Adventitious rooting (AR) is a multifactorial response leading to new roots at the base of stem cuttings, and the establishment of a complete and autonomous plant. AR has two main phases: (a) induction, with a requirement for higher auxin concentration; (b) formation, inhibited by high auxin and in which anatomical changes take place. The first stages of this process in severed organs necessarily include wounding and water stress responses which may trigger hormonal changes that contribute to reprogram target cells that are competent to respond to rooting stimuli. At severance, the roles of jasmonate and abscisic acid are critical for wound response and perhaps sink strength establishment, although their negative roles on the cell cycle may inhibit root induction. Strigolactones may also inhibit AR. A reduced concentration of cytokinins in cuttings results from the separation of the root system, whose tips are a relevant source of these root induction inhibitors. The combined increased accumulation of basipetally transported auxins from the shoot apex at the cutting base is often sufficient for AR in easy-to-root species. The role of peroxidases and phenolic compounds in auxin catabolism may be critical at these early stages right after wounding. The events leading to AR strongly depend on mother plant nutritional status, both in terms of minerals and carbohydrates, as well as on sink establishment at cutting bases. Auxins play a central role in AR. Auxin transporters control auxin canalization to target cells. There, auxins act primarily through selective proteolysis and cell wall loosening, via their receptor proteins TIR1 (transport inhibitor response 1) and ABP1 (Auxin-Binding Protein 1). A complex microRNA circuitry is involved in the control of auxin response factors essential for gene expression in AR. After root establishment, new hormonal controls take place, with auxins being required at lower concentrations for root meristem maintenance and cytokinins needed for root tissue differentiation. Forestry, horticulture, and fruit crops depend to a large extent on the successful establishment of roots in cuttings and other propagules. Clonal propagation is of particular relevance to forestry, since genetic improvement in long lived species with large generation cycles is often limiting. Genetic gains from interspecific hybridization, mutations, and transgenic events can be captured and multiplied faster and more efficiently based on clonal propagation through AR of cuttings. Overall, the main application of AR is propagation by cuttings and its derived techniques adapted to clonal garden greenhouses and ectively . TherefoMost research on AR has been centered on the role of phytohormones, mainly auxins, and cutting physiological conditions. The role of stress responses associated with cutting severance and the relevance of mother plant status has often received less attention, although a shift in focus has been clearly taking place in the last two decades or so. Wound responses associated with cutting severance are integrated, and often necessary, in the steps leading to AR, and mother plant status is a key determinant of rooting propensity of cuttings derived from it. Therefore, the control of environmental variables of stock plants is rather relevant for the clonal propagation process. Clearly, a fundamental aspect governing AR responses to external and internal stimuli is cellular competence to respond. This developmental capacity to respond is responsible for many of the failures to obtain AR in mature cuttings, even upon careful manipulation of environmental variables and phytohormones that can modulate rooting.The concept of adventitious root is based essentially on anatomical origin. Adventitious roots are formed in stems, leaves and non-pericycle tissue in older roots, differing from primary roots, of embryonic origin, and lateral roots, which are derived from the pericycle layer . There aAdventitious rooting is a complex process that can be affected by numerous variables, both internal and external. A large body of evidence has supported the existence of successive physiological phases in the process of adventitious root development, each with specific requirements that can even be antagonistic, but operate in complementary fashion. The most widely recognized AR phases are induction, initiation, and expression .The induction phase in cuttings or detached organs, such as leaves, is generally marked by the immediate consequences of the wounding response caused by severance. It encompasses the first hours after cutting removal, with a local increase in jasmonate, phenolic compounds and auxin at the cutting base, often associated with a transiently lower peroxidase (EC 1.11.1.7) activity, and the establishment of a sink for carbohydrates in the same zone . PeroxidThese overall changes in phytohormone balance, along with other less predominant but not unimportant changes to be discussed ahead, trigger a sequence of gene expression events that leads to proteomic changes, culminating with new root differentiation. Considering different systems and the fragmentary information available, these molecular events of gene expression and gene product accumulation can be putatively summarized in chronological sequence as follows: wounding and water balance stress-related, carbohydrate sink establishment, auxin transport systems, cell wall degradation and assembly, transcription factors involved in cell fate determination, replication machinery, transcription factors with roles in growth and differentiation .At the molecular level, including the participation of various phytohormones, considerably more knowledge is available on lateral root development. Certainly, there is at least some overlap between the processes of lateral root development and AR. Most of the similarities include the requirement for an initial auxin increase, followed by a reduction, the participation of auxin transporters, cell wall dynamics, and the activity of specific transcription factors in these processes. Root growth responses to nutrient gradients, such as nitrate and phosphate , seem toArabidopsis thaliana, have roots formed mostly from the pericycle, which extends from primary roots into the hypocotyls of young seedlings, and do not face stresses capable of disrupting root–shoot correlative influences. A comparison of an intact seedling system with de-rooted older plants or with rooting of petioles of detached leaves showed significant differences, not only in root founding tissues, but also in auxin requirements, sensitivity, and rooting mutant phenotypes  had upregulated light responses and disturbed auxin homeostasis.Auxin endogenous concentration varies over the course of rooting phases, and is needed at higher concentration during the induction phase for proper rooting . In thiseedlings . Light aeedlings . Sorin eEucalyptus globulus plants phthalamic acid (NPA) up to the first 3 days after cutting severance inhibited rooting without affecting auxin concentration or metabolic status at the rooting site, suggesting a role for auxin polarity in rooting capacity that would be different than simply moving auxin to the rooting zone.The molecular basis of rooting competence is an essential aspect of AR. In principle, even if all environmental variables are ideally manipulated so as to favor AR, unless developmental competence is present, responses to the root-promoting signals do not take place and rooting fails. Developmental responsiveness is likely dependent on presence and density of functional phytohormonal receptors and signaling pathways, particularly those for auxin. A detailed investigation on AR of hypocotyls (able to root proficuously upon exposure to auxin) and epicotyls (root poorly even in presence of auxin) of 50-day old seedlings of picotyls . ApplicaPinus taeda stem cuttings, for example  and adult (26 day old) plants of the Landsberg ecotype, it was shown that AR was much slower in adult de-rooted plants and that endogenous polar auxin transport  was crucial for AR  peptides (a family of peptides bearing this signature domain), although these were not sufficient for rooting to occur and had no effect on young plant hypocotyls. The RGD peptides may be important in causing changes to the plasma membrane of plant cells and their interaction with cell walls, perhaps affecting cytological events required for AR in adult plant hypocotyls. Taken together these data indicate that Arabidopsis and in vitro culture systems of tree species are useful tools to study developmental competence to AR.The loss of AR capacity at physiologically mature stages is often associated with the transition to flowering (phase shift from juvenile to adult stage). However, in specific organ parts or under specific culture conditions, loss of rooting capacity can take place and become easily noticeable at much earlier stages of development , providing interesting experimental systems to study this process in trees . Anotherl for AR . These a bolting . A possi bolting . InteresEucalyptus microcuttings exposed to exogenous auxin  and a continuous accumulation of soluble and insoluble carbohydrates during adventitious root formation  plays a central role in determining reactive oxygen species equilibrium in plants and can be induced in response to diverse abiotic and biotic stress conditions . Seconda rooting . Moreovecuttings  and the cuttings .Malus “Jork 9,” thereby promoting AR. The decarboxylation was attributed to the wound response and did not occur to such an extent in non-wounded plant tissues. The action of the phenolic compounds suggests that, at least in part, rooting depends on the inhibition of IAA decarboxylation caused by wounding, so that more auxin is available to induce roots -treated seedlings suggesting that IBA induced overproduction of H2O2 and promoted AR via a pathway involving H2O2. In another study, 2O2-mediated facilitation of adventitious root formation is the early decrease of peroxidase and ascorbate peroxidase activities in IBA and H2O2-treated seedlings. The decrease in activity of these enzymes would be relevant to generate the necessary high level of auxin and H2O2 required for adventitious root induction.Hydrogen peroxide, a form of reactive oxygen, functions as a signaling molecule that mediates various physiological and biochemical processes, as well as controls responses to various stimuli in plants . Li et aPopulus  on plant water status and rooting capacity under controlled environmental conditions, in combination with planting in soils with different water potential. Results clearly showed that soil moisture had a major effect on rooting. Water-stressed cuttings took a longer time to root and formed fewer roots. Pre-soaking of cuttings had a positive effect on rooting, mainly under the drier soil moisture conditions. Although unrooted hardwood cuttings needed moister soil to initiate rooting, once roots were established, they could tolerate somewhat drier conditions. In good agreement, cutting survival and AR were highest in moister substrate for stem cuttings of juniper , azalea (Rhododendron), and holly  of leaves and osmotic potential increased upon formation of root primordia in cuttings . Followicuttings . Hence, Auxins have a rhizogenic action during the root induction phase  and stimulate cells at the cutting base to engage in the establishment of meristemoids . The samE. globulus and verified that IAA level was twofold higher in the easy rooting line, confirming the importance of IAA in AR.In general, free IAA endogenous levels have a transient increase during the induction phase, pass through a minimum at the initiation step and resume an increase in the expression phase . The impArabidopsis seedlings and twofold higher IAA levels compared to the untreated Arabidopsis (superrot2 (sur2) which contains high concentrations of free IAA  and indole-3-butyrylaspartic acid , low stability IAA and moderate stability IBA . IAA cantic acid . The autGH3 family are involved in the maintenance of auxin homeostasis, contributing to regulation of the auxin pool , triggering activation of Ca2+ channels in the plasmalemma. The release of phospholipids promoted by NO would provide substrates for phospholipases, whose activity and released products could further activate Ca2+ release to the cytosol and activate both calcium-dependent protein kinases (CADPKs) and mitogen-associated protein kinases (MAPK). These kinases would in turn lead to cell growth and differentiation associated with AR. NO-promoted AR was also reported for other species, including greenhouse–grown cypress  has also been proposed as a player in the control of AR. In cucumber, AR was favored by NO, acting downstream of auxin, possibly through different transduction pathways . Auxin-s cypress  and E.ggrandis . In thesgrandis . Studiesanthemum  have sugpression . The extArachis hypogaea by blocking cell cycle progression (Gibberellins (GAs) are generally considered inhibitors of AR. This has been shown, for example, in poplar . Moreovegression . Inhibitgression .Polyamines are nitrogen containing, polycationic, low molecular weight aliphatic compounds that can be found in meristematic and actively growing tissues. These metabolites  play various roles, mostly related to control of cell division, development, and stress responses. Because of their positive charges, polyamines are capable of binding to nucleic acids, proteins, and membranes, therefore potentially being able to interfere in processes such as gene expression, cell signaling, membrane stabilization, and modulation of some ion channels . PolyamiFigure 1.A tentative model summarizing some of the main data on phytohormonal control of AR is shown in Given the importance of phytohormones, particularly auxins, to the control of AR, the next three sections will examine fundamental aspects of cell cycle control, root tissue differentiation, auxin transport, metabolism, and action. However, it must be emphasized that most of the knowledge presented in these sections is derived from investigations directed to general plant development or development of primary or lateral roots. Although it is clear that these processes are important in AR, their exact contribution in the specific context of the process is far from complete.Cell divisions in meristems depend on the cell cycle, which involves a mechanism governed by cyclin-dependent kinases  gene is considered a key cell cycle regulator downstream of the SCARECROW (SCR) patterning gene, a member of the GRAS family of transcription factors, acting in the control of cell division, differentiation and cell homeostasis  and B-type (CDKB), the latter being plant-specific. CDKB accumulation depends on the cell cycle period, specifically the B1 subclass in the S phase and after G2 until mid-M and B2 subclass, reaching a peak in G2 and M . Moreoveeostasis . The traectively . Cytokinectively .Arabidopsis, the short hypocotyl 2 (SHY2) gene acts as a negative regulator of auxin signaling (ARF transcription factors and thus avoiding the activation of auxin-responsive genes. SHY2 expression is activated by the presence of cytokinins via the route of AHK3 (Arabidopsis histidine kinase 3) receptor kinase/cytokinin-responsive ARR1 transcription factor, and leads to negative regulation of PIN genes, involved in the efflux of auxin, which consequently causes a reduction in the root meristem size  takes part in maintaining this condition by supporting meristematic identity of the initial cells around it . The QC SCR and SHORTROOT (SHR), also belonging to the GRAS family of proteins, have crucial role in maintaining the meristematic cells pluripotent identity (PLETHORA1 (PLT1) and PLETHORA2 (PLT2) are also involved with meristem maintenance, are induced by auxin, and act in parallel with SHR and SCR, encoding transcription factors AP2-like (SCR expression appears to depend of the gene PDR2 (PLT mRNA is associated with the peak of auxin in stem cells and QC in root meristem (WOX5 (WUSCHEL-RELATED HOMEOBOX 5), root homologue of the shoot WUSCHEL (WUS), also has a function in the stem cell maintenance and signaling . The content of the corresponding mRNA of these genes increased in both species upon auxin exposure within the first 24 h of the rooting process, coinciding with cell reorganization preceding divisions and establishment of defined root primordia. In Pinus radiata, an SHR-related clone was identified with an expression pattern similar to that of SCL, except for the fact that it was auxin-independent, possibly playing a role in root meristem formation and maintenance, as well as in the cambium zone of hypocotyls , a member of the AP2 family of transcription factors, has been shown to be associated with cell division and further establishment of adventitious root primordia in Populus trichocarpa  downregulation of the same gene transcript resulted in delayed AR. A number of genes were co-regulated with PtAIL1 based on microarray and comparative analyses of modified poplar lines up or downregulated for the AP2 transcription factor, included among these additional transcription factors, such as AGAMOUS-Like6 and MYB36  rapid (up to 10 cm per h), often referred to as non-polar, bidirectional transport in the phloem sieve elements, (b) slow (approximately 10 mm per h) or polar, mediated by transporters , mostly Arabidopsis. In stems, the transport is active, polar, and basipetal. According to the chemiosmotic model  protein family are involved with auxin efflux and their asymmetric distribution in the cells is fundamental to the characteristic polar basipetal transport along the stems. The correct localization of PIN proteins is determined by its phosphorylation status, defined by the balance between the kinase protein PID (PINOID) and the phosphatase PP2A. In the case of emerging primordia, the expression of PID is activated, turning PIN protein to a phosphorylated form, leading to its apical localization in the cell. On the other hand, in most situations, PP2A is more active than PID, leading to dephosphorylated PIN protein, resulting in a basal localization in the cell . FurtherBasipetal auxin transport is also affected by the red/far-red (R:FR) light ratio . In openRecent findings revealed the function of a new family of putative auxin transporters, the PIN-LIKES PILS; . These pArabidopsis thaliana multidrug resistance 5). Atmrp5-1 mutants, defective in MRP5 expression, have shown higher free auxin levels and inhibition of root elongation , relatively little is known about transport and metabolism. IBA is more stable than IAA and persists for longer in plant tissues , being by to NAA .pin mutants showed that PIN proteins are the major players in directional distribution networks that mediate auxin maxima and gradients during different developmental processes  are enzymes that participate in limiting ATP content. Polar IAA transport in roots and hypocotyls was reduced in apy2 null mutants when these were suppressed of APY1 (apyrase 1) expression by an estradiol-induced RNAi. Basal portions of APY-suppressed hypocotyls accumulated less free IAA and morphological defects were seen in roots with the same genetic modification. Problems in gravitropic asymmetry of auxin content were detected by means of DR5::GFP constructs in APY reduced plants, either genetically or treated with APY chemical inhibitors. The relevance of apyrase participation in auxin transport during AR is presently unclear and should be object of further investigation.Recent evidence points to a possible role of APY (apyrases) in regulating auxin transport . ExogenoArabidopsis, Auxin gradients are also very important for root organogenesis and both primary and lateral root formation are issues that had good advances in the last decades. Studying root outgrowth in pils2pils5 double loss of function mutant had higher free auxin levels, increased hypocotyl growth and presence of lateral roots, which were longer and more abundant than in the PILS5 gain of function phenotype. This evidence suggests that PILS2 and PILS5 could have specific functions in the cellular regulation of root growth . The control of AR in intact seedlings of Arabidopsis by auxin, for example, involves activation of transcription factors ARF6 and ARF8 , all of which are able to bind auxins specifically and show auxin-enhanced binding to AuxIAA proteins . Upon binding of auxin to the F-Box (TIR1/AFB) subunit of the SCF TIR1/AFB complexes, their affinity toward the domain II of AuxIAA proteins is greatly enhanced with auxin acting as a “molecular glue” bringing the two proteins together; this binding triggers the ubiquitination of the AuxIAA by the SCF complex leading to its destruction by the 26S proteasome . The degand ARF8 . The TIRproteins . Althougafb4 and afb5 mutants are more resistant than tir1 to picolinate auxins such as picloram, suggesting alternative substrate specificity , rise in cytosolic Ca2+ (30 s), phospholipase A activation (2 min), MAPK activation (5 min), among other rapid auxin-triggered responses  in the field of AR may depend on a number of strategies and scientific decisions for possible consideration by researchers. Although model species are a highly valuable tool for unveiling complex developmental processes, it is probably useful to somewhat diversify research objects, at least a couple of species for each general type of plant material  and within these seek for a few genotypes of easier or harder-to-root phenotype, in order to gain a better view of the process. A shift or at least a better balanced focus between research aiming at cuttings and at mother plant status and its implications on subsequent rooting may help achieve a more global understanding/predictable manipulation of AR. The recognition and identification of the main phases of AR should be taken into account in the various materials under investigation, for the process is quite dynamic and requisites and needs change along the process of re-establishing a root system.From the experimental view point, solid associations must be established between structure and function, with a refinement of sampled cell types and tissues , always with a kinetic perspective of the successive phases. Another key association in the realm of methodologies is to maintain an open dialog between the basic and applied research with mutual benefits arising from exchanging operational strategies, investigation methods, and process modulation tools. Finally, a conjunct effort to establish clearer boundaries between lateral and adventitious root development and to seek an integrated look at these two processes within the various plant materials investigated may help clarify some of the contradictory data populating the rooting literature.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Allergic rhinitis (AR) is a common, costly, and troublesome condition, impairing patients' quality of life (QoL), cognitive function, and productivity. Patients with AR report disturbed sleep, fatigue, irritability, and a range of practical problems. However, there is a relative lack of data on how patients with AR perceive their QoL.To better understand how patients perceive AR and their attitudes toward this condition (including QoL) and its treatment options.An online and telephone survey of 3635 people identified as having outdoor and indoor allergies, urticaria, and/or pet allergies was performed in 6 countries.The survey confirmed that patients with allergies perceive their symptoms as causing significant disruptions to their daily lives. Respondents were affected for a considerable part of each day, with the most severe symptoms occurring in the morning. The most important desired effect of medication was the restoration of normal breathing, and the most highly rated attributes of the "ideal" AR drug were efficacy, safety, and freedom from undesirable side effects.The information gathered from allergy sufferers who participated in this survey sheds light on the degree to which people with allergies are affected by their disease and the limitations imposed by associated symptoms. In clinical trials of allergic rhinitis (AR), quality of life (QoL) is assessed by questionnaires that ask subjects about the effects of AR on various domains of their daily lives, including practical functions, social interactions, emotional well-being, and economic activity. These instruments comprise both generic questionnaires that measure physical, mental, and psychosocial functioning, such as the Medical Outcomes Study 36-Item Short-Form Health Survey,,2 and onAn online survey conducted in 2004 to assess QoL in 2002 patients with AR and nasal congestion (or their caregivers) has reinforced these findings . Within Although the typical clinical symptoms of AR might be considered relatively inconsequential, they are consistently mentioned by patients as having a significant impact on QoL. Thus, there may be a disconnect between clinicians' perceptions of AR as a chronic but nonserious medical condition that causes a limited range of symptoms and patients' perceptions of it as a limiting and disabling presence in their lives. This disconnect may in turn lead to suboptimal treatment.To address the way health care providers and patients perceive the effects of AR, a multinational survey, "Understanding the Dynamics of Allergy Suffering and Treatment," conducted by the Forbes Consulting Group, was undertaken. The objective was to better understand patients' attitudes and perceptions about the symptoms and effects of AR, treatment approaches, and available therapies. The survey investigated respondents' product or brand choices and what form of therapy they preferred . Some methodologic flexibility to define concepts was permitted within each country.A questionnaire was administered in July 2005 to respondents in France, Germany, Italy, Spain, the United Kingdom, and the United States, with the goal of obtaining a minimum of 575 complete responses from each country : 500 from people who treated their allergies and 75 from nontreaters. The actual number of complete responses received was 3635. Respondent panels were recruited in a way that the geographic distribution of the panel within each country accurately reflected the population distribution.The survey was conducted by Internet, except in Spain, where it was conducted by telephone. To ensure uniformity of responses and to control for scale-use bias, scale variables were standardized, setting the mean within each variable and within each observation to zero and the SD to 1. A follow-up interview was conducted by Internet in the United States in November 2005.Respondents were included only if they had experienced symptoms related to outdoor, indoor, pet, mold, or dust allergies, including AR and urticaria, during the preceding 12 months. People who primarily had food, insect, or medication allergies were excluded.Survey participants answered questions on the frequency and duration of symptoms, the times of day and the season(s) in which they occurred, and what types of symptoms they experienced. They were given a list of the core allergy symptoms and were asked to note which ones they had: sneezing, itchy/watery eyes, nasal congestion, runny nose, sinus congestion, postnasal drip, sinus pressure, itchy throat, sinus pain, and hives/rash. Patients were also asked to use a list of 9 possible responses to rate the emotional impact of their morning allergy symptoms: more irritable, more fatigued, less motivated, less energetic, hard to concentrate, more frustrated, less alert, more self-conscious, and feelings not affected. There were questions about how disruptive nasal allergies in general  were to various aspects of everyday life, including sleep, participation in social and outdoor activities, and mood. Data were weighted to age, sex, and household income distributions. Respondents in the United States were subdivided according to 3 levels of symptom severity .Lifestyle impact was calculated with index scores based on symptoms and lifestyle disruption. The prevalence of specific symptoms was multiplied by symptom severity and was indexed against the overall average to create individual index scores for the various symptoms. Prevalence of lifestyle difficulties was multiplied by disruptiveness and was indexed against the overall average to create individual scores.Respondent demographics are listed in Table There was considerable intercountry variability with regard to the seasons in which AR was experienced. Forty-four percent of respondents in the United States experienced 3- or 4-season allergies, followed by France (35%), Germany (33%), and the United Kingdom (30%), with much lower incidences reported in Italy 16%) and Spain (11%) . The only exception was in the United Kingdom, where the highest percentage (89%) reported allergies during the summer , followed by the United Kingdom (79), Germany (68), France (62), Italy (37), and Spain (33). Survey participants in the United Kingdom reported the highest number of annual days with indoor allergies (160), followed by the United States (129), Germany (103), France (67), Italy (59), and Spain (36).The Allergic Rhinitis and its Impact on Asthma Workshop recommended that the terms seasonal and perennial AR be replaced with intermittent AR (IAR) and persistent AR (PER). Intermittent AR is defined as the presence of rhinitis symptoms for less than 4 days/wk or for less than 4 weeks, whereas in PER, rhinitis symptoms are present for greater than 4 days/wk and for greater than 4 weeks . Table 2Respondents' rhinitis was largely self-diagnosed in all countries surveyed, led by France (74%), followed by the United States (72%), Germany (62%), the United Kingdom (61%), Italy (60%), and Spain (50%). Spain reported the highest percentage of physician diagnoses (46%), followed by Italy (41%); only in France did less than one third of respondents receive a physician diagnosis. The percentage of respondents who were diagnosed by a friend or a family member varied from 7% in Italy and Spain to 18% in the United Kingdom and France. A negligible percentage was diagnosed by other means; that is, pharmacist, homeopath, physician's assistant, nurse practitioner, or information obtained in a physician's office or from an advertisement. (Respondents could provide more than 1 answer to this question.)A majority of respondents in all countries reported sneezing and itchy/watery eyes to be their most common symptoms . Other symptoms elicited more varied responses. For example, 66% of respondents in the United States reported sinus congestion compared with only 12% of those in Spain , Germany 48%), and the United Kingdom and the United States (both 46%). Lesser percentages were noted for severe afternoon, evening, and nighttime allergies , whereas in the United States it was nasal congestion (56%). More than half of Spanish respondents experienced itchy eyes and runny nose most frequently in the morning; Spain was the only country that reported a 50% or greater response rate for both of these symptoms.Overall, US participants reported more negative emotional effects from morning allergies than those surveyed in the other 5 countries. In general, irritability and fatigue were the most common effects of symptoms, with some variation between the countries. Responders in Italy reported a higher degree of irritability (58%), followed by the United States (55%) and the United Kingdom (53%), compared with only 24% of responders in Spain. French participants noted the highest degree of fatigue (59%), followed by the United States (52%); it was identified as a primary morning concern by 31% to 44% of patients in the other countries.A large percentage of responders reported difficulty in falling asleep without trouble, ranging from 47% in Spain to 26% in Germany and the United Kingdom. When respondents were asked whether this problem was disruptive, 73% in the United States, 71% in France, 68% in Germany, 57% in the United Kingdom, and 52% in Italy said that it was; however, only 24% of those in Spain  said that it was disruptive  and over-the-counter (OTC) treatments, with the highest percentage of drug choices based on physician recommendation occurring in Spain . Spain (66%) and France (62%) had the highest rates of Rx drug use, and the United Kingdom had the highest rate of OTC drug use (41%).In the United Kingdom, 23% of responders selected their own medications regardless of physician input, and 28% of those in this subgroup chose OTC formulations. Friends and pharmacists were also identified as major influences in the selection of OTC formulations, but they had a considerably smaller influence on Rx drug choice. Advertising had some influence when it came to patient choice of OTC medications but was much less influential in Rx drug choice.When questioned about their preferred form of allergy medication Table , most reRespondents from the 5 European countries answered questions designed to identify which brands (both prescription and OTC) they had used and which ones they had heard of and were likely to try in the future. As a rule in all countries, the brand that received the highest percentage of positive responses tended to achieve the highest degree of preference for current and future use.Attributes listed by respondents of the ideal drug to treat allergies were fairly consistent across all countries. Efficacy, rapid onset of action, safety, and lack of side effects were rated as the most important attributes. Table Ideal attributes of allergy medications change over time. In US respondents, attributes that became more significant were relief of symptoms, recommendation by physicians and pharmacists, freedom from sedating qualities and other adverse effects, and not interfering with a good night's sleep. Some remained unchanged, such as efficacy and safety, and others became less significant, including taste, reputation, and use by the whole family.Although there are many treatment options for AR, it remains a largely undertreated condition. The percentage of nontreaters in this survey ranged from 11% in the United States to 21% in Germany. Reasons that respondents in this subgroup did not treat their AR included the following: delaying time to seek treatment because they believed that their symptoms were unimportant, finding the cost of medications too high, or having an inadequate relationship with their physician or health care provider . ImproviThe information gathered from allergy sufferers through the present survey supports the observations of many clinicians that people with AR perceive their symptoms as causing significant QoL disruptions. Allergic rhinitis has an unquestionable impact on QoL, including limitations on daily activity and decreased productivity at work and school.The information provided by QoL questionnaires focusing on AR has also made it clear that patients with AR are impaired in many domains of their daily lives . SymptomP < 0.0001) [P = 0.048). However, the daytime sleepiness could be related to the SAR symptoms as well as the impaired sleep [Respondents in all countries said they had experienced difficulty in falling asleep. Sleep disturbances are commonly noted in patients with AR and contribute to a reduction in QoL . One sur 0.0001) . A smalled sleep .P = 0.0003 vs placebo). Significant decreases in RQLQ subdomain scores were also seen with desloratadine [P ≤ 0.005 vs placebo) [A recent 2-week study of 483 subjects with AR randomized to desloratadine 5 mg or placebo found that treatment with the second-generation antihistamine desloratadine significantly improved total RQLQ scores between baseline and day 14 (ratadine . In anotplacebo) .Given the pervasive adverse effects of AR on function and capacity, it is evident that medical treatment for AR should address issues beyond clinical outcome measures. One of the chief goals of therapy is to improve patients' well-being and their ability to function. Treatment modalities should not only provide optimum clinical benefits; they should also increase patient satisfaction ,21. The Responses to the survey suggest that AR is often perceived as causing daytime fatigue. It is possible that some people with AR could be experiencing the sedating effects of first-generation antihistamines in addition to the impairment and sleep disturbances caused by their symptoms.Parietaria dominate in the Mediterranean region [Although not specifically addressed by the survey administrators, it should be noted that there are differences among the 5 European countries in allergenic pollens. Grass, birch, mugwort, and ragweed pollen predominate in central Europe and in mountainous areas, whereas olive, cypress, and plants in the genus n region . Althougn region . These fn region .Results from this survey highlight the range of allergic symptoms that negatively impact the lifestyle of people with AR. Patients clearly want treatment options that improve their QoL and restore their ability to engage in activities of daily living.Dr. Canonica and Dr. Mullol have received research grants from Schering-Plough Corporation. Dr. Mullol has participated in advisory boards and received honoraria from Schering-Plough Corporation. Dr. Pradalier has no conflicts of interest. Dr. Didier has received honoraria from Schering-Plough Corporation.


In the present study, a series of 4-(4-substituted aryl) semicarbazones were synthesized from substituted anilines and subsequently evaluated for their anticonvulsant activities. The anticonvulsant activities were established by the anticonvulsant drug development (ADD) programme NIH, USA using experimental animal, adult male FCM mice (20–25 g) and adult Sprague-Dawley rats (100–150 g) and screened against electroshock seizure, subcutaneous metrazole and minimal neurotoxicity tests in mice. Compound 7 was found equipotent to carbamazepine in both MES and ScPTZ tests. This study has highlighted the importance of distal alkyl chain which influences the anticonvulsant activity. The antfunctions.et al.[2 group. In continuation, Pandeya et al.[The lipophilic aryl ring with chloro, bromo or nitro groups have been found to be essential for anticonvulsant activity. The distal aryl ring is also implicated at the binding site. The hydrogen bonding in semicarbazone series has been suggested by Dimmock et al.7 to be tya et al.9 suggestya et al.. In thismax, nm) and IR  spectra were consistent with the assigned structures recorded on Jasco Model 7800 and Jasco FT/IR-5300 instruments respectively and 1H NMR  spectra were recorded Jeol FX 90Q FT spectroscope using TMS as internal standard. Chemical shift are quoted in δ (ppm) units relative to TMS. Purity of the synthesized compounds were checked by TLC using silica gel G  and chloroform methanol (9:1) solvent system. 4-substituted semicarbazone derivatives were prepared in three steps as follows:Melting points of the synthesized compounds were taken by the open capillary method and are uncorrected. UV (EtOH-Band λ4-substituted aniline (0.1 mol) was dissolved in 10 ml of glacial acetic acid (GAA) and diluted to 100 ml with water. Equimolar quantity (0.1 mol) of sodium cyanate in 50 ml of warm water was added in previous solution with stirring. The reaction mixture was allowed to stand for 30 min, and then it was filtered, washed, dried and recrystallized from boiling water.To a solution of 4-substituted phenyl urea (0.1 mol) in 20 ml ethanol, an equimolar (0.1 mol) quantity of hydrazine hydrate was added. The reaction mixture was made alkaline by adding 4 g of NaOH, was refluxed for 1-2 h and cooled in ice. The product was filtered under suction and recrystallised from ethanol.A solution of p-phenyl semicarbazide (0.01 mol) and an equimolar (0.01 mol) quantity of appropriate carbonyl compound was refluxed for 30 min in the presence of glacial acetic acid (1-1.5 ml). The product obtained after cooling was filtered and recrystallised from 95% ethanol to give pure compounds . The chaf 0.56; UV  230, 337, 376; IR  3350 (Ar-NH), 1700(C=O), 1610 (C=N), 820 (Ar-H); 1H NMR  5.8 , 6.3-6.5 , 7.6-7.9 , 8.0 .1-(diPhenylmethylene)-4-(4-nitrophenyl)semicarbazone (1); m.p. 123-125°; Yield (%) 70; Rf 0.63; UV  231, 373, 379; IR  3365 (Ar-NH), 1635 (C=O), 1475 (Ar-NO2), 845 (Ar-H); 1H NMR  5.9 , 3.4-3.7 , 6.4-6.7 , 7.6-8.0.1--4-(4-nitrophenyl)semicarbazone (2); m.p. 145-147°; Yield (%) 65; Rf 0.59; UV  283, 372, 380; IR  3610 (Ar-NH), 1638(C=O), 1470(Ar-NO2), 850(Ar-H); 1H NMR  5.7 , 6.9-7.1 , 2.8-2.9 , 8.1 .1-Methylene-4-(4-nitrophenyl) semicarbazone (3); m.p. 230-235°; Yield (%) 60; Rf 0.48; UV  ;225, 376; IR  3370 (CONH), 3270 (Ar-NH), 1720 (C=O), 1610 (C=N), 1525 (Ar-NO2), 855(Ar-H); 1H NMR  6.0 , 4.01 , 3.76 , 3.75 , 8.1 .1--4-(4-nitrophenyl)semicarbazone (4); m.p. 125-130°; Yield (%) 92; Rf 0.76; UV  280, 236; IR  3430(CONH), 3320(Ar-NH), 1735(C=O), 1570(C=N), 1245(Ar-F), 830 (Ar-H); 1H NMR  3.48 , 3.95 , 6.0 , 7.23-7.33 , 7.25-7.59 , 10.09 .1-(4-Methoxybenzylidene)-4-(4-fluorophenyl) semicarbazone (5); m.p. 178-180°; Yield (%) 74; Rf 0.74; UV  328, 228; IR  3370 (sec. NH), 2815 (CH3-N), 1682 (C=O), 1560 (C=N), 1312 (C-N amine), 1035 (Ar-Cl), 845 (Ar-H); 1H NMR  3.42 , 3.92 , 7.85-8.05 , 8.83 , 7.23-7.26 , 8.06 .1-Benzylidene-4-(4-chlorophenyl) semicarbazone (6); m.p. 160-163°; Yield (%) 55; Rf 0.77; UV  228, 251, 374; IR  3420 , 3320 sec. NH, amide), 1735 (C=O), 1570 (C=N), 1245 (Ar-Cl), 830 (Ar-H); 1H NMR  2.29 , 5.8 , 7.2-7.3 , 8.8 .1-(Butan-2-ylidene)-4-(4-chlorophenyl) semicarbazone (7); m.p. 154-157°; Yield (%) 65; Rf 0.49; UV  251, 283, 372, 380; IR ; 3372 (Ar-NH), 1630(C=O), 1575(C=N), 1472 CH2 def.), (840 (Ar-H); 1H NMR  6.1 , 7.35-7.9 , 7.12-7.56 , 8.88 .4-(4-Bromophenyl)-1-(9-oxoanthracen-10(9H)-ylidene) semicarbazone (8); m.p. 207-211°; Yield (%) 30; Rf 0.52; UV  251, 271, 324; IR  3365(Ar-NH), 1572 (C=N), 1478 (CH2 def.), 800 (Ar-H); 1H NMR  5.9 , 6.4-6.6 , 8.80 .4-(4-Bromophenyl)-1-(diphenylmethylene) semicarbazone (9); m.p. 150-156°; Yield (%) 37; Rf0.53; UV  260, 266, 272; IR  3372 (Ar-NH), 1635 (C=O), 1572(C=N), 820(Ar-H); 1H NMR  6.0 , 6.5-6.9 , 8.1 , 9.8 .4-(4-Bromophenyl)-1-semicarbazones (10); m.p. 147-150°; Yield (%) 35; RThe anticonvulsant activities were established by the anticonvulsant drug development (ADD) programme. Adult mInitially all the compounds dissolved in polyethylene glycol were administered intraperitoneally (0.01 ml /g body weight in mice and 0.004 ml /g body weight in rats) at a dose of 30, 100, and 300 mg/kg to all the animals respectively. Compounds were screened for anticonvulsant activity by maximal electroshock seizure (MES), subcutaneous metrazole (ScMet) and Minimal neurotoxicity (TOX) test in mice.MES or maximal seizure pattern test, in which an electrical stimulus of 0.2 s in duration (50 mA in mice and 150 mA in rat at 60Hz) was delivered via corneal electrodes primed with an electrolyte solution containing an anesthetic agent. Mice are tested at 30 min and 4 h following doses of 30, 100 and 300 mg/kg of test compound. Abolition of the hindlimb tonic extensor component indicates the test compound’s ability to inhibit MES-induced seizure spread.The subcutaneous pentylenetetrazol (metrazol) seizure test (scPTZ) was performed using a dose of pentylenetetrazol (70 mg/kg in Sprague Dawley rats) to produce clonic seizures lasting for a period of at least five seconds in 97 per cent (CD97) of animals tested. At the anticipated time of testing the convulsant is administered subcutaneously. The test compound is administered intraperitoneally in mice and orally in rats. Animals are observed over a 30 min period. Absence of clonic spasms in the observed time period indicates a compound’s ability to abolish the effect of pentylenetetrazol on seizure threshold.Minimal neurotoxicity (TOX) was evaluated in rats by examining them for behavioral toxicity using the positional sense test and a gait and stance test. The mice were trained to stand on an accelerating rotarod of diameter 3.2 cm, which rotated at 10 rev/min. Animals were given intraperitoneal injections of the test compounds in the doses of 30, 100 and 300 mg/ kg body weight. Neurotoxicity was indicated by the inability of the animal to maintain equilibrium on the rod for at least one minute in each of the three trials.*transition. K-band of substituted p-nitroaniline was very strong λmax 375–380 nm. The halo substituted derivatives absorbed at 265 nm. The formation of the substituted hydrazones and semicarbazones were confirmed with the formation of C=N band in all spectra from 1500–1620 cm−1. The CONH stretching for amides was observed in the 4-substituted aryl/alkyl semicarbazones at about 3180-3320 cm−1. The chemical shift for aromatic amino group (Ar-NH) was found at 5.82 to 5.89 ppm and 8.86-8.91 ppm for -CONH group present in semicarbazone. The signals for aromatic protons were observed at 7.2-7.6 ppm.The UV spectrum shows presence of chromophores in the 4-substituted aryl/alkyl semicarbazones. The benzene ring absorption is in the range of 200-205 nm pertaining to K-band. The π- πThe series of 4-subsituted phenyl semicarbazones were found to possess significant anticonvulsant activities . The comGenerally MES active compounds are considered to exhibit activity in grandmal epilepsy, contain larger hydrophobic center (phenytoin) and ScMe


Chironex fleckeri and Chiropsella bronzie. During growth, these cubomedusae transitioned from using jet propulsion at smaller sizes to a rowing-jetting hybrid mode of propulsion at larger sizes. Simple modifications in the flexibility and kinematics of their velarium appeared to be sufficient to alter their propulsive mode. Turning occurs during both bell contraction and expansion and is achieved by generating asymmetric vortex structures during both stages of the swimming cycle. Swimming characteristics were considered in conjunction with the unique foraging strategy used by cubomedusae.Evolutionary constraints which limit the forces produced during bell contractions of medusae affect the overall medusan morphospace such that jet propulsion is limited to only small medusae. Cubomedusae, which often possess large prolate bells and are thought to swim via jet propulsion, appear to violate the theoretical constraints which determine the medusan morphospace. To examine propulsion by cubomedusae, we quantified size related changes in wake dynamics, bell shape, swimming and turning kinematics of two species of cubomedusae,  The functional ecology and size of medusae have been shown to be related to and predicted by bell form (Dabiri and others (2007) developed a model to examine the physiological constraints that resulted in the observed morphospace. A premise of this force balance model is that bell shape (quantified as fineness ratio (f) where f = bell height/diameter) affects the force balances necessary for swimming because bell shape influences the propulsion mode of medusae. Medusae with prolate bells (f >1) swim via simple jet propulsion and those with oblate bells (f <1) swim using the more hydrodynamically complex rowing propulsion Few species possess morphologies that do not conform to the constraints model. However, there are some very large prolate cubomedusan species that are effective swimmers despite having bell diameters that surpass model predictions. These medusae swim with a pulse frequency of ∼1 Hz and appear to be jet propelled. If the physiological limits described for jet-propelled medusae are correct, then large cubomedusae must either have a unique muscle structure that increases the forces produced by the muscles or, despite their shape, swim using rowing propulsion. The muscle structure of cubomedusae has been described and shown to have the same basic epitheliomuscular structure as other medusae Cubomedusae have several other traits that are unique among cnidarian medusae. The most well-known unique trait is their complex, lensed eyes. While their eyes have been described previously The goal of this study was to quantify the influence of bell size on body kinematics and fluid interactions of cubomedusae during development. These swimming traits are evaluated relative to other medusae in light of the unique cubomedusan requirements for foraging.Chiropsella bronzie and Chironex fleckeri in January 2009 at James Cook University in Cairns, Australia. C. bronzie were hand-collected from marina docks and C. fleckeri were collected by seining in the intertidal zone along local public beaches. No permits were required for these collections and no permission was needed to sample in these locations. The collected medusae ranged in size  from 0.5–5.6 cm (C. bronzie) and 2.0 to 16 cm (C. fleckeri). All medusae were subsequently transported to the laboratory and maintained in healthy condition in large aquaria during the experiments.We collected −1 using a high-speed digital video camera  placed perpendicular to the laser sheet. The laser sheet illuminated a two-dimensional plane of fluid, and data were collected when the center of the medusa bell was bisected by the laser plane. Fluid velocities were determined using digital particle image velocimetry (DPIV) software package  that analyzed sequential video frames using a cross-correlation algorithm. Image pairs were analyzed with shifting overlapping interrogation windows of decreasing size (64 × 64 pixels then 32 × 32 pixels). This analysis generated velocity vector fields around the swimming medusae. Velocity and vorticity field data were exported from DaVis and later used to calculate various fluid quantities using an in-house Matlab code. Bell kinematics were quantified from the cross-sectional images of the bell and were analyzed using Image-J software (NIH).Swimming kinematics and fluid interactions during swimming were measured for individuals placed into a large aquarium containing filtered seawater seeded with hollow glass spheres (10 µm). Medusae were then illuminated using a 680-nm wavelength laser sheet and recorded at 500 frames sH) and diameter (D). The swimming speed, U, was found by selecting the apex of the bell in each consecutive image, and dividing the bell’s displacement between frames by the inverse of the camera’s frame rate (or 0.002 s).Bell and swimming kinematics were quantified using the same methods as Colin and Costello (2002). Changes in bell shape were quantified using the fineness ratio, f, defined asη, is the ratio of the mechanical work resulting in forward motion to the total work expended during swimming, and can be expressed asdragF is the hydrodynamic drag, U is the swimming speed and lossP is the power loss or the flux of kinetic energy (KE) into the wake per unit time. lossP is the excess KE in the wake divided by the contraction time of the medusae (ct). KE is calculated by revolving one half of the velocity field such thatTo evaluate the propulsive efficiency of medusae, we calculate the Froude efficiency, a common metric used to compare swimming across organisms. The Froude efficiency, dC, byS is the surface area of the bell (estimated as a hemiellipsoid), maxU is the medusae’s maximum swimming speed, and dC is the drag coefficient. Values for dC can be found from empirical models that are dependent upon the Reynolds number (Re = UD/ν) of the swimming medusa, where D is the bell diameter and ν is the kinematic viscosity of seawater (ν = 1.05 × 10−6 m2 s−1 at 20°C). Two empirical models for dC were used to evaluate the hydrodynamic drag. The first model was developed The hydrodynamic drag acting on the medusa’s body can be found from the drag coefficient, 3< Re <106R and H are the radius and height of the medusan bell, respectively. Since the Re for swimming cubomedusae ranges from 10 to 10000, these empirical models can be applied to determine hydrodynamic drag with some certainty.yu). In contrast, the velocity of the fluid entrained from outside the bell past the bell margin had a predominantly x-component (xu). The momentum flux of the jet (pjet) was calculated using the total velocity of the fluid (Tu) and its associated y-component (yu) extracted along a transect extending from the center of the starting vortex to the center of the jet at mid-contraction as:l is the length of the interrogation square of the vector field. To estimate the momentum flux of the entrained fluid (pentrained) the total velocity of the fluid and its x-component were extracted along a transect that extended from the center of the vortex ring to the bell margin at the same period in the contraction cycle as that of the jet (Eq. 7) as:Analyses of wake structures were performed using DPIV velocity vector fields. The contribution of different fluid regions to the starting vortex during bell contraction was measured as the momentum flux (p) of fluid across transects positioned at different locations relative to the bell and starting vortex ring. Due to shadowing, only the vortex ring on one side of each medusa corresponding to the side of incident light was analyzed. Prior to DPIV analysis, the video frames were reoriented so that the trajectory of swimming medusae was parallel to the y-axis. With this orientation, the velocity of the jet of fluid emerging from the subumbrellar cavity had a predominantly y-component (Swimming kinematic profiles illustrate how the bells contracted (increasing fineness ratio) and expanded (decreasing fineness ratio) during consecutive swim cycles and the forward progress of the medusae over time . They reTo examine how bell kinematics change through development we quantified how much different sections of the bell contracted relative to the position at rest . For cubWhile there were not any conspicuous alterations in bell contraction through development, the kinematics of the velarium did differ between small and large cubomedusae . For smaDevelopmental differences in velarium kinematics were accompanied by changes in medusan wake dynamics. The most conspicuous features within wakes of small medusae were strong jet flows expelled from the subumbrellar cavity during contraction . A similThe wake structures of the different propulsive modes used by small and large medusae also highlight key distinctions between jetting and rowing propulsion . VorticiC. bronzie and C. fleckeri result in Froude efficiencies no greater than 40% and many estimates are below 20%  nor the trajectory of the starting and stopping vortices differed on either side of the bell . HoweverThe morphology, sensory capabilities, behavior and propulsion of cubomedusae are unique among medusae. However, their distinctive swimming traits appear well suited to their foraging strategies. Cubomedusae orient toward environmental cues Aurelia spp. and Cyanea spp., and hydrozoans, such as Aequorea victoria, feed as feeding-current foragers For example, rowing scyphozoans, such as Sarsia tubulosa, Aglantha digitale and Leuckartiara sp. involves motionless fishing with extended tentacles while waiting for prey to swim into their tentacles. Consequently, ambush foraging medusae typically feed only when they not swimming Ambush predatory hydromedusae can similarly be considered random foragers. Prey encounter by ambushing medusae such as In contrast to these other taxa, the sensory capabilities of cubomedusae – i.e. image forming eyes – have been shown to enable them to orient to objects in and out of the water A comparison of the swimming efficiency of cubomedusae to other medusan taxa suggests that cubomedusan swimming, while effective, is not efficient . Other mTripedalia cystophora, revealed complex velarium kinematics during which contraction of the velarium on the outside of the turn was initially delayed relative to the inside margin, but subsequently accelerated much more rapidly during the latter part of bell contraction Cubomedusan swimming characteristically entails a high level of maneuverability. Surprisingly, for smaller turns cubomedusae turned primarily during the recovery stroke and only for larger turns did they also turn during the power stroke . PreviouInterestingly, we observed that the largest differences in the relative magnitude of the vortex circulation between inside and outside margins of a turn occurred within stopping vortices during the recovery stroke. This is important because stopping vortices have been shown to contribute forward thrust In conclusion, it has been shown for hydromedusae and scyphomedusae that medusan form and propulsion are tightly coupled with foraging strategy


The inconsistency of predictions from solution concepts of conventional game theory with experimental observations is an enduring question. These solution concepts are based on the canonical rationality assumption that people are exclusively self-regarding utility maximizers. In this article, we think this assumption is problematic and, instead, assume that rational economic agents act as if they were maximizing their implicit utilities, which turns out to be a natural extension of the canonical rationality assumption. Implicit utility is defined by a player's character to reflect his personal weighting between cooperative, individualistic, and competitive social value orientations. The player who actually faces an implicit game chooses his strategy based on the common belief about the character distribution for a general player and the self-estimation of his own character, and he is not concerned about which strategies other players will choose and will never feel regret about his decision. It is shown by solving five paradigmatic games, the Dictator game, the Ultimatum game, the Prisoner's Dilemma game, the Public Goods game, and the Battle of the Sexes game, that the framework of implicit game and its corresponding solution concept, implicit equilibrium, based on this alternative assumption have potential for better explaining people's actual behaviors in social decision making situations. Solution concepts of conventional game theory are mainly based on the canonical rationality assumption that players are rational economic agents who act as if they were maximizing their self-regarding utilities. Nevertheless, increasing experimental data support that decision makers in human society often do not act in accordance with predictions from these kinds of solutions So far, many efforts have been devoted to the modification or extension of the canonical rationality assumption in game theory with the aid of experimental observations. For instance, bounded rationality and other-regarding preference of players are considered in behavioral game theory Although the experimental evidence that rational self-regarding utility maximizers exist in chimpanzee's societies might offer some support for the canonical rationality assumption In this article we try to formally incorporate into the solution concept of game theory the player's character, a parameter encapsulating all diverse factors that may influence his decision making. The characters of players reflect their personal weighting between cooperative, individualistic, and competitive social value orientations. We assume that all players have a common belief about the character distribution for a general player, and rational players act as if they were maximizing their character-related utilities , which turns out to be a natural extension of the canonical rationality assumption. In this procedure, a game is transformed into a Bayesian game, the solution of which, Bayesian Nash equilibrium, is referred to as implicit equilibrium of the original game. An implicit equilibrium provides players with a concrete decision rule which varies with their characters, and no player would feel regret about his decision even though the outcome turns out not to be a Nash equilibrium. The idea behind our framework turns out to be similar to the ERC model, a theory of equity, reciprocity, and competition ad infinitum. It is probably safe to say that this is not realistic in most cases. The concept of bounded rationality in behavioral game theory is proposed to avoid this vulnerability Solution concepts based on the canonical rational assumption also assume that it is common knowledge that each player is rational, each player knows that all players are rational, each player knows that each player knows that all players are rational, and so on The concepts of character and implicit equilibrium we propose here based on this new rationality assumption can better explain people's actual behaviors labeled as irrational by conventional game theory in the experiments of some paradigmatic games. For example, why are at least one third of the dictators willing to offer the recipients more than zero in the Dictator game We begin by defining an We decompose a player's utility into two parts: the outward-looking utility and the inward-looking utility. The former reflects the benefit of the group as a whole gained from playing the game and is the same for all players concerned, whereas the latter reflects the relative advantage of the focal player gained over the utilities of the other players involved in the game. A player's character is defined to reflect his personal weighting between the outward-looking utility and the inward-looking utility, or equivalently, his personal weighting between intergroup and intragroup competition.outward-looking utility of player inward-looking utilityimplicit utility of player character which reflects his personal weighting between the outward-looking utility and the inward-looking utility. When Formally, for any strategy profile A player's character may take any value in the interval Note that the inward-looking or outward-looking utility is distinct from the exclusively self-regarding or other-regarding utility. This is an issue about what factors should be considered as the primitive assumptions about people's instincts related to decision making. We think that the exclusively other-regarding preference as a primitive assumption might contradict the principle of individuals being the unit of natural selection. The definition of implicit utility in (4) is equivalent toThe character of a player, which reflects his personality when facing a game situation, may vary with the contexts in which he needs to make a decision. For example, experimental data show that the cooperation rate usually decreases in repeated public goods games without punishment relative to in those games with punishment when facing one-shot game situations without communication, all players have a relatively stable common belief  about the character distribution for a general player over the interval In other words, for any focal player, implicit game corresponding to implicit equilibrium of By incorporating players' characters and their common belief about the character distribution for a general player, any game can be transformed into a Bayesian game. In particular, let character-related rationality assumption; that is,In contrast to the canonical rationality assumption, the idea underlying this transformation is referred to as the all players are rational economic agents who act as if they were maximizing their implicit utilities.For those players with character The Dictator game is about sharing a pie between two anonymous players, in which the dictator who has complete control over the process of allocation chooses to offer a portion of the pie,  himself . This onAccording to the canonical rationality assumption, the rational dictator who acts as if maximizing his self-regarding utility would take advantage of his dominant position and thus choose to offer the recipient nothing. However, a consistent observation in experiments is that at least one third of the dictators are willing to offer the recipients more than zero across a wide variety of procedures and conditions, although it is true that most of the dictators offer nothing In contrast, the character-related rationality assumption predicts that not all the dictators who act as if maximizing their implicit utilities would offer nothing, as shown in the following proposition.In the Dictator game, (i) a rational dictator with character  would like to offer the recipient any portion of the pie, even as big as the whole pie, and (ii) a rational dictator with character  would offer the recipient nothing.Suppose that the dictator, player 1, offers a portion of the pie, If If As a result of the recipient's inability to influence the allocation procedure, the implicit equilibrium presented in this proposition does not even need the dictator and the recipient to have a common belief It is worth noting that we can get The Ultimatum game is similar to the Dictator game. One only difference is that, in the Ultimatum game, after the proposer offers a portion of the pie,  nothing  [8]. TheAccording to the canonical rationality assumption, this game has a subgame perfect Nash equilibrium where the proposer offers the smallest nonzero amount and the responder accepts it, because a rational proposer knows that the responder who maximizing his self-regarding utility will always accept any positive offer In contrast, the character-related rationality assumption that players act as if they were maximizing their implicit utilities provides a more reasonable explanation for all these experimental observations.In the Ultimatum game, (i) a rational proposer with character  would like to offer any portion of the pie not less than , even as big as the whole pie, where  is the character of the responder, and the responder would accept it, and (ii) a rational proposer with character  would offer  which is never more than half the pie, and the responder would accept it.Suppose that the proposer, player 1, offers a portion of the pie, Because the implicit utility The implicit utility From the proof above we can also infer that the responder with non-negative character would accept any offer As in the Dictator game, the implicit equilibrium presented in this proposition does not also require the proposer and the responder to have a common belief Let us dig deeper into the procedure of the proof of this proposition. The upper bound of the offer that both players will accept, In the Ultimatum game, half the pie is the unique offer that would be always accepted by any two players.Now the characters of both the proposer and the responder fade out completely. Actually, in the case where Fairness, or inequality aversion, is recognized as one of key elements in human strategic interactions The best known model in game theory might be the Prisoner's Dilemma game, which is also played between two anonymous players . Each plAccording to the canonical rationality assumption, one in this scenario would always defect since defecting is a dominant strategy. That is, one can always be better off by defecting than by cooperating, whether the other player defects or cooperates. By backward induction, we know, even in the finitely repeated version, defecting in all steps is the best choice. However, each could achieve a higher utility The predictions given by the canonical rationality assumption contradicts experimental data. There are subjects who cooperate even in the last round of finitely repeated situations, and the experimental studies from 1958 to 1992 show that the cooperation rate of players ranges from 5% to 96.9%, with a mean of 47.4% In the Prisoner's Dilemma game, one would cooperate if his character , and otherwise defect, given that the two players have any common belief about the character distribution for a general player.For player We assume that the two players have a common belief This implicit equilibrium is symmetric in the sense that it works for either player. No player would feel regret even if he chooses to cooperate and the other player chooses to defect, although this outcome is not a Nash equilibrium. From this implicit equilibrium we can also infer that the players would tend to defect when Cooperation is considered as a third fundamental principle of evolution beside mutation and natural selection In the Public Goods game, each of e or not  [30]. ThAccording to the canonical rationality assumption, a rational player in this scenario would free ride since free riding is a dominant strategy. One will incur a loss of In the Public Goods game experiments where players are permitted to contribute any fraction of their endowments, the mean contributions usually end up with between 40% and 60% In the Public Goods game, one would contribute his endowment if his character , and otherwise he would free ride, given that all players have any common belief about the character distribution for a general player.We use For any given player For solving The implicit equilibrium in this proposition is also symmetric and works for all players. For the two-player case, the critical value Note that the implicit equilibria of the Dictator game and the Ultimatum game are not related to the players' common belief In the Battle of the Sexes game  company . When , According to the canonical rationality assumption, this game has two pure strategy Nash equilibria: both watching soccer or both watching opera. In addition, this game has a mixed strategy Nash equilibrium; that is, each player watches his preferred program with probability In the Battle of the Sexes game, one would watch the other player's preferred program if his character , and otherwise his own preferred program, given that both players believe that the character of a general player is uniformly distributed over the interval For two players, We assume that the two players have a common belief Further we assume that both players believe that the character of a general player is uniformly distributed over the interval The procedure of proof shows that the implicit equilibrium in the Battle of the Sexes game is closely related to the players' common belief In the Battle of the Sexes game, one would watch the other player's preferred program if his character , and otherwise his own preferred program, given that both players believe that the character of a general player is uniformly distributed over the interval Under both assumptions about the players' common belief When additional utility The inconsistency of predictions from solution concepts of conventional game theory with experimental observations is an enduring question. By clearly taking the player's heterogeneity into consideration, we have shown in five paradigmatic games, the Dictator game, the Ultimatum game, the Prisoner's Dilemma game, the Public Goods game, and the Battle of the Sexes game, that the concepts of implicit game and implicit equilibrium have potential for better explaining people's actual behaviors in social decision making situations. This theoretical framework is based on the so-called character-related rationality assumption that rational economic agents act as if they were trying to maximize their implicit utilities. The cooperative, individualistic, and competitive social value orientations are incorporated as three special cases where the player's character equals 1, 0, and The concept of character reflects players' heterogeneity, although it does not explicitly consider complicated psychological motivations as in psychological game theory However, as aforementioned, a player's character could be influenced by so many factors that it seems to be a big question how to measure it. Even if this is the case, incorporating the player's character in analyzing games would no doubt be useful to explain people's diverse behaviors in decision making as we shown here. At this point it may be suitable to mention another fact from physics. The temperature of an object is also influenced by many complicated factors, yet now we can conveniently measure it using a thermometer. Likewise, constructing a proper procedure, or even a convenient device, to measure the character of a player facing a game situation is a promising and challenging project. Another interesting issue is how the character of a player (consequently his strategy) evolves in repeated game situations with new information about the strategies of other players in previous plays. The progress in either direction would further extend our understanding about the reason people cooperate in social dilemmas and the way decisions are actually made.


To determine the specific requirements of HSPB2 in heart, we generated cardiac-specific HSPB2 deficient (HSPB2cKO) mice and examined their cardiac function under basal conditions and following cardiac pressure overload.CryAB (HspB5) and HspB2, two small heat shock genes located adjacently in the vertebrate genome, are hypothesized to play distinct roles. Mice lacking both 2/min/mg dry fiber weight, P<0.05), and ATP production in HSPB2cKO hearts was significantly reduced in TAC compared with sham operated mice . Although HSPB2 was not associated with mitochondria under cardiac stress, absence of HSPB2 led to changes in transcript levels of several metabolic and mitochondrial regulator genes.Transverse aortic constriction (TAC) or sham surgery was performed in HSPB2cKO mice and their littermates (HSPB2wt mice). Eight weeks after TAC, we found that expression of several small HSPs  was not markedly modified in HSPB2wt mice. Both cardiac function and the hypertrophic response remained similar in HSPB2cKO and HSPB2wt hearts. In addition, mitochondrial respiration and ATP production assays demonstrated that the absence of HSPB2 did not change mitochondrial metabolism in basal conditions. However, fatty acid supported state 3 respiration rate (ADP stimulated) in TAC operated HSPB2cKO hearts was significantly reduced in compared with TAC operated HSPB2wt mice (10.5±2.2 vs. 12.8±2.5 nmol OThe present study indicates that HSPB2 can be replaced by other members of the multigene small HSP family under basal conditions while HSPB2 is implicated in the regulation of metabolic/mitochondrial function under cardiac stress such pressure overload. The small MW heat shock proteins  are expressed in virtually all organisms, from bacteria to humans. They are evolutionarily related via a conserved sequence domain in the carboxyl region, termed the α-crystallin domain [1002C2]. Functionally, most sHSPs display chaperone-like activity to maintain misfolded proteins in soluble but inactive states and, furthermore, protect cells against stressful conditions. While the selective patterns of expression suggest that sHSPs may impart tissue-specific and specialized roles, the nature of these functions is still under active investigation cryab also named hspb5) and hspb2 form a bidirectional gene pair that reside on chromosome 9 and chromosome 11 in mouse and human genomes, respectively Cryab and hspb2 are the result of a gene duplication event and share a common promoter region, although cryab, but not hspb2, is stress-inducible cryab and hspb2 being deleted in mice The small HSPs αB-crystallin . Our data reveal that the absence of HSPB2 in the heart does not significantly affect the cardiac hypertrophic response to pressure overload stimuli, but that HSPB2 deficiency depresses mitochondrial fatty acid beta-oxidation and ATP production under these conditionsTherefore, beyond the DKO model affecting both CRYAB and HSPB2 expression, a new mouse model that targets specifically hspb2 is required to determine the distinct tissue-specific functions of HSPB2 These studies were approved by the Animal Care and Use Committees of the University of Utah and adhered to the Guide for the Care and Use of Laboratory Animals (NIH).hspb2 locus is depicted in cryab and hspb2, subcloned into pBluescript SK- plasmid vector (pEW32) was a generous gift from Eric Wawrousek . Plasmid EW32 was digested with EcoRI and the ∼6.7 kb fragment containing the entire hspb2 genomic region  with ∼3 kb of downstream sequence was isolated and cloned into pUC18 for subsequent restriction mapping and sequence analysis. The targeting vector was generated by incorporating a loxP oligonucleotide into the unique NcoI site in the first intron of the hspb2 gene approximately 300 bp 3′ of exon 1. The second loxP site and neomycin gene cassettes (loxP-FRT-Neo-FRT) were inserted ∼1700 bp 3′ of exon 2 into a unique NotI site, created after NotI linker incorporation into the unique HpaI site, but in the same orientation as the first loxP site. By eliminating an essential domain for molecular chaperone activity, targeted deletion of the second exon containing the conserved α-crystallin domain by Cre recombinase of this ∼2600 bp fragment completely inactivates the functions of HSPB2. The targeting vector contains a 2.8 kb homologous region upstream of the first loxP site (long arm) and a 1.3 kb homologous region downstream of the loxP-FRT-Neo-FRT cassette (short arm). When crossed with FRT recombinase-expressing mice, the FRT sites flanking the neomycin gene cassette enabled the removal of this positive selection marker and, thus, any potential influences on the targeted or neighboring genes in vivo. The negative selection marker TK1 (thymidine kinase) was incorporated into the targeting vector at the 3′ end of the hspb2 genomic sequence. Southern analyses of the NcoI digested ES cell genomic DNAs were performed with PCR probes generated outside of the borders of the ∼6.7 kb EcoRI fragment used for building the targeting construct to confirm homologous recombination (data not shown).The schematic structure of the wild type, targeted and deleted hspb2 targeted ES cells, we obtained the FLP recombinase129S4/SvJaeSor-Gt(ROSA)26Sortm1(FLP1)Dym/J mice that were crossed with the conditional hspb2 targeted mouse line. Hspb2 conditional targeted animals without the Neomycin cassette were crossed to cardiac specific (α-MHC) Cre expressor mice (CreTG) hspb2 knockout in the heart. Age matched male HSPB2wt and CreTG mice were used as controls.To eliminate the Neomycin cassette used for positive selection for the st Century Biochemicals using the following peptide sequence: CPATAEYEFANPSRLGEQ-amide. This peptide was generated in-house and confirmed by mass spectrometry after HPLC purification. The peptide was injected with multiple protein carriers, using two rabbits. Serum was obtained after favorable titers were reached and HSPB2 antibody used in the present work was the affinity-purified fraction. The working dilution to probe western blots was 1∶5000.Polyclonal antibodies raised against HSPB2 were generated for our laboratory by 21Hearts and soleus, slow-twitch skeletal muscle, from HSPB2cKO and HSPB2wt control mice were isolated, then washed several times in ice cold PBS to remove the blood. The tissue was minced into small pieces and homogenized in standard RIPA buffer. Supernatant was collected after cold centrifugation at 12000 g for 15 min. To prepare cytosolic/mitochondrial fraction, heart tissues were rinsed in cold PBS, homogenized in buffer containing 250 mM sucrose, 1 mM EGTA, 10 mM HEPES and 10 mM Tris-HCl (pH 7.4). The homogenates were centrifuged at 800 g for 7 min to pellet cell and tissue debris and the supernatants were further centrifuged at 4000 g for 15 min at 4°C. Those supernatants containing cytosolic fraction were saved for further use. The pellets containing mitochondria were thoroughly washed and centrifuged 2–3 times with the same buffer and then resolubilized in an EGTA free homogenization buffer. The suspensions were cleared using 0.1% NP40 and incubated for 30 min on ice Total RNA was extracted from ventricles in HSPB2wt and HSPB2cKO mice, using the RNeasy Mini Kit (QIAGEN). The cDNAs were prepared with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The accumulation of the PCR product using QuantiTect SYBR Green PCR Kit (QIAGEN) was monitored in real time by an ABI Prism 7900HT sequence detection system (Applied Biosystems). The following PCR primers  were used to amplifyhsp25/hspb1, hspb2, cryab/hspb5, hsp20/hspb6, and 18 S rRNA. The primer sequences for HIF1- α, IDH2, cox5b, CPT1-ß, MCAD, UCP2 are described elsewhere TAC or sham surgery was performed as previously described At 4 weeks after moderate TAC and 8 weeks after severe TAC, LV pressure and its derivatives (+dP/dt and –dP/dt) were recorded using a Powerlab  and catheter transducer inserted into the ascending aorta and LV chamber via the right common carotid artery.2EGTA, 2.77 mM K2CaEGTA, 6.56 mM MgCl2, 20 mM imidazole, 0.5 mM dithiothreitol, 53.3 mM K-methanS, 20 mM taurine, 5.3 mM Na2ATP, 15 mM PCr and 3 mM KH2PO4, pH 7.1 adjusted at 25°C. The fibers were then washed twice for 10 min in buffer B containing 7.23 mM K2EGTA, 2.77 mM K2CaEGTA, 1.38 mM MgCl2, 20 mM imidazole, 0.5 mM dithiothreitol, 100 mM K-methanS, 20 mM taurine, 3 mM KH2PO4 and 2 mg/ml BSA, pH 7.1 at 25°C.Respiratory parameters of the total mitochondrial population were studied in situ in fresh saponin-permeabilized fibers as previously described 2 min/mg dry fiber weight. State 2 respiration (no ADP) was measured in a 125 mM KCl solution containing the appropriate substrates. State 3 (ADP-dependent) respiration was measured by adding ADP at a final concentration of 1 mM, which stimulates maximum activation of respiration. State 4 respiratory rates were measured by the addition of 1 µg/mL of oligomycin, an inhibitor of ATP synthase. ATP concentration was determined by a bioluminescence assay based on the luciferin/luciferase reaction using the ATP assay kit . The mitochondrial analysis was performed in HSPB2wt and HSPB2cKO mice at 4 weeks after moderate TAC and compared with sham-operated mice.The respiratory rates of saponin-permeabilized cardiac fibers were determined using an oxygen sensor probe in 2 ml buffer B at 25°C with continuous stirring. Studies were performed with 0.02 mM palmitoyl-carnitine and 2 mM malate as substrates All data are presented as mean ±SD. Significance levels were analyzed by Student’s t-test or one factor analysis of variance (ANOVA) followed by Tukey post-hoc test comparison of means, using Prism . A value of P<0.05 was considered statistically significant.hspb2 gene was obtained by using a floxed allele introduced by homologous recombination and the cardiac expression of Cre recombinase driven by αMHC promoter  and one of 15 HSPB2cKO mice  died. Thus, there was no significant difference of survival between HSPB2cKO and HSPB2wt mice. Also, heart weight (HW) and HW/body weight (BW) ratios were not significantly different between 1-year-old HSPB2wt  and HSPB2cKO mice .Because HSPB2 is dispensable for cardiac function under normal/physiologic conditions, we investigated the hypertrophic response of HSPB2 deficient hearts under pressure overload induced by transaortic constriction (TAC). At 4 weeks after moderate TAC, cardiac hypertrophy  and LV fParameters for cardiac hemodynamics, which were evaluated at 8 weeks after TAC, did reflect the cardiac response to pressure overload but again did not reveal any significant variation among the three groups of mice . The mean values for left ventricular systolic pressure (LVSP) and end-diastolic pressure (LVEDP) are graphically presented with +dP/dt, and –dP/dt in hspb2 deletion might be compensated by changes in expression of other members of the small HSP family. Furthermore, the imposed cardiac stress could trigger additional modifications in sHSP expression.The small HSPs form a multi protein family whose members share partially overlapping profiles of expression, molecular structure and interactions To test these hypotheses, we first measured the relative abundance of sHSP transcripts by RT-qPCR. TAC operated hearts exhibited an increased transcript levels of the four sHSPs tested  . Hspb2 dIn contrast to the marked increased in transcripts, the protein levels of HSP25 (HSPB1), HSPB2, CRYAB (HSPB5), HSP20 (HSPB6) insoluble protein extracts from hearts at 8 weeks after sham and TAC procedure were not significantly modified .−/−Hspb2.5 double knockout animals Previously, we had shown that a mouse model deficient in HSPB2 but overexpressing CryAB had slower and less complete recovery of cardiac energetics during reperfusion after ischemia, and that this metabolic defect was similar in We assessed mitochondrial fiber respiration rate and ATP production both under physiological conditions and under pathological conditions of cardiac hypertrophy induced by pressure overload. The mitochondrial respiration rates, using palmitoyl-carnitine and malate as substrate, were indistinguishable between HSPB2wt and HSPB2cKO under normal basal conditions (data not shown), whereas state 3 respiration rates (ADP stimulated) in HSPB2cKO was significantly reduced in TAC-compared to sham operated mice (P<0.05) . Also stBecause mitochondrial state 3 respiration was significantly different in stressed heart lacking HSPB2, we wanted to test the hypothesis that TAC procedure was triggering HSPB2 translocation to mitochondria. This hypothesis was supported by previous data showing that C2C12 cells transfected with recombinant HspB2 construct displayed such association between HSPB2 and mitochondria in vivo  and isocitrate dehydrogenase 2 , OXPHOS such as cytochrome C oxidase subunit b(COX5b), in fatty acid oxidation such as carnithine palmitoyltransferase 1 β (CPT-1β), acyl-coenzyme A dehydrogenase medium chain  and uncoupling protein 2  hspb2 gene (HSPB2cKO). Under normal conditions, HSPB2cKO did not exhibit any obvious cardiac anomaly and this is consistent with the observations made with the DKO animals, which were deficient in both hspb2 and hspb5hspb2, we could not definitively address the role of HSPB2 deficiency in other organs especially in skeletal muscles that harbor high levels of HSPB2 expression.In this study, we describe a new mouse model characterized by the cardiac specific deletion of the hspb1, another sHSP highly expressed during heart development did not provoke any major disturbance in cardiac anatomy or function as evidenced by the normal lifespan of those animals The superfamily of small MW HSPs (∼18 to 32 kDA) has been implicated in diverse functions and biological roles ranging from cellular immunity to oncogenesis to cardiomyopathy and heart failure ex vivoin situ and exvivo ischemic conditions compared with wild-type controls Although prior studies by other investigators have shown that isolated and intact hearts lacking both CRYAB and HSPB2 (DKO) exhibit severe contractile dysfunction and increased myocardial injury in response to ischemia/reperfusion Based on an earlier report on co-localization of HSPB2 How might HSPB2 expression modulate cellular metabolism and mitochondrial function under cardiac stressful conditions without evidence for direct interactions between the chaperone and organelle? The basic mechanism(s) for these intriguing observations are presently unknown but our preliminary studies hint at possible effects of HSPB2 expression on several metabolic regulators. For example, we found that HSPB2 deficiency was associated with up-regulation of CPT-1ß under basal conditions but, unexpectedly, CPT-1ß was down-regulated under cardiac stress using TAC Although the first description of the gene encoding HSPB2 expression was reported in 1998 by Suzuki and collaborators


The androgen receptor (AR) is known to play a critical role in prostate cancer (PC). p53 likely also plays a role given that p53 mutations are commonly found in advanced PC, and loss of wild-type protein function contributes to the phenotype of castration-resistant prostate cancer (CRPC). Nevertheless, the extent of the contribution of p53 dysfunction to PC remains unclear. Here we analyze the effects of p53 inhibition in PC cells and show that it has significant consequences for both the interaction between AR, and chromatin and the proliferative capacity of these cells. Inhibition of p53 expression enabled LNCaP cells to proliferate independently of androgens. Moreover, it modified the genome-wide binding pattern of AR. ChIP-sequnce analyis (ChIP-seq) revealed that fewer AR-binding sites were present in the context of p53 inhibition, suggesting that wild-type p53 is required for stable binding of AR to certain chromatin regions. Further analysis revealed that a lower AR occupancy was accompanied by a reduction in FoxA1 binding at regulatory regions of AR-dependent genes. Our study also identifies a pool of genes that may be transcriptionally regulated by AR only in the absence of p53, and that may contribute to the CRPC phenotype. Overall, our results point to p53 playing an important role in regulating AR activity across the genome. Androgen deprivation therapy is currently the standard treatment for locally advanced, recurrent, or metastatic prostate cancer (PC). However, most patients with metastatic carcinomas progress to a state of disease insensitive to androgen ablation, called castration resistant prostate cancer (CRPC) , 2. The AR is a ligand-dependent transcription factor. In the absence of androgen binding, it is expressed diffusely throughout the cytoplasm, and held in an inactive state through its association with chaperones such as heat shock proteins (HSPs). Androgen binding releases AR from HSPs, facilitating AR homodimerization and nuclear translocation. Activated AR dimers then bind to DNA at specific sequences termed “androgen-response elements” (AREs) within target-gene regulatory regions. Here they serve as a platform for the recruitment of co-activators and the basal transcriptional machinery, modulating gene expression to promote diverse biological outcomes. Transcription factors also have the ability to regulate gene expression by tethering to other response elements through protein-protein interaction with other DNA-bound factors. Thus, AR might indirectly bind to non-ARE sequences in the genome, through a tethering mechanism that involves other transcription factors such as AP-1 and Sp1 as was shown for estrogen receptor , 12. ThuCRPCs were recently reported to feature an altered expression program , accompanied by a novel AR cistrome , which iTo understand the role of p53 in so called “androgen-independent” cell growth of PC, we evaluated the effects of p53 inhibition on the genome-wide chromatin localization of AR in LNCaP. We found that in the absence of p53, the number of AR-binding peaks is significantly reduced, and also that some of those peaks are unique.To investigate the role of p53 in the proliferation of PC cells, we used LNCaP that were stably transfected with sip53 (LNsip53) or siE6 (LNsiE6), as previously described . It is wCells from each of these lines were cultured in CSS for three days, and were then either left untreated or treated with 10 nM DHT for 24 hours. As shown in Fig. in vitro DNA binding of either AR or FoxA1 between LNCaP and LNsip53, before or after DHT treatment. Likewise, FoxA1 bound to the classical response element in the TTS oligonucleotide probe in both sets of untreated cells. Pretreatment of the samples with anti-AR or anti-FoxA1 antibody, however, affected DNA-protein mobility: the application of anti-AR yielded a supershifted band; the application of anti-FoxA1 resulted in significantly diminished DNA binding. These results confirm that the binding observed was specific.Given that FoxA1 is a critical facilitator of DNA binding by nuclear receptors, we examined the binding of FoxA1 to DNA in the same cell lines. First, we carried out an electromobility shift assay (EMSA) to compare AR and FoxA1-DNA binding to oligonucleotides containing classical AREs or TTS (FoxA1 responsive element). As shown in Fig. We next carried out ChIP analysis to investigate the binding of AR and FoxA1 to the PB promoter on our luciferase reporter. To this end, we treated LNCaP-ARE-Luc control, LNsip53-ARE-Luc, C4-2B-ARE-Luc or C4-2Bsip53-ARE-Luc cells with DHT after 3 days of culture in CSS, and performed ChIP using AR or FoxA1 antibodies. We then performed PCR using primers specific to the PB region of this plasmid and quantitated the results by densitometry. As shown in Fig To determine whether eliminating wild type p53 affects the binding of AR to DNA throughout the genome, we performed ChIP-sequence analysis (ChIP-seq). We obtained 12,331,952 and 11,788,138 DNA sequence reads for LNCaP and LNsip53, respectively. These reads were mapped to the human genome (hg18) and corresponded to 4488 [3363 + (1125)] and 1822 [697 + (1125)] unique peaks, respectively Fig. . Of thesIn both cell lines, the majority of peaks were located in either the intron of a known gene or an intergenic region located more than 10 kb from the nearest gene Fig. . NotablyWe next analyzed motif enrichment in AR-bound regions of DNA, using the cis-regulatory element annotation system (CEAS) . Of the ChIP-seq or ChIP-on-ChIP analysis of LNCaP cells was previously reported , 21. We The majority of genes from the shared group had previously been reported as AR regulated. We found AR binding peaks near known AR regulated genes such as KLK3, KLK2, FKBP5, TACC2 and SLC43A1, all of which are reported to be AR-regulated. In both cell lines, AR-specific peaks were found in the enhancer region of KLK3, an upstream enhancer and an intron of FKBP5, and an intron of TACC2 and SLC43A1 Fig. . We confOf the list of LNCaP-specific genes, we examined Acetyl-CoA-carboxylase-α (ACCA) and neuropilin-1 (NRP1). In the case of AACA, we found 4 peaks within intronic regions Fig. . We veriThe NRP1 gene had one peak specific to LNCaP Fig. . We compWe next performed ChIP-PCR on LNCaP and LNsip53, with specific primers flanking these peaks. As shown in Fig. LNsip53-specific AR-binding peaks were the most intriguing group. However the height of AR binding peaks for this group was 3 times lower on average then in LNCaP and this group had a high number of false positive peaks. For example, using the USCS genome browser we could visualize LNsip53 specific peak in an intron of ASH2L Fig. , and co-3H]-thymidine uptake in those cells after treating them with DHT. As shown in Fig. 3H]-thymidine was much higher in LNsip53-Hygro cells than LNsip53-Wig-1 cells, as measured after treatment with DHT at various concentrations. We also measured cell proliferation in these cell lines by carrying out cell counts after culturing them in CSS for 11 days. As can be seen in Fig. Most notably, we found an AR binding peak downstream of Wig-1 Fig . ChIP usThe critical role of p53 in tumor development is well appreciated, but its role in PC is less clearly defined. Alterations in the p53 tumor suppressor are clearly associated with progressive disease, including “androgen-independent” growth of PCs and metastases to bone , 8, 23. Our investigation of the role of p53 in AR signaling in PC took advantage of LNsip53 cells, which had been generated in our laboratory and were described previously . In suppin vitro . Both cell lines were cultured in RPMI 1640, as previously described . To cultThe luciferase reporter vector (pARE-Luc) was provided by Dr. Katerina Gurova  and consists of a cassette containing three AREs from the rat probasin promoter followed by an Hsp70 minimal promoter; it produces almost zero background expression in AR-negative PC cells. Cell lines possessing an integrated ARE-Luc reporter construct were generated by transfecting cells with pARE-Luc and then selecting them using G418. Luciferase activity was measured using a luciferase assay kit (Promega). The values of the luciferase assay were normalized with respect to the values of the protein concentration.Western blot detection of proteins was performed as described previously , using mLNCaP or LNCaPsip53 cells were cultured in CSS for 3 days and treated with 10 nM DHT for 1 hour. Samples for Solexa ChIP sequencing (ChIP-Seq) were prepared using ChIP IT Express Magnetic Chromatin Immunoprecipitation Kit . As recommended by the manufacturer's instructions, 10 μg of DNA were used for each IP probe and incubated with 2 μg of anti-AR mAbs overnight at 4°C. Immunoprecipitations were verified by PCR with primers specific for a known AR regulatory region of the KLK3 (PSA) promoter and enhancer described in The results of ChIP-Seq were obtained using anti-AR or anti-FoxA1  antibodies as described above, followed by PCR with primers specific for AR binding regions derived after analyzing ChIP-Seq results. . These .txt files where then converted to .wig files for uploading to the UCSC genome browser for manual viewing. The .cod files output from the same process as the .bar files were converted to .bed files using the program cod2bed (supplied by cisGenome). Partek GS (Partek Inc St Louis MO) was also used for peak prediction. Eland-aligned sequences were analyzed using default settings and cutoffs. These peaks were compared to the cisGenome peaks, and in general good agreement was found. However, no statistical test was possible due to lack of replicates. The results from Partek were used to generate .bed files, which were uploaded to the cis-regulatory element annotation system (CEAS) for processing, to find enriched regions and motifs ,  was used to identify functional pathway analysis , 35. TheLNCaP and LNsip53 cells were cultured in CSS for 3 days, and were then treated with 10 nM DHT for 1 hour. Nuclear proteins were isolated using NE-Per nuclear and cytoplasmic extraction reagents , and EMSA was performed as described earlier . Double-Cells were cultured in CSS medium for 3 days and treated with 10 nM DHT for 4, 8 or 24 hours. RNA was purified using the RNeasy Mini Kit (Qiagen). Equal amounts (1 μg) of total RNA from control and treated cells were reverse transcribed using the iScript cDNA Synthesis Kit , and the cDNA (5 μL) was amplified with primers specific for selected genes (for primers used in quantitative PCR see Wig-1 from Homo sapiens (NM_022470), and cloned into the expression vector pcDNA3.1/Hygro . Lipofectamine was used for transfection, according manufactures instructions. Empty vector was used to generate control cells resistant to hygromycin.A Wig-1 was made with PCR primers based on the mRNA sequence of The proliferation assay was carried out as previously described .Statistical analysis was performed using the Student's t-test. The statistical significance was determined at p < 0.05. Columns and points in Figures