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During the learning process, a child develops a mental representation of the task he or she is learning. A Machine Learning algorithm develops also a latent representation of the task it learns. We investigate the development of the knowledge construction of an artificial agent through the analysis of its behavior, i.e., its sequences of moves while learning to perform the Tower of Hanoï (TOH) task. The TOH is a well-known task in experimental contexts to study the problem-solving processes and one of the fundamental processes of children’s knowledge construction about their world. We position ourselves in the field of explainable reinforcement learning for developmental robotics, at the crossroads of cognitive modeling and explainable AI. Our main contribution proposes a 3-step methodology named Implicit Knowledge Extraction with eXplainable Artificial Intelligence (IKE-XAI) to extract the implicit knowledge, in form of an automaton, encoded by an artificial agent during its learning. We showcase this technique to solve and explain the TOH task when researchers have only access to moves that represent observational behavior as in human–machine interaction. Therefore, to extract the agent acquired knowledge at different stages of its training, our approach combines: first, a Q-learning agent that learns to perform the TOH task; second, a trained recurrent neural network that encodes an implicit representation of the TOH task; and third, an XAI process using a post-hoc implicit rule extraction algorithm to extract finite state automata. We propose using graph representations as visual and explicit explanations of the behavior of the Q-learning agent. Our experiments show that the IKEXAI approach helps understanding the development of the Q-learning agent behavior by providing a global explanation of its knowledge evolution during learning. IKE-XAI also allows researchers to identify the agent’s Aha! moment by determining from what moment the knowledge representation stabilizes and the agent no longer learns., Region Bretagne, European Union via the FEDER program, Spanish Government Juan de la Cierva Incorporacion - MCIN/AEI IJC2019-039152-I, Google Research Scholar Grant

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an important regulator of extracellular matrix turnover that has been traditionally regarded as a potential tumor suppressor owing to its inhibitory effects of matrix metalloproteinases. Intriguingly, this interpretation has been challenged by the consistent observation that increased expression of TIMP-1 is associated with poor prognosis in virtually all cancer types including lung cancer, supporting a tumor-promoting function. However, how TIMP-1 is dysregulated within the tumor microenvironment and how it drives tumor progression in lung cancer is poorly understood. We analyzed the expression of TIMP-1 and its cell surface receptor CD63 in two major lung cancer subtypes: lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and defined the tumor-promoting effects of their interaction. We found that TIMP-1 is aberrantly overexpressed in tumor-associated fibroblasts (TAFs) in ADC compared to SCC. Mechanistically, TIMP-1 overexpression was mediated by the selective hyperactivity of the pro-fibrotic TGF-β1/SMAD3 pathway in ADC-TAFs. Likewise, CD63 was upregulated in ADC compared to SCC cells. Genetic analyses revealed that TIMP-1 secreted by TGF-β1-activated ADC-TAFs is both necessary and sufficient to enhance growth and invasion of ADC cancer cells in culture, and that tumor cell expression of CD63 was required for these effects. Consistently, in vivo analyses revealed that ADC cells co-injected with fibroblasts with reduced SMAD3 or TIMP-1 expression into immunocompromised mice attenuated tumor aggressiveness compared to tumors bearing parental fibroblasts. We also found that high TIMP1 and CD63 mRNA levels combined define a stronger prognostic biomarker than TIMP1 alone. Our results identify an excessive stromal TIMP-1 within the tumor microenvironment selectively in lung ADC, and implicate it in a novel tumor-promoting TAF-carcinoma crosstalk, thereby pointing to TIMP-1/CD63 interaction as a novel therapeutic target in lung cancer.

Background Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic efficacy of this novel nanostructured system were assessed. Results Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around − 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin microbiota. Additionally, the stability and sterility of developed NPs were maintained along storage. Conclusion TH-NP showed a promising and efficient alternative for the treatment of skin acne infection, avoiding antibiotic administration, reducing side effects, and preventing microbial drug resistance, without altering the healthy skin microbiota. Additionally, TH-NP enhanced TH antioxidant activity, constituting a natural, preservative-free, approach for acne treatment. Graphical Abstract

Although the commensal Streptococcus sanguinis [ S. sanguinis] is isolated from caries-free people, it can ferment carbohydrates producing acids. We aimed to characterize S. sanguinis cariogenic potential as a function of different enamel biofilm formation periods, in vitro. Saliva-coated enamel slabs were inoculated with S. sanguinis to form initial biofilms for 8, 12 or 16 h in presence of sucrose and followed by a period in medium with glucose for 16, 12 or 8 h, respectively, until completion of 24 h. To simulate cariogenic challenges, S. sanguinis biofilms were exposed to 10% sucrose for 5 minutes, 3x/day for 5 days. Biofilm biomass, viable cells, total proteins, intracellular and extracellular polysaccharides production, acidogenicity and enamel demineralization were determined. Biofilms of Streptococcus mutans [ S. mutans ] served as caries-positive control. Biofilms of S. sanguinis forming on enamel for 12 and 16 h showed higher demineralization than those formed during 8 h, but lower than S. mutans biofilms, regardless of the initial biofilm formation time. No differences were detected in the biofilm properties among the different biofilm formation times tested for S. sanguinis . Increased enamel initial biofilm formation time by S. sanguinis appears to induce a cariogenic potential, but lower than S. mutans .

The development of feasible micro/nanoplatforms for various biomedical applications requires holistic research that explores scalable synthesis and design pathways and imposes an interdisciplinary integration of materials science, physical, medical, chemical, and biological knowledge. Thanks to their unique characteristics (i.e., structure, large specific surface areas, tuneability, versatility, and integrity), mesoporous materials have emerged as potential candidates for being part of micro/nanoplatforms for therapeutic, monitoring, and diagnostic applications. In this context, Fe-Pt mesoporous materials are excellent candidates to be part of biomedical micro/nanoplatforms, thanks to their chemical nature, structure, and magnetic properties, which endow them with magnetic locomotion, high cargo capability of therapeutic agents inside the mesoporous cavity, and large surface area for surface functionalization. However, the chemical stability in biological media and cytotoxicity of the Fe-Pt mesoporous material (without considering the effects of architecture and shape) are pivotal elements that determine the suitability of these materials for biomedical applications. This work demonstrates the following: (i) the potential of electrochemical deposition, based on the use of block copolymer micellar solutions as electrochemical media, as an easy, inexpensive, and scalable strategy to synthesize mesoporous Fe-Pt components with tunable chemical composition, porosity, magnetism, and shape (in this case films, but other architectures like nanowires can be easily fabricated using simultaneously hard templates); (ii) the excellent corrosion stability, which is comparable to bulk Au, and minimal chemical dissolution in biological media after 160 h of immersion (∼0.88% of Fe and ∼0.0019% of Pt), which confirms the robustness of Fe-Pt; and (iii) negligible cytotoxicity on HaCaT cells (human immortalized keratinocytes), which reinforces the biocompatibility of Fe-Pt mesoporous structures. Also, the presence of Fe-Pt mesoporous films seems to induce a slight increase in cell viability. These results confirm the biocompatibility of Fe-Pt mesoporous films, making them suitable for biomedical applications.

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

The interconnection between nutrients, metabolites and microbes is a key factor governing the healthy/pathological status of an individual. Thus, microbiota-based research is essential in order to better understand human health and nutrition. Gut bacteria release membrane vesicles (MVs) as an intercellular communication mechanism that allows the direct delivery of factors that prime the host&rsquo, s innate immune system. We have previously shown that MVs from intestinal E. coli activate dendritic cells (DCs) in a strain-specific manner. To gain insights into the regulatory mechanisms involved, here, we have used an RNA deep sequencing approach to identify differentially expressed miRNAs (microRNAs) in DCs which are challenged by the MVs of the probiotic Nissle 1917 (EcN) or the commensal ECOR12. MicroRNAs are post-transcriptional regulatory mediators that permit the fine tuning of signaling pathways. This approach allowed the identification of a common set of miRNAs which are modulated by MVs from both strains and miRNAs which are differentially expressed in response to EcN or ECOR12 MVs. Based on the differential expression of the target genes and subsequent validation experiments, we correlated some of the selected miRNAs with the reported cytokine profile and specific T cell responses. As far as we know, this is the first study to analyze the regulation of miRNAs in DCs by MVs released by gut microbiota.

Background Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. Results Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. Conclusion This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Objectives: Tumor associated fibroblasts (TAFs) are essential contributors of the progression of non-small cell lung cancer (NSCLC). Most lung TAFs exhibit an activated phenotype characterized by the expression of α-SMA and fibrillar collagens. However, the prognostic value of these activation markers in NSCLC remains unclear. Material and methods: We conducted a quantitative image analysis of α-SMA immunostaining and picrosirius red staining of fibrillar collagens imaged by bright-field and polarized microscopy, respectively, using tissue microarrays with samples from 220 surgical patients, which elicited a percentage of positive staining area for each marker and patient. Results: Kaplan-Meier curves showed that all TAF activation markers were significantly associated with poor survival, and their prognostic value was independent of TNM staging as revealed by multivariate analysis, which elicited an adjusted increased risk of death after 3 years of 129% and 94% for fibrillar collagens imaged with bright-field (p = 0.004) and polarized light (p = 0.003), respectively, and of 89% for α-SMA (p = 0.009). We also found a significant association between all TAF activation markers and tumor necrosis, which is often indicative of hypoxia, supporting a pathologic link between tumor desmoplasia and necrosis/hypoxia. Conclusions: Our findings identify patients with large histologic coverage of fibrillar collagens and α-SMA + TAFs to be at higher risk of recurrence and death, supporting that they could be considered for adjuvant therapy. This work was further supported bygrants from the Ministerio de Economía y Competitividad (MINECO/FEDER, UE) (PI13/02368 and SAF2016-79527-R to JA, FIS 12/02040to EM and FIS 12/02534 to EB), Fundació Privada Cellex (to JA),Generalitat de Catalunya AGAUR (SGR 661 to JA, SGR 801 to EM),Junta Provincial de Barcelona de l’Associació Espanyola Contra elCàncer (AECC B16-917 to JA), Fundació Catalana de Pneumologia (toEM), Sociedad Española de Neumología y Cirugía Torácica–SEPAR (PIIOncología Torácica to EM, < /GN6 > SEPAR 437 < /GN6 > to NR),and beca CONICYT (to N.D-V.)

// Rina Ortiz 1, 2, 3, 7 , Jorge Diaz 1, 2, 4 , Natalia Diaz 1, 2, 7 , Lorena Lobos-Gonzalez 1, 5, 6 , Areli Cardenas 1, 2, 7 , Pamela Contreras 1, 2 , Maria Ines Diaz 1, 2 , Ellen Otte 8 , Justin Cooper-White 8 , Vicente Torres 1, 4 , Lisette Leyton 1, 2, 7 , Andrew F.G. Quest 1, 2 1 Center for Molecular Studies of the Cell (CEMC), Advanced Center for Chronic Diseases (ACCDiS), Faculty of Medicine, Universidad de Chile, Santiago, Chile 2 Laboratory of Cellular Communication, Program of Cell and Molecular Biology, Institute of Biomedical Sciences (ICBM), Faculty of Medicine, Universidad de Chile, Santiago, Chile 3 Universidad Bernardo O Higgins, Facultad de Salud, Departamento de Ciencias Quimicas y Biologicas, Santiago, Chile 4 Institute for Research in Dental Sciences, Faculty of Dentistry, Universidad de Chile, Santiago, Chile 5 Andes Biotechnologies SA, Nunoa, Santiago, Chile 6 Fundacion Ciencia & Vida, Nunoa, Santiago, Chile 7 Biomedical Neuroscience Institute (BNI) Santiago, Chile 8 Australian Institute for Bioengineering & Nanotechnology, The University of Queensland, St. Lucia, Queensland, Australia Correspondence to: Andrew F.G. Quest, email: aquest@med.uchile.cl Lisette Leyton, email: lleyton@med.uchile.cl Keywords: Caveolin-1, cancer, dual role, migration, invasion Received: December 04, 2015 Accepted: May 16, 2016 Published: May 31, 2016 SUMMARY Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1.

Human serum albumin undergoes reversible conformational transitions associated with ligand binding or pH changes. Among them, the neutral to base (N → B) transition occurring between pH 7 and pH 9 seems to be relevant for its function as a carrier. Unfortunately, a detailed atomic model for the B-form is still lacking, and several open questions remain concerning the charge distribution of the N-form. In this work, we report comparable molecular models for the N and B conformations that are built using continuum electrostatic calculations of pKa values and extended molecular dynamics (MD) simulations. Our computational models, which are critically assessed in terms of the available experimental observations relative to the N → B transition, reveal interesting similarities and differences between the N- and B-forms of HSA and highlight the importance of setting proper charge configurations in MD simulations.

Streptococcus mutans synthesizes 3 glucosyltransferases (Gtfs) associated with cariogenic biofilms, while commensal Streptococcus sanguinis produces only one; gtfP and hydrogen peroxide (H2O2) by SpxB. The aim was to test the hypothesis that under a sucrose-induced cariogenic challenge, the expression of competition-related genes is differentially regulated depending on whether S. sanguinis or S. mutans primarily colonize enamel. Dual-species biofilms of S. sanguinis and S. mutans were formed under different colonization sequences on enamel slabs and exposed to 10% sucrose for 5 min, 3×/day for 5 days. Biofilms were analyzed for the transcriptional response of competition-related genes encoding gtfB, gtfC, and gtfD for S. mutans and gtfP and spxB for S. sanguinis. In addition, acidogenicity (pH) and viable cells in each of the conditions were determined. For all the genes, a downregulation was observed during simultaneous colonization by both bacterial species. In contrast, gtfB was upregulated when S. sanguinis was the first colonizer (p0.05). Both gtfC and gtfD were upregulated during sequential inoculation with S. sanguinis as the first colonizer. An eleven-fold upregulation of gtfP was observed in biofilms with S. mutans as initial colonizer (p0.05), with a moderate increase in spxB expression. The lowest pH values and viable cells of S. sanguinis were observed when S. mutans first colonized the enamel slabs, compared to the other conditions (p0.05). Demanding sucrose-challenged oral environment requires increased expression of virulence traits to effectively compete and thrive in the dental biofilm, especially when the competitor has already colonized the ecological niche.

International audience; Representation learning algorithms are designed to learn abstract features that characterize data. State representation learning (SRL) focuses on a particular kind of representation learning where learned features are in low dimension, evolve through time, and are influenced by actions of an agent. The representation is learned to capture the variation in the environment generated by the agent's actions; this kind of representation is particularly suitable for robotics and control scenarios. In particular, the low dimension characteristic of the representation helps to overcome the curse of dimensionality, provides easier interpretation and utilization by humans and can help improve performance and speed in policy learning algorithms such as reinforcement learning. This survey aims at covering the state-of-the-art on state representation learning in the most recent years. It reviews different SRL methods that involve interaction with the environment , their implementations and their applications in robotics control tasks (simulated or real). In particular, it highlights how generic learning objectives are differently exploited in the reviewed algorithms. Finally, it discusses evaluation methods to assess the representation learned and summarizes current and future lines of research.

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation.

Background Self-quantification of health parameters is becoming more popular; thus, the validity of the devices requires assessments. The aim of this study was to evaluate the validity of Fitbit One step counts (Fitbit Inc., San Francisco, CA, USA) against Actigraph wActisleep-BT step counts (ActiGraph, LLC, Pensacola, FL, USA) for measuring habitual physical activity among children. Design The study was implemented as a cross-sectional experimental design in which participants carried two waist-worn activity monitors for five consecutive days. Methods The participants were chosen with a purposive sampling from three fourth grade classes (9–10 year olds) in two comprehensive schools. Altogether, there were 34 participants in the study. From these, eight participants were excluded from the analysis due to erroneous data. Primary outcome measures for step counts were Fitbit One and Actigraph wActisleep-BT. The supporting outcome measures were based on activity diaries and initial information sheets. Classical Bland–Altman plots were used for reporting the results. Results The average per-participant daily difference between the step counts from the two devices was 1937. The range was [116, 5052]. Fitbit One gave higher step counts for all but the least active participant. According to a Bland–Altman plot, the hourly step counts had a relative large mean bias across participants (161 step counts). The differences were partially explained by activity intensity: higher intensity denoted higher differences, and light intensity denoted lower differences. Conclusions Fitbit One step counts are comparable to Actigraph step counts in a sample of 9–10-year-old children engaged in habitual physical activity in sedentary and light physical activity intensities. However, in moderate-to-vigorous physical activity, Fitbit One gives higher step counts when compared to Actigraph.

Matrix metalloproteases are multidomain enzymes with a remarkable proteolytic activity located in the extracellular environment. Their catalytic activity and structural properties have been intensively studied during the last few decades using both experimental and theoretical approaches, but many open questions still remain. Extensive molecular dynamics simulations enable the sampling of the configurational space of a molecular system, thus contributing to the characterization of the structure, dynamics, and ligand binding properties of a particular MMP. Based on previous computational experience, we provide in this chapter technical and methodological guidelines that may be useful to and stimulate other researchers to perform molecular dynamics simulations to help address unresolved questions concerning the molecular mode of action of MMPs.

We present the CENCALC software that has been designed to estimate the conformational entropy of single molecules from extended Molecular Dynamics (MD) simulations in the gas-phase or in solution. CENCALC uses both trajectory coordinates and topology information in order to characterize the conformational states of the molecule of interest by discretizing the time evolution of internal rotations. The implemented entropy methods are based on the mutual information expansion, which is built upon the converged probability density functions of the individual torsion angles, pairs of torsions, triads, and so on. Particularly, the correlation-corrected multibody local approximation selects an optimum cutoff in order to retrieve the maximum amount of genuine correlation from a given MD trajectory. We illustrate these capabilities by carrying out conformational entropy calculations for a decapeptide molecule either in its unbound form or in complex with a metalloprotease enzyme. CENCALC is distributed under the GNU public license at http://sourceforge.net/projects/cencalc/.

The effectiveness against radiation of tungsten bismuth caps, used in interventional cardiology is not well known.To determine the degree of radiation protection conferred by these caps in real work conditions.We compared the gross electric charges received at brain lobe levels by three occupationally exposed professionals who participated in 22 consecutive procedures, inside and outside of the tungsten bismuth cap.The median electric charges outside and inside the cap were 3.71 (range 1.46-5.62) and 2.2 (range 1.29-3.93) nC, which correspond to a 40% radiation attenuation. However, the protection was heterogeneous.Tungsten bismuth caps provide an adequate attenuation, but its degree is heterogeneous.

Many researchers have studied somatic symptoms in children. However, its association with severe mood dysregulation (SMD) is poorly known. The aim of this study is to detect the presence of SMD in preschool children and to know the prevalence of somatic symptoms and associations with psychopathology, SMD, and aggressiveness. The study population consists of children between 3 to 6 years of age enrolled in Barcelona's kindergarten schools (n = 319). Their parents completed questionnaires about the presence of somatic symptoms in children, absences from school and pediatric visits, child psychiatric symptoms, presence of symptoms of SMD, and aggressiveness. Teachers were also informed about SMD and aggressiveness. Children who complained frequent somatic symptoms (three or more in the last 2 weeks) were compared with those who did not. Two hundred five children (64.3%) reported at least one physical complaint in the 2 weeks preceding the study. One hundred participants (31.3%) reported frequent somatic complaints. Positive associations were found with anxiety symptomatology, separation anxiety, social phobia, pediatric visits, and school absences, but not with aggressiveness or SMD symptoms. Somatic symptoms are common in a sample of preschool children but do not show a positive association with the symptoms of SMD.

Collagen hydrolysis catalyzed by matrix metalloproteinases is an important and complex process involved in a variety of physiological and pathological conditions. To contribute to its characterization at the molecular level, herein we analyze three different models for the complex formed between the full-length matrix metalloproteinase-2 (MMP-2) enzyme and a synthetic triple-helical peptide (fTHP-5). The considered MMP-2/fTHP-5 complexes mainly differ in the location of the C-terminal hemopexin-like domain, but in all of them, the middle α-chain of the substrate (B-chain) is placed within the active site groove. We performed extended molecular dynamics (MD) simulations to determine the most likely rearrangements of the MMP-2 domains in response to the presence of the triple helix. The relative stability of the MD models is assessed in terms of molecular mechanics Poisson-Boltzmann calculations and approximate estimations of configurational entropy. In addition, the most significant MMP-2···fTHP-5 interactions at the catalytic and noncatalytic domains are also analyzed to gather some clues about the role of the different domains during collagenolysis.

Caveolin-1 is a member of the caveolin family that has been ascribed a dual role in cancer. In early stages of disease the protein functions predominantly as a tumor suppressor, whereas at later stages, caveolin-1 expression is associated with tumor progression and metastasis. Here, some mechanisms associated with caveolin-1-dependent tumor suppression will be briefly discussed before focusing on the role of this protein and particularly phosphorylation of tyrosine-14 in promoting cell migration, invasion and metastasis. Models are provided summarizing possible explanations for these dramatic changes in function, as well as mechanisms by which this may be achieved.

Human activity recognition is a key task in ambient intelligence applications to achieve proper ambient assisted living. There has been remarkable progress in this domain, but some challenges still remain to obtain robust methods. Our goal in this work is to provide a system that allows the modeling and recognition of a set of complex activities in real life scenarios involving interaction with the environment. The proposed framework is a hybrid model that comprises two main modules: a low level sub-activity recognizer, based on data-driven methods, and a high-level activity recognizer, implemented with a fuzzy ontology to include the semantic interpretation of actions performed by users. The fuzzy ontology is fed by the sub-activities recognized by the low level data-driven component and provides fuzzy ontological reasoning to recognize both the activities and their influence in the environment with semantics. An additional benefit of the approach is the ability to handle vagueness and uncertainty in the knowledge-based module, which substantially outperforms the treatment of incomplete and/or imprecise data with respect to classic crisp ontologies. We validate these advantages with the public CAD-120 dataset (Cornell Activity Dataset), achieving an accuracy of 90.1% and 91.07% for low-level and high-level activities, respectively. This entails an improvement over fully data-driven or ontology-based approaches., This work was funded by TUCS (Turku Centre for Computer Science), Finnish Cultural Foundation, Nokia Foundation, Google Anita Borg Scholarship, CEI BioTIC Project CEI2013-P-3, Contrato-Programa of Faculty of Education, Economy and Technology of Ceuta and Project TIN2012-30939 from National I+D Research Program (Spain). We also thank Fernando Bobillo for his support with FuzzyOWL and FuzzyDL tools.

Herein, we present the results of a computational study that employed various simulation methodologies to build and validate a series of molecular models of a synthetic triple-helical peptide (fTHP-5) both in its native state and in a prereactive complex with the catalytic domain of the MMP-2 enzyme. First, the structure and dynamical properties of the fTHP-5 substrate are investigated by means of molecular dynamics (MD) simulations. Then, the propensity of each of the three peptide chains in fTHP-5 to be distorted around the scissile peptide bond is assessed by carrying out potential of mean force calculations. Subsequently, the distorted geometries of fTHP-5 are docked within the MMP-2 active site following a semirigid protocol, and the most stable docked structures are fully relaxed and characterized by extensive MD simulations in explicit solvent. Following a similar approach, we also investigate a hypothetical ternary complex formed between two MMP-2 catalytic units and a single fTHP-5 molecule. Overall, our models for the MMP-2/fTHP-5 complexes unveil the extent to which the triple helix is distorted to allow the accommodation of an individual peptide chain within the MMP active site.

Helicobacter pylori is the etiologic agent of a series of gastric pathologies that may culminate in the development of gastric adenocarcinoma. An initial step in this process is the loss of glandular structures in the gastric mucosa, presumably as the consequence of increased apoptosis and reduced cellular regeneration, which may be attributed to the combination of several bacterial and host factors and to an unfavorable proinflammatory environment. In a previous study, we showed that survivin, a member of the inhibitor of apoptosis protein family, is expressed in the normal human gastric mucosa and that its levels decrease in the mucosa of infected patients and in gastric cells exposed in culture to the bacteria, coincident with increased cell death in the latter case. We investigated the bacterial factors responsible for loss of survivin in gastric cells exposed to H. pylori. The results of this study indicated that the loss of survivin due to H. pylori infection involves proteasome-mediated degradation of the protein. Studies with isogenic mutants deficient in either CagA, VacA, lipopolysaccharide, or gamma-glutamyl transpeptidase (GGT) implicated the latter in H. pylori-induced loss of survivin and cell viability. Moreover, experiments with the GGT inhibitor 6-diazo-5-oxo-l-norleucine and purified recombinant GGT protein indicated that secreted bacterial GGT activity was required and sufficient to induce these effects. © 2013 The Author 2013.

The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells.

Herein we investigate the role played by the so-called "structural metal ions" in the catalytic domain of the matrix metalloproteinase 2 enzyme (MMP-2 or gelatinase A). We performed seven molecular dynamics simulations that differ in the number and position of the noncatalytic zinc and calcium ions bound to the MMP-2 catalytic domain. An additional simulation including the three fibronectin-type modules inserted into the catalytic domain was also carried out. The analysis of the trajectories confirms that the binding/removal of the structural ions does not perturb the secondary structure elements but influences the position of several solvent-exposed loop regions that are placed near the active site cleft. The position of these loops modulates the accessibility of important anchorage points for substrate binding that have been identified in the active site groove. On the basis of semiempirical quantum chemical calculations, we estimated the relative free energies of the MMP-2 models, obtaining thus that the binding of two zinc and two calcium ions to the MMP-2 catalytic domain is energetically favored. In this MMP-2 model, which shows the most compact structure, all of the substrate binding sites are readily accessible. Globally, our results help to rationalize at the atomic level the calcium and zinc dependence of the hydrolytic activity catalyzed by the MMPs.

To investigate the mechanism of the deacylation reaction in the active site of human butyrylcholinesterase (BuChE), we carried out quantum mechanical (QM) calculations on cluster models of the active site built from a crystallographic structure. The models consisted of the substrate butyrate moiety, the catalytic triad of residues (Ser198, Glu325, and His438), the "oxy-anion hole" (Gly116, Gly117, and Ala199), the side chain of Glu197, four water molecules, the side chain of Ser225, and the peptide linkage between Val321 and Asn322. Analyses of the equilibrium geometries, electronic properties, and energies of the QM models gave insights into the catalytic mechanism. In addition, the QM calculations provided the data required to build a molecular mechanics representation of the reactive BuChE region that was employed in molecular dynamics simulations followed by molecular-mechanics-Poisson-Boltzmann (MM-PB) calculations. Subsequently, we combined the QM energies with average MM-PB energies to estimate the free energy of the reactive structures in the enzyme. The rate-determining step corresponds to the formation of a tetrahedral intermediate with a free-energy barrier of approximately 14.0 kcal/mol. The modulation of the BuChE activity, exerted by either neutral molecules (glycerol, GOL) or a second butyrylcholine (CHO) molecule bound to the cation-pi site, does not involve any significant allosteric effect. Interestingly, the presence of GOL or CHO stabilizes a product complex formed between a butyric acid molecule and BuChE. These results are in consonance with the crystallographic structure of BuChE, in which the catalytic Ser198 interacts with a butyric fragment, while the cation-pi site is occupied by one GOL molecule.

The IIA binding site of human serum albumin (HSA) preferentially binds hydrophobic organic anions of medium size (e.g., aspirin, benzylpenicillin, warfarin, etc.) and bilirubin. This binding ability is particularly important for the distribution, metabolism, and efficacy of drugs. In addition, HSA can also covalently link to different IIA substrates owing to the presence of a highly reactive residue, Lys199, which is strategically located in the IIA site. Herein, we present results of three restrained molecular dynamics (MD) simulations of the IIA binding site on the HSA protein. From these simulations, we have determined the influence that the ionization state of the key residue, Lys199, and the nearby Lys195 has on the structure and dynamics of the IIA binding site. When Lys199 is neutral the computed average distances for the most significant interresidue contacts are in good agreement with those estimated from the X-ray coordinates. The analysis of the solvent structure and dynamics indicates that the basic form of Lys199 is likely connected to the acid form of Lys195 through a network of H-bonding water molecules with a donor --> acceptor character. The presence of these water bridges can be important for stabilizing the configuration of the IIA binding site and/or promoting a potential Lys195 --> Lys199 proton-transfer process. These results suggest that both lysine residues located in the IIA binding site of HSA, Lys195 and Lys199, could play a combined and comparable chemical role. Our simulations also give insight into the binding of bilirubin to HSA.

Water solubility is a key aspect that needs to be addressed to obtain drug-like compounds. In an effort to improve the water solubility of our recently reported nanomolar matrix metalloproteinase type 2 (MMP-2) inhibitors based on triazole-substituted hydroxamates, we synthesized a new series of α-sulfone, α-tetrahydropyran and α-piperidine, α-sulfone clicked hydroxamates and determined their inhibitory activities against both MMP-2 and MMP-9. The best results were found for 13e, a water-soluble compound that displays a low nanomolar activity against MMP-2 and is 26-fold less active against MMP-9. This finding allowed us to pursue in vitro permeability through the Caco-2 monolayer and opened the possibility of carrying out further preclinical investigations. Docking and MD simulations have been performed in order to rationalize the biological results. The inhibitory activity of this compound against a panel of ten MMPs was determined showing an interesting MMP-2/MMP-1, -8, and -14 selectivity profile. The cytotoxicity and anti-invasive activity of the compounds on highly metastatic human fibrosarcoma tumor cells (HT1080) were determined, showing, at 10 μM concentration, a decrease in cell invasiveness up to 80%.

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.