled potential electrolyses. The paper also discusses the reaction mechanism of the electrochemical halogenation of 1-substituted azulenes.     

Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.     

Event-driven,pattern-based methodology for cost-effective development of standardized personal health devices

Experiences applying standards in personal health devices (PHDs) show an inherent trade-off between interoperability and costs (in terms of processing load and development time). Therefore, reducing hardware and software costs as well as time-to-market is crucial for standards adoption. The ISO/IEEE11073 PHD family of standards (also referred to as X73PHD) provides interoperable communication between PHDs and aggregators. Nevertheless, the responsibility of achieving inexpensive implementations of X73PHD in limited resource microcontrollers falls directly on the developer. Hence, the authors previously presented a methodology based on patterns to implement X73-compliant PHDs into devices with low-voltage low-power constraints. That version was based on multitasking, which required additional features and resources. This paper therefore presents an event-driven evolution of the patterns-based methodology for cost-effective development of standardized PHDs. The results of comparing between the two versions showed that the mean values of decrease in memory consumption and cycles of latency are 11.59% and 45.95%, respectively. In addition, several enhancements in terms of cost-effectiveness and development time can be derived from the new version of the methodology. Therefore, the new approach could help in producing cost-effective X73-compliant PHDs, which in turn could foster the adoption of standards.     

Energy efficient and reliable data delivery in urban sensing applications: A performance analysis

Urban sensing is an emerging application field for Wireless Sensor Networks (WSNs), where a number of static sensors is sparsely deployed in an urban area to collect environmental information. Data sensed by each sensor are, then, opportunistically transmitted to Mobile Nodes (MNs) that happen to be in contact. In the considered scenario, communications between MNs and sensors require paradigms with a minimal synchronization between devices, extremely fast and energy efficient, especially at the sensor side. To deal with the above issues, in [1] we proposed a hybrid protocol for data delivery from sensors to MNs, named Hybrid Adaptive Interleaved Data Protocol (HI). By combining Erasure Coding (EC) with an Automatic Repeat reQuest (ARQ) scheme, the proposed protocol maximizes the reliability of communications while minimizing the energy consumed by sensors. In this paper, we present an in-depth analysis of the HI performance. We provide an analytical evaluation by defining a flexible model to derive the probability of data delivery and exploiting it to investigate the performance over a wide range of parameters. Moreover, we perform an experimental study to evaluate the HI effectiveness on real sensor platforms. Specifically, we analyze the impact of resource constraints imposed by sensors on data delivery and provide a careful characterization of its actual consumption of resources.     

Driving business performance: innovation complementarities and persistence patterns

Complementarities between technological and non-technological innovation are crucial determinants of firm performance. Although innovation complementarity has been extensively tested in the empirical literature, it has not been analysed in conjunction with innovation persistence. This fact is mainly due to the lack of data sets able to provide adequate longitudinal information. The capacities to develop market-oriented behaviour and introduce new organisational innovations, together with technological innovation, are the drivers of a firm’s productivity and profitability. We find that these activities complement technological innovation and that their impact is greater when they persist over time, thus introducing a more general concept of innovation persistence. We present an empirical model based on a large new panel of Italian manufacturing firms covering the period 2000–2012 which enables us to determine the precise impacts of a firm’s innovative attitude, in a broad definition that incorporates non-technological innovation and persistence, on its productivity and profitability.     

Proteins as biomarkers of oxidative/nitrosative stress in diseases: the contribution of redox proteomics

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) contribute to the pathogenesis and/or progression of several human diseases. Proteins are important molecular signposts of oxidative/nitrosative damage. However, it is generally unresolved whether the presence of oxidatively/nitrosatively modified proteins has a causal role or simply reflects secondary epiphenomena. Only direct identification and characterization of the modified protein(s) in a given pathophysiological condition can decipher the potential roles played by ROS/RNS-induced protein modifications. During the last few years, mass spectrometry (MS)-based technologies have contributed in a significant way to foster a better understanding of disease processes. The study of oxidative/nitrosative modifications, investigated by redox proteomics, is contributing to establish a relationship between pathological hallmarks of disease and protein structural and functional abnormalities. MS-based technologies promise a contribution in a new era of molecular medicine, especially in the discovery of diagnostic biomarkers of oxidative/nitrosative stress, enabling early detection of diseases. Indeed, identification and characterization of oxidatively/nitrosatively modified proteins in human diseases has just begun.     

Parallel enzymatic and non-enzymatic formation of zinc protoporphyrin IX in pork

Zinc protoporphyrin is formed in pork homogenates in both enzymatic and non-enzymatic reactions. Ferrochelatase is active in formation of the highly fluorescent pigment known from Parma ham as demonstrated by inhibition with N-methylmesoporphyrin and by thermal inactivation. A non-enzymatic transmetallisation reaction, exchange of iron in myoglobin by zinc(II), is demonstrated by Pb(II) inhibition of zinc protoporphyrin formation at low Pb(II) concentrations, but promoted at higher Pb(II) concentrations. The non-enzymatic reaction is characterised as a slow bimolecular reaction between protoporphyrin IX and zinc(II) with a second-order rate constant of 0.63 l mol⁻¹ s⁻¹ at 35 °C and a high energy of activation of 98 kJ mol⁻¹ for acetone:water (3:1, v/v) as solvent. Zinc protoporphyrin formation is concluded to be thermodynamically controlled with a formation constant of 4 × 10⁵ M (35 °C, acetone:water (3:1)). An efficient inhibition of formation of zinc protoporphyrin by nitrite is related to myoglobin as substrate and involves both enzymatic and non-enzymatic reactions.