Predicting learner's performance through video sequences viewing behavior analysis using educational data-mining.

This paper analyzes how learners interact with the pedagogical sequences of educational videos, and its effect on their performance. In this study, the suggested video courses are segmented on several pedagogical sequences. In fact, we're not focusing on the type of clicks made by learners, but we're concentrating on the pedagogical sequences in which those clicks were made. We focalize on the interpretation of the path followed by a learner watching an educational video, and the way they navigate the pedagogical sequences of that video, in order to predict whether a learner can pass or fail the video course. Learner's video clicks are collected and classified. We applied educational data mining technique using K-nearest Neighbours and Multilayer Perceptron algorithms to predict learner's performance. The classification results are acceptable, the kNN classifier achieves the best results with an average accuracy of 65.07%. The experimental result indicates that learners' performance could be predicted, we notice a correlation between video sequence viewing behavior and learning performances. This method may help instructors understand the way learners watch educational videos. It can be used for early detection of learners' video viewing behavior deviation and allow the instructor to provide well-timed, effective guidance.

Micelle dynamic simulation and physicochemical characterization of biorelevant media to reflect gastrointestinal environment in fasted and fed states.

The characterization of biorelevant media simulating the upper part of the gastrointestinal tract in the fasted and fed states was investigated by classical determination of physicochemical parameters such as pH, osmolality, surface tension and results were compared to in vivo physiological data. Incorporation of fatty material, in order to better simulate the influence of high fat meal was also performed. Stability and characterization of this medium was studied and compared to classical FeSSIF. Micelle characterization and computer dynamic simulation were performed in order to understand the interaction between lecithin and taurocholate and possible interactions between mixed micelle and drugs. The addition of NaTc, lecithin, and/or fatty materials has no influence on pH and osmolality, whereas the presence of fatty material modifies the surface tension. Values of FaSSIF and FeSSIF are in accordance with in vivo parameters and the presence of micelles can simulate the gastrointestinal environment. Modelization of micelles by computer simulation led to a model of mixed micelles in which structures of NaTc interact either by their hydrophilic or hydrophobic phase to give a bilayer stable model in which the lecithin molecule can insert its long carbon chain. The micelle structure is stable and can enhance dissolution of hydrophobic molecules by hydrophobic interaction with the numerous hydrophobic spaces available in the multilayer hydrophilic/hydrophobic layer.

The neutron structure of urate oxidase resolves a long-standing mechanistic conundrum and reveals unexpected changes in protonation.

Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.

Urate oxidase purification by salting-in crystallization: towards an alternative to chromatography.

BACKGROUND: Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. METHODOLOGY/PRINCIPAL FINDINGS: Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. CONCLUSIONS: The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the current process.

X-ray, ESR, and quantum mechanics studies unravel a spin well in the cofactor-less urate oxidase.

Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid using gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide in absence of any cofactor or transition metal. The catalytic mechanism was investigated using X-ray diffraction, electron spin resonance spectroscopy (ESR), and quantum mechanics calculations. The X-ray structure of the anaerobic enzyme-substrate complex gives credit to substrate activation before the dioxygen fixation in the peroxo hole, where incoming and outgoing reagents (dioxygen, water, and hydrogen peroxide molecules) are handled. ESR spectroscopy establishes the initial monoelectron activation of the substrate without the participation of dioxygen. In addition, both X-ray structure and quantum mechanic calculations promote a conserved base oxidative system as the main structural features in UOX that protonates/deprotonates and activate the substrate into the doublet state now able to satisfy the Wigner's spin selection rule for reaction with molecular oxygen in its triplet ground state.

Near-atomic resolution structures of urate oxidase complexed with its substrate and analogues: the protonation state of the ligand.

Urate oxidase (uricase; EC 1.7.3.3; UOX) from Aspergillus flavus catalyzes the oxidation of uric acid in the presence of molecular oxygen to 5-hydroxyisourate in the degradation cascade of purines; intriguingly, catalysis proceeds using neither a metal ion (Fe, Cu etc.) nor a redox cofactor. UOX is a tetrameric enzyme with four active sites located at the interface of two subunits; its structure was refined at atomic resolution (1 A) using new crystal data in the presence of xanthine and at near-atomic resolution (1.3-1.7 A) in complexes with the natural substrate (urate) and two inhibitors: 8-nitroxanthine and 8-thiouric acid. Three new features of the structural and mechanistic behaviour of the enzyme were addressed. Firstly, the high resolution of the UOX-xanthine structure allowed the solution of an old structural problem at a contact zone within the tetramer; secondly, the protonation state of the substrate was determined from both a halochromic inhibitor complex (UOX-8-nitroxanthine) and from the H-atom distribution in the active site, using the structures of the UOX-xanthine and the UOX-uric acid complexes; and thirdly, it was possible to extend the general base system, characterized by the conserved catalytic triad Thr-Lys-His, to a large water network that is able to buffer and shuttle protons back and forth between the substrate and the peroxo hole along the reaction pathway.

Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: mechanistic implications.

BACKGROUND: Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid and gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide, in the absence of cofactor or particular metal cation. The functional enzyme is a homo-tetramer with four active sites located at dimeric interfaces. RESULTS: The catalytic mechanism was investigated through a ternary complex formed between the enzyme, uric acid, and cyanide that stabilizes an intermediate state of the reaction. When uric acid is replaced by a competitive inhibitor, no complex with cyanide is formed. CONCLUSION: The X-ray structure of this compulsory ternary complex led to a number of mechanistic evidences that support a sequential mechanism in which the two reagents, dioxygen and a water molecule, process through a common site located 3.3 A above the mean plane of the ligand. This site is built by the side chains of Asn 254, and Thr 57, two conserved residues belonging to two different subunits of the homo-tetramer. The absence of a ternary complex between the enzyme, a competitive inhibitor, and cyanide suggests that cyanide inhibits the hydroxylation step of the reaction, after the initial formation of a hydroperoxyde type intermediate.

Oxygen pressurized X-ray crystallography: probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism.

The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.

Recapture of [S]-allantoin, the product of the two-step degradation of uric acid, by urate oxidase.

Urate oxidase from Aspergillus flavus catalyzes the degradation of uric acid to [S]-allantoin through 5-hydroxyisourate as a metastable intermediate. The second degradation step is thought either catalyzed by another specific enzyme, or spontaneous. The structure of the enzyme was known at high resolution by X-ray diffraction of I222 crystals complexed with a purine-type inhibitor (8-azaxanthin). Analyzing the X-ray structure of urate oxidase treated with an excess of urate, the natural substrate, shows unexpectedly that the active site recaptures [S]-allantoin from the racemic end product of a second degradation step.

A preliminary neutron diffraction study of rasburicase, a recombinant urate oxidase enzyme, complexed with 8-azaxanthin.

Crystallization and preliminary neutron diffraction measurements of rasburicase, a recombinant urate oxidase enzyme expressed by a genetically modified Saccharomyces cerevisiae strain, complexed with a purine-type inhibitor (8-azaxanthin) are reported. Neutron Laue diffraction data were collected to 2.1 A resolution using the LADI instrument from a crystal (grown in D2O) with volume 1.8 mm3. The aim of this neutron diffraction study is to determine the protonation states of the inhibitor and residues within the active site. This will lead to improved comprehension of the enzymatic mechanism of this important enzyme, which is used as a protein drug to reduce toxic uric acid accumulation during chemotherapy. This paper illustrates the high quality of the neutron diffraction data collected, which are suitable for high-resolution structural analysis. In comparison with other neutron protein crystallography studies to date in which a hydrogenated protein has been used, the volume of the crystal was relatively small and yet the data still extend to high resolution. Furthermore, urate oxidase has one of the largest primitive unit-cell volumes (space group I222, unit-cell parameters a = 80, b = 96, c = 106 A) and molecular weights (135 kDa for the homotetramer) so far successfully studied with neutrons.

Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site.

Urate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135 kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-azaxanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P2(1) with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P2(1)2(1)2 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (approximately 1.6 angstroms resolution compared with 2.3-3.2 angstroms for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors.

Complexed and ligand-free high-resolution structures of urate oxidase (Uox) from Aspergillus flavus: a reassignment of the active-site binding mode.

High-resolution X-ray structures of the complexes of Aspergillus flavus urate oxidase (Uox) with three inhibitors, 8-azaxanthin (AZA), 9-methyl uric acid (MUA) and oxonic acid (OXC), were determined in an orthorhombic space group (I222). In addition, the ligand-free enzyme was also crystallized in a monoclinic form (P2(1)) and its structure determined. Higher accuracy in the three new enzyme-inhibitor complex structures (Uox-AZA, Uox-MUA and Uox-OXC) with respect to the previously determined structure of Uox-AZA (PDB code 1uox) leads to a reversed position of the inhibitor in the active site of the enzyme. The corrected anchoring of the substrate (uric acid) allows an improvement in the understanding of the enzymatic mechanism of urate oxidase.

The synthetic pentasaccharide fondaparinux: first in the class of antithrombotic agents that selectively inhibit coagulation factor Xa.

Fondaparinux (Arixtra), a synthetic pentasaccharide, is the first in a new class of antithrombotic agents that selectively inhibit coagulation factor Xa. In vitro experiments demonstrated that it is a selective inhibitor of factor Xa. In plasma, fondaparinux selectively binds to antithrombin, catalyzes factor Xa inhibition, and thereby inhibits thrombin generation. Its antithrombotic efficacy has been demonstrated in various animal models mimicking venous and arterial thrombosis. In humans, its pharmacokinetic profile is favorable, with a rapid onset of antithrombotic activity and an elimination half-life allowing a convenient once-daily dosing regimen. In several clinical trials, fondaparinux was more effective than the reference drug, enoxaparin, in preventing venous thromboembolism after hip fracture, major knee, and elective hip replacement surgeries. The overall reduction in the risk of venous thromboembolism ranged between 26.3 and 56.4%, depending on the trial. This superior efficacy was achieved without increasing the risk of clinically relevant bleeding. Fondaparinux also showed promising results in the treatment of patients with venous thromboembolism and acute coronary syndromes. Thus, it is now established that selective factor Xa inhibition is an efficient way to prevent venous thrombosis. The advent of fondaparinux offers an opportunity to improve substantially the management of venous thromboembolism.