N = 231) completed five questionnaires. The results showed that social anxiety was not necessarily associated with all aspects of mental imagery. As suggested by theories, social anxiety was not associated with a preference for verbal processing. However, social anxiety was positively correlated with the visual imagery scale, especially the object imagery scale, which concerns the ability to construct pictorial images of individual objects. Further, it was negatively correlated with the spatial imagery scale, which concerns the ability to process information about spatial relations between objects. Although object imagery and spatial imagery positively and negatively predicted the degree of social anxiety, respectively, these effects were attenuated when socially anxious individuals had high effortful control. Specifically, in individuals with high effortful control, both object and spatial imagery were not associated with social anxiety. Socially anxious individuals might prefer to construct pictorial images of individual objects in natural scenes through object imagery. However, even in individuals who exhibit these features of mental imagery, effortful control could inhibit the increase in social anxiety.According to cognitive theories, verbal processing attenuates emotional processing, whereas visual imagery enhances emotional processing and contributes to the maintenance of social anxiety. Individuals with social anxiety report negative mental images in social situations. However, the general ability of visual mental imagery of neutral scenes in individuals with social anxiety is still unclear. The present study investigated the general ability of non-emotional mental imagery  and the moderating role of effortful control in attenuating social anxiety. The participants ( Cognitive theories suggest that socially anxious individuals generate a negative self-image in social situations, and the negative mental imagery contributes to the maintenance of social anxiety . They imWhile many previous studies have investigated negative mental imagery in individuals with social anxiety, few studies have examined the general ability pertaining to non-emotional mental imagery. It is important to focus on the general ability of neutral mental imagery owing to the following two reasons. First, greater neutral mental imagery ability might be associated with efficient treatment. When assessing mental imagery in social anxiety, A few studies have examined general “non-emotional” imagery use and experience. In Another important aspect of visual mental imagery is the processing subsystem for object and spatial imagery. Based on neuropsychological data, The main purpose of the present study was to clarify the relationships between social anxiety and general non-emotional imagery use and experience  in non-clinical individuals, using self-report questionnaires. According to a previous study , it was The other purpose was to reveal the moderator variables that attenuate social anxiety in individuals with maladaptive mental imagery. The present study focused on effortful control EC; , 2011. ESD = 0.83) years. There were no exclusion criteria in the present study.The participants were 231 undergraduates, of whom 162 were women and 69 were men. All participants provided informed consent and completed a questionnaire in a class for course credit. The age of the participants ranged from 18 to 23 years, with a mean age of 19.62  to 5 (extremely characteristic of me). It assesses the apprehension or distress caused by others’ negative evaluations, which is a core feature of social anxiety . The Japanese version of the scale has high internal consistency (α = 0.92) and test–retest reliability for a 3-month interval  and are asked to rate the clarity and vividness of the images . The VVQ was used to measure the participants’ preference for using imagery  or verbal-logical strategies  when solving problems. r = 0.75 for the object imagery scale and 0.83 for the spatial imagery scale). In the present study, the internal consistencies were adequately high .The VISQ is a self-report questionnaire for measuring object imagery and spatial imagery. The object imagery scale refers to the ability to process visual information about objects or scenes , whereas the spatial imagery scale refers to the ability to process information about spatial relations between objects or their parts . These two scales are related to object and spatial factors in the Object-Spatial Imagery Questionnaire OSIQ; . Each scr = 0.88; The EC scale consists of 35 items on a 7-point Likert-type scale. The EC scale is a part of the Adult Temperament Questionnaire . The EC Table 1. The BFNE was positively correlated with the visualizer scale of the VVQ and the object imagery scale of the VISQ, and was negatively correlated with the spatial imagery scale of the VISQ. The VVIQ and verbalizer scale of the VVQ were not significantly correlated with the BFNE. The EC scale was negatively correlated with the BFNE and was positively correlated with the verbalizer scale of the VVQ. The other mental imagery scales were not significantly correlated with the EC scale.Descriptive statistics and correlations between the BFNE, VVIQ, VVQ, VISQ, and EC scores have been presented in Table 2. The final two-stage model was significant, F = 4.12, p < 0.001, R2 = 0.17, Adj. R2 = 0.13. The object imagery scale of the VISQ was still significantly positively associated with the BFNE, whereas the spatial imagery scale of the VISQ was significantly negatively associated with the BFNE following Step 2 of the analysis. Moreover, the interactions of the VISQ object scale × EC scale and the VISQ spatial scale × EC scale were significant. The results of a simple slope analysis indicated that the VISQ object scale was positively associated with the BFNE when scores on the EC scale were medium  and low , whereas the VISQ object scale was not significantly associated with the BFNE when scores on the EC scale were high . The analysis also indicated that the VISQ spatial scale was negatively associated with the BFNE when scores on the EC scale were medium , and low , whereas the VISQ spatial scale was not significantly associated with the BFNE when scores on the EC scale were high . Multicollinearity was checked by calculating the variance inflation factor (VIF), and all VIF values were less than 3. When gender and age were entered in Step 1 as the covariates, the mental imagery scales and EC in Step 2, and the interactions between mental imagery and EC in Step 3, the results were the same as above.To investigate the interactive effects of the EC scale with the mental imagery scales, hierarchical regression analysis was conducted to predict BFNE scores using the scores on the mental imagery scales and the EC scale. The dependent variable was the BFNE; the independent variables were the VVIQ, visualizer scale of the VVQ, verbalizer scale of the VVQ, object imagery scale of the VISQ, spatial imagery scale of the VISQ; and the moderator variable was the EC scale. First, all independent variables were centered on the grand mean to minimize problems associated with multicollinearity and to scale the variables meaningfully . In StepThe present study investigated the relationship between social anxiety and mental imagery ability, and the interactive effects of EC with mental imagery on social anxiety. A correlation analysis showed that social anxiety was positively associated with a preference for visual mental images and object mental imagery, and it was negatively associated with spatial mental imagery. A hierarchical regression analysis of mental imagery showed that only object mental imagery and spatial mental imagery predicted the degree of social anxiety positively and negatively, respectively. Moreover, scores on the EC scale moderated the effects of these mental imagery scales on social anxiety. High EC could suppress the maladaptive effects of high object mental imagery and low spatial mental imagery on social anxiety.Consistent with the hypothesis, social anxiety was associated with visual imagery, but not with verbal processing. Because verbal processing could attenuate emotional arousal , 2010, vAdditionally, the present findings suggested that not all features of visual imagery were correlated with social anxiety. The vividness of mental images was not associated with social anxiety. The present result was not consistent with those of previous studies, which have shown that anxious individuals had more vivid imagery for neutral or negative situations . There aThe finding indicating a preference for processing visual mental images, especially object mental imagery in social anxiety, was consistent with the hypothesis. Previous studies have shown that high object mental imagery is related to a preference for visualizing scenes when people listen to dialogs . MoreoveAlthough spatial imagery is a subsystem of visual imagery, the present results showed a negative correlation between social anxiety and spatial imagery. Based on cognitive theories , sociallEffortful control is an important factor for protecting against increased social anxiety. Consistent with the findings of previous studies , the preThere are some limitations in the present study. First, the accuracy of participants’ responses is unclear due to the use of self-report scales alone. While socially anxious individuals think that they prefer visual mental images, it is still unclear what they imagine in real situations. Some previous studies used a behavioral measure of imagery in experiments and showed an impaired ability to generate visual mental images in social anxiety . TherefoIn sum, the present study showed that social anxiety was positively related to a preference for visual mental images, especially object imagery. Socially anxious individuals might prefer to construct pictorial images of individual objects in natural scenes, which might increase their anxiety. However, even for individuals who exhibit these features of mental imagery, EC could inhibit the increase in social anxiety. Individuals with low EC and high object imagery or low spatial imagery showed high social anxiety. These results regarding the relationships between mental imagery and social anxiety might be useful to develop efficient mental techniques for treating social anxiety disorder.This study was carried out in accordance with the recommendations of Graduate School of Psychology, Ethics Committee.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.


We explain the anomaly of election results between large cities and rural areas in terms of urban scaling in the 1948–2016 US elections and in the 2016 EU referendum of the UK. The scaling curves are all universal and depend on a single parameter only, and one of the parties always shows superlinear scaling and drives the process, while the sublinear exponent of the other party is merely the consequence of probability conservation. Based on the recently developed model of urban scaling, we give a microscopic model of voter behavior in which we replace diversity characterizing humans in creative aspects with social diversity and tolerance. The model can also predict new political developments such as the fragmentation of the left and the immigration paradox. N as the measure of city size, power law scaling takes the formY can denote material resources such as energy or infrastructure or measures of social activity such as wealth, patents and pollution; Y0 is a normalization constant. The exponent β reflects general dynamic rules at play across the urban system. Similar scale-free, fractal-like behavior has been observed in many human social networks [Formation of cities is the result of socio-economic advantages of concentrating human populations in space outpacing associated costs. Urban agglomeration effects are systematic changes in socio-economic performance, innovation, trade and infrastructure characteristics of all cities as functions of their size. A variety of disciplines including economics –3, geogrnetworks  includinnetworks . Therefonetworks , 18 and networks , and thanetworks –22. In tnetworks , scalingβ > 1 exponents and as a result, larger cities are disproportionally the centers of innovation and wealth. Sublinear scaling β < 1 characterizes material quantities displaying economies of scale associated with infrastructure, where the agglomeration into cities pays off in having to provide fewer roads, shorter cables etc. Thus, material costs related to living in larger cities is disproportionally low. Linear scaling β ≈ 1 is usually associated with individual human needs such as jobs, housing or water consumption [Most urban socioeconomic indicators have superlinear sumption .M of necessary complementary factors must simultaneously be present for an urban phenomenon to occur. For example, to get a patent at least the following six factors should be present: have a technological problem, have a solution, present the idea clearly, apply for a patent, include subsequent corrections from examiners, and satisfy all the legal requirements.Gomez-Lievano, Patterson-Lomba and Hausmann in Ref.  recentlyq ∈ , and it quantifies the complexity of that phenomenon. The fraction of factors that a city provides for an individual is r ∈ . It represents a measure of urban diversity and tends to accumulate logarithmically r = a + b ⋅ log N with the population size, where a and b has been found to be constant across a wide range of urban phenomena. Alternatively, the fraction of factors not present in a city is 1 − r = b ⋅ log N0/N, where log N0 = (1 − a)/b, and N0 ≈ 1.8 ⋅ 1014 is a hypothetical maximal diversity attainable in a city. Given a city with m factors present, the probability that an individual requires any number of the m factors that the city has, but none of the M − m factors that the city does not have is P = (1 − q)M − m ≈ eq(M − m) for q ≪ 1, and the average number of occurrence of the phenomena is Y = N〈P〉N, yielding Y ≈ NeqM(1 − r(N)) = NeqMb log N0/N where we used 〈eqm−〉N ≈ eMr(N)− and averaging goes for cities of population N. Introducing the the scaling exponent β = 1 + Mbq, this scaling curve then takes the universal formN is now the part of population conceivably susceptible to the given urban phenomena.The fraction of factors that an individual does not have and is expected to require from the city in order to be counted into a phenomenon is Scaling laws and universality have been observed in various aspects of the political process and elections –31, thatIn the recent presidential elections in the US it has been noticed that votes for Democrats were disproportionally high in large cities , and in βD ≈ 1.14 and βrem ≈ 1.09, while votes for Republicans and Leave the EU follow sublinear scaling with βR ≈ 0.92 and βlea ≈ 0.91, with high regression coefficients R2 ≥ 0.9 indicating robust urban scaling. While the elections took place in two different political situations, nevertheless they show very similar exponents.First, we analyze data for the votes cast for the two main political parties in urban areas in all post-World War II US presidential elections  and in tβD and of the Republicans βR for the 18 presidential elections in the period 1948–2016. The exponent of the Democrats has an increasing, while the exponent of the Republicans a decreasing historical trend. The Democrat and Republican curves roughly mirror each other in the whole period. The relation of the two exponents becomes apparent when we plot the Republican exponent as a function of the Democrat exponent in In β and the constant Y0 independently from the fits. In Y0 as a function of β. We find a very strong (R2 = 0.96) linear relationα = 12.111 and δ = 11.396. This indicates that the form of the scaling relation is independent of the party and election and has the universal formN is the voter turnout in a city, β is the exponent of the party and log N* = α. The numerical factor eδ − α is equal to 1/2 within numerical error and the parameter N* ≈ 182,000 is the average turnout of a US city of total population 429,000 in 2016. This is about the size of Fort Wayne IN, the 125th Metropolitan Statistical Area of the US.For each election and for each party we can determine the scaling exponent N = N* the parties share the votes equally (YD = YR = N*/2) independent of their exponents βD and βR or of the year of the election and unaffected by historic changes in population. For cities above turnout N* the party with higher β gets the majority of votes, while below this turnout the party with smaller β succeeds in average. While in 2016 already 125 metropolitan areas surpassed the population corresponding to this critical turnout, only 45 did so in 1948.The remarkable property of this scaling relation is that in average at turnout N/2 and by setting N0 = N*/2. There are two discrepancies between our scaling curve (β > 1 (Mq > 0) values only, while in case of elections both superlinear and sublinear exponents arise, and the numerical value of N0 ≈ 1.8 ⋅ 105 is nine orders of magnitude smaller for elections.The observed linear relationship  and the ng curve  and thatYD/N + YR/N = 1 for the sum of the fraction of votes the parties get. Using  could be characterized as comprising of those voters, who don’t accept at least one of those M issues that are necessary for a voter voting for the superlinearly scaling opinion or party.We found that the complexity parameter M values or issues simultaneously. Such values include tolerance and acceptance towards various social groups ranging from women and blacks at the beginning and middle of the 20th century to LGBT communities, immigrants, refugees and various other social minorities recently. In case someone is not able to accept at least one of these, then he or she will probably not vote for the Democrats. That explains why groups of the Republican and the Leave voters look so heterogeneous: they consist of groups that oppose at least one of these liberal values, and that are not held together by a common political agenda otherwise. This also explains the recent steady increase of the Democrat exponent: as more and more M values are introduced, it increases the exponent, making bigger cities having disproportionally more Democrats than smaller ones. This result is in line with the findings of [Here, we can just conjecture based on the agenda of Democrats, that these are liberal values in general, and the Democrat voter typically accepts all of these dings of , who expq as a probability that a voter—left on its own devices—rejects one of the M liberal values, and r(N) is the probability that a city of size N makes a voter tolerant towards those values. Social diversity grows with the city size and voters in cities can face an increasing number of social issues and can develop tolerance towards them. This is in accordance with the immigration paradox in Britain, where voters living near immigrants develop a tolerance, while those who do not are more likely to reject them [r(N)∼log N/N0, but the number of maximal social diversity is reached at N0 ≈ 4 ⋅ 105, which is smaller than the diversity N* ≈ 1.8 ⋅ 1014 observed for the more general type of diversity, characterizing humans in creative aspects. Finally, there is one more consequence of this model: as the number of liberal values M seems to grow continuously, the potential voters who don’t accept one of them also increases, and becomes detrimental for electoral success. This leads to the fragmentation of the political left, since a larger number of smaller parties accepting only a subset of the M values, or even “single-issue” parties can minimize the number of estranged voters and maximize the aggregated votes of all these parties.In this context, we can identify ect them . TherefoWe applied urban scaling theory to the number of votes cast in the Metropolitan and Micropolitan Statistcal Areas in the 1948–2016 presidential elections of the US and the votes cast in the urban areas of the 2016 EU referendum in the UK. We found that out of the two voting options , one always follows a superlinear, while the other a sublinear scaling. Using the historical dataset, we showed that instead of four parameters , the single exponent of the superlinearly scaling party is enough to characterize all processes across the elections and the parties. We derived the other exponent from the superlinear exponent by using the conservation of voting probabilities, and showed that the city turnout distribution determines that this other exponent must be sublinear. We then analyzed the fluctuations around the scaling curve distributions and found that the distribution corresponding to the superlinear exponent is lognormal. We concluded that the two parties play different roles in urban scaling. The party with superlinear exponent drives the process, while the scaling of the party with the sublinear exponent is merely the result of probability conservation. In the context of elections we identified the the elements of the GLPLH model and showed that social tolerance and diversity replaces creative diversity in this context. We pointed to new political consequences of the model. We believe that he model and the calculations could further be extended to metropolitan areas in other countries or to electoral systems with multiple choices.See Sections A-G in S1 FileThe file contains detailed description of the data sources, fitting procedures (power laws and the Kolmogorov-Smirnov test) and calculations.(PDF)Click here for additional data file.


Invasive populations are often associated with low levels of genetic diversity owing to population bottlenecks at the initial stages of invasion. Despite this, the ability of invasive species to adapt rapidly in response to novel environments is well documented. Epigenetic mechanisms have recently been proposed to facilitate the success of invasive species by compensating for reduced levels of genetic variation. Here, we use methylation sensitive-amplification fragment length polymorphism and microsatellite analyses to compare levels of epigenetic and genetic diversity and differentiation across 15 sites in the introduced Australian house sparrow population. We find patterns of epigenetic and genetic differentiation that are consistent with historical descriptions of three distinct, introductions events. However unlike genetic differentiation, epigenetic differentiation was higher among sample sites than among invasion clusters, suggesting that patterns of epigenetic variation are more strongly influenced by local environmental stimuli or sequential founder events than the initial diversity in the introduction population. Interestingly, we fail to detect correlations between pairwise site comparisons of epigenetic and genetic differentiation, suggesting that some of the observed epigenetic variation has arisen independently of genetic variation. We also fail to detect the potentially compensatory relationship between epigenetic and genetic diversity that has been detected in a more recent house sparrow invasion in Africa. We discuss the potential for this relationship to be obscured by recovered genetic diversity in more established populations, and highlight the importance of incorporating introduction history into population-wide epigenetic analyses.


STAT3 mutations in lymphoid cancers leading to hyperactivation of STAT3, which may represent a potential therapeutic target. Using a functional reporter assay, we screened 306 compounds with selective activity against various target molecules to identify drugs capable of inhibiting the cellular activity of STAT3. Top hits were further validated with additional models including STAT3-mutated natural killer (NK)-cell leukemia/lymphoma cell lines and primary large granular lymphocytic (LGL) leukemia cells to assess their ability to inhibit STAT3 phosphorylation and STAT3 dependent cell viability. We identified JAK, mTOR, Hsp90 and CDK inhibitors as potent inhibitors of both WT and mutant STAT3 activity. The Hsp90 inhibitor luminespib was highly effective at reducing the viability of mutant STAT3 NK cell lines and LGL leukemia patient samples. Luminespib decreased the phosphorylation of mutant STAT3 at Y705, whereas JAK1/JAK2 inhibitor ruxolitinib had reduced efficacy on mutant STAT3 phosphorylation. Additionally, combinations involving Hsp90, JAK and mTOR inhibitors were more effective at reducing cell viability than single agents. Our findings show alternative approaches to inhibit STAT3 activity and suggest Hsp90 as a therapeutic target in lymphoproliferative disorders with constitutively active STAT3.Constitutive JAK/STAT3 signaling contributes to disease progression in many lymphoproliferative disorders. Recent genetic analyses have revealed gain-of-function  L) and cell cycle regulators  [STAT3 mutations in a substantial fraction of lymphoid malignancies, including large granular lymphocytic (LGL) leukemia , CD30+ diffuse large B-cell lymphoma (6%), T-cell lymphomas (7%), multiple myeloma (4%), anaplastic large cell lymphoma (10%), natural killer (NK) cell lymphoma (6%) and intestinal T-cell lymphomas (12%) [STAT3 is a transcription factor that participates in tumorigenesis by upregulating expression of cancer promoting genes such as anti-apoptotic BCL2-family members  . Mechaniclin D1) , 8. In aas (12%) . The majas (12%) .Current approaches to inhibit wild-type (WT) STAT3 activation include JAK inhibitors such as ruxolitinib and tofacitinib and direct blocking of STAT3 dimerization with SH2 domain antagonists such as Stattic, LLL12, OPB-51602 and OPB-31121 -24. HoweTo identify targeted drugs that can potentially inhibit constitutively active STAT3 signaling, we assessed the activity of 306 approved and investigational agents in a STAT3 luciferase reporter assay. Positive hits were further validated in different models including STAT3 mutation-containing Ba/F3 cells, NK cell leukemia/lymphoma cells and LGL leukemia patient samples. Besides blocking JAK activity, our results indicate that inhibition of other molecules, such as Hsp90, may have greater impact on mutant STAT3, and could be investigated as therapeutic options for lymphoproliferative diseases with STAT3 mutations.We prescreened 306 compounds with selective activity against various target molecules  to identResults from the initial screen indicated efficacy of several agents against both WT and mutant STAT3 activity (data not shown). Based on these results we designed a smaller panel of 62 agents containing targeted compounds that effectively reduced STAT3 activity, including cyclin-dependent kinase (CDK), mammalian target of rapamycin (mTOR), heat shock protein 90 (Hsp90), and Janus kinase (JAK) inhibitors Figure . Dose reTo confirm the involvement of the drug targets in mediating STAT3 activity, we investigated whether siRNA-mediated knockdown of JAK, Hsp90 and mTOR result in similar reduction of STAT3 activity compared to pharmacological inhibition. As a control, STAT3 knockdown caused over 80% reduction in luciferase activity in both cell line models. In line with the drug screening results, JAK1 knockdown caused over 90% inhibition in IL6-stimulated WT STAT3, whereas mutated STAT3 activity remained unaltered Figure . No cleaTo determine whether the observed drug effects are conveyed through decreased tyrosine or serine phosphorylation of STAT3, the cells were treated with ruxolitinib (JAK1/JAK2 inhibitor), luminespib (Hsp90 inhibitor), INK128 (mTORC1/mTORC2 inhibitor) and temsirolimus (mTORC1 inhibitor) for 8 h. Ruxolitinib effectively inhibited Y705 phosphorylation of WT STAT3 but not mutant STAT3 Figure . NotablyGiven that the compounds appeared to inhibit the transcriptional activity of mutant STAT3 by distinct mechanisms we next investigated whether drug combinations show synergistic effects. All of the tested combinations showed additive inhibition, but no striking synergism was observed . The higTo determine if the observed drug effects on STAT3 function are associated with changes in STAT3-dependent cell growth and viability, we used the murine pro-B cell line Ba/F3. Ba/F3 cells are dependent on IL3 for survival, which activates signal transduction pathways mainly via JAK2 leading to STAT5B phosphorylation . We tranIn a 3-day cell viability assay, mutant STAT3 transformed and IL6-stimulated Ba/F3 cells were found to be sensitive to mTOR inhibition, in contrast to IL3-stimulated parental cells Figure  and 3D. Recent studies have shown that activating mutations to STAT3 frequently occur in NK/T-cell lymphomas, which are dependent on STAT3 activity for growth . To valiThe 7 cell lines were tested against the 62 compounds in a 3-day cell viability assay in the presence of IL2 with only JAK and mTORC1 inhibitors showing differences between mutant and WT STAT3 NK cells . HoweverNext we tested the combinatorial effect of Hsp90, mTOR or JAK inhibition against the mutant STAT3 NK-YS and YT cell lines. Highest synergy was observed between mTOR and JAK inhibitors . LuminesUp to 40% of LGL leukemia patients harbor a STAT3 mutation, and theIn the absence of cytokine stimulation, results from the 3-day cell viability assay demonstrated that purified CD8+ LGL cells with mutated STAT3 were significantly more sensitive to luminespib compared to WT STAT3 LGL cells or healthy donor CD8+ cells Figure . InteresMutation to STAT3 is frequently observed in lymphoproliferative malignancies, suggesting an important role of activated STAT3 in the pathogenesis of these diseases and a potential target for therapy -18. Usinin vivo in STAT3-mutated malignancies through inhibition of microenvironmental cytokine stimulation and STAT3 hyperactivation. A recent clinical investigation has shown the JAK1/JAK3 inhibitor tofacitinib to be a promising salvage therapy for refractory T-LGL leukemia patients with or without STAT3 mutations [From our functional assay, we found that JAK inhibitors were not effective at inhibiting non-stimulated mutant STAT3-driven luciferase reporter activity and STAT3 phosphorylation. In concordance, ruxolitinib treatment of heterozygous mutant STAT3-harboring NK cell lymphoma/leukemia cell lines reduced STAT3 phosphorylation to a smaller extent compared to WT cells. However, in lymphoproliferative disorders, JAK kinases activated by genetic alterations or cytokine stimulation are presumably responsible for mutant STAT3 hyperactivation , 34. We utations . HoweverHsp90 is a chaperone protein involved in the activation and stabilization of a wide variety of client proteins including members of the JAK/STAT and PI3K/AKT/mTOR pathways , 37. We Other drugs classes that reduced STAT3-driven reporter signal were mTOR and CDK inhibitors. A previous study suggested that mTOR can directly phosphorylate STAT3 at the S727 residue, thus enhancing STAT3 activity , 41. HowIn conclusion, our results suggest that STAT3 mutation status can affect drug sensitivity and should be considered when studying targeted therapy options in the spectrum of diseases where STAT3 mutations have been identified. We highlight Hsp90 inhibitors as particularly effective in targeting both mutant and WT STAT3-driven lymphoid malignancies and the combination of Hsp90 inhibitors with JAK or mTOR inhibitors may represent a more potent, long-term therapeutic strategy for STAT3 mutated disease.HEK293 cells containing a luciferase reporter under the control of a STAT3-responsive sis-inducible element  were cultured in high-glucose DMEM containing 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (complete medium). Murine IL3-dependent B-cells  were cultured in complete RPMI medium  + 10 ng/ml mouse IL3 . The Ba/F3-gp130-hIL-6R cell line was a generous gift from Christoph Garber’s laboratory (University of Kiel) and was cultured in complete RPMI + 10 ng/ml human IL6 (Peprotech). NK cell lines KHYG-1, NK-92 and YT were obtained from DSMZ. KAI3, NK-YS and SNK6 were a generous gift from Dr. John Chan  and NKL from Dr. Thomas P. Loughran (University of Virginia). All NK cell lines were cultured in RPMI complete medium + 2.5 ng/ml recombinant human IL2 . Large granular lymphocytic leukemia samples were obtained from eight patients, which had had elevated levels of CD3+CD8+ cells for more than 6 months (patient information is summarized in 5 cells/well using 8 μg/ml polybrene. IL3 was removed after two days causing EP and WT STAT3 transduced cells to die. HEK293-SIE cells were similarly transduced to generate cell lines stably expressing Y640F or WT STAT3.Mutant Y640F and wild type (WT) STAT3 containing plasmids were produced as previously described . AmplifiA panel of 306 compounds including most approved and investigational oncology drugs as well as three small molecule STAT3 inhibitors  was used for the functional assays . All druHEK293-SIE reporter cells stably expressing either WT or mutant Y640F STAT3 were dispensed to prepared 384-well drug plates at 10 000 cells/well using a MultiDrop Combi peristaltic dispenser . Before plating, IL6 was added directly to the cell suspension (100 ng/ml) of WT STAT3 expressing cell lines. The cells were incubated with the drugs for 6 h after which the ONE-Glo luciferase reagent (Promega) was added according to the manufacturer’s protocol. The luciferase signal was measured with a PHERAstar FS plate reader .2, viability was measured with the CellTiter-Glo (CTG) luminescent reagent (Promega) and PHERAstar FS plate reader.Transduced Ba/F3 cells (2 000 cells/well), NK cell lines (3 000 cells/well) and primary CD8+ large granular lymphocytic leukemia cells (10 000 cells/well) were added to the prepared 384-well drug plates. After 72 h incubation at 37 °C, 5% COHEK293-SIE cells stably expressing Y640F or WT STAT3 were plated on 384-well plates in complete DMEM medium. The plates contained pre-plated siRNAs, 3x siRNA/gene in triplicate  and tranCells were lysed in RIPA buffer , the lysates were sonicated and the protein concentrations determined using the Qubit™ Fluorometer , after which 20-50 μg of protein was loaded per sample to 10% or 12% SDS-PAGE gels. After electrophoresis, the proteins were transferred to nitrocellulose membranes , and the membranes blocked with 5% bovine serum albumin (BSA) for 1 h. Primary antibodies  were diluted 1:1 000 in Tris-buffered saline and 0.1% Tween 20 (TBS-T) + 5% BSA and incubated with the membranes 1 h at room temperature (RT) or O/N at +4°C. Secondary infrared antibodies  were diluted 1:15 000 in TBS-T + 5% BSA, and incubated with the membranes for 1 h at RT. The proteins were visualized with the Odyssey imaging system (LI-COR Biosciences) and its application software version 3.0 was used to quantify band intensity levels.U test. P-values of <0.05 were considered as statistically significant.Statistical analyses were performed with Prism software version 6.0 . Differences between responses were calculated by using the Mann-Whitney


This study uses simulation to explore and quantify the potential effect of shifting recombination hotspots on genetic gain in livestock breeding programs.We simulated three scenarios that differed in the locations of quantitative trait nucleotides (QTN) and recombination hotspots in the genome. In scenario 1, QTN were randomly distributed along the chromosomes and recombination was restricted to occur within specific genomic regions (i.e. recombination hotspots). In the other two scenarios, both QTN and recombination hotspots were located in specific regions, but differed in whether the QTN occurred outside of (scenario 2) or inside (scenario 3) recombination hotspots. We split each chromosome into 250, 500 or 1000 regions per chromosome of which 10% were recombination hotspots and/or contained QTN. The breeding program was run for 21 generations of selection, after which recombination hotspot regions were kept the same or were shifted to adjacent regions for a further 80 generations of selection. We evaluated the effect of shifting recombination hotspots on genetic gain, genetic variance and genic variance.Our results show that shifting recombination hotspots reduced the decline of genetic and genic variance by releasing standing allelic variation in the form of new allele combinations. This in turn resulted in larger increases in genetic gain. However, the benefit of shifting recombination hotspots for increased genetic gain was only observed when QTN were initially outside recombination hotspots. If QTN were initially inside recombination hotspots then shifting them decreased genetic gain.Shifting recombination hotspots to regions of the genome where recombination had not occurred for 21 generations of selection (i.e. recombination deserts) released more of the standing allelic variation available in each generation and thus increased genetic gain. However, whether and how much increase in genetic gain was achieved by shifting recombination hotspots depended on the distribution of QTN in the genome, the number of recombination hotspots and whether QTN were initially inside or outside recombination hotspots.Our findings show future scope for targeted modification of recombination hotspots e.g. through changes in zinc-finger motifs of the PRDM9 protein to increase genetic gain in production species.The online version of this article (doi:10.1186/s12711-017-0330-5) contains supplementary material, which is available to authorized users. This study uses simulation to explore the potential of shifting recombination hotspots in the genome to increase genetic gain in livestock breeding. Genetic gain is influenced by four factors: 1) the accuracy of selection; (2) the generation interval; 3) the intensity of selection; and (4) the additive genetic standard deviation. Advances in reproductive technologies, genotyping, sequencing and genomic selection in the last few decades have enabled the manipulation of the first three factors to deliver higher rates of genetic gain in many closed livestock breeding programs  variation, which in turn determines genotypic (genetic) variation. Recombination is advantageous if it uncouples favourable alleles that are tightly linked to unfavourable alleles and, which provides more opportunities for selection. Recombination is disadvantageous if it breaks favourable allele combinations , 5. The A recent simulation study showed that increasing the rate of recombination could increase genetic gain , but achPRDM9 gene has three functional domains: an N-terminal KRAB domain involved in protein–protein binding and interactions, a PR/SET domain involved in histone methylation, and a zinc finger domain involved in DNA sequence recognition and binding. The PR/SET and zinc finger domains are the primary determinants for the specification and initiation of recombination events. Upon binding to a zinc finger DNA recognition site, the PR/SET domain trimethylates lysine 4 of histone H3. This initiates chromatin remodelling to create active chromatin and the formation of a double-stranded DNA break, where the process of repair could involve a recombination event )We assumed that the inheritance of the simulated quantitative trait was fully additive and did not simulate the effects of dominance or epistasis. In our view, dominance would just scale the benefits of shifting recombination up or down. It would not alter the general trends that were observed from a purely additive model because dominance, as with additivity, acts at each QTN independently of actions at other QTN. However, epistasis could greatly alter the general trends. If large epistatic effects exist, they could particularly affect the scenarios where QTN are clustered by function. For example, clustering of QTN could be caused by selection for specific combinations of favourable alleles or could be due to sharing of common regulatory elements, and introducing a recombination hotspot to within these clusters would break up these favourable allele combinations. This would reduce genetic gain but could also have fitness consequences. However, the properties of epistasis are largely unknown and thus difficult to simulate, the impact (if any) of epistasis is not well understood, and the data and theory suggest that epistasis has a minor contribution to the total variation for quantitative traits in livestock populations .We assumed that QTN were either randomly distributed or were clustered in specific regions in the genome. There is some evidence that QTN may be distributed in clusters along the genome. For example, Wood et al.  found 69We assumed that recombination events only occurred inside and never outside hotspots. This extreme scenario was chosen again with the aim of minimising potential sources of noise that would confound the effects due to shifting recombination hotspots alone. Furthermore, empirical studies across a number of species that aim to map recombination events have shown that most, if not all, recombination tends to occur in hotspots –49. FurtThe biggest assumption in our study is that shifting recombination hotspots in livestock breeding programs is biologically, technologically and economically feasible. To effectively shift recombination hotspots, the biological mechanisms controling their exact locations and that initiate and complete a recombination need to be well characterised. As described above, this has been extensively done in model unicellular organisms including many bacterial species and yeast –49.PRDM9 [PRDM9 is highly variable within and across species and even between individuals in the same population, which results in a high diversity in the regions of the genome where the PRDM9 protein will bind and thus the exact locations of recombination events in the genome [PRDM9 paralog, (2) changing the number of zinc finger domains in a single PRDM9 gene, (3) changing the number of zinc finger recognition sites in a region of the genome where PRDM9 already binds, and/or (4) adding or removing new PRDM9 zinc finger recognition sites in the genome in mammals. All of these could potentially be achieved using genome editing technologies such as CRISPR-Cas9 [In many species including many livestock species, the mechanisms that underlie recombination are only partially understood. The major gene that determines the positions of recombination events in most mammal species is PRDM9 , 28, 51.e genome , 52. UsiSPR-Cas9 , provideRecombination is an important biological process for the release of standing allelic variation and could enable a longer sustained response to selection in breeding programs. In this study, we used simulation to show that shifting recombination hotspots to regions of the genome where recombination had not occurred for 21 generations of selection increased genetic gain. However, the benefit of shifting these depended on the locations of QTN and recombination hotspots in the genome. The greatest increase in genetic gain was achieved when QTN were clustered, the number of recombination hotspots was small, and QTN were initially located outside of recombination hotspots. If QTN were initially inside recombination hotspots, shifting them decreased genetic gain. Although currently not technologically possible, advances in genomic technologies such as large-scale sequencing and genome editing over the next decades could make the shifting of recombination hotspots feasible, beneficial and cost-effective for increasing genetic gain in breeding programs.


Metacarcinus edwardsii was analyzed along 700 km of the Chilean coast. The analysis, based on eight microsatellite loci genotyped from megalopae and adult crabs, considered temporal and spatial patterns of genetic variation. The results showed no evidence of spatial patterns in genetic structure, suggesting high connectivity among the sampling sites. The temporal analysis showed no evidence of changes in allele frequencies and no evidence of a recent bottleneck. The lack of spatial structure and allele variation over time could be explained by the interaction of factors such as i) low reproductive variance due to the capability of females to store sperm in the seminal receptacle, which can be used for successive broods, ii) high larval dispersal and iii) high individual reproductive output. Using our data as priors, a genetic modelling approach coincided, predicting this temporal and spatial stability. The same analysis showed that a reduction in population size leads to the loss of genetic variability in populations, as well as of the genetic cohesiveness between populations, pointing out the importance management for species under exploitation, such as M. edwardsii.For marine invertebrates with a benthic adult form and a planktonic larva phase, the connectivity among populations is mainly based on larval dispersal. While an extended larval phase will promote gene flow, other factors such as an intensive fishery and geographical barriers could lead to changes in genetic variability. In this study, the population genetic structure of the commercial crab  Most benthic marine invertebrates are sessile or have little movement in their adult stage; dispersion happens during the planktonic larval period ,2. TypicPatiriella regularis, where upwelling acts as a barrier to gene flow [Littorina sitkana and Nucella lapillus, where larval transport is aided by rafting [Population structure cannot be predicted only by the mode of larval development and the extent of the planktonic period . Speciesene flow . On the  rafting , 9. In t rafting . Therefo rafting ,12, wind rafting .Species with long lived pelagic larvae do not always show homogenous genetic patterns in time and space. For example, temporal variations in allele frequencies have been described ,15 and rFor species facing commercial fishing pressure, population size is an important factor influencing genetic variability and population connectivity . GeneticAnother important point to consider for the genetic structure of populations is the presence of biogeographic barriers and the phylogeographic breaks. Well-known phylogeographic breaks in the Pacific are located at Point Conception (34.5° N) in California , 30–32° Metacarcinus edwardsii, a crustacean inhabiting from the coast of Guayaquil, Ecuador to the Beagle Canal in Chile [M. edwardsii represents around 70% of national crab landings in artisanal fisheries [While these studies focused on phylogeographic analysis with a historical component, for harvested species the actual patterns of connectivity must be understood. In this context, decapod crustaceans are a good model to study connectivity due to their high fertility and extended larval development. In this study we performed a population genetic analysis on the crab in Chile . In Chilisheries ; exploitisheries . The pelisheries  that setisheries . Adults isheries .M. edwardsii.While previous studies have described aspects of the species reproductive biology ,40, larvTo study the spatial genetic structure, 291 adult crabs were sampled from six localities: Concepción (CO), Los Molinos (LM), Ancud (AN), Calbuco (CA), Dalcahue (DA) and Quellón (QU) . SamplesGenomic DNA was extracted from adult crabs using the salt method described by Aljanabi and Martinez (1997) . DNA wasHE) and observed (HO) heterozygosity were estimated with the GENETIX software [The number of alleles per locus, expected  FST with GENETIX software, ii) G”ST with GenAlEx software [ST was evaluated with GENETIX software using 5000 permutations. The GenAlEx software allowed the estimation of the global G”ST and its significance was evaluated with 9999 permutations. The STRUCTURE software infers the most probable number of populations; the analysis was performed using the admixture model and the correlated allele frequencies model. The procedure was run 5 times for each K estimation (from K = 1 to K = 11) with a burn-in of 200000 MCMC iterations. LnP(D) values obtained for each K value were compared using the Structure Harvester software [When highly variable genetic markers are used for the study of population structure, an alternative to the Fommended . One sucsoftware , iii) a software  and iv) software . The stasoftware . FLOCK ssoftware . For ourM. edwardsii among localities, historical migration rates were estimated using the MIGRATE software [6; the swapping interval was 1). Mutation rates of 1 x 10−2 and 1 x 10−3 were used to estimate the migration rate.To assess association between genetic differentiation and geographic distance, a Mantel test was performed using the GENETIX software. The statistical significance was tested with 5000 permutations. Since temporal comparisons within each locality showed no significant difference, data from each locality were pooled for posterior spatial analysis. In order to determine the migration patterns of software . MIGRATEThe effective population size was estimated using two different methods. First, the Bayesian method was implemented in ONeSAMP software  with theBOTTLENECK software  was usedE, HO and departures from HWE were estimated for each year with the GENETIX software. To determine differences among the cohorts analyzed, FST and G''ST were estimated in the same way as for adults. Two analyses were performed: the first comparing the four megalopa cohorts and the second including each megalopa cohort and the adults collected in the same locality. STRUCTURE and FLOCK software were also used to detect differences among megalopa cohorts with same settings as in the adult analysis.Four cohorts of megalopae were sampled in Los Molinos from 2009 to 2012 . The numxy value and the statistical significance was obtained with 1000 permutations in IDENTIX software.Taking into account that larval retention near the coast and the reproductive variance could generate non-random larval settlement in the same area, the likelihood of detecting related individuals in a sample was estimated using the rFor iteroparous species, estimates of effective population size need to consider samples composed of individuals of different ages. Therefore, the estimated population size of each cohort is interpreted as an approximation of the actual number of breeders per season (Nb), instead of an effective population size per generation (Ne) . ConsideM. edwardsii and there was no evidence for a recent bottleneck, we explored the factors that would affect this pattern in a scenario of increased fishing. Currently, around five thousand tons of this species are harvested every year from southern Chile, affecting the population size [For economically important species, exploring the effects of change in both population size and migration rate is relevant in order to understand population connectivity and changes in genetic variability. Considering that no spatial structure was observed in ion size . Migratiion size . Thus, tion size  considerxy values estimated for the groups of individuals collected in the same year showed no statistical evidence  for highly related individuals. This evidence suggests that samples are composed of unrelated individuals  to 25.44 (Cedw4) with a mean of 24 alleles per locus . Departuividuals .ST and G”ST estimated for samples obtained in the same locality but in different years showed no significant differences: LM , DA , AN , CA  and QU . Considering these results, the next analyses were performed with pooled data from each locality.For the temporal genetic structure, FST and G”ST showed no significant differences among sampling sites . The Structure Harvester showed K = 1 had the highest likelihood L(K) mean value and the lowest standard deviation .For the spatial genetic structure, the global Feviation . FLOCK sThe Ne estimated were similar between sites, ranging from 16 (LM 2013) and 475 (DA 2011), except for QU 2013 that presented an extreme value of 1574 . A KruskM) ranged from 1.1 to 3.2  ranged from 0.90 (with a mutation rate of 1 x 10−2) to 0.99 (with a mutation rate of 1 x 10−3).The mutation-scaled historical migration rates  to 12.57 (Cedcrab3) . While 1ST = 0.0017, P = 0.1536 and G”ST = 0.016, P = 0.180). Even when the analysis was performed with the adults collected at the same location, no temporal structure was detected . This result is consistent with that obtained with STRUCTURE  was calculated for each cohort  and the effective population size (Ne) was estimated pooling all megalopa data. Nb and Ne had a similar range of variation and even some values of Nb were higher than values of Ne estimated for the adults sampled in the same locality (Los Molinos) .Ne in generation t = 1 for each of these six sampling sites had an average Ne = 476, similar to the Ne values obtained with our data.The simulation started with 6 sites containing 100,000 individuals and an average of 24 alleles per locus. The estimated ST values (−2) to a rate 1000 times lower. Second, sites with small population sizes showed differences in allele frequencies and lost part of their allelic diversity. After the 10th generation, the sites containing 1,000 individuals and 10% migrants had significantly different FST values. The same was observed for populations composed of 5,000 individuals and a 0.1% migration rate. Finally, in the worst simulated case , a significant value of FST  was observed. This difference was primarily due to changes in allele frequencies, since only one allele per locus was lost during this time. The decrease in the number of alleles appears to be gradual; at the 50th generation the simulation predicted a loss of 4 alleles per locus  and 7 alleles (29%) at the 100th generation  [The traditional h Africa  and the s opilio  and Canc pagurus . This la pagurus  reporteds (2003)  reportedM. edwardsii is an unexpected result considering that Los Molinos shows seasonal coastal upwelling events [Carcinus maenas [Panopea generosa [Due to the long planktonic period, high mortality during this stage of development could be expected , which ag events , and eveg events . While ts maenas  and the generosa  have alsM. edwardsii could explain the observed pattern of genetic structure.The biology of 1. Reproductive variance. Genetic homogeneity over time has been associated with species that have low reproductive variance among individuals that compose each population . As was M. edwardsii, with its long larval period, is a good example of a species showing a lack of genetic structure at a wide spatial scale.2. High larval dispersal. Species with high potential for dispersion given the duration of their pelagic larvae, usually have high levels of connectivity . AccordiM. edwardsii and the lack of evidence of relatedness within each megalopa cohort suggested that larvae mix completely during the planktonic period before benthic settlement. Another study performed in the brachyuran Carcinus maenas also showed genetic stability among cohorts [3. Complete larval mixing during the planktonic period. It has been widely assumed that during the planktonic period, larvae from different locations and/or parents are completely free to mix (e.g. ). Howeve cohorts .Cancer crab species on the east coast of the USA. Interestingly, females of the brachyuran Cancer pagurus produced between 605,600 to 2,310,000 eggs per brood [M. edwardsii, showed values of 500,000 to 1,000,000 eggs per brood.4. Reproductive output. Species with greater reproductive output show more broad-scale connectivity among populations than species with low reproductive output . Brachyuer brood , and hader brood . Our meaNb (model based on semelparous species) [Ne≈G×Nb) [Ne; however, this requires taking into account a correction that considers some life history traits. In our study, the Nb and Ne had similar values, confirming the analysis of Waples et al. (2014) [Bufo calamita [Acipenser fulvescens [Ambystoma opacum and A. talpoideum [Traditionally, estimates of Ne were based on samples of middle age; while samples of one cohort provide information about the species) . In thisNe≈G×Nb) . For iteNe≈G×Nb)  suggeste. (2014) . Similarcalamita , the stulvescens  and the lpoideum .M. edwardsii is the most important artisanal crab fishery in Chile; which makes it important to understand the current state of the species in order to understand the effect of the fishery on its future. This study was conducted in the most intense crab fishing area in the country [ country  (Concepc country  and contS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


To discuss factors related to the financing of the Basic Component of Pharmaceutical Services within the municipal management of the Brazilian Unified Health System.Pesquisa Nacional sobre Acesso, Utilização e Promoção do Uso Racional de Medicamentos no Brasil – Serviços  is a cross-sectional, exploratory, and evaluative study that performed an information survey in a representative sample, stratified by Brazilian regions It considered different study populations in the sampling plan, which represent primary health care services in the cities. Data were collected in 2015 by two methods: in person, by applying direct observation scripts and interviews with users, physicians, and professionals responsible for the dispensing of medicines in primary care services; by telephone interviews with municipal health managers and municipal professionals responsible for Pharmaceutical Services. The results were extracted from the questionnaires applied by telephone. The Componente Básico da Assistência Farmacêutica  to cover demands of other medicines, but only 9.7% believed that these funds were sufficient to cover the demands. The existence of a permanent bidding committee exclusively for acquiring medicines was reported in 40.0% of the cities. Of the sample of 600 eligible cities, we collected 369 interviews (61.5%) with secretaries and 507 (84.5%) with pharmaceutical services managers. 70.8% of the cities have a computerized management system; and 11.9% have qualification/training of professionals. More than half (51.3%) of the cities received funds for the structuring of pharmaceutical services, and almost 60% of these cities performed this type of spending. In 35.4% of cases, municipal secretaries of health said that they use resources of medicines from the  We found serious deficiencies in the public financing of medicines, as well as little concern about the formality in the use of public resources, expenses that meet individual demands to the detriment of the community, insufficient resources allocated to the Basic Component of Pharmaceutical Services, and exhaustion of the financing model. The financing of health systems has become a major challenge before the growing demand for health actions and services. The pharmaceutical spending has been settling as a real threat to the sustainability of public systemsOne way or another, these values are expressive before the set of total spending on health care, either on the account of citizens or Government. In 1996, medicines accounted for 37% of the total spending of Brazilian families with health, becoming 47% in 2008Pesquisa Nacional Sobre Acesso, Utilização e Promoção do Uso Racional de Medicamentos – Serviços Componente Básico da Assistência Farmacêutica  in Brazilian cities.The PNAUM – Services is a cross-sectional, exploratory, and evaluative study, consisting of a information survey in a representative sample of primary health care services in cities. The sample was stratified by the Brazilian regions, considering the different study populations in the sampling planData were collected from July 2014 to May 2015, by face-to-face and telephone interviews. In person, data were collected by applying direct observation scripts, interviews with patients, physicians, and professionals responsible for the dispensing of medicines in SUS primary health care services. By phone, data were collected using semi-structured questionnaires for municipal health managers and professionals responsible for PS in the cities, applied by interviews conducted by trained researchers, with computer-assisted telephone interview technology. All interviews, in-person or by telephone, were preceded by the signing of the informed consent formThe instruments aimed to know the organization of PS, including their financing and the local conduct on the use of financial resources destined to the CBAF. The results relevant to finance-related factors were extracted of the questionnaires applied to municipal secretaries of health and those responsible for the PS of the cities, listed in the following groups of variables: management of financial resources, use of computerized systems, professional training and qualification, ways of acquiring medicines and structuring PS.® software, version 22, using the plan of complex samplesStatistical analysis included estimates of absolute and relative frequencies . Group comparison was held by Pearson’s Chi-square test. Data analysis was performed with SPSSThe research project was approved by the National Research Ethics Committee, by Opinion no. 398,131, September 16, 2013.Of the calculated sample of 600 cities eligible for the study, 369 interviews were conducted (61.5%) with municipal secretaries of health and 507 (84.5%) with those responsible for PS.According to the secretaries, 54% of Brazilian cities have adopted the partial decentralization regime agreeing with the implementation of CBAF resources, followed by 30.8% who have adopted the fully decentralized regime . The PS From the perspective of those responsible for PS, the PS coordination had full autonomy for managing financial resources in 19.7% of the cities, and partial autonomy in 38.2%. They reported that 70.8% of Brazilian cities had a computerized system for PS management, especially in the South region (88.8%). Activities for qualification and/or professional training for workers of PS occurred in 11.9% of the cities .More than half of the cities received, in the year preceding the survey, resources from the states or federal government aimed at the structuring of PS. Just over 50% of the cities made some expense with PS structuring. As stated by the interviewees, 86.4% of cities applied, in the year before the survey, the total value agreed of the corresponding credit of CBAF, compared with 72.7% of states . When asComissão Permanente de Licitação (CPL – Permanent Bidding Committee) – exclusive for the purchase of medicines in the city –, was reported in 37.7% of cities, especially in the Midwest region, with 49%. When asked whether the Programa Farmácia Popular do Brasil (PFPB – Brazilian Popular Pharmacy Program) influenced the purchase of medicines carried out by the cities, 50.9% of them have registered affirmative responses. The North region presented the lowest index of interference (33.2%) and the Southeast, the largest one (61.7%). 66.6% of cities have purchased medicines to meet judicial demands in the year preceding the research. The Midwest region stood out with the highest index (75.3%) and the North region with the lowest (47.5%) (The existence of a  (47.5%) .The association between city and state was the most common partnership for purchasing medicines (26.2%), followed by consortium between cities (15.0%). The South region has adopted the strategy of consortia in 52.2% of the cities . PurchasMedicines are a vital input in health careRelação Nacional de Medicamentos Essenciais , including those related to the diseases and health programs of primary health care, dedicated to meet the individual and collective basic needs of the populationCBAFComissões Intergestores Bipartite (CIB – Bipartite Intermanagement Committees), the form of agreement for using the CBAF resources. According to data collected with secretaries of health, 11% of cities agreed to use CBAF resources centrally by the state manager, who took responsibility for acquisition, logistics, and supplies in the cities. Other 31% agreed on the completely decentralized form in municipal management and became responsible for the direct acquisition of the items. However, more than half (54%) chose the partially decentralized mode, which means that the state management of SUS maintained some responsibility for the medicines purchase, even allowing the cities to adhere to their Price Registration ProceedingsBy establishing the sources of funds intended for financing CBAF, the state and municipal secretariats of health determine, in their respective We observed that the PS financial resource management was held autonomously by those responsible for PS in only 19.7% of cities, although most cities (70.8%) have a computerized system for PS management , which allows monitoring and control by the secretariat of health.Consórcio ParanáRegarding purchases made by cities, 26.2% of them took place in partnership with the state secretariats of health, with emphasis on the Northeast (35.2%) and Southeast (28.9%) regions. We highlight the possibility of states in these regions establishing partnerships with the major public laboratories. The form of the acquisition accomplished by public consortia was adopted by 15% of the cities, especially in the South, where more than 50% of its cities adopted this form of purchase. The Regardless of the scope of SUS management, all purchases of supplies and medicines should be carried out in agreement with the rules that control purchases, biddings, and contracts of the Public AdministrationIt is also the managers’ responsibility to register all financial resources related to PS in the Health Plan, in the Annual Schedule, and in the Annual Management Report, as well as to promote the monitoring of these resources, ensure the balance between responsibility and participation in the financing, and provide analyses with information on prices, quantities purchased, and number of patients metStill regarding the purchase of medicines, we highlight the practice declared by 35.4% of secretaries: using CBAF resources to situations other than those reported in the agreements. Also, the fact that more than 66% of the cities have bought medicines intended to meet judicial demands is noteworthy. In fact, health litigation has the positive effect of ensuring access to medicines in shortage situations, even filling the lack of care or enabling the incorporation of new health technology. However, it represents a great challenge for SUS, as it puts at risk the achievement of the constitutional principles of the system and causes difficulties for PS management, which may compromise the quality of the use of medicines,A significant consequence of litigation is the disorganization of the health systemStill concerning the disorganization of the supplies and medicines purchase, our results showed the interference of PFPB in the acquisition of CBAF medicines. The PFPB was created with the purpose of broadening citizens’ access to essential medicines for the treatment of the most common diseases, by the accreditation of private drugstores, promoting the dispensing of medicines free of charge and/or in co-payment systemThe results related to the funding for structuring and maintaining PS are worrisome. Little more than half of the cities (54.3%) spent in this area, even with the record that 51.3% of cities received some type of financial incentive from the federal or state government. Even lower rates were verified for the investment in qualification and/or professional training for PS workers. This scenario shows a progressive dismantling of the services rendered, in contrast with the fact that, since 2010, it is possible to use up to 15% of the state and municipal resources to finance PS improvements, namely: adjustment of the infrastructure of SUS pharmacies, acquisition of equipment and furniture to support PS actions, and promotion of activities linked to the continuing education and qualification of human resourcesPrograma Nacional de Qualificação da Assistência Farmacêutica no Âmbito do SUS , prioritizing cities of up to 100,000 inhabitants classified in the range of extreme poverty. The program was organized into four areas: infrastructure, education, information, and care. The funding takes place by the transfer of federal resources destined to acquisition of furniture and equipment required for the structuring of public Centrais de Abastecimento Farmacêutico  and Pharmacies in primary health care, in addition to the maintenance of pharmaceutical services. Until 2016, resources were destined to 1,582 cities, totaling 28% of the Brazilian cities. The investment resource, delivered in a single installment, varies, by population, between R$11.2 thousand and R$24 thousand. The total resource cost is R$ 24 thousand/year, delivered in quarterly form. Although it is an important and necessary program, it shows small impact, either as a source of PS financing or by the number of cities covered – mainly by the low population coverage achieved.As a way of supporting the national qualification of PS, the Brazilian Ministry of Health instituted, in 2012, the Política Nacional de Atenção Integral à Saúde das Pessoas Privadas de Liberdade no Sistema Prisional .The PS in SUS are funded with municipal, state, and federal budgets, according to the peculiarities of their components: basic, specialized, or strategic. The CBAF is financed with resources from the three levels of government and has application of minimum values, per inhabitant/year, based on the population of each cityper capita/year of tripartite financing of CBAFper capita expenses with medicines. Between 2010 and 2016, the average percentage of expenses with ASPS jumped from 20.37 to 23.27%per capita expenses of the cities jumped from R$ 15.71 to R$ 21.04Between 1999 and 2016, a 390% increase was registered in the minimum value Despite the freezing of PS resources, the complete exhaustion of the current model of PS financing takes place, derived from the model of SUS financing. The findings of this research base this understanding as well as the law 141/2012The application of the questionnaire by telephone is a limitation of this research, because it could not allow the respondent access or query to auxiliary material. Also, this is a cross-sectional study, which reflects only the moment of the survey, not enabling the establishment of temporal relationships between the results found.Finally, the results observed in this study show serious deficiencies in the financing and management of the CBAF financial resources. Among these, we highlight: little concern with the formality in the execution of public resources, either by evidence of acquisition or destination of resources outside of the regulatory standards or by the non-application by the states and cities of their respective corresponding credits; expenses incurred for meeting individual demands to the detriment of collective ones, and; statement of most managers that the resources intended for the CBAF are insufficient to cover needs and demands of the population. The fact that the main financial incentive of PS is below by more than 50% in its value since 2010 corroborates this understanding, as well as the finding, depending on the results obtained and due to legal regulations, of the exhaustion of the current model of PS financing.Política Nacional de Medicamentos , and the Política Nacional de Assistência Farmacêutica .In the context of the CBAF, major challenges have not yet been overcome. Considering the multiplicity of responsibilities between the management levels of the SUS to ensure the population’s access to medicines in primary health care, addressing these challenges requires revising the PS financing model, the


Deepwater Horizon oil spill devastated large portions of the coastline along the Gulf of Mexico. However, the foundational salt marsh grass, Spartina alterniflora, showed high resilience to this strong environmental disturbance. Following the spill, we simultaneously examined the genetic and epigenetic structure of recovering populations of S. alterniflora to oil exposure. We quantified genetic and DNA methylation variation using amplified fragment length polymorphism and methylation sensitive fragment length polymorphism (MS‐AFLP) to test the hypothesis that response to oil exposure in S. alterniflora resulted in genetically and epigenetically based population differentiation. We found high genetic and epigenetic variation within and among sites and found significant genetic differentiation between contaminated and uncontaminated sites, which may reflect nonrandom mortality in response to oil exposure. Additionally, despite a lack of genomewide patterns in DNA methylation between contaminated and uncontaminated sites, we found five MS‐AFLP loci (12% of polymorphic MS‐AFLP loci) that were correlated with oil exposure. Overall, our findings support genetically based differentiation correlated with exposure to the oil spill in this system, but also suggest a potential role for epigenetic mechanisms in population differentiation.Catastrophic events offer unique opportunities to study rapid population response to stress in natural settings. In concert with genetic variation, epigenetic mechanisms may allow populations to persist through severe environmental challenges. In 2010, the  Classic population genetics studies demonstrate that natural selection in different microhabitats can result in associations of genotypes, or alleles of candidate genes, with habitat type  oil spill  and methylation sensitive fragment length polymorphism (MS‐AFLP) to test the hypothesis that oil exposure in S. alterniflora resulted in genetic and epigenetic signatures of population differentiation. As in previous studies of S. alterniflora ; Grand Isle, LA oiled site 2 (GIO2) (n = 7); and Bay St. Louis, MS oiled (MSO) (n = 8). Nearby uncontaminated reference sites were Grand Isle, LA no‐oil site 1 (GIN1) (n = 9), Grand Isle, LA no‐oil site 2 (GIN2) (n = 10), and Bay St. Louis, MS no‐oil (MSN) (n = 8). Because the minimum number of populations required to detect differences between two groups at the level of alpha = 0.05 is suggested to be n = 7 , which was not affected by the DWH oil spill  and +ACG (HEX) and unlabeled MseI + CAC primers. We sent selective PCR products to the DNA Facility at Iowa State University, IA, USA, where they were electrophoresed on an ABI 3130XL. We scored resulting fragments in duplicate as “1” for present and “0” for absent using Peak Scanner (Thermo Fisher Scientific) and excluded markers that were not supported in duplicate.We used AFLP to assess genetic variation between the field sites using a standard protocol described in Richards et al. . Briefly2.3MspI and HpaII restriction enzymes, which have different sensitivities to cytosine methylation of the same CCGG sequence  and +ACG (HEX) and unlabeled primers HpaII/MspI +TCAC and HpaII/MspI + TCAT. We sent selective PCR products to the DNA Facility at Iowa State University, IA, USA, where they were analyzed on an ABI3130XL. We visualized the resulting electropherograms using Peak Scanner and scored fragments as “1” when present and “0” when absent.We used MS‐AFLP to assess genomewide DNA methylation on the same duplicate DNA extractions used in the AFLP protocol . We also used GenAlEx to calculate estimates of genetic differentiation over all AFLP and MS‐AFLP loci with a hierarchical AMOVA, nesting study sites within oil exposure to compare genetic variation among oil‐contaminated and uncontaminated sites (ΦRT), among sites within contamination level (ΦPR), and within sites (ΦPT). We also used GenAlEx to conduct a locus‐by‐locus AMOVA to characterize genetic and epigenetic differentiation at each locus, using the same hierarchical design. Finally, we performed pairwise AMOVA comparisons to determine which populations were differentiated. For all AMOVA analyses, we used 9,999 permutations to estimate statistical significance and adjusted for multiple comparisons using the sequential Bonferroni method whenever multiple tests were performed.We used GenAlEx version 6.41 . In each analysis, variation in marker profiles was represented by the Euclidean distance matrices as calculated from the binary AFLP and MS‐AFLP methylation data  generated by GenAlEx 6.41. We also used the RDA function within the Vegan package of R , which allows for comparison of nested terms within hierarchical experimental design. Using the Adonis function within the Vegan package of R , and 55 of 56 individuals displayed a unique genotype. There was no difference in genotype diversity between contaminated and uncontaminated sites (p = .262). Bayesian clustering identified two genetic groups ; however, these groups did not clearly reflect either differentiation by oil contamination or geographic separation . Locus‐by‐locus AMOVA revealed 17 loci that varied significantly between oil‐contaminated and unaffected sites , and among populations within site type , and each individual displayed a unique epigenotype. Like the estimates for genetic patterns, there was no difference in epigenotype diversity between affected and unaffected sites (p = .993), and as in our previous studies of S. alterniflora  and redundancy analysis . Locus‐by‐locus AMOVA revealed five loci were significantly differentiated between oil‐contaminated and unaffected sites , it is possible that we were unable to capture a change in genetic diversity among populations if one occurred in response to the oil spill.Following the S. alterniflora is an outcrossing, wind‐pollinated grass with extremely high genetic diversity  in severely polluted habitats show broad genetic differentiation, including an allelic variant of cytochrome CYP1A  in the Gulf of Mexico also showed differential expression of CYP1A among affected and unaffected populations following the Deepwater Horizon oil spill (Whitehead et al., S. alterniflora is robust to heavy oil exposure, we found a signature of genetic differentiation between oil‐exposed and unexposed populations. These results suggest at least some mortality in oil‐exposed populations, consistent with findings of initial losses in live belowground biomass (Lin et al., DWH oil spill, our study adds to the growing number of ecological and evolutionary genomics studies describing population‐level response to pollution.Human‐mediated environmental impacts have been well documented as potential evolutionary drivers of population differentiation. A classic example is the rapid phenotypic change experienced by the peppered moth as a result of coal pollution (Kettlewell, Populations in coastal habitats, and salt marshes in particular, have long been models for phenotypic differentiation across natural environmental gradients (Schmidt & Rand, S. alterniflora in a Georgia salt marsh (Foust et al., DWH oil spill, which resulted in total aboveground dieback, and reduction by approximately 84%–95% of belowground biomass of the leading 5–10 meters of S. alterniflora in heavily oiled Gulf of Mexico marshes (Lin et al., S. alterniflora including natural disturbance events (Pennings & Bertness, Although we found high levels of epigenetic variation among individuals within and among populations, we failed to detect epigenetic differentiation in response to oil contamination. Our previous work showed a weak correlation between environmental conditions and epigenetic variation in Deepwater Horizon oil spill may have acted as a single, discrete event that changed the makeup of the extensive genetic variation present in S. alterniflora rather than inducing a plastic or regulatory response that could be captured by assaying DNA methylation.Epigenetic mechanisms of response, such as DNA methylation, are expected to be evolutionarily favorable when the periodicity of a stressor is short (Lachmann & Jablonka, As studies of epigenetic variation in natural populations move away from quantifying the amount of standing genetic and epigenetic variation in natural populations to describing the role of that variation and the relative contribution of genetic and epigenetic variation to population differentiation, more precise sampling techniques and analyses will be needed. In future studies, a reduced‐representation bisulfite sequencing approach would allow the direct comparison of genetic and epigenetic data sets, and at a much finer scale, with substantially increased statistical power to detect epigenetic differences between populations (van Gurp et al., https://doi.org/10.5061/dryad.pf0s5.Data available from the Dryad Digital Repository:


Many species are shifting their ranges in response to global climate change. Range expansions are known to have profound effects on the genetic composition of populations. The evolution of dispersal during range expansion increases invasion speed, provided that a species can adapt sufficiently fast to novel local conditions. Genetic diversity at the expanding range border is however depleted due to iterated founder effects. The surprising ability of colonizing species to adapt to novel conditions while being subjected to genetic bottlenecks is termed ‘the genetic paradox of invasive species’. Mutational processes have been argued to provide an explanation for this paradox. Mutation rates can evolve, under conditions that favor an increased rate of adaptation, by hitchhiking on beneficial mutations through induced linkage disequilibrium. Here we argue that spatial sorting, iterated founder events, and population structure benefit the build-up and maintenance of such linkage disequilibrium. We investigate if the evolution of mutation rates could play a role in explaining the ‘genetic paradox of invasive species’ for a sexually reproducing species colonizing a landscape of gradually changing conditions.We use an individual-based model to show the evolutionary increase of mutation rates in sexual populations during range expansion, in coevolution with the dispersal probability. The observed evolution of mutation rate is adaptive and clearly advances invasion speed both through its effect on the evolution of dispersal probability, and the evolution of local adaptation. This also occurs under a variable temperature gradient, and under the assumption of 90% lethal mutations.In this study we show novel consequences of the particular genetic properties of populations under spatial disequilibrium, i.e. the coevolution of dispersal probability and mutation rate, even in a sexual species and under realistic spatial gradients, resulting in faster invasions. The evolution of mutation rates can therefore be added to the list of possible explanations for the ‘genetic paradox of invasive species’. We conclude that range expansions and the evolution of mutation rates are in a positive feedback loop, with possibly far-reaching ecological consequences concerning invasiveness and the adaptability of species to novel environmental conditions.The online version of this article (doi:10.1186/s12862-017-0998-8) contains supplementary material, which is available to authorized users. Many species are currently expanding their ranges as a response to increasing global temperatures under climate change . Range eSeveral possible explanations for ‘the genetic paradox of invasive species’ are reviewed in Stapley et al. , particuA species range consists of, more or less connected, separate populations of individuals, in contrast to the investigated single populations in previous theoretical studies , 27–29. We use a spatially explicit individual-based metapopulation model of a sexually reproducing species with discrete generations, which expands its range along a gradient in temperature. We allow the mutation rate to evolve, and investigate its interplay with the evolution of dispersal probability and temperature adaptation during and after range establishment.x-dimension) of 20 patches each (y-dimension). We assume wrapped borders in y-direction, building a tube. Hence, if an individual leaves the world in y-direction during dispersal, it will reenter the simulated world on the opposite side. However, if it leaves the world in the x-direction, it is lost from the simulation. To answer our research questions the model requires a need for local adaptation during range expansion. Thus every column of patches (x-position) is characterized by its specific mean temperature τx. This mean local temperature is used for the determination of the level of local adaptation of individuals. To simulate a large-scale habitat gradient, τx changes linearly from τx=1 = 0 to τx = 250 = 10 along the x-dimension, i.e. by Δτ = 0.04 when moving one step in x-direction.The simulated landscape consists of 250 columns  its optimal temperature τopt, i.e. the temperature under which it survives best, determined by the alleles at its adaptation locus la (see below for details), 4) its genetic mutation rate determined by the alleles at the mutator locus lm (see below under Genetics), and 5) a diploid neutral locus ln, for the sake of comparing the levels of genetic diversity with other loci.Local populations are composed of individuals, each of which is characterized by several traits: 1) its sex, 2) its dispersal probability determined by the alleles at the dispersal locus x, y is therefore assigned a random male from the same habitat patch  and gives birth to a number of offspring drawn from a Poisson distribution with mean population growth rate λ. The offspring’s sex is chosen at random. Density-dependent survival probability s1 of offspring due to competition is calculated as:Local population dynamics follow the time-discrete Beverton–Holt model . Each inK the carrying capacity and Nx,y,t the number of individuals in patch x,y at time t. Finally, the surviving offspring experience a further density-independent mortality risk (1 − s2) that depends on their level of local adaptation, so the matching of their genetically determined optimal temperature (τopt) to the temperature conditions in patch x, y (τx) according to the following equation:with η describes the niche width or ‘tolerance’ of the species. We performed simulations for the species with a niche width of η = 0.5, equivalent to a decrease of survival probability of about 0.032 when dispersing one patch away from the optimal habitat. In this approach we assume that density-dependent mortality (1 − s1) acts before mortality due to maladaptation to local conditions (1 − s2). In addition, each population has an extinction probability ε per generation. Surviving offspring disperse with probability d that is determined by their dispersal locus (see below). If an individual disperses it dies with probability μ, which is 0.2 throughout the landscape. This mortality accounts for various costs that may be associated with dispersal in real populations, like fertility reduction or predation risk .How does the evolution of mutation rates depend on directional selection? For this, a landscape was designed in which the temperature varies in the How does the evolution of mutation rates depend on the process of range expansion, i.e. a series of colonizations? For this, we looked into a stable, non-range expanding population to assess the dependency of such repeated colonization, for which we simulated a non-expanding species’ range under both temperature gradients, so applying a) a non-variable and b) variable temperature gradient in time (as defined in experiment 4). For these experiments the species was initialized in the whole landscape, so not restricted to the native area, and the temperature was steadily increased in the entire landscape. With this, equal selection pressure for the species was forced, but without a range expansion, by changing the temperature at the same rate as experienced by the marginal populations in the spatial scenarios. Global dispersal was applied here.As a follow-up, several other simulation experiments were performed to answer these specific questions:x-position. For the dispersal probability we calculated the arithmetic mean, while the mutation rate was averaged geometrically because the mutator gene codes for the exponent’s value. Genetic diversity was calculated as the variance in allelic values at the adaptation locus, the dispersal locus and the neutral locus, per x-position. The analysis of linkage disequilibrium was done using Genepop version 4.5.1 [la and lm, ld and lm, and ln and lm.The individual phenotypes for the three traits were documented in time and space throughout the simulations and averaged per on 4.5.1  testing s2 is close to one in all simulations, throughout the simulation time and across the entire species’ range. This indicates that colonization of new habitat occurs by pre-adapted individuals only. After the burn-in phase, the species’ range expands across the landscape  with little genetic diversity at the expanding range margin, which increases with the age of the populations Fig. . The maxd = 0.2, panel B), so even if the dispersal probability is fixed to a relatively high value of 0.2, the expansion is slow if the mutation rate is no allowed to evolve. To assess how dispersal rates and mutation rates are connected we look into the results of experiment 2. Under fixed dispersal rates . These show that a fixed, lower mutation rate Fig.  causes ates Fig.  we see tt = 100, with t = 0 is the end of the burn-in period) there is a wide zone of newly colonizing populations at the expanding range margin with no genetic diversity at the temperature  locus  show a clear increase of the dispersal probability and mutation rate compared to the equilibrium conditions in the native area See supplementary file for full legends. Additional file 2:A simple analytical approach to calculate optimal mutation rates. Simple analytical approach showing fitness effects of different mutation rates (PDF 429 kb)