CCNE2 (cyclin E2 gene). Mutational analysis in K562 cells showed that the N-terminal region of p21 is required for repression of gene expression of CCNE2 and other genes. Chromatin immunoprecipitation assays indicated that p21 was bound to human CCNE2 and other p21-repressed genes gene in the vicinity of the transcription start site. Moreover, p21 repressed human CCNE2 promoter-luciferase constructs in K562 cells. Bioinformatic analysis revealed that the CDE motif is present in most of the promoters of the p21-regulated genes. Altogether, the results suggest that p21 exerts a repressive effect on a relevant number of genes controlling S phase and mitosis. Thus, p21 activity as inhibitor of cell cycle progression would be mediated not only by the inhibition of CDKs but also by the transcriptional down-regulation of key genes.It has been previously described that p21 functions not only as a CDK inhibitor but also as a transcriptional co-repressor in some systems. To investigate the roles of p21 in transcriptional control, we studied the gene expression changes in two human cell systems. Using a human leukemia cell line (K562) with inducible p21 expression and human primary keratinocytes with adenoviral-mediated p21 expression, we carried out microarray-based gene expression profiling. We found that p21 rapidly and strongly repressed the mRNA levels of a number of genes involved in cell cycle and mitosis. One of the most strongly down-regulated genes was  CIP1 (p21 herein after) is a member of the Cip/Kip family of inhibitors of cell cycle progression . The first discovered p21 function and so far its best studied biochemical activity was the inhibition of cyclin-dependent kinases p21However, other studies have shown that p21 has activities in addition to cell cycle arrest. Thus, p21 acts as an inhibitor of apoptosis induced by DNA-damaging agents CCNE2 (cyclin E2) is one of the most potently down-regulated genes and p21 was found to bind and repress the human CCNE2 promoter.To address the relevance of p21-mediated gene regulation we have carried out large-scale expression profiling in two different human systems (keratinocytes and myeloid leukemia cells) upon ectopic expression of p21. p21 provokes a rapid and potent down-regulation of genes involved in the execution and control of mitosis in both models. Mutational analysis revealed that the N-terminal region of p21 is required for its transcriptional effects in leukemia cell. 2+ -inducible p21 gene; Kp27-5 cells are K562 carrying a Zn2+ -inducible p27 gene 4 was added (75 µM), the cells were further incubated for 12 h and harvested.Primary human keratinocytes wereprepared from discarded human tissue, mostly from reduction abdominoplasties, with Institutional Review Board approval (MGH#2000-P-002418/3) and were grown as described One million cells per sample were fixed with 80% ethanol and stained with propidium iodide as described previously CDKN1A gene (p21)  and control siRNAs  using Lipofectamine 2000 following the manufacturer's recommendations. 48 h after transfections, cells were analyzed by RT-qPCR Human primary keratinocytes were transfected with 200 nM siRNAs for Total RNA was isolated using TriReagent (Molecular Research Center) kit (Qiagen). Reverse transcription (RT) was performed with iScript reverse transcriptase (BioRad). Quantitative PCR (qPCR) was performed with the SYBRGreen PCR kit (BioRad) in a BioRad MyiQ apparatus. Primers sequences and amplicon sizes used in the RT-qPCR assays are shown in http://biosun1.harvard.edu/complab/dchip/]. The expression data was filtered so as to include genes with expression changes ≥2.4-fold. The data were obtained in compliance with the MIAME guidelines and are deposited in the ArrayExpress database. The accession numbers are E-MEXP-3431 for the expression data with Kp21-4 and Kp27-5 cells and E-MEXP-3430 for expression data of human keratinocytes. The analysis was performed with data from two independent experiments and RNA preparations of each experimental condition or adenoviral infection. The interaction network for differentially expressed genes was generated with the Ingenuity Pathways Analysis software.Total RNA was prepared using RNeasy kit (Qiagen). Biotinilated cRNA was obtained from total RNA and hybridized to Affymetrix HG-U133A chip in the Genomic Facility of Centro de Investigación del Cancer . Data analysis and hierarchical tree clusters were generated using the dChip software 2, 0.5% Tritón X-100 and protease inhibitors). After centrifugation the supernatant was collected as cytoplasmic plus nucleoplasmic fraction. The pellet was washed at maximum setting with CSK buffer, resuspended in CSK buffer, sonicated and saved as chromatin fraction.Total cell lysates and immunoblots were carried out as described ChIP analysis was carried out as described previously 4 for another 24 h. Luciferase activity was then determined with Lysis Solution 1 (Promega), Luciferase Substrate (Promega) and a GloMax 20/20 luminometer (Promega), following the manufacturer's instructions. All transfections were normalized by measuring β-galactosidase activity of the samples. Data are the average of at least three independent experiments and error bars indicate standard deviation.Two million of cells were electroporated with 15 µg of pCyCE1-Luc 4 resulted in induction of p21 that peaked 4–12 h decreasing afterwards . To confirm the repressive effect of p21 we analyzed the level of acetylated-histone H3, a marker of active chromatin The dataset used for the clustering analysis of The fast kinetics of gene down-regulation caused by p21, before any change in cell cycle profile could be detected, suggested that the repression is a direct consequence of p21 activity, rather than an indirect effect. CDK2 inhibition is the best known biochemical activity of p21, and it also occurs in K562 CCNE2, KIF4A) and slower (WEE1) down-regulation kinetics . For further analysis, we focused on the addition . We prevth ZnSO4 . This reth ZnSO4 . We alsopromoter . As addipression . As the pression  we also that p21 , which iCCNE2 and (as a control) CCNE1 promoters. The results demonstrated a modest but significant and reproducible decrease in CCNE2 promoter upon transfection of a p21 expression vector . The results demonstrated the decreased levels of mRNA for all the tested genes, confirming the data of the microarray hybridization . More interestingly, all genes but one (BUB1) contained at least one CDE site in the vicinity of the TSS , topoisomerase IIα CDC25A, MYC4 (in human myeloid cells) or through acute adenoviral infection (in human keratinocytes).Despite the original description of p21 as a CDK inhibitor, a number of reports have described a role of p21 as transcriptional modulator for different genes such as Our results on the gene expression profiling in both cell models showed that p21 provokes a rapid repression of mRNA of genes involved in cell cycle and mitosis control in the two models. Since p21 elicits cell growth arrest this effect could be indirect, i.e., a consequence of the cell cycle arrest. However, in myeloid cells with inducible p21 (Kp21-4 cells), the kinetics of the mRNA down-regulation of most of the analysed genes was almost parallel to the kinetics of the p21 protein up-regulation. This down-regulation of mRNA was also concomitant with the rapid deacetylation of histone H3 at the TSS. The genes repressed by p21 were up-regulated upon p21 silencing, confirming the p21 activity. Moreover, the early p21-induced down-regulation was not reproduced by p27 induction in K562 cells form most genes, despite that p27 also provokes a cell cycle arrest via CDK inhibition. Furthermore, the depletion of CDK2 does not affect the p21-mediated gene repression. Altogether these data argue against the idea that the p21 effects were a direct consequence of the CDK inhibition or of the cell cycle arrest. Thus, it is conceivable that p21 is directly involved in transcriptional repression of a set of genes involved in cell cycle control. Previous unsuccessful attempts to generate a functional p21 fused to the estrogen receptor (so as to get activated by tamoxifen) It is remarkable the similarity in the short-term transcriptional effects of p21 overexpression observed in two very different cell types (keratinocytes and myeloid cells), despite the different long-term consequence of p21 overexpression in each cell type. p21 inhibits epidermal differentiation in keratinocytes CCNE2 (cyclin E2), one of the genes undergoing a fastest down-regulation. ChIP assays revealed that p21 binds in the vicinity of the TSS of human cyclin E2 gene . The results are consistent with previous literature showing chromatin binding of p21 in several models (see Introduction). In contrast, upon induction of p27 for 12 h in Kp27-5 cells, we could not detect significant binding of p27 to CCNE2 promoter.We further analysed the regulation of CCNE2 promoter. The mutational analysis revealed that CCNE2 gene repression depends on the N-terminal region of p21, i.e., the region involved in the binding to cyclin-CDK complexes. The C-terminal domain of p21 is not required, but seems to contribute for full activity of p21 as repressor. It is noteworthy that p21 does not repress CCNE1 promoter (if it does) as efficiently as CCNE2, arguing for a differential role of both cyclins.In full agreement with the results observed at the mRNA level and ChIP assays, luciferase reporter experiments showed that p21 repressed the human WNT4 repression, which depends on the interaction with E2F1 BIRC5, CCNB2, DHFR, KIF4A), despite that a relaxed consensus sequence as E2F's binding site was used for the analysis, which includes E2F4 sites (BUB1) contained at least one CDE site in the vicinity of the TSS. In this context it must be noted that human CCNE2 promoter and first exon are particularly rich in CDE sequences. Thus, p21 may impair transcription of genes in charge of the execution and control of mitosis through the interaction with transcription factors binding to CDE or CDE/CHR motifs.Taken together the data argue for a role of p21 as co-repressor of gene transcription for genes related to cell cycle. As p21 lacks any recognizable DNA-binding domain, the interaction must be indirect, i.e., through another transcription factor or co-factor. It has been reported that p21 binds to specific DNA sequences of some genes. For instance, we have previously shown that this is the case for F4 sites . Our anaAltogether, our results indicate that p21 is a multifunctional protein with the capacity to act through at least two mechanisms to control cell cycle: directly inhibiting CDKs and indirectly regulating genes involved in cell cycle control. The relative importance of each mechanism await further investigation, but it is of note that p21 is able to arrest the cell cycle in CDK2-deficient cells Figure S14. B. Absence of cell cycle profile alteration after short induction times of p21. Cell cycle profile of Kp21-4 and Kp27-5 cells upon induction of p21 and p27 with 75 µM ZnSO4 for 6 and 12 h. The cell cycle profile was determined by flow cytometry of propidium iodide-stained cells. C. p21 induces an irreversible accumulation of G2 and polyploid cells whereas p27 induces a reversible accumulation in G1. Kp21-4 and Kp27-5 were treated with ZnSO4 for 12 h. The cells were then washed to remove the inducers, further incubated for 84 h and the cell cycle profile was determined by flow cytometry (4 days after the induction). The fraction of cells in G1, G2 or polyploidy cells (>G2) is indicated in each case.Cell cycle alterations mediated by p21 and p27 in K562 cells. A. Immunoblots showing the induction of p21 in Kp21-4 cells and p27 in Kp7-5 cells after 6 and 12 h of treatment with 75 µM ZnSO(TIF)Click here for additional data file.Figure S2Interaction networks of genes regulated by p21 in K562 cells. A knowledge-based database  was seeded with the genes regulated by p21 at 12 h of induction . The two(TIF)Click here for additional data file.Figure S34. After 3, 6 and 12 h of induction, total RNA was prepared and expression of the indicated genes was determined by RT-qPCR. The data for Kp21-4 cells are the same than in Comparison of the gene regulation mediated by p21 and p27 in K562 cells. p21 was induced in Kp21-4 cells and p27 was induced in Kp27-5 cells by 75 µM ZnSO(TIF)Click here for additional data file.Figure S44 (to induce p27) were compared to that of cells with induced p21 (Kp21) and parental K562 treated for 12 h with ZnSO4. The heat map shows the hierarchical clustering with those genes with expression variation ≥2.3-fold between uninduced and p27-induced Kp27-5 cells after subtraction of the gene expression changes due to p21 in Kp21 cells(P<0.001). The heatmap shows 179 genes. B. Common genes regulated by both p21 and p27 in K562 cells. Kp21-4 and Kp27-5 cells were treated for 12 h with 75 µM ZnSO4 to induce p21 and p27 respectively. The heat map shows the hierarchical clustering with those genes with expression variation ≥2.3-fold between uninduced cells and Zn2+ –treated cells which are regulated in both cell lines(P<0.001). The heat map shows 90 genes. The genes related to cell cycle according with Gene Ontology are shown in red.A. Gene expression regulation mediated by 12 h induction of p27 in K562 cells. The transcriptome of Kp27-5 cells (Kp27) treated for 12 h with ZnSO(TIF)Click here for additional data file.Table S1Aequorea victoria, encoded in the plasmid pEGFP-C1). An alternative common name of the gene is included for some genes.Primers used in PCR reactions in this work. The forward primer is in the first line. All correspond to human genes except GFP (Green Fluorescent Protein from (DOC)Click here for additional data file.Table S24 (p21 inducer) for 12 h, after subtracting those genes changed by ZnSO4 in parental K562 cells  and in Kp27-5 cells . The 253 genes are included in the heat map of 21.2 (≥2.3-fold) and with a signal difference ≥50 between both experimental conditions (as defined by dChip program and with Affymetrix U133 biochip data). Values are mean of fold changes (expressed as log2) of two independent experiments (P<0.001). For those genes represented by two or three Affymetrix probes, the fold change is the mean between the values of the probes. A negative fold change indicates down-regulation upon p21 induction.Genes regulated by p21 in K562 cells. The table list the genes showing an expression change in Kp21-4 cells treated with ZnSO(DOC)Click here for additional data file.Table S321.2 (≥2.3-fold) and with a signal difference ≥50 between both experimental conditions (as defined by dChip program and with Affymetrix U133 biochip data). Values are mean of fold changes (expressed as log2) of two independent experiments (p<0.001). For those genes represented by two or three Affymetrix probes, the fold change is the mean between the values of the probes. A negative fold change indicates down-regulation upon p21 induction.Genes regulated by p21 in primary keratinocytes. The table list the genes showing an expression change in human primary keratinocytes infected with adenovirus-p21 as described in (DOC)Click here for additional data file.


Women presented themselves as staying in control of their drinking while men described going out with the intention of getting drunk . While women experienced peer pressure to drink, they seemed to have more options for socialising without alcohol than did men. Choosing not to drink alcohol is a behaviour that still requires explanation in early mid-life. Harm reduction strategies should pay more attention to drinking in this age group.Most qualitative research on alcohol focuses on younger rather than older adults. To explore older people’s relationship with alcohol, we conducted eight focus groups with 36 men and women aged 35 to 50 years in Scotland, UK. Initially, respondents suggested that older drinkers consume less alcohol, no longer drink to become drunk and are sociable drinkers more interested in the taste than the effects of alcohol. However, as discussions progressed, respondents collectively recounted recent drunken escapades, challenged accounts of moderate drinking, and suggested there was still peer pressure to drink. Some described how their drinking had increased in mid-life but worked hard discursively to emphasise that it was age and stage appropriate ( There is increasing concern about harmful alcohol consumption in the UK , particue.g. enough to get drunk, enough to be sick) rather than the number of units of alcohol consumed , we conducted focus groups with people who already knew each other and so could draw on existing relationships and shared experiences in the research setting . PreviouIt proved difficult to recruit respondents in this age range. We used a variety of methods including handing out flyers in pubs and on the street, sending email invitations asking people to cascade to friends and colleagues, placing posters on community noticeboards and doctors’ surgeries, phoning community groups and workplaces and advertising on community websites. Once an individual expressed interest, they were asked to recruit a group of up to five people in the same age group who ‘regularly’ drank alcohol. As expected, there was some variation in age in these ‘naturally occurring’ groups; three respondents were younger than 35 and two were over 50 years of age. We also recruited one group of non-drinkers to gain a different perspective on the cultural context of alcohol.In 2009, 36 respondents (15 men and 21 women) participated in eight focus groups . We askeAll respondents were white and lived in the West of Scotland. This was a socio-economically diverse sample. We used the Carstairs score  – an areThe same researcher (CE) facilitated all the groups. After an explanation of the study and assurances about confidentiality, respondents were asked to give written informed consent and permission for the discussion to be tape-recorded. The topic guide included changes in drinking over time, occasions when respondents had drunk more than they intended, reasons for reducing drinking, and men’s and women’s drinking. Respondents were given £20 gift vouchers.Focus groups were transcribed verbatim and checked for accuracy. Fieldnotes were written soon afterwards. Transcripts were read repeatedly and coded thematically; QSR Nvivo was used to facilitate analysis. Following e.g. only drinking a particular brand of gin or preferring certain grape varieties of wine). Some respondents declared that, in contrast to their youth, they would not now feel the need to finish a drink they were not enjoying and felt less pressure from others to drink heavily. Craig’s narrative illustrates this discourse of drinking less as one ages, knowing when to stop and being easily able to resist any pressure to drink more. He contrasted his wilder younger self with becoming a ‘wise old owl’ now:laughs)! When I turned 18, I spent my life clubbing in places like Ibiza where it was just drink constantly and every year I went there until I was 30. And then my boy [son] appeared and it was the same as with Debbie, I started to cool it ... I don’t drink nearly as much. I can’t – the older you get, the less you can drink – maybe three or four glasses of wine and that’s me, done, whereas at one time, I could drink three or four bottles of wine ... I think we’ve all done that once, hide the drink – get rid of it some way or another, not leave it [because of peer pressure]. But nowadays, you can be honest and say, ‘I’ve had too much – I’ve had enough, and don’t even say to me have another one, because I’m not interested’. I can do that now. I know when to stop now .I started drinking about 15 ... cheap and nasty wine , whereas drinking in early mid-life was characterised as being relaxing, sociable and civilised, often accompanied by good food and conversation and by an interest in the taste, rather than the effect, of alcohol . Respondents referred to three demographic stages similar to those discussed by e.g. sharing a bottle of wine over a meal), and drinking during the week (‘drinking on a school night’) was curtailed in order to fulfil work responsibilities, as it was thought to be much more difficult to recover from heavy drinking as one aged. Drinking at weekends was sometimes reduced because leisure time was seen as increasingly ‘precious’. Thirdly, respondents with children were perceived to have many more limits on their drinking. Both fathers and mothers in the sample felt that their drinking had reduced when their children were very young – particularly because of the difficulty of getting up early to look after young children after a heavy night’s drinking – but women tended to introduce the topic and talk about it in more detail, particularly in the two all-female focus groups (FG4 and FG8). For example, Craig (see excerpt above) echoed the point that his female colleague made about reducing her drinking once she had children, but he did not discuss the topic in detail. In contrast, Isobel (mother of three pre-teenage children) described in detail how she and her friends (‘as women’) had a ‘responsible approach’ to alcohol because of childcare responsibilities:Laughing) ... You’re up early in the morning, packed lunches, hangover, school run – it doesn’t work! ... One of the husbands takes the kids, so that ... the mums can sit down minus children, husband, dishwashers and have a drink and a chat ... We actively get dressed, get changed, hair done, makeup on ... what happens at the start of the evening is we all tell each other what we’re doing in the morning. We actually, as women, discuss who’s got what responsibility ... ‘right, we’re not making it late, what time are we stopping at?’ ... In our group, nearly all the husbands either work away from home or work shifts and are on call. So the women always have to have a responsible approach. They can’t just go out, get really drunk and then worry about not going out in the morning .My drinking is controlled by my children. . Self-control and discipline were necessary in order to prioritise responsibilities and counteract the powerful appeal of alcohol, often referred to using metaphors suggesting the natural force of water .I know ... our son is now at a stage where he’s out on a Saturday night at the under-18s disco and comes back about half ten at night ... and although we’re sitting having a drink, I think it’s because we’re now in a situation where, well I know I could get in a taxi and go and get him, whereas when he was younger, I would maybe not see that as an option. But still aware ... I don’t want to be too well oiled if he’s not in yet .Some women were very critical of mothers who consumed alcohol while caring for their children. Women discussed how they tried to avoid, or at least limit, their children seeing them under the influence of alcohol and described how they continued to plan their drinking around older children too. For example, respondents in FG4  had children ranging from 11 to 25 years and discussed how as ‘main carers’ they often monitored their drinking to take account of what their offspring were doing:I think everyone – certainly our age – has had it pushed into us for a long time and I certainly know people who’ve been breathalysed the next day and lost their licence ... it’s a sobering thought ’cause my job depends on my having a licence .We’ve actually got a wee breathalyser in the house ... You maybe get up [the next morning] and take your daughter to choir and you are WAY over and you just can’t, so you send them down in a taxi and it actually makes you stop and think ... But it [drinking] has to be planned, doesn’t it? You can’t just be impulsive in that respect Driving was another ‘responsibility’ which influenced drinking alcohol. Women who lived in the suburbs discussed driving to social occasions in order to save money and for safety reasons, which meant they did not drink alcohol. Both men and women also described ‘taking the car’ as a useful strategy to employ if they did not want to drink. However, most discussions centred on worries about driving the morning after drinking, either to get to work or to transport children to school or leisure activities. Many respondents described how their job depended on having a driving licence and their horror of (being caught) driving over the limit the next morning. Again, this reinforced the idea that drinking in early mid-life needed to be planned rather than spontaneous. Andy described how he planned his drinking around his driving, as he needed his licence to work, while Stella explained how she checked whether she was over the limit before deciding whether to drive the next morning:As the focus groups progressed, different narratives appeared to challenge the idea that respondents drank less as they got ‘older and wiser’. This is perhaps not surprising, given that half of the sample exceeded the ‘recommended’ limits for weekly drinking. Each challenge is discussed in turn.Some of them [work colleagues] are awfully dull and want to leave early .Laughter) .Not in our circle! .Laughing) .Once you’ve had it, you’re like that, ‘Oh, I’ll just have another one. Oh, I’ll just have another wee one’ (Laughing) … It’s like none of us will turn round and say, ‘that’s it, we’ve had enough, let’s go’. It’s like we’re all there to the death and looking for more (Laughing) .Three and four in the morning, and then you’re like that, ‘Who are we going back to? Have you got any wine?’ .In almost every group, respondents told stories about relatively recent drinking exploits which contradicted previous statements about planned, moderate drinking. These included having the intention of ‘going for one’ but then ‘drinking the night away’ and drinking ‘pranks’ . It was clear in the heavier drinking groups  that these stories contributed to a shared identity:e.g. deliberately choosing drinks that would get them drunk faster, blackouts, being asked to leave venues because they were drunk, not knowing where they were or what they were doing).FG5 was unusual in that the respondents were less concerned about presenting an image of themselves as responsible, social drinkers. Their sales jobs required them to work evenings – sometimes promoting alcohol – and they often socialised together after work. Perhaps because of their work culture, they were unusually open about the effects of alcohol .There will be certain nights you’ll decide, ‘yeah I’m getting drunk and I know it, I don’t care, ’cause I’m enjoying it!’ So you know that OK, I can have a few more, I’m still going to get home safe, I’m not going to disgrace myself, I’m going to suffer in the morning, but I’ll still get up and go to work ’cause I’m enjoying myself right now, I’m enjoying the company, so I’ll take the pleasure and pain .Apart from respondents in FG5, while women acknowledged that they did sometimes get drunk, they emphasised that this was not the ‘purpose of their evening’. However, men were more direct about their intentions to get drunk although they often emphasised that there was a reason for this decision and that getting drunk would not mean that they neglected their responsibilities:I don’t drink because I just cannot justify the cost. If I was in Glasgow every weekend, say Friday and Saturday night with my friends, it would be at least £50, £60 a night? ... I just cannot … .Yeah but that’s what you were doing when I first met you! .Laughing) ... you’re sitting, you go to get off the bar stool to go to the toilet and you just feel yourself going ... .And then you get the sort of a dizzy kind of a feeling .I’ve seen you that way, don’t give us that! .I’ve actually went to dominos and not drunk at all, just drunk juices .Is that a fairy tale?! .One strength of focus group data is that people can choose whether to agree with or question others’ accounts. Respondents used humour to disagree with other people’s accounts of mid-life drinking. For example, Alison, a 47-year-old mother of two teenagers, explained how she was now happy to leave half a glass of wine when she felt she had drunk enough. Her colleague, Vicky, laughingly disagreed, whispering ‘blasphemy!’ to indicate that she would not do this. Other interjections more directly challenged the veracity of accounts:men supporting each other in the pub during hard times, while Barbara’s description of her move from non-drinker to ‘party animal’ after her divorce suggested the need (for women) to make up for lost time:For a lot of men the pub is the place that you socialise.. Late thirties and I didn’t really HAVE a social circle anymore. I split up with my ex and it was like finding your way again. My mate said ‘come and meet some people’. It’s just about –‘OK, that person is quite a good guy ... he can come and join our company, he’s probably a bit worse at the moment’ .I got married young, had my son young ... I was a non-drinker until the age of 39 when I got divorced and hit the social scene ... maybe a bit of a party animal for about five or six years and now it’s calmed down ... I still need to feel as if I can party with the best of them! That might be a kind of common theme in amongst women of a certain age, with no men to hold us back .Descriptions of lifecourse changes also called into question the smooth transition from being young with no responsibilities, to being settled in a couple, to having children, and the suggestion that people gradually drank less as they progressed through these lifecourse stages. Respondents who separated from their partner in their thirties described how they started to go out and drink more. Hugh referred to the breakdown of friendships which accompanied separating from his partner and emphasised the importance of We’re mature ladies, we’ve all got personalities and we don’t actually NEED a drink for socialising. We ENJOY a drink but we don’t need it to go and talk to folk … .agreement from others) at night if I’m sitting in the house and the kids are away to bed, I think ... nice glass of wine ... and chill .I find it more as a relaxing thing, not something to get your confidence .agreement from others). Go upstairs and sit with a glass of wine. Be fine after that .That’s exactly what I was going to say. I can if I’ve had a ... bad day with children, just go [gulping] ... and that’s terrible. I need a drink. I don’t actually NEED it, but I do  drinking when their children were young and then drinking more as their children became less reliant. However, the narratives from women in FG4 called this into question. These women were unusual in having dependent children and also being one of the heavier drinking groups. They started off by endorsing the narrative (older people do not NEED to drink) but later in the interview, Jenny and Cath  discussed how the stresses of paid and unpaid work sometimes led to heavier drinking at home:e.g. ‘go on, go on, go, on, just the one’, ‘take one, take one’, ‘just leave the car, just leave the car’ or ‘another one for the road’). Respondents explained that this ‘friendly pressure’ was about everyone being ‘in the same boat’, drinking together and being sociable and relaxed. Alcohol was perceived as a ‘social lubricant’ making the evening more fun and interesting. Respondents described finding it difficult to get into the conversation, feeling like an outsider and being bored by repetitive stories when they went out and did not drink alcohol.Respondents also told stories which challenged the idea that people in mid-life invariably experience much less pressure to drink from friends, and can easily resist this. They described how their intention not to drink alcohol – or to stop drinking – was sometimes just not accepted and illustrated this through the repeated chants of the group (This pressure to drink was illustrated well by respondents who were non-drinkers (FG7). They described how difficult it was for people to understand and accept that they did not drink alcohol; as one woman said, ‘it’s not just socially accepted that people drink – it’s socially EXPECTED’. Most of this group had experience of living in different cities and described receiving a more aggressive reaction from people in the west of Scotland . They found themselves constantly having to justify why they did not drink and described being perceived as judgemental or thinking they were superior to others. They also commented on how much interaction in their age group consisted of people talking about going to the pub. They inverted the common cultural portrayal of drinkers as ‘fun’ and non-drinkers as ‘boring’, so that people who did not drink were characterised as entertaining, creative, witty, making real connections with other people and taking responsibility for themselves, while drinkers were portrayed as dull, having repetitive conversations, having shallow relationships with others propped up by alcohol and being irresponsible and unimaginative.That’s what I always said to them –‘I’m detoxing’, and everybody was fine. It’s a reason [for not drinking], it’s like you’re saying with diets: if you’re on a diet people accept that, don’t they? But you have to give a reason, if you just say ‘I’m just cutting out alcohol’ then people question it .That’s like what I was saying earlier, wasn’t it? People say ‘why?’ .laughter) .Well, they’ve stopped asking us if we’re pregnant, I take it!  drink without an ‘acceptable’ reason. Three women in their late forties demonstrated this point:I think alcohol ... is a way of life. It’s a cultural thing in Scotland definitely. I know there’s serious issues with it, but we’re all – we all drink to socialise ... If you don’t go to the pub, you’d never see anyone ... Fergus and I hardly ever go for a coffee. Do we? .Puts on effeminate voice) Go for a skinny latte . team’s playing about 12.30 kick off ... and I’m just like, ‘ach, it’s too early, I don’t want a drink yet’. I’m in for the craic [banter] and the game ... You walk over with a glass of coke and it’s just … .Oh! Abuse! .laughs) ... Oh God if you walk over with half a pint of beer, ‘what’s wrong with you, are you skint?’ You know. But I can take it! That’s just our company! .‘Oh, here comes the gay boy’, do you know what I mean?  found the idea of men going out for coffee or a meal together, rather than a pint, unlikely or laughable. For example, Graham asserted that he would not see his friends if he did not go to the pub:This extract from the all-male focus group (FG2) also demonstrates the insults and jokes which greeted men’s purchase of soft drinks or half pints and called into question their ‘masculinity’. Although some men felt that there might be less peer pressure in smaller groups, or among more educated people, they still commented on the pervasive influence of the ‘macho’ culture in the west of Scotland which involved drinking until ‘falling down drunk’ or being expected to finish a bottle of whisky once it had been opened.Initially, respondents in this qualitative study of drinking in early mid-life suggested that they only drank alcohol in moderation and instead problematised youthful (binge) drinking. However, these initial assertions that respondents were ‘older and wiser’ were undermined by drinking stories re-told within friendship groups, jokes which questioned stories of responsible drinking, and accounts of continuing peer pressure to drink. Although we did not interview a focus group sample of young people, when we compare our findings with research on younger people there are many parallels .e.g. early parenthood inhibiting opportunities to drink to get drunk), this could sometimes be revisited at a later date . While many respondents in early mid-life did  acknowledge that they sometimes got drunk, they worked hard discursively to make it clear that this was age and stage appropriate (i.e. did not result in them being sick or making a fool of themselves). Their accounts suggested a long list of considerations  in order to (be seen to) meet their responsibilities as workers, parents and/or good citizens before they felt able to embark on the ‘controlled loss of control’ described by There were, however, some features that differentiated these accounts from those of younger people. Norms around drinking were heavily influenced by expectations of appropriate behaviour across the lifecourse; for example, drinking to get drunk was seen as much more acceptable for young people who were learning how to drink than for those in mid-life . DrinkinThese respondents in early mid-life constructed unrestrained and problem drinking as being younger people’s behaviour. Such problematising of other drinkers is reported elsewhere. For example, heavy-drinking older women (>35 units) positioned younger women drinking heavily in public as problematic  while yoIn terms of gender, both men and women saw drinking as pleasurable and a fun way to socialise, although women had more concerns about safety, and explicitly expected to take more responsibility for children. Interestingly, both men and women experienced pressure to drink and referred to alcohol both as a reward and a way to recover from – or survive – the rigours of paid work, unpaid work in the case of women, and other stresses. People manage the tensions between, and attempt to balance, ‘control’ and ‘release’  through a variety of  practices ; thus heIn contrast, male participants were able to state that they were going out with the intention of getting drunk . Excessive and public consumption of alcohol with other men has been a traditional marker of masculinity in Western cultures . The pubOur study has a number of limitations. In focus groups, dominant views (whether strongly expressed or articulated by the majority) can close down discussion. It was notable in this study, however, that respondents frequently challenged others’ views. Focus groups can also show how drinking is linked to constructions of individual and group identity. Like other qualitative studies, our respondents were self-selected and it could be argued that our sample was biased toward respondents with fewer home responsibilities who drank heavily. We advertised for people who drank ‘regularly’ but it could be that those who were most interested were heavier drinkers or that people without young children found it easier to attend a focus group. However, respondents who were parents (20 out of 36 respondents) did reflect back to having younger children. There was also a range of drinkers who participated, including non-drinkers (n=4) and ‘moderate’ drinkers (n=13) as well as hazardous or harmful drinkers (n=19). To some extent our approach managed to get beyond self-presentations as responsible drinkers, but there are likely to be presentational aspects due to the presence of the interviewer, and rhetorical constructions that arise as part of the interview context .Cross-sectional surveys usually find that alcohol consumption reduces in older age groups , but canthe form of “entertainment” and fun for a large group of British people’ and question what alternatives there are for social enjoyment. This question seems particularly pertinent to men, given the lack of opportunity for men to socialise without alcohol identified by respondents in this study.This study provides a starting point for exploring drinking behaviours among adults in early mid-life. Our findings point to three areas which may be of interest to health promoters, policy makers and other researchers. First, our study demonstrates that heavy drinking is normalised in early ‘mid-life’. Drinking excessively is associated with pleasure and sociability and linked to both gender and group identity construction in mid-life as well as in youth. Thus, health promoters and policy makers need to consider this age group more closely. Secondly, health education messages could build on the social meanings of alcohol consumption we have identified for this population. For example, it may be possible to develop the ‘acceptable’ reasons (such as detoxing or driving) identified by respondents for resisting the ‘friendly pressure’ from peers to have a(nother) drink. Thirdly, more research needs to focus on gender and alcohol in order to identify harm reduction strategies which are acceptable to both men and women.


Back in 2003, we published ‘MAX’ randomization, a process of non-degenerate saturation mutagenesis using exactly 20 codons (one for each amino acid) or else any required subset of those 20 codons. ‘MAX’ randomization saturates codons located in isolated positions within a protein, as might be required in enzyme engineering, or else on one face of an α-helix, as in zinc-finger engineering. Since that time, we have been asked for an equivalent process that can saturate multiple contiguous codons in a non-degenerate manner. We have now developed ‘ProxiMAX’ randomization, which does just that: generating DNA cassettes for saturation mutagenesis without degeneracy or bias. Offering an alternative to trinucleotide phosphoramidite chemistry, ProxiMAX randomization uses nothing more sophisticated than unmodified oligonucleotides and standard molecular biology reagents. Thus it requires no specialized chemistry, reagents or equipment, and simply relies on a process of saturation cycling comprising ligation, amplification and digestion for each cycle. The process can encode both unbiased representation of selected amino acids or else encode them in predefined ratios. Each saturated position can be defined independently of the others. We demonstrate accurate saturation of up to 11 contiguous codons. As such, ProxiMAX randomization is particularly relevant to antibody engineering. Saturation mutagenesis  is a core technique within the protein engineer's repertoire. Its importance in engineering non-native ligand-binding domains is undisputed. Thus saturation mutagenesis has played a vital role in creating synthetic zinc-finger-based transcription factors ,2, antibConventional saturation mutagenesis employs degenerate codons NNN, NNK or NNS. Although technically undemanding, degeneracy leads to significant problems that have provoked a drive for non-degenerate alternatives. Traditionally, non-degeneracy was achieved by using trinucleotide phosphoramidites which add whole codons (rather than single bases) during oligonucleotide synthesis . In 2003The advantages and disadvantages of the various approaches to saturation mutagenesis are compared in ProxiMAX randomization is based on iterative cycles of blunt-ended ligation, amplification and digestion with the Type IIS restriction endonuclease MlyI. More specifically, oligonucleotide donors (containing the required codons at their termini) are ligated on to a conserved acceptor sequence. Following ligation, the PCR amplification step provides ample ligated product, while concomitantly diluting non-ligated contaminants. Subsequent digestion removes all but the final codon of the donor sequences to generate a new acceptor, which enters the next cycle . By altehttp://www.biochemsoctrans.org/bst/041/bst0411189add.htm). Bias might result from variant oligonucleotide quality, inaccurate estimations of oligonucleotide concentration, sequence preferences of T4 ligase, amplification bias during PCR or even sequence preference of the restriction enzyme MlyI. To examine and/or exclude these factors, ligations and amplifications were performed in individual parallel reactions. Only after purification and quantification were the individual amplicons combined. Analysis at this midpoint in each of six cycles of saturation  demonstrated that equimolar mixes (5% of each codon) had been generated successfully during each cycle of addition . However, these data offered no insight as to whether subsequent ligations would proceed in an equimolar fashion when those mixtures acted as acceptors. Therefore, after six cycles of saturation mutagenesis, composition of the final assembled library was assessed by Roche 454 pyrosequencing, which demonstrated that, whereas most codons were introduced into the library at the expected ratio, a few were noticeably over- and under-represented. In particular, the histidine codon 5′-CAT-3′ was favoured, whereas codons for lysine and threonine were under-represented .To examine process feasibility, preliminary experiments used a mixture of all 20 donors combined in nominally equal quantities (based solely on manufacturer's data). Results demonstrated that the process worked, but, unsurprisingly, there was bias in the resulting small-scale library . Although there were no discernible rules for the junction preference directly at the point of ligation, there are certainly preferences for 3′-T and 5′-C, which supports the observed over-representation for histidine (CAT), but does not fully explain the reason for histidine being more preferred than arginine (CGT).By eliminating donor concentration and/or quality and PCR preference as potential sources of bias, these data suggest that T4 DNA ligase exhibits some sequence preference in blunt-ended ligations, when presented with a mixture of 5′ and 3′ ends, so that, in this substrate mixture, the enzyme has a different reactivity rate for different codons. To examine this possibility, data from the preceding 6-mer sequence analysis were studied for expected and observed ratios with respect to the last base in the phosphate donor/acceptor junction. A χTo compensate for these apparent sequence preferences, the six cycles of addition were repeated, with the concentration of each donor sequence being adjusted during mixing, to counteract the over- or under-representation shown in Supplementary Figure S2(B). Thus concentrations of donors carrying over-represented codons were reduced, whereas those carrying under-represented codons were increased. Cysteine and methionine codons were also excluded as these are deemed to have liabilities in peptide libraries by being susceptible to oxidation. It is clear from H (variable heavy) chain domain was undertaken. Each position in the loop required a separate mixture of codons so that favourable protein-binding amino acids were included at the correct positions and those that would encode structural or manufacturing liabilities were excluded. The frequencies of the desired amino acids were rounded to the nearest 5% in order to simplify mixing of the codon oligonucleotides and analysis of the final library.Having demonstrated successful equimolar representation, we next sought to create a more relevant library from a protein engineering perspective. Saturation of 11 consecutive codons within the loop of a CDR3 (complementarity-determining region 3) domain of an antibody VFor this longer loop, both right-hand and left-hand acceptors were employed to build two library components in parallel B. The 11in vitro-based approaches is the quality of the antibody library” [Although more involved than conventional degenerate saturation mutagenesis, we believe that the benefits of ProxiMAX randomization easily outweigh the increased outlay on reagents and of time. One to two cycles of ProxiMAX randomization can be achieved in a day and thus our exemplar 11-mer library can readily be prepared in 2 weeks. The bottleneck of directed evolution is library screening  and thuslibrary” . Thus al


Alcohol screening and brief intervention (SBI) is effective in many health-care settings. Previous research has shown significant decreases in harmful drinking due to SBI, but many studies, particularly in emergency/trauma settings, did not use a control group. Thus, it is unclear if observed decreases in harmful drinking are due to the intervention or other factors such as the hospital visit, the substance use assessment, or simply regression to the mean. This project assessed the effectiveness of an SBI program implemented at an urban hospital in the US state of Georgia. Participants were enrolled between January 2009 and April 2010 during an emergency department visit. Intervention patients (n = 120) and control patients (n = 400) were assessed using the Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST) and completed a survey that measured harmful drinking, among other items. Intervention patients received a 10-15 minute motivational-interviewing–based brief intervention aimed at reducing harmful drinking. All patients were contacted six months later for follow-up assessment. Analyses investigated between-group differences in alcohol consumption over time. Among intervention group patients, results show that past 30-day mean drinking days were reduced from 11.7 days at baseline to 5.8 days at six months and mean binge drinking days were reduced from 7.6 days at baseline to 3.0 days at six months. Although the control group also showed significant reductions in mean drinking days and heavy episodic drinking days over time, regression analysis indicated a significantly greater reduction in heavy episodic drinking days for the intervention group. Further analyses examined the impact of the intervention across different risk levels, based on ASSIST score. Preliminary results suggest those with moderate-risk drinking received the most benefit from the brief intervention. These results provide further evidence of SBI’s effectiveness in reducing harmful drinking.


Arteriovenous malformations (AVMs) that involve the mandible are difficult lesions to treat, with traditional options being surgery and embolization. This article describes a large mandibular AVM that was treated with embolization using transarterial as well as direct puncture approaches. Follow-up imaging showed thrombosis of the vascular spaces of the malformation. There were no complications. The patient is doing well and is on follow-up. Arteriovenous malformations (AVMs) of the mandible are rare lesions that have a variety of presentations, including gingival bleeding, dental loosening, swelling, and life-threatening hemorrhage. This artAn 11-year-old boy presented with a few years’ history of gradual generalized enlargement of the mandible. An orthopantomogram  showed aDigital subtraction angiography demonstrated a large AVM within the body of the mandible supplied by branches of both external carotid arteries and draining via a large intraosseous venous sac into the left internal jugular vein . The feeThe patient did well after the embolization procedure and had no complications. An MRI scan of the mandible performed a few days later showed thrombosis within the vascular spaces of the AVM . The patMandibular AVMs are rare high-flow vascular malformations that can present with mandibular enlargement, asymmetry, pain, increased mobility of the teeth and hemorrhage that can be life-threatening. It is diEmbolization of the nidus of the AVM using polyvinyl alcohol particles or N-butyl cyanoacrylate can result in a permanent cure, although treatment failures secondary to recanalization and rebleeding from collaterals have been described.–6 EmboliDirect puncture of the nidus or the draining varix followed by embolization with N-butyl cyanoacrylate or coils has been described.7–9 AlthoMRI can be used for follow-up imaging due to its ability to detect flow voids, thrombus, and reossification. MRI was Intraosseous AVMs are rare lesions that can be treated using endovascular methods on a case-to-case basis. In the treatment of a mandibular AVM, a combined approach with transarterial followed by venous embolization using a direct puncture of the venous sac is an effective strategy.


Injury to the long thoracic nerve with resulting serratus anterior palsy is a typical cause of medial scapular winging. We report a case of a 70-year-old female with scapular winging in the setting of a mildly comminuted midshaft clavicle fracture. The winging persisted for three months after the fracture, which became a nonunion. The winging spontaneously resolved prior to open reduction and internal fixation of the nonunion. The winging recurred after this surgery. The recurrence was attributed to transient irritation and/or inflammatory neuropathy of the brachial plexus caused by the surgical manipulation. This second episode of winging again spontaneously resolved. There are few reported cases of scapular winging in the setting of a clavicle fracture and only one case of recurrent scapular winging. In that case, which was in the setting of an acromioclavicular joint separation, the second episode of winging required long-term use of a brace. By contrast, our patient did not require bracing because the recurrent winging spontaneously resolved, making this a novel case. This case is important because it illustrates that recurrent scapular winging can occur, and spontaneously resolve, in the setting of a mid-shaft clavicle fracture after subsequent reconstruction of a fracture nonunion. Damage to the long thoracic nerve is a classic cause of medial scapular winging , 2. ThisWe report the case of a patient who sustained a closed and minimally comminuted mid-shaft clavicle fracture after a ground-level fall which developed ipsilateral scapular winging that became obvious and clinically significant when she started physical therapy prior to sufficient healing. The winging resolved completely in three months, only to recur after the surgical reconstruction of the nonunion that developed. This second episode of winging also completely resolved three months later. Additional unusual aspects of this case include (1) electrodiagnostic studies revealing evidence of mild injuries to the brachial plexus in addition to the long thoracic nerve and (2) there were no fractures or significant injuries to the ribs or scapular body or to the acromioclavicular, sternoclavicular, and glenohumeral joints. A previously healthy 70-year-old right-hand-dominant female was seen in our clinic three months after sustaining a closed and minimally comminuted mid-shaft fracture of the right clavicle, which resulted from a ground-level fall . She triPhysical examination showed swelling in the mid-clavicular area and crepitance with attempted shoulder motion. Medial winging of her right scapula was obvious and was viewed from behind while the patient was pushing against a wall. Strength with grip, finger abduction, and thumb extension was decreased (4/5) on manual testing. A nerve conduction study and electromyographic evaluation (NCS/EMG) showed evidence of (1) mild brachial plexopathy with involvement of the upper trunk and axillary nerve, in addition to a slightly decreased ulnar motor response and (2) long thoracic nerve injury. The NCS/EMG showed reduced axillary nerve amplitude. There were also motor unit changes in the muscles innervated mainly by the long thoracic and also by the axillary, radial, and ulnar nerves. Based on NCS/EMG criteria, the overall prognosis for recovery was considered good. Recommendations included stopping physical therapy and starting daily treatments with an ultrasound-based bone growth stimulator . Several outcome surveys were completed at each visit . Four moRadiographs six months after the fracture showed a well-established nonunion. Treatment was open reduction and internal fixation with bone grafting . AlthougNine months after surgery, the patient underwent hardware removal because of local discomfort from clothing. Radiographs taken fourteen months after fracture fixation surgery and six months after hardware removal (20 months after injury) show complete healing of the clavicle fracture . She repAlthough medial scapular winging has been reported to occur in association with nonunion and malunions of shoulder girdle fractures , 3, 5, 6Clavicle fractures account for 2.6 to 4% of all fractures, and of those approximately 80% are mid-shaft clavicle fractures , 8. The By contrast, brachial plexus injury (without scapular winging) is more commonly associated with clavicle fractures. For example, of 111 cases of mid-shaft clavicle fracture reported by the Canadian Orthopaedic Trauma Society , eight oBarbier et al.  report aRasyid et al.  report aGonza and Harris  reportedOur interpretation is that our patient sustained a partial neuropraxia of the brachial plexus, which manifested distally as involvement mainly of the long thoracic nerve, and to a lesser extent, the axillary, ulnar, and radial nerves. This interpretation is consistent with her NCS/EMG findings and is more likely than an isolated injury to the long thoracic nerve . The rec Our patient presented with a unique case of medial scapular winging and spontaneous resolution prior to nonunion of her clavicle fracture and with spontaneous and permanent resolution of the recurrent winging after fracture reconstruction. We suspect that the winging was caused by injury the brachial plexus, which indirectly affected the long thoracic nerve. Our case is also unusual because there were no fractures or apparent significant injury to the ribs and scapular body, or to the acromioclavicular, sternoclavicular, and glenohumeral joints.


For example, breathing 100% oxygen can elevate baseline rSO2 and artificially widen the difference between baseline rSO2 and the lowest rSO2 value during a surgical procedure .Optimizing outcome is predicated on understanding what brain oxygen saturation thresholds are worrisome enough to clinically warrant intervention. Reports thus far have substantive inconsistencies as to what the optimal cerebral saturation target should be—which leads us to the question: what is the desaturation threshold, that when crossed, increases the risk of adverse outcomes?  are considered best to optimize rSO2  could also increase the oxygen content. Combined, this would increase overall cerebral oxygen delivery. Conversely, there is the potential for substantive morbidity related to inappropriate transfusion, targeted hypercapnia and/or hypertension. For example, erroneously increasing the PaCO2 above the normal physiologic level in the setting of cardiopulmonary bypass (CPB) could lead to increases of cerebral blood flow in excess of what is needed for cerebral metabolism, with a subsequent increased delivery of CPB-related air bubbles and particulate debris. This is similar to the use of pH-stat blood gas management, where the addition of CO2 to the CPB fresh gas flow (thus correcting the CO2 for hypothermic temperature) has been associated with worse postoperative cognitive outcomes  in cardiac surgery , to determine which outcomes may be related to desaturation. Only then would it be possible to design a randomized controlled trial to investigate the ability of an interventional algorithm, aimed at restoring cerebral saturation, to modify the previously determined target outcomes. This could also serve to delineate a potential saturation threshold to maintain (such as a relative or absolute change). A good example of this is the recent work that we performed in a small observational study (Thus, the field of cerebral oximetry research is still relatively young and contains as many questions as it does certainty. In order to move forward in an expedited and confident fashion, taking advantage of the last 60 years of research in cerebral NIRS, we will need to reconsider our approach regarding further clinical development. Otherwise, we risk wasting this potentially valuable technology, and most importantly, depriving our patients the benefit of improved clinical outcomes with its universal use.


Super Mario Brothers as compared to Call of Duty). Moreover, three factors were significantly related to pathological game use: the use of games to escape daily life, the use of games as a social outlet, and positive attitudes toward the steady accumulation of in-game rewards. The current research identifies individual preferences and motives relevant to understanding video game players' evaluations of different games and risk factors for pathological video game use.A new measure of individual habits and preferences in video game use is developed in order to better study the risk factors of pathological game use . This measure was distributed to internet message boards for game enthusiasts and to college undergraduates. An exploratory factor analysis identified 9 factors: Story, Violent Catharsis, Violent Reward, Social Interaction, Escapism, Loss-Sensitivity, Customization, Grinding, and Autonomy. These factors demonstrated excellent fit in a subsequent confirmatory factor analysis, and, importantly, were found to reliably discriminate between inter-individual game preferences (e.g.,  The video game industry is among the fastest growing sectors of the US economy. This industry generated $25 billion in sales in 2011, and from 2005 to 2009, boasted an annual growth rate of more than 5 times the growth rate of the entire US economy over that same period Siwek, . EvidencCivilization) while another prefers Ken Levine's narrative-based first-person shooters .As video games have grown in universal popularity, they have also grown in diversity. Today's video games constitute a myriad of different studios, developers, and genres. Specific games often contain a variety of options, allowing the player to interact with the game in many different ways. With so many options both between and within video games, it is not surprising that individuals often prefer one game type to another, much like with other forms of popular media. Just as one movie enthusiast may favor Spielberg as another prefers Tarantino, so too might one gamer enjoy Sid Meier's strategy games  tended to better satisfy needs than did critical flops . Importantly, different players found the same critically successful games to be differently satisfying of their SDT needs and thereby differently enjoyable. This phenomenon suggests that individual differences in player preferences may moderate whether a particular game satisfies or stifles SDT needs, and thus, which players will enjoy which games.The intuitive idea that different gamers have different motives and preferences for video games is supported by recent research. For example, Ryan et al.  examinedResearchers, players and game developers alike have long been interested in measuring individual differences in game motivations and preferences. Theories of “player personality” began with Bartle , who speWorld of Warcraft. For example, more Achievement-oriented players had a greater proportion of in-game “achievements” in player-vs.-player combat and cooperative dungeon raids, whereas more Immersion-oriented players had proportionately more achievements related to exploration  . Similarly, some games have lively multiplayer communities  while others are exclusively single-player experiences. Some games allow for the steady accumulation of level-ups and items over time , while other games take place in isolated, non-cumulative games . We expect that players may have preferences for one set of game mechanisms over another, creating meaningful and predictable patterns of covariation between favorite game franchises and motive measurements.Importantly, no measure to date has demonstrated an ability to discriminate between players of different games. The ability to discriminate between game platforms, genres, and titles is tantamount to understanding the differences between diverse video games and their different potentials to inspire pathological use. Thus, a comprehensive and externally valid measure of preferences should be able to discriminate between fans of different styles of game and even different gaming platforms. For example, people who play games primarily through incidental platforms such as Facebook or iPhone  should be measurably different from people who purchase game consoles specifically to play video games. Similarly, fans of different games should vary in their enthusiasm for different game features. For example, some video games have been critically praised for their storytelling  players. By validating this measure through comparisons to preferred video games and gaming platforms, this study is able to explore whether certain game motives, genres of game, or game platforms are associated with greater incidence of pathological game use.www.reddit.com/r/truegaming, www.reddit.com/r/girlgamers, forums.penny-arcade.com, www.rpgcodex.net, www.minecraftforum.net/forum, www.skyrimforums.org, www.conquerorworm.net, and www.badgame.net. Forum posters had the advantage of being plentiful and were willing to volunteer their time for minimal compensation, but self-selection pressures caused these participants to be overwhelmingly male gamers who played daily. Thus, the survey was also distributed to college undergraduates, advertised as a “survey of having fun” rather than a “video game survey,” in order to sample from more females and less frequent players.Participants were collected from two sources. First, Internet volunteers were recruited, with moderator permission, through forum postings at SD = 6.03, range = 10–66.) An additional 300 college undergraduates were recruited from the University of Missouri, who completed the survey in exchange for partial course credit. .The current sample included 1689 individuals recruited from internet discussion forums who completed the survey for a chance to win one of ten $20 Amazon gift cards. , and ethnicity (“Hispanic” or “Not Hispanic”).Participants indicated their age, sex, race (“Very hardcore) to 5  and how frequently they played such games . Participants also indicated how many hours (on weekdays and weekends) they spent playing video games during the following 6 h intervals: midnight to 6 AM, 6 AM to noon, noon to 6 PM, and 6 PM to midnight. They also indicated what proportion of their spare time was spent playing video games on a scale ranging from 1 (Almost none of my spare time) to 5 .Participants indicated how casual they were about playing video games on a scale ranging from 1  and three games that they were currently playing.Final Fantasy franchise were collapsed together, with the exception of Final Fantasy XI and Final Fantasy XIV, which were massively multiplayer online games instead of single-player Japanese role-playing games. Similarly, the 1990s turn-based role-playing games Fallout 1 and Fallout 2 were combined to a single entry, while the 2008 first-person shooter role-playing game Fallout 3 was kept as its own separate entry. The World of Warcraft MMO was kept separate from the Warcraft real-time strategy franchise. Since each successive The Elder Scrolls game has had equally fervent fans and detractors, Morrowind, Oblivion, and Skyrim were each kept as separate entries.To increase statistical power, this item collapsed across games within franchises when individual games were reasonably similar. For example, different entries within the The Legend of Zelda franchise, Final Fantasy franchise (excluding MMOs), Half-Life franchise, Mass Effect franchise, Fallout 1 and 2, Deus Ex 1, Super Mario franchise (excluding spinoffs like Mario Party or Mario Kart), Portal franchise, Skyrim, Halo franchise , Planescape: Torment, Pokemon franchise, Call of Duty franchise, Morrowind, Team Fortress 2, Minecraft, Grand Theft Auto franchise, World of Warcraft, Baldur's Gate 2, and Bioshock franchise.Responses were restricted to the twenty most frequently-indicated favorite games. These included: most typically use to play games .Additionally, participants indicated via checklist which media platforms they Strongly disagree) to 5 (Strongly agree) . Participants were given a “Not applicable” response option in the case that they had no experience with an item. Items were presented in a random order across participants. One survey item requested that participants indicate a “Neither Agree nor Disagree” response. This item served as a proxy for attention. Subjects who failed to mark this item appropriately were excluded.Participants answered 121 video game related questions intended to assess their motives and preferences for such media. Of these items, 20 were taken from the Video Game Uses and Gratifications Instrument developed by Sherry et al.  , as this approach yielded the greatest reliability in previous research (α = 0.78) .After completing the motives and preferences survey, participants completed a measure of pathological video game use developed by Gentile . Particit(1598) = 27.42, p < 0.001], proportionately more female , more casual about video games , played less frequently , and spent a smaller proportion of their spare time on video games . The undergraduate sample thus adds diversity to the study sample, making the following analyses better representative of game use in general rather than game use only by serious players.Compared to the internet sample, the undergraduate sample was younger . We interpret this well-fitting CFA as evidence for the scales' internal reliability, since the relationships between latent factors and their indicator variables were similar across subsets of participants.Results from the CFA demonstrated excellent model fit . Therefore, these participants were returned to the dataset for all subsequent analyses, increasing the total sample size to N = 783.Some participants had responded to all retained items but had been discarded for missing data on other, discarded items. An additional CFA was performed including these participants (Mass Effect) among their favorites should be higher than average on the Story factor, whereas players who enjoy open-world, free-form games  should have higher-than-average scores on the Autonomy factor. Thus, the following analyses examined whether participants' factor scores could be predicted as a function of the games they had listed among their three favorites.If the 9-factor solution represents valid individual differences in game preferences, they should covary with specific game franchises. For example, gamers who list story-based franchises . The influence of each favorite game franchise on each factor is summarized in Table For each of the 20 most-frequently indicated favorite game franchises, a dummy-coded  vector was created to indicate whether a participant listed that franchise among their top 3 favorite video games. A multiple analysis of variance (MANOVA) was then conducted to determine whether GAMES factor scores could be predicted as a function of the vectors of favorite games. Thus, the analysis compared whether gamers who enjoyed a particular game franchise generally scored lower or higher on a particular factor from the 9-factor solution. All results are presented as Type III Sums of Squares, thereby representing the unique variance in each factor after partialing out the variance due to other game franchises. Analysis was restricted to those participants who had indicated at least one of these 20 game franchises as a favorite n = 31. The iSimilarly, if the GAMES factors are externally valid, they should be related to players' choices of gaming platforms. For example, serious players who buy hardware specifically to play games should be different from players who only play games incidentally . Thus, players who indicated typically using certain platforms may be higher or lower on certain factors than other players. As before, dummy-codes were created for each subject for the platforms they reported using to play games. We performed another MANOVA to see whether choice of platform predicted the 9 factor scores. Age was entered as a covariate. Results are summarized in Table A multiple regression was conducted to determine whether factor scores predicted participants' frequency of use, proportion of spare time spent on game use, and self-described attitude toward games . Results are summarized in Table n = 736; thus, all ts represent 734 degrees of freedom. Age was significantly correlated with Catharsis , Loss-Aversion , Social Interaction , Customization , Grinding , and Autonomy . Age was not significantly correlated with Story , Violent Reward , or Escapism .Correlations between age and the GAMES factors were inspected via Fisher r-to-z transformations. Forty-seven participants did not give their age and were excluded from this analysis, leaving a sample of Ms = 3.47 and 2.39, Welch's t(145) = 4.64, p < 0.001] but were not more likely to reach the threshold for diagnosis .Items in the pathology questionnaire were scored as 1 for a “Yes” response, as 0 for a “No” or “Not Applicable,” and as 0.5 for a “Sometimes” response, per the recommendations of Gentile et al., (under review). The item “Have you played video games as a way of escaping from problems or bad feelings?” was discarded based on the results from an item response theory analysis of these items . Items were summed to create a total pathology score for each participant. As recommended by Gentile et al., (under review), the study followed DSM-IV criteria for other disorders and assigned a positive diagnosis to participants who endorsed at least half (7) of the symptoms. The percentage of pathological gamers in the final data was found to be 8.16%, comparable to most similar studies . Additionally, Social Interaction  and Grinding  scores were also significantly associated with increased risk.To determine whether any of the 9 factors were associated with an increase in the odds of exhibiting game pathology, we conducted a multiple logistic regression, using the factors to predict the probability of a positive diagnosis of pathological game use. Players higher on the Escapism scale were much more likely to have a positive diagnosis of pathological game use than individuals lower on this factor .A separate multiple logistic regression was conducted to determine whether participants' reported frequency of play, seriousness about games , and proportion of free time spent on games were associated with the incidence of pathology. Of these, only proportion of free time spent on games was significantly related to pathology , the additional recruitment of 300 college undergraduates helps to diversify the study sample to females and less serious players.This measure improves on previous instruments in a number of ways. First, it builds upon the latent variables of these previous studies by adding new factors, particularly Story, which has become an increasingly important facet to players in the last decade. We also believe that the Grinding factor is of theoretical importance, and may (in combination with Losing) predict how different players differently satisfy SDT needs for competence. It has been said that there are “two kinds of games: those that are won because of skill, and those that are won because of time” Baron, . These tFinal Fantasy, Mass Effect, Planescape: Torment, and Half-Life had greater Story scores than did fans of other games. Similarly, players of free-form RPGs like Skyrim or Fallout had higher Autonomy scores, while players of the carefully-scripted Call of Duty franchise had lower Autonomy scores. The 60-or-more-hours-long RPGs Skyrim and Final Fantasy were each associated with higher Grinding scores. The violent Grand Theft Auto was associated with higher Violence Reward and Violent Catharsis.Our factors demonstrated excellent reliability. Moreover, factor scores were found to be related to participants' favorite franchises in sensible ways. For example, fans of role-playing and story-based franchises like Jetpack Joyride, Tiny Tower, Off the Leash, Punch Quest). Moreover, these games are often “free-to-play,” costing nothing to install and instead being funded by players who convert real-world money to in-game currency in order to purchase these upgrades. Since our Grinding scale measures both attitudes toward earning and paying for in-game rewards, we consider this further evidence for the validity of our scales. However, no phone or Facebook games made it into the 20 favorite titles, so it is yet to be determined whether this business model is the actual cause of the observed relationship between phone games and Grinding.Our factors also seem to represent differences between players of different game platforms. For example, users of the three most conventional video game platforms—Playstation 3, XBOX 360, and personal computers—placed greater value on video game stories, violence, and escapism. However, PC gamers were also noticeably higher in Autonomy, possibly reflecting this platform's tendency for more open-ended, choice-rich, and modifiable video games. They were also much more capable of tolerating losses. By comparison, players of incidental platforms such as phone and Facebook games found losses to be more frustrating. Phone gamers also scored higher on Grinding. Many phone games involve simple, rapid gameplay with progressively earned in-game currency which can then be traded in for various upgrades  that may be more difficult to treat—if someone is using video games to escape these problems, then abstention from video games might only treat those symptoms of video game use while leaving the underlying problem intact. This replicates previous reports of a link between escapism and pathology from Yee . This reWe also found some evidence for a player-game interaction in pathology. Players who have higher social motives for playing games are also more likely to have video game pathology. As mentioned in the introduction, games with multiplayer game mechanics and player-to-player relationships may be difficult to quit, since peer pressure and social obligation contribute to continued play. A relationship was also discovered between Grinding and pathology, supporting our hypothesis that those players compelled to grind for hours and complete 100% of the content of their video games would experience greater problems. In previous research, Yee  suggesteThe evidence that problem video game use is related to emotion regulation or self-escape suggests that pathological game use may be motivated by psychological mechanisms similar to those compelling substance abuse . Games in this style are very different from today's MMORPGs. This study provides initial tools to understand game use across many different genres and styles—even sports and other non-video games.While MMORPGs such as The present research also helps to create a tool for understanding individual differences between players and sources of satisfaction. It has been demonstrated that poor-quality games  are worse at satisfying SDT needs than are high-quality games  . The smaller pool of items will decrease the time required to complete the survey and the likelihood that at least one question will be marked “Not Applicable,” thereby reducing attrition.We conclude by urging researchers to consider specific characteristics of players, their personalities, and the games that they play. A common pitfall in video game research is to treat games as being homogenous machines which convert time into virtual gold and slain dragons, or worse, a vehicle for the delivery of scenes of violence to a passive spectator. Players are active participants in their games and exhibit heterogeneous preferences in the games they play. Players are motivated to play games insofar as those games can provide the fulfillment of psychological needs (Przybylski et al., This research was supported by a Bond Life Sciences Fellowship awarded to Joseph Hilgard.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of the present study was to systematically review fluoride concentration of drinking waters and prevalence of fluorosis in Iran through systematically evaluating results of studies conducted in this regard. In this systematic review study, the required data was collected using keywords including drinking water fluoride, fluoride concentration, Fluorosis, dent*, Iran*, and their Persian equivalents through PubMed, ScienceDirect, IranMedex, SID, MEDLIB, and Magiran databases. Out of 617 articles, 29 articles were finally considered after excluding the remaining articles which were not related to the study objectives. Following precise studying and extraction, the relevant data were summarized in extraction tables and analyzed manually. Excel 2007 software was used to draw diagrams. 4434 samples of surface, ground, and tap water resources collected within 236 months during all seasons in 17 provinces of Iran were used in 29 articles determining fluoride concentrations of drinking water. Average fluoride concentration was estimated to be 0.43 ± 0.17 ppm with zero and 3.06 as minimal and maximal values. The least concentration was seen in tap water. Fluoride concentration of only three provinces was in accordance with the global standard. According to estimations, prevalence of fluorosis was 61% with only 1% as severe fluorosis. Despite lower than standard concentrations of fluoride in drinking water, a relatively high level of fluorosis was seen in Iran. Therefore, the present study aimed at systematically evaluating the studies published on fluoride concentration of different water sources as well as prevalence of fluorosis and providing a clear and comprehensive viewpoint from status of fluoride found in drinking waters and prevalence rate of fluorosis.At present, fluoride concentration of drinking water and the dental caries are regarded as one of the most common health problems and main concerns of dentists,In this systematic review study, the relevant data was collected using keywords including “drinking water fluoride,” “fluoride concentration,” “fluorosis,” “dent*”, “Iran*” and their Persian equivalents through PubMed, ScienceDirect, IranMedex, SID, MEDLIB, and Magiran databases. Articles published from 1990 to 2012, articles reporting fluoride concentration of drinking water and prevalence of fluorosis, and articles published in both Persian and English languages were regarded as inclusion criteria of the study. Articles presented at conferences as well as papers evaluating fluoride in resources other than water, e.g. tea, agricultural products, soil, etc., were excluded from the study. Following database searching, some authentic journals were manually searched in order to identify and cover more published articles. After excluding those articles relating weakly to the study objectives and selecting main articles, references of the selected articles were researched to being assured of identification and evaluation of the available articles and find more related articles. Out of 617 articles, 29 completely related articles were finally considered and the remaining not properly related to the study objecttives was excluded. The selected articles were completely and exactly evaluated . There wIn this study, out of 617 articles, 29 articles completely related to the study objectives were finally selected, exactly studied, and the relevant data extracted , and surface waters (river and pond). The evaluated studies considered 4434 samples of different water resources of the country. Also, 3727 students ranging 7-16 years old were studied to evaluate prevalence of fluorosis. To determine fluorosis severity, Dean Index was used in all studies. As seen, the highest rate of fluoride concentration of different water resources is attributed to the year 2002. The average fluoride concentration of drinking waters was 0.43 ± 0.17 ppm with zero and 3.06 as its minimal and maximal values obtained for Kermanshah and Khorasan Razavi provinces, respectively. In 4434 samples evaluated in the conducted studies, average fluoride concentration was estimated 0.43 ± 0.17 ppm with zero and 3.06 as minimal and maximal values. The highest and lowest concentrations were seen in well, and urban and rural tap waters, respectively. Out of 3727 studied students, only 1% suffered from severe fluorosis. 12 Although fluoride concentration in drinking waters of Iran is less than standard values and their fluoride content should be increased, its disadvantages should also be considered in planning and policy makings, as increasing fluoride concentration above the permitted level would lead to undesirable disorders and complications such as mental disability, change of human chromosome structure, renal damages, and osteomalacia.15 Additionally, the results of global studies have demonstrated that the higher the fluoride concentration of drinking waters, the higher the prevalence rate of fluorosis in the population.18 According to recent reports provided by experts of Iran Fluoride Scientific Association, exceeding fluoride concentration of drinking waters from 0.7 mg/l may increase disadvantages rather than desirable effects of caries prevention.19 Reviewing studies conducted on measurement of drinking water fluoride in the US demonstrated that adding fluoride to drinking waters was not only useless but entailed certain disadvantages and economical costs.20 By precise evaluation of drinking water fluoride and its comparison with standards, authorities and experts should make appropriate decisions and offer proper plans to exactly adjust fluoride concentration of drinking waters. Mean scores obtained from the studies conducted in Iran (0.43 ± 0.17 ppm) is less than standard values of 0.7 ppm for tropical and 1.2 ppm for cold regions recommended by World Health Organization. Therefore, appropriate plans and policies should be designed and executed to standardize fluoride concentration of different water resources of Iran. According to results of the study, most interventions should focus on increasing fluoride of drinking water since fluoride insufficiency leads to caries.Fluoride concentration of drinking waters of every province is demonstrated in 22 Considering fluoride concentration found in the present study, it can be stated that the prevalence of fluorosis in South Africa is higher than Iran. The study conducted on 12-15 year-old students in Khuzestan reported prevalence rate of fluorosis about 51%, which is just below the average value calculated for the country. The results of the present study are in accordance with those of the Hormozgan province.23 Despite low fluoride concentration in drinking waters of Iran, high prevalence rate of fluorosis is seen in this study. Other resources including foodstuff, tea, and toothpastes may be regarded as the most important and justifiable sources of high fluoride dose thath individual receive. Further studies are warranted in order to identify main factors resulting in high prevalence of fluorosis. In researches evaluated in this study, 3727 subjects were studied considering fluorosis. Generally, about 2274 cases (61%) suffered from different degrees of fluorosis. Of these, only 38 students (1%) experienced severe fluorosis. According to reports, the prevalence of fluorosis with fluoride concentration of 0.19 ppm was 47% in South Africa.Lack of meta-analyses of the extracted data due to different ways of data presentation is regarded as one of the drawbacks of the present study. It is recommended to use the same specific procedures in presenting the results of such studies. This was the reason why it was not possible to statistically evaluate the relationship between fluoride concentration and prevalence of fluorosis in this study. The results of the present study demonstrate that although average fluoride concentration in drinking waters of Iran is less than recommended standards, it is still higher than standard level in some regions. Generally, there are different concentrations of water fluoride in Iran due to variable ecological conditions of the country. Considering different effects of variable concentrations of fluoride, setting uniform policies for all regions of the country may result in undesirable complications. Therefore, appropriate policies should be made based on regional conditions as well as specific fluoride concentrations. Also, high prevalence of fluorosis in Iran was noted in spite of low fluoride concentrations, which may have resulted from fluoride uptake from other resources.