Evidence suggests that rehabilitation interventions can improve function and quality of life in survivors of head and neck cancer (HNC), but there is a lack of coordinated, integrated services, and those offered are inconsistent. To address these gaps, we will develop and conduct preliminary evaluation of a rehabilitation consult, built on the theoretical foundations of goal setting and self-management, and composed of a brief functional evaluation, a resource compendium, and collaborative goal-setting and action planning processes.The development of the rehabilitation consult will be guided by intervention mapping, which consists of six steps: 1. Needs assessment; 2. Definition of program objectives; 3. Selection of theory-based intervention methods; 4. Production and pretesting; 5. Adoption, implementation and sustainability planning; 6. Process and effect evaluation. Within the intervention mapping framework, an iterative process of constructing drafts and mini-evaluations with consumers and experts will be used, modifying the rehabilitation consult intervention until a version suitable for formal evaluation is established. The rehabilitation consult will then be evaluated using a prospective, mixed method, single group design with 30 survivors of head and neck cancer. Outcomes will be assessed pre- and post-intervention and at 6-month follow-up.Survivors of head and neck cancer have among the most complex rehabilitation needs of all cancer patients. The rehabilitation consult is expected to improve knowledge and uptake of rehabilitation resources and strategies in survivors of head and neck cancer and thereby improve long-term function and quality of life. If the rehabilitation consult is effective in cancer patients with such high and diverse needs, this project will produce a toolkit that will be adaptable for other types of cancer in other jurisdictions. In contrast with other major chronic conditions such as heart disease and stroke, cancer care does not routinely integrate evidence-based rehabilitation services within the standard continuum. This protocol describes a structured process for the development, implementation and preliminary evaluation of a novel, integrated rehabilitation intervention for survivors of head and neck cancer (HNC) called the rehabilitation consultation (RC). The RC program goals are to increase knowledge about rehabilitation needs and resources to meet those needs, to establish individualized rehabilitation goals for HNC survivors and personalized action plans to meet those goals and to provide support to HNC survivors for the implementation and evaluation of action plans. The goals will be personally important to the individual survivor, and the action plans will be achievable, using resources they can access close to home rather than at the cancer centre, when possible. Additionally, the RC will be integrated into routine HNC follow-up procedures and will be administered as soon as possible following active cancer treatment. It will be led by a professional with a background in one of the traditional rehabilitation professions and will include the following three components: 1. A brief, HNC-specific functional evaluation; 2. A resource compendium; 3. Collaborative goal-setting, action-planning and follow-up processes.Reductions in function and quality of life are particularly high in HNC, as the disease and treatment cause more diverse and serious impairments than many other cancers. Issues may include reductions in swallowing, speech, neck and upper extremity mobility, general deconditioning, fatigue, insomnia, lymphedema, neuropathies, visible facial deformity and psychological distress -9. In adCancer rehabilitation has been defined as coordinated, professional care designed to enable people to maximize physical, social and psychological function within the limits imposed by the disease and treatment effects and to engage in personally valued activities within their social contexts . RehabilThis project employs intervention mapping as an ecologically valid, structured framework to develop, implement and evaluate the RC . Interveneeds assessment, we used information gleaned from focus groups with patients, family members and front-line health care professionals  and from a scoping literature review  to establish the rehabilitation needs of HNC survivors. We conducted six focus groups with 38 survivors, family members and health care professionals from two large cancer centres. Using directed content analysis of the transcripts, we answered specific research questions, including “What are the functional issues following treatment for HNC that might be mitigated with rehabilitation interventions?” Functional issues identified were categorized as impairments or function/participation/health issues and formed the basic list of rehabilitation needs. To augment that list, a scoping literature review was conducted to answer the question, “What is known from the existing literature about outcomes of HNC survivors that are potentially amenable by rehabilitation interventions?” Relevant observational studies published between 2003 and 2013 were identified from MEDLINE, EMBASE, CINAHL, PsycINFO and RehabDATA databases. An initial 1,245 abstracts were screened, 422 full text manuscripts were read and 74 observational studies were identified for inclusion in the review. Observed impairment (body functions and body structures), function, participation and health outcomes deemed as potentially amenable to rehabilitation interventions were added to the list of rehabilitation needs developed from the focus groups. The rehabilitation needs appear as phase 2 and phase 1 in Table In the previously completed step 1, definition of program objectives, the resource requirements that will enable achievement of the program goals described above were specified  and Odette Cancer Centre to assess its feasibility and acceptability. Based on the findings, further modifications will be made, resulting in a beta version of the RC that is ready for implementation and evaluation.planning for adoption, implementation, and sustainability, is a planning step, in which a logic model will be developed to guide the initial implementation and program evaluation. Specific objectives for adoption, implementation and sustainability will be established, and determinants of those will be considered.Step 5, evaluation, will implement the program evaluation plan established in step 5 and will also include a pilot outcome evaluation to estimate the impact of the RC on function and quality of life. We will implement and evaluate the RC using a mixed method, single group study with a convenience sample of approximately 30 HNC survivors post primary cancer treatment, recruited from the PMCC HNC clinics. Eligible participants will be adult survivors of HNC who have completed active treatment  within the past 1–4 months. Exclusion criteria will be lack of English fluency or concurrent major degenerative conditions likely to cause functional deterioration. A research assistant not involved with clinical administration of the RC will assess the following constructs pre- and post-RC intervention and at a follow-up 6 months later: function, quality of life, self-efficacy, community participation, goal attainment and return to work status (if applicable). Table Step 6, r using the formula r2 = z2/N [Quantitative data analysis will be exploratory and descriptive, and effect sizes will be calculated for all outcomes to help plan for a future, controlled trial. We will calculate means, standard deviations and Cohen’s d  effect s2 = z2/N . For qua2 = z2/N . In the Assuming positive outcomes from this single group evaluation, a future, multi-site controlled trial will be designed and implemented. Results from this study will provide feasibility information, such as recruitment rates, will help to define primary and secondary outcomes and will provide data to calculate sample size to ensure an adequately powered trial.This project brings together the diverging views of rehabilitation specialists, focused on long-term real-world function, with those of cancer specialists, focused on acute treatment and episodic symptom management. We have set out to bring those views together to develop a clinically effective and cost-effective rehabilitation intervention that integrates seamlessly with an existing cancer care system. Survivors of HNC have among the most complex rehabilitation needs of all cancer patients because of the anatomical complexity of the head and neck region. The RC is expected to improve knowledge and uptake of rehabilitation resources and strategies in survivors of HNC and thereby improve function and quality of life. The RC will be designed to ensure the components are readily modifiable for use beyond the regional cancer centres within which they were developed. Further, we believe that HNC serves as an ideal incubator for development of the RC. If it is effective in cancer patients with such high and diverse needs, it is expected that this project will produce a toolkit that will be adaptable for other types of cancer in other jurisdictions.


We show that, especially when groups are small, longer-memory strategies make cooperation easier to evolve, by increasing the number of ways to stabilize cooperation. We also explore the co-evolution of behavior and memory. We find that even when memory has a cost, longer-memory strategies often evolve, which in turn drives the evolution of cooperation, even when the benefits for cooperation are low.Complex social behaviors lie at the heart of many of the challenges facing evolutionary biology, sociology, economics, and beyond. For evolutionary biologists the question is often how group behaviors such as collective action, or decision making that accounts for memories of past experience, can emerge and persist in an evolving system. Evolutionary game theory provides a framework for formalizing these questions and admitting them to rigorous study. Here we develop such a framework to study the evolution of sustained collective action in multi-player public-goods games, in which players have arbitrarily long memories of prior rounds of play and can react to their experience in an arbitrary way. We construct a coordinate system for memory- Behavioral complexity is a pervasive feature of organisms that engage in social interactions. Rather then making the same choices all the time – always cooperate, or never cooperate – organisms behave differently depending on their social environment or their past experience. The need to understand behavioral complexity is at the heart of many important challenges facing evolutionary biology as well as the social sciences, or indeed any problem in which social interactions play a part. Cooperative social interactions in particular play a central role in many of the major evolutionary transitions, from the emergence of multi-cellular life to the development of human languageEvolutionary biologists have been successful in pinpointing biological and environmental factors that influence the emergence of cooperation in a population. The demographic and spatial structure of populations in particular have emerged as fundamentally important factors2345679101112Despite extensive progress for simple interactions, an understanding of the evolution of cooperation when social interactions occur repeatedly – so that individuals can update their behavior in the light of past experience – and involve multiple participants simultaneously, remains elusive. Some of the most promising approaches for tackling this problem come from the study of iterated games141516171819202122Nowhere is the complicated nature of the problem more evident than in the discussion surrounding the role of memory in iterated games and the evolution of cooperation222324262328242519313233If we are interested in the evolution of cooperation, however, the stability of some memory-1 strategies against all forms of invaders is not in itself informative. Rather, as we and others have shown, what matters most for the maintenance of cooperation over evolutionary time is the ease with which successful cooperative strategies can evolve3134We study evolving populations composed of individuals playing arbitrary strategies in infinitely iterated, multiplayer games. We focus on the prospects for cooperation in public goods games, and we investigate how these prospects depend on the number of players that simultaneously participate in the game, on the memory capacity of the players, and on the total population size. We then study the co-evolution of players’ strategies alongside their capacity to remember prior interactions, including when such memory capacity comes at a cost. We arrive at a simple insight: when games involve few players and groups are small, longer memory strategies tend to evolve, which in turn increases the amount of cooperation that can persist. And so populations tend to progress from short memories and selfish behavior to long memories and cooperation.n players repeatedly choose whether to cooperate by contributing a cost C to a public pool, producing a public benefit B > C. In each round of iterated play the total benefit produced due to all player’s contributions is divided equally between all players. Thus, if k players choose to cooperate in a given round, each player receives a benefit Bk/n. We study finite populations of N players engaging in infinitely iterated n-player public-goods games, using strategies with memory length m, meaning a player can remember how many times she and her opponents cooperated across the preceding m rounds  strategies, which cannot be invaded by any non-cooperating (or non-defecting) mutants, in the iterated n-player public-goods game. We apply these results to make specific predictions for the effects of group size and of memory capacity on the evolution of collective action through sustained cooperation. Finally we explore the consequences of these predictions for the co-evolution of cooperation and memory capacity itself.We begin our analysis by describing a coordinate system under which the volume of robust strategies can be determined analytically, for games of size Recently, Press and Dyson introduced so-called zero determinant (ZD) strategies in iterated two-player games3738394030N individuals193132Other research has expanded the framework of Press and Dyson to study all possible memory-1 strategies for infinitely repeated, two-player games19313233m strategies in multi-player games of size n, such that all n players’ longterm payoffs are related in a straightforward  way. One essential trick that enables us to achieve this goal is to construct a mapping between memory-m strategies in an n-player game and memory-1 strategies in an associated n × m-player game. We then construct a coordinate system for the space of memory-1 strategies in multi-player games that allows us to easily characterize the cooperating and the defecting strategies that resist invasion. We apply these techniques to the case of iterated n-player public-goods games and we precisely characterize all evolutionary robust memory-m strategies – i.e. those strategies that, when resident in a finite population of N players, can resist selective invasion by all other possible strategies – thereby elucidating the prospects for the evolution of cooperation in a very general setting.Here we generalize this body of work by developing a coordinate system for the space of memory-Our goal is to study the effects of group size and memory on the frequency and nature of collective action in public-goods games. Allowing for long-memory strategies and games with more than two players greatly expands the potential for behavioral complexity, because players are able to react to the behaviors of multiple opponents across multiple prior interactions. And so merely determining the payoffs received by players in such an iterated public-goods game can pose a significant challenge. In order to tackle this problem we develop a coordinate system for parameterizing strategies, in which the outcome of a game between multiple players using long-memory strategies can nonetheless be easily understood.m strategy chooses her play in each round of an iterated game in a way that depends on the history of plays by all n players across the preceding m rounds. In general such a strategy consists of 2n×m probabilities for cooperation in the present round. We write the probability for cooperation of a focal player in its most general form as i. Each m plays for player i, with each entry taking the value c (cooperate) or d (defect). The 2n×m probabilities for cooperation form a basis for m strategies in n-player games. In the n×m vectors that also form a basis for A player using a memory-m strategy in an n-player game as a player who instead uses a memory-1 strategy in an associated n × m-player game. That is, we will think of an n-player game in which a focal player uses a memory-m strategy in terms of an equivalent n × m-player game, which is composed of n “real” players along with m − 1 “shadow” players associated with each real player. The shadow players play the same way that their associated real player did t rounds previously, for 2 ≤ t ≤ m. The focal player’s memory-m strategy is thus identical to a memory-1 strategy in the n × m player game, where the corresponding memory-1 strategy responds to a large set of “shadow” players whose actions in the immediately previous round simply encode the actions taken by the n real players in the preceding m rounds. This trick allows us to reduce the problem of studying long-memory strategies to the problem of studying memory-1 strategies, albeit with a larger number of players in the game.In order to simplify our analysis of long-memory strategies we will conceive of a focal player using a memory-m rounds is correctly recreated at each round of the associated n × m-player game. This construction is straight forward. If the focal player played c in the last round, then we stipulate that her first shadow player will play c in the next round (i.e. it will copy her last move). Similarly her second shadow player will copy the last move of her first shadow player, and so on, up to her (m − 1)st shadow player. The same goes for the shadow players of each of her n − 1 opponents. In this way, all the plays of the last m rounds in the n-player game are encoded at each round in the associated n × m-player game.All that is required is to construct strategies for the shadow players so that the state of the system across the preceding m strategy in an n-player into an associated memory-1 strategy in an n × m-player game, we now describe a coordinate system for memory-1 strategies that allows us to derive a simple relation among the equilibrium payoffs to all players. We define this coordinate system for arbitrary games in the Having transformed an arbitrary memory-k of her opponents receives a net payoff B(k + 1)/n − C, whereas a player who defects while k of her opponents cooperate receives a net payoff Bk/n. That is, the payoff received depends on whether or not the focal player cooperated and on the number of her opponents that cooperated, but it does not depend on the identity of her cooperating opponents. Likewise, if a player has memory of the preceding m rounds of an iterated public-goods game, then her payoff across those rounds depends on the total number of times she cooperated and the total number of times her opponents cooperated, but it does not depend on the order in which different players cooperated nor on the identity of her cooperating opponents. Therefore, rather than studying the full space of 2n×m probabilities for cooperation, we can limit our analysis for iterated public-goods games to strategies that keep track of the total number of times a focal player cooperated, and the number of times her opponents cooperated, within her memory capacity. A focal player’s strategy can thus be expressed as lo denotes the total of number of times the player’s opponents cooperated in the preceding m rounds (which number can vary between 0 and (n − 1)m) and lp denotes the total number of times the player herself cooperated in the preceding m rounds (which can vary between 0 and m).Under a public-goods game, a player who cooperates along with m strategy in an iterated n-player public-goods game, we have developed an alternative coordinate system, defined in κ); (3) The tendency to cooperate in proportion to the payoff received by the focal player (χ); (4) The tendency to punish (i.e. defect) in proportion to the payoffs received by her opponents (φ) and (5) The tendency to punish in response to the specific outcome of the previous rounds  on the prospects for cooperation can be illustrated by considering two extreme cases. When the entire population takes part in a single multi-player game, so that required ; and so i can invade a population in which a defecting strategy is resident only if he receives a payoff Si that exceeds the payoff received by the resident defector. The resulting necessary and sufficient conditions for the robustness of defecting strategies are then:Understanding the expected rate of cooperation in multi-player games requires that we compare the volume of robust cooperative strategies to the volume of robust defecting strategies. A rare mutant smaller values of χ, which for defectors corresponds to more extortionate behavior, such that players try to increase their own payoff at their opponents’ expensen can attenuate the effect of contingent punishment on robustness, they also make it easier to construct an extortionate strategy that is robust; and the latter effect is always stronger, so that larger games permit a greater volume of defecting strategies. Overall, n, tends to increase the volume of robust defectors, in contrast to its effect on robust cooperators.Once again, we see from We confirmed our predictions for the effects of group size on the volume of robust cooperators and defectors by analytical calculation of robust volumes, from C − B/n, and so the incentive to defect grows as the size of the group grows. This of course is the group size paradox, and it is a well known phenomenon for any collective action problemn = N the only hope for robust cooperation is tit-for-tat-like strategies, that are capable of both sustained cooperation and sustained defection, depending on their opponent’s behavior.There is a simple intuition for why larger games make cooperation less robust and defection more robust: In public-goods games with more players, the marginal change in payoff to a player who switches from cooperation to defection is n < N. As such, both cooperation and defection can evolve. Although these robust strategies cannot be selectively invaded by any other strategy when resident in a population, they can be neutrally replaced by a non-robust strategy of the same type, which can in turn be selectively invaded. As a result, there is a constant turnover between cooperation and defection over the course of evolution, with the relative time spent at cooperation versus defection determined by their relative volumes of robust strategies31In general, both cooperators and defectors have positive volumes of robust strategies, provided n we can always find yet larger population sizes N such that robust cooperative strategies are guaranteed to exist. Moreover, increasing the population size N leads to increasing volumes of robust cooperative strategies and decreasing volumes of robust defecting strategies . And so as memory increases, cooperators become more robust relative to defectors, as 2–3 and The change in the efficacy of punishment for rare deviants from the social norm as memory capacity increases is illustrated in all possible invaders, regardless of their memory capacity. However increased memory can nonetheless make robust cooperation easier to evolve, because it allows for more effective contingent punishment. This tends to have a stronger impact when games are small because, as described in N–n, and thus the effect of longer memory on the contributions of Our results on the relationship between memory capacity and the robustness of cooperation raise a number of interesting questions. In particular, memory of the type we have considered does not seem to convey a direct advantage to cooperation (or defection), because a robust cooperative (or defecting) strategy is robust against n = N, there is little evolutionary change in memory capacity and defection continues to be more frequent than cooperation even as strategies and memory co-evolve. In a two-player game, even when memory comes at a substantial cost, an intermediate level of memory evolves, and there is a corresponding increase in the degree of cooperation .


LMTK2) in modulating AR activity, as several Genome Wide Association Studies (GWAS) have shown a marked association of LMTK2 activity with the development of prostate cancer. We confirm that not only is LMTK2 mRNA reduced in prostate cancer tissue, but also LMTK2 protein levels are markedly diminished. Knockdown of LMTK2 protein in prostate cell lines greatly increased the transcription of androgen-responsive genes. In addition, LMTK2 knockdown led to an increase in prostate cancer stem cell populations in LNCaP cells, indicative of increased tumorogenicity. Using multiple approaches, we also demonstrate that LMTK2 interacts with the AR, thus putting LMTK2 as a component of a signaling complex modulating AR activity. Our finding that LMTK2 is a negative regulator of AR activity defines a novel cellular pathway for activation of AR-responsive genes in castrate resistant-prostate cancer. Moreover, pharmacologic manipulation of LMTK2 activity will provide a novel therapeutic target for more effective treatments for patients with castrate-resistant prostate cancer.Progression from early forms of prostate cancer to castration-resistant disease is associated with an increase in signal transduction activity. The majority of castration-resistance cancers persist in the expression of the androgen receptor (AR), as well as androgen-dependent genes. The AR is regulated not only by it associated steroid hormone, but also by manifold regulatory and signaling molecules, including several kinases. We undertook evaluation of the role of Lemur Tyrosine Kinase 2 ( The androgen receptor (AR), a ligand-dependent nuclear receptor, plays a critical role in prenatal development of the prostate –3. For ePP1C) dephosphorylates AR, leading to an increase in AR nuclear retention and increased gene transcription [Kinase-signaling pathways have been implicated in the regulation and modulation of nuclear receptor activity , 17, 1818–20. Hocription . Furthercription . Clearlycription .LMTK2) , a membrane associated kinase  [LMTK2 has a Single Nucleotide Polymorphism (SNP) in intron 9, causing decrease in LMTK2 mRNA levels [LMTK2 might be involved in the development and/or maintenance of prostate gland tumors. However, due to limited understanding of LMTK2 function [LMTK2 has been reported to interact with PP1C  of patients with prostate cancer have identified a genetic variant of Lemur Tyrosine Kinase 2  –32. FurtA levels . These sfunction , 34, itsP1C Fig.  and inhiLMTK2 interacts directly with AR and negatively regulates its activity. Furthermore, a decrease in LMTK2 protein expression, as proposed in prostate cancer, not only results in an increase in androgen mediated AR activity but also increases the androgen-independent activity of AR. Moreover, LMTK2-Knock Down (KD) in prostate cancer cells results in an increase in cell viability and tumorogenecity in the presence and absence of androgen. As such, our study takes observations made in genomic studies and reveals LMTK2 as a novel regulator of AR in prostate epithelium.In summary, the results in this manuscript argue that LMTK2 expression levels with prostate cancer, we initially determined if LMTK2 was expressed in prostate epithelia. We used a model cell line HEK293 as well as prostate cancer cell lines i.e. PTN1A, PC3 and LNCaP for the same. As predicted, immunoblot analysis showed robust endogenous expression of LMTK2 in prostate epithelial and HEK293 cells, which appeared as a single dominant band of ~210 kDa  and AR as expected, but also Lmtk2  containing prostate cancer (n = 48), prostate hyperplasia (n = 8) and normal prostate tissue (n = 14) from a total of 20 individual patients, revealed a marked difference in LMTK2 protein expression levels , low (0–20), medium (20–40), high (40–80) and very high (80–170). A majority, >65% of normal prostate tissue had very high expression of LMTK2, around 13% and 25% of hyperplasia tissue had respectively very high or high expression of LMTK2. In contrast, >67% of tumor tissue contained undetectable or low levels of LMTK2  between a decrease in LMTK2 protein expression and prostate cancer . Significantly higher numbers of LMTK2/AR protein complexes were detected in prostate epithelial cells in comparison to negative control (BAX/CFTR complexes), two known non-interacting protein , a means of assessing protein-protein interaction findings . LMTK2/Aein Fig.  and 4B. LMTK2 in human prostate tissue using co-localization analysis. 16 normal prostate tissue samples were tested using immunostaining for possible co-localization between AR and LMTK2. Fig. LMTK2 appear to co-localize in the glandular prostate epithelial cells. Results obtained from human prostate cancer cell and tissue model together shows that AR and LMTK2 are binding partners suggesting a functional significance of LMTK2 in AR-axis.To rule out cell line artifact, we further explored possible interaction of the AR and LMTK2 expression is associated with human prostate cancer and that LMTK2 and AR are binding partners in prostate epithelial cells. These results prompted us to examine the potential role for LMTK2 in AR-mediated signaling. For this purpose, we created HEK293 cells stably expressing either shRNA against LMTK2, control shRNA (scrambled sequence) or an LMTK2 over-expression plasmid. We also created stable LNCaP cells expressing either shRNA against LMTK2 (LNCaP-KD cells) or control shRNA (LNCaP-Control cells). Knock down or overexpression of LMTK2 in these cell lines was confirmed using immunoblot analysis  enhanced androgen-dependent activation of reporter gene by three fold in comparison to parental cells and by six fold in comparison to cells overexpressing LMTK2  stimulation for times shown in the Fig. LMTK2-KD significantly increased FKBP51 protein levels in comparison to LNCaP-control cells, with the biggest difference of about 5 fold was observed at time zero i.e. during the androgen deprivation stage . LNCaP-KD cells, as expected, showed ~5 times higher cell viability under androgen starvation and ~2.5 times in presence of androgen when compared to LNCaP-control cells , leading to decrease in its mRNA expression, to be associated with prostate cancer [LMTK2 in prostate epithelial cells. In this report, we compared protein expression of LMTK2 in human prostate cancer tissue specimens with normal, hyperplasia and prostate tissue specimens. Our data shows that loss of LMTK2 protein is strongly associated with prostate cancer and prostate hyperplasia, disease states marked by dysregulation of AR. Since, we did not genotype prostate cancer tissues it remains to be determined if SNP rs6465657 associated with prostate cancer results in decrease in LMTK2 protein expression. Furthermore, we also show a high-level expression of LMTK2 in normal prostate epithelial cells (PNT1A), androgen-independent metastatic prostate cancer cells (PC3), androgen-dependent prostate cancer cells (LNCaP) and mouse primary prostate epithelial cells. Since, LMTK2 was expressed in androgen dependent as well as androgen independent prostate cancer epithelial cells, it is likely that function of LMTK2 is not just limited to AR axis and certainly previous studies have shown LMTK2 to be an important binding partner of myosin VI involved in endocytic trafficking pathways in prostate cancer cells [LMTK2, as a negative regulator of AR transcription.Recently, several Genome Wide Association Studies (GWAS) involving prostate cancer patients of Caucasian and East Asian descent identified a genetic variant (SNP rs6465657) of Lemur Tyrosine Kinase 2 , a multi-pass membrane protein belonging to G-protein receptor-1 class undergoes nuclear translocation via importin pathway [It was surprising to find d kinase , 34 thoud kinase –46 and Nd kinase , we predTK2 Fig. , howeverer cells , 49. Sim pathway , 51.LMTK2 is an essential negative regulator of AR transcriptional activity and knocking down its expression in prostate cancer cells leads to significant increase in the AR activity and also an increase in tumorogenecity and cell viability. In contrast, overexpressing LMTK2 leads to repression of AR activity. Hence, it can be deduced that a decrease in LMTK2 expression observed in prostate cancer patient promotes tumor cells proliferation by enhancing AR transcriptional activity.The majority of prostate cancers (PCa) and Castrate Resistant Prostate Cancers (CRPC) have functional AR, which continues to drive the expression of AR-dependent cell proliferative genes and hence the tumor growth despite of low level of systemic androgens , 53. OurLMTK2 in CRPC. Kinases play an important role in driving AR activity in CRPC either by direct activation of AR or by increasing its sensitivity to low level of androgens. FKBP51, an AR-dependent gene, which is also a positive regulator of AR activity, is expressed two fold higher in CRPC compared with primary tumors [LMTK2 expression in prostate cancer cells deprived of androgen results in significantly higher levels of FKBP51 protein as well as increased mRNA levels of AR-dependent genes . These results provided strong circumstantial evidence of a role for LMTK2 in pathogenesis and progression of prostate cancer to castrate resistant stage. In addition, cell viability data showed the strongest role of decrease in LMTK2 in regards to androgen-independent growth in prostate cancer cells, androgen-dependent growth was also affected, although to a lesser degree.One of the most important outcomes from this report was the role of y tumors . Data inLMTK2 might be regulating AR transcription and cell proliferation in the absence of exogenous androgen in prostate cancer cells. There are several possible mechanisms that might be playing a role. Firstly, LMTK2 has been shown to inactivate catalytic activity of PP1, which plays an important role in nuclear retention of AR by dephosphorylating at Ser-650. Hence a decrease in LMTK2 protein express might result in increase in PP1 activity leading to increased nuclear retention and hence increased transcription activity. Secondly, LMTK2 might be directly phosphorylating AR and resulting in increased transcription activity in absence or presence of exogenous androgen. Initial evidence supporting this possibility comes from our study where we show LMTK2 and AR to be binding partners in prostate cancer cells. However, further experiments are needed to support this hypothesis. And lastly, FKBP51, an AR regulated gene is also known to positively regulate AR transcriptional activity. Our study showed increase in FKBP51 protein expression in LMTK2-KD prostate cancer cells. Argument can be made that LMTK2 regulates AR activity through FKBP51. Further experiments are clearly needed to identify possible mechanism through which LMTK2 augments AR transcription and cell proliferation in prostate cancer cells.An important question that arises from our study is the mechanism by which LMTK2 negatively regulates AR activity in prostate cancer cells possibly by directly interacting with AR. Furthermore, loss of LMTK2, associated with prostate cancer can enhance AR transcriptional activity in absence of androgen, suggesting role of LMTK2 in development of CRPC. LMTK2 can now be considered negative regulator of androgen-induced AR-mediated cell growth and transcription. In terms of potential therapeutic target, small molecules that enhance the activity of LMTK2 can decrease AR-proliferative activity in patients with prostate cancer and more importantly with castrate resistant prostate cancer.In conclusion, our findings are the first evidence that The human cell lines, PNT1A  and LNCaP  were cultured in RPMI 1640 medium while PC3  were maintained in F-12K medium. HEK293  were cultured in Advanced Dulbecco's Medium. All culture medium were supplemented with 10% FBS and 1% Pen/Strep  unless specified.LMTK2 cloned in PCI vector was provided as a gift by Dr. Takeshi Inoue, University of Tokyo. Halo tagged Androgen Receptor construct was obtained from Promega . Plasmids expressing and shRNA against human LMTK2 or control shRNA were obtained from DNA 2.0 . Transfections of cells were performed by 4D nucleofector  according to the manufacturer's instruction.Wild type Male mice were euthanized at 8–12 week of age and primary prostate epithelial cells were isolated as described . Cell tyLMTK2 . Coverslips were mounted onto a slide using Prolong Gold with DAPI (Invitrogen). The cellular signal was visualized using a PlanApo 60 ×, 1.42 NA oil immersion objective of an Olympus IX71 inverted microscope  coupled to a VT-Infinity 3 confocal system . For each sample, multiple coverslips were imaged (≥30 cells per coverslip) under identical settings.Cells plated onto poly-L-lysine coated coverslips were fixed according to pH-shift protocol as described  and staiLMTK2 and AR using Immunoblot analyses. Anti-human GAPDH antibody (SC-25778) was used for analyzing non-nuclear fraction while Lamin-A (SC-20680) was used for nuclear fraction.LNCaP cells were starved of androgen for 3 days by replacing complete media containing FBS with Charcoal Stripped Fetal Bovine Serum (CSFBS). On day 3, cells were treated either with DMSO or R1881 (Sigma-Aldrich) as described in results. Nuclear and non-nuclear fractions were separated as per manufacture's recommendation  and were probed for LMTK2 (rabbit anti-LMTK2) using immunoblot assay. AR pull down was confirmed by blotting with rabbit anti-AR N20 antibody. For immunoblot analyses samples were prepared in 4 × LDS sample buffer, resolved on 4–12% gradient Tris-Glycine Gel and transferred on to polyvinyldiflouride (PVDF) membrane. Membranes were probed for several proteins using human anti-AR, anti-LMTK2, anti-β-ACTIN  and anti-FKBP51  antibodies. Proteins were detected and analyzed using Odyssey Sa-Infrared imaging system™ (Li-COR Biotechnology).LNCaP cells were lysed in 1% NP-40 lysis buffer and endogenous complexes of AR were immunoprecipitated using mouse anti-AR 441 antibody. Immunprecipitated complexes were then probed for LMTK2 and AR using a Tyramide Signal Amplification Kit  as per manufacturer's recommendation.Tissue arrays (BC19021a) obtained from US-Biomax  containing normal, malignant and metastatic human prostate tissue was used. The arrays were probed for LMTK2 in LNCaP cells [LMTK2 and mouse anti-AR antibodies to detect AR/LMTK2 interactions. BAX (Bcl2 associated protein-X) and CFTR (Cystic Fibrosis Trans membrane Conductance Regulator) are non-interacting proteins, and hence rabbit anti-BAX (sc-493) and mouse anti-CFTR  were used as negative control. The cellular PLA signal was visualized using the Olympus IX71 inverted microscope as described above and quantified using Image-J software [A Proximity Ligation Assay kit  was used to study the interaction between AR and aP cells . Stainin® III First-Strand Synthesis System (Invitrogen) according to manufacturer instruction. cDNA samples were amplified using SYBR® Green PCR Master Mix on the Applied Biosystems 7500 Detection System. Gene-specific forward and reverse primers . Complementary DNA synthesis reactions were performed with 1 μg of RNA using SuperScript primers  used havPSA (Prostate Specific Antigen) protein levels were detected using an enzyme-linked immunosorbent assay (ELISA) technique [5 per well in RPMI media supplemented with 10% CSFBS. After 3 days, cells were treated for 16 h as detailed in results. PSA in the cultural supernatant and cellular PSA was quantified with an ELISA kit (Abcam) following the manufacturer's instruction. All assays were run in duplicates and repeated 3 times.echnique . Cells w5 per well in 96-well plates) were co-transfected with Halo-AR and/or either AR reporter, negative control or positive control in 2:1 ratio, supplied with cignal androgen receptor reporter kit . Cells were grown in charcoal-stripped media for 2 days and were treated with 1nm R1881 for 16 h. Luciferase activities were measured by using the dual-luciferase reporter gene assay system  following the manufacturer's instruction in plate reader . Final results were normalized for transfection efficiencies using the Renillia Luciferase Assay Value. Luciferase assay were repeated for 3 times.Cells (1 × 105 cells were plated per well onto flat clear bottom white polystyrene 96-well plate (Nunc) in RPMI media supplemented with 10% CSFBS. Cells were treated as discussed in the result section. Cell viability was measured using ATP-GloTM Bioluminometric cell viability assay kit  according to manufacturer's instruction [n = 3). Luminescence was measured using POLARstar Omega.1 × 10truction . Each as5 cells per well were plated onto 6-well Ultra-Low Attachment Plates  in Tumorsphere medium (PrEGM media with supplied growth factors and supplements+1% N2 (Invitrogen) and 1% B27 (Invitrogen). After 7 days number of spheroids  were counted under light microscope. Each experiment was carried out in triplicates.Cells were harvested and resulting pellet was washed in PBS. 1 × 10


Hyperglycemia following solid organ transplant is common among patients without pre-existing diabetes mellitus (DM). Post-transplant hyperglycemia can occur once or multiple times, which if continued, causes new-onset diabetes after transplantation (NODAT).To study if the first and recurrent incidence of hyperglycemia are affected differently by immunosuppressive regimens, demographic and medical-related risk factors, and inpatient hyperglycemic conditions .We conducted a retrospective analysis of 407 patients who underwent kidney transplantation at Mayo Clinic Arizona. Among these, there were 292 patients with no signs of DM prior to transplant. For this category of patients, we evaluated the impact of (1) immunosuppressive drugs , (2) demographic and medical-related risk factors, and (3) inpatient hyperglycemic conditions on the first and recurrent incidence of hyperglycemia in one year post-transplant. We employed two versions of Cox regression analyses: (1) a time-dependent model to analyze the recurrent cases of hyperglycemia and (2) a time-independent model to analyze the first incidence of hyperglycemia.P = 0.018), HDL cholesterol (P = 0.010), and the average trough level of tacrolimus (P<0.0001) are significant risk factors associated with the first incidence of hyperglycemia, while age (P<0.0001), non-White race (P = 0.002), BMI (P = 0.002), HDL cholesterol (P = 0.003), uric acid (P = 0.012), and using steroid (P = 0.007) are the significant risk factors for the recurrent cases of hyperglycemia.Age  with respect to both its first and recurrent incidence, as well as carefully differentiating these two perspectives: a fact that is currently overlooked in the literature. Hyperglycemia is a well-described complication following solid organ transplantation –3. AmongRegarding the first factor, In addition to immunosuppressive drugs, the literature has analyzed other demographic or medical-related risk factors to establish possible statistically significant associations with hyperglycemia and NODAT . The majFurthermore, recent evidence indicates that hyperglycemia occurring in the immediate post-transplant period  is also associated with NODAT , 68.remitting and relapsing hyperglycemia in post-solid organ transplant recipients. Such an understanding can be critical because (1) the insights gained can be quite different from those previously known for the incidence of hyperglycemia and (2) these insights can be extended to other chronic diseases with the possibility of remitting and relapsing, such as cancer and multiple sclerosis. To the best of our knowledge, this is the first study analyzing the first and recurrent incidence of hyperglycemia. In particular, utilizing a population of renal transplant recipients who had no history of DM before transplantation, we undertake a set of analyses to determine which contributing factors are significantly associated with the first incidence, and which ones are significantly associated with the recurrent incidence.In spite of all these efforts, none of these factors  have been analyzed with respect to the time course of post-transplant complications. Specifically, one critical aspect that is overlooked by the literature is an understanding and analysis of After obtaining Mayo Clinic Institutional Review Board (Mayo Clinic IRB) approval (Continuing Review #: PR13-004295-01) and written informed consent from all participating patients, this study conducts an analysis of 292 patients who underwent a renal transplant between 1999 and 2006 in Mayo Clinic Arizona, and who had no history of DM prior to surgery. Briefly, all patients were monitored at the time of transplant as well as month 1, 4, and 12 post-transplant. The available data included (1) demographic data such as age, race, and gender, (2) baseline patient characteristics including body mass index (BMI), blood pressure (BP), total cholesterol (Chol), high-density lipoprotein cholesterol (HDL), low-density lipoprotein cholesterol (LDL), uric acid (UA), and triglyceride (TG), (3) type of immunosuppressive drugs and diabetes medications that were used by the patients, (4) trough level of tacrolimus (as the main immunosuppressive drug used in this study), and (5) results of fasting plasma glucose (FPG) and Hemoglobin A1c (HbA1c) tests as measures of glycemic control. All major abbreviations used in this study are explained in remitting and relapsing hyperglycemia.NODAT was defined as HbA1c ≥ 6.5%, or FPG ≥ 126 mg/dL, or the requirement of diabetes medications  after patient discharge from hospital , 68. We proportional hazard imposed on the response variable (hyperglycemia incidence) by covariates that change over time. For example, the BMI of a patient may change as his/her weight changes . As another example, whether the patient uses an immunosuppressive drug at a specific time or not can be considered as a time-dependent covariate. Therefore, we sought to fully comprehend the effect of these changing behaviors on the recurrent incidence of hyperglycemia. (ii) Cox regression model with time-independent covariates, which measures the proportional hazard imposed on the response variable (the first incidence of hyperglycemia) by covariates at the time of the first incidence of hyperglycemia. (iii) Kaplan-Meier survival analysis to characterize the cumulative probability of experiencing hyperglycemia over time.We now explain the statistical inference methods we employed to analyze the effects of immunosuppressive drugs, the corresponding risk factors, and the inpatient period conditions on the first and recurrent incidence of post-transplant hyperglycemia. The statistical models used were: (i) The Cox regression model with time-dependent covariates, which measures the multiple imputations by chained equations (MICE) [The statistical analyses also include s (MICE) , which wAmong 407 patients in the study cohort, there were 115 patients with the history of diabetes. The remaining 292 patients had no indication of diabetes prior to or at the time of their transplants. The average age of patients who had no diabetes before transplant was 49.7 years, while those who had diabetes before had the average age of 56. remitting and relapsing post-transplant hyperglycemia (and hence the hyperglycemia for the first time). Among these patients, 19+3+1+24 = 47 patients experienced hyperglycemia multiple times, while 20+11+1 = 32 had it just once. As an example of the potential remitting and relapsing nature of post-transplant hyperglycemia, there are 11 patients who developed hyperglycemia at 4 months, which resolved at 12 months.Regarding the definition of remitting and relapsing hyperglycemia, This section sheds light on information about main immunosuppressive medications that have been considered for this study . As mentioned before, we focus on 292 patients with no prior history of diabetes.Tacrolimus (Prograf) is the main immunosuppressive drug utilized in this study. Another important point regarding tacrolimus is the dosage goals and achieved levels at different points of time. Tacrolimus goals are adjusted to avoid toxicity and to the lowest dose possible to avoid rejection per clinical standards of care. This is a standard clinical practice and is based on individual response and pharmacokinetics. Steroid is the second main immunosuppressive drug incorporated in this study. Steroid is usually prescribed by the following mechanism. If after using induction steroids (which last for up to 5 days post-transplant) a patient has an organ rejection, she will receive a taper dose of steroid . Then, by 1 month post-transplant, the patient will be put on the maintenance regimen of 5 mg daily (which is a low dosage), unless the patient has another rejection(s) later and needs possibly extra dosage of steroid therapy. To this end, we observed the following from the data set: (1) Among the 292 patients, only 20 patients had organ rejection at month 1, and hence, had to use a taper dose of steroid at this month. Therefore, there remained 272 patients who had no rejection during month 1. (2) Among 20 patients at month 1, 4 patients at month 4 and 1 patient at month 12 experienced organ rejection . (3) Among 272 patients at month 1, 5 patients at month 4 and 6 patients at month 12 experienced organ rejection . Therefore, according to the mechanism explained before, it can be concluded that 4+5+1+6 = 16 patients (out of 292) had increased dose of steroid  after 1 month post-transplant. Furthermore, as explained before, according to Sirolimus (Rapamune) is the third main immunosuppressive drug incorporated in this study. hazard ratio (HR), such that if mean HR ≥ 1, the corresponding covariate will have a positive effect on the response variable (and vice versa).To address events that may occur repeatedly, such as the repeated occurrence of hyperglycemia, we need to incorporate covariates that change over time . To this end, we employed a Cox regression model with time-dependent covariates and recurrent events, where each event is assumed to occur once a patient meets the criteria defined in According to The reason that the diabetogenic effect of tacrolimus cannot be established when hyperglycemia is occurring repeatedly may be due to the fact that tacrolimus dosage is usually reduced with the passage of time after transplant see . To testAs other observations made in this regard, induction immunosuppressive agents (thymoglobulin and simulect) are significantly associated with lower chance of first hyperglycemia. However, we cannot establish any significant association between using steroid and the first incidence of hyperglycemia. This can be due to the fact that high dosages of steroid were only considered for a small proportion of patients in month 1 post-transplant (see section “Summary of Immunosuppressive Treatment Regimens” for more information).Kaplan-Meier survival analysis to calculate the probability of having hyperglycemia obtained from Kaplan-Meier survival curves.The results of time-independent analysis established by the Cox regression model shows the significant association between the average trough level of tacrolimus and the first incidence of hyperglycemia. Here, we aim to use stratum based on average trough level of tacrolimus classified as “≤10” and “>10” mg/dL. In order to fully comprehend the effect of these levels on the incidence of hyperglycemia, we conduct unadjusted (univariate) analysis as well as ten adjusted analyses for those risk factors mentioned before. However, to incorporate these risk factors into the Kaplan-Meier survival analysis, they should be discretized in classes, which are shown in 2 for obesity). Furthermore, except age, gender, race, and blood pressure, other thresholds have been obtained from [To this end, we consider the main ned from . Regardined from .Trough-level 0 (1). P <0.0001). Specifically, almost all of the former group experience HG by month 4 , while the latter group still have about 80% chance of not experiencing HG by year 1. Although we made these observations for the unadjusted (univariate) analysis, the same behavior can be seen for adjusted analyses: the chance of experiencing HG is significantly different  across groups formed by different risk factors (see 2), and LDL ≥130 mg/dL. Moreover, Trough-level 1 patients with any of the following conditions experience HG by month 1 with a chance not less than 90%: age>50, male, hypertension, Chol ≥200 mg/dL, HDL <40 mg/dL, UA ≥7.3 mg/dL, or TG ≥150 mg/dL.tors see –2K. FurtWe also analyzed the associations of other well-known risk factors for both the first and recurrent incidence of hyperglycemia. To this end, we again applied two types of Cox regression model. The results of these analyses are provided in Combining these results with those in Prior studies have addressed the importance of the inpatient period: what happens to patients during post-transplant hospitalization may have an impact on patient’s conditions after hospital discharge , 68. To Our analyses highlight the complex nature of post-renal transplant hyperglycemia. Some patients never exhibit hyperglycemia, some develop permanent hyperglycemia (NODAT), while for others hyperglycemia may be transient or even recurrent. Hyperglycemia and NODAT have been mostly analyzed for a short period after transplantation , 73. HowThe idea of analyzing hyperglycemia from this perspective  can also be extended to other chronic diseases in which both the first incident and the recurrent ones need to be monitored. For example, prostate cancer and breast cancer are among diseases that may show signs only once or may do so from time to time with periods of remission in between , 81. ForThere are some limitations in our study. First, due to the nature of our study, having patients’ information on a more regular basis  would improve the accuracy of our results. Second, if the data set included patients’ information after the first year post-transplant, we would be able to conduct a more robust Cox regression and Kaplan-Meier survival analysis. Third, although, according to Finally, some of our findings may not be generalizable to other types of solid organ transplants . Therefore, testing our findings can be a fruitful path for future research. By extending the idea of this study and incorporating the time course of complications for other organs, one can establish a holistic framework to analyze (a) the diabetogenic effect of immunosuppressive drugs, and (b) the effect of other risk factors.We analyzed the effects of (1) immunosuppressive drugs, (2) risk factors, and (3) inpatient hyperglycemia on the first and recurrent incidence of post-transplant hyperglycemia in patients who had no history of diabetes mellitus prior to their transplants. We employed two statistical inference methods: (1) Cox regression model with time-dependent covariates to analyze hyperglycemia with recurrence and (2) Cox regression model with time-independent covariates to evaluate the first incidence of hyperglycemia. We also employed Kaplan-Meier survival analysis to characterize the cumulative probability of experiencing post-transplant hyperglycemia over time.Based on the results obtained from these methods, we can state that the diabetogenic effect of tacrolimus (based on its trough level) can be established when hyperglycemia is experienced for the first time. However, in a sharp contrast, this effect cannot be established for the recurrent incidents of hyperglycemia. This difference might be due to the fact that tacrolimus dosage is reduced by physicians over time. As the diabetogenic effect is ruled out, our results show that age, race (non-White), BMI, HDL, steroid use, and uric acid are the only significant risk factors for the recurrent incidence.


Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.Galeterone  is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/AR The recurrence of prostate cancer (PC) with metastases following androgen deprivation therapy (ADT) is a major concern since it is the primary cause of death in patients initially diagnosed with localized PC –3. This Resistance to current therapy has implicated ligand independent signaling and gene amplification of AR, mutations causing promiscuity  and emerIt has been suggested that drugs that target the N-terminal domain (NTD) or regions other than the LBD of AR should be more potent in antagonizing both fAR and AR-Vs signaling . HoweverH-benzimidazole-1-yl)androsta-5, 16-diene  was identified as a selective development candidate which modulates the androgen receptor (AR) signaling pathway at multiple points  inhibits CYP17, antagonizes fAR, and degrades both fAR and AR-Vs in vitro; and also reduces the expression of these proteins in tumor xenografts i) release and induction of endoplasmic reticulum stress response (ERSR) in PC-3 PC cells as a possible mechanism of action of gal [2+-dependent, calpain-mediated breakdown of f-AR in human PC cells [2+ induced by gal treatment enhanced AR depletion.The proteasomal pathway has been described as one of the major mechanisms regulating AR turnover –43. To dn of gal . BecausePC cells –47, we cSince ubiquitination precedes 26S proteasomal protein degradation , we examPI3k activation induces phosphorylation of Akt (at Ser473 and Thr308), Mdm2 (at Ser166) and AR (at Ser210/213 and Ser790) this posttranslational modification can serve as a signal for AR ubiquitylation and subsequent degradation , 43, 49.We then investigated the significance of Akt/Mdm2 phosphorylation in gal/VNPT55-induced AR/AR-V7 depletion. Pharmacological inhibition of PI3k  in LNCaP cells co-treating with gal/VNPT55 suppressed agent-induced AR degradation significantly (~90%) Figure . In CWR2The PI3k-Akt and the mitogen activated protein kinase (MAPK) pathways both crosstalk with the AR and induce its phosphorylation at different serine and threonine residues , 52, 53.It has been reported that both LY294002 and UO126 exhibits off-target effects and decreases AR protein expression –57. In aMdm2 and CHIP are two of the well-studied and established E3 ligases involved in AR proteasomal degradation , 58, 59.Mdm2 has previously been reported to interact and enhance AR ubiquitination preceding its degradation via the 26S proteasome . From ouResults from immunoprecipitation assays were consistent with our siRNA experiments, in that the CHIP E3 ligase is more significant in gal-induced AR-V7 ubiquitylation and degradation. Gal enhanced association between CHIP and AR-V7 , another important focus in this study was also to determine whether these compounds induce apoptosis in PC cells. Apoptotic induction was initially evaluated by the acridine orange/ethidium bromide (AO/EB) dual staining assay [Considering the strong impact that gal or VNPT55 have in promoting the degradation of AR/AR-Vs proteins, and thus, their preclinical anti-prostate cancer activities and their efficacy in the clinic  were stained with annexin-v and propidium iodide, following manufacturers protocol. Interestingly, we observed that at 5 μM, gal and VNPT55 significantly induced apoptosis in CWR22Rv1 cells  [2+ have previously been implicated in the modulation of calpains that also induce apoptosis by cleaving a variety of pro-apoptotic proteins such as calpain 1 which cleaves Bid to enhance cytochrome c release [As stated earlier, we previously reported that gal modulates intracellular Cae (ERSR) . Intrace release . Thus, wSince calpain inhibition did not significantly inhibit caspase 3 and PARP cleavage, we focused on Bax, a proapoptotic protein which could enhance mitochondrial permeability , 67, 68.An increase in Bax and its translocation to the mitochondria has been implicated in cytochrome c release into the cytosol –71, and In summary, gal and VNPT55 induce profound apoptosis in LNCaP and CWR22Rv1 cells. Importantly, these promising and robust data differ from previous reports which suggest that current PC drugs that block AR transactivation do not cause significant apoptosis, which is believed, in part, to be the basis of their failure in the clinic .To demonstrate the specificity of gal/VNPT55 and evaluate any off-target effects, we assessed the effect of gal or VNPT55 on the levels of related nuclear receptor proteins in LNCaP cells, including, progesterone receptor (PR), estrogen receptor beta (ERβ) and retinoic acid receptors (RAR-α and -β), which are known to be regulated by the proteasome-mediated pathway . As showp < 0.0001 vs. vehicle) and 70% (p < 0.0001 vs. vehicle), respectively. In addition, no host toxicity was observed as monitored by changes in body weight throughout the study  [In addition to gal’s well-established triple mechanism of anti-AR activity , 30, we and Bax) . Typicaland Bax) . We obseand Bax) .To confirm that most of these effects seen on the various pathways were not as a result of increased utilized doses, we compared induction of apoptosis at different doses  over 24 and 72 h, using different complementary analytical tools. We also evaluated the effects on AR/AR-V7 at low concentrations with positive results  and F. TRemarkably, gal and VNPT55 had no significant effect on the expression of fAR in immortalized untransformed prostate epithelial cells (PWR-1E). This is very significant as it gives an insight to the low toxic nature of the agent, as observed in the clinic , 34, 35,in vivo and also depleted cyclin D1 and Bcl2, but enhanced the level of Bax. In strong support of their mechanisms of anti-tumor activities, most effects seen in vitro were recapitulated in vivo.Perhaps the most significant piece of data from this study is the anti-tumor efficacy of gal and VNPT55 in fAR/AR-V7 positive CWR22Rv1 xenografts (a difficult-to-treat CRPC model). Gal/VNPT55 significantly inhibited tumor growth compared to vehicle treated groups, with no apparent host toxicity. Gal and VNPT55 also degraded both fAR, AR-V7 v567es that are implicated in all stages of PC progression and resistance settings [in vivo CRPC xenograft data validated the effects observed in vitro. Overall, this study has high translational relevance because it provides strong preclinical validation of phase II results and the proposed pivotal phase III clinical trials of gal in PC patients with AR-Vs.One major mechanism of resistance in CRPC is the overexpression of AR and the emergence of splice variant ARs (AR-Vs). Recently approved PC drugs, i.e., abiraterone and enzalutamide have been reported to cause rapid induction and increased expression of AR-Vs in laboratory and clinical settings , 13, 14.settings , 13, 14.v567es expression plasmid. Mdm2 Monoclonal antibody and polyclonal antibodies against AR N20, ERβ, PR, RARα, RARβ, AR (mouse), calpeptin and ZVAD-fmk were purchased from Santa Cruz. Hsp90, phosphoAR and Hsp70 antibodies were purchased from BD Pharmingen. CHIP monoclonal antibody and MG132 were from Sigma Aldrich, USA. ECL detecting kit was from Thermo Scientific.CWR22Rv1, LNCaP and DU145 cells, and immortalized untransformed prostate epithelial cells (PWR-1E) were purchased from ATCC. PI3k inhibitor (LY294002), MAPK inhibitor (U0126), rabbit polyclonal antibodies against AR, pMdm2, β-actin, Gapdh, pAkt (S473), pAkt (T308), CHIP, PARP, caspase 3, pMdm2, p42/44, phospho p42/44, secondary antibodies, anti-mouse and anti-rabbit HRP were purchased from Cell Signaling. Cell culture reagents  were from Invitrogen. Gal and VNPT55 were synthesized in our laboratory as previously reported , 25. PhoProstate cancer cells were maintained in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics (Invitrogen). For experiments using anti-androgens, cells were cultured in phenol red-free RPMI 1640 supplemented with 5% charcoal dextran FBS and 1% penicillin-streptomycin. PWR-1E cells were maintained in Keratinocyte-SFM (1X) with 1 × 2.5 μg EGF human recombinant and 1 × 25 mg bovine pituitary extract (Invitrogen). Cells were lysed with RIPA buffer (Sigma), supplemented with protease inhibitors (Roche), phosphatase inhibitors (Thermo Scientific), 1 mmol/L EDTA and 1 mmol/L PMSF (Sigma). Lysed cells were clarified by pelleting in a table top micro centrifuge at 13300 rpm for 15 min at 4°C. 50–100 μg total cell lysates were denatured in 5X sample buffers and boiled at 99°C for 5 min. Western blotting was performed as previously described , 25.LNCaP and CWR22Rv1 were treated with gal (10 μM) for 11 and 14 h to elucidate fAR-mdm2/Hsp90 and AR-V7-CHIP/Hsp70 interaction. In ubiquitination assays, cells were treated with 20 μM gal for 24 h with or without MG132 (5 μM) and lysed with RIPA buffer. 1 mg of total cell lysates were pre-cleared with 30 μl of protein A/G sepharose beads (Santa Cruz), for 45 minutes and pelleted for 1minute at 13300 rpm. Supernatant were collected and incubated with 1 μg of polyclonal antibody per 500 μg of total protein and rotated at 4°C for 12 h. Complexes were washed 3X with lysis buffer and 2X SDS loading dye added to elute proteins prior to separation on a 10% tris/glycine gel.v567es expression plasmid, using the Qiagen Effectene transfection reagent following manufacturers’ protocol. Transfection reagents and complexes were washed off 16 h after transfection and replaced with phenol red free media supplemented with 5% charcoal dextran FBS for 20 h. Transfected cells were treated with agents for 24 h and lysed with RIPA buffer. LNCaP and CWR22Rv1 cells were reverse transfected with 100 nM of Mdm2 or CHIP/STUB1 siRNA using lipofectamine RNAiMAX (Invitrogen) for 12 h adhering to manufacturer’s protocol. Scrambled siRNA were transfected as controls. siRNA complexes were replaced with phenol red-free media and treated with agents for 24 h.DU145 cells were transfected with 0.5 μg of AR-V7 or ARMTT assays were performed as described in our previous publications , 25, 30.6 cells per well and treated with indicated concentrations of compounds for 24 h. RNA was isolated with the Qiagen RNeasy reagents following manufacturer’s protocol. 1800 ng of RNA were reverse transcribed into cDNA using high capacity cDNA reverse conversion kit (life technologies). Full length AR primers, AR-V7 and internal control 18S primer sequences used were as reported in Guo et al [t using 18S as internal control.Cells were seeded in 6-well plates at 0.3 × 10uo et al . RelativCWR22Rv1 cells were seeded and grown to 70% confluence in an 8-chamber slide. Cells were then treated with galeterone and VNPT55 at 5 μM and 10 μM in regular media for 24 h. Treated cells were incubated with 125 nM mitotracker red CMXRos  for 30 minutes. Immunostaining was carried out as previously reported . Alexa f® FL Auto Imaging System (Life Technologies).All specimens were kept in 10% buffered formalin for 24 h after which they were embedded in paraffin and 4 μm thick slides prepared and used for immunostaining and hematoxylin-eosin staining. Immunostaining sections were de-parafinised, soaked in alcohol, and antigen retrieval was performed for formalin fixed tissues by heating in citrate buffer (pH = 6). Sections were blocked in 3% hydrogen peroxide for 5 minutes, followed by incubation with the appropriate antibody and epitopes detected using the Ultra-sensitive ABC staining kit . The images were captured with an EVOS6 CWR22Rv1 cells were inoculated into both flanks. Tumor-bearing mice (tumor volume around 50–70 mm3) were randomized into 3 groups  and treated as follows: (i) vehicle control , (ii) galeterone  and (iii) VNPT55 . Tumors were measured twice weekly with calipers and tumor volume was calculated by the formula: length × width2 × 0.5 (mm3). Animals were also weighed weekly and monitored for general health status and signs of possible toxicity due to treatments. Mice were sacrificed after the indicated periods of treatment and tumors and organs excised. Tumors were divided and either flash frozen in liquid nitrogen or placed in 10% buffered formalin for western blot analysis, immunocytochemistry (IHC) and hematoxylin and eosin (H & E) staining.All animal studies were performed according to the guidelines approved by the Animal Care Committee of the University Of Maryland, School of Medicine, Baltimore. Male SCID mice 5–6 weeks of age were obtained from the National Cancer Institute . Surgically castrated mice were housed under complete aseptic conditions, fed autoclaved pellets and sterile water ad libitum. Following a week of acclimatization, approximately 5 × 10in vitro experiments were repeated at least three times and reported as means with standard error where applicable. Western blot on in vivo samples were repeated in at least 2 different tumor sections from different animals. Student T-test and Analysis of variance (ANOVA) were used to determine the significance of deviations or lack thereof.All


Little is known about the natural history of aortic regurgitation (AR) in patients undergoing mitral valve procedures for mitral stenosis.The aim of this study was to evaluate the short- and long-term effects of percutaneous mitral valvuloplasty (PMV) on coexisting AR.A total of 327 patients with rheumatic mitral stenosis  were followed up for between 48 hours and 13 years after PMV. At the time of PMV, 142 (43.3%) patients had no AR, 124 (37.9%) had mild AR, and 61 (18.7%) had moderate AR. After PMV, the follow-up showed that 120 (36.6%) patients had no AR, 103 (31.5%) had mild AR, and 104 (31.8%) had moderate AR.AR progression after PMV and during the follow-up was significant (P < 0.00), but there was no significant increase in aortic valve replacement (AVR) procedures. The rate of AVR was higher in the moderate AR group (3.8%). There were no significant changes in the left atrial size (LA) (P = 0.6), ejection fraction (EF) (P = 0.4), and rhythm (P = 0.4) before and after PMV, respectively.Our findings indicate that among patients with rheumatic mitral stenosis, a considerable number have concurrent AR. Concomitant AR at the time of PMV does not influence procedural success and is not associated with inferior outcomes. Rheumatic aortic insufficiency progresses slowly by nature, and patients with AR and mitral stenosis can safely tolerate PMV without the possibility of undergoing AVR in the near future. Patients with moderate degrees of AR remain good candidates for PMV. Percutaneous mitral valvuloplasty (PMV) was first described by Inoue et al.  and has The aim of this study was to evaluate the short- and long-term effects of percutaneous mitral valvuloplasty (PMV) on coexisting AR.2/m 2 of the body surface area and suitable valve morphology (MV score < 9) with no or mild mitral regurgitation (MR) and no left atrial clot were selected for PMV. All the procedures were performed via the Inoue method.1 Balloons were selected according to patient’s height and body surface area. Hemodynamic parameters, left and right atrial pressures, and gradient changes were recorded during PMV. Mitral valve area, MR, AR, tricuspid regurgitation severity, LVEF, and left atrial diameter were measured via TTE and TEE before the procedure. All the patients underwent TTE 48 hours after PMV and during the follow-up. The EF was defined mostly through the eyeballing method and noting regional wall motion abnormalities and degree of endocardial thickening. The EF and MR before and after PMV were also assessed by ventriculography, which was conducted in most cases before and after PMV.This retrospective study included 328 patients who underwent PMV at a tertiary centre between 1994 and 2007. Patients who had data available regarding the degree of pre-PMV AR were included in the analysis. The clinical and echocardiographic data were acquired from hospital records. All the patients underwent transthoracic echocardiography (TTE) and transesophageal echocardiography (TEE), and atrial size, pulmonary artery pressure, presence of LA thrombus, and MR were recorded. Patients with moderate to severe MS who had a valve area < 1cmColor Doppler, effective regurgitation orifice area, vena contracta width and pressure half time methods were used to define the quality and quantity of AR; and based on these findings, AR was classified into four groups: none, mild, moderate, and severe AR . None ofThe continuous variables are expressed as Mean ± SD, and the categorical variables are expressed as a percentage. The Student’s t-test was employed for the interval variables, Fisher exact test and chi-square test for the categorical variables, and Mann Whitney U test for the ordinal variables . Additionally, the non-parametric test was utilized as Kruskal-Wallis  correlation and one-way Anova (the relationship between age and AR). The Mac Nemar test was opted for form non-parametric methods for the nominal data (rhythm before and after PMV), and the Wilcoxon signed rank test was used to assess the relationship between AR before and after PMV. All the statistical tests were two-sided using α < 0.05 level of significance. The data were managed and analyzed using SPSS 15 for windows . For the confounding factors, repeated measured ANOVA and multiple linear regressions were used. The LV diameters were measured using the paired sample corrections, student t-test, and Friedman test. Data were collected using a personal code for each patient.A total of 328 patients underwent PMV preceded by echocardiography during a 13-year period from 1997 to 2010 in a tertiary cardiovascular center. The study population consisted of 282 (86.3%) women and 45 (13.7%) men. A pregnant woman who suffered from critically severe MS and severe AR and underwent successful PMV and accomplished her pregnancy but refused the follow-up process was excluded. The patients were divided into three groups: no AR, mild AR, and moderate AR. The mean age of our patients was 47.16 ± 11 years . AR was Follow-up data showed that after PMV, 120 (36.6%) patients had no AR, 103 (31.5%) had mild AR, and 104 (31.8%) had moderate AR. Altogether, after PMV, 207(63%) patients had AR. Of the 142 patients, who had no AR before PMV, 120 patients remained the same during the thirteen years of follow-up and 22 patients progressed to mild AR. Forty three (34.6%) patients, who had mild AR initially, showed moderate AR, which was statistically significant. None of our patients had progressed to severe AR. AR progression been mostly prevalent in the women’s group. The follow-up results of the study are depicted in After PMV, the mean EF was 51% ± 5, which was reduced by 1.1%. After adjusting for age and sex as the confounding factors, PMV had no effect on the EF. There was no significant relationship between AR progression and the EF (P = 0.46). In our study there was no significant relationship between AR progression and rhythm changes after PMV (P = 0.4). Sinus rhythm was again more prevalent after PMV, but 32 (9%) of the patients developed AF. The mean LA diameter after PMV was 4.62 ± 0.64mm.The association between increasing age and the LA diameter was measured before and after PMV and was statistically significant (P = 0.001). Four (3.8%) out of the 104 patients with moderate AR after PMV underwent aortic valve replacement (AVR) and concomitant mitral valve replacement (MVR) for mitral valve disease severity as main indication. None of the patients with no or mild AR underwent AVR and no patient underwent sole AVR due to AR severity. There was no significant association between AR progression and the MVR procedure after PMV (P = 0.4) .Rheumatic heart disease (RHD) continues to be a common health problem in the developing world, causing morbidity and mortality among children and adults . ConcurrPatients with severe AR and concomitant MS are usually not referred for PMV. On the other hand, combined aortic and mitral valve replacement is usually associated with higher procedural risks and poorer long-term survival than is the replacement of either of the two valves . Based oSignificant MS and resultant PMV were mostly seen in the women in the present study. The Numboodiri study and other studies also reported predominant patterns in females , 12. As The older patients had a larger left atrial size before PMV and the mean diameter was greater in the men. The diameter of the left atrium after PMV increased 1.4mm, which was not statistically significant. In our study after PMV, the mean diameter of the left atrium in the patients with sinus rhythm was decreased (2mm) but the mean diameter of the left atrium in the patients with AF was increased (2mm). Furthermore, a 1.1% reduction was seen in the EF of the patients during the follow-up in contradiction to our first hypothesis that the EF might increase due to improved preload. This may be in consequence of the increasing age, change of the rhythm, different degrees of AR, and baseline RHD. The mean of the EF before and after PMV was not significantly different between our men and women, but the EF reduction was more predominant after PMV in the females. Although there was no significant relationship between the patients’ rhythm and AR  before and after PMV, it seems that the patients without AR were more likely to have sinus rhythm, which may be due to milder forms of the disease. The patients who underwent MVR were mostly women and older, had larger left atrium and their rhythm was mostly AF. As pointed in results section, in no case was AVR performed as a single procedure. All the patients who underwent AVR had moderate AR before surgery.Most of the patients had mild or moderate AR in our study and there was no assessment of patients with severe AR. One patient with severe AR before PMV tolerated the procedure and her pregnancy well but we do not have long-term follow-up data. Our findings indicate that among patients with rheumatic MS and planned PMV, a considerable number have concomitant aortic regurgitation. However, progression of AR after PMV is slow and PMV does not increase the rate of AVR. Based on these results, we would suggest that PMV is an effective treatment for patients who have MS and concurrent trivial to moderate AR and is not associated with inferior outcomes.


LMNA/C mutations have been linked to the premature aging syndrome Hutchinson’s progeria, dilated cardiomyopathy 1A, skeletal myopathies , Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, autosomal dominant partial lipodystrophy, and axonal neuropathy. Atrioventricular block (AVB) can be associated with several cardiac disorders and it can also be a highly heritable, primitive disease.LMNA/C mutations and cardiomyopathies.One of the most common pathologies associated with AVB is dilated cardiomyopathy (DCM), which is characterized by cardiac dilatation and reduced systolic function. In this case, onset has been correlated with several mutations in genes essential for the proper maturation of cardiomyocytes, such as the gene for lamin A/C. However, no clear genotype–phenotype relationship has been reported to date between DNA and medical histories were collected from (n = 11) members of different generations of one family, the proband of which was implanted with a pacemaker for lone, type II AVB. Exome sequencing analysis was performed on three relatives with AVB, and the mutations therein identified validated in a further three AVB-affected family members.In the initial three AVB family members, we identified 10 shared nonsynonymous single-nucleotide variations with a rare or unreported allele frequency in the 1000 Genomes Project database. Follow-up genetic screening in the additional three affected relatives disclosed a correlation between the lone AVB phenotype and the single-nucleotide polymorphism rs56816490, which generates an E317K change in lamin A/C. Although this mutation has already been described by others in a DCM-affected proband with familiarity for AVB and sudden death, the absence of DCM in our large, AVB-affected family is indicative of genotype–phenotype correlation between rs56816490 and a familial, autosomal dominant form of lone AVB.LMNA/C in patients with lone AVB and their relatives might prevent sudden death in families affected by AVB but without familiarity for DCM. Lone AVB is an age-related disease caused by mutations in LMNA/C gene rather than a complication of DCM.Screening for G613A in The online version of this article (doi:10.1186/s12979-014-0019-3) contains supplementary material, which is available to authorized users. Atrioventricular block (AVB) is a partial or complete block in the transmission of the electrical impulses originating in the atrium or sinus node and directed toward the AV node and ventricles. Depending on the gravity of AVB, cases are grouped into one of three different categories: type I AVB is characterized by prolongation of AV conduction time; type II AVB is characterized by failed transmission of some impulses, with consequent loss of some ventricular contractions; and type III AVB is characterized by complete AV dissociation and loss of many/all contractions or coordination with atrial contraction .AVB can be associated with several cardiac disorders, such as dilated cardiomyopathy (DCM), ischemia, infarction, and fibrosis, and may present as an aggravation of existing cardiac suffering. AVB can also be caused by a highly heritable, primitive disease presenting itself at an early age . Little LMNA/C) is encoded [LMNA/C spans approximately 24 kb, harbors 12 exons, and produces the alternative splice products LMNA (74 kDa) and LMNC (65 kDa) [LMNA/C mutations, reduced LMNA/C expression caused damage to cardiomyocyte nuclei and nuclear membranes, such as focal disruptions, bleb formations, and nuclear pore clustering [One of the most common pathologies associated with AVB is DCM, which is characterized by cardiac dilatation and reduced systolic function. In this case, onset has been correlated with several mutations in genes essential for the proper maturation of cardiomyocytes. For example, autosomal dominant DCM and conduction system diseases are linked to a disease locus at chr1p1-q21, where the gene for lamin A/C ( encoded . The cod(65 kDa) . Lamins (65 kDa) . In patiustering .LMNA/C mutations have also been linked to the premature aging syndrome Hutchinson’s progeria, skeletal myopathies , Charcot-Marie-Tooth disorder type 2B1, mandibuloacral dysplasia, autosomal dominant partial lipodystrophy, and axonal neuropathy [LMNA/C mutations and cardiomyopathies [Apart from dilated cardiomyopathy 1A, uropathy ,11. Howeopathies . TherefoA large family from central Italy was recruited for the genetic study of a familial autosomal dominant form of lone, type II AVB. The pedigree was composed of 31 subjects belonging to five different generations, 10 of which presenting with the pathology. Six affected and five apparently unaffected members participated in the study  and the reported  delay inLMN A/C (rs56816490) was carried in a heterozygous fashion by all affected individuals , we sequenced the whole exome of three affected members of the family: one fourth-generation member  and two third-generation subjects (III:5 and III:8). Filtered results indicated that 10 nonsynonymous and damaging mutations were common to the three subjects Table . We thens Figure . Analysis Figure .Table 2LMNA/C variations are responsible for many different diseases involving, above all, muscle tissue, including DCM with conduction defects [LMNA/C mutations and DCM associated with conduction disturbances. In the present study, we correlate the missense mutation G613A in LMNA/C (SNP ID rs56816490) with a familial, autosomal dominant form of AVB that we classify as “lone” based on the absence of DCM in the family’s medical history and on the absence of any abnormality in ventricular and atrial sizes in the proband. The SNP is located within the 6th exon of LMNA/C, which is located at chr1:156205704, and determines the amino acid change E317K in a segment of the LMNA/C coil 2 that does not interact with proteins or chromatin. defects . HoweverLMNA/C mutations that affected nuclear lamin interactions, whereas the E317K change, present in a non-interacting segment, was responsible for a mild phenotype.Arbustini et al.  have alrIn light of our data, we would re-consider the disease status of the LMNA/C E317K-carrying proband described by Arbustini et al. as affected with lone AVB. Of note, E317K is a rare change in LMNA/C, and both families were from Italy; thus, we cannot exclude a common origin of the mutation.LMNA/C in patients with lone AVB and their relatives. Indeed, in the family we investigated, three subjects  had a medical history of sudden death, as did the E317K LMNA/C carriers reported by Arbustini et al. [LMNA/C inducing accelerated aging, and thus makes appealing the hypothesis that AVB is an age-related disease on its own right, other than a complication of DCM.Our results are important for genetic counselling, pointing to the need for screening G613A in i et al. . FurtherThe study was conducted in accordance with the ethical principles that have their origins in the Declaration of Helsinki. Informed consent was obtained from all subjects in accordance with the Review Board rules of IRCCS Multimedica. Subjects were evaluated, when possible, by medical history, physical examination, and electrocardiography. Reports from echocardiography (ECHO), cardiac catheterization, electrophysiology studies, cardiac surgery, and/or autopsy were reviewed when available. Clinical assessment was performed without knowledge of genotype.http://broadinstitute.github.io/picard/). Post-alignment processing , SNV, and small insertions-deletions (ins-del) calling were performed with Genome Analysis Toolkit (GATK) [Genomic DNA was isolated from whole blood, using the QIAamp DNA Blood Midikit (Qiagen), according to the manufacturer’s protocol. Exonic regions of genomic DNA of three affected subjects  were enriched using either the TruSeq™ Exome Enrichment Kit (Illumina) or the Agilent Haloplex Exome kit based on DNA digestion and capture. Exomes were barcoded and sequenced at multiple sites on the Illumina HiSeq1000 platform, and either 2 × 76-bp (TruSeq) or 2 × 100-bp  PE libraries, using TruSeq SBS Kit v3–HS (Illumina) reagents and a TruSeq PE Cluster kit v.3-cbot-HS flow cell. Average coverage for all the experiments was 70x and at least 20x for 89% of the target. Paired sequencing reads were aligned to the reference genome UCSC, hg19 build) using BWA  and sortt (GATK)  with par build usThe results were first filtered to eliminate common variants (MAF > 1%), variants with low quality score, and variants not shared by all analyzed affected subjects, when covered. Additional frequency filters were used by comparing internal databases of whole exome sequencing data (n = 300). Prioritization was also made based on MAF frequency  methods for selected single-nucleotide polymorphisms (SNPs). The amplified fragments were purified with the Wizard SV Gel and PCR Clean-Up System (Promega) and were sequenced in order to identify the mutation associated with AVB. Primer sequences and PCR conditions are given in Table LMNA/C: a primer targeting wild-type LMNA/C (5′-CAGCTGGCAGCCAAGg-3′) and another primer targeting a mutated variant (5′-CAGCTGGCAGCCAAGa-3′). Direct amplification of genomic DNA of 100 controls was performed using independently both the forward primer and the reverse primer (5′-CGCATCTCGGCCATCTC-3′) followed by visualization on an agarose gel with ethidium bromide staining.Two forward PCR primers were designed for the amplification of the rs56816490 locus in