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Chronic wasting disease (CWD), a prion disease of North American deer, elk and moose, affects both free-ranging and captive cervids. The potential host range for CWD remains uncertain. The susceptibility of the ferret to CWD was examined experimentally by administering infectious brain material by the intracerebral (IC) or oral (PO) route. Between 15 and 20 months after IC inoculation, ferrets developed neurological signs consistent with prion disease, including polyphagia, somnolence, piloerection, lordosis and ataxia. Upon first sub-passage of ferret-adapted CWD, the incubation period decreased to 5 months. Spongiform change in the neuropil was most marked in the basal ganglia, thalamus, midbrain and pons. The deposition of PrP(CWD) was granular and was occasionally closely associated with, or localized within, neurons. There were no plaque-like or perivascular PrP aggregates as seen in CWD-infected cervids. In western blots, the PrP(CWD) glycoform profile resembled that of CWD in deer, typified by a dominant diglycosylated glycoform. CWD disease in ferrets followed IC but not PO inoculation, even after 31 months of observation. These findings indicate that CWD-infected ferrets share microscopical and biochemical features of CWD in cervids, but appear to be relatively resistant to oral infection by primary CWD inoculum of deer origin.

A novel approach for reconciling tuples stored as free text into an existing attribute schema is proposed. The basic idea is to subject the available text to progressive classification, i.e., a multi-stage classification scheme where, at each intermediate stage, a classifier is learnt that analyzes the textual fragments not reconciled at the end of the previous steps. Classifica- tion is accomplished by an ad hoc exploitation of traditional association mining algorithms, and is supported by a data transformation scheme which takes advantage of domain-specific dictionaries/ontologies. A key feature is the capability of progressively enriching the avail- able ontology with the results of the previous stages of classification, thus significantly improving the overall classification accuracy. An extensive experimental evaluation shows the effectiveness of our approach.

The continuous exchange of information by means of the popular email service has raised the problem of managing the huge amounts of messages received from users in an effective and efficient way. We deal with the problem of email classification by conceiving suitable strategies for: (1) organizing messages into homogeneous groups, (2) redirecting further incoming messages according to an initial organization, and (3) building reliable descriptions of the message groups discovered. We propose a unified framework for handling and classifying email messages. In our framework, messages sharing similar features are clustered in a folder organization. Clustering and pattern discovery techniques for mining structured and unstructured information from email messages are the basis of an overall process of folder creation/maintenance and email redirection. Pattern discovery is also exploited for generating suitable cluster descriptions that play a leading role in cluster updating. Experimental evaluation performed on several personal mailboxes shows the effectiveness of our approach.

In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.

The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two ?-d-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl ?-d-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3?,5?-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a "hot spot" in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (k(cat)/K-m) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate delta-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a similar to 6.6 angstrom outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity.

Third International Workshop, NFMCP 2014, Held in Conjunction with ECML-PKDD 2014, Nancy, France, September 19, 2014, Revised Selected Papers

Pyruvate dehydrogenase complex (PDHC) is a key enzyme in metabolism linking glycolysis to tricarboxylic acid cycle and its activity is tightly regulated by phosphorylation catalyzed by four pyruvate dehydrogenase kinase (PDK) isoforms. PDKs are pharmacological targets for several human diseases including cancer, diabetes, obesity, heart failure, and inherited PDHC deficiency. We investigated the inhibitory activity of phenylbutyrate toward PDKs and found that PDK isoforms 1-to-3 are inhibited whereas PDK4 is unaffected. Moreover, docking studies revealed putative binding sites of phenylbutyrate on PDK2 and 3 that are located on different sites compared to dichloroacetate (DCA), a previously known PDK inhibitor. Based on these findings, we showed both in cells and in mice that phenylbutyrate combined to DCA results in greater increase of PDHC activity compared to each drug alone. These results suggest that therapeutic efficacy can be enhanced by combination of drugs increasing PDHC enzyme activity. Electronic supplementary material The online version of this article (doi:10.1007/s10545-014-9808-2) contains supplementary material, which is available to authorized users.

The thermal stability of the Aes acetyl esterase from Escherichia coli has been investigated by means of differential scanning calorimetry and circular dichroism measurements. The calorimetric curves show a denaturation temperature of 68 °C for Aes and 61 °C for the single point mutant V20D-Aes. The same values are obtained from CD denaturation curves of the two proteins recorded in both the far-UV and near-UV regions. Even if the denaturation process is irreversible and characterized by a single calorimetric peak and a single inflection point in both far- and near-UV CD curves, the overall data indicate that the process is more complex than a two-state transition. This is in line with the presence of two structural domains in the 3D model of Aes, according to homology modelling. A comparison of the thermal stability of Aes with those of the homologous thermophilic EST2 and hyperthermophilic AFEST suggests that the optimization of charge–charge interactions should not be so effective in the case of the mesophilic enzyme.

Previously, we reported from theSulfolobus solfataricusopen reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we namedSsoNΔ. Searching the recently reportedSulfolobus acidocaldariusgenome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, namedSsoNΔlong, was 15-fold more active with the substratep-nitrophenyl hexanoate thanSsoNΔ. Furthermore,SsoNΔlong andSsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship betweenS. solfataricusandS. acidocaldariussuggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression ofSsoNΔlong under specific metabolic conditions could be hypothesized.

The human paraoxonase 2 (PON2) has been described as a highly specific lactonase hydrolysing the quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL) and having secondary esterase but not phosphotriesterase activity, in contrast with the related enzymes PON1 and PON3. It has been suggested that PON2 enzyme activity is dependent on glycosylation and its N-terminal region has been recently demonstrated to be a transmembrane domain mediating association to membranes. In the present study we describe a mutated form of PON2, lacking the above N- terminal region, which has been further stabilized by the insertion of six amino acidic substitutions. The engineered version, hence forth called rPON2, has been over- expressed in E.coli, refolded from inclusion bodies and purified, yielding an enzyme with the same characteristics as the full length enzyme. Therefore the first conclusion of this work was that the catalytic activity is independent from the N-terminus and protein glycosylation. The kinetic characterization confirmed the primary activity on 3OC12-HSL; accordingly, in vitro experiments of inhibition of the biofilm formed by Pseudomonas aeruginosa (PAO1) have demonstrated that rPON2 is more effective than PON1. In addition, we observed small but significant activity against organophosphorothiotes pesticides, m-parathion, coumaphos and malathion. The availability of fair amount of active protein allowed to pinpoint, by mass- spectrometry, ubiquitination of Lys 168 induced in rPON2 by HeLa extract and to correlate such post-translational modification to the modulation of catalytic activity. A mutational analysis of the modified residue confirmed the result.

Today's workflow management systems represent a key technological infrastructure for advanced applications that is attracting a growing body of research, mainly focused in developing tools for workflow management, that allow users both to specify the "static" aspects, like preconditions, precedences among activities, and rules for exception handling, and to control its execution by scheduling the activities on the available resources. This paper deals with an aspect of workflows which has so far not received much attention even though it is crucial for the forthcoming scenarios of large scale applications on the Web: Providing facilities for the human system administrator for identifying the choices performed more frequently in the past that had lead to a desired final configuration. In this context, we formalize the problem of discovering the most frequent patterns of executions, i.e., the workflow substructures that have been scheduled more frequently by the system. We attacked the problem by developing two data mining algorithms on the basis of an intuitive and original graph formalization of a workflow schema and its occurrences. The model is used both to prove some intractability results that strongly motivate the use of data mining techniques and to derive interesting structural properties for reducing the search space for frequent patterns. Indeed, the experiments we have carried out show that our algorithms outperform standard data mining algorithms adapted to discover frequent patterns of workflow executions.

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.

The recent developments in technologies and life sciences have paved the way to complex interactions among entities in distributed and heterogeneous environments. As a result, an enormous amount of valuable information is available, spanning from structured to multimedia and spatial or spatio-temporal data. The data mining research community has been focusing on developing specialized techniques and algorithms for the analysis of such data. Some of the problems that have received attention include the analysis of multi-table data, XML data, web data, time series and sequences, graphs and trees. The first International Workshop on New Frontiers in Mining Complex Patterns (NFMCP 2011), held in Bristol UK, on September 24th 2012 in conjunction with the European Conference on Machine Learning and Principles and Practice of Knowledge Discovery in Databases (ECML-PKDD 2012), was aimed at bringing together researchers and practitioners of data mining who are interested in the advances and latest developments in area of extracting patterns from complex data.

Aes, a 36-kDa acetylesterase fromEscherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the α-galactosidase from E. coli. TheE. coli cells harboring plasmid pT7-SCII-aesand, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because α-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.

The esterase genes est2 from Alicyclobacillus acidocaldarius and AF1716 from Archaeoglobus fulgidus were subjected to error-prone PCR in an effort to increase the low enantioselectivity of the corresponding enzymes EST2 and AFEST, respectively. The model substrate ( RS)- p-nitrophenyl-2-chloropropionate was chosen to produce ( S)-2-chloropropionic acid, an important intermediate in the synthesis of some optically pure compounds, such as the herbicide mecoprop. In the case of EST2, a single mutant, Leu212Pro, was obtained showing a slightly enhanced preference toward the ( S) substrate; in the case of AFEST, a double mutant, Leu101Ile/Asp117Gly, was obtained showing an increased preference in the opposite direction. The 3-D structures of the EST2 and AFEST enzymes were analyzed by molecular modeling to determine the effects of the mutations. Mutations were positioned differently in the structures, but in both cases caused small modifications around the active site and in the oxyanion loop.

Nowadays, almost all kind of electronic devices leave traces of their movements (e.g. smartphone, GPS devices and so on). Thus, the huge number of this "tiny" data sources leads to the generation of massive data streams of geo-referenced data. As a matter of fact, the effective analysis of such amounts of data is challenging, since the possibility to extract useful information from this peculiar kind of data is crucial in many application scenarios such as vehicle traffic management, hand-off in cellular networks, supply chain management. Moreover, spatial data streams management poses new challenges both for their proper definition and acquisition, thus making the overall process harder than for classical point data. In particular, we are interested in solving the problem of effective trajectory data streams clustering, that revealed really intriguing as we deal with sequential data that have to be properly managed due to their ordering. We propose a framework that allow data pre-elaboration in order to make the mining step more effective. As for every data mining tool, the experimental evaluation is crucial, thus we performed several tests on real world datasets that confirmed the efficiency and effectiveness of the proposed approach. © 2013 Springer Science+Business Media New York.

The crystal structure of AFEST, a novel hyper-thermophilic carboxylesterase from the archaeon Archaeoglobus fulgidus, complexed with a sulphonyl derivative, has been determined and refined to 2.2 A resolution. This enzyme, which has recently been classified as a member of the hormone- sensitive-lipase (H) group of the esterase/lipase superfamily, presents a canonical alpha/beta hydrolase core, shielded on the C-terminal side by a cap region composed of five alpha-helices. It contains the catalytic triad Ser160, His285 and Asp255, whereby the nucleophile is covalently modified and the oxyanion hole formed by Gly88, Gly89 and Ala161. A structural comparison of AFEST with its mesophilic and thermophilic homologues, Brefeldin A esterase from Bacillus subtilis (BFAE) and EST2 from Alicyclobacillus acidocaldarius, reveals an increase in the number of intramolecular ion pairs and secondary structure content, as well as a significant reduction in loop extensions and ratio of hydrophobic to charged surface area. The variety of structural differences suggests possible strategies for thermostabilization of lipases and esterases with potential industrial applications.

How can we detect communities when the social graphs is not available? We tackle this problem by modeling social contagion from a log of user activity, that is a dataset of tuples (u, i, t) recording the fact that user u "adopted" item i at time t. This is the only input to our problem. We propose a stochastic framework which assumes that item adoptions are governed by un underlying diffusion process over the unobserved social network, and that such diffusion model is based on community-level influence. By fitting the model parameters to the user activity log, we learn the community membership and the level of influence of each user in each community. This allows to identify for each community the "key" users, i.e., the leaders which are most likely to influence the rest of the community to adopt a certain item. The general framework can be instantiated with different diffusion models. In this paper we define two models: the extension to the community level of the classic (discrete time) Independent Cascade model, and a model that focuses on the time delay between adoptions. To the best of our knowledge, this is the first work studying community detection without the network.

EST2 is a novel thermophilic carboxylesterase, isolated and cloned from Alicyclobacillus (formerly Bacillus ) acidocaldarius , which optimally hydrolyses esters with acyl chain lengths of six to eight carbon atoms at 70°C. On the basis of the amino acid sequence homology, it has been classified as a member of the mammalian hormone-sensitive lipase (HSL) subfamily. The crystal structure of EST2, complexed with a sulphonyl derivative, has been determined at 2.6 A resolution by a multiple wavelength anomalous diffraction experiment on a seleno-methionine derivative. EST2 presents a canonical α/β hydrolase core, shielded at the C-terminal side by a cap region built up of five helices. It contains the lipase-like catalytic triad, Ser155, His282 and Asp252, whereby the nucleophile is covalently modified. This allows an unambiguous view of the putative active site of EST2, detecting the oxyanion hole, in whose formation the amino acid sequence motif His81-Gly82-Gly83-Gly84 is involved, and the hydrophobic binding pocket for the acyl chain. The structural model here reported provides the first example of a transition state analogue of an esterase/lipase belonging to the HSL group, thus affording useful information for the design of medical inhibitors. Moreover, as the first X-ray structure of a thermophilic carboxylesterase, the comparison with its mesophilic homologue, the Brefeldin A esterase (BFAE) from Bacillus subtilis , allows the identification of putative determinants of thermal stability.

The esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus is a monomeric protein with a molecular weight of about 35.5 kDa. The enzyme is barely active at room temperature, displaying the maximal enzyme activity at about 80 degrees C. We have investigated the effect of the temperature on the protein structure by Fourier-transform infrared spectroscopy. The data show that between 20 degrees C and 60 degrees C a small but significant decrease of the beta-sheet bands occurred, indicating a partial loss of beta-sheets. This finding may be surprising for a thermophilic protein and suggests the presence of a temperature-sensitive beta-sheet. The increase in temperature from 60 degrees C to 98 degrees C induced a decrease of alpha-helix and beta-sheet bands which, however, are still easily detected at 98 degrees C indicating that at this temperature some secondary structure elements of the protein remain intact. The conformational dynamics of the esterase were investigated by frequency-domain fluorometry and anisotropy decays. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. Remarkably, the tryptophanyl fluorescence emission reveals that the indolic residues remained shielded from the solvent up to 80 degrees C, as shown from the emission spectra and by acrylamide quenching experiments. The relationship between enzyme activity and protein structure is discussed.

GCDFP-15 (gross cysticdisease fluid protein,15 kDa) is a secretory marker of apocrine differentiation in breast carcinoma. In human breast cancer cell lines, gene expression is regulated by hormones, including androgens and prolactin. The protein is also known under different names in different body fluids such as gp17 in seminal plasma. GCDFP-15/gp17 is a ligand of CD4 and is a potent inhibitor of T-cell apoptosis induced by sequential CD4/T-cell receptor triggering. We now report that GCDFP-15/gp17 is a protease exhibiting structural properties relating it to the aspartyl proteinase superfamily. Unexpectedly, GCDFP-15/gp17 appears to be related to the retroviral members rather than to the known cellular members of this class. Site-specific mutagenesis of Asp22 (predicted to be catalytically important for the active site) and pepstatin A inhibition confirmed that the protein is an aspartic-type protease. We also show that, among the substrates tested, GCDFP-15/gp17 is specific for fibronectin. The study of GCDFP-15/gp17-mediated proteolysis may provide a handle to understand phenomena as diverse as mammary tumor progression and fertilization.

The hyperthermophilic Archaeon Archaeoglobus fulgidus has a gene (AF1763) which encodes a thermostable carboxylesterase belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structure predictions and a secondary structure-driven multiple sequence alignment with remote homologous proteins of known three-dimensional structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser160, Asp 255 and His285 as the putative members of the catalytic triad. In this paper we report the building of a 3D model for this enzyme based on the structure of the homologous brefeldin A esterase from Bacillus subtilis whose structure has been recently elucidated. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser160, Asp255 and His285 are located close each other at hydrogen bond distances. The catalytic role of Ser160 as the nucleophilic member of the triad is demonstrated by the [(3)H]diisopropylphosphofluoridate (DFP) active-site labeling and sequencing of a radioactive peptide containing the signature sequence GDSAGG.

A new esterase gene from the hyperthermophilic archaeon Archaeoglobus fulgidus, reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family, was cloned by means of the polymerase chain reaction from the A. fulgidus genome. In order to compare the biochemical properties of this putative hyperthermophilic enzyme with those of the homologous, thermophilic member of HSL group, namely Alicyclobacillus (formerly Bacillus) acidocaldarius esterase 2 (EST2), an overexpression system in Escherichia coli was established. The recombinant protein, expressed in soluble and active form at 20 mg/liter of E. coli culture, was purified to homogeneity and characterized. The enzyme, a 35.5-kDa monomeric protein, was demonstrated to be a B"-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-hexanoate with K(m) and k(cat) values of 11 +/- 3 microM (mean +/- SD, n = 3) and 1014 +/- 38 s(-1) (mean +/- SD, n = 3), respectively, at 70 degrees C and pH 7.1. Inactivation by diethylpyrocarbonate, phenylmethylsulfonylfluoride, diisopropylfosfofluoridate (DFP), and physostigmine, as well as labeling with [(3)H]DFP, supported our previous suggestion of a catalytic triad made up of Ser(160)-His(285)-Asp(255). The sequence identity with the thermostable A. acidocaldarius EST2 was 42.5%. The enzyme proved to be much more stable than its Alicyclobacillus counterpart. The conformational dynamics of the two proteins were investigated by frequency-domain fluorometry and anisotropy decay and the activity/stability/temperature relationship was discussed.

The moderate thermophilic eubacterium Alicyclobacillus (formerly Bacillus) acidocaldarius expresses a thermostable carboxylesterase (esterase 2) belonging to the hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. Based on secondary structures predictions and a secondary structure-driven multiple sequence alignment with remote homologous protein of known three-dimensional (3D) structure, we previously hypothesized for this enzyme the alpha/beta-hydrolase fold typical of several lipases and esterases and identified Ser155, Asp252, and His282 as the putative members of the catalytic triad. In this paper we report the construction of a 3D model for this enzyme based on the structure of mouse acetylcholinesterase complexed with fasciculin. The model reveals the topological organization of the fold corroborating our predictions. As regarding the active-site residues, Ser155, Asp252, and His282 are located close to each other at hydrogen bond distances. Their catalytic role was here probed by biochemical and mutagenic studies. Moreover, on the basis of the secondary structure-driven multiple sequence alignment and the 3D structural model, a residue supposed important for catalysis, Gly84, was mutated to Ser. The activity of the mutated enzyme was drastically reduced. We propose that Gly84 is part of a putative "oxyanion hole" involved in the stabilization of the transition state similar to the C group of the esterase/lipase family.

Organophosphates constitute the largest class of insecticides used worldwide and some of them are potent nerve agents. Consequently, organophosphate-degrading enzymes are of paramount interest as they could be used as bioscavengers and biodecontaminants. Phosphotriesterases (PTEs) are capable of hydrolyzing these toxic compounds with high efficiency. A distant and hyperthermophilic representative of the PTE family was cloned from the archeon Sulfolobus solfataricus MT4, overexpressed in Escherichia coli and crystallized; the crystals diffracted to 2.54 A resolution. Owing to its exceptional thermostability, this PTE may be an excellent candidate for obtaining an efficient organophosphate biodecontaminant. Here, the crystallization conditions and data collection for the hyperthermophilic S. solfataricus PTE are reported.

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B'-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11+/-2 microM (mean+/-S.D., n=3) and 6610+/-880 s-1 (mean+/-S.D., n=3) respectively at 70 degreesC and pH7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic Moraxella TA144 lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (+/-)-3-bromo-5-(hydroxymethyl)-Delta2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity-stability-temperature relationship is discussed in relation to those of the homologous members of the HSL group.

Given a directed social graph and a set of past informa- tion cascades observed over the graph, we study the novel problem of detecting modules of the graph (communities of nodes), that also explain the cascades. Our key observation is that both information propagation and social ties forma- tion in a social network can be explained according to the same latent factor, which ultimately guide a user behavior within the network. Based on this observation, we propose the Community-Cascade Network (CCN) model, a stochas- tic mixture membership generative model that can fit, at the same time, the social graph and the observed set of cas- cades. Our model produces overlapping communities and for each node, its level of authority and passive interest in each community it belongs. For learning the parameters of the CCN model, we devise a Generalized Expectation Maximization procedure. We then apply our model to real-world social networks and in- formation cascades: the results witness the validity of the proposed CCN model, providing useful insights on its signif- icance for analyzing social behavior. © 2013 ACM.

Revised Selected Papers From NFMCP 2012

This paper provides a principled probabilistic co-clustering frame-work for missing value prediction and pattern discovery in users' preference data. We extend the original dyadic formulation of the Block Mixture Model(BMM) in order to take into account explicit users' preferences. BMM simultaneously identifies user communities and item categories: each user is modeled as a mixture over user communities, which is computed by taking into account users' preferences on similar items. Dually, item categories are detected by considering preferences given by similar minded users. This recursive formulation highlights the mutual relationships between items and user, which are then used to uncover the hidden block-structure of the data. We next show how to characterize and summarize each block cluster by exploiting additional meta data information and by analyzing the underlying topic distribution, proving the effectiveness of the approach in pattern discovery tasks. © Springer-Verlag Berlin Heidelberg 2013.

Trajectory data streams are huge amounts of data pertaining to time and position of moving objects. They are continuously generated by different sources exploiting a wide variety of technologies (e.g., RFID tags, GPS, GSM networks). Mining such amount of data is a challenging problem, since the possibility to extract useful information from this peculiar kind of data is crucial in many application scenarios such as vehicle traffic management, hand-off in cellular networks, supply chain management. Moreover, spatial data streams pose interesting challenges for their proper representation, thus making the mining process harder than for classical point data. In this paper, we address the problem of trajectory data streams clustering, that revealed really intriguing as we deal with a kind of data (trajectories) for which the order of elements is relevant. We propose a complete framework starting from data preparation task that allows us to make the mining step quite effective. Since the validation of data mining approaches has to be experimental we performed several tests on real world datasets that confirmed the efficiency and effectiveness of the proposed technique. © 2013 Springer-Verlag.

Clustering XML documents by structure is the task of grouping them by common structural components. Hitherto, this has been accomplished by looking at the occurrence of one preestablished type of structural components in the structures of the XML documents. However, the a-priori chosen structural components may not be the most appropriate for effective clustering. Moreover, it is likely that the resulting clusters exhibit a certain extent of inner structural inhomogeneity, because of uncaught differences in the structures of the XML documents, due to further neglected forms of structural components. To overcome these limitations, a new hierarchical approach is proposed, that allows to consider (if necessary) multiple forms of structural components to isolate structurally-homogeneous clusters of XML documents. At each level of the resulting hierarchy, clusters are divided by considering some type of structural components (unaddressed at the preceding levels), that still differentiate the structures of the XML documents. Each cluster in the hierarchy is summarized through a novel technique, that provides a clear and differentiated understanding of its structural properties. A comparative evaluation over both real and synthetic XML data proves that the devised approach outperforms established competitors in effectiveness and scalability. Cluster summarization is also shown to be very representative. © 2012 Elsevier B.V. All rights reserved.

Lactic acidosis is a buildup of lactic acid in the blood and tissues, which can be due to several inborn errors of metabolism as well as nongenetic conditions. Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. Phosphorylation of specific serine residues of the E1 alpha subunit of PDHC by pyruvate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We found that phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of PDK. Phenylbutyrate given to C57BL/6 wild-type mice results in a significant increase in PDHC enzyme activity and a reduction of phosphorylated E1 alpha in brain, muscle, and liver compared to saline-treated mice. By means of recombinant enzymes, we showed that phenylbutyrate prevents phosphorylation of E1 alpha through binding and inhibition of PDK, providing a molecular explanation for the effect of phenylbutyrate on PDHC activity. Phenylbutyrate increases PDHC activity in fibroblasts from PDHC-deficient patients harboring various molecular defects and corrects the morphological, locomotor, and biochemical abnormalities in the noa(m631) zebrafish model of PDHC deficiency. In mice, phenylbutyrate prevents systemic lactic acidosis induced by partial hepatectomy. Because phenylbutyrate is already approved for human use in other diseases, the findings of this study have the potential to be rapidly translated for treatment of patients with PDHC deficiency and other forms of primary and secondary lactic acidosis.

Because of the widespread diffusion of semistructured data in XML format, much research effort is currently devoted to support the storage and retrieval of large collections of such documents. XML documents can be compared as to their structural similarity, in order to group them into clusters so that different storage, retrieval, and processing techniques can be effectively exploited. In this scenario, an efficient and effective similarity function is the key of a successful data management process. We present an approach for detecting structural similarity between XML documents which significantly differs from standard methods based on graph-matching algorithms, and allows a significant reduction of the required computation costs. Our proposal roughly consists of linearizing the structure of each XML document, by representing it as a numerical sequence and, then, comparing such sequences through the analysis of their frequencies. First, some basic strategies for encoding a document are proposed, which can focus on diverse structural facets. Moreover, the theory of discrete Fourier transform is exploited to effectively and efficiently compare the encoded documents (i.e., signals) in the domain of frequencies. Experimental results reveal the effectiveness of the approach, also in comparison with standard methods.

The study of influence-driven propagations in social networks and its exploitation for viral marketing purposes has recently received a large deal of attention. However, regardless of the fact that users authoritativeness, expertise, trust and influence are evidently topic-dependent, the research on social influence has surprisingly largely overlooked this aspect. In this article, we study social influence from a topic modeling perspective. We introduce novel topic-aware influence-driven propagation models that, as we show in our experiments, are more accurate in describing real-world cascades than the standard (i.e., topic-blind) propagation models studied in the literature. In particular, we first propose simple topic-aware extensions of the well-known Independent Cascade and Linear Threshold models. However, these propagation models have a very large number of parameters which could lead to overfitting. Therefore, we propose a different approach explicitly modeling authoritativeness, influence and relevance under a topic-aware perspective. Instead of considering user-to-user influence, the proposed model focuses on user authoritativeness and interests in a topic, leading to a drastic reduction in the number of parameters of the model. We devise methods to learn the parameters of the models from a data set of past propagations. Our experimentation confirms the high accuracy of the proposed models and learning schemes.

Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus acidocaldarius lactonase, showing low but significant and extremely thermostable paraoxonase activity. This enzyme, that we have named SacPox, is a member of the new described family of phosphotriesterase-like lactonases (PLLs). In this family the binuclear metal centre, which is involved in the catalytic machinery, has been poorly studied up to now. In this work we describe the expression of the protein in presence of different metals showing Mn2+ to support the higher activity. The enzyme has been over-expressed, purified and characterized as a Mn2+-containing enzyme by inductive plasma coupled mass spectrometry (ICP-MS), showing also surprising kinetic differences in comparison with the cadmium-containing enzyme. The Mn2+ containing enzyme was about 30-fold more efficient with paraoxon as substrate and more stable than the Cd2+ counterpart, even though the Mn2+ affinity for the binuclear metal centre is apparently lower. These results increase our knowledge of the biochemical characteristics of SacPox mainly with regard to the metal-ions modulation of function. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 degrees C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni2+ as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=k(cat)/K-M) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-gamma-butyric-lactone (TBBL), and the K-M value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.

In this work we propose a probabilistic hierarchical generative approach for users' preference data, which is designed to overcome the limitation of current methodologies in Recommender Systems and thus to meet both prediction and recommendation accuracy. The Bayesian Hierarchical User Community Model (BH-UCM) focuses both on modeling the popularity of items and the distribution over item ratings. An extensive evaluation over two popular benchmark datasets shows that the combined modeling of item popularity and rating provides a powerful framework both for rating prediction and for the generation of accurate recommendation lists.

We propose two models for improving the performance of rule-based classification under unbalanced and highly imprecise domains. Both models are probabilistic frameworks aimed to boost the performance of basic rule-based classifiers. The first model implements a global-to-local scheme, where the response of a global rule-based classifier is refined by performing a probabilistic analysis of the coverage of its rules. In particular, the coverage of the individual rules is used to learn local probabilistic models, which ultimately refine the predictions from the corresponding rules of the global classifier. The second model implements a dual local-to-global strategy, in which single classification rules are combined within an exponential probabilistic model in order to boost the overall performance as a side effect of mutual influence. Several variants of the basic ideas are studied, and their performances are thoroughly evaluated and compared with state-of-the-art algorithms on standard benchmark datasets.

Here we report a comprehensive analysis through alanine-scanning mutagenesis of the contribution of surface ion pairs to the thermal stability of Alicyclobacillus acidocaldarius esterase 2 (EST2). We produced 16 single mutants, 4 double mutants corresponding to selected ion pairs R31/E118, E43/K102, R58/D130, D145/R148, 2 double mutants (R63A/R98A and E50A/D94A) involving residues of a large ion network on the protein surface and the double-mutant R98A/R148A meant to disrupt the R98 interactions within the said network and, contextually, the interaction between R148 and D145. The double-mutant E43A/E273K was obtained by chance. All selected residues were replaced with alanine except E91, which was mutated to a glycine and K102, which was changed to a glutamine. All 24 proteins were over-expressed in Escherichia coli, purified and characterized with respect to the main features. Structural stability data were compared with an in silico prediction of ΔΔG values. Our study of the individual factors involved in thermostability and their structural interpretation reveals that the great stability of this thermophilic protein can be explained by the contribution of a few residues at the protein surface.

A comparative study aiming at analysing the catalytic activity and stability of four related enzymes belonging to the HSL family in the presence of several water-miscible organic solvents was performed. The studied enzymes were four carboxylesterases: PsyHSL (psychrophilic), Aes (mesophilic), EST2 (thermophilic) and AFEST (hyperthermophilic). The effect of the solvents on catalytic activity was measured by adding solvents up to 20% (v/v) to the assay mixture. For all enzymes at low solvent concentrations (4-5%) the catalytic activity (U/mg) increased up to 3-4-fold; only diethyl ether exerted a negative effect when compared to the reference value without solvent. EST2 and AFEST retained more than 100% of activity for concentrations of solvent >10% (v/v). Similar results were obtained for the stability analysis; in fact these two enzymes appeared more stable than the mesophilic and psychrophilic counterparts. The kinetic parameters of PsyHSL, EST2 and AFEST in all the tested solvents showed an increase in kcat but a negative effect on KM values. In the case of PsyHSL the KM increase was very low resulting in a substantial increase of the specific constant (S); in particular, the addition of dimethyl sulfoxide increased the kcat and the s values about 4- and 3-fold, respectively. We tested the ability of the enzymes to synthesize esters with the aim to discriminate if the decrease of activity in the presence of organic solvents was due to protein denaturation or an inversion of the hydrolytic reaction toward synthesis. While for PsyHSL and Aes the synthesis was very low and completely abolished in presence of solvents, instead for EST2 and AFEST the esters synthesis increased up to 10-fold in the presence of solvents, although the hydrolytic activity dropped about 300-fold.