Procrastination is seen as a severe problem among young people, and many factors have been claimed to be associated with it, playing video games being one of them. One of the reasons why video games might be related to procrastination is their ability to offer instant gratification and feedback, while at the same time offer distractions from less tempting and rewarding tasks. It is not yet agreed on whether or not video game players are more prone towards procrastination and discounting of future rewards.Over 500 participants across two studies completed two surveys on video gaming habits, as well as a measurement of procrastination tendencies. In study 1 participants performed an experiential discounting task, while participants in study 2 performed the 5-trial adjusting delay discounting task, both tasks assessing preference for delayed larger rewards.r(513) = .122.In study 1, hours of videogaming was not significantly related to procrastination or the discount rate. In study 2, hours of videogaming was not strongly associated with procrastination and delay discounting either. However, when asked why they play, those answering to escape reality and to reduce stress had more problems of procrastination than those who play for entertainment, reward or social reasons. Overall, the association between procrastination and hours spent playing video games was weak but positive, Time spent enjoying and engaging in video gaming is done for various reasons, only for a few this is related to procrastination. By using only hypothetical payouts in the discounting tasks, the absence of a relationship between hours spent video gaming, procrastination and delayed gratification requires further investigation. However, playing video games is more than mere procrastination. From the 70s arcade classics Pong and Space Invaders to modern day triple-A games such as Halo and Grand Theft Auto, video games have gone from being a phenomenon at the local arcade-halls to an integral part of the daily lives of millions of gamers around the world. With a multi-billion dollar industry that now far surpasses Hollywood in revenues , game deProcrastination, aka “voluntarily delay an intended course of action despite expecting to be worse off for the delay” , has seeWhile a large body of research on procrastination has investigated the relations between personality traits and procrastination , impulsiWith games becoming more widespread and readily available , games cTo investigate to what degree video gaming is a medium for procrastination, we conducted two studies. In study 1, we explored the connection between gaming hours and procrastination in a Norwegian sample. We also measured sensitivity to delay discounting with hypothetical rewards. Our rational was that highly impulsive individuals should have more problems resisting the “pull” of easily accessible and entertaining games, resulting in more time playing games, and more delay doing other important tasks, i.e. procrastination. In study 2, an online survey, we asked also for the reasons of playing video games, as this can be an important factor for whether or not gaming is a sign of procrastination, or just a relaxing pastime. In this survey we also used two short discounting tasks, one temporal and one effort discounting task. Our rational was that those procrastinating using video games also display cognitive and temporal discounting.If procrastinators are more likely to play video games, and have a stronger need for immediate reward, then one would expect that many hours of video gaming and a high degree of delay discounting is common among procrastinators. That is, we expect that the more one is engaging in activities that provide immediate reward such as video gaming the more one procrastinates. Note that we did not focus on internet gaming, but asked for engaging in any computerized game, offline and online.M = 25.6, SD = 6.8). The low completion rate (17.9%) can be explained by participants having to install the Inquisit Web Player (Millisecond.com) for performing the EDT. Furthermore, of the 119 only 102 had valid EDT results, e.g. completing all rounds.A total of 663 participants were recruited through social media , e-mail and bulletin boards at various Universities in Norway (28.2.-7.3.2016). Survey language was Norwegian. Of those, 393 finished the questionnaire ; and 119  finished both the questionnaire and took the experiential discounting task (EDT). Participants were between 18 and 60 years  has been determined or 5 min elapsed. IP refers to the point where the subjective value of both presented sums is (apparently) identical to the participant. The IP was based on the last six choices, i.e. the average adjusting-option amount. A potential waiting time was added between each trial and after the last trial. Participants were not paid their earned winnings, but were instructed to act as if payment would occur through task instructions. By using only hypothetical rewards, , 47 founN = 393) the PPS had a Cronbach’s alpha of .92, while for the sample with valid EDT results (N = 102), PPS had a Cronbach’s alpha of .928 , simulation, sport, strategy), device used  and age started video gaming. Type of video game was rated on a 5-point Likert scale from 1 – playing this type not at all/rarely to 5 = playing this type of game very often; device used was measured as a percentage.Qualtrics.com). At the end of the questionnaire, each participant was asked to proceed to download the Inquisit Web Player  in order to perform the EDT task.The study was online and took about 15 min to complete. Participants read first a short description of the purpose of the study, contact information, and by proceeding gave informed consent. They were then presented with questions on their video game usage and history, and answered the 5-item PPS. The survey was implemented in Qualtrics (N = 2). The procrastination score was the average score from the five PPS items. We followed the procedure of . A linear regression with age and video gaming hours as predictors explained 1.8% of the PPS score, F = 4.766, P = .009, with video gaming  but not age  statistically significantly contributing.Combining the data from study 1 and study 2 into one statistical analysis, we found a weak but statistically significant positive correlation between the number of hours played video games and the PPS score, F = .333, P = .564, η2 = .001, or the discounting factor k: F = .758, P = .385, η2 = .003 but the co-variate video gaming hours was statistically significant: F = 6.67, P = .01, η2 = .029. Neither in study 1 nor in study 2, did we find a medium effect size of delay discounting and procrastination.Next, we also looked whether delay discounting would be related to procrastination. In an ANCOVA with study 1 and 2 as between subject factor (as we used two different discounting paradigms) yielded no significant main effect for study, Video games are played for various reasons, although the majority play video games for entertainment purposes, some choose it as a break from daily activities, escapism or stress reduction.Our data did not support any strong relationship between hours of videogaming, procrastination, and delay discounting, and effort discounting. In both surveys we found no statistically significant relation between hours spent video gaming and procrastination, nor between delay discounting and procrastination. The associations had a small effect size but were all in the predicted direction. Only in combining the data from both studies could we find a very small, but statistically significant, relation between procrastination and time spent on video games. We caution this result, as we advertised the study as being about videogaming, likely leading to a collider bias i.e. we may have recruited those gamers who are more prone to procrastinate than would be found in the population of all gamers. For example, recent surveys on internet gaming found a low prevalence  of problIndeed, as expected we found that those who indicated that they were playing video games as a mean to escape from reality, or to have a break from stress, had a significantly higher level of procrastination than those who were playing for entertainment, break from everyday life, or for the social value of games. Curiously, even though it seems that these participants were using video games to procrastinate, they did not report more time playing video games than those who were procrastinating less. This strengthens previous findings that hours of video gaming is not related to severity or negative outcomes , 63, butIn this study 2 the English sample spent more hours video gaming than the Norwegian sample. It is possible that some Norwegian respondents took the English version, and we would not expect differences due to the language in which the survey was taken , but thiResults from the effort discounting task showed that of those making errors, those who spent more time, which we treated as proxy for having spent more effort to correctly identify the majority color, had less problems of procrastination. This data is preliminary as a more controlled effort discounting task is required to assess whether procrastination is a general strategy to avoid demanding tasks, or specific to the task that one is postponing. We have chosen this task to avoid a high drop-out rate (compare study 1 vs study 2) but the number of trials are clearly insufficient to draw firm conclusions. However, this preliminary data is in line with the literature that more effortful tasks, and a focus on getting done quickly, is related to more procrastination , 76. FutAs in study 1, we did not find that the discount rate was related to procrastination, contrary to the conclusions of . One posThe purpose of these studies was to examine whether procrastination was related to hours spend video gaming and discounting. Our results indicate no strong relationship between delay discounting, hours spent on video games, and procrastination. Study 2 suggests that not delay but effort discounting might contribute to procrastination. Our data further does not support a strong relationship between video gaming hours and procrastination, but procrastinators indicate that they play for the sake of escaping from reality and to get a break from stress .Our findings are thus in line with newer research showing that amount of time spent on gaming is not necessarily related to negative consequences such as procrastination. For example,  found thFurthermore, both playing video games and procrastinating might be merely symptomatic of other causes, e.g. depression, anxiety ; and peoFirstly, we relied on subjective measures and did not observe hours spent video-gaming. Using smartphone apps  would prThirdly, we did not concomitantly measure depressive symptoms or general well-being, nor included measures on Internet Gaming Disorder.Fourthly, our surveys did not cover all possible factors shown to influence procrastination, e.g. we did not ask whether respondents were students or employees, or unemployed .r = .1) could not be found with 95% power. The sample size rationale was based on [Fifthly, study 2 was statistically insensitive for small to medium effects, and even when pooling study 1 and 2, small effect sizes (based on  but we dbased on , but useThe small effect size between hours playing video games and procrastination severity reported here, needs further investigation. Our pooled data had enough power to detect a small effect size with 90% power. But we did not control for cultural effects and prevalence rates of internet gaming disorder do differ between countries . HoweverIt is possible that if one uses more objective measures of actual hours played excessive gaming may be stronger related to pathological procrastination than found here. On the other hand, by using other scales to measure procrastination, and over a wider age range, there might be no relationship between procrastination and videogaming, as videogaming is just one of many means to procrastinate (e.g. , playingTo our knowledge this is the first study measuring concomitantly procrastination, video gaming habits, and preference for immediate reward. We found no strong support that procrastination is linked to hours of video gaming. By using only hypothetical reward, we also found no association between delay discounting and procrastination. Potentially what and why someone plays video games not the number of hours are a more promising avenue for procrastination research.


Little is known about alcohol consumption among Africans living in rural and urban Africa compared to African migrants in Europe. We compared the patterns of alcohol consumption in a group of Ghanaians living in different locations in Ghana and in Europe and examined the factors associated with drinking alcohol.Data were from a cross-sectional study (RODAM) of Ghanaians aged 25–70 years living in rural and urban Ghana and in Amsterdam, Berlin and London. Information on how often participants consumed at least one standard alcoholic drink in the preceding 12 months, the type of alcoholic beverage and the average serving size was obtained using a food propensity questionnaire. The associations between drinking alcohol and socio-demographic variables, and frequency of attending religious services were investigated using logistic regression models stratified by site and sex. For Ghanaians living in Europe, the number of years since migration and acculturation were also included in the model as covariates.4280 participants (62.2% women) were included in the analyses. In both men and women, the prevalence of drinking and amount of alcohol consumed per day was highest in Berlin  and lowest in urban Ghana (41.4% and 26.8%). After adjustment for age and education in both men and women in Europe, those attending religious services less frequently reported higher levels of drinking alcohol than non-attendants  p-trend with frequency <0.001 in men; 0.002 in women); this association was seen also in men in rural Ghana (p-trend = 0.001) and women in urban Ghana (p-trend = 0.02). The prevalence of drinking was positively associated with years since migration in both men and women in Europe  test for trend p = 0.03 in women; OR 1.29  but no association was found with self-reported measures of acculturation .There are marked differences in alcohol consumption between Ghanaians living in Europe and in Ghana suggesting migration has an important influence of drinking patterns and also suggesting the possibility of requiring different strategies in alcohol reduction campaigns among Ghanaians in different locations. Alcohol consumption is a major public health issue worldwide, accounting for a significant burden of morbidity and mortality annually.–3 The haSeveral factors have been identified at the individual and societal level to affect the magnitude and patterns of alcohol consumption. These include environmental factors such as economic development, cultural and social factors including religious belief, availability of alcohol and the effectiveness of alcohol policies., 10 A wiAlcoholic beverages have been consumed for generations in many sub Saharan African communities but with considerable variation in the pattern and purpose of consumption. Data on Migration and markers of acculturation to the host culture  in the country of settlement have been shown to influence the consumption of alcohol in some but not all studies. AcculturData for the current study are based on the RODAM study, a cross-sectional study of a population of Ghanaians living in three European cities  and in urban and rural Ghana. The sampling process has been described in detail previously. In brieAlcohol consumption patterns were assessed in response to questions on frequency and serving size of alcoholic beverages  in the preceding 12 months. Serving sizes (i.e standard drinks) were given as 1 bottle (0.5L) of beer, 0.25 L of wine  and 8cL of spirits . Average serving size options ranged from half a portion to three portions. Answers to these questions were used to categorise participants as drinkers or non-drinkers , and to investigate frequency of consuming different types of beverage.Grams of ethanol per day were calculated by multiplying beverage specific frequency and portion size with average beverage strengths for Ghana taken from the West-African food composition tables  and for Europe from the German food composition table . Since the maximum portion size that participants could report was 3 portions, this variable was considered as a potential underestimate of participants’ intake if they were heavier drinkers. To understand the relative possible bias due to under-reporting, participants were additionally categorised by whether they reported three or more portions for any beverage  or not.Socio-demographic variables assessed included age, education, and marital status. The level of education based on the highest qualification gained either in Ghana or elsewhere was classified into three groups as elementary (never been to school or primary/elementary schooling only), secondary  and higher education . Marital status was categorised as married, cohabiting, never married, divorced/separated and widowed.Other variables of interest were frequency of attending religious services, psycho-social distress, and smoking status. Smoking status was categorised as never smoker, ex-smoker and current smoker. Participants were asked if they were currently practicing a specific religion and the number of times they had attended a religious service in the preceding 6 months including attendance to church, mosque or synagogue. The level of psychosocial stress was assessed from a combined score measured by a psychological stress scale created by the INTERHEART study which diInformation on the number of years participants had lived in Europe was obtained for those who had migrated based on the response to the following question: “What is your country of birth?” and “What year did you come to live in the UK, Germany or the Netherlands?” Level of acculturation for participants living in Europe was assessed using Berry’s bi-dimensional perspective on three domains–two psychological  and one behavioural . For eacSince the focus of this study was migration, a small number of participants (n = 75) from the European sites who were not first generation migrants or who did not report country of birth were excluded from the analyses.The prevalence of different aspects of drinking pattern  were calculated separately by sex and geographic location.Given that overall prevalence of drinking was low particularly among women, analysis of associations with drinking were investigated only for the outcome drinking alcohol. Factors associated with drinking were investigated by fitting logistic regression models with drinking status as the outcome and age, education, marital status, psychosocial distress, smoking and frequency of attending religious services as exposures. Models were adjusted for 1) age and 2) age and education. Due to small numbers and to limit the number of comparisons, the three sites in Europe were combined together for the regression analysis and site adjusted for as a confounder for the models for the European sites. As a final stage all exposure variables were included in the model to understand which variables were independently associated with drinking however given missing data on co-variates and difficulty of untangling the direction of effects in cross-sectional analyses these models are reported separately as supplementary material. All models were stratified by sex and study site based on a priori interest in whether associations were the same in each location and by gender. For Europe, the number of years since migration and acculturation were also included in the model as covariates. To investigate the validity of grouping the three European sites together interactions between European city and each exposure in the models adjusted for age and education were tested for using likelihood ratio tests. For the logistic regression models a complete case analysis approach was used. Analyses were restricted for all models to those with data on age and education available .For the current analysis 4280 RODAM study participants born in Ghana, aged 25–70 with information on alcohol consumption were included. Of these, 1949 reported consuming any alcoholic beverage in the past 12 month (45.5%). The proportion of participants completing the Ghana-specific food propensity questionnaire were 99.2% Rural Ghana, 98.5% Urban Ghana, 42.6% London; 97.4% Berlin and 63.4% Amsterdam. There were no differences between those completing the food frequency questionnaire in Berlin, urban Ghana and rural Ghana but there was evidence for difference in marital status, frequency of attending religious services and years since migration for the participants from Amsterdam and for age and frequency of attending religious services for participants from London .The distribution of drinking variables in men and women are shown in The distribution of drinking behaviours by geographic location and sex are shown in Among men the prevalence of drinking alcohol and the median amount consumed per day were highest in Berlin, followed by Amsterdam while the lowest levels were in Urban Ghana. London was similar to Urban Ghana while rural Ghana was in between Urban Ghana and the European cities (with the exception of London). In most sites, the most commonly consumed beverage was beer with the exception of London where drinkers were more likely to drink wine. Wine consumption was more common in all three European cities compared to Ghana. In all sites daily drinking of any beverage was rare but in rural Ghana daily consumptions of spirits (9.7%) was higher as compared to all other sites (<2%).Geographic patterns of drinking behaviours were similar for women with a higher prevalence of drinking and median amount of ethanol / day in Berlin and Amsterdam compared to the other sites. The lowest prevalence of drinking was observed for Urban Ghana (26%). In contrast to men, the drinking profile of women in Rural Ghana was similar to Urban Ghana, whilst women in London consumed more alcohol than women in Ghana but less than women in Amsterdam and Berlin. As with men, amongst women drinking wine was less common in Ghana than in Europe, whilst drinking spirits was more common in rural Ghana than in the other sites. Daily consumption of any beverage was very rare in all sites.The findings for women are shown in For both male and female Ghanaians living in Europe years since migration were associated with a higher odds of drinking in age- and education adjusted analysis  test for trend p = 0.03 in women; OR 1.29 . There was no evidence for an association between drinking and any of the three acculturation variables in either sex.The pattern of results remained similar in regression models with mutual adjustment for all co-variables although there was attenuation of the odds ratios . After mThere was no evidence for interaction between drinking status and any co-variates with the exception of years since migration. There was evidence for an interaction (p = 0.02) for both sexes combined between drinking status and years since migration found for the London site  but not for the Amsterdam  or Berlin sites . When stratified by sex the evidence for interaction by site and years of migration was weaker .The RODAM study presented an opportunity to investigate patterns of alcohol use among Ghanaians living in different geographic locations. We found drinking patterns among Ghanaian adults to differ both by geographic location and by gender. Ghanaians living in Amsterdam and Berlin were more likely to consume alcohol and to have higher intakes compared to those living in Ghana. Within Ghana, alcohol consumption was more common in rural than urban areas. In both urban and rural Ghana, the prevalence of drinking alcohol was lower compared to Ghanaians in Europe, particularly in women. This supports the findings of a previous study comparing NCD risk factors in urban, rural and migrant populations showed a significantly lower alcohol consumption in urban compared to rural groups in China and Ghana whereas urban participants in South Africa were significantly more likely to consume alcohol regularly compared to rural participants. In this In line with findings from other migrant populations, we found evidence for a relationship between time since migration and drinking status.  Studies In traditional African societies, alcohol was mainly consumed by male adults, while females and the youth were restrained from drinking, and excess drinking and intoxication attracted negative sanctions. The prevVarious explanations have been suggested for variations in alcohol drinking patterns. These have been reported to differ by age, gender and by country. In this In women in urban Ghana, associations were found between drinking status and education and relationship status . In men, drinking was more common among those who were divorced or separated in rural Ghana but this association was not seen in urban Ghana. These heterogeneous associations are difficult to explain based only on this data but show that alcohol consumption patterns and factors driving them vary within Ghana as well as among Ghanaians who have migrated to Europe. Different patterns between alcohol consumption and socioeconomic status have previously been reported between countries. There arThere are some limitations which should be considered in the interpretation of these findings. Firstly, the recruitment methods between sites were different which may have introduced some selection bias to the study, in particular with respect to the findings from London where participants were recruited from Ghanaian organisations including churches. The questionnaires were either self- completed or completed by face-to-face interviews with the possibility of introducing social desirability bias. This possibility was minimised by using trained interviewers who were not local to the study area and ensuring privacy during the interviews. In addition, not all participants in the study completed the questions in the Ghana-specific food propensity questionnaire (this affected particularly London and Amsterdam) and therefore did not have data on alcohol consumption. Selection bias for the findings from Europe is possible due to this non-response if it was related to drinking status. This seems plausible as there were differences in the characteristics of participants from London and Amsterdam who completed the food-frequency questionnaire compared to those who did not. This could have biased the findings for the European sites but not for Ghana where the amount of missing data on alcohol consumption was extremely small.Measurement of alcohol consumption in this study was limited to questions on frequency and usual volume of ethanol consumed. It was therefore not possible to investigate more detailed differences in drinking patterns such as frequency of heavy episodic or “binge” drinking, or prevalence of alcohol use disorders. In addition, the maximum drink size option available in the questions on usual amount consumed per occasion was “three portions”. To this extent, it was not possible to discriminate heavier drinkers who usually drink more than three portions of alcohol per drinking occasion. There was a small percentage of participants in each site who reported drinking 3 portions of either beer, wine or spirits which suggests some degree of under-estimation of total volume of ethanol consumed is likely. The type of alcoholic beverage consumed under liquor and spirits included home-brewed alcohol with varying alcohol strengths which could potentially have been underestimated. However given the general limitations of using self-reported volume of ethanol from surveys as measures of actual ethanol consumption, where estimates are consistently lower compared with sales data–42, our Despite these limitations, our study provides original data on the drinking of Ghanaians in different locations and factors that may be contributing to these patterns. These findings suggest the need for policy makers to consider the different patterns when addressing the issue of alcohol consumption through public health campaigns and advocacy. Different strategies might be required in alcohol reduction campaigns among Ghanaians in different locations. A potentially effective strategy might include further exploration of the role of religious organisations in alcohol-related public health campaigns and how to reach those not attending religious services.While we have shown migration is associated with differences in drinking behaviour, further work is needed to understand what factors drive these changes and conversely the role of cultural and religious factors in promoting lower alcohol consumption in some migrant groups compared with their host populations.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Compared with wild-type mice, p21 deficient mice showed exacerbation of the fibrosis. Thus we propose that during the initial stage of the renal damage, tubular cells arrest in G2 partially depending on p21, thereby safeguarding kidney functions.Renal fibrosis is accompanied by the progression of chronic kidney disease. Despite a number of past and ongoing studies, our understanding of the underlying mechanisms remains elusive. Here we explored the progression of renal fibrosis using a mouse model of unilateral ureter obstruction. We found that in the initial stage of damage, where extracellular matrix was not yet deposited, proximal tubular cells arrested at G2 of the cell cycle. Further analyses indicated that the cyclin-dependent kinase inhibitor p21 is partially involved in the G2 arrest after the damage. A newly produced monoclonal antibody against p21 revealed that levels of p21 were sharply upregulated in response to the damage during the initial stage but dropped toward the later stage. To investigate the requirement of p21 for the progression of renal fibrosis, we constructed the novel  Epithelial-to-mesenchymal transition (EMT) in proximal tubular cells is one of the key processes in renal fibrosis4. Previous studies indicate that proximal tubular cells are transformed to interstitial fibroblasts or myofibroblasts by activation of various signaling cascades, such as the transforming growth factor-β (TGF-β) and Wnt pathway9.Fibrosis is one of the prominent features of organ disorders where extracellular matrix (ECM) components accumulate excessively. Although numerous studies have sought to uncover the underlying mechanisms in animal models, the molecular details remain unclear. Renal fibrosis is associated with the progression of chronic kidney disease (CKD), for which effective treatments have not yet been developed. Myofibroblasts which mainly express ECMs and contribute to the progression of renal fibrosis are delivered from a variety of origins11. In principle, cell cycle progression is faithfully moderated by the activation of cyclin-dependent kinases (CDKs). CDK inhibitors downregulate the activity of cyclin-CDK complexes via binding12. The CDK inhibitor p21 belongs to the Cip/Kip superfamily and plays multiple roles in the regulation of not only cell cycle control but also cellular senescence15. In the kidney, p21 mRNA is not expressed under unperturbed conditions, but induced after the damage16. Mice lacking p21 show opposite phenotypes, amelioration and exacerbation, depending on the type of damage19. It has recently been reported that p21-deficient mice avoid liver fibrosis because of the elimination of senescent liver stellate cells20. Thus, the role of p21 in the progression of fibrosis appears complex and its critical role is still under debate.A proliferative response to the damage is a hallmark of acute kidney injury (AKI)22. The DNA damage response (DDR) is one of the protective mechanisms against genome instability. The checkpoint kinase Chk1 that is activated upon DSB, phosphorylates various substrates, thereby inducing G2 arrest until the damages are rectified23. Activation of Chk1 is observed in the process of renal damage in rats24. In addition, G2/M cell cycle arrest is also induced after renal damage25. Under sustained damaged conditions, the G2 arrested cells produce pro-fibrogenic factors, including TGF-β and CTGF26, which accelerate renal fibrosis progression26. However, the physiological significance of the cell cycle arrest in response to initial damage is unknown.To maintain genome integrity from various types of damages, such as DNA double strand breaks (DSB), cells also possess checkpoints which comprise a series of signaling pathwaysp21-deficient mice and a newly produced monoclonal antibody. Our data show that in the initial stage of damage, tubular epithelial cells are arrested in G2 of the cell cycle in the mouse model of renal fibrosis. The G2 arrest is induced prior to the DNA damage checkpoint and Wnt/β-Catenin pathway activation, partially depending on the CDK inhibitor p21. In p21-deficient mice, the absence of this CDK inhibitor exacerbates the progression of fibrosis. These data uncover a new insight into cell cycle arrest and p21 in the complex regulation of renal fibrosis.Here we explored the progression of renal fibrosis by implementing new technical tools, the novel 6. In the initial stage (<3d), proximal tubules were expanded and seemed to be damaged by the obstruction and in the later stage (>3d), the area of epithelial cells was decreased and interstitial fibrosis was developed  of renal fibrosis, we conducted unilateral ureter obstruction (UUO), which is widely used as a model of renal fibrosis, and collected sham and damaged kidneys at the various time points after injury Fig. 6. In theped Fig. . As prevped Fig. . The numped Fig. . The areped Fig. . The numped Fig. . These dY15), which corresponds to an inactive form of Cdk1-cyclinB complex27, was dramatically elevated compared to unobstructed kidneys during the initial stage of damage , which is highly phosphorylated in mitosis28, was also increased but Ki67 and p-H3S10 double positive cells amounted to less than 10%  were analysed Figs  and S2. age Fig. . Howeverage Fig. . Furtherlls Fig. . These dlls Fig. , on the lls Fig. . Comparerol Fig. . Approxieys Fig. . In cont10% Fig. . It shoui67 Fig. . These d21. Additionally, activation of the checkpoint kinase Chk1 after renal ischemia/reperfusion injury (IRI) has been reported in rats24. Chk1 activation occurred in a time-dependent manner  foci which accumulate at DNA damage loci22, was increased as well  is implicated in the regulation of Wnt signaling via phosphorylation of β-Catenin29. Indeed, the phosphorylation of GSK-3β at S9, which is the negative form of GSK-3β, and the non-phosphorylated β-Catenin, which is the active form of β-Catenin were both increased accompanied with the development of fibrosis  using cultured human tubular epithelial cells (HK-2). To this end, we added aristolochic acid (AA) that induces aristolochic nephropathyage Fig. . Intrigutro Fig. . Becausetro Fig. . Previounds Fig. . These dnds Fig. . We consp21 mRNA is very low in the control, but is slightly increased in the damaged kidney . Furthermore, in healthy kidneys the p21 band reacting with this antibody was very low level; by contrast, however, it was sharply upregulated by the obstruction . To this end, we took advantages of the i-GONAD  method, in which handling the fertilized egg ex vivo is not required35. Tandem STOP codons were integrated into 60 bases after the first ATG  and p21−/− mice, and sampled kidneys at various time points . Masson’s trichrome staining revealed that fibrosis was more progressive in p21−/− compared to WT mice  and the frequency of apoptotic cells (Cleaved Caspase-3) are not altered  in response to the damage, but in the chronic phase or under sustained damaged, the arrest contributes to the progression of fibrosis via the production of profibrotic growth factors37. Intriguingly, in the reversible UUO model, kidney function can be recovered when the obstruction is removed within 2 days38. In any case, the level of the G2 arrest in proximal tubular cells is one of the features of early stage of renal damage, and is thus a good candidate of biomarker to predict fibrosis in initial stage of damage.Our results indicate that the G2 cell cycle arrest is induced even in the very early stage of damage when fibrosis is not fully developed. Several studies have indicated that the G2 arrest in proximal tubular cells after kidney injury contributes to the progression of renal fibrosisY15. We suppose that the epithelial tubular cells express p21 and subsequently induce the G2 arrest after injury to prevent renal fibrosis progression.We showed that p21 is involved in the G2 cell cycle arrest in the initial stage of renal damage. Our constructed monoclonal antibody detected upregulation of p21 protein in the damaged kidneys. Unfortunately the p21 monoclonal antibody could not be used for immunostaining. Which cell(s) and/or region(s) express p21 after injury is one of the intriguing questions to be explored. The expression pattern of proteins is very similar to p-Cdk1p21 mRNA is increased after kidney injury even in p53 null mice16, the upregulation of p21 during the initial damage stage appears to be independent of p53 in the regulation of renal fibrosis. We further showed that the protein level of p21 in the later stage is reduced compared with the early stage of damage, although the mRNA is expressed. The relationship between p53 and p21 is well established in cancer14. It is a very intriguing question to decipher the molecular mechanism(s) of p53-independent upregulation of p21 in the regulation of renal fibrosis. Previous work showed that the regulation of p21 is bimodal and its level is increased in response to a low dose of DNA damage but transiently degraded upon the higher levels of DNA damage30. The functions of p21 are regulated by multiple phosphorylation events carried out by various kinases including the checkpoint kinase Chk113. We speculate that p21 is upregulated in response to renal damage during the initial stage to protect epithelial tubular cells; however, sustained renal damage causes DNA damage, leading to Chk1-dependent phosphorylation and degradation of p21. Further analysis will clear complex molecular mechanisms of p21 regulation.Consistent with previous studies which the level of p21-deficiency ameliorates the development of the fibrosis and the progression of chronic renal failure after partial renal ablation17. On the other hand, p21 has a protective and beneficial role(s) in renal ischemia/reperfusion injury (IRI)19. In our study, p21 is initially activated and subsequently degraded in the later stage. And p21-deficient mice show severe fibrotic features. It seems that p21 has a protective role(s) in the progression of renal fibrosis. Nonetheless, it is possible that p21 deficiency is beneficial for fibrosis. In a recent study, p21 knockout mice exhibited improved liver fibrosis due to elimination of senescent liver stellate cells20. These paradoxical functions could be attributed to different cell types and the dose of the damage used. In the kidney, a fibrotic injury-induced EMT program in epithelial tubular cells induces a G2 arrest partially dependent on p218. We expect that p21 has bimodal roles, protecting or accelerating the progression of fibrosis, depending on stages of the damage (acute or chronic). Conditional p21 knockout mice, such as cell type-specific or drug inducible, will be useful for understanding these issues. Further analyses will illuminate the role(s) of p21 in the regulation of fibrosis.Previous reports indicate that e.g. handling zygotes ex vivo. The fact that this is time-consuming is also one of the limitations when we wish to perform genetic analysis in animals. In this study, we generated new p21-deficient mice by using the i-GONAD method35, though mice lacking p21 have already existed36. Our constructed mice showed no production of the protein even after injury. Thus, the i-GONAD method will enable us to analyse the phenotypes of knockout mice rapidly and efficiently in the field of animal research.Genome manipulation is a powerful tool for understanding molecular mechanisms in the fields of life science and medical research. Recent advances in genome-editing technology have enabled rapid generation of genome-edited animals with ease. However, the procedure for producing such animals involves multiple complicated steps and also necessitates skillfull techniques, p21-deficient mice were generated by the i-GONAD method as previously described35. In brief, single stranded DNA containing 3 STOP codons and homology sequence against the second exon of Cdkn1a (cggtcccgtggacagtgagcagttgcgccgtgattgcgattgactagctagaattcccgggcgctcatggcgggctgtctccaggaggcccgagaacggt) was injected to the oviductal lumen of pregnant female mice (E0.7) with Alt-R™ CRISPR-Cas9 system (Integrated DNA Technologies) and subsequently electroporated by the electroporator . Guide RNA was designed using CHOPCHOP (http://chopchop.cbu.uib.no/). All materials and equipment were used in accordance with manufacture’s procedures. Correct integration was confirmed by PCR and genome sequence of pups  were obtained from crossing hetero female and male mice.C57BL/6 male mice were used throughout this study. ups Fig. . The p21Renal fibrosis was induced by unilateral ureter obstruction (UUO). Left ureters of age matched male mice were surgically ligated by the 9 mm of string and collected obstructed and unobstructed kidneys at the indicated time points after the operation Fig. . The sam39. Essence is following. Full length of mouse p21 (mp21) was amplified from the cDNA  with the following primers . The amplified fragment was inserted into a pET28a vector with restriction enzymes . His-tagged full length of mp21 was expressed in BL21-CodonPlus-RP cells. Because His-mp21 was insoluble . Images were captured by confocal microscopy  and were processed by Olympus fluoview ver.4.0. Quantified images were taken by using 20x objective lens (512 × 512 pixel image) and analysed using ImageJ. Numbers in figures were per 512 × 512 pixel images. Higher magnification images were taken by using 60x objective lens with immersion oil (Olympus).41. Representative blotting images of each sample are shown in figures. The original scan images are shown in supplementary information  containing protease inhibitor  on ice and lysates were cleared by centrifugation at 15,000 rpm for 15 min at 4 °C. Equal volume of 2xSDS-PAGE sample buffer was added to cleared sample and boiled for 5 min. Samples were stored at −80 °C. Cultured HK-2 cells were directly homogenized in a 2xSDS-PAGE sample buffer. Each sample was separated by SDS-PAGE gel (12.5%) and analysed by western blotting with appropriate 1st and 2nd antibodies. For the detection of p21 phosphorylation, phosphate affinity SDS-PAGE was carried out using 25 µM of Phos-tag acrylamide (NARD Institute)ion Fig. . The figHuman tubular cells HK-2 were obtained from ATCC. HK-2 cells were cultured in keratinocyte serum free medium . Cells were treated with the indicated concentrations of Aristolochic Acid . Dimethyl sulfoxide  was used as a solvent.p21 deficient mice, three different mice of each genotypes were used. Five different regions per mouse were analysed and the averages were used as a value for the mouse. All statistical analyses were carried out using two-tailed unpaired Student’s t-test. P values were indicated by asterisk . NS means “Not Significant”. P < 0.05 was considered as statistically significant.To evaluate comparisons between WT and Supplementary information


Poacea). Leaves of 58 grass species were sectioned transversally, and the sections treated with a 2% sodium hypochlorite solution to clarify the tissue. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips using water. Grass species showing red staining of the bundle sheath cells were considered C4, and species with translucent bundle sheath were considered C3. The results of the histochemical test were then compared with results from carbon isotope composition analysis and the relevant scientific literature. Observations from the histochemical test were congruent with results from δ13C isotope composition analysis, and with data previously presented in the scientific literature. The proposed histochemical test proved efficient for characterization of the photosynthetic pathway in the tested grasses; however, the method should be further tested in a greater number of grass species, encompassing, preferably, all Poacea subfamilies. Future studies may elucidate if the proposed method can effectively be used in other botanical families. Furthermore, additional investigations may determine whether the phenolic compounds indicated by the histochemical test are exclusive to the bundle sheath of C4 grasses and if possible relations exist between these phenolic compounds and the C4 photosynthetic pathway in grasses.An easy and inexpensive method of determining the photosynthetic pathway in grasses using a dye widely used in microscopy. To evaluate the efficiency of a new histochemical test for determination of the photosynthetic pathway in grasses ( The C4 photosynthetic rout evolved in plants tens of millions of years ago in response to environmental changes, characterizing an advantage under high light, hight temperature, or draught conditions  is also used as a distinction criterion between C3 and C4 plants. Values between − 10 and − 14‰ indicate a C4 plant, while values between − 20 and − 35‰ indicate a C3 plant . Leaves of 15 identified forage species and 14 identified bamboo species, of several genera, were collected from plants cultivated at Universidade Federal de Santa Catarina’s Fazenda Experimental da Ressacada, Florianópolis, Santa Catarina, Brazil. Additional leaf samples were obtained from 12 grass species cultivated at Embrapa’s Amazonia Oriental experimental area in Belém, Pará, Brazil, and 13 wild grass species in Santa Catarina state. Dried specimens from grasses collected in Belém were deposited at the Museu Paraense Emílio Goeldi’s herbarium (Herbarium MG), where specimens were identified by means of botanical comparison, and dried specimens of grasses collected in Santa Catarina were deposited at Universidade Federal de Santa Catarina’s herbarium (Herbário Flor), where specimens were also identified by means of comparison.Leaves collected for determination of carbon isotope composition were placed in paper bags for subsequent dehydration, and leaves used in the histochemical test were fixated in 70% ethanol.13C is expressed as the notation delta (δ) and as a value given in parts per thousand (‰), being determined by means of the equation:Rsample and Rstandard are the molar ratios 13C/12C of the sample and the standard, respectively. The employed standard was the fossil limestone PeedeeBelamnite (PDB). At each analysis round reference leaf material of known carbon concentration and δ13C was used . The acceptable analytical error for carbon concentration determination is 0.3% and for δ13C is 0.3‰.Leaf samples were dehydrated in a drying stove . Afterwards, leaves were ground in liquid nitrogen with a mortar and pestle until a fine powder was obtained. Ground leaf samples were analyzed at the Laboratóro de Ecologia Isotópica—CENA/USP . About 2.5 mg of leaf material were conditioned inside aluminium capsules and inserted in an element analyser , for determination of the total carbon concentration. The gases generated by the samples’ combustion were purified and injected directly into a mass spectrometer , for determination of isotope ratios. The natural abundance of Leaves fixed in 70% ethanol were sectioned transversally at their middle portion by hand with stainless steel blades. Leaf sections were treated with 2% sodium hypochlorite solution for tissue clarification. After discoloration, sections were washed with distilled water and double-stained with astra blue and safranin (1% each in 50% ethanol) for 1 min. Sections were then mounted between microscopy glass slides and coverslips, with a drop of water between them. Photomicrographs were obtained using an Olympus BX40 optical microscope attached to an Olympus DP71 camera.Arundo donax and Uruchloa brizantha clearly illustrate the distinction  analysis of all evaluated grasses correlated with the histochemical test. All grasses with isotope composition values between − 26.09 and − 29.26 indicated a C3 plant, and presented a negative result in the histochemical test. In contrast, all plants with isotope composition values between − 9.88 and − 14.21 indicated a C4 plant, and presented a positive result in the histochemical test.Table The histochemical test showed a negative result for all evaluated bamboos, and confirmated the photosynthetic pathway of maize and sugarcane  and rice , and positive to the three photosynthetic pathway grasses  Table .Festuca aurora, Festuca arundinacea, and Holcus lanatus showed negative results. Among the tested wild grasses, Homolepsis aturensis, H. isocalycia, Steinchis malaxum, and A. donax showed negative results, indicating a C3 photosynthetic pathway. All other tested species of forage and wild grasses presented a positive result, indicating they have C4 photosynthetic systems.Among the forage grasses, only Leaf transversal sections that underwent dehydration during performance of the histochemical test presented a slight detachment of the cell wall membrane, and this allowed inferring that the safranin-reactive phenolic substance is located in C4 grasses’ bundle sheath cells’ protoplasts, and not in the cell wall (data not shown).Safranin O is the least specific histochemical indicator of lignin because it not only stains lignin, but all related phenolic substances  attached to cell walls  and intermediary C3–C4 plants. Arundo donax is included in the Arundinoideae subfamily  and within the Arundineae tribe, composed exclusively of C3 species . Additionally, the isolation and identification of the phenolic compounds detected by the histochemical test in bundle sheath cells of C4 grasses may elucidate a possible relation between the presence of these substances and the C4 photosynthetic pathway.


N-acetyl cysteine (antioxidant). We emphasize the importance of the diagnosis of CPFE in early stages through CT in a case of COPD with clinical, laboratory and chest radiographic evidence of fibrosis and the fact that CPFE is associated with pulmonary hypertension, a poor prognostic indicator.Combined pulmonary fibrosis and emphysema (CPFE) is a unique pulmonary condition characterized by simultaneous coexistence of both upper lobe emphysema and lower lobe fibrosis. Pulmonologists should be aware of the entity while evaluating patients with chronic obstructive pulmonary disease (COPD) or pulmonary fibrosis. Airflow and lung volume are relatively preserved but oxygenation is disproportionately impaired in patients with CPFE. We describe a case of an 83-year-old male patient with past history of heavy smoking, in whom the search for the cause of pulmonary arterial hypertension and exercise-induced arterial oxygen desaturation disproportionate to be explained by COPD resulted in a diagnosis of CPFE. He complained of dyspnoea on exertion and non-productive cough. Physical examination revealed basal Velcro rales and clubbing. Chest radiography showed prominent vascular markings, preserved lung volume and subtle fibrosis of the bases. Definitive diagnosis was made on CT scan of the chest, which revealed upper lobe emphysema and lower lobe fibrosis and honeycombing. The patient was managed by long-term oxygen therapy, inhaled corticosteroid, long-acting bronchodilator and antimuscarinic agents, diuretic, pirfenidone (antifibrotic agent), proton pump inhibitor and  Echocardiogram revealed normal chamber dimensions, no regional wall motion abnormality, moderate tricuspid regurgitation, pulmonary arterial systolic pressure of 60 mmHg, normal left ventricular function (left ventricular ejection fraction: 60%) and Grade 1 left ventricular diastolic dysfunction. The patient did not cooperate for pulmonary function test (PFT). Screening tests for pulmonary thromboembolism (colour Doppler of lower limbs and D-dimer assay) were negative. Screening chest X-ray showed prominent vascular markings in the upper and mid zones with subtle fibrosis in the lower zones. A suspicion of fibrotic process in the lungs was raised and further evaluation by high-resolution CT (HRCT) was ordered.An 83-year-old male with a past history of smoking and chronic obstructive pulmonary disease (COPD) presented with persistent non-productive cough, exertional dyspnoea and wheezing that was relieved by inhaler and right-sided pleurisy for 2 days. There were no symptoms of obstructive sleep apnoea. Epworth Sleepiness Scale for obstructive sleep apnoea was performed and score was < 8. There were no symptoms or signs suggestive of connective tissue disorder. Clinical examination revealed bilateral pedal oedema and bilateral basal Velcro rales. SpOExtensive centrilobular and paraseptal emphysema were seen in bilateral lungs with upper lobe predominance  and 2. RN-acetyl cysteine (antioxidant). The patient was advised for regular follow-up.Based on HRCT findings, the patient was diagnosed as a case of combined pulmonary fibrosis and emphysema (CPFE) and managed by long-term oxygen therapy, oral theophylline, inhaled corticosteroid, long-acting bronchodilator and antimuscarinic agents, diuretic, pirfenidone (antifibrotic agent), proton pump inhibitor and Unanimous definition of CPFE is yet to be adopted. Broadly, it includes all patients with simultaneous coexistence of pulmonary fibrosis and emphysema.1 in 1990. But Cottin et al2 were the first to comprehensively describe the syndrome resulting from CPFE in 2005. CPFE was recognized in 8% cases of idiopathic pulmonary fibrosis (IPF) in a study by Ryerson et al.3CPFE as a unique pulmonary condition characterized by the simultaneous coexistence of lower lobe fibrosis and upper lobe emphysema was first described by Wiggins et al2 Agrochemical compounds are also attributed as possible risk factors.4 CPFE patients classically present with progressive shortness of breath, with an average symptom duration of about 3 years before diagnosis.2 As in any fibrotic interstitial lung disease, bilateral basal Velcro rales and clubbing are the main findings on clinical examination.2Most patients with CPFE are elderly males around age 65 years or older with a history of cigarette smoking, the most commonly cited environmental risk factor.3 Diagnosis of CPFE is confirmed essentially on the basis of findings on HRCT. Clinical data and pathological findings are helpful in some cases.It has been reported that CPFE patients are often misdiagnosed as cases of COPD and treated with short-acting bronchodilators and long-acting anticholinergics.5 Reason for this pattern of abnormality in PFT and DLCO is that volume loss and low compliance owing to fibrosis is compensated by hyperinflation and high compliance due to emphysema but pulmonary emphysema and fibrosis have additive effects on carbon monoxide transfer and exercise hypoxemia.5 But this pattern is not specific for CPFE and is also seen in pulmonary vascular diseases, interstitial lung diseases and emphysema.6 (ii) Patients with pulmonary hypertension and abnormal PFTs, especially with a mixed restrictive/obstructive pattern.7 (iii) Patients with normal or near-normal spirometry but with hypoxemia at rest or upon exertion and oxygen requirement disproportionate to spirometric abnormality.5The three main clinical scenarios in which CPFE should be considered are: (i) patients with PFTs showing normal spirometry, or mild obstructive or restrictive pattern and normal lung volumes but with severely diminished diffusing capacity of the lung for carbon monoxide (DLCO).8The exact pathogenesis of CPFE is unknown. Cigarette smoking is quoted as the possible aetiology in majority of the reported cases. Exposure to cigarette smoke led to synchronous development of pulmonary fibrosis and emphysema in the canine model.9 Interstitial fibrotic changes include honeycombing and reticular abnormalities. Ground-glass attenuation areas are also commonly present.2 Sometimes, ground-glass attenuation is the sole abnormality suggesting interstitial lung disease and biopsy is required in this setting10 to differentiate CPFE from other smoking-related lung diseases.Lower lobe interstitial fibrotic changes and upper lobe emphysema are the typical radiologic findings in the CPFE syndrome. Paraseptal, centrilobular emphysematous and bullous changes are seen in CPFE.11 Pulmonary hypertension, acute lung injury and lung cancer are listed as complications of CPFE.Specific treatment does not exist currently for CPFE syndrome. The present guidelines are based on evidence related to the underlying conditions of chronic obstructive lung disease and IPF.12 It does not significantly respond to medical therapy.13 The patients are managed with oxygen therapy and lung transplantation.Pulmonary hypertension is said to occur more commonly and severely in patients with CPFE than in those with isolated IPF.13 In their study, Ryerson et al3 concluded that mortality rate of patients with CPFE is similar to those with isolated IPF, although patients with CPFE had more extensive smoking history, greater oxygen requirements, higher pulmonary artery pressure and poorer gas exchange.3 Similar mortality in IPF as compared twith CPFE was attributed to greater amount of fibrosis in IPF compensating for other mortality increasing factors in CPFE.3Mortality is significant in patients with CPFE. The 1-year survival is as low as 60% in patients with confirmed pulmonary hypertension.We emphasize the importance of suspecting the diagnosis of CPFE in a patient with COPD with evidence of fibrotic process in the lungs on clinical examination or chest radiographic findings with echocardiographic evidence of pulmonary hypertension. We also emphasize that CPFE is a distinct pulmonary condition that, in a classic case, can be definitely diagnosed by HRCT.CPFE is a unique pulmonary condition characterized by the simultaneous occurrence of both upper lobe emphysema and lower lobe pulmonary fibrosis.CPFE is seen mostly in elderly males with significant smoking history.CPFE has a progressive course, with an average duration of symptoms of about 3 years before diagnosis.CPFE patients are often misdiagnosed as cases of COPD and treated accordingly.HRCT is the modality of choice, and it typically demonstrates the characteristic upper lobe emphysema with lower lobe interstitial fibrotic changes.With the classic clinical and HRCT findings, a confident diagnosis can be made without the need for a surgical lung biopsy.Unfortunately, CPFE has no effective treatment options available other than possible lung transplant and overall has a poor prognosis.The prevalence of pulmonary arterial hypertension is high in CPFE, and its presence at diagnosis is by itself a poor prognostic sign.Along with pulmonary arterial hypertension, lung cancer and acute lung injury are listed as complications. Close follow-up is essential.Mortality rate in patients with CPFE is similar to that in patients with isolated IPF.Informed consent was obtained to publish this case (including images and data) and is held on record.


Bacillus amyloliquefaciens ESB-2 on the basis of phenotypic characteristics and 16S rRNA gene sequencing. Response surface methodology was applied to optimize the fermentation conditions for rapid and efficient accumulation of macrolactin A, a pharmacologically important marine antibiotic. Eight fermentation conditions were examined for their significance on macrolactin A production using Plackett–Burman factorial design, where peptone, medium volume and temperature significantly improved production rate. Further optimization was carried out using Box-Behnken design of experiments to study the influence of process variables. The optimized fermentation condition for maximum production was peptone 14.8 mg/mL, yeast extract 1 mg/mL, FePO4 0.01 mg/mL, temperature 26.3 °C, initial pH value 6.0, medium volume 72.4%, rotation speed 150 r/min, inoculation 5% and fermented for 2 days. Under the optimized conditions, the concentration of macrolactin A reached 21.63 mg/L, representing a 2.4-fold increase compared to the original standard condition, which was also 17% higher than previous highest report of 18.5 mg/L and three times higher in terms of daily productivity.In the present work, an antibiotic-producing marine bacterium was isolated from a seawater sample collected from Yuhuan, Zhejiang, China, identified and named as  During the past one hundred years, natural products research has mainly concentrated on the terrestrial biota, and a lot of structurally unique and highly bioactive compounds were found in terrestrial microorganisms, plants and animals. However, for long-term research and development, discovering new and useful compound from terrestrial resources is becoming much more difficult . Meanwhiet al. from a taxonomically undefined deep-sea marine bacterium. It displays a broad spectrum of biological activity, including potent cytotoxicity on B16-F10 murine melanoma cell and antiviral activity against Herpes simplex (types I and II) and HIV virus [Macrolactins are a group of 24-membered macrolides with potent antibacterial or other activities, most of which are produced as secondary metabolites by marine microorganisms . A totalIV virus . Of the Bacillus amyloliquefaciens ESB-2, which was found to rapidly accumulate macrolactins. The aim of the present work was to optimize the culture medium composition for rapid and efficient production of macrolactin A by Bacillus amyloliquefaciens ESB-2 using RSM.An appropriate fermentation medium is very important, because medium composition can significantly affect product yield . Respons1), yeast extract (x2), beer extract (x3), glucose (x4), FePO4 (x5), medium volume (x6), temperature (x7) and initial pH value (x8) for macrolactin A production was investigated by PBD. The effects of these components on the response and significant levels are shown in The importance of the eight variables, namely, peptone (x1), medium volume (x6) and temperature (x7) had confidence levels above 95% (p < 0.05) and were considered to influence macrolactin A production significantly. The others had confidence levels below 95% and hence were deemed insignificant. In addition, R2 = 0.9889 indicated that 98.89% of the variability in the response could be explained by the model.According to statistical analysis of the data by the Design Expert software Minitab 16.0 , peptone1) and medium volume (x6) were positive, while temperature (x7) exhibited a negative effect on macrolactin A production. Therefore, these three variables were selected for further optimization.PBD results also indicated that the effect of peptone (xRSM using Box-Behnken design (BBD) was employed to determine the optimal levels of the three selected variables. The respective levels with the coded levels for the factors are listed in Y) and the tested variables were obtained by application of RSM. By employing multiple regression analysis on the experimental data, the response variable (Y) and the tested variables can be related by the following second-order polynomial equation:Y was the predicted macrolactin A production, x1was peptone, x6 was medium volume, and x7 was temperature.The relationships between macrolactin A production  and low p-value (<0.05) implied the model was highly significant. The fitness of the model could be examined by the coefficient of determination R2 [2-value higher than 0.9, was considered as having a very high correlation. Therefore, the present R2-value reflected a very good fit between the observed and predicted responses, and implied that the model is reliable for macrolactin A production in the present study. The adjusted determination coefficient (R2adj = 97.72%) was also able to confirm the significance of the model.The analysis of variance (ANOVA) data for the selected quadratic polynomial model is listed in ation R2 , which wp-values, are presented in p-value was employed as a tool to check the significance of each coefficient, which also indicated the interactions between the variables [p-value, the more significant the corresponding coefficient was. It was observed from p-values less than 0.05 except for the cross-product coefficient of peptone (x1) and temperature (x7).The coefficient estimates of model equation, along with the corresponding ariables . The smaThe 3D response surfaces and 2D contour plots  generate1 code level was 0.95, x6 code level was 0.62 and that of x7 was −0.74, which were peptone 14.8 mg/L, medium volume 72.4%, temperature 26.3 °C, respectively. To verify the predicted results, validation experiments were performed in triplicate. Under the optimized condition, the observed experimental average concentration of macrolactin A was 21.63 mg/L, suggesting that experimental and predicted values (22.42 mg/L) were in good agreement, which was also 17% higher than previous highest report of 18.5 mg/L [On the basis of medium optimization, the quadratic model predicted that the maximum production of macrolactin A was 22.42 mg/L, when the xBacillus amyloliquefaciens reported in the Genbank (No. JX966407). According to its morphological and physicochemical characteristics, the strain was identified and named as Bacillus amyloliquefaciens ESB-2 [A macrolactins-producing marine bacterium was isolated from a seawater sample collected in Yuhuan, Zhejiang, China. The 16S rRNA gene sequencing result showed that this strain had 97% homology with ns ESB-2 ,28.4, 1 L seawater). Fermentation was performed in two stages: seed growth and macrolactin A production. For the seed growth stage, mycelium from a plate culture was inoculated into 100 mL of seed medium with shaking at 150 rpm at 30 °C for 24 h. Then, 5% (v/v) seed cultures were inoculated into the fermentation medium. The strain was incubated at 30 °C with 150 rpm for 2 days.The bacterium was grown in 2216E medium , the supernatants were analyzed by a Waters Alliance 2695 HPLC system, equipped with a quaternary solvent deliver system, an autosampler, and a 2996 diode array detector . A reverse-phase Waters XBridge C18 column  at 25 °C was applied for all analyses. Methanol–water was used as the mobile phase in gradient elution mode . The effluent was monitored at 280 nm and flow rate was 1.0 mL/min. Then authentic sample of macrolactin A with purity higher than 95% was isolated and purified from Eight fermentation conditions were examined for their significance on macrolactin A production using PBD ,31,32. F4 0.01 mg/mL, temperature 26.3 °C, initial pH value 6.0, medium volume 72.4% and fermented for 2 days. Under the optimized conditions, the concentration of macrolactin A reached 21.63 mg/L. Validation experiments were also carried out to verify the accuracy of the model, and results showed that the predicted value agreed well with the experimental values. Comparing to 9.17 mg/L in original standard condition, 2.4-fold increase had been obtained. This medium also resulted in 17% higher than previous report and three times higher in terms of daily productivity. The present study provides a basis for further study on large scale fermentation for macrolactin A production.This study proved that statistical experimental designs offer an efficient and feasible approach for macrolactin A fermentation medium optimization. A maximum macrolactin A production of 21.63 mg/L was achieved with the following optimized factors: peptone 14.8 mg/mL, yeast extract 1 mg/mL, FePO


Previous research has sought to quantify head impact exposure using wearable kinematic sensors. However, many sensors suffer from poor accuracy in estimating impact kinematics and count, motivating the need for additional independent impact exposure quantification for comparison. Here, we equipped seven collegiate American football players with instrumented mouthguards, and video recorded practices and games to compare video-based and sensor-based exposure rates and impact location distributions. Over 50 player-hours, we identified 271 helmet contact periods in video, while the instrumented mouthguard sensor recorded 2,032 discrete head impacts. Matching video and mouthguard real-time stamps yielded 193 video-identified helmet contact periods and 217 sensor-recorded impacts. To compare impact locations, we binned matched impacts into frontal, rear, side, oblique, and top locations based on video observations and sensor kinematics. While both video-based and sensor-based methods found similar location distributions, our best method utilizing integrated linear and angular position only correctly predicted 81 of 217 impacts. Finally, based on the activity timeline from video assessment, we also developed a new exposure metric unique to American football quantifying number of cross-verified sensor impacts per player-play. We found significantly higher exposure during games  than practices  (p<0.05). In the traditional impacts per player-hour metric, we observed higher exposure during practices (4.7) than games (3.7) due to increased player activity in practices. Thus, our exposure metric accounts for variability in on-field participation. While both video-based and sensor-based exposure datasets have limitations, they can complement one another to provide more confidence in exposure statistics. Concussions are a common injury in contact sports, with an estimated 300,000 sports-related concussions in the United States annually . ConcussTo quantify head impact exposure to players on the field, previous studies have focused on instrumenting American football players with wearable sensors ,9–19 Ta. These sHowever, recent studies have shown that HITS, and many other wearable sensor systems, may suffer from poor accuracy in both classifying head impacts (impact count) –25, and Video assessment represents an option for collecting an independent dataset of head impact exposure. Video assessment has been used extensively to review plays and sports injuries by coaches and medical professionals –33. SeveIn this work, we introduce a video assessment protocol to collect an independent video-based head impact exposure dataset from seven collegiate American football players for a single season. We also equipped players with an instrumented mouthguard ,27,34,35In addition, we combine sensor and video information to introduce a new exposure metric for quantifying head impact exposure unique to American football, which measures the number of head impacts sustained over a single player-play. While traditionally, exposure is quantified as the number of impacts per player exposure (defined as a player participating in a practice or game) or per hour of play time, American football is a unique sport where every player is expected to spend varying amounts of time on the field and in play. As an example, statistics on National Football League games report an average of 133.6 plays per game, of which 10.3 involve punting. Thus, specialized players such as punters are on the field, and thus exposed, far less often than players such as linemen. Our exposure metric accounts for variability in on-field participation, and normalizes exposure by the base unit for American football: a single play. This metric is also a prime example for the complementary analysis of video and sensor exposure because head impact kinematic sensors cannot measure non-impact activities and video does not have sufficient resolution to identify discrete head impacts.To collect a video-based head impact exposure dataset, we relied on a tiered video assessment protocol. Impact exposure and kinematics were also independently measured using the instrumented mouthguard.Data were collected from the Stanford University American football team during the 2015 fall season. Seven players were consented for study participation through the Stanford Internal Review Board IRB #34943. We obtained informed consent from players in written form. Players represented a variety of positions .Players wore instrumented mouthguards ,35 Fig  for the The electronic boards contained a tri-axial linear accelerometer (H3LIS331) and tri-axial gyroscope ITG3701A), both sampling at 1000Hz. The linear acceleration data were filtered at CFC180 (300Hz fourth order Butterworth low pass filter)  while th01A, bothnist.time.gov) prior to an event. A unique “time-sync” mouthguard not built for any players was used to synchronize mouthguard times with the camera times for impact correlation. This was accomplished by having an athletic trainer apply known impacts on the “time-sync” mouthguard in view of all cameras. The real-time stamps on the “time-sync” mouthguard were matched with those on the cameras after the event.Impacts collected on the instrumented mouthguards were time stamped based on an internal real-time clock to within one second. Internal clocks were synchronized to the National Institute of Standards and Technology time  and had IR readings indicating instrumented mouthguards were properly worn. We then cross-verified helmet contact periods and discrete impact events between video-based and sensor-based exposure sets respectively by comparing the synced time-stamps. This differs from previous video confirmation protocols in that we identified helmet contact periods in video independently ,25.Using the independent video-based helmet contact periods, sensor-based impacts, and cross-verified impacts, we computed a traditional exposure metric: the number of helmet contact periods or head impacts per player-hour . We then computed our new exposure metric: the number of cross-verified sensor-based head impacts per player-play. Plays were discretized using the video-based player activity timeline combined from all videos during an event. Helmet Contact, Body Contact, and No Contact activities were associated with players in play, and the Idle and Not in Video activities were associated with players not in play. Sequences of Helmet Contact, Body Contact, and No Contact activities were represented by a single play. Finally, we also computed the number of plays per player-hour.Next, we compared the impact location distributions from cross-verified video-based helmet contact periods and sensor-based head impacts. We defined location vectors representing front , rear , side , oblique , and top  impacts . During First, we integrated sensor linear acceleration and angular velocity data to obtain linear velocity, linear position, and angular position (defined using XYZ Euler angles) during each impact. We also differentiated angular velocity data to obtain angular acceleration ,38. We tTo utilize the peak angular data, we note that if we treat the head and neck as a simple pendulum, the linear and angular motion vectors should be orthogonal. Thus, we define a corrected motion vector by first taking the cross product of the peak linear acceleration, velocity, or position vector with the respective angular acceleration, velocity, or position vector. We then cross the result with the respective angular acceleration, velocity, or position vector to obtain a corrected motion vector, whose location we bin by again finding the location vector with the smallest angle.The cross-verified discrete impacts from the instrumented mouthguard were also used to assess impact severity exposure to compare against previously reported values. We defined severity based on the peak linear acceleration, angular velocity, and angular acceleration magnitude from each impact.Fourteen raters reviewed over 163 hours of video for the seven players spanning 11 practices and 6 games during the 2015 fall season. This represented over 62 hours of mouthguard recordings with over 50 hours of actual in-game or in-practice time. In that time, players participated in 927 plays. Raters identified 1004 Helmet Contact activities after first round video assessment, of which 271 were confirmed following second round video assessment. The instrumented mouthguard recorded a total of 13,034 impacts, of which 10,949 occurred during practice or game periods. 2,032 of these were determined to be in-mouth impacts based on IR readings. When matching time stamps of video-based helmet contact periods and sensor-based head impact events, we cross-verified 193 video-based helmet contact periods and 217 sensor-based impact events .Treating sensor-based and video-based exposure data separately, we observe a large discrepancy in exposure rates . The insNext, we further delineate exposure based on event type (games or practices). When considering sensor-based results independently, the instrumented mouthguard had double the head impact rate in games than in practices. For independent video-based exposure and cross-verified exposure, exposure during practices was higher. Even for cross-verified mouthguard impacts, we found greater head impacts per player-hour exposure in practices  than in games , though the difference is not significant (p = 0.105). When considering the number of cross-verified sensor-based impacts per player-play however, we found that most players sustained more impacts per player-play during games than during practices. Indeed overall, players experienced significantly fewer impacts per player-play during practices  than during games  (p<0.05). This discrepancy is likely due to a difference in amount of play seen, where all players participated in significantly more player-plays per player-hour in practices  than in games  (p<0.05).We quantified the distribution of impact locations in our cross-verified video-based helmet contact periods and sensor-based head impacts . In bothWhen comparing the location determined through video with the location determined used the processed mouthguard kinematics, we found that our best processing technique (corrected linear position) only predicted impact location correctly in 81 of 217 impacts (37.3%). The traditional peak linear acceleration process, however, was least accurate with only 61 of 217 impacts (28.1%) correctly predicted. In general, correctly identifying impact location improved with integration, and improved when assuming orthogonality with angular measures.We also quantified head impacts based on the opposing impact object , which can only be determined from video assessment. We found 64% were to an opponent’s helmet, 27% were to an opponent’s body, 5% were to an opponent’s upper or lower limbs, 3% were to the ground, and 1% were to other objects such as practice pads.2 (95% CI: 1109.1rad/s2-1475.9rad/s2) respectively during practices, and 23.2g (95% CI: 18.5g-26.1g), 12.5rad/s (95% CI: 10.8rad/s-13.9rad/s), and 1435.0rad/s2 (95% CI: 1288.4rad/s2-1723.3rad/s2) respectively during games. While the medians were larger during games, only peak angular velocity magnitude had a significant difference (p<0.05).Impact severity was only quantified in the 217 impacts recorded by the instrumented mouthguard with high IR and cross-verified with video. We delineate impact severity based on event (games or practices). Over all players, the median peak linear acceleration, angular velocity, and angular acceleration magnitude was 18.4g (95% CI: 15.2g-19.8g), 9.4rad/s (95% CI: 8.5rad/s-10.2rad/s), and 1240.5rad/sIn this work, we present a tiered video assessment protocol for obtaining an independent video-based dataset for head impact exposure in American football to compare against sensor-based impact exposure and impact location distributions. This work differs from previous head impact exposure datasets that rely primarily on imperfect head impact sensors, with a few datasets further confirming true positive vs. false positive head impacts in video. Instead, we treat the video assessment as an independent exposure dataset. Exposure datasets collected with impact sensors alone are limited by a high occurrence of false positives due to the sensitivity of motion sensors, and known innaccuracies of many sensor systems. Video assessment of exposure data are limited by the qualitative nature of analyzing video and the inability to quantify impact kinematics. However, in this study, we demonstrate that the two methods can complement one another and reveal directions for improvement in sensor-based and video-based data collection. Combining these information, we also introduce a new American football specific exposure metric based on the number of sensor-based discrete head impacts per video-observed player-play, which account for players varied involvement during events.To demonstrate the limitations of independent video-based and sensor-based head impact exposure, we highlight key differences between between the independent exposure sets and the exposure set determined with impacts cross-verified in both video and sensors.First, we note that the instrumented mouthguard only detected 71.2% of the video classified Helmet Contact events, missing 76 of 271 events. One possible reason for this is that the instrumented mouthguard was set to record an impact event when the linear acceleration magnitude exceeded 10g, which is similar to impact thresholds employed in previous work . In videPreviously, the 10g sensor threshold was chosen to differentiate head impacts from other events resulting in head motion, such as jumping or running . But, asSecond, the instrumented mouthguard alone detected 2,032 impacts, which was an order of magnitude greater than the number of cross-verified impacts. This demonstrates the relatively high rate of false positives that have plagued similar impact sensors in the past, and are commonly mitigated using video confirmation . On the In addition to differences in exposure, we also discuss discrepancies in impact location distributions between video-based and sensor-based data. In video, we found that 93.8% of impacts occurred to the front, front oblique, and side of the head after the second round of video assessment. Processing instrumented mouthguard kinematics found similar trends for impact location with an average of 85.8% of impacts occurring in the front, front oblique, and side locations; however, the mouthguard impact location matched video identified location in only 37.3% of the impacts using the best corrected position process. Current methods for predicting impact location from impact kinematics are relatively primitive, with peak linear acceleration being the most common method . Peak liDespite some improvements using integrated signals and angular kinematics, matching between video-based and sensor-based impact location was still relatively poor. This could be the result of decoupling between helmet impact location observed in video, and head motion as measured by the mouthguard. In a direct impact, the impact location is expected to correspond with the direction of motion, however we note that in some cases, our raters observed impacts in which helmets “slid past one another”, resulting in impact locations that do not necessarily correspond to the measured direction of motion. Furthermore, identifying impact locations in video is subjective in nature. When relaxing the criteria for matching video-based and sensor-based impact locations to allow for neighboring impact locations , all sensor processing methods were able to predict similar video-based impact locations in 140–150 of 271 impacts.In addition to discrepancies in head impact location between video-based and sensor-based datasets, our observed distribution of head impact locations differs with previously reported impact location distributions, which report up to 31.9% of impacts to the rear of the head . The disTo take advantage of both our sensor-based and video-based exposure data, we present a novel head impact exposure metric specific to American football. For our exposure metric, we look specifically at the number of sensor-based head impacts normalized by the number of video-based player-plays, to account for variability in on field activity between different player positions. Schnebel et. al. alluded to head impact exposure per player-play, stating that linemen tend to have head impacts every play whereas skill players have head impacts more rarely . We haveIn the second example, we observe two different linemen participating in the same game. The first lineman is a starting lineman, whereas the second lineman is only used in special packages. We see that while both players were recorded for 75–95 minutes, the starting lineman sustained many more head impacts. In terms of head impacts per player-hour, the starting lineman had a more than eight-fold increase compared with the second lineman. Again, the per play exposure is more consistent across the two linemen. We believe this metric of defining head impact exposure in American football prevents skewing from variable on-field participation. We note that while plays are well-defined during games, they are not as clear during practices. Delineating plays in the video activity timeline was done automatically based on video assessment datasheets and may over-estimate the number of plays in practice due to the use of fast and short individual drills which were commonly counted as a single play. The metric can be further refined to properly count practice drills, though we note that practice drill time is currently counted in per-hour or per-event exposure statistics. Similar metrics accounting for player participation may also be useful for sports like ice hockey and basketball where players also spend varying amounts of time in play.2 (95% CI: 1109.1rad/s2-1475.9rad/s2) respectively during practices, and 23.2g (95% CI: 18.5–26.1g), 12.5rad/s (95% CI: 10.8rad/s-13.9rad/s), and 1435.0rad/s2 (95% CI: 1288.4rad/s2-1723.3rad/s2) respectively during games. Together, these results suggest that head impacts are both more frequent and more severe in games than in practices, which agrees with previous exposure data.With this exposure metric, we found that there were significantly more head impacts per player-play during games  than during practices . From instrumented mouthguard kinematic measurements, we found median head impact linear acceleration, angular velocity, and angular acceleration magnitude was 18.4g (95% CI: 15.2g-19.8g), 9.4rad/s (95% CI: 8.5rad/s-10.2rad/s), and 1240.5rad/sWhile we successfully collected an independent video-based exposure dataset utilizing a tiered video assessment protocol, this study has several limitations. First, video assessment takes a substantial amount of time. Analyzing 160 hours of video required 1000 man-hours, primarily due to the rigor and detail required. This prevents manual video assessment from being viable for everyday use. However, this protocol can be used to collect datasets with which to improve sensor processing algorithms for head impact detection and location identification, and automated video assessment based on these protocols can be developed for a more streamlined post-event analysis ,43. In fSecond, video quality (1080p at 30fps) and quantity (two views per field) also affected the subjectivity of video classification. To account for uncertainty due to poor video quality, we had instructed raters to be sensitive to any potential instance of Helmet Contact, and labeled any helmet overlap with another object as a Contact event. Multiple views were only considered in second round of video review, which accounted for the substantial loss of Helmet Contact events between the first and second round of video assessment . Improved video quality and quantity would increase certainty in video labels. While some institutions can afford superior video quality and quantity, we believe our equipment is a reasonable representation of what many collegiate and lower-level institutions currently use.Third, we have shown previously that the instrumented mouthguard design can be susceptible to lower mandible disturbances, resulting in overestimation of the measured kinematics . DespiteFourth, we note that our sample population of seven players from a single team during a subset of practices and games in a single season is relatively small. Thus, the purpose of this study was not to generalize exposure data but describe differences in video-based and sensor-based datasets. Despite the small sample size, exposure rates and kinematic magnitudes fall within the range of previously reported American football exposure. Because of the small sample population, we did not include delineation of exposure by player position in the main manuscript, but have included it as supporting information  Figs witFinally, we do not consider Obstructed View activities as head impacts. In some activities labeled as Obstructed View, the tracked player is taking part in a large tackle on the field and is hidden beneath other players. This is particularly true of linemen. It is possible that we miss head impacts during these plays, and it is possible that these plays produce more impacts in under-represented locations . In our analysis, there were 29 observed instances of an Obstructed View.In this paper, we have demonstrated through a small field study, the comparison and combination of independent sensor-based and video-based measurements of head impact exposure. Currently in head injury research, many sensor-based approaches are being developed for impact exposure studies. While many sensors have demonstrated promise in enabling on-field measurements of head kinematics, some remaining limitations in kinematic accuracy and impact detection accuracy lower the confidence in estimating impact exposure using a single sensor. As such, many researchers have begun using additional information, such as video verification, to improve the quality of sensor-based measurements, and a working group in the National Institutes of Health are recommending video verification as a component in field deployments . As showIn conclusion, we have developed a tiered video assessment protocol for collecting an independent impact exposure dataset in American football. This video assessment protocol can be implemented in other contact sports. We have also developed a new head impact exposure metric for American football based on the number of head impacts occurring per player-play. We have found players sustain significantly more impacts per player-play in practices (0.20) than in games (0.35) (p<0.05). This method can provide additional independent exposure information to verify exposure data gathered using wearable sensors that are not fully validated. The verified exposure information can be used to better manage players so that they are not over-exposed to potentially dangerous head impact events.All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Stanford University Internal Review Board IRB #34943. Informed consent was obtained from all individual participants included in the study in written form.S1 FigFirst, we compare exposure across player positions. We compared the exposure data defined by (A) cross-verified sensor-based head impacts per hour and (B) cross-verified sensor-based head impacts per play. We use cross-verified sensor-based head impacts as we believe sensor-based head impacts are the best representation of discrete head impacts and cross-verification gives us high confidence for these impacts. We note that linemen experienced higher exposure than other positions. We also note that, as with the aggregated statistics presented in the main manuscript, players generally experienced more impacts per hour in practices, but more impacts per play in games. (C) This is likely because players participated in more plays per hour during practices than games .(TIF)Click here for additional data file.S2 FigWe delineated impact location distributions by player position for both cross-verified (A) video-based helmet contact periods and (B) sensor-based head impacts. We used the most common peak linear acceleration vector method for sensor-based head impact location distribution for comparison to previously reported distributions. Players generally have similar impact location distributions in both datasets, though we note that all positions except the linemen are represented by a single player, and some positions observed relatively low exposure.(TIF)Click here for additional data file.S3 FigFinally, we delineated impact severity by player position. Impact severity were determined from cross-verified sensor-based impacts and were defined with the impact kinematics: (A) peak linear acceleration magnitude, (B) peak angular velocity magnitude, and (C) peak angular acceleration magnitude. The violin plots represent the kinematics distribution observed and were fit using a log-normal distribution. In general, impact severities were higher during games than during practices for all players. Furthermore, while linemen experienced higher exposure rates, they experienced relatively low impact severities.(TIF)Click here for additional data file.