In the UK, men's alcohol-related morbidity and mortality still greatly exceeds women's, despite an increase in women's alcohol consumption in recent decades. New UK alcohol guidelines introduce gender-neutral low-risk alcohol consumption guidance. This study explores how UK newspaper and online news represent women's and men's ‘binge’ drinking to identify opportunities to better align reporting of harmful drinking with evidence.Quantitative and qualitative content analysis of 308 articles published in 7 UK national newspapers and the BBC News website between 1 January 2012 and 31 December 2013.Articles associated women with ‘binge’ drinking more frequently than men, and presented women's drinking as more problematic. Men were more frequently characterised as violent or disorderly, while women were characterised as out of control, putting themselves in danger, harming their physical appearance and burdening men. Descriptions of female ‘binge’ drinkers' clothing and appearance were typically moralistic.The UK news media's disproportionate focus on women's ‘binge’ drinking is at odds with epidemiological evidence, may reproduce harmful gender stereotypes and may obstruct public understandings of the gender-neutral weekly consumption limits in newly proposed alcohol guidelines. In order to better align reporting of harmful drinking with current evidence, public health advocates may engage with the media with a view to shifting media framing of ‘binge’ drinking away from specific groups  and contexts (public drinking) and towards the health risks of specific drinking behaviours, which affect all groups regardless of context. Quantitative and qualitative content analysis of a large, comprehensive, 2-year sample of UK national newspaper news and online news about ‘binge’ drinking.The findings illustrate how media portrayals of ‘binge’ drinking could be harmful, and identify opportunities for these portrayals to be better aligned with evidence.Content analysis facilitates understandings of the messages being presented to the public, but cannot determine the extent to which audiences' understandings are influenced by media representations of specific issues.Some findings may have differed if the data analysed had gone beyond text articles, for example, by incorporating images, video and social media content.While the gap between women's and men's excessive and harmful consumption of alcohol in the UK has narrowed in recent years,As with most health issues, popular perceptions of drinking are likely to be influenced by mass media representations,11et alet alet alet alResearch illustrates a clear gender divide in media portrayals of drinking behaviours, with men's drinking normalised and women's problematised. Day et alet alAs NichollsThis study comprises a comprehensive, mixed-methods content analysis of 2 years of UK media coverage of binge drinking, designed to contribute new insights into a growing body of literature about gendered media representations of alcohol, with a particular focus on binge drinking. Our aim is to improve understandings of UK newspaper and online news representations of women's and men's ‘binge’ drinking, focusing not on how ‘binge’ drinking is defined, but rather on how different types of drinking behaviour are gendered in media content where those behaviours are labelled as ‘binge’ drinking. While the limitations of media content analysis must be taken into account, the improved understandings produced by this research may help to inform efforts to better align media representations of harmful drinking with current evidence, which could in turn improve public understandings of the risks of single-episodic drinking.We selected seven highly circulatedWe selected a search period of 1 January 2012 to 31 December 2013, during which alcohol was subject to heightened media interest due to debates in the UK and Scottish parliaments around MUP.To systematically and comprehensively code and analyse the media content, we used a mixed-methods content analysis. First, we used quantitative content analysis to measure the frequency of content within the articles across the whole sample, and second, we performed qualitative content analysis of the content of a subsample of articles for more in-depth, reflexive analysis.To construct a coding frame with which to code manifest content, SH and CE read randomly selected articles from the sample in batches of 20, recording potential thematic categories relevant to the research topic as they emerged. At the point where a batch of articles was read without any new categories emerging, the list of categories was deemed to have reached saturation. The collected thematic categories were grouped into three broad thematic categories: How is binge drinking described and which sections of the population are associated with this behaviour?; How are the drivers of the binge drinking described and who is to blame?; and What are the consequences of binge drinking? These categories were chosen by a combination of a priori knowledge of the research topic and understandings of the content of the sample that emerged from close reading of the articles.2 tests of whether thematic variables varied by gender focus, publication genre or article format; and paired t-tests comparing the means of thematic variables. The threshold of statistical significance is set at 0.01 throughout to mitigate the risk of type 1 errors.To collect quantitative data, OM read each article in turn, using the coding frame to record whether each theme was present within its manifest content. To ensure consistency of coding, GF coded a random subsample of 39 articles (12.7%). Linearly weighted κ tests of inter-rater agreement between OM and GF were then performed on each variable across those 39 double-coded articles, and variables that returned a coefficient below 0.8 were discarded from the study to ensure that only variables with strong agreement were retained for analysis. The coding frame data were then entered into SPSS for analysis. Statistical procedures comprised: a simple linear regression examining the relationship between publication quarter and the count of articles published; χFollowing quantitative analysis, the thematic category of ‘harms to appearance’ was deemed noteworthy and suitable for qualitative coding and analysis. The decision to focus on that theme was informed by stark gender differences in content that emerged from the quantitative analysis, and an expectation that valuable understandings could be gained from deeper analysis. To analyse the latent content of the 46 articles that had been coded as mentioning ‘harms to appearance’, GF and CP employed a thematic analysis approachBetween 1 January 2012 and 31 December 2013, 308 articles about binge drinking were published in the seven print publications and one website included in the sample. Of these articles, 86 (27.9%) were published in ‘quality’ newspapers, 39 (12.7%) in middle-market tabloids, 75 (24.4%) in tabloids and 108 (35.1%) on BBC News . The majArticles were coded according to whether they mentioned binge drinking in women exclusively , men exclusively , both men and women , or neither men nor women explicitly . To be coded as mentioning both men and women, an article had to include specific discussion of each gender individually. There were no significant associations between gender category and either genre (p=0.382) or article format (p=0.303). Furthermore, neither publication genre nor article format was significantly associated with differences in reporting on any of the thematic categories listed in Articles were coded to identify the characteristics of binge drinkers and the specific drinks associated with binge drinking. Thirty-two (10.4%) of all 308 articles associated mothers (including pregnant women) with binge drinking, which differed significantly (p<0.000) from the 11 (3.6%) that mentioned fathers . There wAlmost two-thirds of articles  described health harms related to binge drinking, while one-quarter (n=77) mentioned risk of death. Mentions of specific categories of harm were recorded , and relAs identified in the quantitative findings, just 8 of the 46 articles that reported on ‘harms to appearance’ mentioned men, and none did so exclusively. These 46 articles were subjected to thematic analysis. A broad conceptualisation of ‘harms to appearance’ was used that included behavioural as well as aesthetic aspects of physical appearance that may be influenced (or perceived to be influenced) by single-episodic drinking. Using this broad definition, two key themes emerged from thematic analysis of the subsample: physical appearance and loss of self-control.Various harms to complexion were reported in relation to women, including the use of make-up to hide grey skin. Articles described specific damage to women's features, including hair, lips, noses and teeth, as well as using less specific descriptions of physical damage such as ‘ravaged’ . An article headlined ‘Lambrini ruined my looks in 5 months’  associated an ‘attractive’ woman becoming ‘a swollen, haggard wreck’ with the consumption of a perry overtly marketed to young women. Weight gain caused by alcohol consumption was cited as both a product of alcohol use and a cause of ‘drunkorexia’, the practice of eating less to offset calories gained from alcoholic drinks.Within the few articles in the subsample that mentioned men, male partial nudity was related to drunken behaviours, such as lost and ripped clothes, and intentional indecent exposure. Typifying this, the Daily Mail described a male student ‘flashing his penis in the street and laughing at his own loutish behaviour’ (19 June 2012). Conversely, partial nudity in women was more typically related to clothing choices presumably made while sober, with descriptions of revealing clothing, particularly miniskirts, and impractical shoes. One article described a fancy dress event in which ‘undergraduate girls take to the streets dressed in little more than their underwear’ . Women were variously described as ‘scantily-clad’ , ‘half-naked’  and ‘nearly bearing all’ . Aside from choices of attire made in sobriety, harms to appearance that could befall women during drinking episodes included smeared makeup, vomit in hair, stained clothes and gravel embedded in knees. Articles also made mention of unintentional exposure of underwear or body parts by women, which was in contrast to men, who were typically described as exposed their genitalia deliberately.Descriptions of women's and men's loss of physical control differed. Some articles depicted drunken women as burdening male partners. For example, two articles described women vomiting on their boyfriends, while one described female students needing ‘to be carried back to their rooms by boyfriends’ . No articles characterised ‘binge’ drinking men as relying on, or burdening, their partners.Women's aggressive behaviours were limited to verbal conflict, with drunken women characterised as noisy, argumentative, emotional and hard to control. An article describing Cardiff nightlife reported that drunken women ‘scream at their boyfriends in shop doorways’ and ‘sob in public’ . Men were more typically associated with aggressive behaviours, including violent language and actions, damaging property and non-specific ‘loutish’  behaviour. While male aggression was presented as endangering others, women were characterised as putting themselves at risk, sometimes of undesirable sexual situations and assault. One article reproduced Office of National Statistics data about fines for drunk and disorderly behaviour, presenting men as much more likely than women to receive such fines, but suggesting that the number of women receiving the fines is increasing more rapidly than men .We examined UK newspaper and online news representations of ‘binge’ drinking, exploring differences and similarities in representations of women's and men's drinking behaviours. Our quantitative analysis identified that women were associated with binge drinking more frequently than men, which conflicts with epidemiological evidenceOur qualitative analysis focused on depictions of the relationship between ‘binge’ drinking and appearance, comprising aspects of physical appearance and behaviour. Women engaged in ‘binge’ drinking were presented as helpless, physically incapacitated and transgressive, and as burdens to male partners, who were sometimes cast as carers for drunken women. ‘Binge’ drinking men were associated with physical and verbal aggression, but not behaviours that marked them as weak or vulnerable; while women were characterised as endangering themselves, men were more likely to endanger others. Articles typically depicted women as less able than men to maintain socially acceptable behaviour during single-episodic drinking. ‘Binge’ drinking was characterised as affecting women's physical appearance more than men's. Articles associated binge drinking with both immediate and long-term damage to physical characteristics and self-presentation. Descriptions of binge drinking women's dress had a moralistic tone that was absent in representations of men, with articles typically focusing on degrees of nudity and implicitly questioning the propriety of women's chosen attire. Our analysis suggests that media representations of women's ‘binge’ drinking do not focus solely on the effects of binge drinking on women, but also reflect broader social expectations about women's public behaviours.st century tabloid newspapers, became the young woman emulating male consumption patterns […] with clothes askew stumbling around the city centre streets at night’.(p. 417)The public face of binge drinking, […] a staple of early 21Measham and ØstergaardThere may be unintended consequences of this disproportionate focus on women's—as opposed to men's—single-episodic drinking in media reports. For example, it may reinforce harmful gender stereotypes by suggesting that drinking is more problematic for women than men, and may encourage victim blaming in relation to sexual assaults after drinking.et al,et alet alsOur findings build on the growing body of literature about gendered media representations of alcohol by focusing specifically on portrayals of binge drinking. Depictions of women as unable to control themselves physically and emotionally echoed the findings of Atkinson et al'sOur analysis supports Herring Our findings have relevance to the development of alcohol guidelines. While the new UK guidelines propose gender-neutral low-risk consumption guidelines, the Department of Health acknowledges that men and women vary in their drinking behaviours and the long-term and short-term health risks they face.Our conclusions are subject to limitations. While mass media's influence on public perceptions of health issues is extensively researched and well established,In conclusion, our analysis suggests that popular representations of binge drinking may be harmful in three ways. First, if public audiences' understandings are influenced by media coverage of ‘binge’ drinking, associations of ‘binge’ drinking with certain demographics and situations could lead the public to underestimate the health risks of single-episodic drinking among those not typically depicted in ‘binge’ drinking narratives, particularly older people. Morally loaded representations of women's binge drinking may reproduce harmful stereotypes and stigma about the vulnerability of drunken women and the social unacceptability of female drunkenness. Finally, media content reinforcing a skewed representation of binge drinking may present a challenge to public health stakeholders seeking to promote evidence-based information, recommendations and policies with the goal of reducing alcohol-related health harms.The misrepresentations identified by this research suggest that there may be a need for health advocates to engage with mass media to promote clear, evidence-informed messages about single-episodic drinking to help better align reporting on harmful drinking with evidence, with the ultimate goal of improving public understandings of harmful drinking behaviours. Media communication strategies may seek to avoid stereotypes of ‘binge drinking’ that implicitly define binge drinking in terms of social contexts and behaviours not directly related to alcohol consumption, instead offering clear, value-free definitions of single-episodic drinking based on specific quantities of alcohol and specific, time-bound episodes. Identifying the need to improve communication of single-episodic drinking is particularly relevant given the recent public consultation about new alcohol guidelines,


However, the combined effect and relationship of these two factors over Cx36-dependent coupling have not been previously studied in detail. Our experimental results in HeLa cells expressing Cx36 show that changes in both pHi and [Mg2+]i affect junctional conductance (gj) in an interdependent manner; in other words, intracellular acidification cause increase or decay in gj depending on whether [Mg2+]i is high or low, respectively, and intracellular alkalization cause reduction in gj independently of [Mg2+]i. Our experimental and modelling data support the hypothesis that Cx36 GJ channels contain two separate gating mechanisms, and both are differentially sensitive to changes in pHi and [Mg2+]i. Using recombinant Cx36 we found that two glutamate residues in the N-terminus could be partly responsible for the observed interrelated effect of pHi and [Mg2+]i. Mutation of glutamate at position 8 attenuated the stimulatory effect of intracellular acidification at high [Mg2+]i, while mutation at position 12 and double mutation at both positions reversed stimulatory effect to inhibition. Moreover, Cx36*E8Q lost the initial increase of gj at low [Mg2+]i and double mutation lost the sensitivity to high [Mg2+]i. These results suggest that E8 and E12 are involved in regulation of Cx36 GJ channels by Mg2+ and H+ ions.Connexin-36 (Cx36) protein forms gap junction (GJ) channels in pancreatic beta cells and is also the main Cx isoform forming electrical synapses in the adult mammalian brain. Cx36 GJs can be regulated by intracellular pH (pH GJ channels serve as an intercellular pathway for ions, small metabolites such as IPj)   family. Structural studies have revealed that Cxs comprise four transmembrane domains (M1-M4), two extracellular loops (E1 and E2), one cytoplasmic loop (CL), and cytoplasmic N- and C-termini (NT and CT). It is well-established that GJs formed of Cxs can be regulated by transjunctional voltage  also has some distinctive characteristics. Unlike other Cx isoforms, junctional conductance (gj) of Cx36 GJs can be upregulated under low pHi  could reveal new modulatory mechanisms of GJs in physiology and pathology.Both pHDurlach, , while p2+]i and pHi on gj between cells expressing Cx36-EGFP. Our data revealed that after gj was reduced by high [Mg2+]i, it could be recovered by intracellular acidification with sodium acetate (CH3COONa). In contrast, after gj was elevated by low [Mg2+]i, both alkalization or acidification with ammonium chloride (NH4Cl) or CH3COONa, respectively, induced a reduction of gj. To consider the most appropriate amino acids which could be involved in Cx36 GJ channel regulation by H+, we used homology modelling to generate a three-dimensional structure of Cx36 using the crystal structure of Cx26  exhibited pKa values which were closest to physiological pH. Thus, we performed experiments with three Cx36 mutants, Cx36*E8Q-EGFP, Cx36*E12Q-EGFP and Cx36*E8Q-E12Q-EGFP, in which negatively charged glutamates were substituted to uncharged glutamines. Experimental results showed that acidification-induced gj increase at high [Mg2+]i in Cx36-EGFP was abolished in Cx36*E8Q-EGFP, while Cx36*E12Q-EGFP exhibited a small gj decrease under the same conditions. The most prominent decrease of gj at high [Mg2+]i during acidification was observed in double mutant Cx36*E8Q-E12Q-EGFP. Therefore, these amino acids could be involved in modulatory mechanisms of Cx36 GJ channels by both, [Mg2+]i and pHi.In this study, we examined the combined effect of i we used pipette solutions containing 0.01, 1 or 5 mM of MgCl2. Differences in osmolarity were compensated with the appropriate concentration of CsCl.Cells were perfused with modified Krebs-Ringer (MKR) solution containing (in mM): 140 NaCl, 4 KCl, 2 CaCl3COONa or NH4Cl. All extracellular solutions were adjusted to pH = 7.4. To reduce pHi to 6.5 and 6.0, we used physiological solution containing 20 and 100 mM of CH3COONa, respectively, and to increase pHi to 7.6, 7.9, and 8.2, we added to the physiological solution 1, 3, and 10 mM of NH4Cl, respectively , which was introduced into the cells through the patch pipettes in a whole-cell voltage clamp mode. The dye was alternately excited with 436 and 500 nm wavelengths, and the emitted light was filtered at 540 nm. The ratios of emitted light collected at excitation wavelengths of 436 and 500 nm (background subtracted) were converted to pHi values based on a calibration curve. The latter was obtained by applying the solutions of different pH  and using ionophore nigericin (20 μM) in the presence of potassium (140 mM) to equilibrate the pHi with an extracellular medium of different pH  software. An excitation filter of 470 nm, and emission filter of 540 nm were used to identify the cell pairs expressing Cx36-EGFP and its mutants. For pHotassium 40 mM to t-test. Differences were considered statistically significant at p < 0.05.Experimental data are reported as the representative result or as mean of at least four independent experiments ± standard error (SEM). Statistical analyses were performed using unpaired Student's 2+]i can substantially modulate gj-pHi dependence of Cx36-EGFP GJs  in pipette solutions ([Mg2+]p). In these experiments, the initial gj  at control pHi = 7.3 was registered immediately after patch opening. Typically, gj increased or decreased at low or high [Mg2+]i, respectively, until it reached a steady-state   before washing out the applied substance. To prepare these solutions, we used modified Ringer's solution in which NaCl was exchanged for equal concentration of CH3COONa or NH4Cl to maintain osmolarity. Extracellular pH (pHo) remained unchanged during the experiments. Solution and pHi measurement protocols are presented in the Methods section. The control experiments in non-transfected HeLa cells showed low conductance in very rare cases (1 in 20), which could be attributed to activity of endogenous connexins in these cells (data not shown).Preliminary data suggested that p. The mean values of -pHi dependence at 0.01 and 1 mM [Mg2+]p were fitted to an equation describing biphasic effects . At [Mg2+]p = 1 mM, acidification to pHi = 6.6 first induced an increase of gj, eff/gj, ss to 1.16 ± 0.08 (n = 8), while further acidification to pHi = 6.0 returned gj, eff/gj, ss to 0.97±0.07 (n = 12). At [Mg2+]p = 5 mM, acidification to pHi = 6.0 increased gj, eff/gj, ss to 1.57 ± 0.15 (n = 4). Figure j, eff/gj, init at different [Mg2+]p, to represent the effect of [Mg2+]i on gj and the consequent changes by altering pHi; at pHi = 7.3 this ratio coincides with gj, ss/gj, init. From Figure j, eff/gj, init)-pHi dependencies shift toward the acidic side as the value of [Mg2+]p increases. Therefore, gj, eff/gj, init reaches a maximum value at pHi range between 7.1–7.5, 6.6–6.9 and below 6.0 for [Mg2+]p of 0.01, 1, and 5 mM, respectively. These experimental results clearly show that pHi and [Mg2+]i regulation of Cx36 is interrelated, particularly at lower pHi values.Figure 2+ ions' interaction with Cx36 protein as they form the path for Mg2+ ions to enter the channel and could be involved in their binding. In order to determine the amino acids which also could be sensitive to pHi, the x-ray crystal structure of Cx26  have pKa values equal to 6.5 and 7.2, respectively. These pKa values were the closest to physiological pHi among other amino acids in the NT domain of Cx36, thus indicating that carboxyl groups at side chains of E8 and E12 may be sensitive to protonation and deprotonation at physiological pHi values. Based on these predictions, E8 and E12 were chosen for point mutations. As substitutes for glutamates, we chose glutamines , which are neutral but polar. Therefore, the substitution of glutamate by glutamine neutralizes the negative charge and abolishes channel protonation, which possibly affects the dynamic behavior of a gate that is pH-sensitive and/or Mg2+-sensitive. Moreover, double mutation, where both E8 and E12 were substituted by glutamines, was also analysed.Some studies have shown that gating polarity and single channel conductance of GJ channels are determined by charged amino acids of the NT domain  was observed at [Mg2+]p = 5 mM. During alkalization to pHi = 8.2 the gj, eff/gj, ss decreased ~70–90% at all [Mg2+]p. Acidification to pHi 6.0 and 6.5 decreased gj, eff/gj, ss of double mutant ~50 and 40% at 0.01 and 1 mM [Mg2+]p, respectively, and ~35 and 5% at 5 mM [Mg2+]p, respectively -pHi dependencies for all mutants were less influenced by rising [Mg2+]p than it is for Cx36-EGFP.The experimental results showed that acidification to pHp Figure , and at y Figure . The uncj, ss/gj, init of Cx36-EGFP, Cx36*E8Q-EGFP, Cx36*E12Q-EGFP and Cx36*E8Q-E12Q-EGFP are presented in Figures 2+]p on gj, ss/gj, init for all mutants is comparable to Cx36-EGFP, with the exception of the E8Q mutation, which abolishes the increase of gj at low [Mg2+]p -pHi between three mutants and Cx36-EGFP is represented in Figures 2+]p causes the strongest uncoupling of cells transfected with Cx36*E12Q-EGFP  and open channel probabilities determined by both acidic and alkalic sensing domains:Here, parameter j curves of pacid and palk under different [Mg2+]i to reproduce experimentally observed gj values. The model parameters at different levels of [Mg2+]i are presented in Table acid of Cx36-EGFP does not depend on pHi at high [Mg2+]i and increases with pHi rising at lower [Mg2+]i  with most prominent difference for Cx36*E8Q-E12Q-EGFP. In addition, overall pacid values were significantly reduced for Cx36*E12Q-EGFP at [Mg2+]i = 5 mM and for double mutant these values were reduced at 1 and 5 mM [Mg2+]i. The dependence of palk on pHi remains comparable to that of Cx36-EGFP and does not significantly change at all [Mg2+]i for all three mutants , located in the channel pore . In Palacios-Prado et al. , residing in the Cx26 GJ channel pore near the extracellular gap could coordinate Ca2+ binding (Bennett et al., 2+. Negatively charged glutamates of the NT domain, which forms the vestibule of the GJ channel (Purnick et al., 2+ ions or facilitate their pass through the channel. At high [Mg2+]i, E8Q and E12Q mutations abolished the stimulating effect of acidification and double mutation of these two amino acids even enhanced uncoupling in the same conditions. In addition, all mutations increased the uncoupling effect at low [Mg2+]i. The effect of [Mg2+]i on gj at control pHi remained comparable to Cx36-EGFP, with the exception of the E8Q mutation, which eliminated the increase of gj at low [Mg2+]i and the most prominent difference was obtained with double mutation, which basically abolished the uncoupling effect of high [Mg2+]i. Our modelling results indicate that both mutations modified the open channel probability determined by the acidic sensing domain, particularly at high [Mg2+]i. In particularly, sensitivity of acidic gating to high [Mg2+]i is gained in mutants with most prominent manifestation in double mutant (Figures i at different [Mg2+]i as compared with Cx36 WT and both single mutations.Mg2+ and H+ ions to E8 and E12. However, the remaining sensitivity of Cx36*E12Q-EGFP to Mg2+ ions at control pHi imply that E12 is not a part of the Mg2+-binding site. Moreover, the lost sensitivity of Cx36*E8Q-EGFP to low [Mg2+]i and the remaining response to high [Mg2+]i at control pHi, suggest that Cx36 could have two or more binding sites with different affinity to Mg2+, similar to TRPM7 channels (Chokshi et al., 2+. These residues are located in the NT domain, which forms the entrance into the channel (Beyer et al., 2+ ions to reach the D47 residue, located at the first extracellular loop. Thus, the substitution of E8 or E12 could disturb local [Mg2+]i at the channel pore, which might explain why the E8Q mutation causes the loss in sensitivity to high [Mg2+]i and the E12Q mutation led to a decrease of gj during acidification at high [Mg2+]i. The loss of double mutant sensitivity to high [Mg2+]i and the increased uncoupling during acidification also imply that these two amino acids are important for Cx36 GJ channels regulation by Mg2+ and H+.The most plausible interpretation of these results might include the direct binding of Mgi and [Mg2+]i effect on gj and the role of the NT domain are unclear. Further investigations are needed to determine the regulatory sites for acidification and their dependence on Mg2+.Overall, the exact mechanism of pH2+]i can modulate the sensitivity of Cx36 channels to pHi. The low sensitivity of Cx36 GJs to low pHi was proposed to act as a preventive mechanism of the function of electrical synapses during brain ischemia (González-Nieto et al., 2+]i. There are a number of studies which have demonstrated therapeutic effect of Mg2+ in the treatment of brain ischemia (Westermaier et al., 2+]i and pHi effect on Cx36 plays at least a partial role in the protective mechanisms. For example, the depletion of ATP during brain ischemia (Sato et al., 2+]i (Henrich and Buckler, Our data show that [MgLR, VS, and FB: conception of the work, design of experiments, collection, analysis and interpretation of data, drafting of manuscript; TK and NP-P: recorded and analysed the experimental data; MS: constructed and applied mathematical models; NP-P and MS: critically revised the manuscript; VJ: performed experiments.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.


Cyanistes caeruleus) videos of demonstrators discovering palatable or aversive prey (injected with bitter-tasting Bitrex) from coloured feeding cups. First we quantify variation in demonstrators’ responses to the prey items: aversive prey provoked high rates of beak wiping and head shaking. We then show that focal blue tits respond differently to the presence of a demonstrator on a video screen, depending on whether demonstrators discover palatable or aversive prey. Focal birds faced the video screen more during aversive prey presentations, and made more head turns. Regardless of prey type, focal birds also hopped more frequently during the presence of a demonstrator (compared to a control video of a different coloured feeding cup in an empty cage). Finally, we tested if demonstrators’ behaviour affected focal birds’ food preferences by giving individuals a choice to forage from the same cup as a demonstrator, or from the cup in the control video. We found that only half of the individuals made their choice in accordance to social information in the videos, i.e., their foraging choices were not different from random. Individuals that chose in accordance with a demonstrator, however, made their choice faster than individuals that chose an alternative cup. Together, our results suggest that video playback can provide social cues to blue tits, but individuals vary greatly in how they use this information in their foraging decisions.Video playback is becoming a common method for manipulating social stimuli in experiments. Parid tits are one of the most commonly studied groups of wild birds. However, it is not yet clear if tits respond to video playback or how their behavioural responses should be measured. Behaviours may also differ depending on what they observe demonstrators encountering. Here we present blue tits ( Potential prey items differ in their nutritional benefits and palatability . TherefoMany bird species show a clear disgust response by vigorously wiping their beaks on a perch . This cuCyanistes caeruleus) use information from foraging conspecifics, and if their response differs depending on the palatability of food that demonstrators encounter. However, when studies involve experimentally manipulating social behaviour, it can be difficult to control what stimuli focal birds observe  females identify specific males that they see in videos (Corvus frugilegus) spend more time looking at a video of their partner, compared to a video of a nonaffiliated conspecific  and blue tit populations in the area and birds have been given British Trust of Ornithology (BTO) ID rings and fitted with small passive integrated transponder (PIT) tags (fitted to a colour ring) since 2012. In January, five sunflower seed feeders were fitted with PIT tag reading antennae and data loggers that scanned birds’ unique PIT tag codes when they landed on a feeder. During the winter, great tits and blue tits form loose fission–fusion flocks that move between food sources , an established study site in Cambridge, UK. There is an ongoing long-term study of great tit  were captured with mist nets (by HMR) in February 2016. Individuals were chosen from the population randomly, but PIT tag records enabled us to calculate association strengths for each observer-demonstrator pair used in the experiment. All captured birds were adults (based on plumage), but their sex was unknown. Birds were housed indoors in individual plywood cages (80 cm × 65 cm × 50 cm) with a daily light period of 12 h. Food  and water were provided ad libitum except prior and during the experiment when food restriction was necessary. This restriction lasted no longer than one hour, following guidelines to the operation of the Animal (Scientific Procedures) Act 1986 (2009), and the Association for the Study of Animal Behaviour’s Guidelines for the treatment of animals in behavioural research and teaching (2012). Birds were kept in captivity for a maximum of four days until released at the capture site, and they were in auditory  contact during housing and experiments. The study was conducted under existing Home Office (PPL 60/4322) and Natural England (2015-6665-SCI-SCI-3) licenses held by HMR.Wild blue tits . Bitrex tastes bitter to humans  and elicWe used six individual blue tits as demonstrators for the videos. Three of these birds were first used as observers in the social information use test before recording them as demonstrators. Birds were moved from their home cage to wooden test cages (66 cm × 50 cm × 50 cm) that differed from the home cage in that they had a front wall made of plexiglass. We coated the plexiglass with tinted film that made it possible to observe and film the birds while minimising effects on their behaviourWe first filmed the demonstrators eating a palatable mealworm in a coloured feeding cup (yellow or green). We then filmed the same individual eating a Bitrex mealworm in a different coloured cup (blue or purple). Because an experience of prey with chemical defences was likely to affect birds’ response towards palatable prey and their willingness to consume it, we always filmed responses towards palatable prey first.From the videos, we quantified differences in demonstrators’ response to palatable and aversive prey from first contact with the prey item until 10 s after eating. We measured (i) how long demonstrators spent wiping their beaks on a perch (in seconds), (ii) the number of beak wipes they performed, and (iii) the number of times the head was shaken. We then used these videos to create standardized videos to present to observers see  and S2.n = 22) two videos, once for each prey type. These two videos were shown on sequential days and the order was randomized among birds. We did not change the demonstrator between different prey types, so observers saw the same demonstrator encounter both palatable and aversive prey. Therefore, we think that any differences in responses of the observers are likely due to the prey type, not the demonstrator’s identity.All videos included 45 s of a demonstrator finding a prey item in a coloured cup and a demonstrator’s response to that prey. In addition, each video included 60 s of a different coloured cup in an empty cage to make sure that observers were familiar with both cup colours and their foraging choice would depend on the information in the video instead of novelty of cups. Thirty seconds of this control video was shown to observers before a demonstrator appeared on the screen and 30 s was shown after a demonstrator, as we predicted that observers might pay attention to the cups differently after seeing a foraging conspecific. We used green and yellow cups when demonstrators encountered palatable prey (randomizing which cup colour was shown with a demonstrator and which in an empty cage), and blue and purple cups when demonstrators encountered aversive prey. We showed each observer  against the plexiglass front wall of the cage for 15 min before showing the video . This diThe entire cage was not in the field of view of the camera, so sometimes birds flying close to the roof of the cage, or holding on near the roof, could not be seen in the video. Therefore, we excluded individuals from our dataset that were visible for less than 30 s during the whole 105 s video (60 s of cups only and 45 s of a demonstrator). After removing these individuals, our dataset included 13 individuals that were shown videos of a demonstrator encountering both palatable and aversive prey, and 3 individuals that were visible only when they were shown a video of a demonstrator encountering palatable prey. The final sample size for video analysis was therefore 16 observers when prey was palatable  and 13 observers when it was aversive .n = 22) used social information from the videos by giving them a choice between two different coloured cups: the cup that a demonstrator fed from, and the cup that was shown in an empty cage without a demonstrator , demonstrating that our network differed significantly from random. We then calculated association between observers and demonstrators within our network of 331 individuals (217 blue tits and 114 great tits). Association strengths were scaled between 0 (never observed in the same flock) and 1 , and in our experiment, these values ranged from 0 to 0.070 . In addition, we calculated the total number of interactions  between demonstrators and observers: these ranged from 0 to 107 . As both association measures gave similar results when analysing observer’s behaviour, we decided to use only association strength in our final analyses.All analyses were conducted using software R 3.2.2 . We used package  to constn = 6). As the time that birds spent on foraging differed among demonstrators  we first divided the time spent on beak wiping and a number of beak wipes and head shakes with the total time foraging, and then compared these rates between aversive and palatable prey.We used a Wilcoxon signed ranked test to analyse differences in demonstrators’ behaviour when encountering aversive or palatable prey, to allow for the small sample size  as a response variable, and prey type , the observer’s association with its demonstrator, and the test order as explanatory variables, using binomial error distribution. The baseline level of the model included the video playback of aversive prey and a first video presentation. Because the distribution of time before birds chose the cup was right-skewed, we modeled it with a negative binomial error distribution, using the time before a choice as a response variable, and an interaction between prey type , social information use , and the test order as explanatory variables. The baseline level of the model included the video playback of aversive prey, a first video presentation and individuals that did not match a demonstrator’s behaviour. Bird identity and a demonstrator video were included as random effects in both models. Most of the birds landed on a cup during the first five minutes after cups were presented. Two individuals, however, were considerably slower at choosing in the aversive prey test, and landed on a cup only after 15 min. We therefore considered them as outliers, and removed them from the final analysis.p = 0.03) as well as the total number of beak wipes  and after  the presentation of palatable prey compared to aversive prey. There were no significant differences between video types in the way observers responded to cups only before the presentation of a demonstrator , suggesting that it was the behaviour of the demonstrator that influenced how long observers faced the video screen. Following presentation of any demonstrators, observers paid overall less attention to the screen showing cups only . In addition, association strength with a demonstrator had a significant effect on focal birds’ behaviour, showing that individuals faced the screen less when they were more closely associated with a demonstrator . The distribution of association scores, however, was skewed, as most of the individuals had low association scores with a demonstrator, and it is therefore difficult to interpret this result. Finally, bird identity (variance = 0.674) and demonstrator video (variance = 1.417), included to the model as random effects, explained some of the observed variation. The final model is presented in The time observers faced the video screen depended on the prey type a demonstrator encountered : observeZ = 0.640, p = 0.52; cups after demonstrator playback * prey type: estimate =0.169 ± 0.310, Z = 0.544, p = 0.59) and their interactions were therefore removed from the final model. The final model showed that a demonstrator’s presence, regardless of prey type , the test order  or the association between observer and demonstrator  did not affect the time that observers were flying during video playback, but random effects explained some of the observed variation , there was no effect of demonstrator presence and prey type on this behaviour  and after  the presentation of palatable prey compared to aversive prey. During the initial cup presentation, observers’ responses did not differ significantly between these video types . The test order  and association between demonstrator and observer  had no effect on the number of head turns performed. In addition, the variance estimates for the random effects were small , so we removed these interactions from the final model. The final model shows that birds were hopping significantly more in the presence of a demonstrator compared to initial cup presentation . Again, the test order  and the association with a demonstrator  had no effect on an observer’s behaviour, and the variance estimates for random effects were small .The number of hops that observers performed did not depend on the prey type a demonstrator encountered , the test order  and the association score with a demonstrator  did not have a significant effect on an observer’s cup choice, but demonstrator video, included as a random effect, explained some of the observed variation . Similarly, after video-playback of a demonstrator’s response towards aversive prey, only 12 birds avoided the cup that the demonstrator fed from, whereas nine birds landed on it first . Only five birds matched our predictions in both tests, choosing the same cup colour as a demonstrator after receiving information about palatable prey, and avoiding that colour after seeing a demonstrator’s disgust response. Again, this was not different from what would be expected if birds foraged randomly .Prey type in the video , or between the first and the second test . Therefore, in both tests birds chose the cup faster when their decision matched a demonstrator’s behaviour. The variance estimates for random effects were small .Click here for additional data file.10.7717/peerj.3062/supp-2Video S2The video shows a demonstrator encountering palatable prey .Click here for additional data file.10.7717/peerj.3062/supp-3Video S3The video shows an observer’s response to different sections of video playback: (i) a control section (a cup in an empty cage) before a demonstrator (30 s), (ii) a demonstrator encountering aversive prey (45 s), and (iii) a control section (a cup in an empty cage) after a demonstrator (30 s).Click here for additional data file.10.7717/peerj.3062/supp-4File S1More comprehensive presentation of the results from all GLM models described in the results section.Click here for additional data file.10.7717/peerj.3062/supp-5Data S1Click here for additional data file.


PI) of cerebral blood flow (CBF), which increases the risk of brain disease. However, the effect of drinking coffee on the PI of the CBF and its interaction with arterial stiffness remain unknown. Against this background, we hypothesized that an acute increase in arterial stiffness induced by drinking coffee augments cerebral pulsatile stress. To test this hypothesis, in 10 healthy young men we examined the effects of drinking coffee on the PI of middle cerebral artery blood velocity (MCAv) and brachial‐ankle pulse wave velocity (baPWV) as indices of cerebral pulsatile stress and arterial stiffness, respectively. Mean arterial blood pressure and baPWV were higher (P < 0.01 and P = 0.02), whereas mean VMCA and mean cerebrovascular conductance index were lower upon drinking coffee (P = 0.02 and P < 0.01) compared with a placebo (decaffeinated coffee). However, there was no difference in the PI of MCAv between drinking coffee and the placebo condition. These findings suggest that drinking coffee does not increase cerebral pulsatile stress acutely despite an elevation in arterial stiffness in the systemic circulation.Previous studies have suggested that the risk of ischemic stroke increases immediately after drinking coffee. Indeed, drinking coffee, that is, caffeine, acutely increases arterial stiffness as well as blood pressure and peripheral vascular resistance. On the other hand, it has been reported that arterial stiffening is associated with elevation in the pulsatility index ( Coffee is one of the most consumed beverages worldwide, but it has been suggested that chronic coffee drinking has a negative influence on cardiovascular function because of caffeine. Caffeine is a central nervous system stimulant of the methylxanthine class  or the pulsatility index (PI) of the cerebral blood flow (CBF), as an index of arterial stiffness or cerebral pulsatile stress, respectively, is associated with cardiovascular disease or acute stroke due to ischemic injury . In addition, all procedures conformed to the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained from all participants prior to the study. Ten healthy men  volunteered for this study. All participants were free of any known cerebrovascular, cardiovascular, or pulmonary disorders and were not taking any prescribed medication. The participants were also not habitual coffee consumers (mean consumption <100 mg of caffeine per day). They were asked to abstain from caffeinated beverages and to avoid strenuous exercise for 24 h before the experiment.Each participant underwent two different conditions in the experimental protocol at random and blind approach: caffeinated (Coffee) and decaffeinated coffee intake (Placebo). Each condition was performed on a different day. The average of days between first and second visit was 4 ± 2 day. During each experimental condition, after a 10‐min baseline measurement, each participant drank 250 mL of caffeinated (containing approximately 150 mg of caffeine) or decaffeinated coffee (containing approximately 4.5 mg of caffeine) at the same temperature (40°C). The participants were seated in a semirecumbent position in a reclining seat and rested quietly for 60 min after caffeinated or decaffeinated coffee intake. We measured hemodynamic variables throughout the experiment and baPWV at 30 and 60 min after caffeinated or decaffeinated coffee intake in a supine position because a plasma caffeine concentration reaches to peak at 0.44 h after oral coffee intake  was sampled using a leak‐free mask and measured using a gas analyzer . The baPWV was measured in a supine position before and at 30 and 60 min after drinking coffee using an automated polygraph apparatus . An electrocardiogram (ECG), a phonocardiogram , and arterial pressure waveforms of the right brachial and posterior‐tibial arteries (using air‐plethysmographic sensors) were simultaneously recorded. Characteristic points of ECG, PCG, and pressure waveforms were automatically detected using a band‐pass filter. Participants changed their position from seated upright to supine and took 15 min of supine rest for the baPWV measurement, as previously reported . Systolic (SBP), diastolic (DBP), and mean arterial blood pressures (MAP) were continuously monitored using finger photoplethysmography . MCAv was measured using transcranial Doppler ultrasonography . A 2‐MHz Doppler probe was placed over the left temporal window and fixed with an adjustable headband. End‐tidal partial pressure of carbon dioxide (PThe cerebrovascular conductance index (CVCi) was calculated by dividing mean MCAv by MAP. Gosling PI was calculated automatically as follows: (systolic MCAv − diastolic MCAv)/mean MCAv. The 1‐min average of hemodynamic variables was calculated before and at 30 and 60 min after drinking coffee. The baPWV was calculated using the following equation:P < 0.05.Data were analyzed using two‐way repeated measures analysis of variance . Following the detection of a significant interaction, post hoc Bonferroni tests were employed to identify the differences. All values are expressed as mean ± SD. The statistical significance for all two‐tailed tests was established at ETCO2 decreased at 30 and 60 min after drinking coffee; however, the Coffee condition did not change pulse MCAv and the PI of MCAv (Table At resting baseline, there was no difference in all hemodynamic parameters between Placebo and Coffee conditions. MAP and SBP were higher at 30 and 60 min after drinking in the Coffee condition than in the Placebo condition Table , whereasIn contrast to our hypothesis, the pulse MCAv and the PI of MCAv did not increase immediately after drinking coffee, despite an increase in baPWV. These findings suggest that drinking coffee causes acute systemic arterial stiffening, but does not increase pulsatile stress in the cerebral circulation. Therefore, the acute increase in the risk of an ischemic stroke immediately after drinking coffee may not be associated with pulsatile stress for the cerebral vasculature.Previous studies have reported that drinking coffee acutely increases the arterial blood pressure, vascular resistance, and arterial stiffness (Mahmud and Feely It has been suggested that an increase in pulsatile stress causes microvascular damage (Webb et al. The diet may affect vascular characteristics. For example, sodium intake was negatively correlated with arterial stiffness, while potassium intake was positively correlated with augmentation index (Garcia‐Ortiz et al. Drinking coffee also has a direct effect on the cerebral vasculature and subsequently causes acute cerebral vasoconstriction Table . This ceA methodological limitation of this study should be mentioned. First, the effect of drinking coffee on cerebral pulsatile stress was assessed from a small sample size in this study. Second, both baPWV and PI values have methodological limitations. The baPWV value may include an influence of change in arterial blood pressure. However, the previous studies (Hajjar et al. We investigated the acute effects of drinking coffee on the PI of MCAv in healthy young men. Unexpectedly, acute arterial stiffening induced by drinking coffee did not affect pulsatile stress in the cerebral circulation. These findings indicate that the acute increase in the risk of ischemic stroke observed immediately after drinking coffee may be caused by other factors rather than an elevation in pulsatile stress in the cerebral circulation.No conflicts of interest for all the authors.


Psychosis seminars enable service users, their carers and mental health professionals to meet outside of a formal care setting, increase understanding of mental illness and help establish a dialogue.To explore feasibility of psychosis seminars in the UK and the experiences of participants.Seven meetings attended by 25 people were held over a 3-month period. An open-ended questionnaire was returned by ten participants. Responses were subjected to content analysis.Benefits experienced were having an open forum for talking freely about mental health issues in a neutral space, learning from others about psychosis and hearing different views. Suggested adjustments were clarifying expectations of participants at the beginning, strengthening facilitation and increasing attendance.Psychosis seminars may help to establish a dialogue among users, carers and professionals and seem feasible in the UK, although adjustment to delivery can help their implementation.None.© The Royal College of Psychiatrists 2016. This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license. Each meeting focuses on a particular topic, which is selected by the participants.,,Psychosis seminars originated from the collaboration between a psychiatric survivor of the German Nazi regime Dorothea Buck, psychologist Thomas Bock and journalist Ingeborg Esterer.In other countries in which psychosis seminars were organised, for example, Ireland and Germany, provision of care is funded through personal health insurance, whereas in the UK the healthcare is funded by government based on taxpayer money. Given the difference in funding and organisation of care, in the UK, there may be different types of challenges for organising independent out-of-hours initiatives outside of regular care provision.The first attempt to establish psychosis seminars in the UK was made by East London NHS Foundation Trust (ELFT). The aim of this study is to examine the participants’ experiences of psychosis seminars in order to explore whether this model is suitable in the UK or requires adaptation for implementation in this country.The project was undertaken as the initiative of a provider of mental health services (ELFT) as a way to increase levels of carer and service user involvement.Service users, carers and mental health professionals in contact with or working for mental health services across the boroughs of City and Hackney, Newham and Tower Hamlets were invited to participate in the seminars. These boroughs are exclusively inner-city areas with high prevalence rates of severe mental health problems and high levels of deprivation.The seminars were held in line with the formal requirements, that is, outside office hours and at a non-hospital venue , which was close to public transport. Topics to be discussed in the seminars were jointly agreed by the participants. There was a rotation between service users, carers and professionals in chairing the meetings.At the end of the project, all participants were asked to complete a brief evaluation form containing the following open-ended questions: ‘What attracted you to attend the psychosis seminars?’ ‘If you did not attend meetings, what prevented you from attending?’ ‘If you did attend meetings, please list three things that you found helpful and three things that you did not like?’ ‘Please list three things that you think would improve the seminars, and how these could be achieved’.–,Participant responses were subjected to content analysis. This allowed the identification of trends and patterns in the data by distilling words into categories that share the same meaning.The steps for inductive content analysis outlined by Elo & KyngäsTwenty-five people attended the seven seminars held over a 3-month period. This included 15 female and 10 male participants with authors S.T. and S.P. among them. Although the number and the compositions of participants from the three groups varied at each seminar, a ‘core group’ of 10 participants attended most seminars. The cost of the project, including venue hire and refreshments, came to less than £1500.Qualitative data about the experiences with attending the seminars were returned by 10 (40%) participants, that is, those who regularly attended the seminars. Four of them were mental health professionals, two carers, three service users and one service user who was also a mental health professional. Respondents did not include authors S.T. and S.P.Three themes were identified in the qualitative data: providing an open forum, enhancing understanding of psychosis and adjusting delivery of seminars. The themes and their subthemes have been illustrated in This theme emerged from the participant’s feedback about why they attended psychosis seminars and what it was they liked about them. Psychosis seminars as an open forum theme encompassed two elements: the provision of inclusive and neutral space and the opportunity for the participants to talk freely.I enjoyed and found it very interesting to meet people from the three groups in a non-hospital environment. (carer 2)The inclusivity of psychosis seminars, the fact that they were not held in a treatment facility and the opportunity to meet people from different stakeholder groups was valued by participants:[I had the] chance to say my piece. (service user 3).It was very good that anyone could ask anybody anything and get a reply. (carer 2).Feeling comfortable to freely share one’s experiences with other people was facilitated by the lack of hierarchy in the groups. It was felt that the open forum was indeed shared by service users, carers and professionals.Talking freely was an important aspect of the open forum provided by psychosis seminars. For example, one professional stated that they attended the seminars because they wished to have an opportunity for open and free discussion focused on service user and carer concerns. Carers and service users appreciated that psychosis seminars enabled them to share their experiences freely and ask questions to professionals:democratic and equal status approach. One professional mentioned among the aspects that attracted him/her to attend was theI want to learn and understand as much as possible about psychosis and available treatment. (carer 2)Thought that I could learn something about the condition that I could use in future work with myself and others in mind. It seems a good way to find out what people want in terms of care, what people feel needs to be considered, as well as how psychosis affects people from each of these groups. Participants reported that they found meetings informative and felt that psychosis seminars helped them to learn more about psychosis and mental health services in general. It seems that participants were indeed keen on learning opportunities as some of them suggested having guest speakers with expertise or experience in mental health at the beginning of each seminar.People were enticed to attend psychosis seminars because they saw them as an opportunity for learning and broadening their horizons:I wanted to get a better understanding of what psychosis is. What it means for those experiencing it, professionals and carers. To help make sense of my experiences (…). (carer 7)Hearing a variety of perspectives and experiences at the seminars significantly contributed to enhancing participants’ understanding of psychosis. This was commonly noted as one of the most helpful aspects of psychosis seminars:To hear different experiences, understanding and opinions regarding mental health. (carer 7)… what I did find really useful was the variety of perspectives. There was also an acknowledgement of and respect for each other’s expertise. Service users and carers appreciated that professionals were involved in the seminars and professionals benefited from listening to carers and users sharing their experiences:I thought to have someone professional there was good. I particularly appreciated the open and clear responses of the professionals. I was interested in the service users’ questions, their knowledge of psychosis and treatment. (carer 2)Getting sense of service user and carer priorities in a general sense. From the outset, participants were interested in learning about others’ experiences at psychosis seminars:The third theme focused on the issues related to delivery of psychosis seminars such as participants’ expectations of the seminars, group facilitation and attendance.The free ranging nature of the meetings did allow interesting things to emerge and too much structure would be a bad thing. (carer 2)On the one hand I like the spontaneity this arrangement allows. On the other hand, it would be nice to know what to expect when, as some topics may be of more interest than others. I think for me, if the sessions had been more structured people could benefit from [the] agenda. Furthermore, it was suggested that ‘shared group expectations’ could be developed as the ground rules were unclear to some of the participants:Could be clearer on expectations of group members (…) – are we allowed to challenge people, or is the expectation more of a didactic model of presentation and question? Participants reported that they did not know what to expect of psychosis seminars in terms of their format. Although some participants liked the open agenda, others had mixed feelings about it or wished to have some form of structure in place:Some people talking a lot; some people not getting their fair share of time to say their piece. (service user 4)Sometimes topics were not discussed fully and I felt there was much more that could have been discussed. (…). Sometimes only a few of the group contributed to discussions and others did not say anything. (carer 7)Carers dominated the seminar. Some participants suggested that psychosis seminars could be chaired professionally, which they thought could give more people a chance to share their experiences.Another subtheme that emerged from the participant’s responses about what could be improved in the delivery of psychosis seminars was the need to strengthen facilitation of groups. Participants felt that there was unequal contribution from people at the seminars with some individuals being more vocal than others. Some people also thought that topics could have been explored in more depth:More people from all three groups but particularly service users. (carer 2)For the next round would it be possible to encourage more even participation of service users, carers and mental health professionals that would involve a senior trainee…and more junior trainees. The frequency of psychosis seminars being weekly and the requirement to commit to all scheduled meetings seemed to have affected group attendance:Maybe weekly is too frequent, not sure. (carer 2)Several of my colleagues had been tempted to join but were put off by the idea of having to commit. I started and then had to commit to something else and finally, when I did get some time, felt that I might have missed too many [seminars] to continue. One participant suggested having an open attendance structure to attract more people with a core group who would be recommended to commit to all seminars:I also think that having an open attendance structure would attract more people. (…) I do, however, also see the value of having a core group which remains constant as that fosters confidence and trust among members. It was also felt that group schedule and topics could be publicised in advance. Furthermore, one participant thought that the recruitment materials for psychosis seminars could be improved:The flyer I saw for the seminars was quite busy and wordy. I think this is brilliant for people who want more information, but can be off-putting for those who don’t know anything about it yet. Once I was shown this flyer I realised I had seen it in the reception area of my service – and did not read it for precisely that reason. Finally, participants reported that the number of people attending psychosis seminars was low and suggested that attendance could be increased with better representation from all three groups:In this study, we explored feasibility and participants’ experiences of psychosis seminars, which were held in the UK for the first time. The themes that have been identified in the open-ended questionnaire data captured three aspects of psychosis seminars: the acceptability of the model which provides an open forum for interaction and discussion between service users, carers and professionals, the benefits of attending seminars in terms of learning and understanding more about psychosis and the issues related to adjusting the delivery of the seminars to improve implementation.Generally, the psychosis seminars model seemed to be well accepted by the participants who reported that they indeed benefited from attending the seminars. This suggests that the seminars may be a promising option to improve communication between users, carers and professionals.However, there were issues related to the delivery of seminars, including unclear expectations about the format and rules, facilitation issues and low attendance. The original psychosis seminars model has an open agenda that is jointly agreed by the participants in the meeting.Furthermore, although psychosis seminars are based on a democratic and equal approach, participants reported unequal contribution of people in the discussions, with some groups dominating the seminar and others not having a chance to speak. This raises questions about how facilitation could be strengthened to ensure that everybody can contribute without undermining the core principles of the equality of psychosis seminars. A suggestion was to have a professional facilitator rather than having a rotating chair from within a group. However, having an external person to run discussions may imply an increase in costs and make the organisation of seminars more complex. Moreover, it is difficult to estimate its effect on the discussion between service users, carers and professionals.Finally, the number of participants of psychosis seminars was relatively low (25 people) and the participants unanimously reported that more people from each of the three groups would have been beneficial. The attendance seemed to have been affected by the requirement for participants to commit to all weekly seminars in the cycle. This needs to be considered when implementing psychosis seminars in the future. One of the suggestions made by participants was having a core group of people who would attend all seminars and then opening attendance to others on a drop-in basis. A less intensive schedule (e.g. fortnightly meetings) could also be considered.The findings of this study are in line with some aspects of the intergroup contact theory, which postulates that equal status, the opportunity to learn about others and the formation of an inclusive group may lead to improved intergroup relations.et alLiterature or evaluative studies of psychosis seminars are lacking. Amering The evaluation of psychosis seminars in Ireland including more than 300 participants (42 of them interviewed) and six sites yielded results consistent with our findings: participants reported that they have learned from each other’s different perspectives on mental health and felt comfortable to speak in an egalitarian meeting, where service users, carers and mental health professionals could listen to each other in a neutral setting.Issues related to facilitation of meetings were also reported by Mac Gabhann and colleagues,Interestingly, the issues related to the format of seminars such as the lack of a structured agenda reported by the participants in this study were not mentioned in other literature. This raises the question whether in our case this was because of the lack of information to the participants prior to seminars (people not knowing what to expect) or because of something specific to the context, that is, people may have been more used to structured meetings.To our knowledge, this is the first study in the UK to explore feasibility and personal experiences of participants in psychosis seminars. Qualitative survey data were collected from all three groups of people who attended the meetings. Content analysis provided a useful framework for analysis in terms of identifying patterns and themes in the data, which helped maximise the information obtained from this exploratory study in an area where systematic research is lacking. Conformability of the analysis was assured by the three authors independently coding the data and refining the themes together. Quotes from the participants’ responses were provided to illustrate the themes and to increase trustworthiness of the results.The quality of the data collected from the open-ended survey (short or missing responses for some questions) and the small sample (only 25 participants and only 10 of them returned the questionnaire) could be considered as the main limitations of the study. It could be argued that future studies could obtain richer data by the use of in-depth interviews or focus groups with the participants.Another limitation of our study is that the initiative was professionally led and service users and carers where not involved in the design, delivery or evaluation of the project, which should be considered when future studies are being developed.It is also important to note that findings could not be generalised to the rest of the UK as psychosis seminars took place only in three boroughs of East London.Psychosis seminars appear to be feasible in the UK and their organisation is not particularly expensive, only requiring room bookings. They may not be an option for all service users, carers or professionals as participation is voluntary and outside working hours.Yet, they were experienced by the participants positively and could potentially lead to improved communication among service users, carers and professionals during mental healthcare delivery, which is advocated by the government policies but may be hindered by power issues. Our findings also suggest that the way seminars are organised and delivered should be considered carefully and agreed by the group in advance.Psychosis seminars could be implemented more widely in the UK, following the example and success of the Mental Health Trialogue Network in Ireland as well as well-established psychosis seminars in other countries.The unconventional nature and philosophy of psychosis seminars may be incompatible with systematic evaluation or experimental trials.


Marsupials diverged from eutherian mammals about 148 million years ago and represent a unique lineage of mammals with distinctive morphological and reproductive characteristics. Marsupials have significantly shorter gestation periods than eutherians. Pregnancy typically ranges from 15 to 35 days, with young being born at a very early developmental stage and lacking differentiated lymphoid tissues and mature effector cells. Recent microbiome studies of the marsupial pouch revealed that marsupial young can face intense microbial challenges after birth, as the pouch contains a broad range of Gram-positive and Gram-negative bacteria. Antimicrobials are believed to play a significant role in the immune protection of marsupial newborns during their pouch life. The skin of the post-reproductive pouch secretes antimicrobial lysozyme and dermcidin, which may contribute to the decreased density of certain bacteria in the pouch. A range of antimicrobial agents, such as immunoglobulins, lysozyme, transferrin, and cathelicidins, have been identified in marsupial milk. Antimicrobial assays have revealed that marsupial cathelicidins have broad-spectrum activity against a variety of bacteria and fungi, including several multi-drug resistant strains. In this article, we will review the action mechanisms of these antimicrobial compounds and discuss how they protect marsupial newborns from potentially pathogenic bacteria inside the pouch. We will also discuss the potential of marsupial antimicrobial compounds as a source of novel antibiotics. Planigale) to over 80 kg in the red kangaroo (Macropus rufus), and life span varies greatly from less than a year for males of several small dasyurid species to up to 27 years for large kangaroos  and give birth to highly underdeveloped young that weigh less than 1% of the mass of the mother . Due to Macropus eugenii), brushtail possum (Trichosurus vulpecula), and Tasmanian devil (Sarcophilus harrisii). Two earlier papers are also available for the koala (Phascolarctos cinereus) (Setonix brachyurus)   and quokchyurus) , both usStaphylococcus aureus representing one of the most common members of the pouch bacterial community at all reproductive stages. Corynebacterium spp. , which represented the most commonly isolated bacterial genus in the tammar wallaby pouch.The tammar wallaby and brushtail possum both have a fully developed pouch that opens anteriorly . Deakin The Tasmanian devil’s pouch opens toward the rear. Sequencing of 16S rRNA gene amplicons on a Roche 454 GS FLX system revealed a highly diverse flora in the devil’s pouch with an average of 1,907 phylotypes  identified in each sample . The obsAll above evidence suggests that the marsupial pouch harbors a wide range of microbes which inevitably include pathogenic species. Additionally, studies also found that the female urogenital tract opening of the tammar wallaby  and abdoKlebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter aerogenes, and Escherichia coli, was detected in the tammar wallaby pouch leading up to and right after (<6 days) parturition   . SeveralE. coli and Staphylococcus aureus  revealed that higher diversity of proteins was produced in the mature reproductive pouch than in the immature or post-reproductive pouch , which belong to Enterobacteriaceae . Therefore, marsupials may still face intense pathogenic pressure after birth and further protective mechanisms are required for pouch young survival.Although pouch secretions significantly reduce the prevalence of certain harmful microbes in the pouch during lactation, evidence found in the Tasmanian devil demonstrates that there are still a large variety of potentially pathogenic bacteria present in the lactating pouch . For exaFigure 2). Starting from the point when the neonate is attached to a teat and starts suckling, phase 2 lasts for a few months until the pouch young begins to generate its own body heat , lysozyme, transferrin, and host defense peptides, have been identified in multiple marsupial species. Most of these components are differentially expressed throughout lactation, in response to different developmental stages of the pouch young .Figure 2), though the second peak does not appear until phase 3 of lactation (Immunoglobulins (antibodies) are commonly found in mammalian milk with the relative abundance of different isotypes varying among species. Ingested antibodies provide the young not only immediate immunity, but also long term resistance against certain infections . In marsactation . It is bactation .Pseudocheirus peregrinus) milk appears to be a late lactation protein and is not detected until phase 3   , indicatin vitro, and their gene expression level is elevated in mammary glands in late involution. These results suggested that the roles of cathelicidins in marsupial reproduction may not be limited to antimicrobial protection of pouch young, but also include regulation of mammary cell proliferation during lactation and mammary gland re-modeling during involution , tammar wallaby, and Tasmanian devil have 12, 14, and six cathelicidin genes in their genomes, respectively . Two peptides, WAM1 and Saha-CATH5, also effectively killed antibiotic-resistant strains, such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistance Enterococcus faecalis (VREF). Hemolytic assays demonstrated that all examined marsupial peptides are not toxic to human red blood cells except at extremely high peptide concentration   . Salt se0 μg/ml) .These studies are the first steps to fully revealing the potential of marsupial cathelicidins as candidates for novel antibiotic development. Further work is required to evaluate the pharmacokinetics of the peptides and to understand the mechanisms of their functions. Moreover, the issue of high cost of peptide production needs to be addressed. Past and current studies of marsupial cathelicidins largely rely on chemical synthesis of peptides, which is more expensive compared to recombinant expression approaches . FurtherFigure 3). Pouch secretions reduce the prevalence of certain harmful microbes in the pouch during lactation, and the milk provides passive immunity for the young at key developmental stages. Immune compounds such as lysozyme, dermcidin, immunoglobulins, transferrin, and cathelicidins play crucial roles in the antimicrobial protection of marsupial pouch young. Studying protective mechanisms in the marsupial pouch will not only improve our understanding on the importance of these components in the mammalian immune system, but will also provide a unique opportunity to discover novel antimicrobials to combat fast-evolving pathogens.Marsupials have developed multiple strategies to protect immunologically naive young in the non-sterile environment of the pouch (summarized in YC wrote the manuscript. KB provided feedback and helped revise the manuscript.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.