In this study, we aim to investigate the effect and underlying mechanism of acupuncture at Taichong (LR3) on renal sympathetic activity in spontaneously hypertensive rats. Unanesthetized rats were subject to daily acupuncture for 2 weeks. Mean blood pressure (MBP) and heart rate variability (HRV) were monitored at days 0, 7, and 14 by radiotelemetry. After euthanasia on the 14th day, blood and the kidneys were collected and subject to the following analyses. Epinephrine and norepinephrine were detected by ELISA. The expression of β-ARs was studied by western blotting and PCR. The renin content was analyzed by radioimmunoassay. 14-day acupuncture significantly attenuates the increase of MBP. The HRV indices, the standard deviation of all normal NN intervals (SDNN), and the ratio of the low-frequency component to the high-frequency component (LF/HF) were improved following acupuncture. Renal sympathetic activation induced upregulation of epinephrine, norepinephrine, and renin content were attenuated by acupuncture. In addition, acupuncture decreased β1-AR expression and improved β2-AR expression. These results indicated that acupuncture relieves the increased MBP via the regulation of renal sympathetic activity and β-ARs.The sympathetic nervous system, via epinephrine and norepinephrine, regulates  Moreover, β1-ARs in the kidney may activate renin release [β-ARs are critical for regulating peripheral resistance and play important roles in the development of hypertension.Enhanced sympathetic nervous system activity is linked to the development and maintenance of hypertension –3. The e release  and, con release . Therefoβ-ARs remains unclear.Acupuncture has been recommended as a complementary therapy for hypertension. In 1996, 64 acupuncture indications were declared by World Health Organization in Milan conference including hypertension . Numerouβ1-AR and upregulation of β2-AR, leading to the decreased BP in SHRs.The present study was conducted to examine the hypothesis that the activation of the renal sympathetic nervous system in SHRs is mediated by acupuncture. Furthermore, we assessed if this renal nerve-mediated effect contributes to the downregulation of Male SHR and WKY rats (12 weeks), weighing 260–300 g, were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing China). The animals were housed in cages at 22 ± 2°C and humidity of 40 ± 5% under a 12-hour light/dark cycle and received standard diet and water ad libitum. The experimental procedures were in accordance with the Guidelines for the Institutional Animal Care and Use Committee of China Academy of Chinese Medical Sciences .The rats were randomly divided into 4 groups, the control group (WKY), the model group (SHR), the acupuncture stimulation group at LR3  (Acu), and nonacupoint acupuncture stimulation (Non-Acu), with 10 rats in each group. Sterilized disposable stainless steel needles  were inserted 5 mm deep at LR3. For the Non-Acu group, the rats received similar treatment as Acu group but the acupuncture site was at a sham acupoint  to replace LR3. The rats in the WKY and SHR groups were given the same time and same level catching-grasping stimulus without acupuncture stimulation. Acupuncture proceeded for 30 seconds each time, once daily for a period of 2 weeks (1 day rest after 6 days of treatment).We measured BP and HRV by radiotelemetry combined with fast Fourier transform analysis of BP and HRV. DSCF-FS01 HRV devices or DSCF-FS02 BP devices  were implanted in rats to follow BP and HRV changes continuously over time. The telemetric techniques and the techniques employed to analyze the autonomic nervous system are described in detail elsewhere . BrieflyData were sampled every 5 min for 10 sec continuously day and night and stored on a hard disk. BP and HRV were recorded using the SP 2006 Lan software. Continuous beat-by-beat values of BP and HRV were recorded during morning hours. Measurements to analyze the autonomic control of BP and HR were performed between 8:00 and 10:00 AM.NE and E contents in the plasma and kidney were measured by enzyme-linked immunosorbent assay (ELISA) kit  according to the manufacturer's instruction. Absorbance in each well was measured using microplate reader  at 450 nm. Concentrations of NE and E in the plasma and renal were determined by interpolation from the standard curve.μL of the cDNA was mixed with primers, 100 nM of probe, and 1x TaqMan Universal Master Mix in each reaction. Predeveloped assay reagents were used and were purchased from Applied Biosystems. Sequence for the primers and TaqMan probes for the rat β1-AR, β2-AR, and renin are shown in −ΔΔCT method using the control group as the calibrator according to the manufacturer's user manual. The value of relative mRNA quantity for control group is 1 with arbitrary units.Total RNA was extracted from the kidney using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. Quantification of the relative amounts of mRNA was performed using the Rotor-Gene6000 1.7 version software (Corbett research). In brief, 2 μg) from each sample was fractionated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane . Membranes were incubated in 5% milk in TBS for 1 h in room temperature. For identification of β-ARs, membranes were exposed to primary antibodies . After washing, the membranes were incubated with secondary antibodies . The bound antibodies were detected using the enhanced chemiluminescent reagent . Data are presented as the β-ARs to actin ratio and then expressed as fold-change compared to WKY group.Total protein in each sample was measured by the BCA assay  using bovine serum albumin as the standard protein. A fixed amount of protein  using a Polytron homogenizer (Kinematica), and centrifuged at 4°C at 140,000g for 5 minutes. Supernatants were incubated with saturating concentrations of rat renin substrate, and angiotensin I generation was assayed by radioimmunoassay (DiaSorin).For determination of PRC, 50 P value < 0.05 was considered statistically significant.Data analysis was performed with SPSS software version 16.0. All values were expressed as the mean ± standard error (SEM). Comparison between the treatment group and the control group was performed by one-way ANOVA test followed by a post hoc least significant difference multiple comparison test. A P > 0.05), but significantly higher than in the WKY rats. After 7 days of acupuncture, the MBP was significantly decreased compared with SHR group and Non-Acu group (P < 0.01), and this decrease was sustained throughout the treatment period (days 7–14). Although the MBP of Acu group was significantly decreased, it was still higher than the WKY group (P < 0.01) over the experimental period. These results suggested that acupuncture could lower BP but is unable to reduce it to normal levels.To confirm the efficacy of acupuncture, BP levels were measured in all groups by the telemetry method after two weeks of acupuncture treatment . Before P > 0.05). In the 14 days of acupuncture, the SDNN was significantly increased compared with the SHR group and Non-Acu group (P < 0.01). However, there was no significant change in RMSSD among the four groups (P > 0.05).To investigate whether the attenuation of BP in SHRs by the treatment with acupuncture is associated with beneficial effects of the automatic nervous system, we used radiotelemetry method to examine the heart rate variability (HRV) in all groups at days 0, 7, and 14 Figures . The valP > 0.05). On the 14th day, acupuncture significantly reduced the ratio of LF/HF compared with the SHR group and Non-Acu group (P < 0.01).The ratio of the low-frequency component to the high-frequency component (LF/HF) shows acupuncture effects on autonomic regulation. LF/HF of the SHR group, Acu group, and Non-Acu group were significantly higher than that of the WKY group at days 0 and 7  and epinephrine (E), acting as neurotransmitters, play important roles in the sympathetic control of arterial BP. Therefore, we tested the contents of NE and E in plasma and renal tissues by ELISA . At the α1A, α1B, α1D, α2A, α2B, α2C, β1, β2, and β3. In this study we focused on two of these receptors, β1 and β2. Both of these receptor subtypes have been strongly implicated in cardiovascular control. To determine whether the antihypertensive effect of acupuncture is associated with beneficial outcome in β-ARs, we detected the mRNA and protein levels of β-ARs after acupuncture treatment by PCR and Western blot, respectively . In contrast, β1-AR protein levels were similar between SHR group and Non-Acu group. The trends of the β-ARs mRNA levels were consistent with its protein levels.Both NE and E act at G protein-coupled receptors of the adrenergic receptor family to mediate sympathetic effects. Within this main classification, there are several subtypes ectively . Compareβ-ARs. Activation of β-ARs directly increases renin secretion. To address the role of acupuncture in the regulation of renin, we used radioimmunoassay to detect the concentration of renin. As shown in The juxtaglomerular (JG) cells in the media of renal afferent arterioles are the major sites of synthesis of the aspartic protease renin, the rate-limiting enzyme in the formation of angiotensin II. The JG cells are in contact with sympathetic nerve varicosities and express postjunctional β1-AR expression and increased β2-AR expression, which means acupuncture has bidirectional regulation effects on β-ARs.In the present study, we show that acupuncture at LR3 reduced MBP and regulated HRV in SHR. And the therapeutic effects were associated with renal sympathetic nervous system, which can be manifested by the significant reductions in the levels of E and NE (an indirect marker of sympathetic activity) and the content of renin  in the plasma and renal by acupuncture. In addition, acupuncture decreased Autonomic imbalance with increased sympathetic activity has been strongly implicated in the pathophysiology of hypertension. Among the different available noninvasive techniques for assessing the autonomic status, HRV has emerged as a simple, noninvasive method to evaluate the sympathovagal balance. In this experiment, we focus on three HRV indexes. SDNN is associated with overall autonomic tone. Therefore, our findings of depressed SDNN in SHR rats suggested a homeostatic disruption in sympathovagal balance. However, because rMSSD is generally associated with vagal pathways, our results with unchanged rMSSD suggest an increase in sympathetic tone in SHR rats. This result is consistent with Schroeder's finding that individuals with low SDNN at baseline were at an increased risk of developing hypertension over 9 years of follow-up . An analβ-ARs expression. Of the two main β-AR subtypes (β1 and β2), β1-AR signaling has been linked to cardiotoxicity. In contrast, β2-AR activates signaling pathways involved in cardioprotection. Gui et al. found that β2-AR has a vasodilatation effect, and Raf kinase inhibitory protein could correct impaired β2-AR to treat hypertension [β1-AR is the “cardiotoxic subtype” whereas the β2-AR is the “cardioprotective subtype.” The two receptors regulate the peripheral resistance which reflects the balance between vasoconstrictor and vasodilator mechanisms. Based on this, we examined the effect of acupuncture on E, NE, and β-ARs. Here we observed that the contents of E and NE of SHR rats were significantly higher compared with the WKY rats. This gives direct evidence that the sympathetic nervous system is hyperreactive in the SHR and is in general agreement with previous studies [β1-AR and increased β2-AR expression in SHR. Therefore, the antihypertensive effect of acupuncture in SHR might be associated with the balance of β1/β2-AR. β-blockers therapy plays a major role in the treatment of cardiovascular diseases. To date, 14 β-blockers have received Federal Drug Administration approval for oral use in patients having systemic hypertension [β-blockers can be divided into three categories, namely, nonselective b-blocker, selective b1- receptor blocker, and b1-receptor blocker with additional α1-receptor blocking activity. Among them, the nonselective b-blocker is used most widely. In general, β-blockers are well tolerated, but serious side effects may occur, which are usually associated with the β2-AR antagonistic activity  [β1 selective blockers lowered BP by a greater magnitude compared to dual receptor beta-blockers [β1-AR expression and increase β2-AR expression, we consider that the low side effects of acupuncture may be related to the increased expression of β2 receptors, which needs to be further investigated.Evidence collected throughout the years has shown that alterations in sympathetic cardiovascular control participate in the development, maintenance, and progression of hypertension. Renal sympathetic nerves induce high BP via E and NE regulates rtension . Thus, s studies , 25. Thertension . By receymptoms) . Wong etblockers . Acupuncblockers . Moreoveblockers . Combineβ1-AR mediated release of renin [β-AR blocker could reduce renal nerve stimulation-induced renin release. In our experiment, we found a significant reduction of renin content after acupuncture. Consistent with our results, Lohmeier et al. demonstrated that renal denervation decreased plasma renin content and abolished the hypertension [The RAAS is an important mechanism in regulating BP. For that reason, an alteration in any molecule that composes the RAAS contributes to developing hypertension . Classicof renin . Therefortension . HoweverThe renal sympathetic nervous system plays an active role in the modulation of BP, and its overactivation can lead to hypertension . The Symβ1-AR and increased β2-AR expression we observed may also be involved in the beneficial effect of acupuncture on hypertension.In conclusion, our results provide evidence that acupuncture at LR3 can significantly relieve the increased MBP through reducing renal sympathetic nervous activity. The effect of acupuncture on renal sympathetic nervous activity is evidenced by the improved HRV and the reduction of NE and E contents. The decreased


Direct reciprocity is a mechanism for cooperation among humans. Many of our daily interactions are repeated. We interact repeatedly with our family, friends, colleagues, members of the local and even global community. In the theory of repeated games, it is a tacit assumption that the various games that a person plays simultaneously have no effect on each other. Here we introduce a general framework that allows us to analyze “crosstalk” between a player’s concurrent games. In the presence of crosstalk, the action a person experiences in one game can alter the person’s decision in another. We find that crosstalk impedes the maintenance of cooperation and requires stronger levels of forgiveness. The magnitude of the effect depends on the population structure. In more densely connected social groups, crosstalk has a stronger effect. A harsh retaliator, such as Tit-for-Tat, is unable to counteract crosstalk. The crosstalk framework provides a unified interpretation of direct and upstream reciprocity in the context of repeated games. Social interactions among people are often repeated, and yet it is assumed that simultaneous interactions are independent from one another. Here, Reiter and colleagues describe a conceptual framework where an action in one game can influence the decision in another. Cooperation is normally opposed by natural selection unless mechanisms for the evolution of cooperation are in place3. One such mechanism is direct reciprocity, which is based on repeated interactions between the same two players5. In repeated social dilemmas, humans often learn to use adaptive rules, telling them when to cooperate, when to defect, and how to motivate others to cooperate8. Cooperation can be achieved if people adopt conditional cooperative strategies such as Tit-for-Tat5, Generous Tit-for-Tat10, or Win-stay, Lose-shift12. Conditional cooperation, paired with some amount of generosity, can maintain a healthy level of cooperation21. It can evolve even if initially rare26.Social dilemmas are situations where mutual cooperation is better than mutual defection and yet there is an incentive to defect30) have either assumed that (i) individuals only engage in one repeated game at a time or that (ii) an individual’s action in one game is independent of all its other interactions. Because humans often engage in many games simultaneously, the first assumption seems to be violated in most practical scenarios. Moreover, evidence from experimental studies suggests that also the second assumption of independence may not always apply38. We say that a player’s decision is subject to “crosstalk” when an interaction that a player has in one repeated game influences how the very same player behaves in another repeated game , or the consequence of a subconscious error . However, the interpretation of our results will often depend on the specific psychological mechanism that gives rise to crosstalk. We revisit this matter in the “Discussion” section.Such crosstalk can result from various psychological processes. For example, experiments on upstream reciprocity suggest that subjects who have received help in their previous interaction often consciously choose to “pay it forward”N individuals. Each individual plays a pairwise repeated prisoner’s dilemma (PD) with each interaction partner. These repeated games occur concurrently. At each time step, we choose a random pair of players for a single interaction , always-defect (ALLD), tit-for-tat . The crosstalk rate γ ∈  specifies how often crosstalk occurs. In the special case of no crosstalk, γ = 0, players perfectly distinguish between all their opponents, and we recover the scenario considered in previous studies of direct reciprocity5. For positive crosstalk rates, cooperative and defective behaviors can cascade in the players’ social network: a player’s action in one game can affect how the co-player acts in a different game, which in turn may influence again other games  and crosstalk occurs, γ > 0, the ALLD player can turn all remaining players into defectors eventually . To illustrate how crosstalk leads to the spread of defection in a generally cooperative society, we place a single ALLD player in a network of rs Figs.  and 3a. lly Fig. , indepenlue Fig. , with thN = 16, the cooperators obtain a higher average payoff than the defector for crosstalk rates up to γ = 0.85 on a circle, and for up to γ = 0.41 on a complete graph. In networks with a low degree, players are more likely to give the adequate response with respect to their current co-player, because, if crosstalk occurs, the current co-player is more likely to coincide with the previous co-player such that crosstalk becomes inconsequential. For this reason, all other explored population structures exhibit crosstalk thresholds between the circle and the complete graph  and q > 0 needs to return to full cooperation after a single defection event. We find that crosstalk leads to a significantly faster recovery  of the population structure and monotonically decreasing with the probability to cooperate after defection  with maximum qM such that N − 1 players with this strategy get at least the same average payoff as the single defector. Analytical calculations for the complete graph and numerical results for all other population structures show that higher crosstalk rates and higher network degrees (that is a higher number of neighbors) require the cooperative players to be less generous  have the highest relative payoff advantage compared to the single ALLD player. The most robust level of generosity exhibits a non-monotonic dependence on the crosstalk rate , but not too much; we find q < qM, with qM depending on the crosstalk rate and on the population structure. Only for the circle, cooperation can prevail even when crosstalk is abundant  with q < qM in fact resist invasion by all possible invading strategies  for the complete graph. This analysis also reveals that there are three classes of strategies in total that are stable against arbitrary invaders , with p sufficiently small  analogous to equalizer strategies of direct reciprocity49. When applied by all residents in the population, equalizer strategies guarantee that the payoff of a single invader is independent of the invader’s strategy.The above analysis is based on a comparison between conditional cooperators and a specific invader, ALLD. More generally, we find that conditionally cooperative strategies 1, , q with 51. In addition, players may occasionally also experiment with new stochastic strategies, which introduces novel behaviors into the population. These two events, imitation and exploration, take the role of selection and mutation in models of biological evolution. We show that a birth-death process as used in many biological applications yields the same results . According to this process, strategies that yield a comparably high payoff are more likely to be imitated by other players qM Fig. . None ofure Fig. . While cTo understand the effect of crosstalk in more detail, we analyzed how easily other strategies fix in a resident population that either consists of ALLD players or GTFT players . While the cooperation dynamics of this model are qualitatively different from our previous results, we again find that higher crosstalk rates impede the stability of cooperation across all population structures  reciprocity. In models of downstream reciprocity, a player’s strategy depends on the co-player’s reputation, and hence on the co-players’ interactions with others64. A combination of direct reciprocity and downstream reciprocity can promote cooperation because a single defection in one game may lead several unaffected co-players to retaliate against the defector29. However, downstream reciprocity makes stronger assumptions on the information players have when making their decision. It requires a player to observe other players’ interactions or reputations to respond accordingly. In contrast, crosstalk is a much more elementary mechanism. It occurs “within” each player and does not rely on additional external information about independent interactions of unrelated players. Our notion of crosstalk is general: it captures that a player’s decision in one game can be affected by the player’s previous experience in another game, but it does not depend on the psychological process responsible for this interdependency.Classical models of direct reciprocity require that players provide a targeted response to each of their co-players66. The framework of crosstalk allows us to explore the consequences when both modes of reciprocity act simultaneously, and possibly interfere with each other. We recover previous results67 that upstream reciprocity alone is most likely to yield cooperation when the population is highly structured. However, our results suggest that cooperation can even be maintained in well-mixed populations when upstream reciprocity is sufficiently coupled with direct reciprocity .Depending on the specific process at work, crosstalk is amendable to different interpretations. Our framework can be taken as a model of upstream reciprocity in the context of repeated games. Under this interpretation, cooperation or defection received from one person is sometimes consciously “paid forward” to another person. Previous analytical models have either focused on direct reciprocity or on upstream reciprocity separately69. Implementation errors only affect the repeated game in which they occur . The population is given by a graph where the nodes represent the players, and the edges reflect all possible interactions between players. Only players connected by an edge can be paired to play the PD. Players use separate two-state automata for each of their neighbors on the interaction graph41. The two states of each automaton are labeled C (cooperation) and D (defection). These states are updated according to the player’s reactive strategy , see Supplementary Figs. p denotes the probability to move to state C if the co-player has cooperated in the previous game (whereas the complementary probability 1 − p gives the likelihood to move to state D). Similarly, q denotes the probability to move to state C if the co-player has defected (and 1 − q is the respective probability to move to state D).To simulate the effect of crosstalk on the cooperation dynamics among players with fixed strategies, we consider a population of size γ a player acts according to the respective automaton state associated with this co-player; with probability γ crosstalk occurs and the player refers to the state of the automaton updated in her last interaction instead. After the game, the automata states are updated according to the game outcome and the players’ strategies. This elementary step is then iterated for a large number of rounds. For the simulation results depicted in Supplementary Fig. 4 realizations to obtain the stationary payoff of GTFT and ALLD players for a given population structure and crosstalk rate.In each round, an edge of the interaction graph is chosen uniformly at random. A single PD is played among the two players adjacent to the chosen edge. With probability 1 − wij are given by the connectivity matrix W (see next section for details).So far, we assumed that every edge in the interaction graph is chosen with the same probability. However, some interactions can occur with a higher frequency than others. To investigate the effects of different interaction frequencies, we studied the spread of defective behavior in a population of GTFT players populating a 5 × 5 lattice . The population structure is given by an N × N connectivity matrix W = (wij). The entries wij give the probability that the next interaction in that population occurs between players i and j. In particular, the connectivity matrix W is symmetric wij = wji, and satisfies wii = 0 and i and j are connected, then wij = w for some constant w > 0 that depends on the network structure, but is independent of the players i and j. For example, because there are N(N − 1) / 2 different links in a complete graph, well-mixed populations can be represented by a connectivity matrix with wij = 2 / (N(N − 1)) for all i ≠ j. As another example, populations on a cycle are represented by wij = 1/N if j occupy neighboring sites, and wij = 0 for all other i, j.To derive an explicit representation for the payoffs that players receive in the long run, we suppose that each player i. Moreover, let i is in state C against player j at time t, and let i is in state C with respect to her previous co-player. We can calculate y yi that player i is in state C with respect to her previous co-player simplifies to i is in state C with respect to a random co-player. In that case, we can rewrite Eq. (N(N − 1) linear equations in the unknowns yij with i ≠ j. By solving this inhomogeneous system, we can calculate the stationary frequency i in state C with respect to player j. Given the stationary frequencies πi of player i by averaging over all co-players,γ, general population structures, and general population compositions . As shown in Supplementary Fig. q) are allowed to be to resist invasion of ALLD )]−1. The parameter s ≥ 0 measures the strength of selection. When selection is weak, p, q), which is uniformly drawn from all reactive strategies. Following the approach of Imhof and Nowak23, we assume that these exploration events are rare. As a consequence, the population is homogeneous most of the time. Only occasionally, a mutant strategy enters the population due to random strategy exploration. This mutant strategy than either goes extinct or fixes before the next exploration event occurs. By simulating this process over a long timespan, we can record how often the population applies certain strategies , and we can compute the resulting average cooperation rate over an evolutionary timescale. In Fig. N = 16, benefit b = 10, cost c = 1, and selection strength s = 1. Other parameter values lead to qualitatively similar results, provided that selection is sufficiently strong and that the benefit of cooperation is sufficiently high to allow for the evolution of cooperation. In the Supplementary Note For the evolutionary simulations, we consider a population with fixed population structure and fixed crosstalk rate g to Eq. . We assuNo data sets were generated during this study.Supplementary Information


ICH). We assessed the association of early glycemic trajectory on ICH mortality and edema growth.Hyperglycemia may be associated with worse outcome after intracerebral hemorrhage (ICH study with glucose measurements at least once between both 0 to 24 and 24 to 72 hours from onset. Hyperglycemia was defined as blood glucose ≥8 mmol/L (144 mg/dL) based on the local threshold for treatment. Glycemic trajectory was defined on maximum values 0 to 24 and 24 to 72 hours after ICH: (1) persistent normoglycemia in both epochs; (2) late hyperglycemia (only between 24 and 72 hours); (3) early hyperglycemia (only before 24 hours); and (4) persistent hyperglycemia in both epochs. Logistic regression with known predictors of outcome estimated the association of glycemic trajectory and 6‐month mortality. A generalized linear model assessed the association of glycemic trajectory and interpolated 72‐hour edema extension distance. A total of 576 patients met eligibility criteria, of whom 214 (37.2%) had persistent normoglycemia, 44 (7.6%) late hyperglycemia, 151 (26.2%) early hyperglycemia, and 167 (29.0%) persistent hyperglycemia. Six‐month mortality was higher in the persistent (51.1%) and early (26.3%) hyperglycemia groups than the normoglycemia (19.0%) and late hyperglycemia (3.6%) groups. Persistent hyperglycemia was associated with 6‐month mortality . Both univariate (P=0.426) and multivariable (P=0.493) generalized linear model analyses showed no association between glycemic trajectory and 72‐hour edema extension distance.We included patients from the Helsinki ICH is harmful if it is persistent. Strategies to achieve glycemic control after ICH may influence patient outcome and need to be assessed in clinical trials.Early hyperglycemia after  Only half of intracerebral hemorrhage patients with baseline hyperglycemia remain persistently hyperglycemic.Association with mortality was only observed in patients with persistent hyperglycemia.Optimizing glycemic status early after intracerebral hemorrhage may improve outcome after intracerebral hemorrhage.The current American Stroke Association guidelines endorse avoidance of hyperglycemia in patients with intracerebral hemorrhage (ICH).There is emerging evidence that secondary injury from perihematomal edema is associated with poor ICH outcome.The aims of this study are to evaluate the impact of early glycemic trajectory on ICH mortality and edema growth. We hypothesized that persistent hyperglycemia is associated with increased mortality and edema growth.Patients from the Helsinki ICH studyHematoma and edema volumes were segmented using semiautomated planimetry.Hyperglycemia was defined as blood glucose ≥8 mmol/L (144 mg/dL) based on the local threshold for treatment. The Helsinki protocol during the study was to provide sliding scale insulin when the glucose was ≥8 mmol/L. Glycemic trajectory was defined on maximum values 0 to 24 and 24 to 72 hours after ICH. Four categories were determined: (1) persistent normoglycemia in both epochs; (2) late hyperglycemia (only between 24 and 72 hours); (3) early hyperglycemia (only before 24 hours); and (4) persistent hyperglycemia in both epochs.−0.927, R2=0.82) utilizing data from HICHS.0.07331−1.478. As the patients were scanned at different time points in routine clinical practice, we interpolated the EED from the observed time of an individual's EED closest to 72‐hour time point, assuming the same proportional growth to derive the expected 72‐hour EED for the individual patient. In other words, if the patient's last scan was at 48 hours and was 70% of the average expected EED at this time point, we assumed the patients to also have 70% of average expected 72‐hour EED.Edema extension distance (EED), a recently reported edema metric, was used in assessing the association of glycemic trends and edema growth.2 or Fisher exact test for categorical variables and Mann–Whitney test for continuous variables. We used mortality at 6 months, the common time point of end point in ICH trials.P<0.1) with increased edema on univariate analysis. A P<0.05 in the multivariable models was considered significant. Propensity‐score matching was performed using R 3.1.0 MatchIt package while all other statistics were performed using SPSS 23 .Standard descriptive statistics were used. Differences in patient characteristics, stroke severity, and imaging metrics between the glycemic trajectories were assessed on univariate analysis using χP<0.001) and they were more likely to be hyperglycemic at 0 to 24 hours . Excluded patients were thus either mild and late, or with characteristics making them prone to early palliation and death  were eligible for the present analysis. Reasons for excluding 437 patients were the following: no planimetric data (n=19), missing 6‐month mortality (n=10), or missing glucose data (n=408) in either or both time windows mostly because of late presentation or early death/palliation, as illustrated in Figure th Table .Of the 576 patients in the analysis, 214 (37.2%) had persistent normoglycemia, 44 (7.6%) late only hyperglycemia, 151 (26.2%) early only hyperglycemia, and 167 (29.0%) persistent hyperglycemia. In 318 (55%) patients with hyperglycemia within 24 hours of ictus, 167 (53%) remained persistently hyperglycemic  deaths by 6 months and factors associated with 6‐month mortality on univariate analysis were older age, prior use of warfarin or antihypertensive medication, evidence of infection, lower GCS, higher NIHSS, higher baseline ICH and absolute edema volumes, irregular hematoma shape, ventricular extension, and 72‐hour EED (Table 3.8% deatP<0.001) adjusted for log‐transformed baseline ICH volume, baseline absolute edema volume, male sex, age, ventricular extension, infratentorial location, NIHSS, and GCS  nor presence of infection  was associated with 6‐month mortality when included in the model or influenced the association of persistent hyperglycemia and mortality  There was also no interaction of surgery (P=0.965) or presence of infection (P=0.594) on the association between glycemic trajectory and mortality. The association with 6‐month mortality in the persistent hyperglycemia group remained significant after excluding the 89 patients with diabetes mellitus .Neither surgery  and 24 to 72‐hour  epochs. There was a significant correlation between the maximum glucose measurements in each epoch . Absolute glucose change between the maximum readings from the 2 epochs was also associated with 6‐month mortality  in the logistic regression analysis adjusting for the predefined covariates and maximum glucose from 0 to 24‐hour epoch. In the 184 (31.9%) patients with baseline (<12 hours from ictus) and follow‐up computed tomography (12–72 hours from ictus) available, introducing hematoma growth into the logistic regression model did not influence the association of persistent hyperglycemia on mortality .In the secondary analyses, glucose as a continuous variable was associated with 6‐month mortality in both the 0 to 24‐hour   with matched baseline characteristics Table . In the 0) Table .P=0.426) or multivariable analyses .A large number of observational studies 48, 9, 10P value not reported). Feng et al assessed the impact of hyperglycemia (mean glucose over 72 hours of ≥150 mg/dL) in 135 ICH patientsP value not reported). Godoy et al reported in 295 patients that those with persistent hyperglycemia in the 72 hours after ICH (n=78) had higher 30‐day mortality (80%) compared with other glycemic trajectory groups on univariate analysis (P<0.001), but no multivariable analysis was reported.P=0.005) in 196 ICH patients.P=0.005). There was no association between hyperglycemia of <24‐hour duration with outcome . These results were derived from a small sample size with a point estimate that approached 1 and a wide confidence interval. The negative association needs to be considered in this context and late hyperglycemia should still be managed as per current best practice guidelines.We did not find a significant association with ICH mortality in the subgroup of patients  classified with late‐only hyperglycemia .The mechanism through which hyperglycemia mediates ICH outcome is uncertain. It is plausible that hyperglycemia reflects more severe brain injury resulting from larger baseline hematoma volume Table . HoweverP=0.37). The Quality in Acute Stroke Care (QASC) study randomized 1126 (51 [4.5%] were ICH) acute stroke patients in stroke units to a set of protocolized interventions for managing glucose, fever, and swallowing dysfunction or guideline management. The intervention group had reduced likelihood of 90‐day death or disability .Two large randomized controlled trials have examined glycemic control on stroke outcome. The UK Glucose Insulin in Stroke Trial (GIST‐UK) enrolled 933 (134 [14.4%] were ICH) stroke patients with admission glucose between 6.0 and 17.0 mmol/L to targeted glycemic control (4.0–7.0 mmol/L) or no intervention.We acknowledge the study limitations including firstly the potential for bias and chance associations in retrospective studies. We tried to minimize bias by predefining our study population and analysis a priori. Secondly, we had to exclude 43% of the patients in the HICHS predominately because of early death, early palliation, or late presentation Figure . The excOver half of ICH patients experienced early hyperglycemia, which is only associated with higher mortality when it persists. Strategies to achieve glycemic control after ICH may influence patient outcome and need to be assessed in randomized controlled trials.Wu is supported by grants from the Neurological Foundation of New Zealand (grant number 1313‐CF) and Royal Melbourne Hospital Neuroscience Foundation; Strbian is supported by grants from the Helsinki University Central Hospital and the Finnish Medical Foundation; Tatlisumak is supported by the Helsinki University Central Hospital and Sahlgrenska University Hospital grants for ICH research; Meretoja is supported by grants from National Health and Medical Research Council , Academy of Finland, and the Finnish Medical Foundation.None.Table S1. Baseline Characteristics Between Included and Excluded PatientsTable S2. Model Fit With Different Logistic Regression ModelsTable S3. Baseline Characteristics in the Propensity‐Score‐Matched PopulationTable S4. Multivariable Logistic Regression Model in Propensity‐Score‐Matched Population on Factors Associated With 6‐Month MortalityTable S5. Generalized Linear Model on Association of Glycemic Status on Extrapolated 72‐Hour Edema Extension Distance (EED) in CentimetersTable S6. Studies Investigating the Association of Glucose and Outcome After Intracerebral HemorrhageClick here for additional data file.


Multiplayer video games promoting exercise-based rehabilitation may facilitate motor learning, by increasing motivation through social interaction. However, a major design challenge is to enable meaningful inter-subject interaction, whilst allowing for significant skill differences between players. We present a novel motor-training paradigm that allows real-time collaboration and performance enhancement, across a wide range of inter-subject skill mismatches, including disabled vs. able-bodied partnerships.A virtual task consisting of a dynamic ball on a beam, is controlled at each end using independent digital force-sensing handgrips. Interaction is mediated through simulated physical coupling and locally-redundant control. Game performance was measured in 16 healthy-healthy and 16 patient-expert dyads, where patients were hemiparetic stroke survivors using their impaired arm. Dual-player was compared to single-player performance, in terms of score, target tracking, stability, effort and smoothness; and questionnaires probing user-experience and engagement.Performance of less-able subjects (as ranked from single-player ability) was enhanced by dual-player mode, by an amount proportionate to the partnership’s mismatch. The more abled partners’ performances decreased by a similar amount. Such zero-sum interactions were observed for both healthy-healthy and patient-expert interactions. Dual-player was preferred by the majority of players independent of baseline ability and subject group; healthy subjects also felt more challenged, and patients more skilled.This is the first demonstration of implicit skill balancing in a truly collaborative virtual training task leading to heightened engagement, across both healthy subjects and stroke patients.The online version of this article (doi:10.1186/s12984-017-0319-x) contains supplementary material, which is available to authorized users. Physiotherapy intensity is a well-recognised determinant of stroke recovery, although questions of method, timing, scheduling, etc., are still debated . Video gRecently, virtual therapy involving two or more players has been proposed as a means of further increasing intrinsic motivation, engagement and social inclusion –12. By pCompared to single-player training games, multiplayer games are more engaging, with the level of impact depending partly upon participant personality traits , 12. To A significant issue in the design of multiplayer games, particularly amongst disabled users, is how to permit differences in skill-levels between players, and allow for effective gameplay, participation and enjoyment by all players , 19, 20.Co-activity characterised by a divisible task that either player can complete independently.Competition each player interacts with the partner to fulfil their own goal and ultimately prevent the other player fulfilling their aim.Cooperation the players work together to complete the task but have different roles .Collaboration the interacting players are assigned the same role and need to work together to complete the task.Inter-player relationships broadly fall into one of four types of human-human interaction , 15. C1. However, collaborative and cooperative interactions have several beneficial properties which can further promote motivation , Why? QE Which game condition did you put the most effort into?  QS During which game condition did you feel the most skilled?  QP During which game condition did you feel the most pressured?  QC Any comments ?I tried very hard on this game [P]I think I am pretty good at this game [P]This game was fun to play [P]I did not put much energy into thisThis game did not hold my attention at allI was pretty skilled at this gameI thought this was a boring game [P]I am satisfied with my performance at this gameI put a lot of effort into thisI enjoyed playing this game very muchI did not try very hard to do well at this gameThis was a game that I could not play very well [P]I thought this game was quite enjoyableIt was important to me to do well at this game [P]I think I did pretty well at this game, compared with the othersThe following questions were asked to assess the intrinsic motivation during both game conditions (single and multiplayer): Note: [P] indicates the questions selected in the patient version. This questionnaire uses a Likert scale from 1 to 7 Figure


In prostate cancer, androgen/androgen receptor (AR) and their downstream targets play key roles in all stages of disease progression. The protein kinase D (PKD) family, particularly PKD1, has been implicated in prostate cancer biology. Here, we examined the cross-regulation of PKD1 by androgen signaling in prostate cancer cells. Our data showed that the transcription of PKD1 was repressed by androgen in androgen-sensitive prostate cancer cells. Steroid depletion caused up regulation of PKD1 transcript and protein, an effect that was reversed by the AR agonist R1881 in a time- and concentration-dependent manner, thus identifying PKD1 as a novel androgen-repressed gene. Kinetic analysis indicated that the repression of PKD1 by androgen required the induction of a repressor protein. Furthermore, inhibition or knockdown of AR reversed AR agonist-induced PKD1 repression, indicating that AR was required for the suppression of PKD1 expression by androgen. Downstream of AR, we identified fibroblast growth factor receptor substrate 2 (FRS2) and its downstream MEK/ERK pathway as mediators of androgen-induced PKD1 repression. In summary, PKD1 was identified as a novel androgen-suppressed gene and could be downregulated by androgen through a novel AR/FRS2/MEK/ERK pathway. The upregulation of prosurvival PKD1 by anti-androgens may contribute to therapeutic resistance in prostate cancer treatment. Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer-related deaths among men in the United States. The initiation and progression of prostate cancer is uniquely dependent on androgen receptor (AR)-induced signaling. Although androgen deprivation therapy provides an initial favorable response in advanced prostate cancer, the more aggressive castration-resistant prostate cancer (CRPC) develops invariably in almost all patients, eventually leading to death. It has become increasingly clear that continuous activation of the AR in CRPC remains the main driving force of tumor progression and metastasis. Thus, understanding the critical events associated with the AR signaling is essential for developing novel and effective therapies to treat CRPC.2+/calmodulin-dependent protein kinase (CAMK) superfamily [PRKD1, the gene encoding PKD1, may drive polymorphous low-grade adenocarcinoma (PLGA), the second most frequent type of malignant tumor of the minor salivary glands [The protein kinase D (PKD) family of serine/threonine kinases belongs to the Caerfamily , 2. To derfamily , 4, PKD2erfamily , and PKDerfamily . In intaerfamily –9, and Perfamily , 11. Emeerfamily , 13. A ry glands . PKD regy glands , 15–20. y glands , skin [1y glands , 21, brey glands , and proy glands . In party glands –26, and y glands .Fibroblast growth factor (FGF) signaling is a highly complex signaling network that comprises 18 ligands, which bind to and activate four highly conserved transmembrane tyrosine kinase receptors . The FGF/FGFR pathway plays an important role in cancer development and progression by modulating a variety of biological processes, including cell proliferation, survival, and migration , 29. FGFPRKD1 gene expression. Further analysis identified FRS2 as a novel mediator of androgen-induced PKD1 repression. The regulation of PKD1 by androgen and AR may have important implications in the therapeutic response to AR-targeted agents.In this study, we report for the first time that PKD1 was tightly regulated by androgen at the transcriptional level in prostate cancer cells and was a novel androgen-repressed gene. Inhibition or knockdown of androgen receptor (AR) blocked androgen depletion-induced PKD1 expression, indicating that AR was required for the repression of Androgen signaling plays a crucial role in prostate cancer initiation and progression. In this study, we sought to determine whether androgen modulated PKD1 expression and signaling. PKD1 was detected in androgen-sensitive LNCaP cells and two castration-resistant LNCaP-derivative cell lines, C4-2 (androgen-hypersensitive) and C81 (androgen-insensitive), but not in androgen-sensitive LAPC4 cells. As shown in Figure The kinetics of PKD1 regulation in response to androgen deprivation or R1881 treatment was examined. As shown in Figure p > 0.5), indicating that R1881 did not impact the stability of PKD1 mRNA. Next, cycloheximide (CHX) was used to inhibit protein synthesis to determine whether the regulation of PKD1 gene expression by androgen involved de novo protein synthesis. CHX induced a nearly 2-fold increase in PKD1 expression and completely blocked R1881-induced PKD1 downregulation, indicating that the suppression of PKD1 expression likely required the induction of a repressor protein  of PKD1 mRNA was determined in the presence of actinomycin D, an inhibitor of gene transcription. As shown in Figure n Figure . This fiAndrogens are important hormones for normal physiology and are responsible for certain disease conditions. Their actions are mediated by the AR, a ligand-dependent nuclear transcription factor. Androgens binds to AR after entering the cells to form an androgen-receptor complex, which then translocates to the nucleus where it binds to androgen response elements (AREs) in the promoter regions and regulates the transcription of its target genes. The actions of AR can be blocked by AR inhibitors, such as bicalutamide (Casodex) or enzalutamide (MDV3100). Bicalutamide is known to bind AR and leads to the formation of a transcriptionally inactive AR complex . In thisTo determine whether AR directly regulated the expression of PKD1, we analyzed the promoter region of PKD1, which led to the identification of two potential AREs upstream of the transcription start site (TSS). The human PKD1 gene spans ~45.7 kb. Analysis of up to 5000 bp of the promoter region upstream from the TSS revealed two putative AREs. . Depletion of FRS2 abolished the R1881-induced suppression of PKD1 transcription, confirming FRS2 as a potential mediator of androgen-dependent PKD1 repression Figure . FurtherA Figure . Thus, Ftrans-Golgi network. Our data indicated that BFA at 5 and 10 μM completely reversed androgen-induced PKD1 suppression  being the best characterized. In contrast, AR-repressed target genes have not been well characterized. These genes constitute a large portion of AR-targeted genes, and some have been shown to play essential roles in prostate cancer progression , 35. DivIn androgen-sensitive prostate cancer cells, depletion of FRS2 blocked R1881-induced PKD1 suppression at both the transcriptional and protein levels. Additionally, inhibition of FGFR and MEK, as well as protein secretion, blocked R1881-induced repression of PKD1. Thus, androgen may repress PKD1 through an AR-induced FGFR/FRS2/MEK/ERK pathway to inhibit PKD1 expression in prostate cancer cells. A previous study showed that FRS2 expression is not regulated by androgen in LNCaP cells . HoweverOur study identified PKD1 as an androgen/AR-repressed gene and uncovered a novel indirect mechanism through which AR regulates PKD1 expression. Although the functional implication of this regulation in prostate cancer progression is still unclear, PKD1 is an important prosurvival signaling protein in normal and cancer cells that functions by regulating multiple signaling pathways, such as stimulating NF-κB, ERK1/2, and Akt and inhibiting JNK and p38 , 25, 40.In summary, our study identified PKD1 as a novel androgen/AR-suppressed gene. The suppression of PKD1 was mediated through an indirect mechanism that involved FRS2, a cell surface adaptor protein that connects FGF/FGFR to the downstream MEK/ERK signaling pathway. Our findings suggested that the prosurvival function of PKD1 may have significant implications in prostate cancer progression and therapy resistance. PKD1 may be targeted to enhance the therapeutic response to anti-androgens in prostate cancer treatment.The synthetic androgen methyl trienolone (R1881) was obtained from Perkin-Elmer Life Sciences , and bicalutamide was purchased from Enzo Life Sciences . Charcoal-treated fetal bovine serum (FBS) was from Hyclone  and Sigma . Other cell culture reagents and media were from American Type Culture Collection . Anti-PKD1, anti-PKD2, and anti-AR antibodies were purchased from Cell Signaling Technology . Antibodies targeting GAPDH and α-tubulin were from Santa Cruz Biotechnology, Inc. . Goat anti-rabbit and goat anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Promega .LNCaP, C4-2, C81, VCaP, and PC-3 cells were obtained from ATCC  and were cultured according to the manufacturer's recommendations. LNCaP cells were discarded after 12 passages. The 23 AR corepressor esiRNAs were obtained from Sigma-Aldrich . The non2, 10% glycerol, 1% Triton X-100, 5 mM EGTA, 20 μM leupeptin, 1 mM AEBSF, 1 mM NaVO3, 10 mM NaF, and 1× protease inhibitor cocktail). Lysate protein concentrations were determined using the Pierce BCA Protein Assay Kit . Cell lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% milk, the membranes were incubated with primary antibodies in blocking buffer at 4°C overnight. After washing, the membranes were incubated with secondary antibodies at room temperature for 1 h. Protein bands were detected using an enhanced chemiluminescence (ECL) kit. Anti-α-tubulin or anti-GAPDH antibodies were used as a loading control. Densitometry analyses were performed with ImageJ software (NIH).Cells were collected and lysed in IP lysis buffer  according to the manufacturer's protocol. One microgram of total RNAs was used to generate cDNA using an iScript cDNA synthesis kit. Real-time PCR was subsequently performed using SsoFast EvaGreen Supermix on a CFX96 Real-Time PCR Detection System . The following primers were used: PKD1, forward primer 5′-CGCACATCATCTG CTGAACT-3′ and reverse primer 5′-CTTTCGGTGCA CAACGTTTA-3′; FRS2, forward primer 5′-ATGG GAATGAGTTAGGTTCTGGC-3′ and reverse primer 5′-GCGGGTGTATAAAATCAGTTCTGTG-3′. Data were normalized automatically by using GAPDH as the loading control, with the following primers: forward primer 5′-GCAAATTCCATGGCACCGT-3′ and reverse primer 5′-TCGCCCCACTTGATTTTGG-3′.p value of less than 0.05 was considered statistically significant .All statistical analyse were carried out using GraphPad Prism IV software. A


AR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a diagnosis of PAIS. We performed extensive molecular studies including whole exome sequencing in a Japanese family with PAIS, identifying a deep intronic variant beyond the branch site at intron 6 of AR (NM_000044.4:c.2450−42 G > A). This variant created the splice acceptor motif that was accompanied by pyrimidine-rich sequence and two candidate branch sites. Consistent with this, reverse transcriptase (RT)-PCR experiments for cycloheximide-treated lymphoblastoid cell lines revealed a relatively large amount of aberrant mRNA produced by the newly created splice acceptor site and a relatively small amount of wildtype mRNA produced by the normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo nonsense mediated decay (NMD) and, if a small amount of aberrant mRNA may have escaped NMD, such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation creating an alternative splice acceptor site resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.Although partial androgen insensitivity syndrome (PAIS) is caused by attenuated responsiveness to androgens, androgen receptor gene ( Affected 46,XY patients frequently exhibit undermasculinized genitalia since birth and gynecomastia with puberty, in the presence of age-appropriate serum androgen values1. Testis development is usually normal, while small testes have been identified in a substantial fraction of pubertal or adult patients with molecularly confirmed PAIS2.Partial androgen insensitivity syndrome (PAIS) is a rare endocrine disorder caused by attenuated responsiveness to androgensAR) mutations on the coding regions and their splice sites have been identified only in <25% of patients with a clinical diagnosis of PAIS, even after excluding other representative causes for PAIS-like phenotypes3. Furthermore, such AR mutations remain undetected in a certain fraction of patients with compromised endogenous AR activity4. These findings imply the possible presence of hidden mutations in non-coding regions of AR or in other genes involved in AR-signaling. Here, we report a deep intronic mutation creating an alternative splice acceptor site at intron 6 of AR.Notably, androgen receptor gene  Fig. . The proClinical findings of the three affected males are summarized in Table AR and for NR5A1, MAMLD1, and SRD5A2 whose mutations often cause PAIS-like phenotypes5 in the three affected males, identifying no pathogenic sequence variant on the coding exons and their splice sites. We next analyzed 25 hypospadias-related genes by target enrichment system7, revealing no pathogenic variant on the coding exons and their splice sites shared by the three affected males.We first performed Sanger direct sequencing for 8. Furthermore, although a total of 20 autosomal, but not X-linked, gene variants (variants 1–20 in Supplementary Table S1) with minor allele frequencies (MAFs) of ≤0.01 in all the four public databases and in-house database were found to be present in the three affected males  and the two carrier females (I-2 and II-2) and absent from the two non-affected males (I-1 and II-1), they were assessed as apparently irrelevant to the PAIS phenotype on the basis of the frequencies in the normal populations, the results of in silico pathogenic analyses, and/or the available information regarding the corresponding genes. Indeed, while missense variants 2, 4, and 15 were completely absent from the public and in-house databases, variants 4 and 15 were evaluated as non-pathogenic by ≥ 3 of the four in silico analyses. Similarly, while missense variants 1, 2, and 12 were evaluated as pathogenic by all the four in silico analyses, variants 1 and 12 were, though extremely rare, registered in the public databases, as were a nonsense variant 5 and a splice donor site variant 14. Furthermore, while variant 2 was completely absent from the public and in-house databases and was assessed as pathogenic by all the four in silico analyses, the tissue expression pattern of the corresponding gene (SOAT1) was apparently inconsistent with the PAIS phenotype.Thus, we carried out whole exome sequencing in all the seven subjects of this family. However, no pathogenic variant co-segregating with the PAIS phenotype was identified on the coding exons and their splice sites (~10 bp of intronic sequences) of all the previously known genes involved in hypospadias or DSDWe also performed aCGH in the seven subjects in this family, to examine the presence or absence of a copy-number variant as an underlying factor for PAIS. However, aCGH identified no pathologic copy number variant co-segregating with the PAIS phenotype in this family.AR. In support of this, the analysis of CAG repeat length polymorphism on exon 1 of AR showed co-segregation of the same allele with the PAIS phenotype  was found to be present in the three affected males and the two carrier females and absent from the two non-affected males, as shown by Sanger sequencing  indicated the presence a pyrimidine-rich sequence and two candidate branch sites near the newly created “AG” splice acceptor motif -rich sequence and a branch site required for splicingn 6 Fig. . Human Stif Fig. . Furtherins Fig. .Figure 2We further studied whether an alternative splicing actually occurs using the newly created “AG” splice acceptor site. For this purpose, we performed reverse-transcriptase (RT)-PCR analysis, using Epstein-Barr virus (EBV)-transformed B-cell lymphoblastoid cell lines (LCLs) established from the affected boys (III-1 and III-2), their 30-year-old non-affected father (II-1), and a 28-year-old healthy adult male with proven fertility. The mRNA samples were obtained after 8-hour incubation with or without the NMD inhibitor cycloheximide (CHX).RT-PCR analysis was performed with primers which could amplify both the wildtype mRNA and the predicted aberrant mRNA  Fig. . ConsequW/Pc) designed to amplify the wildtype mRNA alone and those (PM/Pc) designed to amplify the mutant mRNA alone   was 25–30% in the affected boys and ~100% in the non-affected male and the control male  Fig. . The banns) Fig. . Furtherale Fig. . HoweverAR in a family with PAIS. This new acceptor site was accompanied by a pyrimidine-rich sequence and two candidate branch sites required for splicing and, consistent with this, RT-PCR experiments for CHX-treated LCLs revealed a relatively large amount of aberrant mRNA produced by the newly created alternative splice acceptor site and a relatively small amount of wildtype mRNA yielded by the original normal splice acceptor site. Furthermore, most of the aberrant mRNA was shown to undergo NMD and, if a small amount of aberrant mRNA may have escaped NMD in vivo, as suggested by the weak but discernible bands for the aberrant mRNA obtained from CHX-untreated LCLs of the affected boys (III-1 and III-2), such mRNA was predicted to generate a truncated AR protein missing some functional domains. These findings imply that the deep intronic mutation has resulted in the production of a relatively small amount of wildtype AR mRNA, leading to PAIS.We identified a deep intronic mutation (c.2450−42 G > A) creating an alternative splice acceptor site at intron 6 of AR, except for splice site mutations at the conserved “GT-AG” motif: [1] a > 6 kb deletion at intron 2 involving a putative branch site, which produced ~90% of aberrant mRNA encoding non-functional AR protein due to in-frame skipping of exon 3 and ~10% of wildtype mRNA produced by a cryptic branch site, in a family with typical PAIS11; [2] c.1769−11 T > A at the pyrimidine-rich region at intron 2, which produced two types of aberrant mRNA encoding non-functional AR proteins due to in-frame retention of 69-bp intronic sequence transcribed by a cryptic splice acceptor site and in-frame skipping of exon 3, as well as a very low level of wildtype mRNA, in a family with apparently complete AIS (CAIS)12 1; and [3] c.2450−118 A > G at intron 6, which produced two types of mRNA subject to NMD due to retention of 85 bp and 202 bp sequences of intron 6 respectively, in siblings with CAIS13. These findings, together with the present data, indicate that several types of intronic mutations other than splice site mutations at the conserved “GT-AG” motif do exist in AR, although they remain extremely rare, and that PAIS takes place when a relatively small amount of wildtype AR mRNA is produced. In this regard, it would be worth pointing out that the c.2450−42 G > A identified in this study is the first PAIS-causing deep intronic mutation beyond the branch site.To our knowledge, three intronic aberrations have been identified in AR or other genes.Two points should be made with regard to the present study. First, although we performed extensive molecular studies, possible relevance of other variant(s) has not been excluded formally. Indeed, a true pathogenic variant for 46,XY DSD might have been overlooked by the whole exome sequencing, if it has been registered in the public and/or in-house databases because of its detection in normal females or in the general population due to incomplete penetrance, or if its pathogenic assessment remains more or less equivocal. In addition, such a variant might be hidden in non-examined sequences of AR mRNA expressions among subjects. Thus, it remains to be demonstrated whether the wildtype mRNA expression dosage is actually reduced in the affected males as compared with that in the non-affected males, although the present study revealed markedly reduced relative wildtype mRNA expression ratios in the affected males. This would primarily be due to expression studies being performed for polyclonal EBV-transformed LCLs that could express multiple genes to highly variable degrees, probably depending on the character of clones examined14. For example, it is possible that the LCLs of the affected boy III-1, which showed a high level of AR expression, were primarily composed of clones that efficiently express AR. In addition, since we utilized LCLs of non-affected adult father (II-1) and a healthy adult male for controls primarily because of an ethical issue, the age difference between the affected boys and the control adults may also be relevant to the inter-individual difference in the AR expression dosage.Second, precise comparison of expression dosage between different subjects was impossible even by the quantitative RT-PCR, because of variable AR (c.2450−42 G > A) is the pathogenic variant for PAIS. Further studies will permit to clarify the relevance of AR intronic mutations to the development of PAIS and CAIS.Despite such caveats, the present data strongly support that the deep intronic mutation of This study was approved by the Institutional Review Board Committee at Hamamatsu University School of Medicine, and performed after obtaining written informed consent to participate in this study and to publish molecular and clinical data including external genital findings/images from the adult subjects or from the parents of the affected children. All experiments were performed in accordance with the relevant guidelines and regulations.AR isoforms, transcript variant 1 (NM_000044.4) consisting of eight exons and encoding 920 amino acids was utilized as a reference , as suggested previously15.Of plural Primers utilized in this study are shown in Supplementary Table Genomic DNA was obtained from peripheral blood leukocytes using FlexiGene DNA Kit (QIAGEN).AR, NR5A1, MAMLD1, and SRD5A2 was performed from both directions on the ABI 3130xl Genetic Analyzer (Thermo Fisher Scientific).Sanger direct sequencing for AR, ATF3, BMP4, BMP7, BNC2, CTGF, CYP1A1, CYR61, DGKK, EGF, ESR1, ESR2, FGF8, FGFR2, GSTM1, GSTT1, HOXA4, HOXB6, HSD3B2, HSD17B3, MAMLD1, MID1, NR5A1, SRD5A2, and WT17, was performed using the Haloplex Target Enrichment System (Design ID 10380-1414463301) (Agilent Technologies). After amplification, the coding regions of the 25 genes were sequenced as 150 bp paired-end reads on a MiSeq sequencer (Illumina). Subsequently, nucleotide alterations in the samples were called by the Genome Analysis Toolkit (Broad Institute) and SAMtools 0.1.17 software (http://samtools.sourceforge.net)16.Sequence analysis for 25 causative or candidate genes for hypospadias, i.e., http://genome.ucsc.edu/) using BWA-MEM (Version 0.7.12) with default parameters. Duplicated reads are removed by Picard (Version 1.106), and local realignment and base quality recalibration were performed by GATK Version 3.5. Variants were identified with the GATK HaplotypeCaller, and variants with MAFs > 1% in at least one of four public databases and in-house database shown in Supplementary Table 17.Whole exome sequencing was carried out, using SureSelect Human All Exon V6 (Agilent Technologies). Captured libraries were sequenced by NextSeq. 500 (Illumina) with 150 bp paired-end reads. Reads were aligned to the reference genome (Human GRCh37/hg19)  and ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).Genomewide array comparative genomic hybridization was performed with a catalog human array  (Agilent Technologies), using leukocyte genomic DNA from sex-matched normal subjects as controls. For autosomes and female X chromosomes, log2 signal ratios of <−0.8 and >+ 0.4 were regarded as indicative of heterozygous deletions and duplications, respectively. For male sex chromosomes that appear in a heterogametic condition, log2 signal ratios of −∞ and around +1.0 were interpreted as hemizygous deletions and duplications, respectively. When ≥three consecutive probes showed abnormal log2 ratios, the corresponding region was regarded as copy number variants (CNVs). The genomic position was based on human GRCh37/hg19. CNVs were regarded as normal variants if they have been registered in the public databases such as Database of Genomic Variants , the affected boys (III-1 and III-2), and an adult healthy control male with proven fertility by a standard methodhttp://www.imagej.com-about.com/). The RT-PCR products were also subjected to direct sequencing. RT-PCR with PW/Pc or PM/Pc primers was similarly carried out with one μg of total RNA. Quantitative RT-PCR with PW/Pc or PM/Pc primers was also performed for mRNA samples extracted from CHX-treated LCLs by the SYBR Green methods on StepOnePlus system with Software v2.2.2 (Thermo Fisher Scientific), using GAPDH and ACTB as internal controls. We calculated the relative mRNA expression of each sample based on its threshold cycle (Ct) in comparison to the Ct of GAPDH or ACTB. Then, we calculated the ratio of the wildtype AR mRNA expression to the total (the sum of wildtype and mutant mRNAs) AR mRNA expression obtained from CHX-treated LCLs.Total RNA samples were isolated from immortalized LCLs cultured in media containing dimethyl sulfoxide or cycloheximide (Sigma) for 8 hours, using RNeasy Mini Kit (QIAGEN). RT-PCR with P1/P2 primers was performed with one μg of total RNA, using ReverTra Ace qPCR RT Kit (TOYOBO). Then, RT-PCR products were subjected to electrophoresis, and the band intensity was measured with ImageJ version 1.49 software (Supplementary information


Spasticity is a major complication after stroke, and botulinumtoxin A (BoNT-A) injection is commonly used to manage focal spasticity. However, it is uncertain whether BoNT-A can improve voluntary motor control or activities of daily living function of paretic upper limbs. This study investigated whether BoNT-A injection combined with robot-assisted upper limb therapy improves voluntary motor control or functions of upper limbs after stroke.Two subacute stroke patients were transferred to the Department of Rehabilitation.Patients demonstrated spasticity in the upper extremity on the affected side.BoNT-A was injected into the paretic muscles of the shoulder, arm, and forearm of the 2 patients at the subacute stage. Conventional rehabilitation therapy and robot-assisted upper limb training were performed during the rehabilitation period.Manual dexterity, grip strength, muscle tone, and activities of daily living function were improved after multidisciplinary rehabilitation treatment.BoNT-A injection in combination with multidisciplinary rehabilitation treatment, including robot-assisted arm training, should be recommended for subacute spastic stroke patients to enhance appropriate motor recovery. However, it is unclear whether BoNT-A can improve voluntary motor control or ADL functions of upper limbs. However, multidisciplinary rehabilitation therapies using RAT in combination with BoNT-A injection have rarely been applied to subacute poststroke spasticity. Thus, we report on 2 cases showing the beneficial effects of RAT in combination with BoNT-A injection on upper limb spasticity in the subacute phase of stroke.Recently, task-specific high-intensity training with a multidisciplinary team approach has become an important concept in stroke rehabilitation therapy, and robot-assisted arm training (RAT) has been shown to allow well tolerated and intensive task-specific repetitive training of the paretic arm.2The study was approved by the Ethics Committee of Jeju National University Hospital. Written consent was obtained from both patients.2.1On April 27, 2016, a 37-year-old woman visited the emergency room of OO hospital because of hemiparesis in the right side. Brain magnetic resonance imaging revealed a left basal ganglia and corona radiata infarction. Antiplatelet medication , antihypertensive drugs , and atorvastatin (20 mg/d) were prescribed. On the 16th hospital day (HD), the patient was transferred from the department of neurology to the department of rehabilitation. Conventional rehabilitation therapies, including physical therapy and occupational therapy, were provided twice per day, 5 times a week. On HD 44, the muscle tone of the paretic arm had increased to a modified Ashworth Scale (MAS) of 2 for the wrist, finger joints, and elbow joint, and antispastic medication (baclofen 30 mg/d) was prescribed. On HD 77, the muscle tone of the shoulder, elbow, hand, and fingers was aggravated to MAS 2 and 3, and the patient could not extend the clenched hand. One vial containing 200 U of neu-botulinium toxin A  was diluted with 8 mL normal saline, and a total of 287.5 U was injected by 1 experienced physiatrist into the subscapularis, biceps brachii (Bi), brachioradialis (BR), brachialis (Bra), pronator teres, flexor carpi radialis (FCR), flexor digitorum profundus (FDP), flexor digitorum superficial (FDS), and flexor pollicis longus (FPL) muscles, under electrical stimulation and ultrasound guidance. There were no side-effects and 2 types of exoskeletal upper limb robot were then applied. The program of the Neuro-X system  was progressed from passive mode to active-assisted exercises of the wrist, elbow, and shoulder joints in 2 dimensions through games that depended on the patient's functional status and that consisted of horizontal rotational movements at an angular velocity of 30 degrees per second, allowing shoulder abduction–adduction and elbow flexion–extension. The tasks of the ReoGo system  consisted of virtual reality-based execution of 3D movements with mobilization of the shoulder and elbow joints and hand gripping, with these movements resembling real ADL tasks (reaching and cup-to-mouth maneuvers). The movement mode can be progressed from completely passive to completely active, allowing the robot's arm to exert variable degrees of training , manual function test (MFT), box and block test (BBT), Korean version of the modified Barthel index (K-MBI), Fugl-Meyer Assessment (FMA-UL), and grip strength of the paretic arm during the rehabilitation therapy period Table .2.2On October 28, 2015, a 51-year-old man visited the emergency room of OO hospital because of dysarthria, dysphagia, and right hemiparesis, and was diagnosed with intracranial hemorrhage in the left basal ganglia; stereotactic aspiration of an intracerebral hematoma was conducted. On HD 17, the patient was transferred from the department of neurology to the department of rehabilitation, and physical therapy, occupational therapy, and speech therapy were conducted. On HD 149, the muscle tone of the paretic shoulder, elbow, finger, and wrist had increased to MAS 2 and 3, but the patient could not extend the wrist. A total of 350 U neu-BoNT-A was injected into the subscapularis, Bi, BR, Bra, FCR, FDS, FDP, and FPL, and 2 types of exoskeletal robot-aided arm therapy were initiated  in the subacute phase may have altered the natural process of motor recovery. However, the Copenhagen study reported that 80% of stroke patients had reached their best ADL function within 6 weeks postonset, and 95% had completed functional recovery within 12.5 weeks postonset. The Copenhagen 2 stroke durations of the patients in this report were 11 weeks and 21 weeks, and they were considered to have achieved the plateau of ADL functions at the BoNT-A injection. Indeed, high-intensity repetitive task-oriented robotic arm and hand training was performed to enhance neuroplasticity, and performances in BBT  and handgrip strength were improved at discharge. Although the paretic arm functions were not fully satisfactory for the independent performance of ADL, they permitted increased chance of using the paretic upper limb in ADL, such as in grasping and holding objects or bimanual manipulation activities.4BoNT-A injection at the subacute stage and combined conventional rehabilitation therapy and RAT contributed to improvements in voluntary hand grasp power, manual dexterity, and spasticity. Therefore, a multidisciplinary rehabilitation treatment in combination with a BoNT-A injection and RAT should be recommended for enhancing appropriate neuroplasty in subacute spastic stroke patients.


Despite the diverse physiological activities of androgens and glucocorticoids, the corresponding receptors are very close members of the nuclear-receptor super family. Their action mechanisms show striking similarities, since both receptors recognize very similar DNA-response elements and recruit the same coactivators to their target genes. The specificity of the responses lies mainly in the tissue-specific expression of the receptors and in their ligand specificity. In cells, where both receptors are expressed, the mechanisms leading to the difference in target genes are less obvious. They lie in part in subtle variations of the DNA-binding sites, in cooperativity with other transcription factors and in differential allosteric signals from the DNA and ligand to other receptor domains. We will highlight the different suggestions that might explain the DNA sequence selectivity and will compare the possible allosteric routes between the response elements and the different functions in the transactivation process. The interplay of androgen and glucocorticoid receptors is also highly relevant in clinical settings, where both receptors are therapeutically targeted. We will discuss the possibility that the glucocorticoid and androgen receptors can play partially redundant roles in castration-resistant prostate cancer. The physiological roles of androgens and glucocorticoids are very different, and this is reflected in their clinical applications. Androgens are the male sex steroids mainly involved in development and maintenance of reproductive organs and spermatogenesis. Clinically, they might provide benefit as hormone replacement therapy for hypogonadal men or in cases of severe cachexia or osteoporosis , 2. On tAndrogen and glucocorticoid effects are mediated by their corresponding receptors, the AR and the glucocorticoid receptor (GR), which are both nuclear receptors. Nuclear receptors are ligand-inducible transcription factors; they have a centrally located DNA-binding domain (DBD) which is connected via a hinge region to a carboxyterminal ligand-binding domain (LBD) and an amino-terminal activation function (NTD) Fig. a. The DBThe LBDs of all nuclear receptors are folded in a typical 3-layered, 12-helical structure with a ligand-binding cavity . While tThe NTD of the nuclear receptors is very diverse both in length and sequence. In addition, between the NTDs of AR and GR, there is little or no conservation, although both have transcription activating properties . A comparison between the NTD of AR and GR has been extensively reviewed elsewhere –9.Here, we will compare how the AR and GR interact with DNA and chromatin; how they evoke tissue-specific transcriptional responses and in how far they have interchangeable functions, for instance, in prostate cancer , 11.AR and GR are steroid receptors which form a specific subfamily of the nuclear receptors. Steroid receptors can be divided based on their sequence specificity into two groups: the estrogen receptors (ER) and ER-like receptors on one hand and the glucocorticoid, mineralocorticoid (MR), progesterone (PR), and androgen receptors on the other hand. The latter are also called the oxosteroid receptors.The structures of the DNA-binding domains of nuclear receptors were solved by X-ray crystallography , 13 and A detailed comparison of the DBDs from AR and GR reveals many structural similarities, but also some remarkable differences. The amino acids in the DNA-binding helix, which make the sequence-specific contacts with the bases, are identical in GR and AR, but small nuances in the three-dimensional structure of the α-helix indicate a slightly stronger affinity of the AR-DBD for its 5′-AGAACA-3′ motif . For theThe amino acids of the second zinc finger which constitute the dimerization surface of the DBDs are also conserved between the oxosteroid receptors Fig. b. Nevertin vitro. Indeed, the P-box of the ER-DBD makes sequence-specific contacts with the 5′-AGGTCA-3′ hexamer half sites of the estrogen response elements (ERE), while the oxosteroid P-box recognizes their cognate 5′-AGAACA-3′ hexamer. However, despite these differences, the AR is able to bind EREs and this has been proposed to be a mechanisms by which it interferes with ER functioning in breast cancer [In conclusion, although the two zinc fingers of the AR and GR-DBDs differ only in 12 out of 65 residues and even when most of the substitutions are conservative, small variations in structure seem to have functional consequences. These subtle differences in the way steroid receptors bind DNA correlated with slightly different consensus binding sites and overlapping but distinct sequence specificities between oxosteroid receptors, which will be discussed further , 20. Surt cancer . ObservaThe lack of receptor specificity of the first identified response elements for glucocorticoids, progestagens, mineralocorticoids, and androgens has been confusing. Indeed, in binding and transactivation assays, all glucocorticoid response elements (GRE) are recognized by AR, MR, and PR and vice versa; many androgen response elements (AREs) are recognized by the other oxosteroid receptors –24. In ain silico analyses of the ARBS lead to the idea that the AR might also bind monomeric motifs or dimeric motifs with variable spacing and orientations [The first systematic discovery of genomic AR binding sites was based on ChIP-on-chip and ChIP-seq experiments on AR-positive prostate cancer cell lines. While the earlier, limited ‘old school’ biochemical analyses indicated that AR binds DNA motifs that are organized as 5′-AGAACA-3′-like repeats, initial ntations –31. Howentations , 32. A sntations . Chen etntations .While the data for monomeric binding are less convincing for AR, monomeric binding for the GR has been reported to some of its enhancers . MoreoveIt should be noted that in this review, we do not discuss the possibility of indirect DNA binding, which has been well documented, certainly in case of the GR. Indeed, such GR tethering to DNA via other transcription factors could involve monomeric receptors and will result in receptor-specific effects on gene activation and/or repression –40. In hDespite the high similarity between AREs and GREs Fig. a, we andA comparison of the sequences of the first selAREs with that of the first known classical AREs led us to propose that the selAREs could be organized as direct repeats, rather than inverted repeats of 5′-AGAACA-3′-like hexamers , 24. ThiTo answer the question how common selective AREs are and whether this type of selectivity has any in vivo relevance, we developed the SPARKI mouse model. In this model, the exon encoding the second zinc finger of the AR was replaced for the corresponding exon of the GR gene. The resulting mutant AR can still bind to classical AREs, but no longer binds to or activates via selAREs; hence the name specificity affecting AR knockin (SPARKI) . The SPAThe evaluation of the AR binding sequences in SPARKI and WT tissues allowed for the construction of a consensus sequence for selAREs (AR binding lost in SPARKI) and for classical AREs (AR binding retained in SPARKI and present in WT) Fig. b. These Therefore, how can we explain the variation in sequence preference between AR and GR? For both receptors, it was postulated that the affinity for the upstream, more conserved 5′-AGAACA-3′ hexamer is high and binding to this hexamer results in a DNA-dependent dimerization with a second monomer that will bind hexamers that can diverge more from 5′-AGAACA-3′ –16. It iUnfortunately, in the only crystal structure of the AR-DBD bound to DNA that has been published until now , the motDespite their name, hinge regions are more than the flexible connection between the DBDs and the LBDs. For the AR-, PR-, GR-, and ER-DBDs, it is clear that residues immediately carboxyterminal of the second zinc finger are involved in DNA binding Fig. a 50, 5151. IndeeLike for most proteins, allostery plays an integrative role in the functioning of nuclear receptors. For the ligand-binding domains of NR, there are clear allosteric pathways identified between the ligand-binding pockets and the activation functions, where coregulators interact . Many daPhysiological evidence for allosteric effects in the AR comes from the functional analysis of AR-NTD mutations, and detected in androgen insensitivity patients that affect the functioning of AR in an ARE-specific way . In addiThe recent discovery of many thousands long noncoding RNA-encoding genes in the human genome will affect the study of virtually all biological processes . These lIn vitro, this RNA motif can titrate MR, PR, and AR activity in cellular overexpression experiments [Another action mechanism was reported for ‘growth arrest-specific transcript’ called gas5. GR was shown to bind to a motif in this lncRNA, and this was proposed to squelch the receptor away from the genome . The receriments . It willIn conclusion, over the coming years, we can expect a dramatic increase in our insights on how lncRNAs affect NR biology in many different physiological processes and disease mechanisms.The first AREs in eukaryotic genes were described for the androgen-dependent, prostate-specific C3 component of the prostatic binding protein , 75, theDespite near identity of the DBDs, there are large differences between AR and GR in mobility reflecting kinetics of chromatin binding as measured by fluorescence recovery after photobleaching. These differences of residence at the MMTV enhancer array are compound effects of DNA binding as well as differential effects of other binding transcription factors, histon modifiers, and chromatin remodeling complexes , 87.While AR can be targeted in castration-resistant prostate cancer with ADT or anti-androgens, glucocorticoids can be used to relieve pain or suppress androgens. A first clinical observation relevant for this discussion is that AR-targeting therapies can lead to the appearance of mutations that change ligand specificity of the AR and allows glucocorticoids to act as agonists for the AR .In addition, the high similarity between the DBDs of AR and GR as well as their response elements led to the hypothesis that the receptors could be interchangeable . In normIn prostate cancer cell lines, many genes that are androgen responsive can also respond to glucocorticoids, provided that the GR is expressed . In suchOver the last years, major steps forward have been taken in our knowledge of the transcription activation process. Surprisingly, we are still far from understanding the tissue-specific actions of AR and GR, two very similar steroid receptors with very divergent biological roles. Indeed, although general roadmaps of the activation are well described, now, we need to search for the details that explain the different rules of engagement of these two receptors.The description of structures of full size receptors or larger receptor fragments bound to DNA, ligand, and coregulator peptides continues to reveal more details on the allosteric pathways within the receptors , 97. The


AR) expression is suggested by the upregulation of choline kinase-alpha in prostate cancer. Recently, detection of AR aberration in cell-free DNA as well as early 18F-fluorocholine positron emission tomography/computed tomography (FCH-PET/CT) were associated with outcome in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone and enzalutamide. We aimed to make a direct comparison between circulating AR copy number (CN) and choline uptake at FCH-PET/CT. We analysed 80 mCRPC patients progressing after docetaxel treated with abiraterone (n = 47) or enzalutamide (n = 33). We analysed AR CN from plasma samples using digital PCR and Taqman CN assays and total lesion activity (TLA) and metabolic tumor volume (MTV) on FCH-PET/CT at baseline. A meaningful correlation was showed among AR gain and TLA/MTV compared to AR non-gained cases , independently from type of treatment. Multivariate analysis revealed that AR CN and only TLA were associated with both shorter PFS  and OS . AR gain appeared significantly correlated with choline uptake represented mainly by TLA. Further prospective studies are warranted to better address this pathway of AR-signalling and to identify multiplex biomarker strategies including plasma AR and FCH-PET/CT in mCRPC patients.The association between choline uptake and androgen receptor ( Androgen receptor (AR) signalling axis is the most important actor in PC pathogenesis and in the progression to castration resistant PC (CRPC), representing a fundamental target of androgen deprivation therapy2. In PC, multiple direct mechanisms can stimulate AR signalling and often lead to therapeutic resistance, including AR copy number (CN) gain or somatic point mutations6 and constitutively active AR splice variants such as AR-V77. Among the indirect mechanisms responsible of increased AR protein expression, there is also the up-regulation of choline kinase alpha (CHKA). It binds directly the AR ligand-binding domain (LBD), fulfilling different roles involved in the tumour growth and disease progression9. Classical function of CHKA in Kennedy pathway is the production of phosphatidylcholine, essential for plasma membrane biogenesis10. In addition, CHKA has a “nonclassical” function, interacting with cytoplasmatic AR and promote its stability. CHKA plays a role as a chaperone for AR, resulting in the ubiquitination and activation of AR–dependent transcription. Recent genome wide approaches11 suggested this dual interaction between CHKA and AR in cytoplasm, promoting AR overexpression and increased signalling, which in turn may provide more CHKA production.Prostate cancer (PC) is the most common cancer type for the estimated new cancer cases and the third tumour for the estimated deaths among men in 2015 worldwide14.Some studies have documented the overexpression of CHKA protein in multiple cancer types including breast, colorectal, endometrial, lung, ovarian, and prostate, providing an increased uptake of choline18, especially PC22, by mainly using 18F-fluorocholine (FCH), its radiopharmaceutical analogue.Choline is a relatively new radiopharmaceutical for positron emission tomography (PET)/computed tomography (CT) imaging, and its utility in visualizing and staging different tumours has been published22 in metastatic CRPC (mCRPC) patients treated with novel anti-AR therapies, such as abiraterone23 and enzalutamide24, have strengthened the hypothesis of a relationship between choline uptake and AR.Recent studies, investigating the early response assessment on FCH-PET/TC imaging26. We aimed to identify an association between circulating AR CN and choline uptake to better understand the complex AR signalling axis and alternative pathways of the resistance mechanisms, such as choline metabolism, and to improve multiplex biomarker strategy in mCRPC patients.In this study, we evaluated the choline uptake in mCRPC patients measuring the total lesion activity (TLA) and metabolic tumour volume (MTV) which have shown much greater prognostic value than conventional PET measurements such as the maximum standardized uptake value (SUVmax)AR gain prevalence in the two treatment groups. Table Between October 2011 and November 2015, 80 mCRPC patients  were enrolled for this study. Overall median age was 74 years (range 42–90). All patients previously received a docetaxel-based regimen, and 35 (43.8%) patients received previous treatments . At baseline, all patients had evidence of mCRPC and were categorized according to the extent of metastases in a binary categorical variable, defined as bone or lymph node only (low spread) including 40 cases, and visceral involvement or combined bone and lymph node disease (extensive spread) including 40 cases without differences between abiraterone and enzalutamide group. On the contrary, the median number of metastatic lesions on FCH-PET/TC is significant different between two groups . Androgen receptor was gained in 24 cases (30%) with no significant difference in AR gain  (Table AR CN gain (A) and low choline uptake/AR CN normal (B), respectively.The reliability of the presented data has been shown in the Fig. y) Table . Figure At the time of analysis, 77 (96.2%) out of 80 patients had progressive disease (PD) and 51 (63.7%) patients had died. Median follow-up was 22 months (range 1–42). The median progression-free survival (PFS) and overall survival (OS) were 6.4 months .AR CN and FCH-PET/CT and outcome. In the univariate analysis, AR status, all PET parameters, and number of metastatic lesions predicted significantly PFS and OS  2.03 , and HR 2.68 , respectively] and OS  42, in order to better define tumour biology, mechanism of treatment resistance and potential therapeutic targets in mCRPC patients.In conclusion, this study firstly combined two biomarker approaches of imaging and genomics may implement the patient treatment selection, avoiding useless overtreatment and toxicity. This work increases the prospects for personalized CRPC treatment by stratifying patient cohorts according to their AR CNs and FCH-PET/CT imaging studies were obtained within 30 days of therapy initiation. During treatment, PSA response and toxicity were evaluated on a monthly basis. FCH-PET/CT was repeated after 3 to 6 weeks. A CT scan was performed after 3 months of abiraterone and enzalutamide therapy. Disease progression was defined according to Prostate Cancer Working Group 2 (PCWG2) criteria43. The study included patients participating in a protocol approved by the Institutional Review Board of Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Italy (REC 2192/2013). The study was approved by the Local Ethics Committee (Ethics Committee Area Vasta Romagna and IRST) and informed consent for the use of biological material for research purposes was obtained from all patients before plasma sample collection. All experiments were performed in accordance with relevant guidelines and regulations.Eighty consecutive patients with mCRPC without neuroendocrine differentiation in progression after docetaxel and treated with abiraterone and enzalutamide were included in this retrospective study. Selection criteria included histological confirmation of adenocarcinoma of the prostate, baseline serum testosterone <50 ng/dL, Eastern Cooperative Oncology Group (ECOG) performance status ≤2, ongoing androgen deprivation therapy. Exclusion criteria were renal insufficiency and/or concomitant therapy with proton pump inhibitors which could influence the CgA levels and so potential onset of neuroendocrine differentiation. All patients had completed previous chemotherapy at least 4 weeks before to allow baseline examinations for abiraterone and enzalutamide to be performed. Metastatic disease was documented by bone scan, computed tomography or magnetic resonance imaging. In the group of patients treated with abiraterone, therapy consisted of 28-day cycles of daily abiraterone acetate 1000 mg with twice-daily prednisone 5 mg. In the group of patients treated with enzalutamide, treatment consisted of enzalutamide 160 mg once daily. The choice of therapy was at the discretion of the treating physician. In each therapy group, treatment was continued until there was evidence of PD or unacceptable toxicity. Peripheral blood samples for plasma DNA extraction for the investigation of cell-free FCH-PET/CT scans were performed on an integrated PET/CT system  in 2D acquisition mode for 3 minutes per bed position. PET scanning was performed 45 minutes after intravenous injection of 18F-Methilcholine . The field of view comprised the skull to mid-femurs. Low dose CT  without contrast agents was carried out for attenuation correction and as an anatomical map. The emission data were adjusted for scatter, random coincidence events, and system dead time utilizing the provided software.44. Each patient had the MTV calculated as the sum of each three- dimensional volumes of interest. Moreover, for each lesion volume and SUV mean was multiplied and then summed to generate TLA. The first estimate MTV is a purely volumetric entity, while TLA also takes the metabolic activity of the lesion into account, thereby producing an estimate of the tumour activity45. FCH-PET/CT images were read sequentially by using a Xeleris III workstation . PET, CT, and PET/CT fused images provide to properly assess the scans in axial, sagittal, and coronal cuts.The reading and interpretation of PET/CT scans were assigned to two nuclear medicine physicians with consolidate experience. Criteria to define PET/CT positivity included the presence of focal areas of increased tracer uptake with or without any underlying lesion identified using CT. Semiquantitative criteria based on SUVmax and the target to background ratio were used to aid the visual analysisAR CN assays were performed using AR gene and at least two different reference genes: RNaseP, NSUN3, ElF2C1, and AP3B1 and ZXDB at Xp11.21 as a control gene, as previously described5.Peripheral blood samples were collected within 30 days of each treatment initiation, maintained at room temperature, processed within 30 min, and stored at −80 °C. Circulating DNA was extracted from 1 to 2 ml of plasma with the QIAamp Circulating Nucleic Acid Kit (Qiagen) according to the manufacturer’s instructions. DNA quality and concentration were estimated using spectrophotometry quantification . We performed copy number of androgen receptor (AR) by duplex TaqMan quantitative real-time PCR (qPCR) assay  and multiplex droplet digital PCR on a QX200 ddPCR system (Bio-Rad). AR gain was performed using median test (Wilcoxon). PFS and OS were estimated using the Kaplan-Meier method and survival curves were compared using the logrank test. Cox regression model was used to investigate potential predictors of PFS and OS and to estimate HR and their 95% CI. All P-values were two-sided and a P < 0.05 was considered as statistically significant. Statistical analyses were performed with SAS 9.4 software .Progression-free survival was defined as the time between the first day of abiraterone and enzalutamide treatment and the date of PD or death (whichever came first). Patients who had not progressed at database closure were censored at the last tumour evaluation or discontinuation of treatment due to toxicity. Overall survival was defined as the time between the first day of treatment and the date of death from any cause or the date of the last follow-up visit. Receiver-operating characteristics (ROC) analysis was performed to estimate accuracy of TLA and MTV, considered as continuous variables. The area under the ROC curve with 95% CI was calculated. The comparison of MTV and TLA median values in patients with or without Results of this study have been presented in part at the European Society of Medical Oncology (ESMO) Cancer Congress 2015 in Vienna, Austria, 25 September–29 September, 2015 (poster presentation session).Supplementary Table S1