The triad is a particular structure in which an ambivalent social relationship takes place. This work is focused on the search of behavioral regularities in the practice of motor games in triad, which is a little known field. For the detection of behavioral patterns not visible to the naked eye, we use Theme. A chasing games model was followed, with rules, and in two different structures (A↔B↔C↔A and A → B → C → A) on four class groups (two for each structure), for a total of 84, 12, and 13 year old secondary school students, 37 girls (44%) and 47 boys (56%). The aim was to examine if the players' behavior, in relation to the triad structure, matches with any ludic behavior patterns. An observational methodology was applied, with a nomothetic, punctual and multidimensional design. The intra and inter-evaluative correlation coefficients and the generalizability theory ensured the quality of the data. A mixed behavioral role system was used (four criteria and 15 categories), and the pattern detection software Theme was applied to detect temporal regularities in the order of event occurrences. The results show that time location of motor responses in triad games was not random. In the “maze” game we detected more complex ludic patterns than the “three fields” game, which might be explained by means of structural determinants such as circulation. This research points out the decisional complexity in motor games, and it confirms the differences among triads from the point of view of motor communication. Motor games with rules enclose players behavior regularity due to the expectations of the roles. The regularities of these behaviors also depend on the communication structure to which the players are subjected. Play behaviors are an orderly way of communicating in games with rules. The relationships between the roles of the games and its time are two key aspects of the analysis of the relationships showed by the players, in this way triadic relationships are defined. Triad motor games are composed of three decisional units , in a context of motor communication , while circulation is the sequence of connections from one source to the same starting point, completing one cycle. Structure 2 does not have reciprocity, but with one-way circulation, however, structure 1 has 3 reciprocities and two-way circulation, that is, it has two origins. Their reciprocity is affected in the confrontation between the three teams, because each duel would annul itself reciprocally. In relation to the circulation property in structure 2, in simultaneous capture games, if the players of team A take prisoners of the players of team B, it means that most of the players of team C will remain as free players because there are scarce possibilities for team B players to get some catch from team C. That is, team A, following the rules of the game and fulfilling their pretensions, hinder their chances of winning the game. These situations, scarcely studied in the motor game, were already analyzed in depth with a social approach by Caplow . It shouThe internal logic of the motor game leads to focus attention on the role as it is a route of play when players act. Role is a structural indicator that helps to operationalize the motor game, as well as to order the strategic procedures that the players put into practice to reach their objectives. In an apparently uncontrolled context in a game, each driving situation is unique and unrepeatable. Role represents the path, the structure of communication support, and T-patterns (detected with Theme) emerge from the observed behaviors. In this sense, the T-patterns found are not rules of decision for the game, but seemingly random events which are timely and behaviorally organized, though. For this reason, an ecological methodology , two communication conditions are tested when developing the game strategy; the game “the maze” is more complex due to the number of reciprocities, while “the three fields” does not have any reciprocity. In addition, the one-way communication of the game “the three fields,” directly activates a structural paradox with consequences on the strategy of teammates and/or rivals.In this line of research, aimed at the motor game, the objective was to look for temporary regularities in two triad games, under two different communication structures, through game roles and their observable behaviors in practice. For this study, the following generic and specific objectives were considered, respectively.- Identifying systemic properties in two triadic games and analyzing their influence in practice.- Discriminating the scope of triadic behavior according to the roles of the game.Hypothesis:- The increase in decisional complexity and strategic regularity (T-patterns) is greater in favor of the triadic game with a greater number of reciprocities in its communication.An observational methodology design was selected for the study. It is a relatively recent methodological approach  with written informed consent from all parents or legals tutors of all participants (Declaration of Helsinki).The number of players was 84, consisting of 37 (44%) girls and 47 (56%) boys between 12 and 13 years old  from two secondary schools in the Canary Islands (Spain). The two institutes were located in different cities and islands. The cities were middle-class urban places. Both the groups and the institutes were selected according to accessibility and intentionality  and “the three fields” (modification), which are both chase games. In “the maze” (A↔B↔C↔A), three teams are formed, with the same strengths, and all players try to capture each other simultaneously, under action conditions that regulate only one part of the body for contact. The captured player assumes the role “prisoner,” remaining crouched in the place where he/she was captured; “prisoners” can be released if they are saved by a free player, under the role of “savior” Navarro, . The teaTo analyze these games, the motor interactions of the roles were taken as a reference Table . In addi“The maze” (modified) has a total of 42 motor interactions . That is, for each cooperative interaction, six antagonists were found. In contrast, when quantifying the motor interaction in “the three fields” game (modified) relational values decrease to 24 motor interactions . Therefore, a structural difference between these two games comes from doubling antagonism over cooperation. Consequently, these are two games structures with marked differences.For the construction of the registration tool, a previous exploratory study  is identified because it bears the initiative of the game, and it is observable through effective captures (CA) or pursuit actions (PA), which brings together at least two players involved with negative emissions. Sometimes, (C) tries to defend players who are already prisoners and it is then when (DEF) takes place, while passive was the player who was not involved in the game (P). When it was observed that a player belonging to different teams pursued an opponent player, it was considered (ALZAAC), being an ambivalent behavior. Although the previous role had more initiative, the “dodger” role (E) offered a reciprocal response to the catch action (C). In this sense, the dodger (E) will act by dodging on the opponent (EA) but he/she can also flee (HA) or move to free places (DLL). When it is observed that a fellow player intends to interfere in a chase between a catcher and a fleeing player, it was considered (AC). When the action is developed cooperatively between players  of different teams, it was considered (ALZAE). If a player was caught and did not admit it, this conduct was considered (NR). The prisoner role (P) occurs when a player has been previously captured. If the player is simply standing without facilitating his/her release we say that he/she is not in attention (A), but other times the player facilitates the saving action (CE). The fourth role, liberator (L), tries to save fellow prisoner players (TUFC) but sometimes the release was verified between players of different teams (TUFA).The images, video recordings, were taken in the educational centers of each group. In order to have a clear observability of each record Anguera,  and to hThe measurements related to the itineraries of the roles and properties that attend the two play structures served as an a priori platform, thus systematizing the recording of motor behaviors through a system composed of four criteria and 15 categories. The follow-up of an observational methodology , a pattern tree graph with a greater verticality than in group 2 could be observed. The most dependent relations of group 1 were intragroup and not intergroup. The behavior of the TR team player  links its appearance in the game to the captive player . The TG player  is imprisoned a prisoner and timely attached to the dodge action . The same player  is captured and related to the player's catch action .The player of the TG team  performs release actions related to fellows that are captured . Finally, from the pattern tree graph, we find a group of three behaviors that link the release of partners  to the existence of fellow prisoners  and , similar to the previous case.In the second group of the same game, the teams  showed through the pattern tree graph more intergroup behaviors with recurrent time dependence than intragroup ones. A prisoner of team Y  with a catcher of team B . We observed a group of three behaviors of the NC team of the prisoner role in attention  with displacements to free places  and fleeing . Also,  and , like  with  and .The two most complex T-patterns detected for the groups 3 and 4 are displayed in Figure The group 3 most complex pattern detected, displayed in Figure Group 4 shows a greater time dependence on game behaviors than group 3. In this group, the captive player of the TG team  caused captures  in the player belonging to the TR team. The two previous behaviors relate to the captures of a player of the TB team . Also, the dodging of the player belonging to the team TR  caused that same player to fall prisoner . The two responses above were linked to the TG team reaction .The most obvious time recurrences were established with team B players. First, catches of team B  with releases that another player from the same team performed . Also between the player  of the same team and . Another linkage found corresponded to behaviors within team B and player  and . Other relationships that were part of the behavioral cluster, although less remarkable, would be those shown among the last four behaviors mentioned, to cause the identification of the behavior of team O  as a captured player. When team O freed teammates , actions were activated on team B to capture , while prisoners on hold . Finally, the flight behavior of team B  was activated.A powerful data pattern detection technique was used, Theme 6.0 , is determinant in the triad analysis because it hinders circulation fulfillment. That is to say, it is inversely proportional: to more reciprocity between the sum of existing duels, less circulation in one direction. This property justifies the structural paradox when the relation is fulfilled in a single sense, as happens in “the three fields” (modified). In this sense, it is a property of the circulation network, transformed into a triad constrains  was found to be TUFA . According to this, the need for releases in the maze mentioned in the previous section seems to be reinforced due to the communication structure , is the release of opponents made by the player  linked to the player of the same team when performing dodges . It may be this criticality what leads the teams to partnership with rivals. Based on the above, its positive or negative good value Heider,  is vitalWe have already alluded to a smaller elaboration of the T-patterns in the three fields in front of the game the maze, backed by the number of T-patterns but also by a greater strategic and temporary structuring of the motor action. The collaborations or release actions between players in the first game showed different behaviors regarding the previous game. Release actions among fellow players (TUFC) were only identified in group 4. That is, the lower number of T-patterns identified in the three fields affected the savior role. In group 3, there was no collaboration to make releases between fellow players or opponents, which could indicate a state of normative incomprehension, culturally understandable in the players for a lack of triadic experiences that group 4 put into practice. The property of circulation and its paradoxical effects on the motor decision, could explain the high strategic disorganization identified by THEME in the third triad group. Again, structure and play roles described the pattern of the motor decision, showing the situational demands that were brought by the groups and found by the relevant detection of temporary recurrences in view of previous studies in sports (Jonsson et al., In conclusion, T-patterns have solved the underlying complexity of the two different game structures and their groups, beyond the systemic solution, showing how players are temporarily confronted in natural contexts of practice.This study has demonstrated the advantages of using a technique pattern detection to address the internal complexity of the motor triad. The inclusion of a time dimension has meant an advance for the interpretation and analysis of data from ecological contexts, confirming the properties of the ludic structure.The decisional complexity of the T-Patterns was different in “the maze” and “the three fields” triadic games. It was also reinforced by the number and composition of T-patterns. The relational strategies identified in the four groups were different, since no similarities were found; which confirms the high complexity of each game developed by each group. This high complexity shows a specific variability for each triadic motor game.Among the limitations that accompanied the study, the inclusion of the spatial criterion as a facet of it would be worth mentioning. Similar studies could replicate this research, with different populations and age groups, to have a thorough knowledge about the triadic effects. Performing analysis aimed at detecting the inhibition and activation of particular play behaviors would add an important explanatory value. The theory of motor play needs to put on hold the circle of appreciation and move on to research, in order to know what these and other motor games hide, as exceptional formulas of social interaction.MA and VN-A have contributed to the theorical and methodological development of the article while GJ has contributed with the data analysis. Results and discussion have been prepared by all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arsenic contamination of drinking water affects more than 140 million people worldwide. While toxic to humans, inorganic forms of arsenic (arsenite and arsenate), can be used as energy sources for microbial respiration. AioX and its orthologues (ArxX and ArrX) represent the first members of a new sub-family of periplasmic-binding proteins that serve as the first component of a signal transduction system, that’s role is to positively regulate expression of arsenic metabolism enzymes. As determined by X-ray crystallography for AioX, arsenite binding only requires subtle conformational changes in protein structure, providing insights into protein-ligand interactions. The binding pocket of all orthologues is conserved but this alone is not sufficient for oxyanion selectivity, with proteins selectively binding either arsenite or arsenate. Phylogenetic evidence, clearly demonstrates that the regulatory proteins evolved together early in prokaryotic evolution and had a separate origin from the metabolic enzymes whose expression they regulate. With oxygen or nitrate (at pH > 9) as the terminal electron acceptor, arsenite (+III) can be used as an electron donor with some organisms clearly gaining energy from its oxidation3. In all cases of arsenic metabolism, the metabolic enzymes involved in the oxidation or reduction reactions are members of the DMSO reductase family of molybdenum-containing enzymes8. The most well characterised arsenic-metabolising enzyme is the arsenite oxidase (Aio) purified from aerobic arsenite oxidisers, with two X-ray crystal structures resolved10. Aio consists of two heterologous subunits, a catalytic molybdopterin guanine dinucleotide-containing subunit (AioA) that also contains a 3Fe-4S cluster, and a small Rieske subunit (AioB)9. The respiratory arsenate reductase (Arr) has been purified and characterised from three organisms11 and like the Aio consists of two heterologous subunits, a molybdenum-containing subunit (ArrA), which based on sequence analysis contains one 4Fe-4S cluster and a small subunit (ArrB) with four 4Fe-4S clusters8. The alternative arsenite oxidase, Arx, which operates in an anaerobic respiratory chain with nitrate reductase as the terminal reductase, has not been studied biochemically but is thought to consist of four subunits based on the arx loci  can serve as a terminal electron acceptor coupling its reduction to the oxidation of a variety of organic and inorganic electron donors16. The expression of Aio in the Proteobacteria is regulated by a regulatory gene cluster adjacent to the arsenite oxidase genes (aioBA). The regulatory cluster consists of three genes, aioX, aioS and aioR, which encode a periplasmic-binding protein (PBP), a sensor histidine kinase and a response regulator, respectively. All three genes are essential for arsenite oxidase gene (aioBA) expression19 and in the case of Rhizobium sp. str. NT-26 are co-transcribed20. AioX is thought to be the sensing protein as it has been shown to bind arsenite in vitro14. Arsenite-bound AioX is thought to act as a ligand for AioS, the sensor histidine kinase that upon auto-phosphorylation, phosphorylates AioR, the response regulator, which binds to a conserved sequence upstream of a RpoN promoter19.In some arsenic-metabolising bacteria, expression of the molybdoenzyme is strictly regulated, with gene expression only detected when the organism is grown in the presence of the arsenic oxyanionHere we report the identification and characterisation of a new subfamily of arsenic oxyanion-binding proteins from phylogenetically distant bacteria that have evolved to regulate bioenergetic arsenic metabolism. These proteins, which demonstrate substrate selectivity, could be used as novel sensors for both toxic arsenic oxyanions.aioXSR regulatory gene cluster is found in some arsenite oxidisers of the Alpha- and Betaproteobacteria. Searches of the GenBank database, revealed that the XSR regulatory module was conserved in a sub-set of other phylogenetically distant arsenic-metabolising bacteria including the anaerobic arsenite oxidiser, Alkalilimnicola ehrlichii str. MLHE-1 (Gammaproteobacteria) (encode Arx – genes are arxB′ABC) and the arsenate respirer, Chrysiogenes arsenatis (Chrysiogenetes) (encode Arr – genes are arrAB) , which contain a AAA+ ATPase domain. Like in NT-26, a putative RpoN promoter (tggcaaggctattgct) and ArxR-binding site (gtaacaattcaagc) (based on homology to the NT-26 AioR-binding site), were identified upstream of the arsenite oxidase genes. ArrR, is a member of a different response regulator family, namely the CitB-like family, similar to the response regulator for citrate sensing in Klebsiella pneumoniae22 and unlike AioR/ArxR does not possess an AAA+ ATPase domain. ArrR has sequence similarity to the DNA-binding, helix-turn-helix portion of FixJ23, with a putative FixJ-like binding site motif24 (tcaggtttttccctga) identified upstream of arrA.AioR and ArxR are the response regulators controlling expression of the arsenite oxidase genes and are members of the AtoC family of response-regulators  to ArxR and AioR than to the ArrR from the Firmicutes and Chysiogenetes, which is a further example of how the XSR phylogeny is more congruent to the 16S rRNA gene than to that of the molybdenum-containing enzyme.The phylogeny of the XSR regulatory modules are congruent with the 16S rRNA gene phylogeny Fig. . By conters Fig. . The ArrarxB’ABC) together with the regulatory cluster (arxXSR) are conserved in anaerobic arsenite oxidisers, all of which are members of the Alpha-, Beta- and Gammaproteobacteria. The aioBA genes are more widespread, with homologues in both prokaryotic domains of life and in the following phyla of the Bacteria, Proteobacteria, Chloroflexi and Thermus-Deinococcus. The aioXSR regulatory module, however, is present only in some arsenite oxidisers in the Alpha- and Betaproteobacteria. In the arsenate respirers, the arrAB genes are present in members of the Firmicutes, Proteobacteria (Gamma- and Epsilonproteobacteria), Chrysiogenetes and Deferribacteres phyla while the arrXSR genes are present in all of these except the Gammaproteobacteria and the Deferribacteres.The genes that encode the alternative arsenite oxidase  electron density maps generated from the crystals of Se-Met labelled protein there was an unaccounted electron density in the putative ligand-binding region near residue Cys106. The electron density was consistent with the presence of a phosphate ion, from the crystallisation conditions, and its location would suggest that Cys106 might be involved in binding arsenite.The crystal structure of the phosphate-bound AioX was used to solve the structures of the apo and arsenite-bound forms of the protein by molecular replacement. Figure Furthermore, conformational rearrangement upon arsenite binding would inevitably result in changes to the molecular surface of AioX, including its electrostatic potential Fig. . InteresSuperposition of the phosphonate-bound structure of PhnD and the phosphate-bound structure of AioX emphasizes the structural similarity between the two proteins, while close inspection of the ligand-binding sites identifies a set of conserved residues that are engaged in interactions with two similar ligands Figs  and 5A. The presence of a phosphate ion in the binding site of AioX leads to the hypothesis that the phosphate ion mimics arsenite binding. A comparison of the X-ray structures however, indicates that while AioX can bind phosphate, the resulting structure does not stabilize the 53–61 loop conformation seen in the arsenite-bound form. Furthermore, even though the structure is similar to the arsenite-bound form, with the main conformation of Tyr88 akin to that of the arsenite-AioX Fig. , electroThe residues that constitute the binding pocket of the arsenite- and phosphate-bound structures of AioX, are Tyr88, Cys106, Tyr131, Ser161, Ser163, His192 and Asp210. Alignment of AioX with ArrX, ArxX and PhnD sequences . ArrX bound arsenate and also phosphate with a Kd of 1 and 150 μM, respectively  ely Fig.  while no25 and a member of the D-cluster of substrate-binding proteins26. Proteins of this type predominantly bind tetrahedral oxyanions26 and while the binding of arsenate and phosphate by ArrX conforms to this trend, the binding of arsenite by AioX and ArxX is different as arsenite has a trigonal pyramidal geometric structure. The AioX orthologues are the only members of this family that function as signalling proteins. The other members of the family, including the structural homologue PhnD, are part of ABC transporters. Other PBPs shown to be involved in signal transduction include LuxP and TorT, which bind autoinducer-229 and trimethylamine27 and regulate the lux30 and tor28 operons, respectively. LuxP and TorT however are structurally and phylogenetically distinct to AioX, indicating independent evolutionary origins.AioX is a class II PBPThe arsenite-bound crystal structure of AioX showed significant conformational changes compared with the apo and phosphate-bound structures, specifically in the loop of residues 53–61 and the shape, as well as electrostatic potential, of the protein surface. It is possible that these modifications allow its interaction with the sensor histidine kinase, AioS, leading to its activation. The fact that the arsenite-bound and apo-AioX crystals displayed differences in molecular packing  supports the idea that loop conformation change might result in the generation of a new protein interaction surface.d). Phosphate concentrations that organisms encounter will depend on the environment. A. ehrlichii MLHE-1 is from an environment extremely rich in phosphate (up to 1 mM)31, while gold mine tailings often don’t contain enough phosphate to support microbial growth32, and eutrophied lake sediments may contain 7 µM phosphate33 (both Rhizobium sp. str. NT-26 and C. arsenatis were isolated from mining environments). A phosphate ion was identified in the crystal structure of AioX, however, its binding does not stabilize the putative signalling loop as is the case with arsenite binding. As AioX and ArxX did not bind phosphate in conditions tested in ITC, we conclude that AioX and ArxX do not bind phosphate under biologically relevant conditions.Binding experiments performed by ITC showed that AioX and ArxX could only bind arsenite from the potential ligands selected, while Arr bound arsenate and phosphate  are also conserved in ArrX, which did not bind arsenite but did bind arsenate and phosphate albeit the latter with about 150-fold less affinity. This finding indicates that binding residues alone do not confer ligand specificity but other factors, such as overall conformation of the protein, may play a role in selectivity. The K34 and it is hypothesised that Aio is the common ancestor to all other members of the DMSO reductase family of molybdoenzymes35. Studies have also shown that while respiratory molybdoenzymes are principally inherited by vertical gene transfer, they are also subject to duplication events and divergence, particularly in the Firmicutes and Proteobacteria phyla36. Such a scenario is apparent for divergence of Arx and Arr, where the ‘older’ enzyme Arr, which uses menaquinone to reduce arsenate, has diverged to form Arx which uses ubiquinone to oxidise arsenite37.Based on phylogenetic studies, some molybdenum-containing enzymes were present in the last universal common ancestor38.The congruency of the AioX, AioS and AioR phylogenies to the 16S rRNA gene indicates that the regulatory genes emerged early in prokaryotic evolution and their functions adapted to that of the molybdoenzymes, whose gene expression they regulate. This is supported by the similar GC content of the regulatory genes and the molybdoenzyme genes (data not shown). Adaptation to conform to the function of a cognate molybdoenzyme is also observed in the NarJ family of proteins, which function as chaperones and are essential for cofactor insertion in several families of molybdoenzymesaioXSR genes are present in some arsenite oxidisers of the Alpha- and Betaproteobacteria. In Polaromonas sp. str. GM1, the Aio is constitutively expressed and a putative phage integrase is present immediately upstream of aioB suggesting that this insertion event may have led to the deletion of the aioXSR genes39. We have identified other examples of gene loss including in the putative arsenite oxidiser Verminephrobacter aporrectodeae40 where a 188 bp fragment of the aioX gene remains, with the rest of the regulatory cluster missing. The putative arsenite oxidisers of the genus Burkholderia (member of the Betaproteobacteria) also show how the aioXSR genes may have been lost. The aioXSR genes are present in B. cenocepacia but absent in B. vietnamensis, B. multivorans and B. ubonensis. Typically, AioA and AioB phylogenies resemble those of the 16S rRNA gene consistent with vertical inheritance35 which is the case for B. cenocepacia but not the other Burkholderia species. In the other Burkholderia species, the AioA subunit clusters with six Pseudomonas  species. Transposases and integrases are located within 10 kb of the aioBA genes in these Burkholderia species, which may indicate loss and horizontal gene transfer of the aioBA genes from Pseudomonas to Burkholderia accounting for the absence of aioXSR in those species.The XSR gene cluster is conserved in all arx-containing organisms but it appears that it has been lost in some that contain aio. Phylogenetic analyses of the arrAB genes suggests both vertical and horizontal transfer of these genes35 and it’s possible that the regulatory genes were lost after such a horizontal gene transfer event.The In conclusion, we propose that the protein AioX represents a new subfamily of PBPs that bind arsenic oxyanions.aioX gene from Rhizobium sp. str. NT-26 and its orthologues, arxX from A. ehrlichii str. MLHE-1 and arrX from C. arsenatis were cloned without their signal sequences into pPROEX-HTb+ (Invitrogen) for cytoplasmic protein expression in E. coli str. JM109. The aioX and arrX genes were amplified from genomic DNA using PCR with the following primers: aioXF, aioXR, arrXF and arrXR  and the pellets suspended in 40 ml binding buffer . Cells were centrifuged at 16,000 × g for 10 min (4 °C), suspended in 10 ml/gm (wet weight) binding buffer and broken at 14,000 PSI in a pressure cell homogenizer (Stansted Fluid Dynamics). Debris and unbroken cells were removed by centrifugation at 30,000 × g for 30 min (4 °C). Total cell extract was applied to a 1 ml His Gravitrap  Ni-charged column which was then washed with 100 ml binding buffer. Bound proteins were eluted from the column in 5 ml elution buffer . The eluate was filtered through a 0.22 µm filter (Millipore), concentrated using a 10,000 MWCO centrifugal filter (Millipore) and loaded onto a Superdex 75 gel filtration column  pre-equilibrated with either 20 mM Tricine, pH 7.5 (AioX and ArxX) or 50 mM Tris-HCl, pH 8 (ArrX). Chromatography was carried out at 0.8 ml/min and fractions corresponding to the protein peaks were pooled and concentrated with the 10,000 MWCO centrifugal filter (Millipore), snap frozen in liquid nitrogen and stored at −80 °C until required. Protein concentrations were determined with the absorbance at 280 nm, measured with a NanoDrop 2000 spectrophotometer (Thermo), and a predicted molar absorbance coefficient 42; ε (M−1 cm−1) for AioX, 42860, ArxX 44350, ArrX 25330. Protein purity was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis.AioX, ArxX and ArrX were heterologously expressed in 1 l lysogeny broth (LB) (in a 5 l flask) containing 100 µg/ml ampicillin and 40 µM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 21 °C with shaking at 180 rpm for 20–24 h. Recombinant proteins were purified from JM109λFor crystallisation, the His-tag was removed from AioX using AcTEV protease (Invitrogen) according to the manufacturer’s instruction. The cleaved AioX was then separated from uncleaved protein and the protease using affinity chromatography as described above.43. JM109 harbouring pPROEX-HTb + -aioX was grown at 37 °C overnight in 50 ml of M9 medium44 containing ampicillin (100 μg/ml). The 50 ml was inoculated into 1 l M9 medium and incubated at 37 °C with shaking (170 rpm). Cells were grown until OD600 nm ~0.5 followed by the addition of an amino acid mix consisting of 100 mg each of lysine, threonine, phenylalanine; 50 mg each of leucine, isoleucine, valine and 60 mg of L-selenomethionine and the cells grown for 15 minutes prior to induction with IPTG. The SeMet-AioX protein was purified as described above with one modification, all buffers contained 5 mM of dithiothreitol.The sulphur atoms of methionine residues in AioX were replaced with selenium by providing selenomethionine in the growth medium during protein expression using a modified protocol2PO4 and 0.91 M K2HPO4 pH 7.2, (2) apo-AioX crystals grew in 20% PEG 3350, 0.2 M sodium malonate and 0.1 M Bis-Tris propane, pH 8.5 and, (3) AioX-arsenite crystals grew in 25% PEG 3350 and 0.1 M Tris-HCl, pH 7.6. Single crystals were collected and mounted in a nylon cryo-loop using 25% glycerol as a cryo-protectant and flash cooled directly in liquid nitrogen prior to data collection.For crystallisation of the SeMet-AioX, apo-AioX and AioX-arsenite, the recombinant AioX protein was concentrated to 20 mg/ml. Crystallization screening was performed at 16 °C, using a mosquito (TTP Labtech) and the crystallisation screens from Hampton Research (Index) and Molecular Dimensions . Conditions that yielded crystals were as follows: (1) SeMet-AioX crystals grew in 0.49 M NaH45. SAD data were collected from one SeMet-AioX crystal and processed with CCP4 software. The MTZ reflection file obtained was used to determine the position of five selenium atoms through SHELX C/D/E (CCP4). BUCCANEER was used to generate the AioX model. The final AioX model was generated through interactive cycles of model building in Coot46 and a restrained refinement routine in REFMAC547. The structures of apo-AioX and arsenite-bound AioX were solved using molecular replacement using the phosphate-bound structure as the model, but with the phosphate ion omitted. Phases were obtained with PhaserMR48 and model building was performed in Coot46. Refinement of the new structure was carried out in REFMAC547. The data collection, processing and refinement statistics are listed in Table 49. The following PDB entries were used for structural comparisons: 3N5L (a binding protein component of an ABC phosphonate transporter from Pseudomonas aeruginosa) and 3P7I (a PhnD in complex with 2-aminoethyl phosphonate from E. coli)50. Three structures and the associated structure factors were submitted to PDB with the following IDs: 6ESK, 6EU7 and 6ESV for apo, arsenite-bound and a phosphate-bound AioX.Crystals were screened and X-ray diffraction data collected at the following synchrotron radiation sources: Diamond Light Source, beamlines I04 and I02 and Soleil synchrotron, beamline Proxima 1. The diffraction images were integrated and scaled using the XDS software package and merged using Aimless in the CCP4 program suited, ΔH, and ΔS. Antimonite , arsenate (Na2HAsO4), arsenite (NaAsO2), molybdate (Na2MoO4), phosphate (KH2PO4), phosphonate 2NH2), selenate (Na2SeO4), sulphate (Na2SO4), tetrathionate (K2S4O6), thiosulphate (Na2S203) were tested as potential substrates by titrating 1–10 mM substrate into 100 µM protein in 1.5–2 µl injections. Protein-substrate combinations that tested positive for binding were repeated with three independent protein preparations. AioX and ArxX titrations were performed in 20 mM Tricine, pH 7.5 and ArrX in 50 mM Tris-HCl, pH 8. Substrates were prepared in the same buffer as the protein into which they were titrated.Binding of potential substrates by AioX, ArxX and ArrX was determined by ITC using a MicroCal200 ITC instrument  at 25 °C with constant stirring of 750 rpm. Binding parameters were calculated using Microcal Origin software with a one-site binding model to give estimated values of N, K51 with NT-26 sequences as queries. Phylogenetic analyses were performed using MEGA52. Multiple sequence alignments were generated using MUSCLE53. Phylogenetic trees were constructed with the maximum-likelihood method, using the Jones-Taylor-Thornton model, and tested by 200 bootstrap resamples. Accession numbers used to construct trees are in Supplementary Table Sequences from confirmed arsenic metabolisers and other representatives were retrieved from GenBank using BLASTAll data generated or analysed during this study are included in this published article (and its Supplementary Information file).Supplementary information


Previous studies have indicated that inefficient energy utilization may play a pivotal role in hypertrophic cardiomyopathy (HCM). However, whether plasma free fatty acid (FFA), a main energy substrate of heart, has an effect on HCM remains unclear. Besides, several studies have suggested sex-related differences in HCM features and FFA metabolism. Here, we aimed to explore the association between plasma FFA levels and HCM and potential effects of sex on this relation.A total of 412 patients ) with HCM were recruited. Complete medical history was collected. Echocardiography and cardiovascular magnetic resonance imaging (CMRI) were performed. Fasting plasma FFA was determined by clinical laboratory. Left ventricular mass (LVM), maximum wall thickness (MWT), and left atrium diameter (LAD) were assessed with CMRI.p = 0.005). Compared with women, men had greater LVM index (LVMI) . FFA levels in male patients correlated positively with LVM, LVMI, LAD, cholesterol levels, high-density lipoprotein-cholesterol (HDL-C) levels, heart rate, and systolic blood pressure (SBP). However, none of these variables were significantly associated with sqrt (FFA) in female patients except a borderline correlation of LAD (p = 0.050). Multiple linear regression analysis was performed in male patients and revealed that HDL-C , heart rate , SBP , LVMI , and LAD  were independently associated with increasing FFA levels.The median FFA levels were 0.38 (interquartile range (IQR) 0.27–0.52) mmol/L in men and 0.40 (IQR 0.30–0.59) mmol/L in women. The FFA levels were significantly lower in men compared with those in women (In patients with HCM, LVMI, LAD, HDL-C, SBP, and heart rate were independently associated with increasing plasma FFA levels in males, whereas not in females. These results suggest that sex may affect the pathogenesis of HCM through influencing FFA metabolism. And these sex-related differences should be taken into account in therapeutic approaches to influence myocardial FFA metabolism in HCM. Hypertrophic cardiomyopathy (HCM), a common cause of sudden death in young people, is characterized by primary asymmetric left ventricular hypertrophy without an alternative cause , 2. WithSeveral studies have suggested that inefficient energy utilization resulted from sarcomeric mutations plays a pivotal role in HCM , 3. CircHowever, to date, few studies have been performed to evaluate the associations of FFA with cardiac structural and functional parameters. Hence, here we sought to explore the relation between FFA levels and HCM with a large cohort of HCM patients. Considering aforementioned sex-related differences in HCM features and FFA metabolism, we determined potential effects of sex on the relation between FFA and HCM. Owing to its three-dimensional tomographic imaging with high spatial resolution, cardiac magnetic resonance imaging (CMRI) was employed in the diagnosis and morphological characterization of HCM in the present study .The protocol of this study was approved by Fuwai Hospital  ethics committee and complied with the Declaration of Helsinki. The informed consents were obtained from all participants.2), concomitant neoplasma, connective tissue disease, and pregnancy. Finally, a total of 412 patients with HCM were recruited in the present study. Relevant clinical variables including complete medical history, physical examination, 12-lead electrocardiography, 24-h ambulatory electrocardiographic monitoring, blood examination, transthoracic echocardiography, and CMRI were collected.Consecutive patients with HCM, evaluated at Fuwai Hospital  from December 2012 to December 2015, were enrolled. The diagnosis of HCM was based on a maximum left ventricular wall thickness ≥15 mm , as measured by echocardiography or CMRI, in the absence of other cardiac or systemic diseases capable of producing such magnitude of hypertrophy . PatientAll transthoracic echocardiography were performed by experienced cardiologists as recommended by the American Society of Echocardiography , using aCMRI was performed using a 1.5-T speed clinical scanner . The imaging protocol and analysis have been described previously . BrieflyFasting blood samples were collected for all enrolled subjects within 2 days of TTE and 1 week of CMRI examination. Then the laboratory measurements were conducted by the clinical laboratory. All assays were performed within 4 h of blood collection by medical technologists who were unaware of any clinical information about the studied patients. Plasma FFA was measured with enzymatic assay  on a Beckman-Coulter LX20. The intra-assay coefficient of variation was 1.03%, and the inter-assay coefficient of variation was 1.11%. Serum levels of triglycerides, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were measured by routine laboratory methods using an Olympus AU-5400 auto-analyzer . Plasma levels of N-terminal proB-type natriuretic peptide (NT-proBNP) were measured using an electrochemiluminescent immunoassay . Hyperlipidemia was defined as those with serum LDL-C ≥3.37 mmol/L or triglycerides ≥1.70 mmol/L.t test or Mann-Whitney U test depending on the distribution of variables. Categorical variables were compared with χ2 test, and Fisher’s exact test was used when the expected frequency was <5. Pearson’s correlation test or Spearman’s correlation test was used to examine correlations between two continuous variables when indicated. Stepwise multiple linear regression analysis  was conducted to identify independent variables that might determine FFA levels. In order to obtain normal distribution, square root transformation was applied to plasma FFA in correlation tests and multiple linear regression analysis. Variables with a p value <0.10 in the univariate analysis were included in the multiple regression analysis. A two-tailed p value <0.05 was considered as statistically significant. Statistical analysis was performed with SPSS version 19.0 software .The values were expressed as mean ± SD or median (interquartile range (IQR)) for the continuous variables and as the number (percentage) for the categorical variables. Comparisons of continuous variables between two groups were assessed using independent Student’s p = 0.005). Besides, men were younger  than women. There were more patients with maximum wall thickness ≥30 mm and resting LVOTG ≥30 mmHg in men. In contrast, men had lower NT-pro-BNP levels. Of note, serum HDL-C levels were significantly lower in men . Additionally, men had higher BMI and diastolic blood pressure and were more likely to smoke .The clinical characteristics of studied population stratified by sex are summarized in Table p < 0.001) and maximum wall thickness (p < 0.001). In addition, in comparison with women, men had greater LV end-diastolic volume, LV end-systolic volume, stroke volume, cardiac output, and LV mass. However, after being adjusted with body area, only LV mass index (LVMI) was still higher in men .Echocardiographic and cardiovascular magnetic resonance imaging data of the patients with HCM are presented in Table p = 0.028). In women, greater FFA levels were found in those who had palpitation (p = 0.025), non-sustained ventricular tachycardia (p = 0.015), and hyperlipidemia (p = 0.040). Notably, smoking and statin therapy did not affect FFA levels both in men and women.Table r = 0.134, p = 0.037), HDL-C , heart rate , and systolic blood pressure . Furthermore, there were also significant correlations between sqrt (FFA) and maximum wall thickness (MWT) , LVM , LVMI , heart rate , SBP , LVMI , and LAD  were independently associated with increasing sqrt (FFA).Then multiple linear regression analysis was performed to identify independent determinants of FFA levels in male patients Table . HDL-C  , 22, whiThere is considerable evidence that high FFA levels increase cardiac sympathetic activity , 41. In Of note, men were much more likely to be smokers in our study, which may be a confounder. However, our data showed that smoking did not affect FFA levels in both men and women. Besides, statin therapy may be a major confounder. Barbarash et al. studied 210 patients with acute myocardial infarction (AMI) and reported that atorvastatin resulted in a decrease in FFA levels 12 days after AMI . HoweverAiming at inefficient myocardial energy utilization in HCM, several drugs are introduced into the management of HCM, such as perhexiline and trimetazidine , 49, whiThere are some limitations in our study. First, plasma FFAs were measured once and totally. Serial and classified measurements of FFAs might provide more implications and increase sensitivity for correlation. Even so, a significant correlation between plasma FFA levels and variables reflecting severity of HCM was detected in our study with large sample size using CMRI. Second, in an attempt to elucidate the role of FFA in the pathogenesis of HCM, genetic testing for HCM may be helpful. Third, in order to determine whether the relationship between FFA and HCM is causal or circumstantial, it is intriguing to investigate plasma FFA levels in mutation carriers without left ventricular hypertrophy in comparison with subjects with overt hypertrophic cardiomyopathy. Finally, this is a cross-sectional and single-center study. Considering that elevated FFA levels are shown to be an independent predictor of sudden cardiac death in patients with other heart diseases, the potential prognostic values of plasma FFA for cardiovascular events in patients with HCM are needed to be revealed in further investigation with follow-up.In patients with HCM, LVMI, LAD, SBP, and heart rate were independently associated with increasing plasma FFA levels in males, whereas not in females. These results suggest that sex may affect the pathogenesis of HCM. And these sex-related differences should be taken into account in therapeutic approaches to influence myocardial FFA metabolism in HCM.


We assessed the recovery of 2 face transplantation patients with measures of complexity during neuromuscular rehabilitation. Cognitive rehabilitation methods and functional electrical stimulation were used to improve facial emotional expressions of full-face transplantation patients for 5 months. Rehabilitation and analyses were conducted at approximately 3 years after full facial transplantation in the patient group. We report complexity analysis of surface electromyography signals of these two patients in comparison to the results of 10 healthy individuals.Facial surface electromyography data were collected during 6 basic emotional expressions and 4 primary facial movements from 2 full-face transplantation patients and 10 healthy individuals to determine a strategy of functional electrical stimulation and understand the mechanisms of rehabilitation. A new personalized rehabilitation technique was developed using the wavelet packet method. Rehabilitation sessions were applied twice a month for 5 months. Subsequently, motor and functional progress was assessed by comparing the fuzzy entropy of surface electromyography data against the results obtained from patients before rehabilitation and the mean results obtained from 10 healthy subjects.At the end of personalized rehabilitation, the patient group showed improvements in their facial symmetry and their ability to perform basic facial expressions and primary facial movements. Similarity in the pattern of fuzzy entropy for facial expressions between the patient group and healthy individuals increased. Synkinesis was detected during primary facial movements in the patient group, and one patient showed synkinesis during the happiness expression. Synkinesis in the lower face region of one of the patients was eliminated for the lid tightening movement.The recovery of emotional expressions after personalized rehabilitation was satisfactory to the patients. The assessment with complexity analysis of sEMG data can be used for developing new neurorehabilitation techniques and detecting synkinesis after full-face transplantation.The online version of this article (10.1186/s12984-018-0356-0) contains supplementary material, which is available to authorized users. Understanding the rehabilitation of facial muscles and nerves after facial transplantation has remained a challenging problem in neurorehabilitation –8 since Functional electrical stimulation is a noninvasive technique that is used for rehabilitation of patients after stroke , spinal We propose a new individualized rehabilitation method with selective FES for patients after full-face transplantation and an assessment of the results of rehabilitation by comparing complexity measures from multichannel sEMG data. Neuromuscular stimulation modality was enhanced with visual stimuli to improve the cognitive aspects of the rehabilitation process. We chose a fuzzy entropy based method as a complexity measure; this measure was used to analyze sEMG data in various ways –26 and tFunctional electrical stimulation was performed on both transplantation patients during primary facial and facial expression movements to improve these movements. A battery powered 8-channel electrical stimulator with a peak amplitude of 10 mA was used . The frequency and the pulse width of the stimulation were fixed as 30 Hz and 60 μs, respectively. The duration of the stimulation was 40 s for each movement in a session. Facial sEMG signals  collected from healthy individuals and patients were used to investigate active muscles in each movement. The selected healthy individuals were male (31 ± 5 years). Fourteen bipolar electrodes (9-mm diameter and 20-mm interelectrode distance) were used to collect sEMG data and apply electrical stimulations. The electrode locations of the measurements are described in the Supplementary Material to Ref. , 29. A hSemmes-Weinstein’s monofilament test (SWMT) was used to investigate sensory innervation.A 37-year-old male had lost his facial muscles in a burn accident when he was 3 years old. He was not able to perform facial expressions when he was admitted to the polyclinic. He underwent full-face transplantation in May 2012. All muscles for facial expression and eyelids were transplanted, but those for mastication were not . RehabilPatient B was a 22-year-old male who had burns and lost his facial muscles due to exposure to boiling water at 7 months of age. He was also not able to perform facial expressions when he was admitted to our plastic and reconstructive surgery polyclinic. He underwent face transplantation (excluding eyelids) in January 2012. The infraorbital, supraorbital, and mental nerves were coapted. The lower branches were also coapted to the donor’s facial nerve trunk . Rehabilu(1), u(2), …, u(N) from the measurements, form a sequence of vectors x(1), x(2), …, x(N − m + 1) in Rm for the selected embedded dimension m, defined by xm(i) =  for each i, 1 ≤ i ≤ N − m + 1. Then, the distance xm(i) and xm(j) should be the following:W. Chen et al. developed fuzzy entropy (FuzzyEn)  to measuA family of exponential functions is used as the fuzzy function, which measures the similarity degree of two vectors:φm is defined as follows:where the parameter of the exponential is chosen as n = 2. The family of exponential functions is convex and continuous. The function FuzzyEn parameter can be defined from the time series as follows:r = 0.15 and m = 2, respectively [The tolerance value r and embedded dimension m were fixed as ectively , 27, 33.Fuzzy entropy values were calculated for each movement for the patient group before and after rehabilitation. Fuzzy entropy values smaller than 0.01 were removed to clean the muscle resting state; subsequently, the mean values of fuzzy entropy measures were calculated for each channel to evaluate the effects of the rehabilitation sessions. The results are given for primary facial movements and simple facial expressions.Overall, we observed improvements in symmetry and similarity between the patient group and healthy individuals during the 4 primary facial movements after rehabilitation. Moreover, synkinesis was eliminated for the lid tightening movement by using our rehabilitation method.For the lid tightening movement, synkinesis in the lower face of patient A was eliminated with rehabilitation. Similarity in the pattern of fuzzy entropy for this primary motion between patient A and healthy individuals increased after the rehabilitation process. Fuzzy entropy data for the lid tightening facial movement in all subjects are illustrated in Figure For the lip funneling movement, the activity of the orbicularis oris muscle in the patient group became more symmetric, but the patient group showed synkinesis in their frontalis muscle, and this deficiency remained after the rehabilitation process.For the lip puckering movement, the patient group showed more symmetric muscle activity in their lower face, but synkinesis remained in their upper face after rehabilitation.For outer brow raising, patient A showed improvements in the left side of his upper face. Synkinesis remained in his lower face after rehabilitation. Patient B showed a similar pattern to that of healthy individuals before and after rehabilitation.Patient A showed 1.4±0.1 between 19 and 39 months postoperatively and patient B showed 1.2±0.2 improvements between 23 and 43 months postoperatively according to SWMT


Mindreading refers to the ability to attribute mental states, including thoughts, intentions and emotions, to oneself and others, and is essential for navigating the social world. Empirical mindreading research has predominantly featured children, groups with autism spectrum disorder and clinical samples, and many standard tasks suffer ceiling effects with neurologically typical (NT) adults. We first outline a case for studying mindreading in NT adults and proceed to review tests of emotion perception, cognitive and affective mentalizing, and multidimensional tasks combining these facets. We focus on selected examples of core experimental paradigms including emotion recognition tests, social vignettes, narrative fiction (prose and film) and participative interaction , highlighting challenges for studies with NT adult cohorts. We conclude that naturalistic, multidimensional approaches may be productively applied alongside traditional tasks to facilitate a more nuanced picture of mindreading in adulthood, and to ensure construct validity whilst remaining sensitive to variation at the upper echelons of the ability. Mindreading describes the ability to attribute mental states to oneself and others, and is essential for predicting behavior , which depicts belief-states through social vignettes. In the traditional object-transfer paradigm, participants must identify a target agent’s mistaken belief about the location of an object, through understanding that the agent lacks knowledge that the object has moved. For example, A wrongly believes that the sweets are in the opaque jar, because they did not witness B move them to the cupboard (first-order); B wrongly believes A will look for the sweets in the jar, unaware that A secretly watched them being moved (second-order).Cognitive mentalizing entails setting aside one’s own perspective to attribute states to other agents. Both children and adults demonstrate automatic egocentric bias in verbal and visual perspective-taking tasks e.g., , however beliefs , led to False-belief tasks have been applied to child development studies  and why (mindreading) questions about human behaviors depicted through photographs. Designed for fMRI studies, the Why/How Task also generates reliable behavioral (accuracy and response time) data. Whilst simple social images do not reflect the complexity of real-world mindreading stimuli, they present opportunities to examine the brain basis for behavioral differences between participants.In the original study, success was higher on affective compared to cognitive trials, a finding replicated by How Task  – an altStrange Stories Task assessed comprehension of short, naturalistic narratives including joke, lie, appearance/reality, and contrary emotions. The range of narratives proved more sensitive to subtle between-group differences than FBTs, paving the way for more complex narrative-based approaches.Naturalistic narrative stimuli allow mindreading targets to be contextually embedded e.g., , which mSST; Participants in the Short Story Task SST; , read a after reading, so future studies should vary task order to control for potential priming effects.The original SST demonstrated sensitivity to variation among NT adults (scores ranged from 2 to 14 of 16 points), and concurrent validity with the Eyes Test and Interpersonal Reactivity Index fantasy subscale IRI;  supportefaux pas.Film stimuli enable researchers to present dynamic interactions e.g., , but canIn the validation study, the MASC converged with three extant mindreading measures: a basic emotion recognition task, the Eyes Test and Strange Stories Task (shortened). However, MASC scores predicted Strange Stories Task performance in participants with Asperger Syndrome, and emotion recognition in controls, which indicated that verbal strategies may have compensated for facial processing difficulties. The authors recommended future studies vary mental state complexity and part of the face focused on (eyes/mouth). Additionally, both groups performed at ceiling on the control questions, so future revisions should incorporate more challenging questions to account for other cognitive processes . NotablyVersions of the MASC include the original German and dubbed English editions [dubbing did not interfere with participants’ task focus  and geneEmpathic Accuracy Paradigm  technology enables the construction of more complex interactive scenarios. The rld Task  requiresTraining is required prior to participation in VEs, however, this could prove a beneficial tradeoff for studying live, interactive mindreading whilst incorporating the range of variables available to fiction approaches. As with fiction tasks, processes associated with interpreting the narrative features of VEs may impact mindreading performance; research has shown that in-game storytelling enhances affective mindreading e.g., . While tand simpler measures, to assess concurrent validity and probe mindreading variation both within and between participants. In this way, multidimensional stimuli need not problematize construct validity, but could prove fruitful to the development of multi-systems approaches, and studies of the neural architecture underlying mindreading in adulthood, which in turn may expand the wider social cognition literature.Extant mindreading research has primarily focused on children, ASD, and clinical populations, and standard measures can suffer ceiling effects with NT adults. However, several classic tests have been adapted for research with adult cohorts. Tasks measuring specific mindreading dimensions such as emotion perception and cognitive mentalizing risk subtracting out key processes, particularly as social displays can be suppressed, and so interpreting mental states may require integration of verbal, physical and contextual information . In contFF: Concept, design, literature review , writing . RT: Writing, literature review.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Treatment with androgen-targeted therapies can induce upregulation of epithelial plasticity pathways. Epithelial plasticity is known to be important for metastatic dissemination and therapeutic resistance. The goal of this study is to elucidate the functional consequence of induced epithelial plasticity on AR regulation during disease progression to identify factors important for treatment-resistant and metastatic prostate cancer. We pinpoint the epithelial plasticity transcription factor, Snail, at the nexus of enzalutamide resistance and prostate cancer metastasis both in preclinical models of prostate cancer and in patients. In patients, Snail expression is associated with Gleason 9-10 high-risk disease and is strongly overexpressed in metastases as compared to localized prostate cancer. Snail expression is also elevated in enzalutamide-resistant prostate cancer cells compared to enzalutamide-sensitive cells, and downregulation of Snail re-sensitizes enzalutamide-resistant cells to enzalutamide. While activation of Snail increases migration and invasion, it is also capable of promoting enzalutamide resistance in enzalutamide-sensitive cells. This Snail-mediated enzalutamide resistance is a consequence of increased full-length AR and AR-V7 expression and nuclear localization. Downregulation of either full-length AR or AR-V7 re-sensitizes cells to enzalutamide in the presence of Snail, thus connecting Snail-induced enzalutamide resistance directly to AR biology. Finally, we demonstrate that Snail is capable of mediating-resistance through AR even in the absence of AR-V7. These findings imply that increased Snail expression during progression to metastatic disease may prime cells for resistance to AR-targeted therapies by promoting AR activity in prostate cancer. AR gene or alternative splicing of AR pre-mRNA [Prostate cancer is responsible for over 80 deaths per day in the US . Androgepre-mRNA –8. The bpre-mRNA –17. Notapre-mRNA , 18, 19.pre-mRNA . HoweverPreclinical models have shown that AR-FL and AR-V7 overexpression is associated with induction of a mesenchymal or stem-like phenotype known to promote metastasis –24, and in vitro model of enzalutamide resistance using LNCaP95 cells. These cells were originally derived from the LNCaP model through passaging in low androgen conditions [Enzalutamide is a potent inhibitor of AR signaling that is currently used as a first line therapy to treat prostate cancer patients with metastatic CRPC. Although most patients initially respond to enzalutamide treatment, all eventually progress within several years , and somnditions , 35, 36,nditions . We furtnditions , 38, we LNCaP95 Enza-R cells have a significantly higher proliferation rate over DMSO-treated, passage control cells when treated with enzalutamide Figure . InteresNext, we examined whether knocking down AR-FL or AR-V7 impacted the growth of control and Enza-R cells when treated with enzalutamide. We were able to specifically target AR-FL or AR-V7 each with two independent shRNAs Figure . LNCaP95Based on our observation that both Snail and AR are increased in enzalutamide-resistant cells, we sought to assess whether Snail played a role in the induction of AR in Enza-R cells and the impact of Snail knockdown on enzalutamide resistance. Control cells maintained expression of AR-FL in the presence of Snail knockdown and exhibited relatively low levels of AR-V7 that was further reduced with Snail knockdown. Stable knockdown of Snail did not further sensitize control cells to enzalutamide. In contrast, Snail depletion with shRNAs inhibited both AR-FL and AR-V7 expression in EnzaR cells Figure . ConsequZEB1 and VIM and repression of CDH1  was sufficient to induce nuclear localization of Snail Figure . Nuclear1 Figure . Importa1 Figure . These rOur results indicate that Snail over-expression induces a more migratory and invasive phenotype in prostate cancer cells. Based on this, we hypothesized that Snail would be associated with clinically aggressive disease. To determine whether Snail is associated with aggressive prostate cancer in clinical disease, we analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) and found that Snail RNA levels significantly correlated with increasing Gleason score . Snail Rin vitro. We next asked whether AR-V7 was expressed in clinical samples of prostate cancer. To assess the levels of AR-V7 expression, we used RNA in-situ hybridization (ISH) to measure AR-V7 mRNA in our metastatic biopsies [As discussed above, we observed high nuclear staining of both Snail (100%) and total AR (94%) in our metastatic samples  and demobiopsies . Given tIn our metastatic biopsies, we commonly observed high nuclear expression of Snail and AR, and both Snail and AR were upregulated in our pre-clinical model of enzalutamide resistance. Consequently, we examined AR regulation in the presence of activated Snail in our tamoxifen-induced model. We found that both AR-FL and AR-V7 were upregulated when Snail was activated Figure . In addiGiven that AR expression and nuclear localization were increased upon Snail activation, we measured the impact of Snail activation on enzalutamide resistance. Compared to vehicle-treated cells, Snail activation with 4OHT in LNCaP95 cells significantly (p<0.001) increased resistance to enzalutamide treatment Figure  at conceTo determine whether Snail-mediated enzalutamide resistance was due to upregulation of AR expression and nuclear localization, we used shRNA to stably knockdown either AR-FL or AR-V7 in LNCaP95 cells with inducible Snail . In the in vitro data indicate that Snail does not require AR-V7 to mediate enzalutamide resistance. We hypothesize that AR-V7 is important for enzalutamide resistance only if it was previously selected upon during castrate conditions.Our results suggest that both AR-FL and AR-V7 are important in promoting Snail-mediated enzalutamide resistance. We hypothesized that Snail may require AR-V7 to facilitate enzalutamide resistance. Therefore, we tested the ability of Snail to induce enzalutamide resistance in the absence of AR-V7. The original LNCaP cell line does not have detectable levels of AR-V7 and expression of inducible Snail in LNCaP cells was not sufficient to induce AR-V7 expression Figure . Conversin vitro.Our results suggest that enhanced expression of Snail is important not only in the context of metastatic disease, but also in the setting of human CRPC by contributing to AR expression in response to AR-targeted therapies and thus resistance to AR blockade. We identify a novel function of Snail as a key factor in mediating enzalutamide resistance through the regulation of AR expression and cellular localization to the nucleus. Indeed, depletion of Snail reduces AR-FL and AR-V7 expression and restores enzalutamide sensitivity. These results highlight the potential clinical utility in co-targeting the mechanisms that contribute to the expression and activity of AR-FL and AR-V7 to increase the efficacy of currently used AR-targeted therapies. We also demonstrate that Snail is clinically associated with metastases as compared to localized prostate cancer, and Snail activation is sufficient to enhance the aggressiveness of prostate cancer cells AR transcriptional start site. Interestingly, increased AR transcription rates are directly linked with AR-V7 generation [AR transcription. A previous study reported that Zeb1, a known Snail target gene, could recognize the E-boxes in the AR promoter and drive AR transcription [Snail is a transcription factor involved in the regulation of epithelial plasticity through controlling expression of genes involved in migration and invasion. These genes contain E-box elements required for Snail recognition and transcriptional regulation. One hypothesis is that Snail can regulate AR-FL and AR-V7 transcription through the recognition of two E-boxes located near the cription . Howevercription , and thecription , 45.in vitro model of enzalutamide resistant CRPC, inhibition of Snail decreased AR-FL and AR-V7 expression. Together these results from clinical samples and cell-based models, suggest that Snail is important for early invasion and metastasis in hormone-sensitive disease. However, during further disease progression to CRPC, Snail additionally functions in regulating expression of AR, thereby becoming important for induction of treatment resistance to AR-targeted therapies such as enzalutamide.In clinical samples, we show that both Snail and AR-V7 expression are increased in metastatic biopsies as compared to primary prostate cancer biopsies, with Snail upregulation observed even in the non-castration resistant metastatic setting. Prior work has detected low levels of AR-variants in localized prostate cancer using RNA-sequencing methods . Our cliRecent studies have highlighted the heterogeneity between and within each prostate cancer patient , 47. In Another model-dependent effect is that of the relationship between AR-FL, AR-V7, and enzalutamide resistance. Yamamoto et al. (2015) observed that AR-FL drove enzalutamide resistance in LNCaPs harboring the F876L agonistic mutation, while AR-V7 mediated enzalutamide resistance in the 22Rv1 model, in which AR-V7 is expressed at higher levels than full-length AR . These svice versa, one could envision a scenario in which either of these factors initiates gene changes that feed into the other, thereby leading to an aggressive, treatment-resistant tumor. In this scenario, the result would be the same whether the feedback loop started with AR induction or activation of Snail , a tissue microarray (TMA) on a random subset (n=188) of patients in the Shared Equal Access Research Center (SEARCH) database treated at the Durham VA was developed as previously reported .For metastatic prostate cancer samples (n=31) and a second cohort of localized prostate cancer samples (n=29), formalin fixed metastatic tissue was collected from the Duke University pathology department and Duke Cancer Institute Biorepository Core under a separate Duke IRB approved protocol. Clinical data on prior therapy and metastatic site were collected.LNCaP95 cells were kindly provided by Dr. Scott Dehm (University of Minnesota) and are reported in previous studies . LNCaP95LNCaP95 control and LNCaP95-Enza-R cells were cultured in media containing DMSO (Sigma) or 50 μM enzalutamide for at least one week. Cells were counted using the Countess II (Life Technologies). Average cell number/mL and standard error of the mean (SEM) were calculated from triplicate wells. For LNCaP95 control an Enza-R cells stably expressing shRNAs, cells were treated with DMSO or 50 μM of enzalutamide and counted as above. For LNCaP95 Snail expressing cells, ethanol (EtOH) or 4OHT pre-treated LNCaP95 cells were treated with DMSO or 6.25, 12.5, 25 and 50 μM of enzalutamide. Cell viability was measured after 10 days of drug treatment using the WST-1 assay reagent (Roche). For LNCaP95 Snail cells stably expressing shRNAs, cells were treated with DMSO or 50 μM of enzalutamide for one week. Cells were then fixed in 4% paraformaldehyde  and stained with Hoechst dye (Sigma). Wells were then imaged on an inverted Olympus IX 71 epifluorescence microscope. Total area was calculated from three representative images per well using ImageJ (Version 2.0.0-rc-41/1.50d).Total RNA was isolated using the Quick-RNA Miniprep kit (Zymo Research). Total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies). Aliquots of 5-fold diluted reverse transcription reactions were subjected to quantitative (q)PCR with KAPA SYBR FAST master mix using the Vii7 real time-PCR detection system (Applied Biosystems). GAPDH mRNA levels were measured for normalization, and the data are presented as “Relative Expression”. A complete list of primer sequences is provided in the Supplementary Text.For immunoblot analysis cell extracts were mixed with SDS sample buffer and submitted to SDS-PAGE. Following electrophoretic transfer onto nitrocellulose, the filters were blocked, incubated with antibodies and developed using the Odyssey-FC imager (LI-COR).For nuclear and cytoplasm fractionation, nuclear and cytoplasmic extraction was performed according to the manufacture's protocol using the NE-PER kit (ThermoScientific). A complete list of primary antibodies and their dilutions is provided in the Supplementary Text. All immunoblot images are of cropped blots to improve the clarity and conciseness of the manuscript and retain all important bands.For immunofluorescence (IF), cells were fixed in 4% PFA, permeabilized with 0.2% Triton X-100, and stained with Hoechst. Cells were blocked with 5% bovine serum albumin  prior to incubation with primary antibodies. Cells were incubated in Alexa Fluor secondary antibodies (Life Technologies) and then imaged on an inverted Olympus IX 71 epifluorescence microscope or using Cellomics Software for nuclear to cytoplasmic quantification.Culture medium was added to the lower wells of Boyden chambers (BD Bioscience), and cells were added to the upper chambers. Migratory cells attached to the lower side of the inserts were fixed with 4% PFA, permeabilized with 0.2% Triton X-100, and stained with Phalloidin (Life Technologies). The inserts were washed with 1× PBS (Life Technologies) and imaged using a 4× objective on an inverted Olympus IX 71 epifluorescence microscope and analyzed with ImageJ. Matrigel invasion assays were performed using Matrigel-coated 24-well transwell plates (BD Biosciences) as described above.We performed antibody optimization and analytic validation for all antibodies as previously described . An expeFor AR-V7 RNA-ISH, formalin fixed paraffin-embedded metastatic tumor slides were incubated with the AR-V7 probe (401221) using the RNAscope 2.0 HD Brown Assay from Advanced Cell Diagnostics according to the manufacturer's protocol. An expert pathologist scored AR-V7 mRNA expression using a 0-4 scale following the guidelines outlined in the manufacture's protocol.Data are shown as means ± SEM. Student's t-test or multiple group comparison was performed by one-way ANOVA followed by the Sidak method for comparison of means. P≤0.05 is considered significant. Differences in Snail and AR-V7 expression between localized and metastatic samples were analyzed using a Chi-square test. Data available through the TCGA was analyzed using the Kruskal Wallis test using JMP (version pro 12). All other analyses were performed using Prism (version 6.0d).


Casino Royale. Bond's poker skill reflects his abilities as a spy: Spotting lies and deception, and thinking one move ahead of his opponent. But like other domains of human skill, poker has been affected by the rise of the machines. In 2015, a supercomputer with 48 CPUs running for 68 days “solved” heads-up limit hold'em poker, the simplest poker game played for money in casinos and online , calling , or by raising (doubling the bet). The strategy involves finding a single threshold point: All hands weaker than this are folded, and those stronger are played by raising (raise-or-fold). Calling, as a potential strategy, is never considered . Now, the first player's first round strategy must specify play in one additional scenario. If the second player re-raises, then the first player is revisited with the fold, call, raise trilemma.  In this case folding is inadvisable from a risk-reward perspective . So the first player should raise-or-fold , and then always-call. The entire first round strategy boils down to a single hand: The worst hand worth raising. An effective strategy could not be simpler.Here is a simple strategy as the first player on the first round reflecting these principles . This player can play any individual hand by Matt Hawrilenko followed this strategy in the 2008 match Newall, . Over 1,Polaris from 2008 is significantly weaker at poker and used fewer computational resources than “Cepheus,” the unbeatable 2015 agent (Bowling et al., In conclusion, the expert's strategy in 2008 in this key situation closely matched the unbeatable 2015 computer's strategic frequencies. This is another example of expert performance approaching perfect game theoretic strategy (Walker and Wooders, The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study aims to investigate (1) motives, motivations and expectations regarding the choice for a specific rehabilitation setting after herniated disc surgery and (2) how rehabilitation-related motivations and expectations are associated with rehabilitation outcome  three months after disc surgery.The longitudinal cohort study refers to 452 disc surgery patients participating in a subsequent rehabilitation. Baseline interviews took part during acute hospital stay (pre-rehabilitation), follow-up interviews three months later (post-rehabilitation). Binary logistic regression and multiple linear regression analyses were applied.(1) Motives, motivations and expectations: Inpatient rehabilitation (IPR) patients stated “less effort/stress” (40.9%), more “relaxation and recreation” (39.1%) and greater “intensity of care and treatment” (37.0%) regarding their setting preference, whereas outpatient rehabilitation (OPR) patients indicated “family reasons” (45.3%), the wish for “staying in familiar environment” (35.9%) as well as “job-related reasons” (11.7%) as most relevant. IPR patients showed significantly higher motivation/expectation scores regarding regeneration (p < .001), health (p < .05), coping (p < .001), retirement/job (p < .01), psychological burden (p < .05) and physical burden (p < .001) compared to OPR patients. (2) Associations with rehabilitation outcome: Besides other factors  rehabilitation-related motivations/expectations were significantly associated with rehabilitation outcome measures. For example, patients with less motivations/expectations to achieve improvements regarding “physical burden” showed a better health-related quality of life (p < .01) three months after disc surgery. Less motivations/expectations to achieve improvements regarding “psychological burden” was linked to a better mental health status (p < .001) and a greater satisfaction with rehabilitation .Rehabilitation-related motivations and expectations differed substantially between IPR and OPR patients before rehabilitation and were significantly associated with rehabilitation outcome. Taking motivational and expectation-related aspects into account may help to improve allocation procedures for different rehabilitation settings and may improve rehabilitation success. Musculoskeletal disorders are the major indication group for a rehabilitation treatment in Germany , includiAccording to Paragraph 9 of the German Social Code IX (SGB IX) patients who apply for AHB have a “Wunsch- und Wahlrecht” meaning “the right to individual wishes and choice relative to assessments, services and institutions as well as to the various benefits…concerning every aspect of the implementation of these services.” . This alA successful rehabilitation outcome after medical conditions does not only depend on the severity of illness, nor the quality of the rehabilitation treatment itself. The achievement of a rehabilitation programme is also linked to the individuals`motivation and compliance regarding the rehabilitation process . FindingWhat are motives, motivations and expectations regarding the choice for a specific rehabilitation setting (inpatient or outpatient) after herniated disc surgery?How are rehabilitation-related motivations and expectations associated with rehabilitation outcome  three months after disc surgery?This study is a longitudinal cohort study. Detailed information on study design and sample is published elsewhere . A totalThe study has received ethics committee approval of the University of Leipzig (Ethik-Kommission an der Medizinischen Fakultät der Universität Leipzig). At the initial contact, participants were verbally informed on the purpose of the study (including handing out a study information form) and provided written consent to take part in the study.This study contains information about age, gender, family status and educational level.Besides the length of acute care hospital stay (when disc surgery was conducted), disc location  and the presence of other chronic diseases were assessed. Pain intensity was measured by using a verbal numeric pain scale . The Composite International Diagnostic Interview   was usedPatients were asked whether they were employed within the last 3 months before baseline and for their subjective prognosis of gainful employment  ,8. The SThe baseline interview included the FREM-17  , or were still on certified sick leave or were already receiving an early retirement pension (NATW).The Short Form 36 Health Survey (SF-36) was used to assess health-related quality of life . The relPatients were asked about their overall satisfaction with rehabilitation using a 5 point Likert-Scale which was later on dichotomized into a binary satisfaction variable: “yes”  and “no” .All calculations were carried out using the Statistical Package for the Social Sciences (SPSS) Version 20.0  and STATA detailed comparison of socio-demographic, illness-related and work-related characteristics in IPR and OPR patients is published elsewhere . AccordiThe qualitative approach to find individual motives behind patients´ preferences for a certain rehabilitation setting (inpatient vs. outpatient) revealed 16 motive categories which are presented in All patients (N = 452) who participated in subsequent rehabilitation provided information about their ability to work status at follow-up interview. Thus, ability to work rates were significantly higher in OPR patients (62.8%) compared to IPR patients (29.6%) . The results of binary logistic regression analysis  show thaOPR = 41.1 (11.3); mean (SD) IPR = 36.3 (9.6); p < .001), but also a significantly better mental health status (mean (SD) OPR = 55.8 (9.7); mean (SD) IPR = 52.5 (12.5); p < .01) three months after disc surgery. The results of multiple linear regression analysis regarding the physical health status are shown in 451 (99.8%) of the patients who participated in subsequent rehabilitation provided information about their health-related quality of life at follow-up interview. OPR patients did not only report a significantly better physical health status compared to IPR (mean (SD) 448 of the patients (99.1%) provided information about their satisfaction with subsequent rehabilitation. Thus, the majority of the study sample recorded to be satisfied with AHB . There were no statistically significant differences between different rehabilitation settings . The results of binary logistic regression analysis  revealedThe role of patients´ motivations and expectations is an under-researched topic , even thPatients´ motivations and expectations before rehabilitation differed greatly depending on the specific rehabilitation setting. Thus, IPR patients had more expectations and therefore were motivated to achieve greater improvements regarding regeneration, health, coping, retirement/job, psychological burden and physical burden compared to OPR patients. A possible explanation for this difference in motivations and expectations might be a “pre-selection” of patients with worse health status into IPR . Hence, In contrast to this, OPR patients stated significantly more often a better “location and accessibility” of the rehabilitation clinic, “negative expectations towards the other setting”, “independency/flexibility” and “age structure” (similar age) as important reasons for their choice. Of course the availability of outpatient rehabilitation facilities within urban areas is much higher compared to rural areas. Hence, it makes sense that the choice of a certain rehabilitation setting is also dependent on the local conditions. OPR patients were significantly younger than IPR patients in the present study. Hence, younger patients might prefer to stay independent and flexible rather than to focus on relaxation and recreation. They also might prefer a younger patient population within OPR setting for this reason.The present findings suggest that gaining knowledge about patients´ motives regarding their setting preferences may help to find the most perfect fit between individuals`expectations and the chosen rehabilitation setting.Results regarding rehabilitation outcome three months after disc surgery were drawn from examinations of ability to work status, health-related quality of life and patients´ satisfaction with rehabilitation. While there were no setting-specific group differences regarding rehabilitation satisfaction, IPR and OPR patients differed significantly regarding ability to work rates and physical as well as mental health status. Thus, OPR patients´ ability to work rate was twice as high as in IPR patients. Consistently, OPR patients reported significantly better physical and mental quality of life scores than IPR patients. In addition to this, OPR turned out to be a positive predictor variable for ability to work and a better physical health status three months after disc surgery. These findings should not be misinterpreted as quality differences between both settings. In fact these differences can be explained by greater physical as well as mental impairments prior to rehabilitation treatment . In lineLindell et al.  found loOPR = 40.7 years, mean age IPR = 43.4 years). This may be leading to differing results with regard to other studies.Unlike to the results of Lillefjell et al.  and LindFurthermore, a better educational level was showing a weak association with ability to work three months after disc surgery. Dionne et al.  hypothesRegarding physical quality of life three months after disc surgery the findings of the presented study show that lower pain intensity and a shorter length of acute care hospital stay are weakly associated with a better physical health status. Moreover, the absence of other chronic diseases at the time of acute hospital stay was moderately associated with a better physical health status. A better overall health status at the time of disc surgery therefore seems to be important in order to predict a better physical health status three months after disc surgery. In line with Johansson et al. , women tOn the other hand, a better mental quality of life was strongly associated with a better educational level (10th grade vs. 9th grade). This corresponds well with the study of Bjelland et al.  who founPatient satisfaction is an important outcome measure in rehabilitation research ,36. ThusBesides the longitudinal study design and a large sample size, a great strength of the study is that disc surgery patients were already approached during acute hospital treatment. Patients`individual answers regarding their motives, motivations and expectations regarding rehabilitation treatment and rehabilitation setting may therefore not be biased by social desirability towards rehabilitation clinic.A limitation of the study is that the presented findings regarding the choice of a certain rehabilitation setting only rely on patient´s own reports regarding their preferences, motivations and expectations. This does not necessarily say anything about the concrete allocation procedure that lead to the decision for a certain rehabilitation setting. Hence, the physician´s opinion, the advice of the hospital social service and structural factors such as the lack of outpatient rehabilitation facilities in the patient´s neighbouring region may all contribute to the allocation procedure in different ways. Future studies should take all these allocation factors into account that allow making detailed conclusions on allocation procedures regarding different rehabilitation settings. Using mixed-method-designs of qualitative and quantitative survey techniques are strongly recommended in this context. Another limitation of the study is that, although taking patients´ pain intensity into account, it does not give information about the chronicity of neck and back pain problems and its influence on motivational and expectation factors. Regarding this matter, Skatteboe et al.  discuss Little is known about the role of patients´ motivations and expectations regarding the choice of an inpatient or outpatient rehabilitation setting. The present study, for the first time, provides information about individual motives, motivations and expectations behind this decision in a sample of patients undergoing herniated disc surgery. Thus, this study contributes to a better understanding of the patients`perspective within decision-making process for a specific rehabilitation setting. Rehabilitation-related motivations and expectations before rehabilitation had impact on physical and mental quality of life as well as on satisfaction with rehabilitation treatment three months after acute hospital stay. There were also several other associations with rehabilitation outcome parameters: Thus, higher age, a better educational level, the absence of other chronic diseases, a shorter length of acute care hospital stay, a better subjective prognosis of gainful employment and OPR were associated with improved ability to work. Male gender, the absence of other chronic diseases, lower pain intensity, a shorter length of acute care hospital stay, employment within the last three months, a better subjective prognosis of gainful employment and OPR was significantly associated with a better physical health status. A higher educational level, a lumbar disc herniation and the absence of psychiatric comorbidity before surgery were significantly associated with a better mental health status. In addition, male gender was associated with a greater satisfaction with rehabilitation. The present study therefore suggests the following clinical implications for patients undergoing herniated disc surgery. First, the assessment of rehabilitation-related motives, motivations and expectations is strongly recommended at the beginning of rehabilitation treatment, preferably already at the end of the preceding acute hospital stay. Taking motivational and expectation-related aspects of patients undergoing herniated disc surgery into account at an early stage may help to find the perfect match between rehabilitation patient and rehabilitation setting. Another advantage of such an assessment could be that special motives, such as the need for psychological counselling, could be addressed during rehabilitative care and would be prevented from being overseen. Second, increasing the level of information regarding similarities and differences of IPR and OPR on the one hand and providing material regarding general treatment procedures on the other hand might help to improve allocation processes and satisfaction with rehabilitation. Third, assessing patients´ self-prediction of return to work after disc surgery on a regular basis and in the same context individual counselling regarding realistic goal setting may help to improve work-related rehabilitation outcome. Fourth, the establishment of self-help groups may increase feelings of connectedness and social support, especially for female patients, and may help to reduce gender differences regarding health-related quality of life. Longitudinal studies incorporating qualitative and quantitative methods are strongly recommended to investigate if these measures help to improve rehabilitation outcomes.