STAT5 is an essential transcription factor in hematopoiesis, which is activated through tyrosine phosphorylation in response to cytokine stimulation. Constitutive activation of STAT5 is a hallmark of myeloid and lymphoblastic leukemia. Using homology modeling and molecular dynamics simulations, a model of the STAT5 phosphotyrosine-SH2 domain interface was generated providing first structural information on the activated STAT5 dimer including a sequence, for which no structural information is available for any of the STAT proteins. We identified a novel intramolecular interaction mediated through F706, adjacent to the phosphotyrosine motif, and a unique hydrophobic interface on the surface of the SH2 domain. Analysis of corresponding STAT5 mutants revealed that this interaction is dispensable for Epo receptor-mediated phosphorylation of STAT5 but essential for dimer formation and subsequent nuclear accumulation. Moreover, the herein presented model clarifies molecular mechanisms of recently discovered leukemic STAT5 mutants and will help to guide future drug development. D816V-positive systemic mastocytosis (SM)649S710F into mice identified STAT5 as a putative oncogene capable of inducing tumor initiation in many hematopoietic cell types, independent of dysregulated upstream kinasesSTAT5, represented by the highly homologous isoforms STAT5A and STAT5B, is an essential transcription factor for the proliferation, survival and differentiation of myeloid cells24In addition to these functions in physiologic and malignant myelopoiesis, STAT5 represents a master regulator of lymphopoiesis, controlling the proliferation and differentiation of T and B cells through various cytokines including IL-2 and IL-7121415161718The addiction to STAT5-mediated signal transduction processes has been considered to be a potential Achilles heel in leukemic signaling networks, resulting in intense research aiming at the pharmacological inhibition of STAT5. Hence, prevention of STAT5 dimer formation is thought to interfere with tumor cell proliferation and survival2021Oncogenic somatic STAT5 mutations are characterized by stimulation-independent activation and predominantly map to the SH2 domain  of the transcription factor. However, constitutive STAT5 activation is additionally promoted by somatic mutations located in the sequence following phosphotyrosine 694 , a region for which only limited or no structural information is available for any of the STAT dimers .Therefore, the structural determinants of the STAT5A dimer interface were predicted by bioinformatics approaches and molecular dynamics (MD) simulations. The modeled system includes the SH2 domain (residues 589–687) and the C-terminal tail segment , which consists of a short flexible linker , the phosphotyrosine motif  and the N-terminal part of the transactivation domain  . The ini24Next, 2,000 ns-long MD simulations were performed  on the h2324M2) interacts with the SH2 domain of M1 with the same orientation as pY694M1. Salt bridges formed by pY694 and the highly conserved and invariant R618 located in the βB strand of the opposite SH2 domain are key interactions in both intermolecular PTM-SH2 domain interfaces. Indeed, they are fully preserved during the MD simulations with occupancies of 100% (pY694M1) and 97% (pY694M2), respectively. Moreover, the coordination of pY694 in both monomers is supported by the formation of transient hydrogen bond interactions with K600, S620, S622 and T628 in the opposite monomer during the MD simulations  in the CTS of monomer 1 (M1) interacts with the SH2 domain of the opposite monomer (M2) in a direction orthogonal to the strands of the central β-sheet . In a syulations . Notablyulations 2425.Our model further identified two novel residues involved in the recognition of pY694. These are the βD residues N642 (βD5) and K644 (βD7), which stabilize the aromatic ring of the phosphotyrosine by hydrophobic interactions . Finally24M1 and K700M1 with Q701M2, and I699M2 and Q698M2 with E705M1 . In the opposite monomer, F706M2 forms aromatic interactions with F633M2, Y665M2 and W631M2 with occupancies of the hydrophobic contacts of 41.4%, 41.3% and 21.7%, respectively. Such a network of hydrophobic interactions substantially contributes to the recognition of F706 in their respective interfaces /JAK2-mediated phosphorylation of Y694, dimer formation and nuclear accumulation of STAT5A being mutated at residue F706 (F706A) or in the hydrophobic acceptor interface . These mutants were studied in comparison to STAT5A wild-type and the non-functional R618Q mutant. HeLa T-REx cells stably expressing eYFP-tagged STAT5A constructs and the EpoR revealed a severely impaired tyrosine phosphorylation of all mutants in response to Epo stimulation. Intriguingly, the inhibition of phosphatases by sodium vanadate treatment rescued the Epo-induced phosphorylation of STAT5A mutants F706A, F633A or Y665A demonstrating that the reduced phosphorylation of these mutants in the absence of vanadate is caused by phosphatase activity rather than dysfunctional receptor recruitment . Of noteTo study the impact of these mutations on STAT5A dimerization, blue-native PAGE was performed. Since phosphorylation at Y694 is a prerequisite for the formation of STAT5A dimers, lysates were analyzed after treatment of cells with Epo and vanadate. Phosphatase inhibition resulted in a 2.15-fold increase in dimerization of STAT5A wild-type in response to Epo stimulation, illustrating that the EpoR/JAK2/STAT5A signaling pathway in steady-state is highly counter-regulated by phosphatases. Surprisingly, while in response to vanadate treatment STAT5A proteins carrying the F706A mutation appeared to be phosphorylated markedly above reference levels (Epo stimulation of STAT5A wild-type in the absence of vanadate), their dimerization was severely impaired (−2.37-fold) . LikewisIn contrast, vanadate treatment failed to restore the receptor-mediated phosphorylation of the STAT5A mutants F633A/Y665A, W641A and R618Q to reference levels , suggestIn resting cells, STAT5 is evenly distributed between cytoplasm and nucleus. Formation of phosphorylated dimers is a requirement for nuclear accumulation of STAT5. Confocal imaging of fluorescently labeled STAT5A revealed that vanadate treatment resulted in a forced nuclear enrichment of the wild-type protein in erythropoietin-stimulated cells, while the investigated STAT5A mutants did not accumulate in the nucleus reflecting their impaired capability to form dimers . Taken tIn a defined subset of aggressive lymphoid cancers, the somatic mutations N642H (βD5) and T628S (β-sheet C) have recently been identified, which map to the phosphotyrosine-binding pocket in interface 1 of the STAT5 SH2 domain , and 4b.15171416N642H and STAT5AT628S by the detection of dimers that appeared 2.10-fold and 1.48-fold increased in Epo-stimulated cells, suggesting that both mutations interfere with appropriate deactivation of the JAK-STAT signaling pathway  demonstrating that this mutation confers a stronger constitutive activation to STAT5 proteins compared to T628S . Strikingly, phosphatase inhibition failed to restore STAT5A dimerization back to reference levels confirming that the phosphorylation of STAT5A is necessary yet not sufficient for dimerization . These fN642H, mutations in the PTM resulted only in a weak factor-independent constitutive activation, illustrating that both mutants are more sensitive to dephosphorylation similar to STAT5AT628S. The introduction of the F706A mutation into I699L and Q701L mutants again significantly diminished dimerization in stimulated cells compared to the corresponding single mutants (3.13-fold and 2.54-fold), despite boosted phosphorylation levels caused by vanadate treatment  appears at a lower frequency of 2.0% in lymphomas of NK or γδ T-cell origin (1/94)reatment . Withoutreatment . Notably of F706 . Neverthed cells .Y665F showed a significant 1.82-fold (p = 0.0020) increase in the amount of dimers in stimulated cells compared to reference levels, while vanadate treatment remained again insufficient to rescue pSTAT5AF706A or pSTAT5AF706G dimerization rization .Y665F carrying the F706A (3.90-fold) or F706G (3.50-fold) mutation, suggesting that the elimination of the Y665-hydroxyl group is sufficient to enable appropriate dimerization of STAT5AF706A/G. Nevertheless, these dimers remained highly susceptible for phosphatases. Therefore, it appears evident that besides its role in the dimerization process, binding of F706 to the hydrophobic interface is critical to protect STAT5A dimers from rapid inactivation  and 6.16-fold (p = 0.0007) in stimulated cells compared to the Y665F single mutant . Surpristivation . In accotivation .The crystal structures of the activated STAT1 and STAT3 dimers were used to predict the structural determinants of the fully assembled SH2 domain interface of STAT5A by the combination of homology modeling and MD simulations. The resulting model, validated with experimental data, revealed novel and unique features of the dimerization interface of STAT5A and was used to unravel the impact of disease-related STAT5 mutations on protein structure and function.Not unexpected, the model corroborates reciprocal phosphotyrosine-SH2 domain interactions as a conserved driver of STAT dimerization resulting in a grossly symmetric dimer2428The binding interface selectivity of SH2 domains is mediated by individual binding pockets, additional to the phosphotyrosine-binding site, that primarily recognize residues in the pY+1 and pY+3 positions located C-terminal to the phosphotyrosine residue. In most SH2 domains, the βD5 residue critically contributes to selectivity by recognition of the pY+1 position3124In STAT1 and STAT3, the PTM forms a pair of cross-over connections with the opposite PTM significantly increasing the intermolecular contact area at the dimer interface24In recent years, an increasing number of somatic STAT5 mutations has been identified in leukemia patients. The vast majority of lymphoma-associated STAT5 mutations such as N642H, T628S, I699L, Q701L and Y665F map to the SH2 domain and PTM of the transcription factor and are characterized by an activating phenotype1415161718T628S directly strengthens phosphotyrosine coordination by the SH2 domain. Importantly, the improved phosphotyrosine-binding ability of both SH2 domains did not suffice to rescue dimerization of STAT5A proteins lacking F706, further strengthening the relevance of intramolecular interactions for STAT dimerization (The mutations N642H and T628S reside in the phosphotyrosine-binding interface and peptide precipitations confirmed an increased binding affinity for both of the mutated SH2 domains suggesting that constitutive activity is a consequence of an impaired inactivation by STAT5 phosphatases . MD simu1734rization .I699L/F706A and STAT5AQ701L/F706A exhibited a weaker dimerization defect compared to the F706A mutant providing evidence that L699 (pY+5) and L701 (pY+7) significantly support the dimerization of STAT5A. Hence, constitutive activation of STAT5AI699L and STAT5AQ701L is most likely a consequence of increased binding affinities between the two PTMs . These are bacteria CobB sirtuin  were initially obtained by using homology modeling. The latter was based on the correspondent domains in the crystal structures of activated STAT1 dimer  scoring functionErythropoietin  was used at a concentration of 1 U/ml. Anti-pY694-STAT5  and anti-GFP  were used for immunoblotting. Anti-rabbit and anti-goat antibodies conjugated to horseradish peroxidase (HRP) were ordered from, DAKO .® XL Site-Directed Mutagenesis Kit according to manufacturer’s instructions .The cDNA of murine STAT5A was either cloned into pLentiLox  or pcDNA3.1  expression vectors and fused to the cDNA of the enhanced yellow fluorescent protein (eYFP). The cDNA of the human erythropoietin receptor (EpoR) was cloned into the pcDNA5/FRT/TO expression vector and fused to the cDNA of the hemagglutinin tag (HA). Fluorescent proteins were fused to the C-terminus of the target proteins, whereas the HA-tag was fused to the N-terminus of the human EpoR. Point mutants of STAT5A were generated with PCR based QuikChange®-LT1  according to manufacturer’s instructions. Stable transfection of HeLa T-Rex cells for the inducible expression of HA-EpoR was performed with the Flp-In system  using 250 μg/ml hygromycin B  and 15 μg/ml blasticidin  for selection. The expression of HA-EpoR was induced with 5 ng/ml doxycycline  for 24 h. All media were supplemented with 10% FCS  and 25 U/ml penicillin/streptomycin . Cells were incubated at 37 °C in a water-saturated atmosphere with 5% CO2. Phosphatase inhibition was carried out by sodium vanadate (Na3VO4) treatment (1 mM) for 2 h.HeLa T-Rex  cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/GlutaMAX . Transient transfection of HeLa T-REx cells was performed using Trans ITTo achieve stable and constitutive expression of STAT5A constructs in inducible HeLa T-Rex cells, self-inactivating lentiviral constructs were used according to the guidelines of the RNAi consortium. Briefly, the cDNA of STAT5A-eYFP and the point mutants of STAT5A-eYFP were cloned into the lentiviral pLentiLox expression vector downstream of the CMV promotor . HEK293-T packing cells were seeded on 6-cm dishes on to be 80–90% confluent at the time of transfection. On the second day, HEK293-T cells were transiently co-transfected with the lentiviral vector, psPAX2 (packing plasmid) and pMD2.G (envelope plasmid). Eighteen hours after transfection the culture medium was replaced by high serum growth medium containing 30% FCS. Viruses were harvested 24 hours after medium exchange. For the infection of HeLa T-Rex cells, virus-containing supernatant was supplemented with 8 μg/ml polybrene  prior to infection.2HPO4, 1.5 mM KH2PO4, adjusted to pH 7.4) once and lysed with RIPA lysis buffer  supplemented with phosphatase/protease inhibitors , 5 μg/ml aprotinin, 2.5 μg/ml leupeptin) according to standard procedures. The proteins were separated by SDS-PAGE and transferred to a PVDF membrane with subsequent immunodetection using specific antibodies. Primary and HRP-conjugated secondary antibodies were diluted in TBS-N buffer . Membrane-bound antibody complexes were detected by chemiluminescence .HeLa T-REx cells were washed with PBS  supplemented with 2% digitonin . The lysates were cleared by centrifugation and incubated with Coomassie brilliant blue G-250 and separated over night at 4 °C using NativePAGETM Bis-Tris gel system with a gradient polyacrylamide gel (4–16%) according to the manufacturer’s instructions. Dimerization of STAT5A-eYFP constructs was analyzed by detection of the eYFP fluorescence with a typhoon fluorescence scanner  by excitation with a 488 nm laser line. The emission was detected using a 515–555 nm bandpass filter.Transiently transfected HeLa T-REx parental cells were lysed with storage buffer  supplemented with phosphatase/protease inhibitors. Lysates were cleared by ultracentrifugation . Peptides corresponding to the phosphotyrosine motif of STAT5 (b-tat-AKAVDGpYVKPQIKQVVPEFVNASADAG-a) were synthesized with >95% purity, modified by N-terminal biotinylation (b-) and C-terminal amidation (-a) and contain the HIV tat protein derived GRKKRRQRRRPQ (tat) sequence at the N-terminus. 25 nmol of the peptides were coupled to NeutrAvidin Agarose Resin  for 2 h at 4 °C, washed twice with storage buffer and incubated with 1 mg of the lysate over night at 4 °C. Peptide/protein complexes were washed 3 times with storage buffer for 10 minutes and the precipitates were analyzed by SDS-PAGE and immunoblotting.HeLa T-REx cells expressing fluorescently labeled fusion proteins were grown on glass cover slips. After paraformaldehyde (3.7%) fixation cells were stained with DRAQ5  and Phalloidin-Atto 550  to label nuclei and actin, respectively. Cover slips were mounted with ImmuMount  as has been described previouslyInformation on statistics and sample numbers is given in the corresponding figure legends.How to cite this article: Fahrenkamp, D. et al. Intramolecular hydrophobic interactions are critical mediators of STAT5 dimerization. Sci. Rep.6, 35454; doi: 10.1038/srep35454 (2016).


To date, no short scale exists with established factor structure that can assess individual differences in competition. The aim of the present study was to uncover and operationalize the facets of competitive orientations with theoretical underpinning and strong psychometric properties. A total of 2676 respondents were recruited for four studies. The items were constructed based on qualitative research in different cultural contexts. A combined method of exploratory structural equation modeling (ESEM) and confirmatory factor analysis (CFA) was employed. ESEM resulted in a four-factor structure of the competitive orientations and this structure was supported by a series of CFAs on different comprehensive samples. The Multidimensional Competitive Orientation Inventory (MCOI) included 12 items and four factors: hypercompetitive orientation, self-developmental competitive orientation, anxiety-driven competition avoidance, and lack of interest toward competition. Strong gender invariance was established. The four facets of competition have differentiated relationship patterns with adaptive and maladaptive personality and motivational constructs. The MCOI can assess the adaptive and maladaptive facets of competitive orientations with a short, reliable, valid and theoretically underlined multidimensional measure. Research on individual differences in competitive orientations is mostly based on a unidimensional concept of competition , 2009. FIndividual differences in competitive orientations can have critical effects on performance in achievement situations  and theyUntil the 1990s, the major ruling paradigm in competition research conceptualized competitive orientation as a unidimensional construct . This apHowever, in the last three decades, there has been a paradigm change regarding competitive attitudes and orientations in the field of personality psychology , 2009, sSimilarly to enjoyment of competition which refers to personal attitudes toward one’s competitive behavior , whereas the second dimension is contentiousness which refers to attitudes toward avoidant behavior in arguments and conflicts.Apart from the distinction of self-image concerns and it refers to disliking competition because of the fear of failing or being perceived negatively. The second is called as performance concerns, referring to disliking competitive situations because other people are inclined to have high expectations for the self. The third dimension, called distraction of attention, directly refers to the detrimental effects of evaluation on attention. Complementing and further developing the existing models with two dimensions, Another typology was created by personal-development competitive attitude (PDCA), the primary focus is on personal growth and on the enjoyment and mastery of the task in a competitive situation. The goal attainment and competition outcome  is important, but not at the expense of the derogation of other competitors (Regarding the petitors . Individpetitors . PDCA ispetitors .hypercompetitive attitude (HCA) on competition avoidance, was also based on “the need of individuals to check their ruthless ambition because of excessive fear of losing the affection and approval of others as a consequence of either being successful in competition with others or through failure in such competition”  can measure the most important facets of competition, (b) which can be applied to one’s competitive orientation in different fields , (c) which is short and thus can be applied in combination with a battery of other instruments, and (d) which has strong psychometric properties in terms of factor structure, validity and reliability.Therefore, we constructed the MCOI by generating items on the basis of previous qualitative studies. Then, in four studies, the inventory was validated by using a construct validity approach that includes both within-network and between-network validity analyses . Within-In the first study, our goal was to develop a pool of items that correspond to each of the competition facets described in the “Introduction” section. These items were created based on previous interviews and responses to open-ended questionnaire carried out by In several studies with high school students, college students, teachers, business people of different nationalities open-ended questionnaires requiring free descriptive answers and semi-structured interviews were applied in order to reveal the qualitatively different concepts of competition of the respondents . ParticiM = 30.51, SD = 0.69). Sample 2 was representative in terms of gender, age, level of education among those Hungarians who used Internet at least once a week. As the data gathering was online, this sample was not representative among those who do not use internet at least once a week. For this reason, we use the term of comprehensive sample for these sorts of samples. The participants were selected randomly from an Internet-enabled panel including 88000 members with the help of a Hungarian publisher company in July 2015. For the preparation of this sample, a multiple-step, proportionally stratified, probabilistic sampling method was employed. Individuals were removed from the panel if they gave responses too quickly  and/or had fake (unused) e-mail addresses.In the present study, two different samples were used for the purpose of validation and cross-validation. For Sample 1, the item set was tested on a sample of 525 university students , aged between 18 and 92 , education , and place of residence .This final comprehensive sample of 500 participants had the following characteristics: gender , age . Participants were informed about the content of the questionnaire. They volunteered for the study, they did not receive any kind of compensation for the participation.For the statistical analyses, SPSS 22 and Mplus 7.3  were useIn the next part, exploratory structural equation modeling ESEM;  was condFollowing common guidelines , multiplInternal consistency was measured with Cronbach’s alpha  with itsTable 1). By following these criteria, a total of eight items were eliminated, resulting in 12 items that were retained for the subsequent analyses. Skewness and kurtosis scores were under ±2  corrected item-total correlations, (2) normality in terms of skewness and kurtosis, and (3) content validity compared to the other items and competition in general 1. The results indicate strongly target loadings  and minimal cross-loadings . The latent inter-factor correlations, reliability indices and descriptive statistics can be seen in Table 2.Next, ESEM was performed to investigate the factor structure of the remaining items. This solution yielded adequate fit to the data . This solution confirmed that the scale has appropriate factor structure.Similarly to the ESEM model, the first-order CFA model indicated good fit to the data in the comprehensive sample  consisted of 445 male and 455 female respondents . Regarding the highest completed level of education, 16.2% (n = 146) of the respondents have primary level of education, 23.9% (n = 215) have vocational school degree, 37.5% (n = 338) graduated from high school, and 21.2% (n = 190) have higher education degree. Regarding the place of residence 21% (n = 189) of the respondents live in the capital, 20.2% (n = 182) live in the county towns, 34.3% (n = 309) live in towns and 24.5% (n = 220) live in villages.This comprehensive sample . One school was located in a Hungarian village , one was in a town , one was in a county town , and one was in the capital  of Hungary. Two pupils did not report the location of their school.This sample of elementary school students consisted of 216 participants  from four elementary schools, aged between 12 and 15 years (N = 192) consisted of 80 female and 104 male students (eight of them did not respond this question) from a Hungarian high school, aged between 14 and 20 years . The students were living in a town  or in a village .The high school student sample . This solution corroborates our findings. In the next step, the results were cross-validated on the high school and elementary school samples which also demonstrated good fit to the data: elementary school sample , high school sample 2. Standardized factor loadings and inter-factor correlations can be seen in Figure 1.Similarly to the results of Study 1, the first-order model indicated good fit to the data on this second comprehensive sample (χTable 3), giving further strong support for the validity and reliability of the MCOI.All internal consistency and reliability indices  demonstrated good values  model where factor loadings were constrained to be equal across the groups; third, strong  model where intercepts are set to be equal; fourth, strict  model where error variances needed to be equal. Moreover, latent variances-covariances and latent factor means could also be investigated. Therefore, the following goal of this study was to test gender invariance on the MCOI.Multiple levels of invariance testing can be distinguished: first, For the purposes of measurement invariance testing, three adult samples were combined from previous samples: the university sample of 525 participants from Study 1, the comprehensive sample of 500 participants from Study 1 and the comprehensive sample of 900 participants from Study 2. For further sample characteristics, see previous studies.For measurement invariance, baseline models were identified for the different subsamples. Next, parameter constraints were gradually imposed from the least restrictive to the most restrictive one e.g., : configuTable 4. First, configural model with no parameters constrained to equality in the two groups was estimated. This model provided adequate fit to the data . All subsequent models  were successfully estimated with the gradually imposed constraints. While most χ2 and Δχ2 test were significant, other model fit indices  did not decrease more than the recommended cut-off values. In many cases, parsimony-corrected fit indicators  even improved with the addition of invariance constraints. In sum, the present results provided strong support for the conclusion for the gender invariance of the MCOI on the level of latent means.Goodness-of-fit indices for the estimated models can be seen in Previous studies of this research demonstrated that the MCOI had strong within-network validity  in termsself-developmental competitive orientation would be positively related to personality traits as perceived resilience as an indicator of psychological health  with 214 females and 129 males. From participants under the age of 18 (n = 130), parental consent was obtained prior to the beginning of data collection. With regards to their highest level of education, 188 participants (54.8%) completed primary education, 85 (24.8%) had secondary education, and 66 (19.2%) completed college/university level education. 4 participants (1.2%) did not specify their level of education. 53 participants (15.5%) lived in capital city, 78 (22.7%) lived in county capitals, 96 (28%) lived in cities, 57 (16.6%) lived in villages and 59 (17.2%) lived abroad.A total of 343 participants were recruited for the study from high schools and universities based on a call distributed by teachers. The participants filled in an online questionnaire in the educational institutions. The age of the participants ranged from 13 to 57 , and Discrepancy . Similarly to The APS-R  is desigPerfectionism drives me to be motivated.”) and four items measuring negative consequences . Participants provided their responses on a five-point scale ranging from 1 (extremely untrue of me) to 5 (extremely true of me).The COPS  was usedThere is satisfaction in a job well done.”), Mastery , and Competitiveness . Responses are rated on a 5-point scale ranging from 1 (strongly disagree) to 5 (strongly agree).Work and Family Orientation Questionnaire  is a 19-Table 5, while the associations between the different competitive orientations and adaptive-maladaptive outcomes are presented in Table 6. Partial correlations were performed to control for the covariance between the different facets: when conducting the partial correlations between the competitive factors  and the outcomes, all other competitive factors  are controlled for. Generally, most associations were in line with the expectations. Self-developmental competitive orientation had a positive association with resilience, positivity, the discrepancy factor of perfectionism, the positive consequences of perfectionism, and all achievement motivations. Hypercompetitive orientation was only associated with the high standards factor of perfectionism, the negative consequences of perfectionism and the work-related achievement motivation. Although the lack of interest and anxiety-driven competitive factors had high associations in the previous studies of this investigation, they are rather distinct on the basis of their associations with the correlates. More specifically, while the lack of interest factor had a small positive correlation with resilience, it was negatively related to the discrepancy factor of perfectionism and the work-related achievement motivation. On the other hand, while the anxiety-driven factor was inversely related to resilience and the discrepancy factor of perfectionism , it was also positively related to the high standard factor of perfectionism and the negative consequences of perfectionism.Descriptive statistics and internal consistency indices are presented in “I have been up against tough competition all my life. I wouldn’t know how to get along without it.” which is fundamentally different from Béla Bartók’s view on competition “Competitions are for horses, not artists.” These quotations refleca dominant competition orientation , these orientations can co-exist. Therefore, our goal was to build upon unidimensional competition measures  were performed to investigate the factor structure of the MCOI. All analyses suggested that MCOI has an adequate factor structure. These results were corroborated by other reliability and validity indices. The Cronbach’s alpha, the composite reliability and factor determinacy of the four factors also underscored that MCOI is a valid and reliable measure. To our best knowledge, no prior measure focusing on different aspects of competitive orientations was examined by invariance testing. In the case of MCOI, gender invariance on the level of latent means was achieved, giving strong support for the generalizability of the MCOI across male-female subgroups. In sum, on the basis of these results MCOI is a brief measure that, has good factor structure, with reliable subscales and high level of gender invariance.The MCOI model is compatible with the theoretical background of previous multidimensional models. However, compared to the models of The MCOI can distinguish competition approach, competition avoidant and competition neutral orientations. It includes two competition approach orientations: hypercompetitive and self-developmental competitive attitudes. The main difference between these two is that the hypercompetitive orientation is strongly result-oriented, while the self-developmental competitive orientation is strongly process-oriented. In the MCOI model, the competition avoidance is anxiety-driven. Finally, the neutral dimension is related to the interest vs. uninterest toward competitive situations. Therefore, this measure can assess both approach and avoidant aspects of competitive orientations, it can differentiate between the result- and process-orientation, and it can assess the general interest toward competitive situations.In terms of between-network validity, we examined whether the factors of the scale were associated with other factors in expected directions. Self-developmental competitive orientation was positively related to hypercompetitive orientation and negatively related to anxiety-driven and uninterested competitive orientations. This might be explained along the approach vs. avoidance dimensions. The positive link between self-developmental and hypercompetitive orientations can be explained by that both belong to the competitive approach dimension. The negative links between self-developmental orientation vs. competitive avoidant and uninterested facets can be attributed to the notion that these latter orientations express an avoidant or neutral standpoint regarding competition. Hypercompetitive orientation was negatively or non-significantly related to anxiety-driven competition avoidance and lack of interest toward competition. The negative correlations can be explained by the approach vs. avoidance and neutral distinction. However, in this case we might suppose that hypercompetitive orientation can appear more easily simultaneously with anxiety-driven competitive orientation. More specifically, considering both the relatively strong negative relationship between self-developmental competitive orientation and anxiety-driven competitive orientation and the weak or non-significant link between hypercompetitive anxiety-driven competitive orientation, the probability of being simultaneously hypercompetitive and being anxiety-driven competition avoidant can appear as more probable than being self-developmental competitive and anxiety-driven competition avoidant at the same time. Future studies should explore this supposition with person-centered methods. Finally, it is important to mention that the anxiety-driven and uninterested orientations were positively related. This link is also reasonable, as both of these represent disengagement regarding competition, but for different reasons. This can also be the subject of further research.Self-developmental competitive orientation is the self and ability improvement. In line with prior studies  in competitive situations in which the end may justify the means. On the basis of prior work of Anxiety-driven competition avoidance orientation focuses on the general anxiety deriving from the process of competition. We expected that it would show an inverse negative correlational pattern compared to self-developmental competitive orientation. The results partially supported our expectations: anxiety-driven individuals who avoid competition might be less resilient, have higher standards in perfectionism and have a higher discrepancy between their perfectionist ideals and their actual state. Interestingly, this avoidant behavior is also associated with negative consequences of perfectionism. In sum, anxiety-driven competitive orientation, similarly to Lack of interest in competitive orientation is related to the disinterest in competitive situations and the lack of any approach or avoidance motivation regarding competitive situations. Concerning this facet, we expected similar correlational pattern as in the case of anxiety-driven competition avoidance, but with smaller correlational coefficients. The results were not in line with our expectations: this factor was weakly related to resilience and perfectionism discrepancy, while moderately and negatively related to work-related achievement motivation. Indeed, when individuals are not interested in competitions (regardless of it being overt or hidden), they are less likely to put additional effort in their work. Individuals characterized by this orientation may be less concerned with expectations of others in competitive or other achievement situations in which a “well-done job” is required.To summarize, these convergent validity results suggest that competition avoidance and the lack of interest toward competition do not appear to have strong adaptive personality and motivational links. Their correlates showed a slightly less adaptive correlational pattern than in the case of hypercompetitive orientation. In contrast, self-developmental competitive orientation had a generally positive personality and motivational correlational pattern. These results can have both theoretical and practical implications.Prior self-reported assessments of individual differences in competition had shortcomings in terms of not using the factor-analytic approach , 2009 orFrom the broader theoretical perspective, it might be important to consider the potential positive aspects of competition and competitive orientations. The present results regarding self-developmental competitive orientation fit a broader research stream emphasizing potential positive aspects of competition in terms of performance, interpersonal relationships, resource control, intrinsic and extrinsic motivations . The preFurther research should explore whether individuals with dominantly self-developmental orientation behave similarly to individuals with other dominant competitive patterns. It would be especially important as in a relatively recent meta-analysis, On the basis of prior studies and the present correlational pattern, we can expect less adaptive personality and motivational outcomes from the strongly result-oriented, competition-approach hypercompetitive orientation . However, considering the overall correlational pattern, lack of interest in competition and especially anxiety-driven competition avoidant orientation shows even less adaptive personality and motivational profile. These patterns lead to the most important practical implication.Competitive orientation refers to one’s thoughts, emotions, and anticipated or actual behaviors regarding competitive situations. On the basis of the first two studies, this perception of one’s competitive orientation can be diverse. On the basis of Study 4, this diversity is related to fundamentally different personal and motivational profiles. Institutions as schools or workplaces can shape these orientations and if these institutions create norms in which competitiveness in itself should be diminished, adaptive self-developmental competitive orientation will be less likely to be reinforced. At the same time, the lack of interest toward competition and anxiety-driven competition avoidance can be reinforced. Therefore, based on the results of the present multidimensional measure, it might be more beneficial to create such norms that promote mastery-based, learning-, and process-oriented self-developmental competitive orientation instead of eliminating every form of it. In sum, it would be better to create constructive competitive institutional norms ,b that cAlthough the present study has several strengths , it is not without limitations. All measures were completed on self-report scales; therefore, they could lead to possible biases . In future studies, these scales should be complemented with objective tools that could directly measure not only self-reported competitive behavior while, at the same time, respecting the privacy of the individuals. Despite convergent validity was established, the present study did not focus on assessing discriminant validity of the MCOI. Future research is needed to overcome this shortcoming. As correlational design was used in the present research, causality cannot be inferred. Moreover, within the framework of the current research, only cross-sectional studies were conducted which did not allow the examination of different life events that might influence competitive orientations. A longitudinal design would be beneficial in examining how potential life events  could have an impact on the individual’s competitive attitudes. Furthermore, from both theoretical and practical perspectives, it would be beneficial to examine the adaptive and maladaptive blend of competitive orientations. Replications in other cultures could also be performed as different cultural and economic characteristics might also influence one’s competitive orientations. Regarding the MCOI itself, further examination is needed to assess its temporal stability as well as its convergent, divergent and predictive validity.On the basis of four studies, we could identify four main aspects of competitive orientations: hypercompetitive, self-developmental, anxiety-driven competition avoidant, and lack of interest toward competition. High levels of gender invariance were established. The Multidimensional Competitive Orientations Inventory is short, and grasps the multifaceted nature of individual differences in competition. For the first time, this measure can allow the identification of adaptive and maladaptive blends of competitive attitudes.GO and MF contributed to the study design, literature review, data gathering, manuscript writing, and to the data analyses and interpretation. IT-K and BB contributed to data analyses and interpretation, and to the manuscript writing. NB and KI contributed to the literature review, data analyses, and to the manuscript writing. All authors commented on the draft and contributed to the final version, approved the publication of the manuscript, and agreed to be accountable for all aspects of the work.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.


Padina boergesenii. The phytochemical investigation resulted in the isolation of a new secondary metabolite, padinolic acid (1), along with some other semi-pure fractions and sub-fractions. The planar structure was confirmed through MS and NMR (1D and 2D) spectral data. The NOESY experiments coupled with the biogenetic consideration were helpful in assigning the stereochemistry in the molecule. Compound 1 was subjected to enzyme inhibition studies using urease, lipid peroxidase, and alpha-glucosidase enzymes. Compound 1 showed low to moderate α-glucosidase and urease enzyme inhibition, respectively, and moderate anti-lipid peroxidation activities. The current study indicates the potential of this seaweed and provides the basis for further investigation.In continuation to our study of the chemical and biological potential of the secondary metabolites isolated from Omani seaweeds, we investigated a marine brown alga,  Man has been using the oceans as a useful resource for producing economically important materials . During The secondary metabolites from organisms living in the marine environment have been found to possess ameliorative potential in the exploration of drugs for various human-associated diseases. Ailments such as diabetes are regarded serious metabolic disorders in the human body. This is normally shown as high blood sugar, insulin resistance, and a relative lack of insulin . The figPadina is found along the Omani coastal areas, and some of the Padina species are considered important sources of iodine and mannitol + and m/z 455 [M − H]− for protonated and deprotonated molecular ions, respectively. The molecular formula was further confirmed through high resolution mass spectrometry (HR-ESIMS), which showed the deprotonated molecular ion peak at m/z 455.3513 (M − 1). The seven indices of hydrogen deficiency were assigned to the tetracyclic system with two olefinic moieties and one carbonyl group in the molecule. These observations were fully supported by 13C NMR spectral data.Compound m/z 133, 203, and 248 indicated the Δ7-terpenoid skeleton. The EI-MS also showed fragment ion peaks at m/z 438, 412, 286, and 83 corresponding to the hydroxyl group, the carboxylic group, and the alkyl chain in compound 1. The evidence of retro Diels-Alder fragmentation was provided by the fragment ion peaks at m/z 316 and 122, which further supported the Δ7-terpenoid skeleton in 1. The IR spectrum showed the absorptions for a hydroxyl (3460 cm−1), a carboxylic acid (1705 cm−1), and a trisubstituted double bond . The 1H NMR spectrum , 1.03 , 1.73 , and 0.88 , respectively. Another tertiary (Me-30) and a secondary (Me-28) methyl group appeared as overlap signals at δ 0.97 to 0.99. These methyl groups were further confirmed through 13C NMR spectral data, displaying the signals at δ 15.1 (Me-19), 16.1 (Me-29), 16.6 (Me-28), 16.7 (Me-21), 19.5 (Me-27), and 28.6 (Me-30). The olefinic proton (H-7) appeared at δ 5.25 in the form of a multiplet, whereas the H-3 proton  appeared as a multiplet at δ 3.16. The terminal methylene protons were observed at δ 4.62 and 4.73 . These observations were indicative of the terpenoid skeleton in the molecule.The prominent fragments in the EI-MS  at m/z 1spectrum  of 1 ind13C NMR data for compound 1 (7-terpenoid skeleton + (12), 438 [M+ − H2O] (3), 412 [M+ − CO2] (2), 286 (6), 248 (100), 203 (47), 133 (44), 83 (16); (+)FAB-MS: m/z 457 [M + H]+; (−)FAB-MS: m/z 455 [M − H]−; HRESI-MS: m/z 455.3513 [M − H]− .Colorless powder (5.5 mg); Saccharomyces cerevisiae) inhibition assay was performed as described in the method of Oki et al. [p-nitrophenyl α-d-glucopyranoside  as a substrate, 2.0 units/mL α-glucosidase enzyme and sodium phosphate buffer  was prepared with or without sample. The reaction initiated by incubation at 37 °C for 30 min. The rate of hydrolysis in PNP-G by α-glucosidase was recorded on ELISA every 30 s.The α-glucosidase enzyme  and various concentrations of compound (10–100 μg/mL) were incubated at 37 °C for 30 min in 96-well microplate. An indophenol method was used, where the ultimate product (ammonia) was used to monitor urease inhibition activity of the tested compound. The reaction further continued by adding the phenol reagent (1% phenol (w/v) and 0.005% sodium nitroprusside (w/v); 45 μL) and alkali reagent  into the wells. The mixture was incubated at 37 °C for 30 min which was followed by recording the absorbance at 630 nm on ELISA microplate reader . All the enzyme inhibition assays were repeated three times. The inhibition percentage was calculated through: Inhibitory activity (%) = 100 − (ODtest well/ODcontrol) × 100. Thiourea and acrabose were used as the standard inhibitor for urease and α-glucosidase enzymes, respectively, whereas a blank was used as a negative control. The same concentrations of compound 1 and the standard were used to compare the activity.Besides that, the inhibition potential of the isolated compound for another important enzyme urease was undertaken according to the method of Golbabaei et al. . In briet/Ao) × 100; where At and Ao are the absorbances for compound and control. The experiment was repeated three times.In addition to enzyme inhibition, the isolated compound was also analyzed for inhibiting the level of lipid peroxidation. This was performed by a modified method of thiobarbituric acid reactive substances (TBARS) . Using ip < 0.05) of concentration vs. activity using GraphPad Prism v6.01 .A two-way ANOVA analysis of the mean values of three replicates was also performed to understand the significance (P. boergesenii was studied comprehensively to investigate the presence of biologically active secondary metabolites. Subsequent to chromatographic extraction and spectroscopic identification, compound 1 was evaluated for potential enzyme inhibition studies. Results indicated that compound 1 possesses low to moderate urease, anti-lipid peroxidation and α-glucosidase inhibition activity, respectively, as compared to the known standards. These findings suggest that seaweed species can be considered as a potential resource for novel bioactive secondary metabolites and future therapeutic agents which cannot be synthesized easily but possess high activity against human ailments.The search for new sources of natural products is one of the important steps in the drug discovery process. In the current study, the macroalga


The ecto-5’-nucleotidase CD73 plays an important role in the production of immune-suppressive adenosine in tumor micro-environment, and has become a validated drug target in oncology. Indeed, the anticancer immune response involves extracellular ATP to block cell proliferation through T-cell activation. However, in the tumor micro-environment, two extracellular membrane-bound enzymes (CD39 and CD73) are overexpressed and hydrolyze efficiently ATP into AMP then further into immune-suppressive adenosine. To circumvent the impact of CD73-generated adenosine, we applied an original bioinformatics approach to identify new allosteric inhibitors targeting the dimerization interface of CD73, which should impair the large dynamic motions required for its enzymatic function. Several hit compounds issued from virtual screening campaigns showed a potent inhibition of recombinant CD73 with inhibition constants in the low micromolar range and exhibited a non-competitive inhibition mode. The structure-activity relationships studies indicated that several amino acid residues  located at the dimerization interface are involved in the tight binding of hit compounds and likely contributed for their inhibitory activity. Overall, the gathered information will guide the upcoming lead optimization phase that may lead to potent and selective CD73 inhibitors, able to restore the anticancer immune response. Nucleotidases play a central role in maintaining the nucleotide pool homeostasis and the only extracellular member of this family, CD73, has become an attractive target in oncology because of its high expression level on immune and cancer cells. In the tumor microenvironment, CD73-generated adenosine prevents the pro-inflammatory response and is considered as a potent immune suppressor. The current study aimed at developing new CD73 inhibitors by targeting an allosteric binding site in order to block the enzyme dynamics and therefore its enzymatic function. Most of the existing inhibitors have been elaborated on the basis of the substrate skeleton and act as competitive inhibitors. Here, four non-competitive compounds are presented with an inhibition constant in the low micromolar range. This study confirms the existence of an allosteric binding site located at the dimerization interface allowing modulation of the enzyme activity by small molecules, similarly to a previously described monoclonal antibody. The immune response constitutes a major barrier for preventing cancer progression through the activation of T cells and subsequent release of pro-inflammatory cytokines. This process is initiated and tightly regulated by extracellular ATP which impacts a large variety of cells  through the binding to P2X and P2Y receptors, inducing persistent inflammation and regulatory cell inhibition –3. In hein vivo development of carcinoma /NHA, where NHA is the number of heavy atoms excluding hydrogens and expressed in kcal.mol-1.HA-1) [Ki−cLog P, where pKi = -log(Ki)), BEI or binding efficiency index (BEI = pKi / molecular weight in kDa), SEI or surface-binding efficiency index (SEI = (pKi) / (Polar surface area /100 Å) [Identification and characterization of druggable cavities were achieved with Fpocket or MDpocket software . Selectiosa.php)  and by cosa.php)  by usingel 2.4.1 . DespiteLC-1.1.2  of the ALC-1.1.2 . A confiLC-1.1.2 ) in ordeLC-1.1.2  and stru-1.HA-1) ,68, LLE  /100 Å) .NT5E gene encoding for the human soluble sCD73 protein (residues 27–549) was kindly provided by Prof. N. Scrutton [TM vector 1 (ThermoFisher Scientific) after PCR amplification to include EcoRI/NotI restriction sites. Protein was expressed in Sf9 insect cells (Life Technologies) using the pFastBac baculovirus system (ThermoFisher Scientific) according to the manufacturer’s instructions. Insect cells were grown in suspension with stirring at 110 rpm in EX-CELL 420 medium at 28°C (Sigma) up to a density of 4×106cells per mL and then infected with baculovirus encoding CD73. The cellular supernatant was harvested by centrifugation  48 h post-infection, filtered (0.22 μm), supplemented with protease inhibitors  and concentrated on crossflow cassette (Vivaflow 200 Sartorius). The concentrate was centrifuged  and purified on HisTrap Excel column connected to a FPLC Äkta purifier system . The enzyme purity, size and activity were assessed by SDS-PAGE, Western blot and steady-state kinetics with various AMP concentrations. An extinction coefficient of 56,310 l.mol-1.cm-1 was used for determining protein concentration at 280 nm.The plasmid with Scrutton . This coScrutton . The inswww.molport.com) gathering all compounds from various suppliers. The purity and structural integrity of the purchased chemical compounds have been evaluated by NMR and mass spectroscopy  using a Partisphere 5-SAX column (AIT France) and 10 mM ammonium phosphate buffer pH 5.5 as mobile phase. For non-water soluble compounds, DMSO was used and the final percentage did not exceed 0.5% in order to preserve the full enzyme activity (enzyme tolerance for DMSO was determined up to 2%). The commercial human CD73 enzyme produced in eukaryotic cells (Interchim) was used to compare the kinetics parameters of both batches and to confirm the inhibition promoted by hit compounds.The 33 hit compounds were purchased from MolPort compound order service (troscopy . Adenosikcat), Michaelis (KM) and inhibition (Ki) constants. Recombinant enzyme (2.5 nM), substrate  were mixed in a thermostated beaker at 37°C in the presence or in the absence of inhibitors and reaction was stopped every 5 s by acid quenching before HPLC analysis (as mentioned above). Quantification of adenosine and AMP was achieved by integrating peaks  and raw data were analyzed using Grafit 7 (Erithacus software) and fitted with four different equations describing either a competitive, uncompetitive, non-competitive or mixed inhibition mode. The best model  fitting the experimental data was considered as the inhibition mode and used for determining Ki. All experiments were carried out using three different inhibitor concentrations and Lineweaver-Burk plots were drawn to illustrate the inhibition modes.For the most interesting compounds, the inhibition mode was determined by steady state kinetic assays to obtain apparent catalytic  on the purified recombinant enzyme (means +/- SD of three independent experiments) and inhibition constants (Ki) and mode for most active compounds (NC for non-competitive) are indicated. In addition to clog P, different metrics for ligand efficiency are included: LE, LLE, BEI and SEI Click here for additional data file.S1 MovieLarge collective motions observed during the dynamics of CD73 from the open to the closed conformation. The volume changes observed for targeted cavity are shown in mesh representation.(MPG)Click here for additional data file.S1 FigA) Root mean square deviation (RMSD in Å) of backbone atoms from C-domains between conformers issued from targeted molecular dynamics simulations and crystal structures (C-domains were defined by residues 337–549). B) Overlay of crystal structures  and conformers . All structures were aligned onto both C-domains of 4H2G. C) Zoom-in view of the superimposed C-domains from all modeled and x-ray structures (the targeted cavity at the dimerization site is depicted in grey mesh).(TIF)Click here for additional data file.S2 FigStructure quality assessment using ProSA II Z-score calculation .(TIF)Click here for additional data file.S3 FigDiagrams were computed using the Rampage program . Percent(TIF)Click here for additional data file.S4 FigComparison of the docking poses obtained with the five conformers for the most active compounds: (A) RR3, (B) RR4, (C) RR6, (D) RR9, (E) RR11, (F) RR16, (G) RR18 and (H) RR20. The binding pose of each compound is shown in stick representation with a color code according to the conformer used for docking .(PDF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.


Delphinapterus leucas) in conjunction with multi-decadal patterns of sea-ice in the Pacific Arctic to investigate the influence of sea-ice on spring migration and summer residency patterns. Substantial variations in sea-ice conditions were detected across seasons, years and sub-regions, revealing ice–ocean dynamics more complex than Arctic-wide trends suggest. This variation contrasted with a highly consistent pattern of migration and residency by several populations, indicating that belugas can accommodate widely varying sea-ice conditions to perpetuate philopatry to coastal migration destinations. However, a number of anomalous migration and residency events were detected and coincided with anomalous ice years, and in one case with an increase in killer whale (Orcinus orca) sightings and reported predation on beluga whales. The behavioural shifts were likely driven by changing sea-ice and associated changes in resource dispersion and predation risk. Continued reductions in sea-ice may result in increased predation at key aggregation areas and shifts in beluga whale behaviour with implications for population viability, ecosystem structure and the subsistence cultures that rely on them.There is increasing concern over how Arctic fauna will adapt to climate related changes in sea-ice. We used long-term sighting and genetic data on beluga whales ( How such declines reverberate through marine ecosystems is largely unknown . In uppeBeluga whales predictably return to specific coastal locations each spring and summer . GeneticWe used genetic data to investigate the population of origin of whales returning to four traditional coastal sites in the Alaskan and Canadian Arctic between 1988 and 2007. We compiled detailed beluga sighting and harvest data for the same period to assess inter-annual variation in timing of return. Finally, we analysed sea-ice data in the Bering, Chukchi and Beaufort seas to determine seasonal and regional patterns of sea-ice from 1979 to 2014. We addressed two specific questions: (i) how does the pattern of annual return to a particular coastal area correlate with inter-annual variation in sea-ice and (ii) do anomalies in beluga migration behaviour correlate with sea-ice anomalies?2.Tissue samples from 978 beluga whales were collected over a 30-year period from subsistence harvests and from live whales via biopsy. Total DNA was extracted from each sample and screened for variation within 410 bp of the mitochondrial genome and for polymorphism within eight hypervariable microsatellite markers, according to previously published methods . Populath) were based on sightings of whales by native hunters and field biologists, from aerial surveys and from the timing of subsistence harvests  data  for the 3.h) near shore waters each summer. For the years 1988–2007, beluga presence based on sightings ranged from as early as 20 June (1997) to as late as 16 July  (a). Mean June SIC for this region varied considerably from 5.2% (1997) to 83.7% (2006) and was weakly correlated with the timing of whale sightings near shore . A consistent genetic profile was found for both mtDNA (figure 2b) and nDNA (figure 2e) for beluga whales occurring off Kasegaluk Lagoon in June and July, indicating that the same, distinct population returned at roughly the same time each year over the course of the study despite high inter-annual variation in sea-ice. Similar patterns were found for the Mackenzie Delta .Beluga whales occur in the eastern Chukchi Sea  a. Mean Jh) each summer with a peak in mid-June. The genetic signature across years denoted a distinct Kotzebue population (c). After 1983, the occurrence of whales in Kotzebue Sound decreased dramatically. Two exceptions to this occurred in 1996 and 2007 when several hundred whales entered the sound. The genetic profile of these whales was distinct from the earlier period (c). The 1996 event occurred within the same timeframe (10–28 June) as pre-1983 occurrences. The 2007 event primarily occurred five weeks later (23–28 July).Prior to 1983, beluga whales returned to Kotzebue Sound in the southeastern Chukchi Sea map, h each supulation c. After r period c. The 19d) except for 1996 when whales from another population entered the northern part of the Sound (Norton Bay) in May. The microsatellite pattern was less distinct as Norton Sound does not form a distinct nDNA cluster (e).In Norton Sound in the eastern Bering Sea a consistent mtDNA profile was observed in most years d except  cluster e.b–e). Both sexes occurred in all three events although the Kotzebue Sound 2007 event comprised males primarily (more than 90%), differing significantly from a sex ratio of near parity for most other years sampled (p = 0.018).The genetic analysis determined that all three atypical events involved whales most likely from the eastern Beaufort population which winters in the western Bering, migrates through the Chukchi and summers in the Beaufort Sea b–e. Bothp < 0.05) and there are more negative anomaly years in the latter half of the record.The springtime sea-ice regime of the northern Bering, eastern Chukchi and southern Beaufort seas is characterized by a northward ice retreat from regions 1 to 7, but there is considerable variability both inter-annually and between regions . In liner = 0.66, p = 0.05, χ2p = 0.047; region 1—Kotzebue: r = 0.65, p < 0.01, χ2p = 0.016; region 2—Norton: r = 0.66, p = 0.05, χ2p = 0.047).Among the years identified as individually anomalous for each region certain years stand out as being anomalous throughout the study area. For example, 1983 was a positive anomaly in all regions except region 2, while 2003 was a negative anomaly in all regions except region 7. 1996 was a negative anomaly during April and May in regions 1–4, corresponding to an early break-up in Norton and Kotzebue sounds; whereas 2007 was a negative anomaly from May to July in regions 5–7. Ice conditions in Kasegaluk Lagoon (region 5) show especially strong inter-annual variability, with 26 out of 36 years being either positive or negative anomalies. A significant association was found between the atypical whale migration events recorded in Norton and Kotzebue sounds and negative ice anomalies in the northern Bering Sea  in the northern Bering Sea and in summer (June–July) in the Chukchi and Beaufort seas could be key. Early break-up should facilitate rather than impede migration of Beaufort belugas to their traditional summering area in the Mackenzie Delta, and indeed no anomalies in the period of occurrence of whales in the delta were detected for 1996 or 2007 , which mUnusual sea-ice patterns may have had a more indirect effect where beluga whales altered their behaviour to pursue associated shifts in prey or to avoid changing predation risk. Recent warming is causing changes in fish communities in the Arctic . As yet,It is possible that other unknown proximate factors, including anthropogenic activities, could have contributed to the altered whale migration patterns. Here we proffer an ecosystem-level, basin-wide perspective of likely ultimate and proximate causes. Our findings reveal a complex relationship between a UTL species and the Arctic cryosphere, where responses to changing sea-ice are influenced by the changing physical challenges and by biological dictates. Beluga whales exhibit great resolve in reaching critical stationary resources such as coastal moulting and breeding sites. However, changes in prey availability and predation pressure will require an adjustment in movement and habitat use patterns. Decreasing sea-ice will likely increase predation risk as temperate apex predators including killer whales have easier and longer access to the Arctic. Behavioural and ecological responses of beluga whales to all these changes may alter the pattern of return to traditional coastal sites, with possible cascading effects throughout entire marine ecosystems and the subsistence communities that rely on them.


Both theoretical and experimental evidence indicate that synaptic excitation and inhibition in the cerebral cortex are well-balanced during the resting state and sensory processing. Here, we briefly summarize the evidence for how neural circuits are adjusted to achieve this balance. Then, we discuss how such excitatory and inhibitory balance shapes stimulus representation and information propagation, two basic functions of neural coding. We also point out the benefit of adopting such a balance during neural coding. We conclude that excitatory and inhibitory balance may be a fundamental mechanism underlying efficient coding. Neural information coding is one of the central topics in neuroscience. The brain utilizes some features of action potential sequences (spike trains) to encode sensory and cognitive information. The algorithm operating within those features is called neural code. Half a century ago, Perkel and Bullock noted that a potential neural code must serve at least four functions: stimulus representation, interpretation, transformation, and transmission  plots. In cortical neurons, the reversal potential of EPSCs is around 0 mV, whereas for GABAA-receptor-mediated IPSCs, it is around −75 mV. The average synaptic currents reverse at around −35 mV  for cortical neurons. The electrode contained 2 M caesium acetate and 50 mM QX-314 to minimize the contribution of K and Na currents. The recorded synaptic current is defined asGlobal balance is quantified by using global measures of excitatory and inhibitory synaptic currents, including measuring spontaneous or ongoing excitatory and inhibitory postsynaptic currents (mEPSC and mIPSC) and the field potential, which is considered a rough signature of the relative timing and magnitude of excitation and inhibition Froemke, . In factgsyn is composed of the sum of the excitatory conductance (ge) and inhibitory conductance (gi) as follows:where the total measured synaptic conductance Ie = ge(V − Ee) and inhibitory synaptic current Ii = gi(V − Ei),In addition, the total synaptic current is the sum of the excitatory synaptic current Ee = 0 mV is the reversal potential for excitatory synaptic current, and Ei = −75 mV is the reversal potential for inhibition for cortical neurons. To solve the above equations, we havewhere ge and gi) , researchers have tried to simultaneously record the time series of both the excitatory and inhibitory currents and then obtain the correlation between them. Since adjacent neurons in the cortex generally receive strongly correlated synaptic inputs, researchers can record both excitatory and inhibitory currents separately and simultaneously, each in a single neuron in a pair of neighboring cells, and the correlation between the excitatory and inhibitory currents onto a single cell can be inferred from the correlation between the time series from the two cells , Dehghani and colleagues found that excitatory and inhibitory ensembles are well-balanced and co-fluctuate instantaneously in all states of the wake-sleep cycle  at different temporal scales  neurons in the network should be connected sparsely  and randomly; (2) the strength of the inhibitory connections should be high enough to balance network in which feedback excitation and inhibition could be canceled  to achieve a more fine-scale E/I balance  and activity (high level of energy consumed); however, an E/I ratio that is too low leads to suppressed activity with low information content. Therefore, the tradeoff between these two aspects requires the balance of the excitatory and inhibitory currents. On the other hand, based on recent theoretical studies, the E/I balance also plays a vital role in determining the fidelity of spiking activity propagation and gating of the multiple signal pathways. More experimental investigations are expected to test theoretical hypotheses and predictions in the near future. As discussed above, the implementations and functions of the two types of E/I balance, global balance and fine-scale balance, are different from one another. More studies are also needed to demonstrate the exact differences between the effects of these two types of balance on neural coding.Here, we mainly discussed the roles of E/I balance in neural coding, while many additional studies have focused on the role of E/I balance in other brain functions, e.g., the formation of memories (Vogels et al., YY and SZ: designed research, wrote the paper, and performed research. All authors reviewed the manuscript.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.


This is a morphometric study to examine the proximity of the T12 vertebra to the thoracic aorta. Our aim was to define the appropriate length of the pedicle screw to be used in the 12Methods: Randomly selected cases from the same ethnic group in a specific age group were examined in terms of the length from the anterior vertebral body and the screw entry point of the T12 vertebra to the thoracic aorta. In light of these data, a statistical analysis was made for the selection of the most appropriate screw length.Results: A statistically significant difference was detected in the distance from the T12 left screw entry point to the aorta between males and females (p=0.001). No statistically significant correlation was found between age and the distance between the left screw entry point and the aorta (p=0.105). Also, no statistically significant difference was detected between the T12 vertebral body-aorta distance in males and in females (p=0.212). The relationship between the shortest aorta-vertebral body distance and age was not statistically significant (p=0.7). Similarly, there was no statistically significant difference between the left screw entry point-aorta distance and the aorta-vertebral body shortest distance (p=0.731).Conclusions: Significant differences were observed between males and females in terms of the distance between the T12 vertebra left screw entry point and the thoracic aorta (p=0.001). Thus, we can assert the need for the preoperative evaluation of patients with computed tomography in selecting the appropriate screw length and avoiding complications. The spinal fixation method based on transpedicular screws was first described by Roy-Camille et al. . Since 1The thoracolumbar region is the area of transfer of biomechanical movement in the spine, therefore, it is the area where pathologies are seen most often. These pathologies include trauma, degenerative diseases, osteoporotic compression fractures, instabilities, neoplastic diseases, and infections . The insThe size of the screw should be determined by the compatibility of the entry point with the anatomical guides, such as the proximity of the thoracic aorta to the vertebral body and particularly at the level of the T12 vertebra. This is extremely important in preventing mortal complications, such as aorta injury.Our aim in this study was to determine the optimum screw length by examining the T12 vertebra pedicle morphology with computed tomography (CT) and, thereby, to preoperatively calculate its depth.The study was designed as a prospective, randomized, and morphometric study to examine the proximity of the T12 vertebra to the thoracic aorta using a CT scan. The CT sections of the T12 vertebrae of patients aged between 25 and 40 years who presented at the Emergency Department or clinic, for whom a thoracic CT was requested for any possible pathology, were examined. The slices were obtained from only an Anatolian population. Patients with spinal deformity, tumor, fracture, infection, congenital anomaly, or history of surgery in the lumbar region were excluded. The study was completed with 30 males and 30 females who met the inclusion criteria and had a mean age of 31.9±3.9 (range: 25 to 40) years.First, axial images at 3-mm thickness were obtained. Following reconstruction in the bone window, the measurements of three images where the pedicle thickness was the greatest were taken, and the thickest measurement of the pedicle was selected from these images. Then, the distance between the screw entry point and the thoracic aorta and the distance between the anterior vertebral body and the thoracic aorta were digitally measured on this image with a magnetic resonance imaging (MRI) scanner . Axial slices of 3-mm thickness from the thoracic vertebra were taken using a Philips MX 8000 CT unit  and examined.Tomography images of the T12 vertebra were taken from the pedicle isthmus at 3-mm thickness (mid-pedicle slice) in the axial plane. The following parameters were examined on the CT slices: the distance between the screw entry point and the thoracic aorta and the distance between the anterior vertebral body and the thoracic aorta. For the length between the left screw entry point and the aorta, the measurement of the closest point of the T12 left screw entry point to the thoracic aorta was made . The measurement results were expressed as mean±standard deviation (SD) and range of values. Statistical analyses were performed with the student’s t-test and Pearson’s correlation coefficient and using the PASW Statistics v.18 software .A statistically significant difference was detected in the distance from the T12 left screw entry point to the aorta between males and females. The distance in the males was 47.12±3.38 (range: 40.01 to 54.00) mm and in the females, it was 43.70±3.00 (range: 37.99 to 49.26) mm (p=0.001)  years) and the distance between the left screw entry point and the aorta (45.41±3.61 (range: 37.99 to 54.00) mm) (p=0.105)  mm) and in females (1.94±0.52 (range: 1.02 to 3.56) mm) (p=0.212)  mm) and age (31.93±3.91 years (range: 25 to 40) years) was not statistically significant (p=0.7)  mm) and the aorta-vertebral body shortest distance (1.85±0.54 (range: 0.78 to 3.56) mm) (p=0.731) (Table Pedicle screw stabilization in the thoracic region is a widely used method applied in the treatment of several pathologies, such as degenerative diseases, deformities, tumor, and traumatic instability. Knowledge of the vertebral body and pedicle morphology provides great advantages to the surgeon in the application of the pedicle screw during surgery.During spinal instrumentation, the surgeon must decide on the screw length and its path to obtain the strongest fixation without damaging the neurological and vascular structures adjacent to the pedicle and vertebral body. The ideal techniques for screw advancement are obvious, however, the surgeon generally makes the decision of appropriate thickness and length of screw based on previous experience. When compared to the lumbar vertebra, the placement of the thoracic pedicle screw is more difficult due to its small size, varying entry points, and proximity to neurovascular structures and the spinal cord . Any kinThe ideal pedicle screw should have the maximum diameter and length that will not impinge on the pedicle cortical layer and the vertebral body . RecentlComputed tomography is the easiest method that gives the most accurate results in the evaluation of vertebral morphometry. Therefore, the majority of studies examining screw pedicle compatibility have used CT scanning, as in the current study -19. The This study shows the importance of the length and safety of T12 pedicle screws, evaluates the average values of pedicle screw length, and increases awareness of it. But, the length of the pedicle screws should be measured in the patient’s CT images or plain X-ray before surgery and determined based on the morphology of each patient. This is the major limitation of our study.In conclusion, regardless of the technique applied, pedicle-screw-based instrumentation is a strong fixation method for the thoracolumbar spine. When placing the pedicle screw in the T12 vertebra, the length of the screw is just as important as the screw entry point and orientation. Therefore, preoperative radiological planning and distance measurement will reduce iatrogenic vascular injuries.


Drug development involves pharmacometric experiments in animals. Such experiments should limit animal pain and stress. Conventional murine models of atopic dermatitis (AD) used in drug development are generated by weekly painting of hapten on dorsal skin for 5 weeks. The present study aimed to develop a protocol that involves less animal distress. The experiments focused on serum total IgE levels, which are a marker of AD. The conventional protocol induced ever rising IgE levels. Experiments with extended intervals between sensitizations showed that IgE peaked ~5 days after the second sensitization, after which it returned to the control level within 12–19 days. An additional third sensitization on day 28 further increased the serum IgE level. In the 4–5 days after the second sensitization, the dorsal skin exhibited typical AD-like lesions with edema, scabs, epithelial-cell hypertrophy, marked mast-cell and lymphocyte infiltration of dermis, and increased IL-4, IL-6, IL-10, IL-1β, IL-17A, IFN-γ and TNF-α expression. Thus, two 2,4-dinitrofluorobenzene sensitizations yield a murine AD model in less than 20 days. This study shows that animal model protocols used in drug development can be fine-tuned so that they remain effective yet cause animals less stress and pain. During drug discovery, it is necessary to use animals for various experiments, including for evaluating the efficacy and safety of the candidate drug. However, we should minimize the number of animals used in such experiments and reduce their stress and pain as much as possible. This can best be achieved using simple standard methods that generate reliable results while imposing the smallest possible burden on the animals. The development of such methods is thus an important research priority.2. AD is a serious skin disease that is characterized clinically by eczema and itching4. Its key immunological feature is high levels of serum total IgE6. These high IgE levels are induced by some leukocytes; including T helper type 2 (Th2) cells that are specific for allergens such as certain foods and insect proteins. When these cells in the circulation encounter these allergens, they produce inflammatory cytokines such as interleukin (IL)-4. In turn, Th2 cells stimulate B cells to produce IgE in spleen10, after they have been presented to the antigen by leukocytes. The serum IgE then activates a variety of immune cells that bear IgE receptors on their cell surface, including mast cells, basophils, and eosinophils. The binding of antigen by receptor-bound IgE induces the cells to secrete inflammatory cytokines, chemokines, histamine, and leukotriene. These molecules in turn promote other inflammatory reactions that eventually lead to further exacerbation of the clinical manifestations of AD11. As a result of this immune cascade, serum total IgE concentrations generally correlate positively with AD symptoms12, although further research on this relationship is needed.There are many patients with atopic dermatitis (AD) all over the world13. In particular, AD phenotypes can be reproducibly induced by painting haptens, including 2,4-dinitrofluorobenzene (DNFB) and 2,4-dinitrochlorobenzene (DNCB) on the dorsal skin and ears of mice. More than 130 papers have described a protocol that generates a murine model of AD with DNFB or DNCB painting .Current murine models of AD are generated by repeated painting of DNFB or DNCB on the dorsal skin and ears  challenge following OVA pulsed basophil injection through the tail vein and subcutaneous immunization with papain, a protease allergen, respectively. These immunizations induced Th2 cell differentiation, which was promoted by IL-4 stimulation, and led to atopic dermatitis in lesional skin. Perrigoue, J. G. et al. showed that Th2 cytokine-dependent resistin-like molecule-β (RELMβ) expression was higher on days 12–18 after Trichuris. muris infection27. The presence of Th2 cells in peripheral tissue is a clinical feature of atopic dermatitis.Protocols 2–4 were inspired by previously reported allergen exposure experiments. Talay, O. N. brasiliensis and T. muris, suggested that it is possible that, if sufficient time elapses after the first sensitization, a second sensitization may efficiently generate AD-like reactions. Third, the Protocol 1 findings and allergen immunization experiments indicated that the first and second sensitizations should be separated by more than 7 days. This led to experiments to determine the optimal duration between the first and second sensitizations that allows IgE levels to peak and then return to the control level before the third sensitization is administered.These observations, together with the Protocol 1 findings, indicate that, when constructing a protocol for generating an animal model of AD that involves fewer painting sessions than conventional methods, several aspects must be considered. First, it is clear that one DNFB painting session is not sufficient to induce AD-like reactions. Second, infection with helminth parasites, including Thus, an experiment with Protocols 2, 3, and 4 was performed. Six mice underwent weekly painting sessions for 3 weeks, as in Protocol 1. They were then painted for the fourth time 9 days later (day 23). The mice were then divided into three groups of two. The mice in Protocols 2, 3, and 4, respectively, underwent a fifth painting session 12, 19, or 26 days after the fourth session  Fig. .Figure 27 days after the fourth session and then started to wane. The fifth session was then performed: it rapidly induced a second peak 5–9 days later. The IgE levels then started to wane , the IgE levels peaked ane Fig. .7 days after the fourth session and then waned to a steady state. The fifth sensitization was then administered: the IgE levels peaked 12 days later. After the latter peak, the IgE levels waned slightly before spontaneously rising again. As a result, the highest IgE levels in the Protocol 3 mice were on the last day of observation (day 93) , the IgE levels peaked 93) Fig. .3–7 days after the fourth session and waned to a steady state. The fifth sensitization was then administered, and the IgE levels peaked 5 days later, followed by slight waning , the IgE levels peaked ing Fig. .on average ~5 days after DNFB sensitization.These results indicate that the most optimal interval between painting sessions is >12 days and <19 days. In addition, it seems that IgE concentrations start rising P = 3.4 × 10−6, day 19 and P = 0.04, day 26).These results led us to Protocol 5, which involved three DNFB sensitizations at 14 day intervals Fig. . In thrend DNFB painting  staining of the DNFB-painted skin revealed initiation of epidermal hypertrophy on day 1 and clear inflammation, characterized by exacerbated intracellular oedema of the epidermis, on day 2. During days 2–3, the stratum corneum thickened and partially disrupted Fig. . The epind DNFB painting  and 24 h after the second painting sessions were subjected to quantitative PCR and the expression of inflammatory cytokines and AD-related genes was measured. As shown in Fig. These results indicate strongly that two DNFB sensitizations 14 days apart are sufficient to induce AD signs and symptoms. Thus, this protocol, which uses two sensitizations rather than the five that are conventionally used, reliably and effectively generates a murine model of AD. Not only does this model place a lower burden on the mice in terms of pain and stress than conventional methods; it is simpler and the mouse model is generated faster.33, and decreases the skin level of the AD-related filaggrin gene34  yields continuously rising IgE concentrations35 , the IgE levels had reached steady state levels. The second infection induced a second IgE peak 10 days later24. The fact that DNFB-induced IgE levels take ~5 days to peak may reflect the splenic response to hapten sensitization: it takes several days for B cells to start producing IgE. Indeed, in animals immunized with OVA, it takes at least 5 days for IgE production to become detectable and peak IgE levels are only detected ~10 days after the immunization39. These and our observations indicate that the most appropriate interval between hapten sensitizations is 14 days, as this gives the B cells enough time to become activated, produce IgE, and then become inactive. We are currently testing this notion with cell staining and quantitative PCR assays of the B cells in the spleen. Our approach might accelerate investigations into how and where IgE production is regulated as well as into ways to reduce stress and pain in mice.Many studies show that weekly sensitization with hapten, insect extracts, or foods over a period of more than 1 month  and water were freely available. All experiments were conducted in accordance with the 2006 Japanese Association for Laboratory Animal Science guidelines for the care and use of experimental animals. The experimental protocols were also approved by the Animal Experiment Committee of Osaka University .i.e., five painting sessions; Fig. i.e., Protocols 2–4 also involved five painting sessions; Fig. After anesthetization, the dorsal skin of the BALB/c mice was shaved and tape stripped to disrupt the skin barrier. DNFB  was then applied on a 1 × 1 cm square of the dorsal skin of each mouse according to one of five protocols. DNFB or vehicle patch was removed 2 h later. Protocol 1 is a conventional regimen where the dorsal skin was painted once weekly for 5 weeks (About 100 µl of blood were collected from the legs using Goldenrod ANIMAL LANCET (Bio Research Center) and transferred into BD Microtainer Tubes . The blood was allowed to stand for 15–30 min at room temperature before centrifugation at 7,900 g for 2 min. Sera were obtained and stored at −80 °C prior to analysis. The total IgE antibody levels in the sera were quantified by ELISA (BD Biosciences).Scratching behavior of mice was recorded for 15 min. The record was performed 2 h after DNFB sensitization in the observation chamber using a digital video camera (CASIO EXILIM). Mice scratched themselves too many times for 1 s to count them. Therefore, multiple scratching behavior around the rostral shaved area using hind paws was counted as one event. The movie was played back at the slower speed using Kinovea.In the Protocol 5-treated mice, dorsal skin tissue was removed before and 1–5 days after the second painting session. The excised skin specimens were fixed in 10% neutral formalin  or 4% paraformaldehyde  and embedded in paraffin . Sections (6 µm thick) were prepared and stained with hematoxylin and eosin  or toluidine blue (Sigma). Toluidine blue specifically stains mast cells. The definition of lymphocyte is described in the legend of Supplementary Fig. 2 and one HPF  equipped with a CCD camera . The images were displayed using FLUORESCENCE DIGTAL MICROSCOPE CAMARA CONTROLLER VB-7000 (KEYENCE). All images were analyzed using ImageJ. Epidermal hypertrophy of H&E stained images was calculated by using a scale bar  and Direct-zol RNA MiniPrep (Zymo Research) according to the manufacturers’ instructions. Subsequently, 50–100 ng of RNA was reverse-transcribed to first strand cDNA using Superscript III Reverse Transcriptase (Invitrogen). RT-qPCR was then performed by placing 5 μL of a 1:~20 dilution of the cDNA into a well that contained 15 μL of reaction mix: the reaction mix included 0.04 μL of 100 μM primer sets, 0.4 μL of Rox High reagent, and 10 μL of KAPA SYBR Fast qPCR Kit (Nippon Genetics). Reactions were run in at least duplicate on a StepOnePlus real-time PCR machine (Life Technologies) with the following protocol: 95 °C (30 s) followed by 40 cycles of 95 °C (5 s) and 60 °C (30 s). Data were normalized for the amount of Spt5 using the ΔΔCt method and a sample from the vehicle group was used as a calibrator. Primer sequences are listed on supplementary Table https://www.R-project.org/). Statistically significant differences were determined using one-way analysis of variance (ANOVA) with Tukey honestly significant difference (HSD) post hoc analysis. P values of less than 0.05 were considered significant and statistical significances at P < 0.05 and <0.01 are indicated by respective symbols in the figures.Statistical comparisons among groups were made by using R software (All data generated or analyzed during this study are included in this published article and its Supplementary Information files.Supplementary Information