The acute antiprotozoal effect of hederagenin derivatives was more pronounced than that of cholesterol and cholic acid derivatives. Modifications in the structure of hederagenin, cholesterol, and cholic acid derivatives resulted in compounds with different biological activities in terms of acute effect and stability, although those which were highly toxic to protozoa were not always the most stable over time. Most of the hederagenin bis-esters, and in particular hederagenin bis-succinate (TSB24), hederagenin bis-betainate dichloride (TSB37) and hederagenin bis-adipate (TSB47) had a persistent effect against rumen protozoa in vitro, shifting the fermentation pattern toward higher propionate and lower butyrate. These chemically modified triterpenes could potentially be used in ruminant diets as an effective defaunation agent to, ultimately, increase nitrogen utilization, decrease methane emissions, and enhance animal production. Further trials in vivo or in long term rumen simulators are now needed to confirm the in vitro observations presented.The antiprotozoal effect of saponins is transitory, as when saponins are deglycosylated to the sapogenin by rumen microorganisms they become inactive. We postulated that the substitution of the sugar moiety of the saponin with small polar residues would produce sapogen-like analogs which might be resistant to degradation in the rumen as they would not be enzymatically cleaved, allowing the antiprotozoal effect to persist over time. In this study, we used an acute assay based on the ability of protozoa to break down [ The manipulation of the rumen microbial ecosystem using plant secondary compounds has proved to be a useful strategy to increase the efficiency of feed utilization by ruminants . Plants Saponins are plant secondary metabolites which consist of one or more sugar moieties glycosidically linked to a less polar aglycone or sapogenin . The sugin vitro.Saponins can form irreversible complexes with cholesterol in the protozoal cell membrane causing cell rupture and lysis . Rumen pHedera helix) fruits were collected from several locations around Bangor , dried at 50°C for 2 days and milled. Ivy fruit meal (3.79 kg) was extracted with ethanol (15 L) for 6 h, leading to a crude extract (541 g) comprising triglycerides, saponins, oligosaccharides, and pigments (anthocyanins). The crude extract was then washed with petroleum ether (3 × 500 mL) and dried overnight at 50°C under vacuum, obtaining a fine powder (368 g) which comprised mainly mixed saponins and oligosaccharides. Then an additional extraction with n-butanol was carried out, obtaining a refined extract comprising saponins (15% DM). Hederagenin, the aglycone part of the saponins, was obtained via hydrolysis of ivy fruit refined extract in ethanolic solution with aqueous HCl.Ripe ivy  of the defatted fruit extract. Fractions containing hederoside B were concentrated and subsequently washed with methanol. Nuclear magnetic resonance data (pyridine-d5) of the obtained compound was in agreement with that reported in the literature  were synthesized from the aglycone hederagenin produced above as described in patent application PCT/EP2016062383  were synthesized following the same methods for esterification of organic molecules, described in patent PCT/EP2016062383 (Cholesterol and cholic acid derivatives (16062383 . HederagThe purity of the synthesized compounds was established by quantitative nuclear magnetic resonance (qNMR) spectroscopy using a Bruker Ultrashielded 400 spectrometer  confirming purities of 80–99% for most derivatives except TSB37 and TSB38 which had a purity of 66 and 58%, respectively. It should be noted that the antiprotozoal activity of compounds TSB37 and TSB38 may be either over or indeed underestimated due to the impurities present.in vitro as the breakdown of [14C] labeled bacteria by rumen protozoa as described by Streptococcus bovis in Wallace and McPherson media (14C] leucine (1.89 μCi/7.5 mL tube) as the sole nitrogen source, for 24 h. Cultures were centrifuged , supernatant discarded and pellets re-suspended in 7 mL of simplex type salt solution . This process was repeated three times to prevent re-incorporation of released [14C] leucine by bacteria. The labeled bacterial suspension was sampled to determine its radioactivity and then it was used as the substrate in the incubations with rumen fluid.The effect of hederagenin, cholesterol, and cholic acid derivatives on protozoal activity was measured on media  containiion STS;  containibis-sulfate disodium salt (TSB38), cholesteryl succinate (TSB39), and lithocholic acid succinate (TSB42) were dissolved in dimethyl sulfoxide (DMSO) at 1% of the incubation volume. The rest of the derivatives and Hederoside B were solubilized in ethanol at 1% of the incubation volume as it has been shown that such concentration of ethanol in rumen fluid should not impair fermentation  were acidified [by adding 125 μL of 25% (wt/vol) trichloroacetic acid and centrifuged . Supernatant (200 μL), was diluted with 2 mL of scintillation fluid to determine the radioactivity released by liquid-scintillation spectrometry , fed at maintenance level . Animal procedures were carried out in accordance with the Animal Scientific Procedures Act 1986 and protocols were approved by the Aberystwyth University Ethical Committee. Rumen digesta was obtained before the morning feeding and strained through two layers of muslin and diluted with STS (1:1). Diluted rumen fluid (7.5 mL) was then incubated with labeled bacteria (0.5 mL) in tubes containing no additive (control) or 0.05, 0.1, 0.5, or 1 g/L of the modified triterpenes or steroids; hederoside B, a natural saponin isolated from ivy fruit, was also incubated at 0.05, 0.1, 0.5, and 1 g/L. Hederagenin entation . Two conbacteria .Entodinium, 8% Epidinium, 21% Diplodinium, 3% Isotricha, and 3% Dasytricha.The initial protozoal population in the inoculum used in the incubations was quantified by optical microscope using the procedure described by 2 mesh screen. Treatments consisted of control incubations (0.3 g of diet only), with either ethanol or DMSO added at 1%, and incubations with the synthesized compounds  at 0.5 or 1 g/L of the incubation. To compare the antiprotozoal effect of the synthesized compounds against that of a natural saponin from ivy, hederoside B (dissolved in ethanol) was incubated at 1 g/L. Bottles were incubated at 39°C under CO2 receiving a gentle mix before every sampling time. Samples at different time points  were collected for visual assessment of protozoa motility. Ciliate protozoa motility was assessed in 30 μL of sample against a common scale when examined at low magnification (100×) using light microscopy. This evaluation was conducted in less than 1 min/sample to avoid the cell damage originated by the oxygen and temperature exposure. A score between 0 (no whole protozoa evident) and 5  was given according to the scale described by To estimate the stability of the antiprotozoal effect and measure the influence of the modified triterpene and steroids on fermentation parameters, strained rumen fluid from each cow was diluted 1:2 in artificial saliva solution . Aliquot14C-bacterial inoculum) and the time (from 0 h to 5 h), as well as its correlation coefficient. The slope of this trend-line indicated the bacterial degradation rate (as % h-1) by the rumen protozoa and ultimately their activity. Trend line slopes as well as fermentation parameters were analyzed statistically by randomized block ANOVA, with individual cows as a blocking term. Inhibition of protozoa activity (% with respect to the control) was analyzed using ANOVA with treatment, dose and their interaction as fixed effects and cow as blocking term. When significant effects were detected across the different doses, means were compared by Fisher’s unprotected LSD test.A simple linear regression was conducted to model the relationship between the percentage of radioactivity released  was used.Protozoal motility was analyzed as a Repeated Measures Design, with treatment as main factor and incubation time as subject factor. A stability index, to estimate the persistence of the saponin effect over time, was calculated as the percentage of the motility at 8 h that remained at 24 h. Interaction between treatment and time as a measure of differential temporal dynamics between treatments was also considered. Differences were declared significant at R2 > 0.99) over the 5 h of incubation with both control treatments (with ethanol or with DMSO). For each derivative, the rate of bacterial degradation at different doses as compared with the control is shown in Supplemental Table Table 1) was significantly different between compounds and doses (P < 0.001). Derivatives TSB44, TSB45, TSB46, TSB47, TSB52, and TSB42 were more effective in inhibiting protozoa activity than hederoside B, the major ivy saponin. Among the cholesterol and cholic acid derivatives, TSB39, TSB40, and TSB43 were less effective against protozoa than the natural saponin (P < 0.001).The amount of bacteria degraded by protozoa increased linearly  and hederoside B at 1 g/L, were tested over 24 h in Figures 3, 4). The effect of hederagenin derivatives when added at 0.5 g/L or 1 g/L is shown in Figures 3A,B, respectively. Although, 1 g/L of hederoside B decreased protozoa motility at 4 and 8 h of the incubation , there was a strong treatment × time interaction (P = 0.05), and protozoal motility recovered afterward (reaching a score of 4.26 at 24 h), suggesting the expected degradation of the saponin during the incubation. Some of the derivatives, TSB45 and TSB46, showed the same effect as the natural saponin, initially decreasing protozoa motility but with motility recovering after 24 h . Other derivatives, TSB24, TSB47, and TSB52, added at 1 g/L, however, resulted in a greater decrease in protozoa activity over time  with no sign of recovery in motility. Indeed, vacuoles were visible at 24 h suggesting protozoal death (scores of 2.15–2.9). Only few of the hederagenin derivatives  did not show an effect on protozoa motility (P > 0.05) at any of the concentrations tested. Cholesterol and cholic acid derivatives did not seem to be effective in reducing protozoa motility over time as shown in Figure 4. Only TSB42 when added at 1 g/L showed a slight decrease in protozoa motility after 8 and 24 h of incubation . A stability index, to estimate the persistence of the saponin effect over time, was calculated as the percentage of the motility at 8 h that remained at 24 h . Whereas the compounds located above the origin on the y-axis were stable , those below the origin on the y-axis showed a loss of effect on protozoal motility . The derivatives close to or on the origin of the y-axis  correspond to those compounds that were less effective against protozoa (scores of about 4.5 at 8 h) but with an effect that was maintained at 24 h.Cell motility, measured as an index of protozoa viability, remained unaltered (score of 4.8) over the 24 h incubation period in control incubations with ethanol or DMSO ; indeed, pH was slightly greater in the presence of TSB35 and TSB37 at 0.5 and 1 g/L (P < 0.001) in comparison to the control. Similarly, no effect on the concentration of total VFA was observed in incubations with hederoside B or with most of the derivatives . Only TSB35 and TSB36 caused a reduction in the concentration of VFA (P < 0.05) when added at 0.5 and 1 g/L. Also, some of the derivatives decreased the molar proportion of acetate . Almost all treatments caused shifts in the molar proportions of VFA toward lower butyrate and higher propionate (P < 0.05), to different extents depending on the compound .Neither the natural saponin, hederoside B, nor the modified triterpenes or steroids caused a decrease in pH (bis-glutarate), TSB37 (hederagenin bis-betainate dichloride) and TSB47 (hederagenin bis-apidate) which, when added at 1 g/L, decreased the molar proportion of acetate and butyrate by 11–13.5% and 35.5–52.7%, respectively, with an increase in propionate of 64.5–84.2%. Cholesteryl succinate (TSB39) and cholic tri-succinate (TSB41) did not have any effect on the molar proportions of VFA. Cholic succinate (TSB40) caused only a slight decrease in butyrate (P = 0.013) at 1 g/L, as compared to the control. TSB42 and TSB43 also resulted in decreases in acetate and butyrate and increases in propionate although to a lesser extent than those caused by hederoside B. Molar proportions of branched-chain VFA  decreased (P < 0.05) in incubations with TSB24 (–13%) and TSB38 (-16%) at 1 g/L and TSB50, TSB51, TSB52, and TSB58 at 0.5 and 1 g/L . TSB43, however, resulted in an increase (P = 0.044) in BCVFA when added at 1 g/L . This was mainly due to changes in isovalerate rather than isobutyrate , and five cholesterol and cholic acid based derivatives carrying one to three succinate residues  was evaluated. Our 5 h in vitro incubations results showed that, irrespective of their resistance to degradation, some of the hederagenin derivatives were more effective in reducing protozoa activity than the natural saponin hederoside B. The greatest effect was shown with TSB45, TSB46, and TSB52 which reduced protozoa activity by 63–75% when they were incubated at 0.05 g/L. Interestingly among the cholesterol and cholic acid derivatives, TSB39 (cholesteryl succinate) had the lowest antiprotozoal effect and, TSB42 (lithocholic acid succinate) was one of the most effective compounds tested, decreasing protozoa activity by 75% when added at 0.05 g/L. These results agree with the observations of in vitro incubations were carried out sampling at 0, 4, 8, and 24 h to assess the stability of the derivatives in a mixed rumen population. Derivatives TSB24, TSB47, and TSB52 seemed to be very effective in causing a decrease in protozoa motility over time without recovery after 24 h, contrary to the results observed for hederoside B and the rest of compounds. Surprisingly, none of the cholesterol and cholic acid derivatives showed an effect on protozoa motility. Although TSB42 had a strong effect in bacterial breakdown by protozoa over 5 h of incubation, little effect on protozoa motility was observed in 24 h in vitro batch cultures. These results may suggest a quicker degradation, and thus the loss of activity, of this compound by rumen bacteria as compared with other derivatives tested. It is apparent that the compounds that showed a high level of acute toxicity against protozoa were not always the most stable ones over time. A stability index was calculated as the percentage of the 8 h activity that remained after 24 h . Even though TSB35 reduced protozoa activity by 93% when added at 1 g/L, this compound was among the least stable derivatives. TSB24 and TSB47, however, showed both high toxicity (reduction of protozoa activity of 95–100%) and stability over time.Although the antiprotozoal effect of saponins has been consistently shown in  studies , it was  studies . This tr studies . Makkar Most of the hederagenin derivatives did not influence total VFA concentration. However, shifts in the molar proportions of VFA toward lower acetate and butyrate which was compensated by a higher propionate were observed. These changes have been previously reported when using different sources of saponins . The shiin vitro. Whereas some compounds (TSB24) were more effective in reducing protozoa activity and motility, others (TSB37) caused a substantial increase in propionate. If the effect of these compounds can be confirmed in vivo, the use of these modified triterpenes in ruminant nutrition will have the potential to improve the efficiency of nitrogen utilization and decrease methane production thus potential boosting productivity.Clearly modifications in the structure of hederagenin resulted in compounds with different biological activities bis-esters, and in particular hederagenin bis-succinate (TSB24), hederagenin bis-betainate dichloride (TSB37), and hederagenin bis-adipate (TSB47) had a persistent effect against rumen protozoa in vitro, shifting the fermentation pattern toward higher propionate and lower butyrate. The confirmation of these effects in vivo would help to determine if these novel chemically modified triterpenes could potentially be used in ruminant diets as an effective defaunation agent to, ultimately, increase nitrogen utilization, decrease methane emissions, and enhance animal production.Most of the hederagenin ER-M, SD, CW, MB, ML, DP, RB, and CN contributed to the conception and design of the work; ER-M and GdF conducted the research; ER-M wrote the manuscript; ER-M, GdF, SD, CW, MB, ML, DP, RB, and CN reviewed the manuscript. ER-M and CN had primary responsibility for the final content. All authors read and approved the final 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.


However, much healthcare leadership education focusses on the training and competence of individuals and little attention is paid to the interprofessional workplace and how its inherent complexities might contribute to the emergence of leadership. Underpinned by complexity theory, this research aimed to explore how interprofessional healthcare teams enact leadership at a micro-level through influential acts of organising. A whole  team workplace-based study utilising video-reflexive ethnography occurred in two UK clinical sites. Thematic framework analyses of the video data (video-observation and video-reflexivity sessions) were undertaken, followed by in-depth analyses of human–human and human–material interactions. Data analysis revealed a complex interprofessional environment where leadership is a dynamic process, negotiated and renegotiated in various ways throughout interactions . Being able to “see” themselves at work gave participants the opportunity to discuss and analyse their everyday leadership practices and challenge some of their sometimes deeply entrenched values, beliefs, practices and assumptions about healthcare leadership. These study findings therefore indicate a need to redefine the way that medical and healthcare educators facilitate leadership development and argue for new approaches to research which shifts the focus from  Regulatory bodies endorse the importance of incorporating leadership education at all stages of healthcare professionals’ careers Garling . FurtherThere is growing opinion that traditional individualistic understandings of leadership are no longer sufficient to explain successful leadership  and thus it becomes an Prevailing organisational researchers seek to reduce and simplify the complex environments they study focussing on top-down leadership Hosking . From a Using complexity as a theoretical lens, we can pay attention to the way in which leadership emerges across multiple levels of a healthcare organisation and within multiple timescales  and one hospital ward (Site B). Local Deaneries  provided details of potential sites and key contacts were approached by email and face-to-face discussion. Site A is a medium-sized community general practice providing primary care services for a UK town and rural district. Site B is a 30-bed elderly rehabilitation ward in a district general hospital in a small UK city.All members of the interdisciplinary teams working directly in both sites were invited to participate. Recruitment occurred face-to-face and all potential participants were approached on an individual basis and given a ‘cooling off’ period to consider their possible participation. Multi-level written consent was sought by the lead author for: observation in the workplace; observation in the workplace using video; use of video in the reflexivity sessions; participation in the video-reflexivity sessions; and use of video-recordings for educational and dissemination purposes. Most consents were obtained at this point. However, some were obtained during later stages as other staff members became involved in the study serendipitously. Table The primary author (an ex-physiotherapist) undertook data collection for this study. Whilst an outsider to the workplaces studied, her previous clinical experiences afforded some ‘insider’ knowledge, which facilitated her comfort within both clinical settings and allowed her to ‘understand’ the ‘language’ of healthcare practices and discuss aspects of working practices with participants using this language  a period of familiarisation and observation within the workplace; (2) video-footage was recorded of real workplace practice (video-observation stage); and (3) this footage was then edited and the edited footage played back to the interprofessional team, providing them with the opportunity to reflect on and discuss their practices (video-reflexivity stage). Note that only staff-to-staff interactions were observed and recorded.Familiarisation of the researcher to the environment and the environment to the researcher allowed for the identification of possible points in the working week where video-observation could best take place . Familiarisation was achieved through informal discussion with participants at various points during the working day alongside basic field notes discussed later with the co-authors. During familiarisation, the primary author attended each site for short periods (30–180 min) during normal working hours (8–6 pm) in site A on five different occasions and in site B on seven different occasions .The observation without video stage involved watching practices that had been identified by the participants within the familiarisation stage as points of interprofessional interaction (with non-patient contact). In site A, the primary author was onsite for periods of between 2 and 5 h on four occasions ; in site B the primary author was onsite for periods of 1–4 h on four occasions . During this time, basic field notes were taken which provided outline details pertaining to working practices, notes from one-to-one conversations with various participants and more detailed descriptions of the format of more formal settings, for example, multidisciplinary team meetings.The focus of the video-observation stage was to capture leadership interactions as they occurred within the context of the interprofessional workplace. A small ‘handi-cam’ with a wide-angled lens was used. The initial intention was to video using a ‘fly-on-the-wall’ style and the majority of footage was captured in this way. However, in some footage participants would interact with the researcher behind the camera discussing their thoughts and practices whilst being videoed, with this style of videoing becoming ‘expert-apprentice’ (i.e. with participants as ‘experts’ explaining to the ‘apprentice’ researcher what they were doing and thinking). In total, 12 h, 37 min of video-observational data were collected. Table The purpose of the video-reflexivity stage was to provide participants with the opportunity to reflect on leadership practices and discuss these as they saw them happening. The first step in this process was to edit the video-observational footage collected into short clips that could be discussed within the reflexivity sessions. Decisions about what to edit were made as a research team who met regularly during this phase to discuss the footage and how it might be edited. Editing decisions were based on several factors including: (1) range of participants and their roles; (2) researcher field notes; and (3) the researchers’ knowledge of the literature and healthcare leadership roles Gordon . Thus, tThese edited clips were then played within VRE group sessions to which all participants were invited. Within these sessions the edited clips (pertaining to that site only) were played and the lead author facilitated discussion focussing on leadership. Each VRE group began with an introduction to the session and explanation of the layout. Following this, the researcher would show an edited clip, then provide participants with the opportunity to discuss the clip generally, in terms of leadership and followership and any other aspects they particularly wanted to discuss in relation to the clips. The researcher’s role within these sessions was to facilitate the discussion through prompt questions, to ensure the discussion stayed on topic (i.e. leadership) and probe some issues in more depth. Each session was videoed using the hand-held camera on a tripod, with wide-angled lens. Unlike the video observation footage, the researcher was visible within the video-reflexivity footage. Of the raw video-data outlined in Table A team-based approach to data analysis was undertaken whereby each author watched a selection of edited video clips and VRE group data independently. Following this, the research team met to discuss overarching themes. A coding framework was developed, which formed the basis for broad analysis of the video-observational and VRE group footage and detailed analysis of the edited video clips. In order to assist with managing the large volumes of visual data, computer assisted qualitative data analysis software (CAQDAS) was used Lewins . For theAcross the raw video-observational footage, we identified points at which the process of leadership occurred in context , that is, leadership interactions where the focus of the IAOs were decisions about ongoing patient care. Other IAO types included: educational leadership, where the IAO occurred during an educational activity; administrative leadership, for example, decisions about bed management; and change leadership.We derived from the data common features of an IAO to include some or all of the following processes: (1) information exchange, typically found across all leadership interactions and used to facilitate an IAO; (2) leadership non-negotiation  or leadership negotiation, in which it was observed that team members “granted” leadership to the most appropriate member; such granting occurring either overtly through direct designation of leadership or covertly through questioning or non-verbal interactions; and (4) discussion and agreement of a plan (following information exchange and leadership negotiation) or passive compliance of a plan, without offer of opinion, planning or discussion .IAOs of all types were generally led by trained medical staff (i.e. GPs and consultants), in particular during formal contexts such as ward rounds and multidisciplinary meetings. For example, in Fig. leadership emergence included staff outside traditional professional and interprofessional hierarchies, undertaking shared leadership. For example, in Fig. In contrast, our analysis also revealed IAOs in informal contexts and IAOs where leadership was shared by more than one member of the multidisciplinary team. These interactions were characterised by leadership appearing to be negotiated and re-negotiated throughout an interaction. Thus, leadership could be described within these scenarios as emergent as it happened in immediate response to unpredictable situations. Across the video-observational data, this Exploring human–human and human–material interactions during these IAOs allowed us to examine in greater detail how leadership was enacted. These included non-verbal interactions; control and/or use of material artefacts; and the use of language.Physical positioning and eye contact was an important part of the formal leadership interaction between David and Douglas see Fig. , still 1Less clear-cut is the physical positioning between Jason and Fiona in their interaction. In Fig. Control and/or use of artefacts was an important factor in leadership IAOs across our data. Patient data in various forms was a key human–material interaction across the footage. Control of materials like, for example, who had sight of patient and nursing notes; computer screens and the keyboard; and who was deciding which patient to discuss on the hospital ward’s white board were key strategies in the negotiation of leadership. Those that were in ‘control’ of these artefacts were the participants who tended to be in the position of leader within the interaction.For example, in Fig. In contrast, in Fig. Directives were typical across the data and implied leadership, particularly in more formal interactions where leadership was less ambiguous. For example, in Fig. In contrast, whilst directives are not used throughout the interaction between Jason and Fiona, questioning was used as a way of carefully negotiating professional boundaries and leadership identities. In Fig. Finally, personal pronouns and the words collocated with pronouns were also a way in which leadership identities and relationships were negotiated. For example, in Fig. In Fig. When the interaction in Fig. David also emphasised the importance of other interprofessional team members’ roles in clinical leadership IAOs. Table When this interaction was discussed in VRE group 9 with a higher stage specialty trainee (‘Katie’), she also recognised the clear role of consultants as leaders and the different ways in which their leadership was enacted. She also noted physical positioning . Our research suggests that leadership in healthcare is enacted within complex adaptive systems (CASs), something that is often discussed within the healthcare literature but seldom researched  and through our analysis we identified how each level interacts and influences leadership at a micro-level. Leadership was enacted through the local policies and procedures present within each site, through paperwork and IT systems to which participants continuously interacted within the video-observational footage, linking the micro-level system to those at the meso-level  and macro-level .The use of VRE allowed for engagement with leadership complexity, enabling us to explore the whole workplace as a dynamic learning system. It allowed us to visibilise practice in the moment rather than discussing general leadership behaviours that were simplified or reassembled through field notes and memory . Finally, using video-reflexivity provided formal accountability for our own analysis and provided rich opportunity for the co-construction of meaning which served to enrich our study findings (Iedema et al. This research highlights both professional hierarchies and the complex emergence of leadership in the healthcare workplace. Thus, we argue that our research emphasises the need for leadership education at all levels including both intra- and interprofessional activity. Current healthcare literature asserts the need to shift to distributed patterns of leadership that are adaptive to complex healthcare systems (Royal College of Physicians Canada We particularly suggest that VRE, in addition to being used as a research methodology, could be employed within leadership education. By viewing themselves in practice, participants could explore opportunities for change and improvement in leadership practices through open discussion about their everyday but often unseen work. VRE could help develop the ability of teams to lead their way out of problems through reflexive discussion.Our study explores one snapshot in time with respect to workplace-based leadership practices. Longitudinal research is now key to provide insights into how healthcare leadership develops over time. Moreover, longitudinal work would allow additional exploration of how macro-, meso- and micro-systems interrelate with different leadership activities that are enacted. Repeated VRE ‘cycles’ or a participatory action research approach would help to address this (Bleakley and Cleland


ACF7 leads to aberrant microtubule organization, tight junction stabilization and impaired wound closure in vitro. With the mouse genetics approach, we show that ablation of ACF7 inhibits intestinal wound healing and greatly increases susceptibility to experimental colitis in mice. ACF7 level is also correlated with development and progression of ulcerative colitis (UC) in human patients. Together, our results reveal an important molecular mechanism whereby coordinated cytoskeletal dynamics contributes to cell adhesion regulation during intestinal wound repair and the development of IBD.In the intestinal epithelium, the aberrant regulation of cell/cell junctions leads to intestinal barrier defects, which may promote the onset and enhance the severity of inflammatory bowel disease (IBD). However, it remains unclear how the coordinated behaviour of cytoskeletal network may contribute to cell junctional dynamics. In this report, we identified ACF7, a crosslinker of microtubules and F-actin, as an essential player in this process. Loss of  The cytoskeleton plays a key role in cell/cell junction formation, but how the coordinated behaviour of the cytoskeleton contributes is not known. Here the authors show that actin-microtubule crosslinker ACF7 plays a key role in tight junction stabilization and wound healing in intestinal epithelium. To address the molecular mechanism, we show that ACF7 is essential for orchestrating microtubule dynamics in intestinal epithelial cells. By X-ray crystallography, we further delineate the structural basis underlying ACF7 interaction with microtubules, and corroborate the role of this interaction in tight junction dynamics and wound healing. With conditional gene targeting to ablate ACF7 expression in the intestine of mice, our findings have uncovered essential roles for ACF7 in intestinal tissue morphogenesis and epithelial cell migration in vivo. In addition, the conditional knockout (cKO) animals are susceptible to experimental colitis, and the expression of ACF7 is strongly correlated with development of ulcerative colitis (UC) in human patients. Taken together, with a comprehensive approach ranging from mouse genetics to molecular and cell biology, our work identified a hitherto unappreciated mechanism by which tight junction dynamics and intestinal wound repair are regulated by cytoskeletal coordination.In this report, we demonstrate that depletion of endogenous in vitro model of ACF7 knockout (KO) with CRISPR  technologyCas9 (CRISPR associated protein 9) and an ACF7-specific gRNA (guide RNA). Clones of infected cells were selected upon antibiotics treatment, and ablation of ACF7 was confirmed by the western blotting analysis  test, and treatment of cells with nocodazole or KO of ACF7 dramatically delayed the recovery  for fluorescence recovery in both nocodazole-treated cells and ACF7 KO cells  analysis. The SEM of the small intestine showed that there were substantially fewer villi per unit area of the small intestinal epithelium in the ACF7 cKO mice compared with the WT mice. Moreover, the villi of the ACF7 cKO mice exhibited a hypertrophic and irregular form compared with those of WT mice  gene is only expressed in differentiated enterocytes of the small intestine. To determine the potential effects of an intestinal ACF7 deficiency on the terminal differentiation of enterocytes, the small intestine was stained for ALP. Compared with the control, the small intestine of the ACF7 cKO mice showed no significant alterations in ALP staining . Loss of ACF7 did not lead to a significant difference in the colon staining of CA1, either  and killed 4 or 24 h later for immunohistochemical examination. The migration rate of the epithelial cells, as defined by the height of the BrdU-positive cells along the crypt–villus axis, was significantly diminished in the intestines of the ACF7 cKO mice compared to those of the WT littermates . Strikingly, 6 days post DSS treatment, all of the cKO mice died, whereas more than 80% of WT littermates survived the same treatment  and tumour necrosis factor-α (TNF-α) (0.163±0.011 versus 0.118±0.009 ng ml−1) were dramatically increased in the colon of ACF7 cKO mice compared with WT organ (−1) and TNF-α  were observed in the ACF7 cKO mice compared with ACF7fl/fl mice  and tumour necrosis factor (TNF)-α, were measured in a colonic organ culture system. Before DSS treatment, we did not detect any significant difference in the basal level of the inflammatory mediators . HoweverWT organ . Meanwhi/fl mice . TogetheP<0.05), including CLIP2 and ACF7  level and higher rate of faecal occult blood in the human patients  in UC patients in comparison to the control group . Six genand ACF7 . The dimpatients . To furtts  phosphorylation of cingulin, which plays a critical role in epithelial morphogenesis Drosophila embryo as a model, studies have indicated that microtubules can enhance the assembly of adhesions by sustaining Par3 and E-cadherin level at adherence junctions through a dynein-dependent apical restriction2It remains elusive how guided microtubule dynamics may participate in cell/cell junction dynamics. It has been shown that microtubules may regulate adherence junction through the polarized delivery of cadherins. With in vitro and in vivo. With cKO of ACF7, we demonstrated that loss of ACF7 can exacerbate experimental colitis in mice. Consistently, reduced expression level of ACF7 correlates with the development of UC in human patients. Together, this reveals an intriguing connection between spectraplakin-controlled cytoskeletal/cell adhesion dynamics and the development of IBD. However, the exact mechanisms whereby cytoskeletal and cell adhesion coordination contributes to colitis remain unclear at present. Interestingly, aside from ACF7’s well-established role in cytoskeletal orchestration, ACF7 can also participate in cellular signal transductionACF7 affects the expression of various Wnt downstream targetsACF7 cKO epithelium. Future studies will be essential to delineate the potential signalling cascade involved in this process.Abnormalities in cell junctions and aberrant cell movement and wound healing are critically involved in the pathogenesis of IBD1232441In closing, our findings provide critical insights into the mechanics behind ACF7-dependent cytoskeletal coordination in tight junction dynamics and intestinal cell movement, paving the way for probing more deeply into the intricate signalling network orchestrating the crosstalk between F-actin and microtubule cytoskeletal networks in intestinal epithelium, as well as its relevance in the physiology and pathology of human gut.Rabbit serum against BrdU was produced by BD Pharmingen . Rabbit serum against pan-ACF7  was produced by Covance company . Rabbit polyclonal Abs against HA  and His tag  was obtained from Santa Cruz Biotechnology, Inc. . BrdU was produced by Sigma. DSS  was obtained from MP Biomedicals company. Fluorescein isothiocyanate-dextran (FITC-Dextran)  was obtained from Sigma-Aldrich (USA). Rabbit polyclonal anti-CD3 was obtained from Epitomics  and anti-CD68  was produced by Abcam company . Enzyme-linked immunosorbent assay kits of TNF-α and MIP-2 were produced by EIAab Science Co., Ltd . EDTA-containing tubes were produced by Axygen, Inc., Union City . Complete blood count determined in an Sysmex xs-800i blood analysis system . Analysis for FITC-Dextran concentration with a Synergy HT Multi-Mode Microplate Reader  at an excitation wavelength of 485 nm and emission wavelength of 535  nm.fl/fl alleles (cKO) were born in the expected Mendelian numbers. The cKO mice were further crossed with vil-RFP-ZO1 mice for intravital imaging of intestinal epithelium and tight junctions. The mice were housed under specific pathogen-free conditions in the University of Chicago and Affiliated Sixth People’s Hospital of Shanghai Jiaotong University and consumed a standard sterile diet and filtered water ad libitum under a 12-h light–dark cycle. The experimental protocol was approved by the Animal Care and Use Committee and the Ethics Committee of University of Chicago and Shanghai Jiaotong University.Mice homozygous for the germline floxed  insertions were bred to Villin-Cre (Vil-Cre) recombinase transgenic mice fl/fl (n=6) and ACF7 cKO (n=6) mice were exposed to 2.0% DSS for 7 days. Meanwhile, 10- to 14-week-old female ACF7fl/fl (n=6) and ACF7 cKO (n=6) mice were exposed to 2.0% DSS for 5 days followed by normal drinking water for 5 days. The mice were checked each day for morbidity, and weight was recorded. Induction of colitis was determined by weight loss, faecal blood, diarrhoea, and, upon autopsy, weight and length of colon. To quantify induction of colitis, a DAI was calculated as reported53545556DSS  was dissolved in the drinking water of mice. Fresh DSS solution was provided every day. 8- to 12-week-old male ACF7−1 body weight, 50 mg ml−1). After 1 h, 120 μl of blood was collected from the tip of the tail vein in mice before DSS treatment. After DSS administrated, we collected 120 μl blood by cardiac puncture after given FITC-Dextran 1 h when end of the experiment or time of dying. The blood was centrifuged at 4 °C, 10,000 g for 5 min. Plasma was diluted in an equal volume of PBS (pH 7.4) and analysed for FITC-Dextran concentration with a Synergy HT Multi-Mode Microplate Reader  at an excitation wavelength of 485 nm and emission wavelength of 535 nm. Standard curves were obtained by diluting FITC-dextran in non-treated plasma diluted with PBS (1:3 v/v).This measure is based on the intestinal permeability towards fluorescein isothiocyanate-dextran (FITC-Dextran)  as described5859For each animal, histological examination was performed on the colon; samples were fixed in 10% formalin before staining with H&E. All histological quantitation was performed blinded using a scoring system as described5657ZO1 was excited and bleached using the DPSS 561 laser. For wound-healing analysis, mice were anaesthetized and the abdomens were opened by a midline incision. Electrocautery was used to open a 2-CM loop of jejunum along the antimesenteric border. The abdominal cavity was closed under the loop of jejunum while protecting the neurovascular supply. The mucosal surface of the jejunum was placed against the coverslip bottom of a petridish containing HBSS. Mice were imaged with multiphoton confocal microscope in the light imaging facility of University of Chicago.Intestinal wound-healing and FRAP analysis were carried out in live animals3462Formalin-fixed, paraffin- embedded colon sections were deparaffinized in xylene and rehydrated with alcohol. Samples were boiled for 10 min in antigen retrieval solution  and left at room temperature for 30 min. Slides were then incubated  with primary antibodies for CD3 , CD68 . After three times washing with PBS, the slides were incubated for 45 min, room temperature with FITC labelled secondary antibodies . Slides were microscopic examined and photographed .2. After 24 h, supernatant fluid was collected, centrifuged and stored at –80 °C. Supernatants were analysed for TNF-α, macrophage inflammatory protein-2 (MIP-2) content in duplicate using commercially available enzyme-linked immunosorbent assay kits . In some experiments, serum was obtained by centrifugation, stored at –80 °C, and then cytokine concentration was measured.Colon tissue (about 1 cm) from rectum to caecum were excised, opened and cut longitudinally washed in cold phosphate-buffered saline (PBS) supplemented with penicillin, streptomycin and amphotericin B and incubated in 1.5 ml serum-free RPMI 1,640 medium, penicillin, streptomycin and amphotericin B cultured in 12-well, flat-bottom culture plates  at 37 °C in 5% COHundred microlitres of blood was collected from the tip of the tail vein on fourth day of 2% DSS treatment, blood was drawn into EDTA-containing tubes . After vigorous mixing, complete blood count was determined in an Sysmex xs-800i blood analysis system  using mouse specific settings. White blood cell (WBC) population was determined by blood smear, and corrected by WBC count.The project was approved by the institutional review board of the Shanghai Tenth People’s Hospital . Written informed consent was obtained from all the patients and tissue sample donors, and anonymity was assured by tracking the patients through their clinical history numbers.http://www.xybiotech.com) using the Human Cytoskeleton Regulator PCR array platform with the company’s proprietary primer set.Total RNA was isolated with the RNA Extraction Kit . The first strand synthesis of cDNA was performed with the Reverse Transcription Kit . Reactions were run on the FTC-3000 Qrt-PCR machine . Relative changes in mRNA and expression were determined using the 2–ΔΔCt methodg, and the supernatant was collected for protein quantification and SDS gel electrophoresis. Following protein transfer, the membrane was immunoblotted with different antibodies. The signals were detected bychemiluminescence.Proteins were extracted by scraping the luminal side of the intestine onto a clean glass slide using another clean glass slide. The scraped tissue was then homogenized in lysis buffer containing 50 mM tris-HCl (pH 6.8) and 2% sodium dodecyl sulfate (SDS). Insoluble material was removed by centrifugation at 20,0002O2 to block endogenous peroxidase activity, then microwave antigen retrieval was performed for 20 min in citrate buffer (pH 6.0) determined. After washing in 10 mM Tris buffer (pH 6.0), the slides were incubated with 3% normal sheep serum to eliminate non-specific staining and then incubated with primary antibody for 30 min at 37 °C. For immunostaining, the peroxidase-conjugated secondary antibody reagent was used and the peroxidase activities of staining were visualized with 3,3′-diaminobenzidine-tetrahydrochloride . The nuclei were counter-stained with haematoxylin. MACF1 antibodies were purchased from Sigma. The immunostaining grading was performed based on the staining extent  and the staining intensity . The combination of extent (E) and intensity (I) of staining (E × I) was gained varying from 0 to 9.Dissected tissues were fixed in 4% paraformaldehyde solution and paraffin-embedded (FFPE). Five-micrometre sections were dewaxed in xylene and rehydrated in graded alcohol baths. The slides were treated with methanol with 0.3% HThe slides were stained with H/E for histological examination before immunostaining. To evaluate the disease severity of UC patients, the Geboes grading system was used2. For CRISPR KO, cells were infected with lentivirus encoding both Cas9 and gRNA specifically designed for human ACF7. Potential contamination with mycoplasma was screened using the ATCC universal mycoplasma detection kit.Caco-2 cells were obtained from Dr. Jerrold Turner lab and maintained in DME (4.5 g glucose per liter) with 10% FBS. Cells were cultured at 37 °C incubator with 5% COACF7 KO caco-2 cells were fixed and stained. Images were taken on Leica SP8 confocal system. 3D reconstruction and surface 2D plot were carried out with NIH Image J software.WT or Escherichia coli BL21(DE3) cells. Briefly, cells were grown in 10 l of LB medium containing 100 μg ml−1 ampicillin at 30 °C. The culture was induced with 0.4 mM isopropyl-β, D-thiogalactopyranoside (IPTG) at an OD600  value of ∼0.5. Cells were collected within 5 h of induction. The collected bacteria were resuspended in ice-cold buffer containing 20 mM Na–Hepes (pH 7.5), 100 mM NaCl and 1 mM PMSF. The cells were then lysed in a French pressure cell. Cell debris was removed by centrifugation for 1 h at 4 °C and 25,000g. The protein solution was then purified with Ni2+-affinity and G-200 sizing columns. The purity of the protein (>95%) was judged using 10% SDS-PAGE stained with Coomassie Brilliant Blue. The N-terminal sequence of the ACF7-GAR protein was confirmed by sequencing. The purified ACF7-GAR protein was then concentrated to 20 mg ml−1 in a buffer containing 25 mM Hepes pH 7.5, 150 mM NaCl, 2 mM CaCl2 for crystallization.The ACF7-GAR containing a 6 × His tag at the N-terminus was overproduced in −1 ACF7-GAR protein was mixed with 1 μl of reservoir solution containing 0.1 M sodium cacodylate trihydrate pH 6.5, 0.2 M (NH4)2SO4, 25%(v/v)PEG4000. The resultant mixture was equilibrated against 500 μl of the reservoir solution. Typically, the dimensions of the crystals were 0.2 mm × 0.1 mm × 0.1 mm. All crystals were flash frozen with 25% glycerol in mother liquor for checking and data collection. Crystals were derivatized in mother liquor solution by soaking with 0.1 M Hg(AC)2 for 2 h before flash frozen, back soaking was also applied to improve the clarity.Crystals of the 6 × His ACF7-GAR were obtained using hanging-drop vapour diffusion. The GAR crystals were grown at 4 °C in 24-well plates with the following procedures. One-microlitre protein solution containing 20 mg mlGAR crystals were tested and screened on an in-house X-ray source of Rigaku MicroMax-002+ system equipped with Saturn 944+ CCD detector (Rigaku) and Oxford Cryo-system. The data sets were integrated and processed using Rigaku crystalclear-1.44 for in-house data sets and HKL2000 for Synchrotron. High-resolution native data sets were collected at 100 K on beamline BL17U of Shanghai Synchrotron Radiation Facility, the crystal structure of GAR was determined by single isomorphous replacement with anomalous scattering of mercury derivative and refined to 1.5 Å.SHELXC/D/E and atomic model was auto-built PHENIX suite, model improvement and refinements were finalized in COOT and REFMAC in CCP4. In the final model, the electron density is clear and continuous, 98.5% residues located in favoured region, 2.5% located in allowed region and no residues located in outlier region by Ramachandran plot. All crystallographic figures were drawn in Pymol.Heavy atoms searching and initial Phasing were undertaken by Microtubule binding was examined using microtubule binding protein spin-down assay kit from Cytoskeleton , according to manufacturer’s instructions. In brief, about 0.1–0.9 nM of assembled microtubules were incubated with about GAR wt/mutant molecules (∼1 μg) in a 200 μl reaction. The bound and unbound proteins were separated by ultracentrifugation. Western blotting analysis was performed as described previouslyt-tests (two-tailed) were performed as appropriate. Correlation analyses were performed using Pearson’s test. Statistical significance was defined as P<0.05.Statistical analyses were performed using the GraphPad Prism software  and SPSS for Windows 15.0.0  statistical software. Survival curves were estimated using the Kaplan–Meier method, and the resulting curves were compared using the log-rank test. For comparisons, a one-way analysis of variance and Student’s For this study, we will make our data available to the scientific community, which will avoid unintentional duplication of research. All the research data will be shared openly and timely in accordance with the most recent NIH guidelines. The structure results have been deposited to Protein Data Bank (PDB) with accession code 5 × 57.How to cite this article: Ma, Y. et al. ACF7 regulates inflammatory colitis and intestinal wound response by orchestrating tight junction dynamics. Nat. Commun.8, 15375 doi: 10.1038/ncomms15375 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures and Supplementary Table3D reconstruction of confocal imaging of WT caco-2 cells for tight junction (ZO-1) and microtubules.ACF7 null caco-2 cells for tight junction (ZO-1) and microtubules.3D reconstruction of confocal imaging of in vivo.FRAP analysis of WT colonic epithelium ACF7 cKO colonic epithelium in vivo.FRAP analysis of


Inspired by the recent spatio-temporal action localization efforts with tubelets (sequences of bounding boxes), we present a new spatio-temporal action localization detector Segment-tube, which consists of sequences of per-frame segmentation masks. The proposed Segment-tube detector can temporally pinpoint the starting/ending frame of each action category in the presence of preceding/subsequent interference actions in untrimmed videos. Simultaneously, the Segment-tube detector produces per-frame segmentation masks instead of bounding boxes, offering superior spatial accuracy to tubelets. This is achieved by alternating iterative optimization between temporal action localization and spatial action segmentation. Experimental results on three datasets validated the efficacy of the proposed method, including (1) temporal action localization on the THUMOS 2014 dataset; (2) spatial action segmentation on the Segtrack dataset; and (3) joint spatio-temporal action localization on the newly proposed ActSeg dataset. It is shown that our method compares favorably with existing state-of-the-art methods. Joint spatio-temporal action localization has attracted significant attention in recent years ,16,17,18Despite the successes of the prior methods, there are still several limiting factors impeding practical applications. On the one hand, a large number of methods ,3,5,13 cAlthough there are several tubelet-style (which outputs sequences of bounding boxes) spatio-temporal action localization efforts ,12,22, tWith applications in untrimmed videos with improved spatial accuracy in mind, we propose the spatio-temporal action localization detector Segment-tube, which localizes target actions as sequences of per-frame segmentation masks instead of sequences of bounding boxes.The proposed Segment-tube detector is illustrated in We conduct experimental evaluations  of the proposed Segment-tube detector and existing state-of-the-art methods on three benchmark datasets, including (1) temporal action localization on the THUMOS 2014 dataset ; (2) spaThe spatio-temporal action localization detector Segment-tube is proposed for untrimmed videos, which produces not only the starting/ending frame of an action, but also per-frame segmentation masks instead of sequences of bounding boxes.The proposed Segment-tube detector achieves collaborative optimization of temporal localization and spatial segmentation with a new iterative alternation approach, where the temporal localization is achieved by a coarse-to-fine strategy based on cascaded 3D ConvNets  and LSTMTo exactly evaluate the proposed Segment-tube and to build a benchmark for future research, a new ActSeg dataset is proposed, which consists 641 videos with temporal annotations and per-frame ground truth segmentation masks.In summary, the contributions of this paper are as follows:The remainder of the paper is organized as follows. In The joint spatio-temporal action localization problem involves three distinctive tasks simultaneously, i.e., action classification, temporal action localization, and spatio-temporal action localization. Brief reviews of related works on these three topics are first provided. In addition, relevant works in video object segmentation are also introduced in this section.The objective of action classification is to determine the presence of a specific action  in a video. A considerable amount of previous efforts are limited to action classification in manually trimmed short videos ,13,28,29Many methods  rely on Temporal action localization aims at pinpointing the starting and ending frames of a specific action in a video. Much progress has been made recently, thanks to plenty of large-scale datasets including the THUMOS dataset , the ActThe sliding window-based methods typically exploit fixed-length temporally sliding windows to sample each video. They can leverage the temporal dependencies among video frames, but they commonly lead to higher computational cost due to redundancies in overlapping windows. Gaidon et al.  used sliThe frame-wise predictions-based methods classifies each individual video frame , and aggregate such predictions temporally. Singh et al.  used a fThe action proposals-based methods leverage temporal action proposals instead of video clips for efficient action localization. Jain et al.  producedThere are many publications about the spatio-temporal action localization problem ,43,44,45In contrast, we propose the spatio-temporal action localization detector Segment-tube for untrimmed videos, which can provide per-frame segmentation masks instead of sequences of bounding boxes. Moreover, to facilitate the training of the proposed Segment-tube detector and to establish a benchmark for future research, we introduce a new untrimmed video dataset for action localization and segmentation , with temporal annotations and per-frame ground truth segmentation masks.Video object segmentation aims at separating the object of interest from the background throughout all video frames. Previous video object segmentation methods can be roughly categorized into the unsupervised methods and the supervised counterparts.Without requiring labels/annotations, unsupervised video object segmentation methods typically exploit features such as long-range point trajectories , motion Differently, supervised video object segmentation methods do require user annotations of a primary object , and the prevailing methods are based on label propagation ,53. For However, all the above video object segmentation methods assume that the object of interest (or primary object) consistently appears throughout all video frames, which is reasonable for manually trimmed video dataset. On the contrary, for practical applications with user-generated, noisy untrimmed videos, this assumption seldom holds true. Fortunately, the proposed Segment-tube detector eliminates such a strong assumption, and it is robust to irrelevant video frames and can be utilized to process untrimmed videos.T frames, our objective is to determine whether a specific action V, and if so, temporally pinpoint the starting frame k. Simultaneously, a sequence of segmentation masks Given a video k from an untrimmed video, as illustrated in A coarse-to-fine action localization strategy is implemented to accurately find the temporal boundaries of the target action Coarse Action Localization. The coarse action localization determines the approximate temporal boundaries with a fixed step-size . We first generate a set of U saliency-aware video clips V , and prk is present in video clip The proposal network is action class-agnostic, and it determines whether any actions  to score each video clip A classification network  is further trained to predict a Fine Action Localization. With the obtained per-clip specific action labels Subsequently, we average specific action scores ith superpixel in frame With the obtained temporal localization results, we further conduct spatial action segmentation. This problem is cast into a spatio-temporal energy minimization framework,ing SLIC , due to Data Term. The data term V. With a segmentation B for V, we estimate two color Gaussian Mixture Models (GMMs) and two location GMMs for the foregrounds and the backgrounds of V, respectively. The corresponding data term V. Similarly, V, i.e., Equation  is definSmoothness Term. The action segmentation labeling B should be spatially consistent in each frame, and meanwhile temporally consistent throughout video V. Thus, we define the smoothness term by assembling an intra-frame consistency model and an inter-frame consistency model.The intra-frame consistency model enforces the spatially adjacent superpixels in the same action frame to have the same label. Due to the fact that the adjacent superpixels either have similar color or distinct color contrast , the welEquation  is definThe inter-frame consistency model encourages the temporally adjacent superpixels in consecutive action frames to have the same label. As the temporally adjacent superpixels should have similar color and motion, we use the Euclidean distance between the motion distributions of temporally adjacent superpixels along with the above contrast-dependent function in Equation  to constw (HOOF)  of the sOptimization. With B for video V.raph cut  to minimV using saliency [k from an untrimmed video V by a coarse-to-fine action localization strategy, and then the spatial action segmentation further produces the spatial per-frame segmentation B by focusing on the action frames identified by the temporal action localization. With the new segmentation B of video V, the overall optimization alternates between the temporal action localization and spatial action segmentation. Upon the practical convergence of this iterative process, the final results B are obtained. Naturally, the temporal action localization and spatial action segmentation benefit each other. In the experiments, we terminate the iterative optimization after practical convergence is observed, i.e., the relative variation between two successive spatio-temporal action localization results are smaller than With an initial spatial segmentation saliency , the temWe conduct extensive experiments on multiple datasets to evaluate the efficacy of the proposed spatio-temporal action localization detector Segment-tube, including (1) temporal action localization task on the THUMOS 2014 dataset ; (2) spaThe average precision (AP) and mean average precision (mAP) are employed to evaluate the temporal action localization performance. If an action is assigned the same category label with the ground truth, and, simultaneously, its predicted temporal range overlaps the ground truth at a ratio above a predefined threshold . Such temporal localization of an action is deemed correct.The intersection-over-union (IoU) value is utilized to evaluate the spatial action segmentation performance, and it is defined asTraining the proposal network. The proposal network is to predict each video clip k  in the original video or not (negative label). If four or more positive labels are recorded, the original video is accepted in the ActSeg dataset and the time boundaries of the action are determined as follows. Each accepted video is independently distributed to 3~4 undergraduate students for manual annotation  and an additional quality comparison is carried out for each accepted video by a graduate student and the best annotation is selected as the ground truth.The complete ActSeg dataset contains 641 videos in nine human action categories. There are 446 untrimmed videos and 110 trimmed videos in its training split, 85 untrimmed videos and no trimmed video in its testing split. Mixed Dataset. To maximize the number of videos in each category  in To summarize, the experimental results on the above three datasets reveal that the Segment-tube detector produces superior results to existing state-of-the-art methods, which verifies its ability of collaboratively and simultaneously implementing spatial action segmentation and temporal action localization with untrimmed videos.The segment-tube detector is highly computational efficient, especially comparing with other approaches that fuse multiple features. Most video clips containing pure background are eliminated by the proposal network, thus the computational cost with the classification network is significantly reduced. On a NVIDIA  Tesla K80 GPU with 12 GB memory, the amortized time of processing one batch (approximately 40 sampled video clips) is approximately one second. Video clips have variable length and 16 frames are uniformly sampled from each video clip. Each input for the 3D ConvNets is a sampled video clip of dimension We propose the spatio-temporal action localization detector Segment-tube, which simultaneously localizes the temporal action boundaries and per-frame spatial segmentation masks in untrimmed videos. It overcomes the common limitation of previous methods that either implement only temporal action localization or just  video object segmentation. With the proposed alternating iterative optimization scheme, temporal localization and spatial segmentation could be achieved collaboratively and simultaneously. Upon practical convergence, a sequence of per-frame segmentation masks with precise starting/ending frames are obtained. Experiments on three datasets validate the efficacy of the proposed Segment-tube detector and manifest its ability to handle untrimmed videos.The proposed method is currently dedicated to spatio-temporal localization of a single specific action in untrimmed videos, and we are planning to extend it to simultaneous spatio-temporal localization of multiple actions with per-frame segmentations in our future work. One potential direction is the generation of multiple action category labels in the classification network of the coarse action localization step, followed by independent fine action localization and spatial action segmentation for each action category.


Episodes of malnutrition in early childhood can produces alterations in the salivary glands. The investigation of mechanisms that can reduce the impact of malnutrition on the defenses of the organism is of the utmost important and interest to public health. The aim of this study is to evaluate the effect of low-level laser on the saliva of children aged 1 to 5 years with energy-protein malnutrition.Mandatory inclusion criteria are diagnosis of malnutrition. The sample will consist of 50 men and women malnourished children aged 12 to 71 months. Saliva will be collected and the volume of saliva will be measured and the salivary flow rate will be determined (mL/min). Concentrations of salivary IgA in all samples will be measured using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit. Low-level laser (laser diode) will be administered in the region of the parotid glands bilaterally as well as in the regions of the submandibular and sublingual glands.This study will be the first that investigate the effects of local laser therapy on the salivary glands of malnourished children.Clinical.trials.gov as NCT03355313, first received in 21 November 2017. Thus, malnutrition means that cells do not receive the nutrients necessary to perform their functions of energy production, tissue formation/repair, and the regulation of their own functioning.,9Energy-protein malnutrition occurs when there is an imbalance between food intake and the physiological expenditure of energy and nutrients in all cells of the body, including those responsible for the formation of dental tissue, saliva, and the oral epithelium.,11Malnutrition in the early years of life leads to growth deficits in childhood, as reflected by anthropometric indicators. This condition is also associated with a higher child mortality rate, greater probability of acquiring infectious diseases, impaired psychomotor development, and poorer academic performance as well as lower productivity in adulthood.,13Episodes of malnutrition in early childhood, with consequent calcium, phosphate, and vitamin A, C and D deficiencies, can increase one's susceptibility to dental caries through 3 probable mechanisms: defects in tooth formation (odontogenesis), delayed tooth eruption, and alterations in the salivary glands.–16 As saliva is the main defense factor of the oral cavity, a reduction/change in its physical properties (secretion rate and buffering capacity) can cause immunological disorders that affect an individual's defense capacity.It is likely that the significant increase in susceptibility to caries in malnourished individuals stems from alterations in the salivary secretion rate, since a reduction in salivary flow  increases the susceptibility to both dental caries and dental erosion.–20 Indeed, patients with IgA deficiency can experience recurring upper airway , lower airway (pneumonia), and gastrointestinal (diarrhea and parasitosis) infections.Studies have demonstrated that salivary Immunoglobulin A (IgA) also plays an important role in the immunity of the oral mucosa. and is found in serum and organic fluids, such as saliva. There are 2 types of this antibody. IgA1 is produced by B cells of the bone marrow and IgA2 is produced by B cells of the mucosa, accounting for approximately 80% of the total production. Once produced by plasmocytes (differentiated from activated B cells) in the lamina propria of the epithelium of the salivary glands, IgA binds to a polymeric Ig receptor in the base of the epithelial cells, where it is transported and released through proteolytic cleavage to join other substances that compose the salivary mucus.,23IgA was first identified in 1959 by HeremansStimulated and non-stimulated salivary flow rates can be reduced significantly in individuals who have suffered severe malnutrition in early childhood as well as those with continued nutritional stress. Thus, malnutrition can exert a continual effect on the reduction in salivary gland function in adolescence as result of malnutrition in early childhood, which suggests that exocrine systems can be compromised for long periods after exposure to malnutrition. The conversion of laser light into useful energy for cells through photochemical and photophysical reactions can simulate the production of mitochondrial adenosine triphosphate (ATP), cell proliferation, and protein synthesis.,27The investigation of mechanisms that can reduce the impact of malnutrition on the defenses of the organism is of the utmost important and interest to public health. Among such mechanisms, low-level laser therapy has demonstrated effectiveness in the treatment of diverse conditions and disease through the promotion of the biomodulation of the cell metabolism and due to its analgesic and anti-inflammatory properties with no mutagenic or photothermal effects. Regarding an increase in salivary IgA, low-level laser intensifies the activation of B lymphocytes, which differentiate into plasma cells, thereby contributing to the increase in immunoglobulin levels.Laser stimulation of the major salivary glands to produce more saliva occurs through the increase in local circulation due to vasodilatation, the induction of the proliferation of glandular cells and cell respiration/ATP synthesis as well as the release of growth factors and cytokines to stimulate protein exocytosis.It is important to study the impact of malnutrition in children because its long-term consequences could be reduced which implies promoting positive effects on oral and general healthy.The primary aim of this trial is to evaluate the effect of low-level laser on the saliva of children aged 1 to 5 years with energy-protein malnutrition. Secondary outcomes are to establish the degree of malnutrition in children aged 1 to 5 years and to analyze the saliva of children aged 1 to 5 years with energy-protein malnutrition.22.1An experimental cross-sectional study is proposed, which will be conducted at the Center for Educational and Nutrition Recovery (CREN) in the city of Maceió, state of Alagoas, Brazil.2.2https://clinicaltrials.gov/ct2/show/NCT03355313.Clinical.trials.gov as NCT03355313, first received in 21 November 2017, 2.32.3.1This cross-sectional study will use a sample of children aged 12 to 71 months of the 7th Administrative Region of Maceió. This region is one of the poorest region in Maceió. The children's parents/guardians will be interviewed at the CREN in the city of Maceió, state of Alagoas, Brazil. The sample size was estimated considering a prevalence of malnutrition of about 10% in children under 6 years old, with statistical power of 80% and 95% significance, resulting in a sample of 50 children.2.4The allocation sequence will be generated by CREN. The number/symbol of each child will be sent by the computer by the registration model in CREN which is based on the distribution by learning classes. Assessments regarding saliva and nutritional status will be blinding for trial participants and data analysts.2.5Children aged 1 to 5 years enrolled at the CREN in the city of Maceió whose parent/guardians signed as statement of informed consent agreeing to the participation of the children.2.6Children aged 1 to 5 years not enrolled at the CREN in the city of Maceió and children whose parents/guardians did not sign a statement of informed consent, teachers, caregivers, health professionals, and voluntary interns.2.72.7.1Z score) between +1 and –1 standard deviations (SD) of the median indicative of the ideal range, Z between –1 and –2 SD indicatives of mild malnutrition, Z between –2 and –3 SD indicatives of moderate malnutrition and Z >–3 indicative of severe malnutrition.The children will be weighed on a duly calibrated electronic anthropometric scale  barefoot and wearing light clothing in the presence of the mother or caregiver. Height will be determined using a non-flexible metric tape measuring 2 m in length (precision: 0.1 cm). The measurement will be performed twice and the mean will be used for the calculation of the indices. Using these measures, height for age and weight for age will be determined using the nutritional reference standards recommended by the World Health Organization, with a cutoff point . Salivary flow values will be analyzed based on the values below  using the drainage method. The children will have not ingested food or beverages (except water) 1 hour before the procedure and will not have performed oral hygiene within 2 hours before the procedure. The child will be instructed to swallow prior to the collection and then instructed not to swallow, allowing the saliva to drain between the lips (which will be separated) into a test tube (aspirator) connected to a 15-mL Flacon tube positioned near the mouth.One aliquot (1 mL) of saliva will be transferred immediately to a small tube (capacity: 5 mL) for titration with hydrochloric acid 0.005 Ne. The tube will be shaken for 15 seconds and buffering capacity will be measured using a glass electrode (Orion) coupled to a pH meter . Titration under partial carbon dioxide gas is considered the standard method for determining the buffering capacity of saliva. The reading of this test is performed based on the following parameters: pH 3.0 to 4.0 = very low to low buffering capacity; pH 4.5 to 5.0 = intermediate buffering capacity; and pH ≥5.5 = normal/good buffering capacity. and will be analyzed  kit  Fig. . Salivarα = 0.05). The Statistical Package for the Social Sciences (SPSS)  version 15.0 will be used for all analyses.The data will be analyzed using Analysis of Variance (ANOVA) and Pearson's correlation test . Laser will be administered for 10 seconds on 4 intraoral points and 4 extraoral points in the region of the parotid glands bilaterally as well as 1 intraoral point and 1 extraoral point in the regions of the submandibular and sublingual glands. The first session will occur after the collection of saliva. The second and third sessions will be performed 7 and 14 days after the first session, respectively. The final saliva collection will be performed after the third laser session.2) = 2500; beam profile = multimodal; beam spot on target (cm2) = 0.04; irradiation at target (mW/cm2) = 2500; exposure time (s) = 40; Radiant exposure (J/cm2) = 100.0; radiant energy (J) = 4; number of points irradiated = 10; irradiated area (cm2) = 0.40; application method = Contact; number of treatment sessions = 3; frequency of treatment sessions per week = 1; Total radiant energy (J) = 40.The laser will be adjusted according to the following parameters: Central wavelength (nm) = 808; spectral band width (FWHM) (nm) = 2; operational mode = continuous; mean radiant power (mW) = 100; polarization = randomized; aperture diameter (cm) = 0.2; irradiation at aperture  and correlation with the malnutrition will be established. The results will be analyzed using ANOVA and Tukey's contrast test. The level of significance will be 5%.3The study of salivary aspects in malnourished children and possible treatments that can be used to improve salivary quality and quantity in these children has significant social relevance, as saliva is one of the main mechanisms against infection and participates in essential functions of life, such as swallowing and the maintenance of oral health.Assistance provided by all the CREN employees was greatly appreciated. Also, the authors would like to show their gratitude to the children and parents.Conceive and design the study: Karlla Almeida Vieira, Alessandro Melo Deana, Sandra Kalil Bussadori. Will perform the experiment: Karlla Almeida Vieira, Clarissa Moraes Bastos, Marilya Gabriella Correia Vitor, Karlla Almeida Vieira, Maria Fernanda Setúbal Destro Rodrigues, Sandra Kali Bussadori. Will analyze the data: Karlla Almeida Vieira, Vanessa Christina Santos Pavesi, Maria Fernanda Setúbal Destro Rodrigues, Kristianne Porta Santos Fernandes. Will perform the statistical analysis: Alessandro Melo Deana, Sandra Kalil Bussadori. Write the paper: Karlla Almeida Vieira, Alessandro Melo Deana, Kristianne Porta Santos Fernandes, Sandra Kalil Bussadori.Conceptualization: Karlla Almeida Vieira, Alessandro Melo Deana, Sandra Kalil Bussadori.Data curation: Karlla Almeida Vieira, Kristianne Porta Santos Fernandes, Sandra Kalil Bussadori.Formal analysis: Karlla Almeida Vieira, Alessandro Melo Deana, Maria Fernanda Setúbal Destro Rodrigues, Vanessa Christina Santos Pavesi, Sandra Kalil Bussadori.Investigation: Karlla Almeida Vieira, Maria Fernanda Setúbal Destro Rodrigues.Methodology: Karlla Almeida Vieira, Clarissa Moraes Bastos, Marilya Gabriella Correia Vitor, Alessandro Melo Deana, Maria Fernanda Setúbal Destro Rodrigues, Sandra Kalil Bussadori.Project administration: Karlla Almeida Vieira, Sandra Kalil Bussadori.Supervision: Karlla Almeida Vieira, Sandra Kalil Bussadori.Validation: Karlla Almeida Vieira.Writing – original draft: Karlla Almeida Vieira, Alessandro Melo Deana, Sandra Kalil Bussadori.Writing – review and editing: Karlla Almeida Vieira, Alessandro Melo Deana, Kristianne Porta Santos Fernandes, Sandra Kalil Bussadori.


Nigeria has high levels of alcohol consumption, and little or no regulation of the alcohol industry. There is a dearth of studies exploring young adults’ drinking in a Nigerian context with only a few predominantly quantitative surveys. These do not explore the social meanings attached to drinking practices nor do they shed light on potential gender differences and how these are mediated by popular media. This qualitative study addresses this gap with semi-structured interviews involving 31 undergraduate students. It identifies that media consumption shapes drinking behaviour in ways which are highly patterned and gendered. Participants with high consumption of both Hollywood films and popular American reality television series associate heavy alcohol consumption with high social status, economic independence and gender equality. By contrast, Nollywood  films which are intended to act as moral tales and warn of the dangers of drinking appear paradoxically to support participants’ views of alcohol as positive . Nigeria currently has no serious regulation of alcohol on television which is embedded in everyday life. Attempts to develop wider public health campaigns and policies should take this saturated media landscape into account to develop harm reduction strategies which are linked directly to media literacy programmes. Mass media portrayals of alcohol are pervasive, and their impact on adolescents and young adults are significant , 2. The According to Miller , ‘audienand providing models and information about alcohol use that may not be available in peer group or family’ . So they had a baby, and somehow they just had to marry, and that was how the guy’s life changed, and he became influential. Interestingly, their views on alcohol in Hollywood films were shared by all female participants whose views not only highlighted the positive representation of alcohol but also showed how characters drink freely in contrast with the reality of alcohol consumption in Nigeria where young people’s drinking would be considered taboo. In the excerpts below, female participants explain the role they imagine that alcohol plays in the everyday life of ‘typical’ Americans and how alcohol representation in Hollywood differs from portrayals in locally made movies:In all these movies I watch, I noticed that these outsiders [foreigners] take red wine all the time. Like even if they are just eating… lunch or dinner, you’ll see them pouring themselves a glass of wine, and they are always drinking … In Nigerian films, alcohol use among young people is kind of restricted … I see Nigeria as more of a religious kind of country than Americans because most ‘‘Janded’’ [foreign] movies I watch, they tend to drink alcohol, especially the New Yorkers and those New Jersey people… The youth there drink alcohol …; most of their scenes are always in clubs … The participants were not only aware of the positive images of alcohol in Hollywood films, but also reality television shows such as Jersey Shore and the America’s Next Top Model . Both male and female participants demonstrated their perceptions of how alcohol consumption among young people is normalised in American television and movies, unlike those produced by, and about Nigeria.Participants were invited to share their views on the type of movies they watch, the reasons for their preference, whether or not they typically see onscreen alcohol portrayals in the movies and their perceptions of how alcohol was represented. Many of the male participants indicated that they watch Hollywood movies, and all reported seeing alcohol depicted. When they were asked to explain how it was depicted, their accounts show that alcohol appeared to be normalised within different genres such as drama, epic and High School films as evidenced in Boniface’s and Peter’s words: ‘it’s kind of rare where you watch American films, and you don’t see drinking in that film’; ‘I just noticed that they enjoy it, and ladies drink alcohol too…They enjoy drinking’. The majority of the male participants demonstrated awareness of alcohol portrayals, showing how it is used in the performance of masculinity:…There is this drama that I saw; the guy does this funny thing of always having a bottle or a can of beer in his pocket … and he always drinks. Sometimes he opens the bottle with the head of his belt or he hits it on his head to open the bottle before drinking …; you really want to be like him. The guy is always the happening guy [popular] … I think I would like to go to a bar one day so that I can feel like this guy … Chike said he watches television for up to 21 hours a week and typically drinks four bottles of beer or three bottles of stout  on each drinking occasion. One significant aspect of his account is his plan to go to a bar to practise what he saw in the movie. Another is his reference to ‘the guy is always the happening guy’. Nearly all of the male participants demonstrated their perception of how drinking without showing signs of intoxication is related to ‘fame’  what they are not… Seeing a celebrity drinking Heineken in a movie … maybe he drank it in a very unique style, you’ll now want to keep that same position when you drink. You just want to be like him. Therefore, you make Heineken your brand. Like if this ‘big man’ can really like Heineken, why don’t I try Heineken? … So you’ll go to a bar and say, ‘please give me a bottle of Heineken,’ because you just want to have those feelings that you’re like him. Of particular interest, is his reference to the ‘Heineken brand’. In Nigeria, any product that is imported from Western countries is regarded as superior to its local counterparts. For example, despite the fact that ‘Nigerian Breweries Plc’ (owned by Heineken) localised the production of Heineken beer since the early 2000 s, many Nigerians still mistake the brand as a foreign product. In this vein, some of the male participants mentioned that Heineken brand is a beer for the ‘upper class’, and also indicated that they drink Heineken whenever they want to show off their ‘class’ to their peers in public drinking spaces. Cranwell and colleagues  let’s do the same thing and have fun too. Larry is among the participants who preferred Hollywood films, and he spends up to 42 hours weekly watching television. Within our sample, he engaged in drinking that was heavier than many participants and it appears that this heavy television viewing normalises his consumption of large quantities of alcohol. Additionally, all the male participants revealed that they had taken part in drinking games, and some described learning about these from ‘movies high school movies’. For example:For those games they do at parties, I think they learned it from scenes in High School movies. I mean ‘the first to drink and drop the bottle’… .The positive portrayal of alcohol in Hollywood movies is also perceived to have influenced our female participants. For example, some believe that onscreen drinking by movie characters could lead to alcohol initiation while others have learned how to play drinking games from Hollywood films:I’ve seen movies where friends do ‘drinking competitions’; they’ll give you a glass of wine and you’ve got to gulp it down all at once. If you take it twice, you lose the game… Even if it’s a bottle, you have to drink it without removing the bottle from your mouth until the drink finishes… Sometimes they will give drinks that have higher alcohol content, and if you vomit you lose the game. So … it’s [learned] in movies. That is where we see different competitions, and that’s where I saw for the first time that you could drink alcohol without removing the bottle from your mouth … The women’s accounts also revealed nuanced, sophisticated aspirational drinking which appears to have been influenced by their viewing of Hollywood movies and other American reality television programmes:A character said in one American movie … that red wine is good for the heart because it helps pump blood to the heart. I have not really asked a doctor, but since then, I just started taking it… Interviewer: So are you saying that you started to drink red wine because you watched the movie?Yeah it’s more like ‘professional’ because these people [movie characters] always use it for ‘professional things’… they are the ones that actually made me develop a liking for red wine. Because … if they go to meetings, or they are celebrating something, they will say, ‘this calls for wine’, and you’ll see them just open wine and pour it, and they take it with wine glasses. So it looks very ‘professional’ and ‘mature’. So that’s why I just like it. Female participants described in detail how not only their drink of choice but their comportment during drinking are influenced by imitating movie characters who were considered to be sophisticated and professional. Chichi consistently uses the terms ‘professional’ and ‘mature’, and highlights her admiration of Hollywood movie characters, stating that they often comported themselves in parties, conferences or business meetings by queuing up or sitting down to be served by waiters, who pour wine into their glasses. In her words, ‘as they are talking, they sip, and they drop [put down the glass], so it’s very clean and classic’. Her perception of these actors shows that their onscreen behaviour is quite different from what she regularly sees in Nigeria, where poor queuing culture or the act of drinking directly from the bottle prevails. She also references what she considers to be ‘sophisticated’ drinking in popular reality franchise:Another thing that stimulated me was the ‘American Next Top Model’. So one of the tests these models had to pass was wine testing … just to show you how these people value wine. They are blindfolded, and then they bring two different types of wines … and tell you to taste this one. After taking it, you’ll taste the other one. After testing them, they will be like, can you differentiate these two wines or can you name the one that is French or an American wine? …And then you’ll see some of the models getting it right. At least … even though they don’t get both of them, they will get one right just because … wine is something they are used to … As a self-reported Hollywood film addict, who spends over 50 h weekly watching television, it appears that the consumption of American films may have a significant influence on Chichi’s drinking behaviour and she consumes at least two bottles of red wine per drinking occasion. The media may have also influenced her perception of social reality, in terms of social inequality and women’s subordination in Nigeria. During the interview session, Chichi argued that women should be free to consume large quantities of alcohol of their choice like men do if they wish. Other participants in the study supported the view that seeing alcohol represented in a positive light in a movie could engender students’ drinking. Both male and female participants stated that many youths identify with Hollywood movie characters, seeing them as ‘significant others’. Thus, they often copy their drinking (and non-drinking) behaviours.The data also revealed that Hollywood’s High School films are popular on this campus and offer a particularly aspirational view of drinking amongst students:The [unequal drinking] orientation is changing… The media are displaying alcohol and this time around, it is no longer restricted [to males]… When you come to an environment like this university…, you meet people, you mix with friends, and you hear this orientation of equality between male and female genders; so everybody wants to try out new things. ‘Nobody wants to be left behind just because you are tagged a woman’. Our data reveal that while the men who view Hollywood films consume four or more bottles of beer/stout on a drinking occasion, the majority of their female counterparts also use large quantities of wine or other flavoured alcoholic beverages, highlighting the globalization of the culture of intoxication among youths : we are the ‘big girls’ or the ‘big boys’. The data also revealed some nuances on the way in which Nigerian movies portray how alcohol consumption can be used to ameliorate anxiety and depression. For example, male participants described characters in Nollywood movies using heavy drinking in a bid to cure depression. Although this is clearly a negative portrayal, our male participants indicated that these portrayals are powerful and could possibly influence behaviour, particularly for young people facing relationship difficulties:…Under normal circumstances, if somebody breaks your heart you’ll think over it and get rid of it, but if you are watching those videos where they normally create the impression that when people are depressed or heartbroken, they will prefer to drink, you will do that…. Even the women maybe her boyfriend annoys her so she will go to the bar and start drinking. That is the scenario they create… When you are depressed, the next thing you’ll do is go to the bar and start drinking. Kelly revealed how he turned to alcohol to help his depression and how he learnt this practice through the media:… It’s from the [local] media. I have heard a lot of people talk about that. Initially I didn’t believe the whole thing, but when I started drinking I discovered that it worked for me…, so now I have formed the habit of taking alcohol when I am depressed. Kelly views local television for over 50 h weekly, and this appears to have influenced his drinking practices, in that he consumes three bottles of beer per drinking occasion, in addition to the large quantities of locally-made ‘palm wine’ .Interestingly, the participants also noted that Nollywood films show how alcohol consumption is used to construct superior masculinity or femininity among young people:Among our female participants, it was found that local media influenced their brand preference with positive attributes being associated with certain brands. Ada, for instance, recalled that local television influenced her preference for ‘Guinness stout’ which she found to be particularly helpful during menstruation because: ‘‘it cleanses out the rubbish in our stomachs… if you have it with milk, it gives blood, and it also gives energy’’. Many other participants also revealed that this practice is popular among Nigerian women who use stout to ameliorate menstrual pain and perceived it as useful to enhance energy or regain blood . When sThese accounts have shown that although the Nigerian media depict alcohol mainly in a negative light, some of the participants were nonetheless influenced to consume alcohol in diverse ways. While the men learnt to use the number of bottles an individual can drink to determine how manly he is, and to ameliorate depression, they also learned to associate specific brands with affluence and worldly status. The women also learnt to associate drinking with a higher social status and to ameliorate period pain.The findings of this study highlight how young people’s drinking in Nigeria is mediated by media representations and aspirational ideas fuelled not only by Western images but also by local media depictions of alcohol. Our analysis found evidence of alcohol images depicted in a positive light in Hollywood movies, reinforcing previous research , 21 whicThe lived experience of the participants showed that their viewing was gendered . It alsoInterestingly, the fetishization of foreign alcoholic beverages evident here tends to reflect practices which refer back to Nigeria’s colonial history. During the colonization of Nigeria 1914–1960) by the British Colonial Government, imported alcoholic beverages were reified by the British Officials, who consumed foreign beverages and distanced themselves from the locally-made brands  by the B. ConsequThis was reinforced even when beer importation began to grow. Again, drinking British, Danish and German beers became ‘an emblem of European lifestyles and values’ . The asset al.'s [Our findings also demonstrated sophisticated aspirational drinking among the participants, especially the women, to the extent that they not only copied the brand and admired the drinking postures of Hollywood movie characters, but they used their drinking to perform non-traditional femininity . The actet al.'s  assertioet al.'s [The women’s perception of drinking as a marker of female independence was also mediated by the American media, but this appears to be a deviation from the traditional femininity that is anchored in the Nigerian culture, which discriminates against women’s drinking , 46. Indet al.'s  assertioOur data also identified participants’ perception of the negative representations of alcohol in Nollywood films, and how male participants in particular, described this as an exaggeration of social reality. Paradoxically, the negative depiction was also perceived to influence drinking with male participants described learning to drink to enhance their low mood from these supposed moral tales. This indicates that audiences have a complex and often ambivalent relationship with media that can be difficult to predict. Our data supports other research that shows how participants may reject or subvert health related messages in popular media .In this article, we explore how drinking is embedded in the everyday culture of a group of young Nigerians at University. It is a small study and relied on the accuracy of self-reported accounts of how our participants consume alcohol and local/foreign media. However, it represents a rare qualitative account of how media and alcohol are part of University life for our participants. Although, this may also be the case for most students in other countries, a focus on the Nigerian context shows a particular aspirational outlook which we argue could be based on a specific colonial past. Our research suggests that local and imported media may be important conduits of messages concerning drinking– moral tales from local media can be easily discounted in light of more aspirational messages from US media. At present, there is no regulation of alcohol via policies in Nigeria  and impoHowever, we argue that it is vital that media literacy education is aimed at facilitating the critical engagement with ‘media messages that promote risky behaviors’ . Because