Double Rashba Quantum Dots Ring as a Spin Filter

We theoretically propose a double quantum dots (QDs) ring to filter the electron spin that works due to the Rashba spin–orbit interaction (RSOI) existing inside the QDs, the spin-dependent inter-dot tunneling coupling and the magnetic flux penetrating through the ring. By varying the RSOI-induced phase factor, the magnetic flux and the strength of the spin-dependent inter-dot tunneling coupling, which arises from a constant magnetic field applied on the tunneling junction between the QDs, a 100% spin-polarized conductance can be obtained. We show that both the spin orientations and the magnitude of it can be controlled by adjusting the above-mentioned parameters. The spin filtering effect is robust even in the presence of strong intra-dot Coulomb interactions and arbitrary dot-lead coupling configurations.     

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using ³²P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.     

Residue Folding Degree—Relationship to Secondary Structure Categories and Use as Collective Variable

Recently, we have shown that the residue folding degree, a network-based measure of folded content in proteins, is able to capture backbone conformational transitions related to the formation of secondary structures in molecular dynamics (MD) simulations. In this work, we focus primarily on developing a collective variable (CV) for MD based on this residue-bound parameter to be able to trace the evolution of secondary structure in segments of the protein. We show that this CV can do just that and that the related energy profiles (potentials of mean force, PMF) and transition barriers are comparable to those found by others for particular events in the folding process of the model mini protein Trp-cage. Hence, we conclude that the relative segment folding degree (the newly proposed CV) is a computationally viable option to gain insight into the formation of secondary structures in protein dynamics. We also show that this CV can be directly used as a measure of the amount of α-helical content in a selected segment.     

N-Glycosylation as a Tool to Study Antithrombin Secretion,Conformation, and Function

N-linked glycosylation is a crucial post-translational modification involved in protein folding, function, and clearance. N-linked glycosylation is also used therapeutically to enhance the half-lives of many proteins. Antithrombin, a serpin with four potential N-glycosylation sites, plays a pivotal role in hemostasis, wherein its deficiency significantly increases thrombotic risk. In this study, we used the introduction of N-glycosylation sites as a tool to explore what effect this glycosylation has on the protein folding, secretion, and function of this key anticoagulant. To accomplish this task, we introduced an additional N-glycosylation sequence in each strand. Interestingly, all regions that likely fold rapidly or were surrounded by lysines were not glycosylated even though an N-glycosylation sequon was present. The new sequon in the strands of the A- and B-sheets reduced secretion, and the B-sheet was more sensitive to these changes. However, the mutations in the strands of the C-sheet allowed correct folding and secretion, which resulted in functional variants. Therefore, our study revealed crucial regions for antithrombin secretion and could potentially apply to all serpins. These results could also help us understand the functional effects of natural variants causing type-I deficiencies.     

A hybrid of bovine pancreatic ribonuclease and human angiogenin: an external loop as a module controlling substrate specificity?

A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 63–74 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' – 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 59–70 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 1875–1884].Thus, the external loop in residues 63–74 of RNase A appearsto behave, at least in part, as an interchangeable ‘module’that influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein.     

Mutational analysis of DNase I-DNA interactions: design, expression and characterization of a DNase I loop insertion mutant with altered sequence selectivity

A mutant of bovine pancreatic DNase I containing two additionalresidues in a loop next to C173 has been expressed in Escherichiacoli, purified and characterized biochemically. Modelling studiessuggest that the inserted arginine and glutamate side chainsof the modified loop sequence C173-R-E-G-T-V176 could contactthe bases 3' to the cleaved bond in the major groove of a boundDNA, and that up to 10 bp could interact with the enzyme andpotentially influence its cutting rate. The loop insertion mutanthas an 800-fold lower specific activity than wild-type and showsoverall cleavage characteristics similar to bovine pancreaticDNase I. Compared with the wild-type enzyme, the mutant showsa strongly enhanced preference for cutting the inverted repeat:5'-GACTT A AAGTC-3' CTGAA T TTCAG or close variants thereof.Unexpectedly for a minor groove binding protein, the preferredcutting sites in opposite strands are staggered by 1 bp in the5' direction, causing the cleavage of a TA and a TT step, respectively.This finding demonstrates that the sequence context is relativelymore important for the cutting frequency than the nature ofthe dinucleotide step of the cleaved bond, and clearly showsthat base recognition is involved in determining the sequenceselectivity of the mutant. The importance of the sequence 5'to the cleaved bond for the cutting rate suggests that the additionalmajor groove contacts may require a distortion of the DNA associatedwith a higher energy barrier, resulting in an increased selectivityfor flexible DNA sequences and a lower overall activity of themutant enzyme.     

Virtual Drug Repositioning as a Tool to Identify Natural Small Molecules That Synergize with Lumacaftor in F508del-CFTR Binding and Rescuing

Cystic fibrosis is a hereditary disease mainly caused by the deletion of the Phe 508 (F508del) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. Cystic fibrosis remains a potentially fatal disease, but it has become treatable as a chronic condition due to some CFTR-rescuing drugs that, when used in combination, increase in their therapeutic effect due to a synergic action. Also, dietary supplementation of natural compounds in combination with approved drugs could represent a promising strategy to further alleviate cystic fibrosis symptoms. On these bases, we screened by in silico drug repositioning 846 small synthetic or natural compounds from the AIFA database to evaluate their capacity to interact with the highly druggable lumacaftor binding site of F508del-CFTR. Among the identified hits, nicotinamide (NAM) was predicted to accommodate into the lumacaftor binding region of F508del-CFTR without competing against the drug but rather stabilizing its binding. The effective capacity of NAM to bind F508del-CFTR in a lumacaftor-uncompetitive manner was then validated experimentally by surface plasmon resonance analysis. Finally, the capacity of NAM to synergize with lumacaftor increasing its CFTR-rescuing activity was demonstrated in cell-based assays. This study suggests the possible identification of natural small molecules devoid of side effects and endowed with the capacity to synergize with drugs currently employed for the treatment of cystic fibrosis, which hopefully will increase the therapeutic efficacy with lower doses.     

Electrochemistry as a Tool to Monitor Lewis Acid-Base Reactions between Methacrylonitrile and Metallic Surfaces: A Theoretical and Experimental Proposal

This paper presents a tentative extension of the Lewis acid-base concept to the case of an organic molecule interacting with a polarized metallic surface. Towards this aim, we make use of the Density Functional Theory (DFT) viewpoint on Lewis acid-base interactions. This theory has been shown to be relevant to describe adhesion processes at a molecular scale. It allows the introduction of three key parameters, for the molecule as well as for the metallic surface. These are the DFT chemical potential, μ, the absolute hardness, η and the Fukui function, f(inag hear). In the present paper, we show that the DFT chemical potential, μ, of the metallic surface is linearly related to the electrode potential drop, Δε, imposed between this surface and a reference electrode in an electrochemical cell. Thus, while the chemical potential of the molecule is only determined by its chemical structure, that of the metallic surface can be monitored continuously. This means that the Lewis acidic or basic character of the metallic surface towards the molecule can, in principle, be chosen. We present experimental results arguing in the sense of this model by studying the interaction of 2-methyl 2-propenenitrile (methacrylonitrile) alternatively with a metallic cathode and with a metallic anode. The two different transient molecule surface interactions are frozen thanks to an anionic electropolymerization of the monomer on the cathode and to a (first reported) cationic electropolymerization of the monomer on the anode. A detailed analysis of the molecular structures of the two resulting polymer/metal interfaces shows results which are in agreement with the theoretical predictions.

This paper is dedicated to Professor Jacques Schultz as an acknowledgement of his constant interest in our work.     

Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD) simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA) method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π-π, CH-π and CH-CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. IV. Combinations of Asn20->Cys/Ala27->Cys, Ser30->Pro, Gly137->Ala, 311314 Loop, Ser411->Ala and Ser436->Pro

Six previously constructed and nine newly constructed Aspergillusawamori glucoamylases with multiple mutations made by combiningexisting single mutations were tested for their ability to produceglucose from maltodextrins. Multiple mutations have cumulativeeffects on glucose yield, specific activity and thermostability.No general correlation between glucose yield and thermostabilitywas observed, although mutations that presumably impede unfoldingat high temperatures uniformly increase thermostability andgenerally increase glucose yield. Peak glucose yields decreasewith increasing temperature. The best combination of high glucoseyield, high specific activity and high thermostability occursin Asn20Cys/Ala27Cys/Ser30Pro/Gly137Ala glucoamylase.     

Fighting the Huntington’s Disease with a G-Quadruplex-Forming Aptamer Specifically Binding to Mutant Huntingtin Protein: Biophysical Characterization,In Vitro and In Vivo Studies

A set of guanine-rich aptamers able to preferentially recognize full-length huntingtin with an expanded polyglutamine tract has been recently identified, showing high efficacy in modulating the functions of the mutated protein in a variety of cell experiments. We here report a detailed biophysical characterization of the best aptamer in the series, named MS3, proved to adopt a stable, parallel G-quadruplex structure and show high nuclease resistance in serum. Confocal microscopy experiments on HeLa and SH-SY5Y cells, as models of non-neuronal and neuronal cells, respectively, showed a rapid, dose-dependent uptake of fluorescein-labelled MS3, demonstrating its effective internalization, even in the absence of transfecting agents, with no general cytotoxicity. Then, using a well-established Drosophila melanogaster model for Huntington’s disease, which expresses the mutated form of human huntingtin, a significant improvement in the motor neuronal function in flies fed with MS3 was observed, proving the in vivo efficacy of this aptamer.     

Microscale Thermophoresis as a Tool to Study Protein Interactions and Their Implication in Human Diseases

The review highlights how protein–protein interactions (PPIs) have determining roles in most life processes and how interactions between protein partners are involved in various human diseases. The study of PPIs and binding interactions as well as their understanding, quantification and pharmacological regulation are crucial for therapeutic purposes. Diverse computational and analytical methods, combined with high-throughput screening (HTS), have been extensively used to characterize multiple types of PPIs, but these procedures are generally laborious, long and expensive. Rapid, robust and efficient alternative methods are proposed, including the use of Microscale Thermophoresis (MST), which has emerged as the technology of choice in drug discovery programs in recent years. This review summarizes selected case studies pertaining to the use of MST to detect therapeutically pertinent proteins and highlights the biological importance of binding interactions, implicated in various human diseases. The benefits and limitations of MST to study PPIs and to identify regulators are discussed.     

Metabolomics as a Tool to Elucidate the Sensory,Nutritional and Safety Quality of Wheat Bread—A Review

Wheat (Triticum aestivum) is one of the most extensively cultivated and used staple crops in human nutrition, while wheat bread is annually consumed in more than nine billion kilograms over the world. Consumers’ purchase decisions on wheat bread are largely influenced by its nutritional and sensorial characteristics. In the last decades, metabolomics is considered an effective tool for elucidating the information on metabolites; however, the deep investigations on metabolites still remain a difficult and longtime action. This review gives emphasis on the achievements in wheat bread metabolomics by highlighting targeted and untargeted analyses used in this field. The metabolomics approaches are discussed in terms of quality, processing and safety of wheat and bread, while the molecular mechanisms involved in the sensorial and nutritional characteristics of wheat bread are pointed out. These aspects are of crucial importance in the context of new consumers’ demands on healthy bakery products rich in bioactive compounds but, equally, with good sensorial acceptance. Moreover, metabolomics is a potential tool for assessing the changes in nutrient composition from breeding to processing, while monitoring and understanding the transformations of metabolites with bioactive properties, as well as the formation of compounds like toxins during wheat storage.     

Adhesion to zirconia as a function of primers/silane coupling agents,luting cement types,aging and test methods

This study evaluated the adhesion of resin cements to zirconia with different primers/silane coupling agents using two test methods with and without aging. Zirconia discs (Cercon) (N = 900, n = 15 per group) were ground finished to 2000 grit silicone carbide and randomly divided into seven groups: (a) C: No treatment (Control), (b) SG: Signum, (c) CL: Clearfil Ceramic Primer, (d) AP: Alloy Primer, (e) Monobond Plus, (f) ES-R: ESPE-Sil after Rocatec and (g) ES-C: ESPE-Sil after CoJet. Methacrylate (Variolink II-VL) and MDP based (Panavia F2.0-PN) dual-polymerized and self-adhesive resin cements (RelyX Unicem-RX) were adhered and polymerized accordingly. The specimens were further randomly divided into two groups to be tested after (a) 24-h dry storage at 37 °C and (b) thermocycling (×5000, 5–55 °C). Macroshear (MSB) and macrotensile bond tests (MTB) were conducted in an universal testing machine (crosshead speed: 1 mm/min) and failure types were analyzed after debonding. Data were analyzed using Univariate analysis and Tukey’s tests (α = 0.05). Two-parameter Weibull modulus, scale (m) and shape (0) were calculated. While primer/silane (p < 0.001), cement type (p < 0.001) and aging (p < 0.001) significantly affected the bond results, test method did not show significant difference (p = 0.237). In MSB test, Weilbul moduli were more favorable for MP-VL (4.2) and AP-PN (6) combinations and after aging for MP-VL (4.2) and AP-PN (5.66). In MTB test, after aging, Weilbul moduli were more favorable for AP-PN (5.41). Bond strength results mostly decreased with SG (24–92%) after aging. Cohesive failures in the cement were more frequent with PN (252) compared to VL (83).     

Hydrido‐Cobalt Catalyst as a Selective Tool for the Dimerisation of Arylacetylenes: Scope and Theoretical Studies

A simple hydrido‐cobalt complex efficiently catalyses the highly regio‐ and stereoselective dimerisation of various terminal arylacetylenes under mild conditions. The corresponding (E)‐1,4‐enynes are obtained as sole isomers with good to excellent yields. DFT calculations revealed that the reaction proceeds via a C H activation/hydrocobaltation pathway.