Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer. Prostate cancer is the most frequently diagnosed cancer and the second leading cause of cancer mortality in men living in the developed world . The US Two large scale studies examined the chemopreventive potential of finasteride and dutasteride, both of which inhibit 5α-reductases, the enzymes that convert testosterone to the more potent androgen DHT in prostate tissue. The prostate cancer prevention trial (PCPT) demonstrated that finasteride reduced the overall risk of prostate cancer in low risk men  . There hMultiple retrospective studies have shown that long-term use of warfarin prior to diagnosis is strongly associated with a reduced incidence of prostate cancer. One study examined the use of warfarin in the 5 years preceding diagnosis of urological cancers and discovered that at least 2 years of warfarin use reduced the risk of prostate cancer , but not other urological cancers . Two othWarfarin is an anticoagulant that belongs to the class of vitamin K antagonists, which inhibit vitamin K epoxide reductase (VKOR), a key enzyme in the vitamin K cycle Figure  10]. Vi. Vi10]. The mechanism underlying the association between warfarin use and the reduced risk of prostate cancer is unclear. Because of its role in anticoagulation and the risks associated with that activity, warfarin is not a suitable candidate as a chemopreventive agent. Therefore, it is important to understand the mechanism of action of warfarin and determine if it is possible to separate its chemopreventive properties from its anticoagulation effects. Previously, we identified warfarin and other vitamin K antagonists as AR inhibitors in a high-throughput screen for novel AR regulators . BecauseWarfarin, scopoletin and other coumarins were identified as AR antagonists in a high-throughput screen in HEK 293 kidney cells expressing AR . In addiWarfarin and other vitamin K antagonists are known to bind and inhibit the VKOR protein. Therefore, we performed siRNA knockdown of VKOR using two different siRNAs and determined the effect on AR activity using a luciferase reporter assay and qPCR of AR target genes. Although knockdown was not complete Figure , the siRin vivo in mouse prostate cells as it does in cultured cells, we treated mice for 4 weeks with sub-lethal concentrations of warfarin, as warfarin is a known rodenticide. Mice were also castrated as a positive control for inhibition of AR target genes. The effect of warfarin was confirmed by measuring the clotting time of blood in treated animals   as well et al, study compared men with 6 months warfarin usage with 6 weeks of warfarin usage, thus mitigating any potential ascertainment bias [Overwhelming evidence indicates that long term warfarin usage is associated with a decreased risk of PC. While this data is necessarily retrospective in nature as a prospective study testing the effects of the anticoagulant warfarin is not feasible due to safety concerns, the four largest experiments contain over 2.5 million subjects combined, and each reaches a very similar conclusion: warfarin usage of more than a half of a year significantly reduces the risk of a PC diagnosis. Although men on warfarin tend to be less healthy, and therefore less likely to undergo aggressive screening for PC, the association between warfarin use and decreased risk of PC is not likely attributable to ascertainment bias. Each study carefully controlled and adjusted for potential confounders and most importantly, the Schulman ent bias . Recall Even though warfarin has not been studied prospectively in the context of prostate cancer prevention or treatment, coumarin, a related compound of the same class, has been shown to have some therapeutic properties against prostate cancer. Treatment of rats with high dose of coumarin was shown to significantly shrink the growth of prostate tumors, and at even caused the regression of the prostate, suggesting that coumarins such as warfarin could potentially have some antiandrogen properties . When usBecause of the primary role that vitamin K plays in the γ-carboxylation of proteins and our initial observation that warfarin can inhibit AR activity, we first determined if there were γ-carboxylation sites on AR. We utilized a previously described methodology specifically for identifying the location of γ-carboxylation on proteins . Due to Warfarin is the most widely used anticoagulant but dosing of warfarin is particularly difficult as the effective dose is highly variable between individuals  and warfBecause the anticoagulatory and chemopreventive effects of warfarin appear to be separable, it is likely possible to identify safer drug targets downstream of warfarin and develop novel chemopreventive agents against them. To identify potential candidates, we used RNA-seq to identify the signaling pathways affected by warfarin treatment in benign mouse prostate tissue. In addition to a downregulation of AR signaling, we observed very significant decreases in several pathways, many of which were related to PPAR signaling. Here we show that warfarin can inhibit PPAR signaling and that inhibition of PPARγ causes decreased AR activity. PPARγ is a well characterized regulator of AR activity, through its effects appear to be cell-type dependent , and exaBecause warfarin has been documented to have chemopreventative properties distinct from treatment with 5α reductase inhibitors, namely decreased detection of high grade cancers in addition to low grade cancers, it could very well be that warfarin's effects on both PPARγ inhibition and other signaling pathways contributes to chemoprevention by mechanisms in addition to AR inhibition. Vitamin K signaling is known to regulate lipid biosynthesis through binding and activation of the steroid and xenobiotic receptor (SXR) . ActivatDihydrotestosterone was purchased from Steraloids. Vitamin K antagonists were purchased from: warfarin sodium , scopoletin (Sigma), brodifacoum (Sigma). Bicalutamide was purchased from Sigma. PPAR agonists and antagonists were purchased from: GW1929 (Sigma), Pioglitazone (Santa Cruz), GW9662 (Sigma).Human embryonic kidney cell line (HEK 293) and prostate adenocarcinoma cell lines (LNCaP) were purchased from ATCC. BPH-1 cells were a gift from Ann Donjacour. HEK 293 stably expressing fluorescent (CARY) and HA-tagged AR were generated previously . Mouse pt-test  was performed and association was considered significant when P < 0.05 and indicated by an asterisk.Cells were transfected using Lipofectamine Plus (Thermofisher) with PSA-luciferase , MMTV-lut test was used to determine significant differences between DHT treated and drug treated populations.For growth curves, LNCaP cells were transferred to charcoal stripped (C/S) media 3 days before they were split and plated at a density of approximately 20,000 cells/well in 48-well plates, in quadruplicate. The following day, medium with or without DHT and drugs was added to the cells. Proliferation was determined by measuring the DNA content of the cells in each well. Cells were fixed in 2% PFA, followed by staining for 5 min at room temperature with 0.2 ng/mL DAPI in PBS solution. The cells were washed with PBS solution, then read on a fluorescence plate reader using 365/439 excitation/emission wavelengths. A Student Site-directed mutagenesis was performed using PCR with primers containing the desired mutations. AR expression plasmid was amplified with mutant primers using KAPA High Fidelity polymerase (Kapabiosystems) using manufacturer's protocol. Parent plasmid was digested with DpnI (Agilent) for 1 hour. Mutant plasmids were transformed into NEB 5-alpha competent cells (NEB). Mutations were screened by Sanger sequencing.LNCaP cells were treated with drugs for 24–72 hours and lysed in TBS, 0.1% Triton X-100, protease and phosphatase inhibitors (Roche). Immunoprecipitation was performed using anti-AR  or anti-GLA antibody . Western blot was used to detect AR , GLA or ART-27 .All animal experiments were conducted with approval from the institutional Animal Care and Use Committee of City of Hope. Warfarin treatment was performed by dissolving warfarin in drinking water at the desired concentrations. Male nude mice, aged 8 weeks, were obtained from the NCI breeding program. Mice were either treated with warfarin, surgically castrated or left untreated as controls. Mice were euthanized after 4 weeks and the prostate was harvested. Coagulation time was measured by sliding newly acquired blood in a glass capillary tube until it no longer slides or remained uncoagulated after 10 minutes.t-test was performed and association was considered significant when P < 0.05 and indicated by an asterisk.Total RNA was extracted from cells or homogenized tissue using GeneJet RNA purification kit (ThermoFisher). Reverse transcription was performed using M-MLV reverse transcriptase (ThermoFisher). Gene expression was quantified using SYBR green (ThermoFisher) with Rox reference dye (ThermoFisher) on a StepOne Real Time PCR System (ThermoFisher). Relative gene expression was calculated by ΔΔCT. Student's p-values, and genes were only considered if the false discovery rate (FDR) was < 0.25 and the absolute value of the fold change was > 1.5. Gene ontology analyses were performed using GSEA [RNA sequencing was performed by the City of Hope Integrative Genomics core facility. cDNA synthesis and library preparation was performed using TruSeq RNA Library prep kit in accordance with the manufacturer supplied protocols. Libraries were sequenced on the Illumina Hiseq 2500 with single read 40 bp reads. The 40-bp long single-ended sequence reads were mapped to the human genome (hg19) using TopHat and the frequency of Refseq genes was counted with customized R scripts. The raw counts were then normalized using trimmed mean of M values (TMM) method and compared using Bioconductor package “edgeR”. The average coverage for each gene was calculated using the normalized read counts from “edgeR”. Differentially regulated genes were identified using one-way ANOVA with linear contrasts to calculate ing GSEA , DAVID [Immunohistochemistry was performed using standard protocols. Antigen retrieval was performed on paraffin-embedded sections using citrate-based antigen unmasking solution . Slides were blocked with 10% normal goal serum, and then stained with VKOR antibody developed by Berkner  (dilutedImmunoprecipitated AR was separated by SDS-PAGE and stained with SimplyBlue (Themofisher). Gel bands were excised and destained in ammonium bicarbonate. After disulfide bond reduction with 10 mM tris(carboxyethyl)phosphine and thiol alkylation with 50 mM iodoacetamide, gel bands were incubated with trypsin/Lys-C or chymotrypsin (Promega) overnight. Peptides were extracted with 0.1% TFA/70% acetonitrile and lyophilized. Methyl esterification was performed by incubating in 2M methanolic HCl for 1 hour at 20°C to improve detection of γ-carboxyl groups . Samples


The first edition of Enabling Recovery was a much-welcomed arrival for the discipline of rehabilitation psychiatry as it emerged from its marginal ‘resettlement’ function in the late 1990s. The discipline is now accepted as part of mainstream psychiatric practice.This second edition takes into account new data and developments, and adds an international perspective. The book covers a wide range of topics over 31 chapters, comprehensively written by 42 contributors from diverse backgrounds, including researchers, clinicians, patients, managers and policy makers. To start with, there are historical accounts of altered mental states and how cultures through the ages have understood them and protected/treated people. The authors integrate themes of recovery, personalisation, social exclusion and disability with contemporary practice. Interventions are described – medication, a range of psychological therapies, physical healthcare and guidance around challenging behaviour and substance misuse. The section discussing service delivery in a range of settings presents the rehabilitation pathway as a whole-system approach, moving through different settings as people become more independent. It usefully details what is required for effective in-patient care, community care and supported accommodation, housing and work. Also included are special topics such as brain injury, autism, risk management and international psychiatry and, finally, a chapter on the expanding evidence base for the practice of specialist rehabilitation.Although the book focuses on rehabilitation psychiatry, its appeal is wider and it is useful to multidisciplinary mental health teams, medical students and other healthcare practitioners. It is easy to read – the diversity of topics allows each chapter to stand alone so readers can dip in and out of the book as they please. It also functions as a ‘toolkit’ to support shared decision-making when faced with complexity. The chapters ‘Assessment for Rehabilitation’ and ‘Rehabilitation at the Coalface’ are particularly handy as they describe practical approaches to developing formulations, engaging with people and developing goals and treatment plans.


The Lazarillo of Tormes’ picaresque novel introduces a story where two subjects sequentially extract  tokens from a common pool in an asymmetric information framework (the first player cannot observe her partners’ actions). By introducing a reward for both subjects in case that in every period at least one subject had taken one single token, we define an interesting coordination game. We conduct an experiment with 120 undergraduate students to study their behavior in this framework. We find that if the second player is allowed to take more tokens than her partner, then the frequency of cooperators does not seem to be affected by the informational asymmetry. Nevertheless, this asymmetry (i) incentives the second player to use her ‘power of extraction’ while the social externality is still available, (ii) yields to more asymmetric profit distributions when subjects win the social externality and (iii) delays the breach period in case of coordination failure. Furthermore, the first choice of the first player is determinant for getting the reward. Now I want to be generous with you: we’ll share this bunch of grapes, and you can eat as many as I do. We’ll divide it like this: you take one, then I’ll take one. But you have to promise me that you won’t take more than one at a time. I’ll do the same until we finish, and that way there won’t be any cheating.” The agreement was made, and we began. But on his second turn, the traitor changed his mind and began to take two at a time, evidently thinking that I was doing the same. But when I saw that he had broken our agreement, I wasn’t satisfied with going at his rate of speed. Instead, I went even further: I took two at a time, or three at a time—in fact, I ate them as fast as I could. And when there weren’t any grapes left, he just sat there for a while with the stem in his hand, and then he shook his head and said, “Lázaro, you tricked me. I’ll swear to God that you ate these grapes three at a time.” “No, I didn’t”, I said. “But why do you think so?” That wise old blind man answered, “Do you know how I see that you ate them three at a time? Because I was eating them two at a time, and you didn’t say a word.”“The Lazarillo of Tormes, his Fortunes and Misfortunes , a major exponent of Spanish picaresque literature of the Golden Age, which presents a clear overview of the society of the sixteenth Century through the eyes of Lázaro of Tormes. Lázaro is a boy who has to earn a living in Salamanca as the apprentice of different masters, the first one being a cunning blind man.The above paragraph reproduces the original text (translated into English) of In spite of the huge development in science and technology and the deep changes in the society occurred over the last centuries, this parable maintains all its essence in present days. The story illustrates how the lack of transparency may have an important role in breaking agreements, behaving as a non-cooperative short-term profit maximizer and triggering corruption bubbles . There is a long list of examples where agents forget about social externalities and deviate from social norms if they do not feel controlled, e.g., contributors in a typical tax declaration problem, departments charging expenditures on a common budget, firms extracting natural resources, countries deciding about fiscal policies within a monetary union, risk managers deciding about their risk exposure, etc. Nevertheless, in such situations the non-cooperative behavior might not be necessarily a consequence of the lack of transparency or supervision.To shed light on this issue, we experimentally investigate the effect of asymmetric information between heterogeneous subjects that exploit a common pool  through sequential extractions. The topic of voluntary sharing/contribution on common pool resource systems or public goods provision has been recently revisited in experimental literature and explained by indirect reciprocity—e.g. –3. The eThe effects of transparency on conditional cooperation have been studied in other contexts, e.g. public goods  or auctiFurthermore, since we deal with heterogeneous subjects (heterogeneity in the sense of different extraction power from the common pool), our work is also related to those studies that investigate the role of asymmetries in sequential settings—see, for instance ; 18], ,  or 20]  20] ThisIn order to disentangle which of these two asymmetries (information or extraction power) is more relevant, we design an experiment inspired by the aforementioned Lazarillo’s tale where two subjects have to share a given endowment (60 tokens) by means of sequential decisions. In this framework, we introduce incentives for holding a certain sharing rule by giving a final reward (20 extra tokens) to both subjects in case the rule is satisfied in every round of the experiment. The rule requires that at least one of the two subjects has taken a single token, which captures a certain type of “sustainability” in the extraction as in a natural resources problem. Furthermore, and with the purpose of analyzing which of the two aforementioned asymmetries is more relevant, the second player is allowed to take one, two or three tokens in every round whilst the first player is only allowed to take either one or two tokens. The final payoff of every subject is the bunch of tokens taken throughout the experiment plus the final reward in case of success in holding the rule. We consider two treatments: Control treatment, where the first player can also observe the decisions of the second player; and Impunity treatment, where the first player is unable to observe the decisions of her matched partner.This game involves a subgame perfect Nash equilibrium (SPNE) where in every round the first mover takes two tokens and the second mover responds by taking one single token. However, the capacity of taking three tokens by the second player may represent a credible threat and induce different sharing schemes.Our results show that most players deviate from the SPNE. In particular, we find that in both treatments second players take advantage of their power of extraction and achieve higher profitability than first players. This occurs not only when coordination fails and they lose the final reward, but also, and more surprisingly, when cooperation holds. This means that the power of extraction seems to be more relevant than the extraction order.breach period (the period in which the condition to win the prize does not hold anymore) compared to the Control treatment.Regarding transparency, we find that non-observability does not seem to distort the level of cooperation . However, the blindness of the first player has a significant impact on the final distribution of payoffs since second players of cooperators present higher profits in Impunity than Control treatment. Furthermore, Impunity delays the first choice of the first player is a key determinant for the success in coordination/cooperation. Most of first players that started cooperating (taking one single token in the first period) succeeded in winning the final reward, and, on the contrary, most of first players that started aggressively (taking two tokens in the first round) failed. In fact, both treatments present a positive correlation between the first choice of the first player and the following choices of both players, i.e. the more aggressively first player starts, the more aggressively both subjects behave throughout the experiment, increasing the likelihood of coordination failure.Finally, we also find that, regardless of the treatment, the The rest of the paper is structured as follows: Section 2 describes the experiment and the different strategies and solutions of the game, Section 3 discusses the results of the experiment and, finally, Section 4 gathers the main conclusions.N tokens in an undetermined number of rounds, T, by sequentially extracting them from a common pool . The power of extraction of both players is different. Type A (first mover) can extract either one or two tokens in every round while Type B is allowed to take one, two or three tokens, after Type A has already made her decision, in every period. Therefore if we denote the decision undertaken by subject i ∈ {A, B} in period t ∈ {1, 2, …, T} as dit∈ Dit, then DAt = {1, 2} and DBt = {1, 2, 3}. Note that, although not explicitly considered in the second player’s options, if in the last round there are no more tokens left for the second player, she is also allowed (forced) to ‘extract zero’.Two players have to share NA + NB = N. In addition, both players may win a reward of R tokens in case that at the end of the game the following condition is being satisfied: “In every period t at least one of the players had taken one single token”. Note that R≥16 is a necessary condition to make both subjects consider coordinating their strategies to win the prize. Therefore the payoff function for player i = {A, B} is:The game ends when no token remains and the final payoffs of both players depend directly on the number of tokens extracted during the whole game, denoted by The strategy profiles of every subject may be characterized by a sequence N = 60 and R = 20. We applied a between-subjects design to investigate the impact of the informational asymmetry implied by the opacity/transparency of the decisions of first player (Type A) on the decisions of the second player (Type B) and its implications on the coordination/cooperation to win the reward and the final playoffs. For this purpose, we consider two treatments:Our experimental design implements exactly the framework described in previous section but setting Control treatment (CT): Both players can observe the decisions of their matched partners in each round. However, as the game is sequential, Type A only observes the tokens taken by Type B after she has already made her own decision.Impunity treatment (IT): The first player (Type A) cannot observe the decisions of Type B. We denote it as IT because Type B can take advantage of her power of extraction without being detected by Type A. As a consequence, in this treatment, Type A does not know how many tokens are left in every round. That is, Type A does not know the period in which the game will be over.In this particular setting we highlight the following strategy profiles as possible outcomes of the experiment:R = 20, the final SPNE payoffs would be . If Type B deviates from SPNE by extracting three tokens in all rounds, final payoffs would be . Thus, if R is larger than 16, Type B has no incentive to deviate from SPNE.The SPNE strategy profile is solved by backward induction. This profile is  in every period, i.e. Type A extracts two tokens and thus, Type B takes a single token so as to fulfil the compensation rule the end of the experiment. That is, Type A should always take advantage of being the first mover because he anticipates the fact that in this case Type B would be forced to take one single token. Considering coordination strategy profile to every strategy profile such that both agents win the final reward of 20 tokens. Within these profiles payments may vary from  =   =  depending on the times where each subject only takes one token. For instance, We denote by ∏A, ∏B) = ). Other cases of coordination profiles are those in which both subjects succeed in winning the reward and yield to an egalitarian share  = . It is noteworthy that playing this strategy profile might represent a credible threat by player B to avoid that player A exploits her advantage of being the first mover, since Type A has much more to lose than Type B if the Lazarillo’s strategy profile is the final outcome of the game. In fact, the effect of this ‘threat’ on the first mover’s advantage is one of the contributions of this game. These profiles are those consistent to the story of the Lazarillo of Tormes, i.e., Type A and Type B take, respectively, two and three tokens during 12 rounds. The expected payoff of such a profile is  and, at the same time, they have impunity for their own actions and are allowed to overreact by extracting more than the others. This makes us formulating the following second hypothesis.Hypothesis 2The second player exploits her extraction power taking more tokens than the first player and getting higher profitability.We also hypothesize that, by reciprocity, transparency makes second players less aggressive (i.e. more cooperative). Consistently, the final distribution of tokens should be more egalitarian in CT than in IT.Hypothesis 3The lack of transparency leads to more unequal distribution of payoffs.On the other hand, CT should accelerate the breach period (period in which the reward is no longer available) when coordination fails, since the aggressive behavior is observed and partner’s tolerance to it reduces.Hypothesis 4The lack of transparency delays the breach period when coordination fails.The first observation is that the 20 extra tokens reward is won in 18 (19) out of the 30 groups in IT (CT) treatment. Therefore, and regardless of the treatment, around the 62% of the subjects succeed in coordinating their decisions to achieve the social benefit. It is noteworthy that the coordination mechanism rarely follows the SPNE. As a matter of fact, it only happened once (3.3%) in each treatment. Furthermore, the opacity of the second player’s decisions does not seem to significantly hinder cooperation. This finding leads to our first result.Result 1The one-sided lack of transparency does not reduce the levels of cooperation to get the final reward.Although the blindness of Type A does not seem to worsen coordination, the lack of transparency has an impact on the decisions of the players and, consequently, on the final distribution of payoffs. The results of Result 2Second players exploit their power of extraction and get a higher profitability than first players. However, transparency smoothens such behavior while the reward is still available.This treatment effect is reinforced by the two additional findings. The first one is that in IT the distribution of profits is less egalitarian for the successful groups in IT (Type A/Type B average profits are 45.722/54.278) than in CT (Type A/Type B average profits are 46.737/53.263). More importantly, 7 out of the 19 succeeding groups (37%) achieved an egalitarian distribution of profits (50 tokens each) in CT whilst only 2 out of the 18 groups (11%) coordinated their strategies to yield the same outcome in IT. The second one is that the average breach period in IT (4.917) is almost two times that of CT (2.818) as a consequence of the opacity of the second players’ actions. The next result summarizes these findings:Result 3The one-sided lack of transparency yields to a more asymmetric profit distribution for coordination profiles. When coordination fails, the one-sided lack of transparency delays the breach period.treatment (a dummy variable scoring one in IT and zero in CT) reduces the probability of having an egalitarian share and increases the breach period. Additionally, we also find that both the probability of egalitarian distribution of profits and the breach period depend positively on the variable patience (the number of consecutive times in which second player takes one token after observing that first player has taken two tokens). Regression analyses and tests in Finally, we investigated on the determinants of the players’ choices. Result 4The first choice of the first player is positively correlated with the choices of both players during the game and represents a signal for the willingness to cooperate.Furthermore, Type A’s choices in both treatments depend positively on Type B’s actions in the previous period  and on the errors in guessing Type B’s choices (i.e. deception on Type B’s expected choices makes Type A play harder). The latter assessment is based on the guesses about partner’s choices that were elicited in every period during the experiment (before taking their decisions subjects were asked to guess the other player’s action). These guesses were not incentivized. Observe that the positive correlation between the choices of Type A and the lagged choices of Type B in IT implies that Type B is capable of predicting the actions of Type A to some extent (as in the Lazarillo’s story). The choices of Type B, however, depend negatively on the contemporaneous choices of Type A in both treatments since Type B can always observe Type A’s movement and her decision is conditioned on it. The negative correlation is likely induced by her attempts to keep the reward available. Finally, misperceptions about the expected choices of Type A affect negatively to her choices .There exist many different situations where if agents coordinate their actions according to a certain rule of sustainability they get a social externality . In all of these situations it seems that the asymmetric lack of transparency makes the agents more aggressive on their decisions (short term profit maximizers) and put the social benefits of coordination/cooperation under jeopardy. We designed a new experiment to study the effects of the asymmetric information on the actions of individuals facing such problems. Unlike other related games based on the prisoner’s dilemma, the induced game is sequential, although we endowed the second player with the power to play harder than her opponent (by taking more tokens) to offset to some extent her disadvantage of being the second mover. This is a key issue of our experiment since we focus on the reactions of the second player on the first player’s actions when her own actions can be hidden.The experiment, inspired by the tale of the Lazarillo of Tormes, asks two agents to sequentially extract tokens from a finite common pool until there is no token left. Since we are interested in analyzing which asymmetry is more relevant, either the advantage of moving first or the larger capacity of extracting, we let the second player extract more tokens than the first player in every round . In the Control treatment there is full information about the actions but in the Impunity treatment only the second player can observe the decisions of her partner. We also include a valuable reward for both subjects  in case subjects coordinate their actions in such a way that in every round at least one of them had taken a single token.breach period in case of coordination failure. Furthermore, we find that the first choice of the first player determines the behavioral dynamics of the game. Most of first players that started cooperating (aggressively) succeed (fail) in winning the prize.Under these conditions we observe that the larger capacity of extracting outweighs the advantage of being the first mover and thus second players achieve higher profitability. This occurs regardless of the failure or success in coordinating to get the final reward, as well as in both treatments. Although, the one-sided lack of transparency does not seem to significantly affect the level of cooperation, it plays an important role in three different dimensions: (i) It increases the ‘abuses’ of the second player while the reward is still available, (ii) it yields more asymmetric profit distributions when subjects succeed in coordination (iii) it delays the In summary, the Lazarillo’s game results to be a useful scheme to understand cooperation in contexts of asymmetric informational conditions and highlights the importance of transparency in decision-making. The experimental evidence reflects that transparency reduces the abuses of the more informed subjects and induces more egalitarian profit distributions.Thank you for participating in this experiment. The purpose of this experiment is to study how people make decisions in a particular context. The instructions are simple and if you follow them carefully you will be able to win money in cash and confidentially (nobody will know the payments of the other participants). In the experiment there are neither correct nor incorrect answers. Do not think that we expect a particular behavior. However, notice that your decisions will affect your payments. If you raise your hand someone will answer your questions, but no other communication is allowed during the experiment.In this experiment there is a participant A and a participant B. Each participant A will be randomly matched with the same participant B during the whole experiment.At the beginning of the experiment you will know whether you are a participant A or B, which will be randomly assigned by the computer. The identity of your matched partner will be anonymous during and after the experiment.The experiment has a number of rounds in which both partners will have to share 60 tokens.Every round has two steps.Step 1: Participant A may take either one or two tokens in every round.Step 2: Participant B, after observing the action of participant A, may take either one, two or three tokens in every round.in every round at least one of the participants had taken one token, then both participants will get 20 extra tokens at the end of the experiment.If Participant A will never know the number of tokens taken by participant B in each round.At the end of the experiment you will be privately paid your earnings. Your final payment will be the sum of the tokens you had accumulated during the experiment. The tokens will be converted to Euros at the rate 1 token = €0.35.If you are the Participant A… you can take one or two tokens per round.If you are the Participant B… you can choose one, two or three tokens per round and you can observe the tokens taken by Participant A.In every round if at least one of the participants have taken only one token then both participants will receive 20 extra tokens at the end of the experiment.To make sure that you have understood these instructions and before starting the experiment you have to answer a simple questionnaire and then you will be able to proceed with the experiment in your computer.Participant A = ____ tokensParticipant B = ____ tokensIf Participant A has taken a total of 22 tokens and Participant B has taken a total of 38 tokens and, at least in one round none of the participants had taken just one token, then which is the final payoff (in tokens) of both participants?Participant A = ____ tokensParticipant B = ____ tokensIf Participant A has taken a total of 22 tokens and Participant B has taken a total of 38 tokens and, in all rounds at least one of the participants had taken only one token, then which is the final payoff (in tokens) of both participants?S1 FileData obtained from the experiment.(ZIP)Click here for additional data file.


Aortic valve regurgitation (AR) imposes a volume overload (VO) to the left ventricle (LV). Male rats with a pathological heart overload usually progress more quickly towards heart failure than females. We examined whether a sexual dimorphism exists in the myocardial transcriptional adaptations to AR. Adult Wistar male and female rats either underwent a sham operation or were induced with AR and then followed for 26 weeks. Female AR rats gained relatively more LV mass than males (75 vs. 42%). They had a similar increase in LV chamber dimensions compared to males but more wall thickening. On the other hand, fatty acid oxidation (FAO)-related LV enzyme activity was only decreased in AR males. The expression of genes encoding FAO-related enzymes was only reduced in AR males and not in females. A similar situation was observed for the expression of genes involved in mitochondrial biogenesis or function as well as for genes encoding for transcription factors implicated in the control of bioenergetics and mitochondrial function . Although females develop more LV hypertrophy from severe VO, their myocardial gene expression remains closer to normal. This could provide survival benefits for females with severe VO. Women are more likely than men to develop HF with preserved ejection fraction (HFpEF) associated with diastolic dysfunction and concentric left ventricle (LV) remodelling. Unfortunately, no treatment has been proven effective for HFpEF. LVH becomes more prevalent in women after menopause and constitutes the strongest mortality predictor from HF2.In pressure overload (PO) diseases such as aortic valve stenosis or arterial hypertension, female patients usually develop more LVH, although they have better EF and less myocardial fibrosis than male patients4. Hypertensive women also develop more LVH compared to men even for similar blood pressure levels4. Volume overload (VO) typically induces eccentric LV hypertrophy. Sex differences in patients with volume overload (VO) diseases  remain mostly unstudied.Cardiac hypertrophy (H) is used as a prognostic indicator of progression towards heart failure (HF). HF occurs in women as frequently as in men but later in life and less often from ischaemic causes6. For instance, male mice with transverse aortic constriction  develop concentric LVH sooner than females7. LV remodelling in these male TAC mice later evolves towards eccentric LVH and HF symptoms8. Genes associated with extracellular matrix (ECM) production or with mitochondrial function are also more strongly modulated in males. Similar observations have been made in several other animal models of LVH and/or HF6. Two VO models have been studied in rats. Females with arteriovenous fistula (AVF) develop a similar amount or more LVH than males12. Progression towards HF is slower for females, and their survival rate is higher13. In the aortic valve regurgitation (AR) rat model, we observed that females developed more LVH than males and that removing oestrogens by performing an ovariectomy did not influence LVH development14.In pre-clinical heart disease models in rodents (either rat or mouse), oestrogens have been shown to slow the development of LVH, delay HF and improve survivalIn both pre-clinical PO and VO rodent models, the development of HF seems to be slower in females, but this may not be directly linked to levels of cardiac hypertrophy. Therefore, we wanted to investigate sex differences in myocardial transcriptional adaptations to severe VO from severe AR. We demonstrate that LV gene expression in rats with severe AR is significantly less altered in females, although they developed significantly more cardiac hypertrophy than males.All sham-operated animals were alive at the end of the protocol. Three male AR rats and one female AR rat died suddenly overnight before the end of the six-month follow-up period. No animals developed signs of heart failure, defined as dyspnoea, peripheral oedema or excessive weight gain. Table AR caused a significant gain of cardiac mass for both male and female rats Table . This ga15 resulting from AR was moderately higher in males than in females compared to sham controls . The myocardial performance index or MPI  was similar between male and female AR animals.As expected, AR caused marked LV dilatation, as illustrated by an increase in the end-diastolic dimensions Table  that wasHeart rates were lower in AR groups Table . Stroke We measured the variation of different echocardiographic parameters during the 26 weeks of the study by comparing values at the end of the protocol to those at baseline in order to emphasize both the contributions of the volume overload and normal growth on cardiac remodelling. As illustrated in Fig. We did not observe any significant differences between groups after 6 months in terms of interstitial LV fibrosis as estimated by Masson’s trichrome staining (not shown). Cardiomyocyte cross-sectional area (CSA) was increased in AR animals compared to sham animals, as illustrated in Fig. As expected, Anp and Bnp gene expression was elevated in AR animals Fig. . BaselinFatty acid oxidation (FAO) capacity was impaired in the LV of AR rats, as illustrated by the decreased hydroxyacyl-Coenzyme A dehydrogenase (HADH) enzymatic activity Fig. . This wa16. The expression levels of most of these genes were significantly decreased in male AR rats, whereas this decrease was only minimal in females  of these genes, as illustrated in Fig. We previously identified a group of 54 genes that were associated with mitochondrial function and down-regulated in male AR ratsThe main observation of this study is that the female sex is associated with a myocardial transcriptional profile that is closer to normal despite developing more hypertrophy in response to volume overload. There was more LV wall thickening in female AR animals, whereas chamber dilation was relatively similar, resulting in less eccentric LV remodelling compared to males. This remodelling was accompanied by lower levels of wall stress in females despite higher levels of hypertrophy and a similar degree of aortic valve regurgitation.18. The different LV remodelling pattern  also seems to result in lower wall stress, which may play a role in maintaining normal myocardial bioenergetics. On the other hand, the increase in LV wall stress caused by VO in female rats remained important. However, the moderate transcriptional and metabolic responses of the myocardium to this significant stress are surprising.Most bioenergetics and mitochondrial function markers we measured remained in the normal or near-normal range in female AR animals, whereas they were clearly lowered in males. This suggests that the cardiac hypertrophy level cannot be considered to have a similar predictive value for the progression towards HF for both sexes. Relatively normal myocardial bioenergetics in females in spite of the presence of more cardiac hypertrophy may explain why their progression towards HF is slower than in males with VO21. Dent and collaborators demonstrated the presence of sex differences in neurohormonal activation in the arteriovenous fistula (ARF) VO model. After 16 weeks of VO from ARF, they observed that the protein levels of both the β1 and β2-adrenergic receptors were decreased in male rats but increased in females. Catecholamine plasma levels were also significantly lower in females. This was accompanied with increased adenylate cyclase activity, suggesting that β-AR levels and function remained mostly normal in female ARF animals9. This could likely contribute to the maintenance of relatively normal energy metabolism and mitochondrial function as we observed in our study, considering the importance of this system in regulating metabolism23. We did not observe marked sex differences in the expression of the adrenergic receptors at the gene level  led to decreased LV hypertrophy in the AR rat model and better survivall Figure  between 26. In that PO model, the mRNA levels of Anp, Myh7 and Serca2a were all more altered in males than in females. In mice with TAC, males also developed similar levels of hypertrophy compared to females but displayed more myocardial fibrosis and progressed faster to HF27. Interestingly, many genes associated with mitochondrial function were more down-regulated in males than in females27. In our study, we observed after 6 months a general reduction in the expression of many mitochondrial function markers in males, while these markers remained mostly unchanged in females. We showed in a previous study that the amount of LV mitochondrial DNA in our model after 9 months was similar to controls16. However, this result did not prove that mitochondrial morphology was normal in our rats. Although we did not thoroughly evaluate mitochondrial function in this study, our transcriptional profile indicates that mitochondrial function is worse in male AR rats. Moreover, we observed that the activity of succinate dehydrogenase, an enzyme implicated in both the citric acid cycle and the respiratory chain, was reduced again only in male AR rats28. Pgc1α, an important factor in the control of mitochondrial biogenesis and fatty acid oxidation (FAO), followed the same expression pattern27. Pgc1α was previously demonstrated to be positively modulated by oestrogens, which could explain our observation. In a mouse model of doxorubicin-mediated cardiotoxicity, similar sex differences were observed. The male myocardial transcription profile of genes associated with mitochondrial function was more altered than the female profile. This correlated with poorer LV systolic function and survival29.Sex differences in the development of cardiac hypertrophy have been studied in several animal models. In Wistar rats with ascending aortic banding, LV hypertrophy was shown to be similar between the sexes, but progression towards HF was faster in males30. We also know from previous µPET studies performed in male rats with chronic AR that glucose utilization is also increased at this stage of the disease19. Our current results suggest that this energy substrate shift to glucose may have not taken place in female AR rats. Eccentric cardiac hypertrophy does not seem to trigger changes in myocardial substrate preference in female rats. It is interesting to note that myocardial capillary density seemed less affected in female AR rats, suggesting a better myocardial angiogenic response to severe VO31. This may result in not only better nutrient availability to the cardiac cells but also better oxygen availability, facilitating FAO, a highly oxygen-consuming process compared to glycolysis32. These observations support the need to investigate further and to study myocardial energy substrate avidity by µPET in animals of both sexes33.We observed that FAO was impaired in male AR animals. This is in accordance with previous observations made in this model35. Their impact is probably even more important than that of Pgc1α, which is not down-regulated in patients with HF36. Errα is also a known downstream target of the oestrogen receptor α, which could explain the maintained mRNA levels of various genes in females37.In addition to Pgc1α, the expression of both oestrogen receptor-related (Err) transcription factors (α and γ) was reduced in AR males but remained unchanged in females. Err transcription factors regulate genes involved in FAO, mitochondrial biogenesis and oxidative phosphorylation14. This previous study was too small to address whether oestrogen removal influences survival. The current results suggest that the female sex provides benefits, but we are not able to determine whether oestrogens are responsible for these benefits during the evolution of eccentric LVH. Dent and collaborators reported in the ARF model that ovariectomy was associated with less hypertrophy but more LV dilation and that exogenous oestradiol administration reversed this effect11. Interestingly, the presence or absence of oestrogens had important effects on the myocardial β-AR content, plasma catecholamine levels and adenylate cyclase activity in ARF rats. Oestrogens can also stimulate calcineurin degradation downstream of these signalling pathways, attenuating the evolution of cardiac hypertrophy towards HF38. In this study, oestradiol replacement therapy in ovariectomized TAC mice was also shown to limit cardiomyocyte elongation without affecting width, suggesting a capacity of oestrogens to delay eccentric hypertrophy, decompensation and possibly HF.The role of oestrogens in the rat AR model of LV remodelling is still unclear. Ovariectomy does not influence the level of LV hypertrophy in the rat AR model8. Heart adaptations to VO in women (with the exception of pregnancy) have received very little attention so far. Observations made in animals cannot necessarily be transposed to humans, and caution must be used. This study did not take into account ageing and menopause, factors that are highly relevant to heart disease in women. The effect of gonadectomy and hormone replacement therapy was not assessed in this study. We did not perform a thorough evaluation of mitochondrial function or neurohormonal status, but limited ourselves to primarily studying gene expression. It is likely that an analysis performed at the level of proteins, enzymatic activity or function would have provided more definitive conclusions.Our results support that the female sex provides benefits in keeping closer to normal myocardial function and bioenergetics in presence of severe VO and this despite important LV remodelling. This is in line with other observations relating to cardiac hypertrophy made in womenIn this study, we showed that the abnormal transcriptional pattern usually associated with pathological cardiac hypertrophy does not occur in female rats with severe aortic valve regurgitation, despite an important gain in heart mass. We observed that cardiac remodelling in AR female rats leads to moderately less LV eccentricity and wall stress, better capillary density and more stable expression of many myocardial bioenergetics and mitochondrial function markers. The precise role of oestrogens during LV eccentric hypertrophy development will need to be addressed in upcoming studies.40. Only animals with >60% regurgitation were included in the study. The regurgitant fraction was estimated by the ratio of the forward systolic flow time–velocity integral (VTI) to the reversed diastolic flow VTI measured by pulsed Doppler in the thoracic descending aorta.Wistar male (350–375 g) and female (225–250 g) rats purchased from Charles River  were studied for 180 days (26 weeks). The animals were divided into four groups: male sham-operated animals , female sham , males with aortic regurgitation  and female AR . The protocol was approved by the Université Laval’s Animal Protection Committee and followed the recommendations of the Canadian Council on Laboratory Animal Care. Severe AR was induced by retrograde puncture of the aortic valve leaflets under echocardiographic guidance as previously described41. A complete echo exam was performed before AR induction and at the end of the protocol as previously described43. LV mass was calculated by the following formula: 1.04((EDD + PW + SW)3 − EDD3). EDD, PW, and SW are end-diastolic diameter, posterior wall thickness, and septal wall thickness, respectively. Left ventricular mean wall stress was estimated using the formula developed by Quinones et al. SAP x ((EDD + ESD)/2 × (PWd + PWs)). SAP, ESD, PWd, and PWs are systolic arterial pressure, end-systolic diameter, diastolic posterior wall thickness, and systolic posterior wall thickness, respectively15. The hearts were harvested as previously described24.Left ventricular and arterial pressures and dP/dT (positive and negative) were measured invasively using a dedicated catheter under isoflurane anaesthesia 44.Sections from paraffin-embedded mid-LV portions were stained using Masson’s trichrome staining. Three sections per slide were studied for the evaluation of cross-sectional area (CSA) of the cardiomyocytes as described previouslyBandeiraea simplicifolia coupled with horseradish peroxidase . Capillary density was analysed in the subendocardial region of the LV myocardium (inner third) as described elsewhere31.Sections of 8-µm thickness were cut from the frozen LV and were stained with isolectin B4 from 45. QuantiTect (Qiagen) and IDT  Primer Assays  enzyme activities. Small pieces of LV (20–30 mg) were homogenized in a glass-glass homogenizer with either 9 or 39 volumes of ice-cold extracting medium pH 7.4  depending on the enzyme activity assayed. Enzymatic activities for hydroxyacyl-Coenzyme A dehydrogenase (HADH), hexokinase (HK), citrate synthase (CS) and succinate dehydrogenase (SDH) were determined as previously described46.Left ventricle samples were stored at −80 °C until assayed for maximal . All methods were carried out in accordance with the relevant guidelines and regulations.Supplementary table and figures


People with disabilities face challenges accessing basic rehabilitation health care. In 2006, the United Nations Convention on the Rights of Persons with Disabilities (CRPD) outlined the global necessity to meet the rehabilitation needs of people with disabilities, but this goal is often challenged by the undersupply and inequitable distribution of rehabilitation workers. While the aggregate study and monitoring of the physical rehabilitation workforce has been mostly ignored by researchers or policy-makers, this paper aims to present the ‘challenges and opportunities’ for guiding further long-term research and policies on developing the relatively neglected, highly heterogeneous physical rehabilitation workforce.The challenges were identified through a two-phased investigation. Phase 1: critical review of the rehabilitation workforce literature, organized by the availability, accessibility, acceptability and quality (AAAQ) framework. Phase 2: integrate reviewed data into a SWOT framework to identify the strengths and opportunities to be maximized and the weaknesses and threats to be overcome.The critical review and SWOT analysis have identified the following global situation: (i) needs-based shortages and lack of access to rehabilitation workers, particularly in lower income countries and in rural/remote areas; (ii) deficiencies in the data sources and monitoring structures; and (iii) few exemplary innovations, of both national and international scope, that may help reduce supply-side shortages in underserved areas.Based on the results, we have prioritized the following ‘Six Rehab-Workforce Challenges’: (1) monitoring supply requirements: accounting for rehabilitation needs and demand; (2) supply data sources: the need for structural improvements; (3) ensuring the study of a whole rehabilitation workforce (i.e. not focused on single professions), including across service levels; (4) staffing underserved locations: the rising of education, attractiveness and tele-service; (5) adapt policy options to different contexts , even within a country; and (6) develop international solutions, within an interdependent world.Concrete examples of feasible local, global and research action toward meeting the Six Rehab-Workforce Challenges are provided. Altogether, these may help advance a policy and research agenda for ensuring that an adequate rehabilitation workforce can meet the current and future rehabilitation health needs.The online version of this article (doi:10.1186/s12960-017-0182-7) contains supplementary material, which is available to authorized users. There is an estimated one billion people with long-term or residual disabilities around the globe: 15% of the world’s population , as mediPeople with disabilities can experience secondary health conditions resulting from their impairments , 11 and This paper focuses on the state of the physical rehabilitation workforce globally and the challenges people face in accessing physical rehabilitation workers. People with rehabilitation need or demand typically include those with long-term physical, cognitive and/or development impairments contributing to limitations in mobility, self-care, other daily activities and/or restricted social participation. People with temporary physical impairments  are also, for a period of time, in need for physical rehabilitation.Access to needed rehabilitation can be problematic for many reasons. First, in lower income countries, where the vast majority of people with disabilities live , 22, 23,The study and monitoring of the rehabilitation workforce, and how people with disabilities access them, has been mostly ignored by researchers and policy-makers , 32–35. Universal health coverage, a commitment of Member States of the United Nations and a Sustainable Development Goal frequently seen as an ‘ultimate expression of fairness’ , 40, canThe rehabilitation health workforce supply consists of many different configurations of professions. This includes physicians specialized in physical medicine and rehabilitation, physical therapists (PTs), occupational therapists (OTs), speech-language pathologists, prosthetic and orthotic practitioners, and PT/OT assistants, among a wide array of other health workers and family supplying the population’s physical rehabilitation needs. In addition to that heterogeneity in its whole composition, the existence, practices, education and competencies of any of those rehabilitation health workers often vary widely across countries, and even within the same country , 25.Phase 1: critical review of the rehabilitation workforce literature, focusing on the AAAQ framework: the availability, accessibility, acceptability and quality  of the pPhase 2: integration of reviewed data into a SWOT framework  to identThis paper aims to identify long-term ‘challenges and opportunities’ for advancing the global study, monitoring and development of the relatively neglected, highly heterogeneous, physical rehabilitation workforce. To do so, we have conducted a two-phased investigation:Searches for the relevant literature were conducted in PubMed, covering the period between March 2006 and March 2016, using the following MeSH terms: ‘Manpower’ OR ‘Health Manpower’ AND rehabilitation-related terms, abstracted from previous studies finding physical rehabilitation content in PubMed , 44. AddSecondary searches  were also performed. The World Report on Disability  was alsoPapers were primarily selected, and their content abstracted, if published in English and potentially fitting into any category of the AAAQ framework . Table 1Papers finally included in the review were selected, at the synthesis stage, according to the following criteria: more recent since 2008), specific for the (sub-)topic addressed, and whose content was not synthesized/addressed by any included systematic review. Additional file 008, specA SWOT analysis  was condTable Originally from the management literature , SWOT anThe rehabilitation workforce literature commonly reports important limitations in the supply data sources , 50–53.First, mandatory professional registration/licensing mechanisms for rehabilitation workers are absent in many countries, especially lower income countries , 54–57. Second, the Global Atlas of the Health Workforce provides no data on a specific category of rehabilitation workers, who are typically aggregated under ‘other health workers’, with unrelated professions such as ambulance workers .All of this is complicated by the lack of uniform international definitions/classifications of who are rehabilitation health workers, and by policies that continue to place the monitoring of rehabilitation workers low on the health agenda, in turn related to how societies often interpret and react to disability , 24, 50.Finally, terminologies used to describe the same profession  vary. More importantly, their competencies, education, credentials and typical practices also vary within and across countries or practice locations, for the same profession , 58, 59.Determining rehabilitation workers’ supply requirements is made on the basis of population size , 53, 60,Substantial needs-based shortages of rehabilitation workers are documented and projected in many places around the globe , 62, 63.Although scarcely studied, international migration appears to aggravate global inequalities: in the United States of America (USA), foreign-educated, recently licensed PTs came predominantly from the Philippines and India . SingapoAmong high-income countries, supply variability also exists , 24, 25:Few programs exist for educating qualified rehabilitation workers in lower income countries , 25, 56.Access to rehabilitation services and workers is usually harder in rural or remote areas –71. ThisIn low-income countries, people in need are significantly challenged to access any rehabilitation health workers , 82. LacHome , communiIn some high-income countries , patient’s self-referral to rehabilitation workers has been increasingly implemented . BesidesCoverage gaps typically affect more the socially vulnerable people with disabilities, under- or uninsured, resource-poorest, belonging to disadvantaged race/ethnic groups and those living in rural or remote areas: this phenomenon of ‘double disparities’ accentuate the vicious circle of disability and social disadvantage , 95.Finally, rehabilitation services delivered outside hospitals are typically less funded, less attractive to rehabilitation workers, financially and academically, and thereby less available to those in need , 96, 97.Some initiatives report promoting culturally competent rehabilitation workers. These include studies focused on tailoring approaches to indigenous populations in Oceania , 99 or tThe need for same-sex provider applies to rehabilitation in some cultures . Female Understanding and changing families’ perceptions of disability and rehabilitation may enhance children with disabilities’ access to rehabilitation . CommuniThe competencies, skills and practices of rehabilitation workers can vary substantially across countries or practice locations.PTs successfully take on advanced competencies such as ordering X-rays or making musculoskeletal ‘diagnosis’ in some jurisdictions within high-income countries  , 111–113PTs/OTs with more advanced practices increasingly delegate high-volume, less-skilled tasks to PT/OT assistants , 117, inWhere demand clearly exceeds supply, a cross-disciplinary assimilation of practices occurs more among rehabilitation workers , 25, 59.A SWOT analysis was made of the critical review results Table .Major strengths (S) relate to some specific research: the literature broadly identifies where higher unmet needs for rehabilitation health workers exist, e.g. in lower income countries, and in rural regions elsewhere. The literature also identifies distance education and international clinical education and service placements, from higher to lower income countries.Major weaknesses (W) are the sub-development of supply data sources and of monitoring mechanisms. A uniform, international classification for defining different competencies, constituents and practices of rehabilitation workers is lacking. Also, there is no agreed strategy to determine rehabilitation supply requirements.Opportunities (O) include possibilities for global scaling-up of some exemplary initiatives, for example from Canada and Australia, aimed at attaining rehabilitation workers willing and capable of working in underserved areas. Locally tailored policy solutions (e.g. outreach programs) and innovative service delivery models like tele-rehab are also increasingly tested to enhance access in underserved areas. Solutions for undersupply and inadequate skill mix of rehabilitation workers  may be widely applicable.Threats (T) emanate from the inherent complexity of the physical rehabilitation workforce, which may assume multiple configurations of professions, varying in education, competencies and practices, even within the same profession. The risk is to try to overcome this complexity by oversimplification, e.g. only monitoring specific professions instead of monitoring also the whole physical rehabilitation workforce. Finally, in the face of that complexity, and the low policy priority given to disability and rehabilitation, there is a risk of a continued negligence in the study and development of the rehabilitation workforce.This section focuses on presenting six rehabilitation workforce challenges identified through the literature review and SWOT analysis. As a whole, identification of these challenges could advance the rehabilitation workforce agenda and inform researchers and policy-makers on advancing rehabilitation workforce studies and policies.The challenges are displayed in Fig. Many studies reviewed did not account for rehabilitation supply requirements beyond population size , 32, 60.Toward that end, the concepts of ‘need’  and ‘demand’  might be considered, with their relative pros and cons. For example, using demand requirements can lead to perpetuating systems’ inefficiencies, by allocating more resources where least needed. On the other hand, using need indicators may not account for whether population will actually use rehabilitation services, e.g. by lack of financial coverage or population unawareness that these resources are available , 121–123Finally, the most suitable metrics and measures of need and demand to be used as rehabilitation supply requirements also need to be determined, inclusively considering recent practices and advancements in disability measurement –126.Studies commonly report important limitations in the availability of accurate, reliable, comprehensive, disaggregated (by profession and working sector) and comparable supply data , 24, 32.The monitoring of the rehabilitation workforce would need (i) investments in the collection and analysis of national rehabilitation workforce data, facilitated by the utilization of a minimum data set and registration of practitioners; (ii) agreed professional definitions, classifications and credentials; and (iii) improved availability of data, in formats that do not aggregate rehabilitation health workers with other occupational groups. For any international comparisons, definitions and classifications should be globally standardized, as much as feasible.The rehabilitation workforce is principally composed of professionals specifically trained to provide rehabilitation care, but many other health workers, even non-health workers, sometimes meet the physical rehabilitation needs of the population. Examples are physicians, nurses, community health workers, athletic trainers and even special education teachers.When meeting those needs, all these groups, within their specific competencies and practices, need to be considered when estimating the supply of rehabilitation workers.Finally, for a comprehensive determination of the rehabilitation workforce and of the unmet needs, studies can include how this workforce is allocated across employment sectors , the healthcare continuum  and practice locations  , 96, 97.Lower income countries and rural and remote regions are typically underserved by health workers and more so by rehabilitation health workers. Policy options to address this challenge may include a number of possibilities which can and should be mutually complementary.First, some rehabilitation education programs might focus on the competencies for working in underserved contexts, while clinical education and field experience in those locations are also useful –103, 111Second, for those already working in underserved locations, it is important to design and implement retention measures, like opportunities for distance learning, career development and support systems . Other eA third option is to develop remote services delivered by tele-means , 91, 129Finally, health workers such as physician, nurses or community health workers also can be trained to acquire and apply rehabilitation competencies within their practices , 136.Currently, few examples exist for any of those initiatives, and encouraging countries to introduce them is a major challenge.Meeting rehabilitation workforce needs, and ultimately the population rehabilitation needs, may require different solutions for not only different countries but also within a country  , 41, 59.The key challenge in ‘well-served’ areas (e.g. metropolitan areas in high-income countries) is to create conditions for the entire rehabilitation workforce to perform at the top of their credentials, to achieve the best outcomes at the lowest cost.That may include policy action such as (i) designing and implementing mechanisms  for higher accountability for the value of care ; (ii) reAll of this can be productive if well-planned, studied and supported by adequate training, supervision or decision-making algorithms , 119.Savings from efficiency gains in ‘well-served’ locations can be re-allocated to ensure that the more vulnerable population has access to the rehabilitation health workers they need , 33, 137While policies for universal rehabilitation coverage might be in place, supplying underserved areas may also require implementing (i) trans-disciplinary models of rehabilitation practice , 77, 111While locally tailored solutions are certainly appropriate, that does not mean that global, integrative solutions do not apply.For instance, international migration of rehabilitation workers and its determinant have been under researched. Such studies are best achieved when using data from both sending and receiving countries. Besides, policies may aim to take benefit from international mobility, instead of just mitigating potential perverse effects of the so-called brain drain , 127.For example, international clinical placements and temporary exchange programs of clinicians and students, inclusively among countries of high and low income , 140, caFinally, international health technical aid is required on a regular basis , but morAlthough using a structured PubMed search, the critical review does not reflect a systematic or scoping review approach, so these results should not be understood as such. Besides, exclusion of non-English papers and the absence of a structured search for the grey literature can turn the literature reviewed under-representative of certain countries’ scenarios (e.g. Latin America countries). Also, to provide a notion of the range of findings on each point being made, we often provide examples from high- and low-income countries. This does not obviate the need to study and develop the many countries’ realities in between.Global development policies, such as the Sustainable Development Goals, aim to improve the lives of marginalized groups, by reducing the burden of diseases and poverty. Meeting the rehabilitation needs of people with disabilities who are marginalized, have lower health status, lower healthcare access, often live in poverty and are limited in their social functioning would contribute to all these goals. Inspired by the Six Rehab-Workforce Challenges, which seem aligned with the global strategy on human resources for health , action Local policy-makers might (i) determine the local needs and/or demand for rehabilitation; (ii) develop mechanisms to monitor the available rehabilitation workforce, including across geographies and service levels; (iii) take action to reduce needs-based shortages of rehabilitation workers ; and finally (iv) promoting rehabilitation systems strengthening, including the implementation of cost- and supply-effective service delivery models  and response outreach programs cutting across traditional silos.Global policy-makers might (i) assure that more resources are allocated, equitably, to the study and development of the rehabilitation workforce; (ii) define international standards for assessing rehabilitation needs; (iii) develop uniform classifications of rehabilitation workers, competencies and practices; and finally (iv) support partnerships across countries for scaling-up local training and even for the cross-national service provision.Lastly, human resources for health researchers and their funders must include rehabilitation workers in their agenda. If this is not done, the growing advances in the rehabilitation science and practice , 148 wil


Airway smooth muscle (ASM) hyperplasia is a key feature of airway remodeling in development of lung diseases such as asthma. Anomalous proliferation of ASM cells directly contributes to ASM hyperplasia. However, the molecular mechanisms controlling ASM cell proliferation are not completely understood. Semaphorins are versatile regulators of various cellular processes including cell growth and proliferation. The role of semaphorins in ASM cell proliferation has remained to be addressed. Here, we report that semaphorin 3A (Sema3A) receptor, neuropilin 1 (Nrp1), is expressed on human ASM cells (HASMC) isolated from healthy and asthmatic donors and treatment of these cells with exogenous Sema3A inhibits growth factor-induced proliferation. Sema3A inhibitory effect on HASMC proliferation is associated with decreased tyrosine phosphorylation of PDGFR, downregulation of Rac1 activation, STAT3 and GSK-3β phosphorylation. Bronchial sections from severe asthmatics displayed immunoreactivity of Nrp1, suggestive of functional contribution of Sema3A-Nrp1 axis in airway remodeling. Together, our data suggest Sema3A-Nrp1 signaling as a novel regulatory pathway of ASM hyperplasia. Asthma is a chronic inflammation of conducting airways in association with airway hyperresponsiveness (AHR) and remodeling . StructuProliferation of ASM cells induced by a wide spectrum of mitogens  has been proposed as a primary mechanism underlying increase ASM mass . SeveralSemaphorins, identified as a family of conserved secreted or membrane-bound axon guidance proteins, are involved in orchestration of various cellular processes including cell proliferation beyond the nervous system , 8. It hSemaphorin 3A (Sema3A) can function as a chemorepulsive cue inhibiting axonal outgrowth during neural development . Sema3A in vitro and ex vivo; and evaluated effect of Sema3A treatment on human ASM cell (HASMC) proliferation. We revealed that HASMC constitutively express Nrp1 in vitro. ASM bundle within bronchial biopsies from mild, moderate and severe asthmatics display Nrp1 expression. Sema3A significantly inhibited HASMC proliferation induced by platelet-derived growth factor (PDGF) via suppressing PDGFR, STAT3 and GSK-3β phosphorylation, and Rac1 GTPase activation. Our data suggest that Sema3A and its receptor are involved in the regulation of ASM hyperplasia in chronic airway diseases such as asthma.In this study we investigated the expression of Sema3A binding receptor Nrp1 on HASMC It has been previously shown that Sema3A binds directly Nrp1 expressed on the surface of target cells , 18. Expin vivo expression of Nrp1, we stained bronchial tissue sections obtained from healthy, mild and severe asthmatics using specific mAb. As shown in Figure in vitro and ex vivo.To evaluate n = 3, P < 0.01) and asthmatic donors . As demonstrated in Figure n = 4, P < 0.01). These results were further confirmed by performing manual cell count and trypan blue exclusion in HASMC isolated from healthy . Collectively, our studies suggest that Sema3A negatively regulates HASMC proliferation that involves Nrp1 receptor.To determine whether Sema3A inhibitory effect on HASMC proliferation is mediated via Nrp1, cells were treated with recombinant Nrp1 and stimulated with PDGF or PDGF combined with Sema3A , 23. SinReceptor tyrosine kinase, e.g. PDGFR β, phosphorylation plays a pivotal role in intracellular signal transduction activated upon growth factor stimulation . Therefon = 4, P < 0.01). Sema3A alone did not significantly affect HASMC proliferation which may explain its non-significant effect on basal HASMC proliferation in response to Sema3A.Rac1 GTPase has been previously shown as a key mediator which contributes to PDGF-induced ASM cell proliferation . TherefoAs reported previously, PDGF induced phosphorylation of both GSK3-β  and STATDysregulated ASM cell proliferation leads to increased ASM mass which is associated with airway narrowing in some airway disorders including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis . Deciphein vivo. However, comparison between healthy and asthmatic HASMC revealed similar levels of Nrp1 mRNA and protein surface expression. Expression of Nrp1 has been extensively studied in visceral smooth muscle cells but not in those of ASM cells [Here, we revealed that the Sema3A receptor, Nrp1, is expressed on HASMC from both healthy and asthmatic subjects suggestive of a functional contribution in ASM biology. We further confirmed expression of Nrp1 on human ASM bundles in healthy and asthmatic lung tissue sections indicating a potential unknown role of Sema3A-Nrp1 axis SM cells -32. SeleBlockade of Sema3A inhibitory effect by exogenous human recombinant Nrp1 treatment suggests that Sema3A signaling might be initiated through this receptor on HASMC surface. It should be mentioned that the repulsive role of Sema3A in growth cone guidance during development of nervous system is mediated through Nrp1 and plexinA1 as the ligand-binding and signal transducing elements, respectively . In factInteraction of PDGF-BB with the PDGFR induces dimerization which is essential for the activation of tyrosine kinase receptors . SubsequPrevious studies have demonstrated the non-redundant role of GSK-3β pathway in HASMC proliferation . In addiSTAT3 is also a key signaling element of HASMC proliferation which has been shown to physically interact with Rac1 GTPase in these cells . As a noIt has been demonstrated that Sema3A inhibits dendritic cell (DC)-induced T cell proliferation via affecting polymerization of cytoskeletal filamentous (F)-actin and early events of T cell activation . In factThe finding that the Sema3A-Nrp1 inhibitory axis is an upstream effector of the signaling cascade Rac1, GSK-3β and STAT3 provides an additional regulator of HASMC proliferation which had not been shown previously. In this study, we established Sema3A as a novel regulator of HASMC proliferation by suppressing Rac1, GSK-3β and STAT3 signaling through direct binding to Nrp1. However, other signaling pathways involved in regulation of HASMC proliferation such as PDGFR, MAPK/ERK and PI3K/Akt may also be negatively affected upon Sema3A treatment. Future studies may elucidate the potential role of other intracellular signaling components in Sema3A-inhibited HASMC proliferation.in vivo strategies to treat ASM hyperplasia based on Sema3A-Nrp1 axis and its downstream signaling. In summary, our study established Sema3A as a novel regulator of HASMC proliferation by suppressing Rac1, GSK-3β and STAT3 signaling mediated by Nrp1. Our findings raise the possibility of using Sema3A as a new therapeutic target to inhibit HASMC proliferation leading to minimize airway remodeling.Indeed, Sema3A and signaling mediators studied here are ubiquitously expressed in several cell types beyond HASMC and play diverse roles at both homeostatic and pathological conditions in other tissues. Therefore, specific targeting of HASMC should be carefully considered when designing novel Bronchoscopy was performed according to procedures approved by the Human Research Ethics Board of Laval University, Quebec, Canada. Written informed consent was obtained from each individual. Bronchial biopsies were obtained from healthy, mild/moderate, and severe asthmatic individuals fulfilling the American Thoracic Society selection criteria. The clinical characteristics are depicted in Table Primary HASMC were obtained from healthy non-asthmatic and asthmatic individuals in accordance with procedures approved by the Human Research Ethics Board of the University of Manitoba, Winnipeg, Canada and the Ethics Committee at University Hospital Basel, Switzerland. The clinical characteristics of asthmatic patients used to isolate HASMC have been mentioned in Table Recombinant human Sema3A, Nrp1 and PDGF-BB as well as APC-conjugated mouse anti-human Nrp1 monoclonal antibody and also sheep anti-human Nrp1 affinity purified polyclonal antibody were purchased from R&D Systems  and FITC-conjugated anti-human Nrp1 was from Abcam . Cell culture media were obtained from Invitrogen . FBS and Insulin-Transferrin-Selenium-X (ITS-X) were purchased from HyClone Laboratories  and Invitrogen, respectively. All other reagents are from Sigma-Aldrich unless otherwise indicated.HASMC were isolated from lower tracheal section of healthy non asthmatic donors from whom recipient cannot be matched. Asthmatic bronchial HASMC were isolated from dissected ASM bundles of the bronchial biopsies from mild asthmatics. The patients did not manifest exacerbations and received no medication 1 week before bronchoscopy. HASMC were used at passage 2-5 in all experiments. HASMC were grown on plastic dishes in DMEM containing 10% FBS to reach about 70% confluency and then serum starved to synchronize for 48 h in Ham's F12 supplemented with ITS.-ΔΔCt method. All RT- and q- PCR experiments were performed using the following primers. The forward and reverse-specific primer sequences and the size of the amplicon for NRP1 were FWD5'TATTCCCAGAACTCTGCCC3'and REV5'TGTCATCCACAGCAATCCCA3’, 253 bp and for GAPDH were FWD5'AGCAATGCCTCCTGCACCACCAAC3'and REV 5’-CCGGAGGGGCCATCCACAGTCT-3’, 137 bp. The annealing temperature and the number of cycles for both NRP1 and GAPDH were 60°C and 30 cycles.Total RNA was extracted from HASMC using TRIzol™ (Life Technologies). 2 μg of RNA was subjected to MultiScribe™ Reverse Transcriptase to synthesize cDNA. Expression of NRP1 and GAPDH was analyzed by conventional RT-PCR in healthy primary HASMC. Expression of NRP1 was also compared between healthy and asthmatic HASMC by using qPCR. GAPDH was used to normalize mRNA quantification. Expression values and statistical significance were calculated using the 2® . 1x105 HASMC were incubated with either APC-conjugated mouse anti-human Nrp1 or IgG2a as isotype control for 30 min at 4°C. The cells were washed twice, resuspended in flow buffer and acquired on FACSCanto™ II flow cytometer . Nrp1 surface expression was analyzed by FlowJo software .Primary cultured HASMC were detached using VerseneHASMC were seeded onto sterile glass coverslips, fixed in 4% paraformaldehyde; permeabilized and then non-specific Ab binding was blocked. Next, cells were labelled with sheep anti-human Nrp1 Ab or either sheep IgG and incubated overnight at 4°C. HASMC were incubated with biotin labelled rabbit anti-sheep IgG followed by addition of streptavidin-alkaline phosphatase for 1 hour. Development and counterstaining were performed by using Fast-red and modified Mayer's haematoxylin , respectively. Finally, mounted coverslips were visualized by AxioVision software .Paraffin embedded sections were dewaxed in xylene and then rehydrated through graded concentrations of alcohol to water. Antigen retrieval was performed by boiling for 10 min in sodium citrate buffer . Sections were incubated with blocking buffer  for 1 h at room temperature. After 3 washes with TBS, sections were incubated with rabbit anti-human Nrp1 (10 μg/ml) at 4°C overnight. For double immunofluorescence staining, sections were incubated with both rabbit anti-human Nrp1 and mouse anti-human alpha smooth muscle actin (αSMA) (4 μg/ml) Ab. After washing, sections were incubated with either goat anti-rabbit Alexa 488 alone or with goat anti-mouse Alexa 568 for 1 hr at room temperature, followed by washing and mounting with an anti-fade containing 4′,6-diamidino-2-phenylindole (DAPI). Sections were visualized with Zeiss microscope by using AxioVision software .® 488). Finally, EdU incorporation into newly synthesized DNA as an indicator of cell proliferation was measured by flow cytometry. To further confirm EdU incorporation data, serum-starved HASMC were treated with different concentrations of Sema3A  with or without PDGF (10 ng/ml) in a different set of experiments. In some EdU incorporation experiments, a control group was included in which HASMC were stimulated with the same Fc subclass as Sema3A-Fc to demonstrate that Fc portion does not influence HASMC proliferation. After 48 h, HASMC were counted manually using a hemocytometer and cell viability was determined by trypan blue exclusion in a randomized manner.Serum-starved HASMC were stimulated with Sema3A at presence or absence of PDGF and cell proliferation was assessed using Click-iT™ EdU flow cytometry assay kit (Life Technologies). Briefly, 10μM of a thymidine analogue 5-ethynyl-2′-deoxyuridine reagent was added to HASMC 16 h after stimulation and cells were harvested 24 h later. Cells were fixed, permeabilized and then stained with Click-iT reaction cocktail containing copper sulphate and fluorescent dye azide . Snap-frozen HASMC lysates in liquid nitrogen were subjected to Rho binding domain of Rac1 which was pre-coated in a Rac-GTP affinity 96-well plate. Then, Rac1-GTP as the active form was detected using primary and HRP-conjugated secondary Abs followed by chemiluminescent development.Protein lysates obtained from G-LISA were used for PDGF receptor (PDGFR), GSK-3β and STAT3 signaling studies. 10 μg of each lysate was loaded on 10% SDS-PAGE and transferred to PVDF membranes. Non-specific binding sites were blocked by adding non-fat milk followed by overnight incubation with phosphorylated GSK-3β (S9) and STAT3 (Y705) at 4°C. PDGFR β phosphorylation was studied by using an antibody sampler kit . It contains specific monoclonal antibodies against total form of PDGFR β as well as five tyrosine phosphorylation sites  on PDGFR β. Then, blots were incubated with HRP-conjugated secondary Abs and bands were visualized with ECL reagents. Total anti-PDGFR β, GSK-3β and STAT3 were used as loading controls. Densitometric analysis was performed by using Image Lab™ 4.1 software  and integrated density value was presented as the fold-increase in phosphorylated over total forms of GSK-3β and STAT3 compared to time zero.t test, one-way or two-way ANOVA. Bonferroni's multiple comparison post-hoc analysis was also performed. Differences were considered to be statistically significant at # and * P < 0.05, ## and ** P < 0.01.GraphPad Prism 5.0 software was used for statistical analysis and values were presented as the mean±SEM of at least three independent experiments. Data were analyzed by