Past research has found that playing a classic prosocial video game resulted in heightened prosocial behavior when compared to a control group, whereas playing a classic violent video game had no effect. Given purported links between violent video games and poor social behavior, this result is surprising. Here our aim was to assess whether this finding may be due to the specific games used. That is, modern games are experienced differently from classic games  and it may be that playing violent video games impacts prosocial behavior only when contemporary versions are used.Experiments 1 and 2 explored the effects of playing contemporary violent, non-violent, and prosocial video games on prosocial behavior, as measured by the pen-drop task. We found that slight contextual changes in the delivery of the pen-drop task led to different rates of helping but that the type of game played had little effect. We failed to find evidence that playing video games affects prosocial behavior. Research on the effects of video game play is of significant public interest. It is therefore important that speculation be rigorously tested and findings replicated. Here we fail to substantiate conjecture that playing contemporary violent video games will lead to diminished prosocial behavior. Video games proliferate most contemporary Western cultures and are one of the most commercially consumed forms of media. Indeed, major titles in the video game category regularly outsell the most successful titles in other media formats. For example, according to US and UK sales, the highest grossing video game over five days through to the end of 2011 was Call of Duty: Modern Warfare 3 ($775 m). This eclipses the highest grossing movie over a similar period in 2010, The Dark Knight ($20 c). However, not only are video games pervasive throughout contemporary culture, they are typically violent in nature and have thus become a target of public concern. Indeed, it is common for the behavior of those perpetuating extreme acts of violence, such as that by Anders Breivik, James Holmes, and Adam Lanza, to be linked to video game play  took part in Experiment 1for course credit. Participants were mostly Caucasian (88%) with a minority reporting Asian ethnicity (12%). Participants gave written informed consent to participate in the experiment. Ethical clearance was granted by the Behavioural & Social Sciences Ethical Review Committee at the University of Queensland.Sixty-four undergraduate students  at a large metropolitan university  available at the start of the session. Grand Theft Auto was included as an exemplar of an anti-social game. Since, intent is an important component of the standard definition of aggression Call of Duty was selected as a violent control game. In Call of Duty, which is a first-person shooter game, players assumed the role of various soldiers who wield firearms and explosives, and can engage in close quarters combat. Participants played the ‘zombie’ mode, where they needed to simultaneously solve puzzles to progress through a series of rooms, while also killing zombies with a variety of guns and weapons. As previously mentioned, the violence in Call of Duty reflects a morally defensible intent to survive, or avoid death. Many games that could be considered violent employ a similar style of ‘self-defense violence’ (killing others to avoid being killed). To this end, Call of Duty served as a violent control to the deliberately anti-social content of Grand Theft Auto. Call of Duty qualified as a violent game because the zombie deaths were often quite extreme and grotesque (e.g. zombie corpses could be blown apart). We also selected the zombie mode because the gameplay was reasonably linear, meaning that each participant had a similar experience while playing the game.In World of Zoo players needed to create a successful zoo exhibit, which was achieved by taking care of animals by feeding, cleaning, and playing with them. Unlike the other games described here, World of Zoo is not explicitly marketed towards adults. It is, however, one of the few commercially available games that requires prosocial behavior and does not contain violent or adult themes.This is a non-violent puzzle game where the player used a gun that shoots entry and exit points of a portal, allowing them to access areas they would not normally be able to. This game acted as a non-violent control condition because, like Call of Duty, the player uses a gun-shaped tool to interact with the virtual world. The gun in Portal 2, however, shoots portals instead of bullets.Participants were informed that the session would comprise two unrelated studies that were bundled together in the interest of time. The first study was described as a pilot study that asked for participants’ reactions to pre-selected games so we could assess their suitability as stimuli for future experiments. Participants were randomly assigned to play one of the four previously outlined games for 20 minutes and then asked to fill out a questionnaire ostensibly aimed at assessing their experience of playing the game. This questionnaire contained questions regarding the participant’s levels of interest, frustration, and arousal experienced, and was used to test whether the games differed on factors other than the presence or absence of violent content. The questionnaire contained 12 Likert items . The second study was described as a distinct investigation of social attitudes. To this end, participants were asked to complete a series of questionnaires unrelated to the current study to reinforce the ostensible reason for the second study and to minimize suspicion about the true hypothesis. Once participants finished filling out these questionnaires the experimenter said he had to rush to the other side of campus for an ostensible appointment he had forgotten, and that he would debrief the participant via email. The experimenter gathered his belongings, namely some folders, some pens balanced on the folders, and a cup of coffee, all to emphasize that he was fully laden. He then opened the door and, as the participant moved past the experimenter, ‘accidentally’ tipped the folders such that the pens fell to the floor, muttering under his breath as he did so. As per Greitemeyer and Osswald p = .220, two-tailed) and the games without violence . Since there was no difference between similar games, and in order to increase power, we then collapsed the conditions into two broad categories (Violence-Present vs. Violence-Absent) and calculated a Chi-Square. The test on the collapsed data found no difference in prosocial behavior between Violence-Present games and the Violence-Absent games, χ2  = 0.00, p = 1.000, V = 0.00.For all experiments we report whether the self-report measures differed according to levels of the video game type variable, and follow up significant differences with hierarchical logistic regressions to determine if the self-report measures predict variance above and beyond video game type.F = 3.72, p = .016. Follow-up analyses showed that the violent game was significantly more frustrating  than the non-violent game  and the prosocial game , ps<.010,but not the anti-social game , p = .126.In experiment 1, a series of one-way ANOVAs established that our video game stimuli differed beyond presence or absence of violence. Analyses revealed a main effect of self-reported frustration, F = 13.23, p<.001. The prosocial game was significantly less arousing  than all of the other games; violent , anti-social , and non-violent , ps<.001.There was also a main effect of self-reported arousal, F = 14.96, p<.001. Participants found the non-violent game significantly more interesting  than all of the other games; violent , anti-social , and pro-social , ps<.003.Finally, the games differed on self-reported interest, R2 = .13, χ2 = 2.42, p = .491. None of the three self report measures were significantly linked to prosocial behavior, Wald tests<.88, ps>.348.We conducted a hierarchical logistic regression to determine if the self-report variables could account for additional variance in prosocial behavior above and beyond the video game manipulation. We entered the video game variable at Step 1 and the three self-report measures at Step 2. As a set of predictors, the self-report measures do not account for additional variance above and beyond the video game manipulation, Nagelkerke Greitemeyer and Osswald The prosocial game World of Zoo was marketed primarily as a children’s game and, thus, inherently differed from the other games in more ways than just the presence of violent/prosocial content (e.g. significantly less arousing), a problem acknowledged in other research In Experiment 1, participants filled out questionnaires directly after playing the game, whereas in Greitemeyer and Osswald M = 21.63, SD = 5.50). Most were Caucasian (77%), though some reported Asian ethnicity (14%), or other (9%). Participants provided written informed consent and either received course credit for participating in the experiment or a small monetary reimbursement. Ethical clearance was granted by the Behavioural & Social Sciences Ethical Review Committee at the University of Queensland.We recruited 64 undergraduate participants  from the first-year participant pool at a large metropolitan university. Participant ages ranged from 17–43  and Portal 2 (non-violent).As per Experiment 1, participants were instructed that the experimental session comprised two ostensibly unrelated studies; the first to gather participants’ opinions of pre-selected games to determine whether they would be suitable stimuli for a future experiment, and the second to pilot test various measures of social attitude. Participants were randomly allocated to play either Grand Theft Auto or Portal 2for 20 minutes. Following the game, and to keep the story of the first ostensible study believable, the participant rated the games on the same measures as in Experiment 1. This was the only task the participant completed between the video game and pen-drop task, taking no more than 60 seconds (12 items). We deemed this short questionnaire necessary for the cover-story of the first study.We then manipulated the context in which the pen-drop task was administered; having it follow the video game prime and ending the session there (Session-Ends), or following the video game prime but with the session continuing into the second ostensible experiment (Session-Continues).In the Session-Ends condition, following the participant completing the ostensible first study, the experimenter ‘realized’ that he forgot to bring the materials for the second study and would have to end the experimental session early. As in Experiment 1, the experimenter gathered his belongings, making him appear sufficiently laden, and opened the door for the participant. The experimenter dropped the pens as the participant moved past him, waiting for the participant to help gather the pens. Participants were then probed for suspicion and debriefed.In the Session-Continues condition, participants finished the questionnaire for the ostensible first study and handed it to the experimenter. The experimenter then reached for the materials for the ostensible second study, knocking over a tin of pens placed at the end of a table, equidistant from both the experimenter and participant. The experimenter waited for the participant’s reaction; did they pick up the pens or not? Once the pens had been gathered the experimenter began the second study. Participants completed the second study, were probed for suspicion, and then debriefed.χ2  = 12.30, p<.001, V = .438.R2 = .03, χ2 = 0.59, p = .444 or the Session-Continues condition , Nagelkerke R2 = .12, χ2  = 2.76, p = .096.Within each level of the timing variable we conducted logistic regressions to see whether the rate of helping differed according to the game played. The type of video game played did not have an effect in either the Session-Ends condition , Nagelkerke t(62) = 0.13, p = .895), arousal (t(62) = 1.04, p = .300), or interest (t(62) = 0.50, p = .617).We ran t-tests to determine if the video games were experienced differently. Between the two games, we found no differences in terms of frustration . There was, however, a main effect of the context in which the pen-drop was administered: rate of helping was significantly higher in the Session-Continues condition (75%) than in the Session-Ends condition (31%).χ2  = 0.80, p = .777, V = .035.In order to account for possible lack of power given the relatively small cell sizes used here and in Experiment 1, we collapsed the Session-Ends data with the anti-social and non-violent data from Experiment 1. Despite doubling participant numbers, differences in rates of helping between anti-social and non-violent conditions remained non-significant, Experiment 2 was designed to evaluate whether manipulating the contextual administration of the pen-drop task would help reveal an effect of playing an anti-social game. While subtle contextual differences in the administration of the pen-drop task are able to move base-rates of helping behavior, they were not sufficient for revealing the anticipated violent video game effect. Given the failure to show an effect of violent video games, it was necessary to attempt a procedural replication of Greitemeyer and Osswald In Experiments 1 and 2 we failed to find any effect of playing a violent video game on prosocial behavior. It is conceivable that the motive for violence in classic games is much less ambiguous than contemporary games . Thus, we decided to replicate Greitemeyer and Osswald We recruited 32 undergraduate participants  from the first-year participant pool at a large metropolitan university. Participant ages ranged from 17–26 . Again, most were Caucasian (81%), with small minorities reporting Asian ethnicity (16%), or other (3%). Participants provided written informed consent and received course credit for participating in the experiment. Ethical clearance was granted by the Behavioural & Social Sciences Ethical Review Committee at the University of Queensland.Following Greitemeyer and Osswald The general prosocial aim of this game is to prevent a colony of lemmings from mindlessly marching over cliff edges or into hazards by assigning them useful roles . This gets progressively more taxing as players proceed through the increasingly difficult levels. A player’s score is determined by how many lemmings they save from mindless self-death.Lamers is a violent parody of Lemmings, with the goal being to kill as many of the characters as possible before they reach their goal. Players have various weapons at their disposal, including guns and explosives.Following Greitemeyer and Osswald χ2  = 0.53, p = .716, V = .129.Here we adopted the exact protocol reported by Greitemeyer and Osswald t(30) = 0.88, p = .387), arousal (t(30) = 1.10, p = .282), or interest (t(30) = 0.80, p = .430).Again, we conducted a series of t-tests to determine whether our video game stimuli differed on potential variables of interest. As with Experiment 2, we found no difference between the video game stimuli on frustration . We find this an unlikely reason for our failure to replicate, however, given that past research, including Greitemeyer and Osswald Other criticisms could be aimed at our choice of stimuli. First, we only selected single exemplars of contemporary video games to represent each game category. Other studies use multiple exemplars or even pilot test multiple exemplars, finally settling on two that score similarly on experience measures  is most strongly affected by violent video game habits (mediated by decreased empathic concern)


Alcohol misuse has been linked to intimate partner violence (IPV). However, this association is not usually examined in Russia. Moreover, more investigation is required as to whether specific drinking contexts are also associated with IPV. The objectives of this study are: to investigate whether alcohol misuse is associated with IPV and to further examine whether specific drinking contexts among drinkers are associated with IPV.A questionnaire was used to collect information on demographics, health status, alcohol use, and violence involving sexual partners among 440 participants who were recruited from an STI  clinic center in St. Petersburg, Russia for a cross-sectional study from 2008 to 2009. Multivariate logistic regression was used for analysis.Overall, 47.0% participants were classified as misusing alcohol and 7.2% participants perpetrated IPV in the past three months. Participants with alcohol misuse were 3.28 times  as likely as those without alcohol misuse to perpetrate IPV. Among participants who had consumed alcohol in the past three months, those who usually drank on the streets or in parks  were more likely to perpetrate IPV.Both alcohol misuse and certain drinking contexts  were associated with IPV. The association between drinking contexts and IPV needs further investigation, as do the underlying mechanisms for this association. IPV prevention initiatives might benefit from reducing alcohol misuse. Drinking contexts such as drinking on the streets or at parks as well as the factors related to the use of alcohol in these contexts may also need to be addressed. Alcohol consumption, particularly at hazardous drinking levels, is highly prevalent in Russia. The estimates based on 2001-2003 data revealed that, on average, each Russian aged 15 years and older consumed 15.2 L of pure ethanol alcohol per year, among the highest rates in the world [Alcohol misuse has also been linked to intimate partner violence (IPV) in both experimental and empirical studies ,5. In a A limited number of studies on IPV in Russia demonstrated that IPV is common in the country -10. A suThe association between alcohol misuse and IPV is much less investigated in Russia, particularly in comparison to countries in North America and Western Europe . MoreoveThe examination of drinking context and its association with IPV in Russia is also important because it may provide valuable information for IPV intervention programs. Thus, we conducted a study to investigate whether alcohol misuse is associated with IPV among participants from a sexually transmitted infection (STI) clinic in St. Petersburg, Russia. As a secondary aim, we examined whether certain types of drinking settings are associated with IPV among alcohol users.From June 2008 to June 2009, a cross-sectional study was conducted among patients who visited a public STI clinic in St. Petersburg, Russia. The city of St. Petersburg is comprised of 18 districts, nearly all containing a public STI outpatient clinic . The current study was conducted at the genitourinary department of the Kalininsky clinic. The clinic provides services free of charge or for a nominal fee to patients, the majority of whom are local residents. Consecutive adult patients aged 18 years and older who reported genitourinary complaints or had a need for STI-related services  were invited to participate in the study. In total, 500 patients were eligible; 440 of them agreed to participate and completed a self-administered questionnaire. The study was approved by the institutional review boards of the Biomedical Center in St. Petersburg, Russia and Yale University in Connecticut, USA.The questionnaire contained 58 items and took about 15 minutes to complete. Demographics, alcohol use and behaviors towards sex partners were collected in the questionnaire. Demographic variables included age, sex, education, employment status, marital status and monthly income. Alcohol use information included frequency of alcohol use, frequency of having six or more drinks on one occasion as well as drinking contexts . Behaviors towards sex partners included history (lifetime and in the past three months) of insulting, swearing at or threatening a sexual partner, history of pushing, grabbing, slapping, punching, beating up or choking a sexual partner, and history of physically forcing a sexual partner to have sex or forcing a sexual partner to do something sexually that he/she did not want to.Using a modified version of the three-question based Alcohol Use Disorders Identification Test (AUDIT-C), participants were asked the following three questions:1) How often have you had a drink containing alcohol in the past three months?2) On average, how many servings of drinks containing alcohol do you typically consume on a day when you are drinking?3) How often, in the past three months, did you have six or more drinks on one occasion?Each option for each question has been allotted a score between zero and four and thus the score range of AUDIT-C is between zero and 12. A score of four or more in men and three or more in women were considered positive . AUDIT-C has been widely accepted as a practical and valid screening test for alcohol misuse ,15.Participants were also asked three drinking context questions. The three questions and responses were as follows: "Which alcohol beverages do you usually drink ?" The possible responses were beer, gin and tonic or other alcoholic cocktails, wine/champagne, liquor/sherry/vermouth, vodka, brandy and other strong beverages, and other. "At what venue do you usually drink ?" The possible responses were home, bar, restaurant, night club, friend's place (party), street/park, staircase, other, and anywhere. "With whom do you drink most often?" The possible responses were friends, spouse, parents or other relatives, colleagues, sexual partner(s), alone, other, and with anyone.The measurement of IPV was based on the revised version of the Conflict Tactics Scale . In the Logistic regression models were used to determine if alcohol misuse was associated with IPV among all participants. Among the subgroup of participants who had drunk alcohol in the past three months, we examined whether drinking contexts were associated with IPV. If two or more drinking contexts were found to be significantly associated with IPV, these variables were later included in the same model to determine if each drinking context might still have an independent effect on IPV. The significance level was defined as p < 0.05, and SAS software  was used to analyze the data.Among the 440 patients who participated in the study, 286 (65.0%) were men and 154 (35.0%) were women  and drinking in the street or park  were significantly associated with IPV. These ORs were adjusted for significant covariates including alcohol misuse, marriage status and monthly income. Age, gender, education and employment status were not independently significantly associated with IPV among drinkers.Considering that these two drinking context variables (drinking liquor/sherry/vermouth and drinking in the street or park) may confound each other, we examined their associations with IPV in the same model, further adjusting for AUDIT-C, marriage status and monthly income Table . We founAlthough violence against women has been pervasive for a long time in Russian culture , controvIn the present study, we found that alcohol misuse was significantly associated with IPV among patients attending an STI clinic in St. Petersburg, Russia, which is consistent with results from previous studies conducted in Russia ,19,20 anThe observed association is of concern given the high level of alcohol misuse among both Russian men and women. Although the prevalence of alcohol misuse observed in the present study is lower than that observed in another study conducted in Arkhangelsk, Russia by using AUDIT criteria , it remaA striking finding of this study is that participants who usually drank on the street or at parks were more likely to perpetrate IPV than those without this preference. It should be noted that this association was observed after controlling for important confounders in the present study such as alcohol misuse, marital status, and monthly income. Previous studies have reported that drinking at bars is independently associated with violence ,29. For An alternative explanation for the association between drinking on the streets or at parks and IPV is that drinking on the streets or at parks may have a distal influence on IPV. That is to say, certain factors underlying this drinking preference may be a risk for IPV. For example, those who usually drink on the street or in parks may share similar cultural environments in which IPV may be more common compared to those who drink at other locations. It has been reported that in some cultures, drinking may serve as an excuse for IPV . If thisThe study has several limitations. First, the prevalence of IPV in the past three months might be underestimated because self-reported IPV measurements are likely to be affected by a social desirability bias. To minimize such bias, self-administered questionnaires were used to collect information. Second, the method used to identify significant drinking contexts may increase the chances of detecting a significant association because a series of logistic regression models were conducted to examine the relationship between different drinking contexts and IPV. Third, the confidence interval for the association between drinking context and IPV is relatively wide; this suggests that future studies with larger sample sizes may be needed to better understand such correlations. Fourth, the results may not be generalized to other populations or clinical settings. Fifth, this is a cross-sectional study and thus a causal relationship cannot be established.The present results demonstrate that both alcohol misuse and certain drinking contexts  are associated with IPV. Future research needs to investigate whether drinking on the street or in parks represents contexts that facilitate IPV or whether this depicts a sub-group of individuals at risk for IPV. IPV prevention initiatives might benefit from the reduction of alcohol misuse. This might include targeting contextual levels of drinking or addressing factors that are associated with drinking on the street or in parks.The authors declare that they have no competing interests.NA: main contributor to conception, statistic analyses and writing. WZ: contributed to data analysis, interpretation, and drafting this manuscript. AS, contributed to data acquisition, coordination and management, as well as quality control and interpretation of data. RS, contributed to data acquisition, coordination and management, as well as quality control and interpretation of data. AK, contributed to conception and review of the manuscript. TK, contributed to data acquisition, quality control and interpretation and management of data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/11/629/prepub


Recent technological advances have enabled investigators to characterize the molecular genetics and genomics of hepatic neoplasia in remarkable detail. From these studies, an increasing number of molecular markers are being identified that correlate with clinically important tumor phenotypes. This paper discusses current knowledge relevant to the molecular classification of epithelial primary hepatic tumors that arise in adults, including focal nodular hyperplasia (FNH), hepatocellular adenoma (HCA), hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), and combined HCC-CC. Genetic analysis has defined molecular subtypes of HCA that are clinicopathologically distinct and can be distinguished through immunohistochemistry. Gene expression studies have identified molecular signatures of progression from dysplastic nodules (DNs) to early HCC in cirrhosis. Analyses of the mutational spectra, chromosomal aberrations and instability, transcriptomics, and microRNA profiles of HCC have revealed the existence of biologically distinct subtypes of this common malignancy, with prognostic implications. Molecular characterization of biliary and hepatic progenitor cell phenotypes in liver cancer has shed new light on the histogenesis of these tumors and has focused attention on novel therapeutic targets. In coming years, the molecular classification of hepatic neoplasms will be increasingly valuable for guiding patient care, as targeted therapies for liver cancer are developed and brought into clinical practice. Over the past decade, tremendous advances have been made in the technologies available for characterizing the molecular genetics, genomics and epigenetics of neoplasia. These advances have greatly accelerated basic research aimed at elucidating the molecular mechanisms underlying tumorigenesis. In addition, they have led to the identification of molecular markers that correlate with important biological characteristics of tumors. Such markers are increasingly valuable in clinical practice as tools for facilitating the diagnosis and categorization of tumors, determining their aggressiveness and, in some cases, predicting their responses to particular forms of therapy. The ideal classification system for any group of neoplasms would be based on our understanding of their ontogeny and would integrate histologic features with molecular data in a clinically meaningful way. In this paper, we will discuss the molecular biology of primary hepatic tumors, with particular emphasis on the roles of molecular characterization in clinical practice. We will focus on primary hepatic neoplasms that arise in adults, including hepatocellular adenoma (HCA), hepatocellular carcinoma (HCC), cholangiocarcinoma (CC), and the rare entity, combined hepatocellular and cholangiocarcinoma (HCC-CC). Focal nodular hyperplasia (FNH), although thought to be a nonneoplastic lesion, is often considered in the differential diagnosis of other hepatocytic tumors and, therefore, has also been included in this discussion. However, consideration of mesenchymal, hematopoietic and childhood liver tumors is beyond the scope of this review.FNH is the most common of the benign hepatocellular tumors, arising in approximately 3% of adults , 2. AlthFocal nodular hyperplasia (FNH) is a common, benign hepatocellular lesion that is believed to arise in response to localized hyperperfusion of the liver parenchyma, typically in association with a microscopic arterial malformation , 7. FNH Traditionally, FNH has been considered nonneoplastic and, accordingly, it is thought to have no malignant potential. However, molecular analyses of the clonality of FNH have yielded conflicting results, with some studies finding that up to 50% show X chromosome inactivation patterns suggestive of monoclonality , 8. In gβ-catenin, HNF1α, or p53 genes in classic FNH [Given the uncertainty surrounding the issue of neoplasia in FNH, several investigators have searched for genetic alterations that might define these lesions. If a genetic abnormality characteristic of FNH were found, it would greatly support its neoplastic nature, and the finding could be diagnostically useful in clinical practice. However, the results of such studies thus far have been uniformly negative, with no mutations detected in the APC, axin, ssic FNH , 10–12.Both FNH and HCA consist predominantly of well-differentiated hepatocytes. In FNH, there are intervening fibrous bands that radiate from a central scar and contain abundant, proliferating bile ductules. In contrast, HCA have only rare bile ductules, if any, and typically show much less fibrosis. However, the regions of fibrous scar in FNH are variable in abundance and are not always adequately sampled in needle biopsies, sometimes making the histologic distinction from HCA difficult. We have found that the expression patterns of biliary markers, as determined by immunohistochemistry, are frequently different in FNH and HCA, not only within regions containing bile ductules, but also in hepatocellular areas . Antibodβ-catenin activation are expanded [β-catenin gene occur in a subset of HCA. In adenomas with β-catenin mutation, GS is diffusely expressed, whereas in adenomas with normal β-catenin, GS is absent in large areas of the lesion. Unfortunately, however, in the latter type of HCA, irregular and/or centrizonal expression of GS can be present focally, particularly at the periphery of the tumor [Immunohistochemical staining for the cytoplasmic enzyme, glutamine synthetase (GS), has also been proposed as a technique for differentiating between FNH and HCA . GS is aexpanded , and thiexpanded . In HCA he tumor . TherefoFirst described a half-century ago, HCA is a rare, benign liver tumor that most commonly occurs in women and has been associated with oral contraceptive use . It is aIn 2002, Bluteau et al. published the results of a genome-wide search for tumor suppressor genes in HCA . Microsaβ-catenin gene in a fraction of these tumors. Earlier work by many independent groups had implicated Wnt pathway activation in liver carcinogenesis, and β-catenin mutations have been found in a significant percentage of HCC. To investigate the possibility that aberrant β-catenin signaling plays a role in very early hepatic neoplasia, Chen et al. performed directed LOH analysis and genomic sequencing of several Wnt pathway genes [β-catenin gene, and to express truncated forms of the protein that are predicted to be constitutively activated.Shortly after genetic alterations of HNF1 in HCA were reported, a group in Taiwan published their discovery of activating mutations of the ay genes . Three oβ-catenin mutation status as well as genotype-phenotype correlations in these tumors [β-catenin mutation and HNF1 inactivation were mutually exclusive in these tumors, indicating the existence of at least two distinct, nonoverlapping molecular subtypes of HCA.The French group that had originally identified HNF1 inactivation in HCA next expanded their study to include assessment of e tumors . In a coIn the 1990s, a class of histologically unusual hepatic tumors was described and termed “telangiectatic FNH” (T-FNH) , 20. Theβ-catenin were further subdivided into two groups on the basis of the presence or absence of inflammatory infiltrates. The subgroup showing inflammation, named inflammatory HCA (I-HCA), was found to include all lesions formerly classified as T-FNH. Thus, four subtypes of HCA were proposed: (1) HNF1-inactivated HCA (H-HCA), (2) β-catenin-mutated HCA (β-HCA), (3) I-HCA, and (4) nonmutated, noninflammatory HCA [β-catenin mutation was discovered to occur in a fraction of I-HCA as well as in noninflammatory HCA [In the genotype-phenotype study described above, HCA that were found not to harbor genetic alterations in either HNF1 or tory HCA . A subsetory HCA .The hypothesis that I-HCA represents a biologically distinct entity was greatly strengthened with the finding of a marked overexpression of acute-phase reactants by the hepatocytes of these lesions . At bothβ-catenin mutations are sometimes found [In summary, HCA can be assigned to one of four categories . The fires found . This tyβ-catenin but are noninflammatory; it has been reported that approximately 10% of adenomas belong to this β-HCA subtype. Beta-catenin activation is most easily assessed through immunostaining for glutamine synthetase, which is diffusely overexpressed in these lesions. Alternatively, immunohistochemical staining for β-catenin itself may be used to distinguish between tumors without mutation, in which β-catenin has a membranous localization, and those with mutation, in which an aberrant, activated form of β-catenin accumulates within nuclei. In both I-HCA and β-HCA, β-catenin mutation has been associated with a high risk of malignancy. These tumors are common in men, and they often show features, such as pseudoacini and hepatocellular dysplasia, that are frequent in HCC. In fact, because β-HCA may be histologically indistinguishable from well-differentiated HCC [β-HCA in a collection of 41 adenomas subjected to molecular analysis [β-catenin mutation in association with morphologic features suspicious for malignancy should warrant a pathologic diagnosis of either outright or suspected hepatocellular carcinoma, rather than HCA. This is an area in need of further research to determine the molecular differences, if any exist, between β-HCA and HCC. Such findings would help to elucidate the pathogenesis of HCC and might serve as the basis for a diagnostic test of malignancy in histologically equivocal cases.The third category of HCA is somewhat controversial and consists of lesions that harbor activating mutations of ated HCC , their eanalysis . We feelβ-catenin, or gp130. This is the smallest group of HCA, accounting for only about 5% of the total. Lesions that cannot be fully evaluated due to extensive necrosis and/or hemorrhage are also classified in this group. Whether these noninflammatory, nonmutant tumors represent a truly distinct subtype of HCA, or whether they are biologically related to one or more of the other three subtypes, is an open question that awaits further investigation.The fourth and final category of HCA are those that are noninflammatory (negative for acute phase markers) and do not harbor mutations in HNF1, HCC almost always arises in the context of chronic liver disease, usually in patients with cirrhosis. Cirrhosis of any etiology constitutes the predominant risk factor for HCC. Worldwide, infection with hepatitis B virus (HBV) accounts for the majority of cases, whereas in the United States, chronic hepatitis C is the most common predisposing factor. HCC is often diagnosed late in the course of disease, but in developed countries, surveillance programs that use improved radiologic techniques for monitoring liver lesions in patients with cirrhosis are leading to increased rates of early HCC detection.It is now well accepted that in cirrhotic livers, the vast majority of HCC arise from benign precursor lesions called dysplastic nodules (DN) . AlthougSeveral groups have used gene expression profiling to identify “molecular signatures” that can accurately distinguish between DN and early HCC –37. PerhThe gold standard for diagnosis of DN and early HCCs is still histology; in fact, internationally agreed upon histologic definitions of these lesions and a standardized nomenclature have only recently been formulated . This moMany immunohistochemical markers have been assessed individually, but what has emerged as most useful currently is a panel of three markers: GPC3, GS, and HSP-70. Glypican-3 was first identified as a gene whose mRNA is frequently expressed in HCC but not in benign, adult liver, HCA or CC . Numerouβ-catenin signaling [In an effort to augment the sensitivity of GPC3 immunostaining as a molecular test for HCC, and to retain its specificity in distinguishing DN from malignant lesions, Di Tommaso et al. examined the value of adding other tumor markers to the analysis . Heat-shignaling , as discignaling . When thignaling . This reAlthough immunohistochemical staining for the GPC3/HSP70/GS panel is currently recommended as the best ancillary technique to aid in the diagnosis of early HCC in difficult needle biopsy specimens , the seaThe overall incidence of HCCs in patients without cirrhosis is difficult to estimate and varies with geography and the prevalence of risk factors in the population , 47. MosHepatocellular carcinomas that arise in the absence of chronic liver disease or known risk factors for malignancy are almost always a histologic variant known as fibrolamellar HCC (FL-HCC). The great majority of these tumors occur in patients under the age of 35, and they display a characteristic histology, with nests of large, oncocytic, malignant hepatocytes surrounded by thick bands of layered fibrosis. Although the rarity of these tumors has hampered efforts to elucidate their molecular pathogenesis, FL-HCCs have been found to contain unique molecular alterations that distinguish them from the more common forms of HCCs (reviewed in ). The diHepatocellular carcinoma has long been known to display extraordinary genetic complexity and molecular heterogeneity. However, it is not clear which of the many genomic, genetic, and epigenetic alterations found in HCCs are the most critical in driving its molecular evolution and/or defining its biological behavior. For this reason, there is currently no molecular subclassification of HCCs that is widely accepted and routinely implemented in clinical practice. Nonetheless, a host of molecular genetic markers have been found to correlate with clinical parameters, and in some instances, to have independent prognostic value in particular circumstances.Although in recent years, surveillance of those at high risk of hepatic malignancy has initiated a trend toward earlier diagnosis of liver cancer, a substantial majority of HCC patients still present with locally advanced or metastatic disease . Even amHCCs generally show a high level of chromosomal instability, and this is a phenotype that is acquired early in the process of carcinogenesis . Thus, cAlthough high-resolution mapping of chromosomal alterations in cancer is valuable mostly as a research tool, a group in Japan has demonstrated that HCCs can be subclassified into distinct, clinically meaningful groups based solely on their patterns of chromosomal alterations . In thisThe quantitative assessment of genomic alterations as an indicator of tumor aggressiveness has proven useful in predicting the risk of HCC recurrence after liver transplantation. In an effort to identify molecular markers of recurrence risk in HCC patients who had undergone transplantation, Marsh et al. analyzed explanted tumors for loss of heterozygosity (LOH) of microsatellite markers at nine tumor suppressor gene loci . They foβ-catenin (CTNNB1), which is mutated in approximately 30% of HCC, is the oncogene most frequently activated in this cancer. HCC bearing β-catenin mutations are more likely than their nonmutant counterparts to show chromosome stability, an absence of HBV infection [β-catenin mutation in HCC is associated with favorable or unfavorable prognosis [HCCs have been extensively analyzed for the detection of somatic mutations affecting known oncogenes and tumor suppressors (reviewed in ). The genfection , a well-nfection . Howeverrognosis , 68. In rognosis . In the rognosis .A large number of studies have examined global gene expression patterns in HCCs using DNA microarray technology . ThIn 2004, a group led by Dr. Thorgeirsson at the National Cancer Institute published the results of a genome-wide expression study of 91 HCCs showing that the tumors fell into two subclasses with distinct mRNA expression profiles and dramatically different patient survival . The pooThe existence of two broad categories of HCCs, one with and the other without a “high-proliferation” gene expression signature, has been confirmed in multiple, independent studies . Most haα-fetoprotein (AFP) status (positive or negative for elevated serum AFP) was shown to define four prognostic categories, each with a characteristic gene expression pattern. Importantly, this result was replicated in three independent cohorts of HCC , in one of which the EpCAM analysis was performed by immunohistochemistry, rather than mRNA hybridization. Thus, the EpCAM-AFP classification system has the potential for easy application in clinical practice. It is not yet clear whether the stratification of AFP-positive cases according to EpCAM expression will be clinically useful as a prognostic indicator, since AFP positivity is already well-known to be associated with aggressiveness in HCC [β-catenin pathway inhibitors, and/or stem-cell targeted agents such as anti-EpCAM antibodies [The epithelial cell adhesion molecule, EpCAM, is a known marker of fetal hepatoblasts and adult hepatic progenitor cells as well as biliary epithelium. In 2004, Kim et al. performed microarray studies of cirrhotic liver and found that EpCAM is dramatically overexpressed in premalignant lesions as well as in a subset of HCC . Moleculs in HCC . Howevers in HCC , Wnt/β-ctibodies .Gene expression profiling has yielded novel insights into the biological heterogeneity of HCCs and has particularly highlighted the importance of hepatic progenitor cell/stem-cell characteristics in defining its aggressiveness. In addition, these studies have focused attention on the roles of “cancer stem-cells” in HCCs . HoweverMicroRNAs (miRNAs) are short, noncoding RNA molecules that regulate gene expression by binding to specific messenger RNAs and preventing their translation into protein. Because each type of miRNA is able to downregulate hundreds of genes at a time, miRNAs often control entire transcriptional programs that determine fundamental cellular properties and behavior. Accordingly, miRNA profiling has emerged as an extremely valuable method for phenotyping and subclassifying tumors . CompareMany independent groups have conducted comprehensive analyses of miRNAs in HCC, and a plethora of informative miRNA markers have been identified. Many of these miRNA signatures correlate with important biological parameters, such as metastasis –83, diffIt has been shown recently that the reduced expression (silencing) of miRNA-26 in HCC tumors of male patients is associated both with poor survival and response to interferon-alpha adjuvant therapy . InteresCholangiocarcinoma (CC), defined as carcinoma of biliary-type epithelium, can arise in the liver parenchyma or anywhere along the extrahepatic biliary tract. The extrahepatic type (which includes “Klatskin tumors” of the liver hilum) is much more frequent, accounting for up to 90% of CC cases . The hisCC generally presents at a late stage, when curative resection is no longer possible. Unfortunately, this malignancy is highly aggressive and resistant to standard chemotherapeutic regimens. Molecular characterization of CC is in the early investigative stages (reviewed in ), and usper se. The fact that many HPC markers are also expressed by biliary epithelium and the recent findings on progenitor cell marker expression by HCC greatly complicate this field. Thus, although there is evidence that HCC-CC arises from a bipotential hepatic progenitor cell, the histogenesis of this rare neoplasm is still debated (Combined HCC-CC is a rare primary liver tumor characterized by the intimate intermingling of histologically unequivocal HCC and CC components . Most ca debated .Only a few studies have examined the molecular characteristics of HCC-CC. Zucman-Rossi's group in France used microsatellite-based LOH analysis to assess chromosome stability in 9 CC, 15 HCC-CC, and 3 HCC/CC collision tumors . CombineIn recent years, remarkable progress has been made in elucidating the molecular pathology of hepatic tumors. In the area of benign hepatocellular lesions, advances in our understanding of molecular subtypes have led to a new classification system of HCA, with important genotype-phenotype correlations and easy application to routine clinical practice. In the field of early hepatic neoplasia, microarray studies of gene expression have identified simple molecular signatures that can distinguish dysplastic nodules from early HCCs in a background of cirrhosis. Although such expression signatures can be determined by quantitative RT-PCR, the use of immunohistochemistry, which allows the simultaneous assessment of histology and protein expression, is preferred in needle biopsies. Molecular measures of prognosis in patients with HCCs and CC are emerging; in retrospective studies, a variety of molecular techniques have successfully identified subgroups of HCCs with distinct clinical associations, such as prognosis, risk of recurrence after attempted curative treatment, and response to adjuvant interferon therapy. Although these assays are not yet developed for widespread clinical application, their refinement and use for the stratification of patients undergoing treatment in clinical trials will lead to further advances in therapeutic options for liver cancer. The current system of classifying hepatic tumors based on histologic architecture and patterns of differentiation has many limitations. Molecular studies have supported a model of hepatic tumorigenesis in which neoplasms recapitulate various lineages and stages of hepatic progenitor cell differentiation. These discoveries are likely to have an important impact on liver tumor classification and to lead to a newer classification system, based on histologic and molecular features, with improved prognostic and therapeutic significance.


Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT. Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini The p53 upregulated modulator of apoptosis (PUMA), a p53 target, is necessary for stress-induced apoptosis The immortalized MCF10A cell line was obtained from American Type Culture Collection  and cultured as previously described Growth factor-reduced Matrigel was purchased from BD Transduction Laboratories . DMEM/F12 medium, donor horse serum, To-Pro-3 nucleus dye, and anti-mouse antibody conjugated to fluorophore 488 were purchased from Invitrogen . Hydrocortisone, insulin and cholera toxin were purchased from Sigma . Recombinant human epidermal growth factor (EGF) was purchased from Peprotech . Normal goat serum was purchased from Jackson ImmunoResearch .To produce p21 or PUMA shRNA under the control of the U6 promoter, two 62-base oligos were annealed and then cloned into pBabe-U6 shRNA expression vector To generate MCF10A cell lines with stable knockdown of ΔNp73 in combination with p21 or PUMA, the following plasmids, pBabe-U6-shΔNp73, pBabe-U6-shp21 or pBabe-U6-shPUMA, were co-transfected into MCF10A cells. The resulting knockdown cell lines were selected with puromycin and confirmed by RT-PCR and/or Western blot analysis. The shRNA oligos used are listed with the siRNA targeting region shown in uppercase .Actin gene was chosen as a loading control and detected with primers 5′-ctgaagtaccccatcgagcacggca-3′ (sense) and 5′-ggatagcacagcctggatagcaacg-3′ (antisense).Total RNA was extracted from cells using TRIzol  according to manufacturer’s instructions. cDNA was synthesized using M-MLV Reverse Transcriptase Kit (Promega Corporation) according to manufacturer’s protocol. The primers to detect ΔNp73 are sense 5′-gatccatgccctcgtcccac-3′ and antisense 5′-ctgctcatctggtccatgg-3′. The Western blotting was performed as described 2HPO4, 3.5 mM NaH2PO4, 0.1% BSA, 0.2% Triton X-100 and 0.05% Tween-20) containing 10% normal goat serum for 2 h and further blocked in a secondary blocking buffer B  for 1 h. The structures were incubated overnight with primary antibodies at 4°C, washed thoroughly with buffer A with gentle shaking, and stained with FITC-conjugated secondary antibody (diluted 1∶200 in buffer A) for 1 h. The structures were nuclear stained with 5 µg/mL of To-Pro-3 in PBS for 15 min at room temperature. The To-Pro-3 stain was removed by washing the chamber slide with PBS for 5 min and then the slide was mounted under a glass cover slip with 0.1% para-phenylenediamine D (PPD) and 90% glycerol in PBS. Microscopic analysis was performed using a confocal microscopy system  and images were acquired using the software for Carl Zeiss Laser Scanning Microscope (LSM-510). The images of acinus structures were captured by the Z-stacking function for serial confocal sectioning at 2 µm intervals. Experiments were conducted in triplicate.3-D structures in Matrigel were fixed in 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.5% Triton-X100 in PBS for 30 min at 4°C. After quenching with 100 mM glycine in PBS, the structures were pre-blocked in a primary blocking buffer A  followed by staining with 0.1% crystal violet. Experiments were performed in triplicate.For wound healing assay, cells were grown in a 6-well plate for 24 h. The monolayers were wounded by scraping with a P200 micropipette tip and washed two times with PBS. At specified time points after the scraping, cell monolayers were photographed using a Canon EOS 40D digital camera . Migration rate of cells was measured by averaging the time required to close the borders of cells. Six regions were analyzed in each well, and the result was expressed as the mean ± SD. Experiments were conducted in triplicate.Data were presented as Mean ± SD. Statistical significance was determined by Student’s t test. Values of P<0.05 were considered significant.in vivoMCF10A cells in 3-D culture undergo various biological events, such as apoptosis, proliferative suppression, polarization and cell adhesion, to form an acinus structure with a hollow lumen similar to the normal acinus Next, to examine the role of p21 in mammary morphogenesis, we generated multiple MCF10A cell lines in which p21 was stably knocked down by shRNA , a, irreThe above observations suggest that lack of PUMA or p21 leads to partial lumen clearance but is not sufficient to completely disrupt morphogenesis of MCF10A cells. Thus, we explored whether PUMA cooperates with p21 to regulate morphogenesis of MCF10A cells. To test this, we generated MCF10A cell lines in which both PUMA and p21 were stably knocked down , a and iLoss of cell polarity induces EMT Here, we found that PUMA-KD or p21-KD led to irregular acinus-like structures with filled lumen –3. PreviNext, we tested whether ΔNp73 counters the effect of PUMA-KD or p21-KD on acinus formation. We found that MCF10A cells with Np73&PUMA-KD exhibited normal cobble-stone-like epithelial cell morphology in 2-D culture , a and fNext, we examined whether ΔNp73-KD counters the effect of PUMA-KD or p21-KD on EMT. We found that the expression levels for E-cadherin were nearly restored to near-normal levels, whereas the levels for β-catenin and laminin V were decreased, in MCF10A cells upon knockdown of ΔNp73&PUMA or ΔNp73&p21 as compared to the cells upon knockdown of p21 or PUMA alone . In addiMammary gland is an intact, well-ordered architecture. One event during mammary morphogenesis is acinus formation. This process starts with establishment of epithelial cell polarity, which then modulates cell proliferation and apoptosis required for acinus maturation and lumen formation EMT plays an important role in embryogenesis and development. During EMT, epithelial cells lose their epithelial features and acquire a fibroblast-like morphology, accompanied with up-regulation of mesenchymal markers and enhancement of migratory properties, contributing to pathological processes such as fibrosis and cancer ΔN isoform of p73 possesses a dominant negative activity towards TAp73 and possibly p53


Since there is evidence that Mg2+-deficient groups were allowed to freely access a 0.005% Mg2+-containing diet while control mice were fed a normal, 0.2% Mg2+-containing diet. The emotional behaviour of Mg2+-deficient mice was assessed in a battery of anxiety tests including the open field test, the light/dark test, the stress-induced hypothermia test, and the hyponeophagia test. Markers of HPA axis function including CRH gene expression and plasma ACTH levels were quantified. Neuronal activation patterns in the HPA system were investigated using mapping of the immediate early gene c-Fos as a marker of neuronal activation in response to an anxiety-provoking situation.Mice assigned to Mg2+-deficient mice did indeed display enhanced anxiety-related behaviour in numerous anxiety tests. The enhanced anxiety-related behaviour of Mg2+-deficient mice was sensitive to chronic desipramine treatment in the hyponeophagia test and to acute diazepam treatment in the open arm exposure test. Mg2+ deficiency caused an increase in the transcription of corticotropin releasing hormone in the paraventricular hypothalamic nucleus (PVN), which coincided with elevated ACTH plasma levels, pointing to an enhanced set-point of the HPA axis. Chronic treatment with desipramine reversed the identified abnormalities of the stress axis. Functional mapping of neuronal activity revealed hyper-excitability in the PVN of anxious Mg2+-deficient mice and its normalisation through diazepam treatment.Compared to controls, Mg2+ deficiency model as a mouse model of enhanced anxiety, showing sensitivity to treatment with anxiolytics and antidepressants. It is further suggested that dysregulations in the HPA axis may contribute to the hyper-emotionality in response to dietary induced hypomagnesaemia.Overall, the present findings demonstrate the robustness and validity of the Mg


One objective of the expertise-based randomized controlled trial portion of a current developmental center grant is to determine which of three control groups would be most appropriate for a larger scale study concerning the effectiveness of Activator Methods chiropractic technique (AMCT) for temporomandibular disorders. A video evaluation instrument was developed to assess the equivalence of doctor interactions with participants in the active and sham AMCT groups.One doctor of chiropractic (DC) delivered the chiropractic intervention to the active and sham AMCT groups while being video recorded. The evaluation instrument codified DC communications into 4 domains: therapeutic , procedural , effectiveness , and affective  interactions. Activator Adjusting Instrument (AAI) clicks, encounter duration, touch orientation, and evaluator assessment of treatment group were documented. A trained video evaluator, blinded to treatment group, coded 34 active and 30 sham treatment videos by placing a hash mark in the appropriate category for each interaction. Descriptive statistics included medians and interquartile ranges.DC-initiated verbal communications were similar between active and sham AMCT in the procedural and affective domains. Notable differences were observed in the medians of the number of DC-initiated verbal communications between active and sham AMCT sessions in the therapeutic and effectiveness domains. More AAI clicks were recorded for active (42) vs sham (22) AMCT. Encounter duration also differed between active and sham AMCT (13 vs 11 minutes). The video evaluator correctly identified 66% of active AMCT, but only 31% of sham sessions.Definitive conclusions about how differences in DC behaviors may have impacted study results cannot be drawn until we have completed data analysis for the primary endpoint. Investigators may want to consider adding this type of analysis in manual therapies when sham or other control groups are used.


Garcinia is a plant under the family of Clusiaceae that is commonly used as a flavouring agent. Various phytochemicals including flavonoids and organic acid have been identified in this plant. Among all types of organic acids, hydroxycitric acid or more specifically (−)-hydroxycitric acid has been identified as a potential supplement for weight management and as antiobesity agent. Various in vivo studies have contributed to the understanding of the anti-obesity effects of Garcinia/hydroxycitric acid via regulation of serotonin level and glucose uptake. Besides, it also helps to enhance fat oxidation while reducing de novo lipogenesis. However, results from clinical studies showed both negative and positive antiobesity effects of Garcinia/hydroxycitric acid. This review was prepared to summarise the update of chemical constituents, significance of in vivo/clinical anti-obesity effects, and the importance of the current market potential of Garcinia/hydroxycitric acid. In the United States, IASO reported that 35.5% of men and 35.8% women were obese (BMI ≥ 30) -5-HT release in the brain [A typical reduction of food appetite and an increased serotonin availability were observed in all the weight control studies of HCA-SX on both animal and human subjects. These were associated with reduced levels of total cholesterol, LDL, triglycerides, and serum leptin as well as increased HDL level and urinary excretion of fat metabolites , 84, 87.he brain  are invoIn relation to this, gene expression profiling carried out by a research group demonstrated the modulation of a specific set of genes (about 1% of 9960 genes and ESTs) in the adipocytes by dietary HCA-SX supplementation . FurtherG. cambogia and G. atroviridis associated with high-fat diet- (HFD-) induced obesity where dyslipidemia, fatty liver, insulin resistance, and hyperleptinemia were acquired along with the overexpression of leptin, TNF-α, resistin, PPARγ2, C/EBPα, and SREBP1c genes in epididymal adipose tissue. The effect of G. cambogia was largely attributed to its HCA content [G. cambogia/HCA resulted from the combined actions of several mechanisms including suppressing de novo fatty acid biosynthesis and appetite [Garcinia/HCA based on their distinct mechanisms: (1) serotonin regulation and food intake suppression, (2) decreased de novo lipogenesis, (3) increased fat oxidation, and (4) downregulation of a spectrum of obesity-associated genes. Obesity, particularly caused by accumulation of visceral fat, is a serious risk factor of various life-style diseases such as coronary heart disease, diabetes, hyperlipidemia, hypertension, and cancer , 4. Huma content , 91. Subappetite , 60 and appetite , subsequappetite , 38, 92.G. cambogia, G. atroviridis, and G. indica [G. cambogia, Gymnema sylvestre, Zingiber officinale, Piper longum, and resin from Commiphora mukul on the modulation of food intake by serotonin modulators in female mice. The results obtained were compared with fluoxetine, a drug that was reported to enhance 5-HT neurotransmission [P < 0.05) antagonized the hyperphagic effect of p-chlorophenylalanine (PCPA), 8-hydroxy-2-(di-N-propylamino)-Tetralin (8-OH-DPAT), cyproheptadine, and 2-deoxy-D-glucose (2-DG) which further instigate possible serotonergic involvement in the effects of OB-200G on food intake in female mice. Preuss et al. [G. cambogia extract, suggesting that the ability of HCA in body weight gain reduction was most probably due to its combined effects on the metabolic and serotonin pathways. In addition, Roy et al. [HCA, the primary acid in the fruit rinds of . indica , has bee. indica , 93. ATP. indica , 94–97. . indica , 99. Mor. indica . However. indica  demonstr. indica  conductesmission . Both OBs et al.  reporteds et al.  They repy et al.  reportedLeonhardt et al.  reportedP < 0.01) in both groups. In the study, the rats underwent restrictive feeding for 10 days prior to ad libitum feeding . The control groups of both experiments had compensated the body weight loss, whereas the HCA-fed rats groups regained only 68 ± 4% (1% fat diet) and 61 ± 8% (12% fat diet) of the body weight regained by their respective control groups after 22 days of such ad libitum feeding. Despite significant reduction in weight regain in rats fed with 1% and 12% fat diet, long-term suppression of HCA on food intake was only detected in combination with 12% fat diet (Experiment 2). This was in line with the results obtained by Leonhardt et al. [β-hydroxybutyrate, and insulin, hepatic fact, and glycogen concentrations) in both experiments, except decreasing plasma triacylglycerol levels and increasing the liver fat concentration in Experiment 2 (rats fed with 12% fat diet). The fact that HCA did not affect plasma β-hydroxybutyrate (BHB) levels did not support the hypothesis that HCA suppressed food intake via increased hepatic fatty acid oxidation. Leonhardt and Langhans  then extt et al.  who suggHowever, contradicting results were obtained by Kovacs et al. , 71 who Several factors might contribute to the controversial results of the efficacy of HCA in human studies. One of contributing factors is the highly variable doses used in the human trials which ranged from 5 to 250 mg/kg of HCA per day , 70. Besin vivo and in vitro rates of lipogenesis in several tissues reported to convert carbohydrate into fatty acids , in which HCA was predominantly given to rodent models [in vivo fatty acid synthesis in rat liver. The rats were placed on chow diet for 7–10 days, followed by 45 h of fasting prior to a scheduled diet high in fructose or glucose for 10 to 15 days. The sodium salt of HCA at dose levels of 2 to 20 mM was administered by intraperitoneal injections 45 min before injection of 3H2O. Fatty acid biosynthesis in rat liver (μ moles 3H2O incorporated/g liver/h) was measured 3.5–5 h after starting of the final feeding. Profound decrease in fatty acid synthesis by 25 to 30 days was obtained with an intraperitoneal dose of 0.1 mmole per kg of body weight . In addition, 50% of inhibition was detected at a dose level of 0.28 mmole per kg body weight. The reduction of the acetyl-CoA by HCA and thus limiting the availability of building blocks required for fatty acid and cholesterol biosynthesis has led to suggestions that HCA inhibited lipogenesis. Several studies conducted by Sullivan and colleagues had confirmed the inhibition of t models , 97, 108G. cambogia/HCA affected respiratory quotient (RQ) and EE in rats and human. Lim et al. [de novo lipogenesis in rats, which is consistent with the findings of Westerterp-Plantenga and Kovacs [It was reported that m et al. , 110 shom et al.  further d Kovacs  in humande novo lipogenesis were investigated. A double-blind, placebo-controlled, randomized, and crossover design experiment was conducted on 10 sedentary male subjects. The subjects performed glycogen depletion exercise, followed by a 3-day high-fat low-carbohydrate  intake in order to create a similar glycogen storage capacity. Subsequently, a 7-day high-carbohydrate diet  supplemented with either 500 mg of regulator HCA (HOB Ireland Ltd.) or placebo was administered. Each intervention ended with a 60 h stay in the respiratory chamber (days 9 and 10). De novo lipogenesis occurred as indicated by RQ > 1.00 in all subjects. Significantly, lower 24 h EE , resting metabolic rate , and RQ at night  were detected with HCA as compared to placebo. Fat balance and thus net fat synthesis as de novo lipogenesis tended to be lower (P < 0.1) with HCA as compared to placebo. Taken all together, Kovacs and Westerterp-Plantenga concluded that the administration of HCA during overfeeding of carbohydrates may reduce de novo lipogenesis. In this respect, Kovacs and Westerterp-Plantenga  further However, opinions differ widely with respect to this issue. The mechanism underlying the anorectic effect of HCA is still unclear. Furthermore, whether the suppression of body weight regained was solely due to reduced food intake or whether there was involvement of increased EE remained unknown. Contradictory results were reported on the effects of HCA on EE. Previous reports by Leonhardt and colleagues , 102 andGarcinia extract and insulin were added simultaneously, the number of larger droplets markedly decreased while the smaller droplets (10–20 μm2 or <10 μm2) increased in 3T3-L1 cell. The activity of cytosolic glycerophosphate dehydrogenase (GPDH) which converts dihydroxyacetne phosphate to glycerol 3-phosphate (predominant substrate for triglyceride synthesis) increased from undetectable levels to between 100 and 187 U/mg of cytosolic protein after adipose conversion. However, no significant decrease in enzymatic activity was detected after administration of the Garcinia extract. Taken together, the authors therefore suggested that Garcinia extract interferes with lipid synthesis in fat cells via fatty acid supply inhibition without affecting adipose conversion. Blunden  reportedμL of a 0.48 mol/L HCA solution per mice significantly increased (P < 0.05) serum free fatty acid levels and concentration of glycogen in the gastrocnemius muscle, even though the respiratory exchange ratio was not different from that in the control group. On the other hand, chronic administration of 10 mg HCA twice a day significantly lowered (P < 0.01) the RQ during resting and exercising conditions in mice. Lipid oxidation, calculated from RQ, and oxygen consumption were significantly enhanced, and carbohydrate oxidation was significantly less in these mice during the early stages of running (P < 0.01). Taken all together, the authors therefore suggested that chronic administration of HCA augmented the endurance exercise performance in mice via the attenuation of glycogen consumption caused by the promotion of lipid oxidation during running exercise. Furthermore, Ishihara et al. [Ishihara et al.  conductea et al.  suggeste2 max for 60 min followed by 80% VO2 max until exhaustion. The results obtained showed that the respiratory exchange ratio (RER) was significantly lower in the HCA trial than in the control trial (P < 0.05). Fat oxidation was significantly increased by short-term administration of HCA, and carbohydrate oxidation was significantly decreased (P < 0.05) during exercise in athletes. In a continuation of their study, Lim et al. [P < 0.05). In addition, Lim et al. , 110 alsm et al.  conducteβ-oxidation of fats in body tissues [A more recent approach for determining fat metabolism by HCA was conducted by measuring urinary concentration of malondialdehyde (MDA), acetaldehyde (ACT), formaldehyde (FA), and acetone (ACON) of the tested subjects. The urinary excretion of these four metabolites was proposed to be a consequence of enhanced  tissues . The eff tissues , 64 as w tissues  and 32–1 tissues , 64. γ, and C/EBPα, are highly expressed in the adipose tissue and actively participate in the lipid metabolism of adipocytes by coordinating lipogenic and adipocyte-specific gene expression [γ interacts with several other transcription factors. C/EBPα and PPARγ interact via a positive feedback loop in the differentiated adipocytes, to induce each other's expression [γ with SREBP1c increases the transcriptional activity of PPARγ [Lipogenic transcription factors, including SREBP1c, liver X receptors, PPARpression . PPARγ ipression . Besidesof PPARγ . aP2  were increased in the adipose tissues of the obese rodents and humans [α suppressed transcription factors such as PPARγ and C/EBPα which, in turn, activated the GLUT4 gene [Fatty acid synthase, acetyl-CoA carboxylase 1, and SREBP1c mRNA concentrations were decreased in the adipose tissue of the obese animal models . On the d humans . A high UT4 gene , 122.G. cambogia fruit rind extract containing 60% (–)-HCA on serum leptin and insulin in mice. G. cambogia extract reduced serum total cholesterol, triacylglycerol, and nonesterified fatty acids in mice. Nevertheless, the body weight gain and fat pad weight were not affected in the treatment. No significant difference in blood glucose level was detected between groups, but a significant reduction of serum insulin (P < 0.05) was detected, suggesting that the G. cambogia extract efficiently improved glucose metabolism in the treated animals. In addition, the treatment decreased serum leptin levels and the leptin/WAT ratio. Besides, the changed ratio of body weight correlated positively with leptin levels in their study. Furthermore, it had been reported that leptin suppressed the signal transduction of insulin via cytokine interactions [G. cambogia extract on serum insulin in their study occurred through leptin-like activity. Hayamizu et al.  evaluateractions , 125. Haractions  suggesteGarcinia on visceral fat mass, lipid profiles in the serum and liver, serum adipocytokine levels, and regulation of the expression of multiple adipose tissue genes were reviewed. Kim et al. [G. cambogia, soy peptide, and L-carnitine  on rats rendered obese by high-fat diet (HFD). An HFD  with identical composition of the high-fat control diet (CD) applied in the study was fed to five-week-old male Sprague-Dawley rats for 9 weeks to create an obese conditions in rats that mimic to human obesity. Body weight gain, visceral fat-pad weight, and serum and hepatic biochemistry of rats were measured. The 0.38% mixture-supplemented HFD (D + M) reduced the total body weight and the accumulation of visceral fat mass and lowered the blood and hepatic lipid levels, which led to the improvement of insulin resistance in the HFD-induced obese rats. Moreover, the mixture of G. cambogia, soy peptide, and L-carnitine improved dyslipidemia in rat models with HFD-induced obesity. Downregulation of the expression of leptin, tumor necrosis factor-alpha, and sterol regulatory element binding protein 1c genes in the epididymal fat tissue of rats fed with CD + M diet was obtained. In contrary, upregulation of the uncoupling protein 2 (UCP2) gene in epididymal adipose tissues was induced with CD + M diet. No effect on the food intake of the animals was observed in the study, suggesting that the mixture exerted antiobesity effect via modulation of the metabolic derangement induced by HFD during which interactions between the multiple genes implicated in the process of adipogenesis might be involved, rather than simply suppressing appetite. A similar observation was obtained by Kim et al. [G. cambogia diet administrated group than in the HFD mice, implying less efficient transformation of the feed mass into body mass. The antiobesity effects of m et al.  reportedG. cambogia in terms of promoting weight loss and lowering cholesterol level were extensively studied. However, evidence for the effectiveness of G. cambogia or its derivative products was largely derived from animal studies [G. cambogia from in vitro and animal studies, more equivocal results were obtained from randomized double-blind placebo-controlled experiments dealing with human subjects [G. cambogia extract in 44 healthy volunteers  and concluded that G. cambogia is generally safe to be consumed. Several equivocal findings of RCTs were reported on the effectiveness of supplements containing HCA  since 1976 (search of US Patents and Trademark Office in year 2012 using Google patent search). These patents are on various aspects, including HCA enrichment from Garcinia rind, HCA and food products/dietary supplements prepared therefrom, methods of production, and their use. The majority of the patents are related to G. cambogia/atroviridis and/or HCA derived from Garcinia on obesity and weight loss. The patent numbers are as follows: 8,197,867, 8,097,286, 8,017,147, 8,003,138, 7,943,186, 7,943,183, 7,927,636, 7,858,128, 7,846,970, 7,772,428, 7,741,370, 7,687,082, 7,550,161, 7,507,421, 7,431,951, 7,335,651, 7,311,929, 7,230,131, 7,214,823, 7,208,615, 7,189,416, 7,179,488, 7,063,861, 6,982,098, 6,899,891, 6,875,891, 6,855,358, 6,770,782, 6,706,899, 6,676,977, 6,610,277, 6,565,847, 6,541,026, 6,489,492, 6,485,710, 6,476,071, 6,447,807, 6,428,806, 6,426,077, 6,413,545, 6,399,089, 6,395,296, 6,277,396, 6,221,901, 6,217,898, 6,160,172, 6,147,228, 6,113,949, 6,054,128, 5,972,357, 5,911,992, 5,783,603, 5,656,314, 5,612,039, 5,536,516, 4,007,208, 4,006,166, 4,005,086, 7,119,110, 3,994,927, 3,993,668, 7,153,877, 3,993,667, 4,028,397, 7,153,528, 7,015,250, 6,638,542, 6,579,866, 6,482,858, 6,441,041, 6,383,482, 6,207,714, 5,817,329, 5,626,849, and 3,965,121.The claims on enhanced human health associated with inia/HCA . SeveralGarcinia/HCA supplementation on promoting human health. A multitude of metabolic functions had been reported for HCA or HCA-containing Garcinia extract, such as reducing blood lipids, inducing weight loss, suppressing appetite, and reducing food intake based on results obtained in both animal trials and human clinical trials (Garcinia extract and HCA were generally safe to be consumed. Collective results from 17 clinical studies which involved 873 subjects demonstrated the safety of HCA and HCA-SX for human consumption [Garcinia/HCA supplements are efficient tools against various health problems especially obesity remain to be proven in larger-scale and longer-term clinical trials, despite substantial public interest in such supplements. Many diet supplements containing Garcinia/HCA marketed as weight management products are the combination of active ingredients rather than containing a single agent. Thus it is difficult to evaluate the effectiveness of single agents when the combination products are tested. In addition, awareness of the safety and efficacy of the weight management supplements available in the market should be raised among health care providers in order to assist their patients in analyzing the risks and benefits of the dietary supplements. Thus, scientific investigations on the potential health promoting effects of herbal preparations as diet supplement are prerequisites for new discoveries of alternative therapies.The nutraceutical industry is flourishing, and interest in establishing scientific credibility has attained importance for many companies and scientists. In the recent years, more clinical trials had been conducted to elucidate the functional effects of l trials . These dsumption . These ssumption . Based osumption . However


Alcohol-related liver disease (ALD) remains a leading cause of liver-related morbidity and mortality. Age, fibrosis stage, MELD score and continued alcohol consumption predict outcome in everyday clinical practice. In previous studies increased hepatocyte nuclear area and hepatocyte expression of p21, both markers of senescence, were associated with increased fibrosis stage and a poor outcome in non-alcohol-related fatty liver disease, while increased hepatocyte nuclear area was related to liver dysfunction in ALD cirrhosis. This study, therefore, investigated the pattern of hepatocyte cell cycle phase distribution and hepatocyte p21 expression in relation to outcome in ALD.Liver sections from two cohorts were studied. The first comprised 42 patients across the full spectrum of ALD. The second cohort comprised 77 patients with ALD cirrhosis. Immunohistochemistry assessed hepatocyte expression of cell cycle phase markers and p21. Regenerating liver (n=12) and “normal” liver sections (n=5) served as positive and negative controls, respectively.1/S phase and increased hepatocyte p21 expression (p<0.0001), which correlated independently with fibrosis stage (p=0.005) and an adverse liver-related outcome (p=0.03). In the second cohort, both hepatocyte p21 expression (p<0.001) and MELD score (p=0.006) were associated independently with an adverse liver-related outcome; this association was stronger with hepatocyte p21 expression  than with MELD score . Further, hepatocyte p21 expression co-localised with increased hepatic stellate cell activation.In the first cohort there was little cell cycle progression beyond G1/S phase in ALD. The striking independent associations between increased hepatocyte p21 expression and both fibrosis stage and an adverse liver-related outcome in both cohorts suggests hepatocyte senescence plays an important role in ALD. Measuring hepatocyte p21 expression is simple and cheap and in this series was a useful measure of long-term prognosis in ALD.The findings are consistent with impaired cell cycle progression beyond the G Alcohol-related liver disease (ALD) encompasses a broad spectrum of liver injury ranging from simple steatosis through alcohol-related hepatitis to cirrhosis and hepatocellular carcinoma –3. The pThe validity of clinical criteria in predicting outcome in ALD has been established, but less is known of liver histology in relation to prognosis. In one study increased hepatocyte nuclear area was related to hepatic dysfunction (prolonged prothrombin time and increased serum bilirubin) and hepatic decompensation  . IncreasIrreversible cell cycle arrest that limits the proliferative potential of cells is a feature of cellular senescence and is mediated by p21, a universal cell cycle inhibitor . p21 alsA recent study demonstrated that markers of senescence including hepatocyte p21 expression predicted an adverse liver-related outcome in patients with non-alcohol related fatty liver disease . This prTwo cohorts of patients with ALD were studied. All patients gave a history of sustained excessive alcohol consumption . Other recognised causes of liver disease including chronic viral hepatitis, autoimmune liver disease, hereditary hemochromatosis, α1-antitrypsin deficiency and Wilson’s disease were excluded with appropriate investigations. All patients had routine haematology and biochemistry blood tests performed at the time of liver biopsy and were reviewed at least every 6 months until death, an adverse liver-related outcome or the censor point. Only those patients with complete follow-up data were included.The first cohort comprised 42 patients within the full spectrum of ALD who underwent liver biopsy and remained under long-term follow-up at Cambridge University Hospitals. A second cohort comprised 77 patients with biopsy confirmed alcohol-related cirrhosis under long-term follow-up at University Hospital, Southampton, which was studied to confirm the findings in relation to outcome in the first cohort.A composite primary endpoint was used for the analysis of an adverse liver-related outcome. An adverse liver-related outcome event was defined as liver-related death, liver transplantation or the development of hepatocellular carcinoma. Survival was determined as the time from liver biopsy; survivors were censored at last clinic appointment.Liver biopsy specimens were obtained from 12 patients during the regenerative phase of acute ischaemic-reperfusion injury following liver transplantation to serve as positive controls for expression of the full range of cell cycle phase markers. All of these patients made a rapid recovery after transplantation with normal liver function at discharge from hospital. All the liver biopsy specimens showed evidence of regeneration on H&E-stained sections.Liver biopsy specimens were also obtained from background liver from five patients with colorectal cancer metastases, which served as negative controls for cell cycle phase markers. None of these patients had received chemotherapy prior to liver biopsy. Needle biopsies were distant to metastases with normal histology on H&E-stained sections.All liver tissues were obtained from formalin-fixed paraffin-embedded liver needle biopsy specimens, in accordance with Cambridge University Hospitals and University Hospital, Southampton local research ethics committee guidelines and approval. All participants provided informed, written consent for the liver tissue to be used for research purposes. Patients were only included in the study if the biopsy exceeded 1.5cm in length.A Bond™ machine (Leica Microsystems), was used to perform automated immunohistochemistry. Unconjugated mouse monoclonal anti-Mcm-2 served as a marker of cell cycle entry . Unconjugated mouse monoclonal anti-Cyclin A  and unconjugated mouse monoclonal anti-PH3  served as markers of S phase and M phase respectively. Unconjugated mouse monoclonal anti-p21 served as a marker of cell cycle arrest .2=0.93; p<0.0001). Mcm-2 and p21 positive hepatocytes were expressed as a percentage of the total number of hepatocytes; Cyclin A and PH3 were expressed as percentage of the number of hepatocytes expressing Mcm-2.Hepatocytes with brown-stained nuclei were considered positive for Mcm-2, cyclin A, PH3 and p21 whereas hepatocytes with blue-stained nuclei were considered negative; differences in the intensity of staining were not taken into consideration. Immunohistochemistry was assessed quantitatively to ensure objectivity. All images obtained from DotSlide digital microscope were analysed twice - using ScanR analysis software and manually. Photomicrographs of at least 20 lobular fields were analysed for each section (at least 1000 hepatocyte nuclei were counted). There was close correlation between the semi-automatic ScanR analysis and manual counting , a marker of activated hepatic stellate cells. Qualitative assessment examined the geographic association between hepatic stellate cell activation and hepatocyte p21 expression.Liver samples were assessed and scored using a modified Kleiner scoring system by specialist liver histopathologists (SD & AB), blinded to the immunohistochemistry results and clinical outcome. Architectural staining was performed using Gordon & Sweets’ reticulin and Chromotrope Aniline Blue (CAB) for assessment of fibrosis stage. Hematoxylin and Eosin (H&E) staining was used for quantification of steatosis grade and inflammation. Fibrosis was scored from 0 (none) to 4 (cirrhosis) and steatosis scored 0  to 3 . The histological diagnosis of alcohol-related steatohepatitis was based on evidence of hepatocellular injury and inflammation.Data are shown as median (range) or number (percentage) unless otherwise stated. A p-value of <0.05 was considered significant. Non-parametric analysis  utilising the Mann–Whitney U test or 1-way ANOVA  was applied where data were not distributed normally.Logistic regression analysis was performed using SPSS 15.0 for Windows to study factors associated with fibrosis stage. Input variables were age, sex, presence of alcohol-related steatohepatitis and MELD score.Log-rank test  and Cox-regression model were used to identify predictors of clinical outcome. For the purpose of the Kaplan-Meier analysis alone the following parameters were divided into two groups based on their cohort medians: amount of alcohol consumed, hepatocyte p21 expression and MELD. In all other instances these parameters were treated as continuous variables. Only variables with a p-value of <0.10 on the univariate analysis were subjected to analysis multivariate analysis (Cox-regression).The clinical and histological characteristics of 42 patients within the spectrum of ALD (first cohort) are summarised in The clinical characteristics of 77 patients with histology confirmed alcohol-related cirrhosis (second cohort) are summarised in Expression of hepatocyte Mcm-2, cyclin A, PH3 and p21 were negligible in normal liver tissue .Mcm-2 expression was higher in hepatocytes in ALD and regenerative liver compared to normal liver, indicating cell cycle entry of hepatocytes in both conditions  in relation to the proportion of hepatocytes that were Mcm-2 positive was much lower in ALD compared to regenerating liver  or with the presence of alcohol-related steatohepatitis .Hepatocyte p21 expression demonstrated no correlation with either MELD score (p=0.11 and p=0.36) or the amount of alcohol consumed at the time of liver biopsy (p=0.20 and p=0.94) in the first and second cohorts, respectively. In addition, in the first cohort, there was no difference in hepatocyte p21 expression between those who were consuming and those not consuming alcohol at the time of liver biopsy (p=0.07). A similar analysis was not carried out in the second cohort, as only one patient was not consuming alcohol at the time of liver biopsy.In the first cohort, hepatocyte p21 expression correlated positively with prothrombin time  in the first cohort revealed that patients with higher hepatocyte p21 expression (above the median) were more likely to develop an adverse liver-related outcome than those with lower p21 expression p=0.03; . Age  in the second cohort revealed that patients with higher hepatocyte p21 expression  versus 40 months (1-90); p=0.04).1/S phase and increased hepatocyte p21 expression. The association between hepatocyte p21 expression and a subsequent adverse liver-related outcome prompted further investigation. Thus, a further well-defined group of patients that had undergone liver biopsy and long term follow-up were recruited to study the association between hepatocyte p21 expression and clinical outcome. Since hepatocyte p21 expression was higher in advanced fibrosis and an adverse liver-related outcome is more common in patients with advanced fibrosis, the second cohort was selected such that all patients had ALD cirrhosis. The strong relation between increased hepatocyte p21 expression and an adverse liver-related outcome was confirmed in this second cohort. These data are consistent with previous data in patients with non-alcohol-related fatty liver disease which revealed a close link between hepatocyte p21 expression, fibrosis stage and increased liver related morbidity and mortality [In this study, the distribution of hepatocyte cell cycle phase markers and hepatocyte p21 expression were examined in the first cohort, which was selected carefully from a larger series such that alcohol was the only known risk factor for liver disease, long-term follow-up was assured, liver sections were of adequate size and the patients covered a broad spectrum of liver histology. This revealed impaired hepatocyte cell cycle progression beyond Gortality .1 phase of cell cycle [1/S phase in ALD. This is corroborated by a marked increase in hepatocyte p21 expression in ALD compared to regenerating liver, since p21 is a universal cell cycle inhibitor which exerts its effect at the G1/S phase transition [Oxidative stress, such as ethanol-induced oxidative stress, causes double-strand DNA breaks , which ill cycle . The preansition . It was ansition  to preveThere was progressive increase in hepatocyte p21 expression with increasing fibrosis stage in ALD. This observation, together with the increase in hepatocyte nuclear area in ALD cirrhosis described previously  and prevHepatocyte proliferation is critical for liver regeneration . AccumulThe relation between hepatocyte p21 expression and outcome is noteworthy. Indeed in this series with ALD, the relation between hepatocyte p21 expression and outcome was stronger than that for MELD score, fibrosis stage, alcohol-related steatohepatitis or continued alcohol consumption. Hepatocyte p21 expression can be assessed readily by immunohistochemistry (much more readily than nuclear area) and has the potential to be useful for assessing prognosis in day-to-day clinical practice, stratification in future clinical trials in ALD and even when liver transplantation is being considered and the prognosis remains uncertain.The first cohort comprised a consecutive series of patients with all stages of fibrosis and all manifestations of alcohol-related steatohepatitis. Although patients were selected for single aetiology, tissue quality and extended follow-up, in every other respect, this cohort reflects ‘real world everyday practice’. The second cohort included only patients with cirrhosis but with all grades of alcohol-related steatohepatitis. Both cohorts were selected carefully only to include those with biopsy-proven ALD, those with adequate tissue for meaningful analysis, with adequate long-term follow-up and all necessary data available including information on alcohol consumption during follow-up.Information on the quantity of alcohol consumed during follow-up was self-reported and may be less accurate and this remains a limitation of the study. A further limitation was that information regarding nutritional status, which may have influenced the clinical outcome, was not available in a sufficient number of patients for analysis.In conclusion, this study demonstrated that impaired cell cycle progression in hepatocytes, hepatocyte senescence and progressive fibrosis in ALD were inter-related. Further, there was a strong relation between the proportion of senescent hepatocytes and an adverse liver-related outcome. Similar findings were demonstrated in non-alcohol-related fatty liver disease . It is p