Action video game training may hold promise as a cognitive intervention with the potential to enhance daily functioning and remediate impairments, but this must be more thoroughly evaluated through evidence-based practices. We review current research on the effect of action video game training on visual attention and visuospatial processing, executive functions, and learning and memory. Focusing on studies that utilize strict experimental controls and synthesize behavioral and neurophysiological data, we examine whether there is sufficient evidence to support a causal relationship between action video game training and beneficial changes in cognition. Convergent lines of behavioral and neurophysiological evidence tentatively support the efficacy of training, but the magnitude and specificity of these effects remain obscure. Causal inference is thus far limited by a lack of standardized and well-controlled methodology. Considering future directions, we suggest stringent adherence to evidence-based practices and collaboration modeled after clinical trial networks. Finally, we recommend the exploration of more complex causal models, such as indirect causal relationships and interactions that may be masking true effects. In September 2013, action video game Grand Theft Auto V broke all previous entertainment sales records by grossing $1 billion in just three days . At presd = 0.46, 95% CI , where expert video game players are compared to video game novices (hereafter referred to as “experts” and “novices”), as well as in true experiments, d = 0.45, 95% CI , where participants train with a video game for a fixed period of time and are compared to their own initial performance or a control group. Yet non-experimental studies, by nature, preclude the possibility of strict causal inference, and a lack of rigorous and standardized methodology in the experimental training studies makes those findings vulnerable to possible confounds.Are cognitive benefits a direct consequence of video game training? In the only comprehensive meta-analysis on video games to date, To the extent that video games improve cognitive functioning, the critical next step is to determine what aspects of video games drive cognitive benefits, how this works, and what it targets in the brain. Additionally, different game types may have various effects on cognition or interact with specific domains. While evidence of neuroplastic change is necessary to establish causality, it is not sufficient. We must know what aspects of gameplay drive the change, as well as how and where it manifests in neural circuitry and observable behavior. Examining the literature, The current review attempts to qualitatively address the issue of causality by adopting a strict focus on studies whose methodology provides the elements necessary for causal inference. We consider only experimental studies that include some form of training paradigm. We will not consider non-experimental, quasi-experimental studies, or correlational studies in this analysis. Within these studies we give the highest priority to those which include one or more of the following design elements: experimental control in the form of active and/or passive comparison groups, neuropsychological data to assess the transfer of training, and neurophysiological evidence to identify the structural or functional correlates of differences in cognitive performance. Unfortunately, there are few studies that include all of these design elements. Insufficient evidence exists to specify the exact “active ingredient” within video games; therefore we use a broad focus on games with an action component. For the purposes of this review we use a broad definition of the term action so as to not exclude games based on thematic or esthetic design elements. We summarize findings with respect to improvement in three cognitive areas: (1) visual attention and visuospatial processing, (2) executive functions, and (3) learning and memory. Lastly, we illustrate areas in need of further investigation and provide commentary for future directions.Perhaps one of the more intriguing mechanisms of cognitive change relates to whether training on action video games can enhance one’s ability to efficiently learn novel tasks. The process of developing skills that facilitate learning in other contexts, referred to as “learning to learn” , may undWhile attention and executive functions play a key role in learning to learn, it is less clear whether other aspects of cognition, such as memory, contribute toward this process. In particular, findings from training studies examining working memory have been inconsistent. Working memory is unique in that it requires not only maintaining information in short-term storage, but also places heavy demands on attention and continual response inhibition . BehavioSeveral recent experimental studies support the idea that training increases various components of executive functioning e.g., . AngueraSuper Mario 64 for 30 min a day over a period of 2 months showed significant post-training group differences in gray matter volume in the right DLPFC, right hippocampus, and cerebellum , have substantial variance in presentation of visual stimuli and in cognitive load. The multi-faceted nature of video games creates difficulties in appropriately categorizing game titles, and may be contributing to contradictory and confusing results in both the cognitive and neurophysiological studies of video games.Additionally, researchers have not yet classified video games in a way that fully accounts for titles that blur genre lines. For example, MarioKart is a racing game in which players use various weapons and abilities to disrupt their opponents’ progress. Lastly, it is crucial to consider the potential effects of individual differences. Future research should devote more attention to investigating what factors, if any, are more predictive of cognitive enhancement success. Affective domains such as motivation and reward sensitivity are underexplored, and, apart from a few studies e.g., , baselinAction video games deliver dynamic, multi-sensory stimulation that requires users to navigate tasks that are equally challenging and entertaining. This medium provides not only a unique tool for investigating human cognition and neuroplasticity, but also the means for potentially counteracting cognitive decline and remediating cognitive impairments. Yet such promise and opportunity must not supersede the need for rigorous and unbiased scientific evaluation. Action video game training may indeed lead to enhancements in attention, visuospatial processing, and executive functioning. However, the magnitude and specificity of these effects remain unclear. Future research should not only adopt methodologies based upon best practices from clinical trials, but also incorporate evidence from both behavioral and neurophysiological approaches.All authors participated in the literature review, drafted the initial manuscript sections with exception to the discussion, and provided critical revisions on the initial draft. Joseph Bisoglio, Timothy I. Michaels, and Joshua E. Mervis wrote the discussion and all authors contributed revisions to the final version of the manuscript, which all authors approved for submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Risky patterns of alcohol use prior to pregnancy increase the risk of alcohol-exposed pregnancies and subsequent adverse outcomes. It is important to understand how consumption changes once women become pregnant.The aim of this study was to describe the characteristics of women that partake in risky drinking patterns before pregnancy and to examine how these patterns change once they become pregnant.A sample of 1577 women from the 1973–78 cohort of the Australian Longitudinal Study on Women’s Health were included if they first reported being pregnant in 2000, 2003, 2006, 2009 and reported risky drinking patterns prior to that pregnancy. Multinomial logistic regression was used to determine which risky drinking patterns were most likely to continue into pregnancy.When reporting risky drinking patterns prior to pregnancy only 6% of women reported weekly drinking only, whereas 46% reported binge drinking only and 48% reported both. Women in both binge categories were more likely to have experienced financial stress, not been partnered, smoked, used drugs, been nulliparous, experienced a violent relationship, and were less educated. Most women (46%) continued these risky drinking patterns into pregnancy, with 40% reducing these behaviors, and 14% completely ceasing alcohol consumption. Once pregnant, women who binged only prior to pregnancy were more likely to continue (55%) rather than reduce drinking (29%). Of the combined drinking group 61% continued to binge and 47% continued weekly drinking. Compared with the combined drinking group, binge only drinkers prior to pregnancy were less likely to reduce rather than continue their drinking once pregnant .Over a third of women continued risky drinking into pregnancy, especially binge drinking, suggesting a need to address alcohol consumption prior to pregnancy. Heavy alcohol use during pregnancy can have detrimental effects, such as Fetal Alcohol Spectrum Disorders Binge drinking episodes during pregnancy have been found to increase the risk of adverse outcomes such as poor neurodevelopment, Considering the complexity regarding the effects of alcohol consumption during pregnancy and the inability to define a safe level of alcohol use, a number of alcohol guidelines worldwide have recommended abstinence for pregnant women. It would be useful to establish if risky drinking patterns prior to pregnancy are modified once women become pregnant and if not, identify the characteristics of women engaging in these risky drinking patterns before pregnancy to enable early intervention. Some studies have reported the proportions of these drinking behaviors before and during pregnancy. The aims of this study were to: define the characteristics of women partaking in risky drinking patterns prior to pregnancy; investigate if women modify their risky drinking patterns once they become pregnant; and identify risky drinking patterns prior to pregnancy that increase a woman’s risk of continuing the behavior into pregnancy.Ethical clearance for the Australian Longitudinal Study on Women’s Health (ALSWH) was obtained from the Universities of Newcastle and Queensland, Australia . Women provided written informed consent to participate in the study.This study uses data from the ALSWH, which commenced in 1996. Using the national health insurance database which provides universal healthcare to all Australian citizens and permanent residents (Medicare), women were randomly sampled, with those from rural and remote areas sampled at double the rate of women from urban areas. Born between 1973–78, 1946–51, and 1921–26, three age cohorts of women were sent mailed invitations to participate. After the baseline survey in 1996, each cohort was mailed a survey on an approximately three-year interval basis. More detailed methods can be found on the longitudinal study’s website The 1973–78 cohort data was used for this study. This cohort was broadly representative of similarly aged Australian women at the time of recruitment. Pregnancy status was determined using a prospective measure at every survey which asked “Are you currently pregnant?” Participant characteristics prior to pregnancy (i.e. the survey before the index pregnancy) were examined in relation to risky drinking patterns at that time. The sociodemographic and health-related characteristics that were measured at the survey before pregnancy included: participant’s age, partner status, highest educational attainment, area of residence, possession of private health insurance, level of stress about money to gauge income management, ever having experienced a violent relationship with a partner, having had a previous live birth, having had a Pap test in the last two years, ever having smoked or ever having used illicit drugs. The final response categories for these characteristics can be seen in Alcohol use items were measured at the survey when the woman was pregnant and at the survey prior to her pregnancy. Weekly drinking was measured by collapsing the answers to the question “How often do you usually drink alcohol?” into only two responses - ‘at least once a week’ versus ‘less than once a week’. The ‘less than once a week’ category was a combination of the response options ‘less than once a month’ and ‘less than once a week’. The ‘at least once a week’ category included response options ‘on 1 or 2 days a week’, ‘on 3 or 4 days a week’, ‘on 5 or 6 days a week’, and ‘every day’. Binge drinking was measured by the survey item “How often do you have five or more standard drinks of alcohol on one occasion?” with responses categorized into ‘never’ versus ‘ever’. The latter included the responses: ‘less than once a month’, ‘about once a month’, ‘about once a week’, and ‘more than once a week’. The usual quantity of alcohol consumption was measured by the item “On a day when you drink alcohol, how many standard drinks do you usually have?” Responses to this item were ‘1 or 2 drinks per day’, ‘3 or 4 drinks per day’, ‘5 to 8 drinks per day’, and ‘9 or more drinks per day’.The primary outcome was change in risky drinking patterns from before pregnancy to pregnancy. Risky drinking patterns before pregnancy were defined as drinking behaviors that had been found in previous studies to increase a woman’s risk of consuming alcohol during pregnancy. The three levels used to categorize the primary outcome of change in risky drinking patterns from before pregnancy to pregnancy were ‘stopped’, ‘reduced’, or ‘continued’. A change to complete abstinence from alcohol during pregnancy was defined as ‘stopped’. A ‘reduced’ change varied per risky drinking group. For those in the binge only group, a change of drinking pattern from bingeing to alcohol use without bingeing was classified as ‘reduced’. A change from drinking at least once a week to drinking less than weekly was labeled as a ‘reduced’ change for the weekly drinking only group. For the combined drinking group (binge and weekly), the term ‘reduced’ referred to some alcohol use where either or both risky drinking patterns were ceased. Participants that continued their risky drinking patterns were used as the reference group in multivariate analyses. They were chosen as the reference group because they were considered to be most in need of intervention, as they did not report a change in risky alcohol consumption patterns once becoming pregnant.All statistical analyses were run using SPSS . Descriptive statistics were reported for socio-demographic and health-related characteristics in relation to the three risky drinking patterns prior to pregnancy  and were assessed using chi-square tests and Analysis of Variance (ANOVA), as appropriate. The distribution of usual quantity of alcohol use prior to pregnancy was calculated for each risky drinking pattern to examine drinking habits within groups. Characteristics that significantly differed between the three groups (p<0.05) were considered in the following multivariate analyses.The association between risky drinking patterns prior to pregnancy and change in drinking behavior from before pregnancy to pregnancy was examined using multinomial logistic regression. The outcome for the regression was the change in drinking patterns, modeling the risk of stopping or reducing the risky drinking pattern versus continuing such behavior into pregnancy. Unadjusted odds ratios were initially calculated. Then the model was adjusted for participant characteristics, building the model by controlling for characteristics significantly related to risky drinking patterns prior to pregnancy. A final multinomial logistic regression model was conducted controlling for all significant characteristics. Although it was not a main focus of this analysis, the final model was adjusted to see if the change in Australian alcohol guidelines for pregnant women  Of the 1577 participants included in the analysis, 19% reported a pregnancy in 2000, 23% in 2003, 32% in 2006 and 26% in 2009. Ninety-nine (6%) reported that before pregnancy they consumed alcohol at least weekly without any binge drinking, 725 (46%) reported only binge drinking during this time, while 753 (48%) reported both weekly and binge drinking patterns prior to pregnancy. The majority (94%) of participants that were weekly drinking usually consumed no more than two drinks on a drinking day, with the remaining 6% reporting three to four drinks per drinking day. Of the participants in the binge only drinking group, on a drinking day 37% drank up to two drinks, 35% drank three to four drinks, while the remaining 28% drank five or more. The majority (51%) of participants in the combined drinking group reported drinking up to two drinks on a drinking day, with 36% drinking three to four and 13% drinking five or more drinks. Regardless of risky drinking patterns before pregnancy, fewer than 17% of the women completely stopped these behaviors once they became pregnant, with most women (46%) continuing these risky drinking patterns. By utilizing data from a population-based prospective cohort study, the results provide a strong level of evidence to suggest that Australian women who participate in risky drinking patterns before pregnancy are likely to continue these drinking patterns into pregnancy. There is only a small likelihood that these women will completely abstain from alcohol during pregnancy. Less than one in five women stopped consuming alcohol once becoming pregnant, with no difference in stopping between the three drinking categories. However, a substantial proportion of women made the move in the right direction by reducing these risky drinking patterns when pregnant. Interestingly, women partaking in both binge and weekly drinking were more likely to reduce their drinking compared to those who only did one or the other. This may be due to the fact that they had more opportunity to reduce as there were two behaviors they could change rather than just one. However, further investigation is needed to understand why this was the case.Although some women took a positive step in reducing risky alcohol patterns once they were pregnant, women who participated in binge drinking prior to pregnancy were the least likely to do so. Even the women who partook in both risky drinking patterns (i.e. weekly and binge) prior to pregnancy were less likely to reduce their binge drinking rather than their weekly drinking. These findings lend support to previous research from France which found that binge drinking was more common than weekly drinking in pregnant women, Women in the current study who binge drank prior to pregnancy appeared to be of a lower socio-economic status as reflected by their lower education status and lack of private health insurance. Binge drinking in this group could be due to a difference in knowledge and views, as previous examination of women’s perceptions of safe levels of alcohol consumption found that the mean number of alcoholic drinks believed to be acceptable on any one occasion seemed to reduce with higher socioeconomic advantage. Also of interest was the finding that women who consumed alcohol before pregnancy through weekly drinking only were found to be significantly less likely to reduce their drinking behavior only after illicit drug use and smoking status were taken into account. The findings from this group need to be interpreted with caution given the small sample size (n = 99). Previous research found that the chances of continuing concurrent alcohol use and smoking into pregnancy increased if women were heavier smokers prior to pregnancy. The use of a self-report questionnaire lends itself to the potential for social desirability bias. However, a previous study found that pregnant women accurately reported their smoking, a behavior considered socially unacceptable, when compared to biological measurements. The findings of this study highlight the need for a primary prevention strategy to reduce prenatal alcohol use by addressing risky drinking patterns, particularly binge drinking, prior to conception. This study provides further support to existing clinical guidelines which promote alcohol consumption being addressed before pregnancy occurs. The majority of women with risky drinking patterns before pregnancy continued these behaviors once they became pregnant. Although a number of women modified their drinking habits by reducing risky drinking patterns, less than one in five women in this sample completely abstained from alcohol once becoming pregnant, as currently recommended by a number of guidelines worldwide.


Both selenium and non-steroidal anti-inflammatory drug (NSAID) sulindac are effective in cancer prevention, but their effects are affected by several factors including epigenetic alterations and gene expression. The current study was designed to determine the effects of the combination of selenium and sulindac on tumor inhibition and the underlying mechanisms.We fed the intestinal tumor model Apc/p21 mice with selenium- and sulindac-supplemented diet for 24 weeks, and found that the combination of selenium and sulindac significantly inhibited intestinal tumorigenesis, in terms of reducing tumor incidence by 52% and tumor multiplicities by 80% (p<0.01). Mechanistic studies revealed that the combination of selenium and sulindac led to the significant induction of the expression of p27 and p53 and JNK1 phosphorylation, and led to the suppression of β-catenin and its downstream targets. Impressively, the data also showed that demythelation on p21 promoter was associated with tumor inhibition by the combination of selenium and sulindac.The selenium is synergistic with sulindac to exert maximal effects on tumor inhibition. This finding provides an important chemopreventive strategy using combination of anti-cancer agents, which has a great impact on cancer prevention and has a promising translational potential. APC) gene [Apc gene is inactivated by a mutation [p21WAF1/Cip1 gene could enhance intestinal tumorigenesis in the Apc mouse model (i.e. Apc/p21 mice) [Apc+/−/p21+/+ mice, but not in the mice in which even a single p21 allele was inactivated (i.e. Apc+/−/p21+/−) [Apc/p21 mice with diets supplemented with selenium or combination of selenium and sulindac in present study to determine the intestinal tumor inhibition.Sulindac is a non-steroidal anti-inflammatory drug (NSAID) that has shown to be effective in preventing intestinal tumors in familial adenomatosis polyposis patients that inherit a mutant allele of the adenomatosis polyposis coli (PC) gene ,2, in inmutation -5, and imutation . Our pre21 mice) . Most in/p21+/−) , because/p21+/−) . To detein vivo studies have demonstrated that dietary selenium supplementation can reduce cancer incidence in animal models of melanoma and cancers of colon, breast, liver, esophagus, head and neck, kidney and lung [in vivo and in vitro through a novel anti-cancer mechanism - activating JNK1 and suppressing β-catenin signaling [Selenium, an important trace element, is essentially involved in different physiological functions in mammalian and human body. Selenium has significant activity as a chemoprevention agent for cancer. Epidemiological and experimental studies have suggested that intake of dietary selenium is inversely related to overall cancer risk. The effect was most pronounced in gastrointestinal and prostate cancer -11. In aand lung ,11. The and lung . While tignaling , in addiignaling ,15.Apc/p21 mouse model of intestinal tumor was applied in the current student. We found that selenium was synergistic with sulindac and exerted maximum tumor inhibition efficacy through inhibiting p21 promoter methylation, inducing p53, p27 and phosphorylation of JNK1, and suppressing Wnt/β-catenin signaling, although selenium alone showed slight inhibitory effect in the Apc/p21 mice.The unique Apc+/−/p21+/− mice spontaneously developed intestinal tumors when they fed with the AIN-76A defined diet, at average multiplicities of 1.95 per mouse  and tumor multiplicity decreased to 1.66, the differences were not significant (P> 0.05), in comparison with the mice fed the AIN-76A diet.In consistent with our earlier report , loss ofApc+/−/p21+/− and Apc+/−/p21−/− mice although sulindac inhibited tumorigenesis in the Apc+/−/p21+/+ mice in which both P21 alleles were wild-type [Apc+/−/p21+/− mice.Our earlier study also reported that sulindac did not exert tumor inhibition in ild-type . HoweverApc/p21+/− mice [Apc/p21+/−[To elucidate underlying mechanisms of tumor inhibition by selenium and sulindac, the potential changes of molecules involved in Wnt-β-catenin signaling pathways were determined. As shown in Figure +/− mice , and thepc/p21+/−,8, no fuApc+/−/p21+/− mice, the impact on p21 promoter methylation status was evaluated using the isolated intestinal epithelial cells from different mouse groups, and the methylation-specific PCR was performed. Consistent with our previous report, mouse p21promoter distal CpG region was hypermethylated in intestinal epithelial cells of the Apc+/−/p21+/− mice. Selenium and sulindac synergistically impacted mouse p21 promoter methylation, although selenium alone did not  has been recently found to phosphorylate and destabilize hypoxia-inducible factor 1α (HIF-1α). Plk3 can also phosphorylate and stabilize PTEN phosphatase, a known regulator of HIF-1α and tumor angiogenesis . WhetherApc/p21+/− mice through impacting methylation, inducing the expression of p27, p53 and phosphorylation of JNK1, and suppressing β-catenin signaling. Current finding provides an important chemopreventive strategy using combination of anti-cancer agents instead of a single one. Therefore, the finding from this study has a great impact on cancer prevention and has a promising translational potential.In conclusion, selenium and sulindac are synergistic to inhibit intestinal tumorigenesis in the Apc1638+/− mice were mated with p21−/− mice to produce Apc1638+/−/p21+/− mice. At weaning (approximately 3–4 weeks), littermates were randomized to genetic/dietary groups and fed ad libitum AIN-76A diet,AIN-76A diet supplemented with 0.00004% (4.0 ppm) sodium selenite or the AIN-76A diet supplemented with 0.00004% (4.0 ppm) sodium selenite and 0.02% (200 ppm) sulindac . The mice were weighed weekly and maintained on diet for 24 weeks or until they exhibited significant weight loss or other signs of extensive tumor formation. The animals were sacrificed by CO2 overdose, followed by cervical dislocation, and rapidly dissected for evaluation of tumors and fixation of tissues, as described previously [As described previously ,7,31, theviously ,34. ThisThe gastrointestinal tract from stomach through rectum was removed. The entire intestinal tract was opened longitudinally, and the stomach opened along the greater curvature to expose the mucosa. The full length of the intestinal tract was immediately examined for neoplastic lesions under a dissecting microscope. The location and the macroscopic features of all mucosal growth were recorded. Tumor incidence (percentage of mice with tumor), multiplicity (number of tumors per mouse), and size (tumor volume) were analyzed.Intestinal epithelial cells from each strain were isolated by incubating opened mouse intestine from duodenum as described recently ,35. Prot′-TGGTTTGAGAATTGGATTTAATTTT-3′, reverse (−641 to −665) 5′-TCCCAAAAAATCCCACTATATCTAA-3′, as this region was reported to play a critical role in the response to sulindac in our previous study [Genomic DNA was isolated using a Dneasy Tissue Kit  from the intestinal epithelial cells. Cytosine methylation was determined using EZ-DNA methylation kit  according manufactory instruction. Briefly, under these conditions, unmethylated cytosine was converted to uracil, but 5’-methylcytosine remained as cytosine. The primers for methylation-specific PCR were designed using MethPrimer as reported previously . The prius study . The theApc/p21 mice fed with AIN-76A control diet, AIN-76A plus sodium selenite, or AIN-76A plus sodium selenite and sulindac, to determine whether there was a significant effect of selenium alone or synergistic effect of selenium and sulindac on tumor prevention.Values for the average of intestinal tumor numbers were expressed as means ± SD. Analysis of variance was used to compare data derived from Combination of selenium and sulindac significantly inhibited intestinal tumorigenesis, which was linked to induction of the expression of p27 and p53 and JNK1 phosphorylation, suppression of β-catenin signaling and demythelation on p21 promoter.APC: Adenomatosis polyposis coli; p21: p21WAF1/cip1; MS-PCR: Methylation-specific polymerase chain reaction.All authors have no competing interests to declare.YW designed and guided the experiments, BX, PN and DH conducted the experiments, BX and YW analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.


Paenibacillus polymyxa SQR-21, which consists of one circular chromosome of 5,828,438 bp with 5,024 coding sequences (CDS). The data presented highlight multiple sets of functional genes associated with its plant-beneficial characteristics.Here we report the complete genome sequence of a plant growth-promoting rhizobacterium (PGPR),  Paenibacillus polymyxa (formerly Bacillus polymyxa), a Gram-positive, sporulating bacterium, is considered to be a plant growth-promoting rhizobacterium (PGPR)  (P. polymyxa SC2 (NC_014622) (P. polymyxa M1 (NC_017542) (P. polymyxa CR1 (NC_023037)  , 2. Origm (PGPR) , 4, P. pediation –12. Thus_014483) , P. poly_023037) , have beibiotics , 17 and ibiotics , SQR-21 ibiotics , 20. HerP. polymyxa SQR-21 was performed with Roche 454 sequencing technology. The constructed library was sequenced by the GS FLX Titanium series chemistry. A total of 1,000,000 sequence reads with average read lengths of 350 to 450 bp (resulting in up to 400 Mb of sequence data) were obtained, representing an average of 70-fold coverage of the genome. The reads were assembled into 19 scaffolds, and the 54 sequence gaps both within and between the scaffolds were filled by sequencing PCR products using an ABI 3730 capillary sequencer.Whole-genome sequencing of P. polymyxa SQR-21 is composed of a 5,828,438-bp circular chromosome, with a mean GC content of 45.64%. Based on the genomic data, 5,024 coding sequences (CDS) were predicted by GeneMark  gene clusters, including pmx and fus gene clusters responsible for biosynthesis of polymyxin and fusaricidin, respectively, which reveal high similarities to the published gene clusters (–P. polymyxa SQR-21 provides useful information for both basic and applied research, which also facilitates the understanding of the functions and evolutions of the Paenibacillus polymyxa genome.clusters –26. One Paenibacillus polymyxa SQR-21 has been deposited in NCBI’s GenBank under the accession number CP006872.The complete sequence of


Research into neural mechanisms of drug abuse risk has focused on the role of dysfunction in neural circuits for reward. In contrast, few studies have examined the role of dysfunction in neural circuits of threat in mediating drug abuse risk. Although typically regarded as a risk factor for mood and anxiety disorders, threat-related amygdala reactivity may serve as a protective factor against substance use disorders, particularly in individuals with exaggerated responsiveness to reward.We used well-established neuroimaging paradigms to probe threat-related amygdala and reward-related ventral striatum reactivity in a sample of 200 young adult students from the ongoing Duke Neurogenetics Study. Recent life stress and problem drinking were assessed using self-report. We found a significant three-way interaction between threat-related amygdala reactivity, reward-related ventral striatum reactivity, and recent stress, wherein individuals with higher reward-related ventral striatum reactivity exhibit higher levels of problem drinking in the context of stress, but only if they also have lower threat-related amygdala reactivity. This three-way interaction predicted both contemporaneous problem drinking and problem drinking reported three-months later in a subset of participants.These findings suggest complex interactions between stress and neural responsiveness to both threat and reward mediate problem drinking. Furthermore, they highlight a novel protective role for threat-related amygdala reactivity against drug use in individuals with high neural reactivity to reward. Increased amygdala reactivity to threat has been consistently associated with heightened risk for mood and anxiety disorders . In contConsistent with these findings, a recent study has linked a genetic variant conferring increased risk for drug abuse  with relHere, we explore the interactions of recent life stress, threat-related amygdala and reward-related VS reactivity in predicting variability in self-reported problem drinking in a sample of 200 young adults. We focused on drinking because alcohol is the most commonly used and abused drug in adolescents and young adults , and its2=0.035, b=−0.26, p=0.012). Among participants with low VS reactivity . There was no such interaction for right VS or amygdala reactivity , and no significant main effects of either amygdala or VS reactivity on problem drinking .As expected , there w2=0.033, b=−0.25, p=0.011). Furthermore, childhood trauma did not interact with amygdala or VS reactivity to predict problem drinking . Finally, the same three-way interaction emerged in a subsample of participants (N=85) who completed a three-month follow-up assessment of stress and problem drinking . The temporal stability of this interaction suggests that stress-related problem drinking reflects rather than affects the relative neural responsiveness to threat and reward.Demonstrating the specificity of these findings to recent, as opposed to early, life stress, the three-way interaction remained significant when total scores from the Childhood Trauma Questionnaire  were addAn important caveat to consider when interpreting these findings is the possibility that participants drinking more alcohol may experience more stressful life events partially as a result of their increased drinking, rather than the other way around. Since our measures of stress and problem drinking are based on retrospective self-report spanning the past 12 months, the directionality of the association between stress and drinking cannot be determined on the basis of these analyses. Thus we cannot rule out the alternative interpretation that individuals with high VS reactivity and low amygdala reactivity are more likely to experience highly impactful stressful life events in the context of problem drinking. This interpretation would be consistent with a heightened drive to pursue immediate rewards, coupled with a reduced ability to recognize and avoid threat in those individuals.Limitations notwithstanding, we provide novel evidence that recent life stress is associated with increased problem drinking only in individuals with higher reward-related VS reactivity and lower threat-related amygdala reactivity. Consistent with the relative temporal stability of amygdala  and VS  reactiviAUDIT: Alcohol Use Disorder Identification Test; BOLD fMRI: Blood oxygen level dependent functional magnetic resonance imaging; LESS: Life Events Scale for Students; VS: Ventral striatum.YSN and ARH declare no competing interests.YSN and ARH designed this study. YSN conducted the statistical analyses and drafted the manuscript. ARH edited the manuscript. Both authors read and approved the final manuscript.


Escherichia coli O157:H7 (EcO157), an agent of life threatening hemolytic-uremic syndrome, resides in ruminants and is released in feces at numbers as high as 10 million cells/gram. EcO157 could survive in manure for as long as 21 months, but we observed a 90% decrease in cells of an outbreak strain of EcO157 within half a day in wastewater from dairy lagoons. Although chemical, environmental and biological factors may be responsible for this decrease, we observed an 11-fold increase in native protozoa when wastewater was re-inoculated with 2×107 cells of EcO157/mL. These protozoa engulfed the green fluorescent protein labeled EcO157 within 2 hours after inoculation, but expelled vacuoles filled with live EcO157 cells within 3 days into surrounding wastewater, whereas other protozoa retained the EcO157-filled vacuoles for 7 days. EcO157 was not detected by confocal microscopy either inside or outside protozoa after 7 days. Mixed cultures of protozoa enriched from wastewater consumed EcO157 preferentially as compared to native aerobic bacteria, but failed to eliminate them when EcO157 cells declined to 104/mL. We isolated three protozoa from mixed cultures and typed them by 18S sequencing as Vorticella microstoma, Platyophyra sp. and Colpoda aspera. While all three protozoa internalized EcO157, only Platyophyra and Colpoda acted as predators. Similar to mixed cultures, these protozoa failed to eliminate EcO157 from PBS containing no other supplemental nutrients or prey. However, spiking PBS with cereal grass medium as nutrients induced predation of EcO157 by Platyophyra sp. after 3 days or enhanced predation by Colpoda after 5 days. Therefore, attempts to enrich protozoa to decrease EcO157 from dairy lagoons, may correspond to an increase in protozoa similar to Vorticella and possibly facilitate transport of bacterial pathogens to food crops grown in proximity. Escherichia coli O157:H7 (EcO157) infections were responsible for 5% of the 684 produce associated outbreaks during the same period and 19% of all EcO157 associated outbreaks were due to consumption of contaminated produce Produce was linked to 23% of 117,136 foodborne illnesses associated with outbreaks occurred during 1998 and 2007 in the United States Although highly prevalent in some cattle herds in the US Salmonella enterica serotype Typhimurium phage type DT104 Legionella pneumophilaMycobacterium aviumE. coli) and other pathogenic bacteria demonstrated increased resistance to chlorine disinfection Tetrahymena pyriformisE. coliPathogenic EcO157 often escapes detection because of its low numbers or, possibly, because it is concealed in alternate hosts such as flies, birds, dogs, cats, rodents, horses 7 per gram of feces E. coli in co-culture with Acanthamoeba polyphagaProtozoa constitute half of the microbial biomass in rumen and are essential in providing nutrients to the host through a symbiotic relationship with other rumen inhabitants Protozoa from 10-fold serial dilutions of wastewater samples were determined using the most-probable number (MPN) method The populations of rifampin and nalidixic acid resistant EcO157 were monitored by plating 100-µL portions of serial dilutions of wastewater in 0.01M PBS on LB agar supplemented with 100 µg/mL rifampin, and 50 µg/mL each of nalidixic acid and cycloheximide . Cycloheximide was used to control fungal growth on enumeration media. The populations of GFP-EcO157 strain MM123 were monitored by plating the serial dilutions on LB agar supplemented with 100 µg/mL rifampin, and 50 µg/mL each of cycloheximide and kanamycin. The plated media were incubated overnight at 37°C and counted, except that the fluorescent colonies of MM123 were counted on a UV Transilluminator .One-mL portions of serial dilutions of wastewater were plated on Petrifilm count plates  to enumerate aerobic and coliform bacteria 5 and 8.6±0.7×107 CFU/mL. Overnight growth of EcO157 in LB broth supplemented with 100 µg/mL of rifampin and 50 µg/mL of kanamycin was centrifuged and resuspended in 0.01M PBS was used as a food source for protozoa. The flasks were incubated at 25°C on a gyratory shaker operated at low speed (50 rpm) to simulate circulating aeration in the lagoons, and samples were monitored at various intervals for GFP-EcO157, protozoa and aerobic bacteria. The microcosms containing the high inoculum were re-inoculated with 1.7±0.2×107 CFU/mL after 7 days.Native protozoan populations were monitored in wastewater supplemented with or without EcO157 strain MM123 as alternate prey. MM123 is a spontaneous rifampin-resistant (100 µg/mL) mutant of GFP-labeled strain RM2315 , which has been described previously c. 4,000,000 mol wt, Sigma-Aldrich) were stained with 40 mM propidium iodide and 10 mM SYTO 59  for 1 h to stain dead and live bacteria, respectively, then visualized by scanning on a Leica TCS-NT confocal microscope. Polyox enabled visualization by slowing down the protozoan movement. The internalization of EcO157 was determined by scanning through the total depth of protozoa as 5 to 10 µm optical sections. The sections were compiled using the Leica TCS NT Software and the fluorescence by GFP, propidium iodide, and SYTO 59 were assigned green, red and blue colors, respectively, for compiled images.Wastewater samples from the experiment described above were examined at various intervals to determine the diversity of native protozoa that engulf GFP-EcO157. Five-hundred-microliter samples of wastewater containing 0.3% Polyox  and acridine orange , and diluting samples with an equal portion of glycerol to decrease protozoan motility.https://www.atcc.org/Attachments/4018.pdf, Accessed 6 October 2010) supplemented with 5% of 0.2-µm-filter-sterilized wastewater . A 7-day old growth of protozoa from CGWM was concentrated at 300×g and suspended in 0.01M PBS as the inoculum to test predation. Tests were conducted in triplicate 250-mL Erlenmeyer flasks containing 50-mL of CGWM inoculated with 2×107 cells of EcO157 and 8×103 cells of enriched protozoa per mL . CGWM inoculated with only MM123 was used as a no-predation control. The populations of GFP-MM123, protozoa and aerobic bacteria were monitored at various intervals.Consumption of EcO157 strain MM123 by protozoa was determined using natural protozoa enriched from wastewater. One liter of wastewater was filtered through a 250 µm sieve, centrifuged  and the pellet containing protozoa was transferred in 10 mL of 0.01M PBS to 250 mL of Sonneborn medium  with each well containing 100-µL cereal grass medium without the supplemental wastewater (CGM) and incubated at 25°C. Purity of protozoan cultures in 96-well plates was checked microscopically at periodic intervals and purified further by transferring 10-fold serial dilutions at 7-day intervals to fresh 100-µL of CGM until they were confirmed pure by microscopy.Protozoa were grown in 100 mL-portions of CGM, concentrated by centrifugation at 300×g for 10 min and the cell pellet was used to extract DNA used for the amplification and sequencing of 18S rRNA as described in 7 cells/mL of EcO157 and 1.1×103 cells/mL of Vorticella microstoma or 1.5×102 cells/mL of Platyophyra sp or 2.1×103 cells/mL of Colpoda aspera. These treatments provide ∼10,000, 70,000 and 5,000 cells of EcO157 as food for each one of the Vorticella, Platyophyra and Colpoda cells, respectively. The survival of EcO157 in the absence of predation was monitored in PBS inoculated with only EcO157. As protozoa appeared to slow down in PBS, the treatments were supplemented with 5% CGM on day 3 for Vorticella and Platyophyra and on day 6 for Colpoda. The populations of GFP-EcO157, protozoa and aerobic bacteria were monitored at various intervals.Consumption of EcO157 strain MM123 was determined using three cultures of protozoa isolated from wastewater. Protozoan growth from 250-mL CGM supplemented with 50 µg/mL of tetracycline was centrifuged , the pellet was washed twice in 100 mL of 0.01M PBS to remove bacterial carry-over, and re-suspended in 100 mL PBS as the inoculum to test predation. Tests were conducted in triplicate 250-mL Erlenmeyer flasks containing 50 mL of PBS inoculated with 1.1×106 and 6.6±0.9×108 CFU/mL) of EcO157 strain MM149 as a supplementary food source . MM149 was isolated from dairy manure in northwestern Oregon and was selected for rifampin (110 µg/mL) and nalidixic acid (50 µg/mL) resistance to aid in discriminating the colonies from the bacteria in wastewater during enumerations In an attempt to increase the numbers of native protozoa that consume EcO157, the wastewater was supplemented with or without 10% (w/v) LB broth and inoculated with two different concentrations (7.3±0.7×10P<0.05) between various treatments.Repeated-measures 2-way ANOVA  was used to compare differences in growth of protozoa, EcO157, coliforms, and aerobic bacteria in wastewaters. All comparisons were in triplicate. Bonferroni post hoc test was used to determine the earliest interval at which the decline of EcO157 or growth of protozoa differed significantly  after the start of the experiment. EcO157 cells decreased from 2.1×107 to 1.5×104 (a 99.9% decrease) during the same period and resulted in a decline in ratio of EcO157 to protozoa from 122∶1 to 0.04∶1. The spike in aerobic bacterial counts (P<0.001) at zero and 7 days was a result of enumerating the inocula as aerobic bacteria. At the low inoculum level of 105 CFU EcO157/mL, the populations of protozoa and aerobic bacteria remained essentially the same as in uninoculated wastewater (not shown).These studies and microscopy experiments were facilitated with a GFP-labeled strain of EcO157 (MM123). After an initial 1-day lag, EcO157 populations declined rapidly with a decrease of 90% populations within 0.5 day for both inoculum levels, and also after the second re-inoculation to samples treated with the higher inoculum of EcO157 . MPN coucO157/mL , a repea5 CFU/mL) and could not be detected inside protozoa (GFP-EcO157 (MM123) cells engulfed in a variety of predatory protozoa were observed by confocal laser scanning microscopy of wastewater samples. GFP-EcO157 cells were sparsely distributed in the surrounding wastewater at the lower inoculum level  in the absence of protozoa and maintained at an elevated level of 5×107 CFU/mL after 4 days. The number of aerobic bacteria remained stable during the first 3 days (3×107 at day 3), whereas the cells of EcO157 were consumed preferentially (1×105 at day 3). After 3 days, the protozoa switched to grazing aerobic bacteria after 99.6% of EcO157 cells were consumed. The ratio of aerobic bacteria to protozoa on the 3rd day was 189,000∶1, whereas the EcO157 cells to protozoan ratio declined to 630∶1. However, protozoan numbers also declined within a day, but maintained at 79±3 cells/mL after the initial decline.The number of EcO157 declined rapidly during the first 4 days in the presence of protozoa , whereasVorticella microstoma (GenBank accession # DQ868347), Platyophyra sp. (EU039905) and Colpoda aspera (EU039892). All the sequences obtained from 5 clones selected for each protozoan matched 100% with the GenBank sequences.Protozoan diversity was observed to decrease from 8 to 10 morphological types to 5 types during the transition from natural wastewater to CGM supplemented with 5% filtered wastewater. Purification by serial dilutions and culturing in CGM resulted in the isolation of three ciliates characterized by 18S rRNA sequencing as Platyophyra and Colpoda consumed EcO157 cells in preference to the aerobic bacteria . Addition of LB resulted also in a 3-fold increase in protozoan numbers (1.6×105 with LB vs 5×104 with out) after a 3-day incubation (P<0.05). However, there was approximately a 4-log and 2-log higher decrease in EcO157 and coliforms at day 3, respectively, compared to the experiments with LB, indicating a corresponding increased fitness in E. coli and coliform bacteria in an environment with adequate nutrients  inoculated four times sequentially into wastewater from dairy lagoons over a 45-day period 7 EcO157 CFU/mL. Rates of consumption of bacteria by protozoa of 9 to 266 per hour for a flagellate and 200 to 5,000 per hour for a ciliate have been reported previously 4 consuming from 106 to 109 bacteria per day and, therefore, is consistent with protozoan increases after the second inoculation due to acclimation of protozoa to the EcO157 strain tested in this study. These results are in agreement with a recent observation of increased growth of protozoa in co-culture with EcO157, S. enterica and Listeria monocytogenesE. coli ONT:H32 cells from 9.7 to 0.9 days after the 4th re-inoculation E. coli by specific protozoa We demonstrated incremental growth of native protozoa with sufficient populations of EcO157. This would be consistent with our previous study with To distinguish protozoan predation from other biotic and abiotic factors, we confirmed that protozoa enriched from wastewater and grown in CGWM were responsible for a significant decrease in the population of outbreak strain of EcO157 (MM123). Furthermore, EcO157 grew in the absence of protozoa and maintained at ∼8 log CFU/mL. Direct evidence for predation of EcO157 in wastewater has not been reported, however, extended survival has been reported for EcO157 in filtered well water 5 cells/mL after a 3-day incubation. If the predation is merely density dependent Protozoa enriched from wastewater consumed EcO157 preferentially  in compaV. microstoma, Platyophyra sp. and C. aspera by culturing serial dilutions of the protozoan enrichments in CGWM. Apparently these protozoa in wastewater from dairy lagoons did not originate from the ruminants Entodinium and Epidinium that are most prevalent in the cow rumen C. aspera consumed simultaneously both EcO157 and aerobic bacteria carried over from wastewater, in contrast to Platyophyra sp. requiring a supplemental addition of 5% CGM as nutrients to initiate consumption of EcO157 and aerobic bacteria. However, V. microstoma, a common inhabitant of activated sludge systems Vorticella  E. coli and predatory protozoa as observed in nutrient and alternate prey rich wastewater supplemented with CGM occurs in nature, the protozoa could be an effective refuge for bacteria in general, and foodborne pathogens in particular, thus serving as an environmental reservoir, as has been reported for coexisting M. avium and A. polyphagaV. microstoma ingested EcO157 without digesting them  and different cows. This complex ecology could involve specific interactions between protozoa and pathogens, elimination or protection of pathogens in protozoa, and protozoa adaptations specific to predominant bacteria in the agricultural environment. Further studies will be required to determine the reproducibility of these complex interactions, the detection of other factors and, potentially, the identification of novel interventions beneficial for food safety.Our study provides new information about the risks associated with run-off from dairies and ranches Method S1Characterization of monocultures of protozoa by 18S rRNA sequencing(DOC)Click here for additional data file.Figure S15 CFU of GFP-EcO157/mL. The compiled image was obtained with transmitted light, propidium iodide and GFP. Protozoa did not contain any internalized GFP-EcO157 cells.Confocal image of a protozoan taken 2 h after treating the wastewater with 1×10(TIF)Click here for additional data file.Figure S27 CFU of GFP-EcO157/mL. The flagellate ingested and retained live cells of EcO157 within food vacuoles although the surrounding wastewater contained little or no GFP-EcO157.Image of a flagellate taken 4 days after inoculating the wastewater with 9×10(TIF)Click here for additional data file.Figure S37 CFU of GFP-EcO157/mL.A protozoan with GFP-labeled cells attached to the surface. Confocal image was taken 2 h after inoculating the wastewater with 9×10(TIF)Click here for additional data file.


Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity.DOI:http://dx.doi.org/10.7554/eLife.00102.001 A central feature of the immune system is the ability to detect bacteria, viruses and other pathogens so that they can be repelled or neutralized before they cause lasting damage to an organism. Cells employ a number of different receptors that can detect these pathogens or the molecules they produce. Many of these are called pattern recognition receptors because they recognize certain signatures of microorganisms such as nucleic acids or carbohydrates. An important class of pattern recognition receptor is the toll-like receptor: there are many different families of the receptors, each recognizing a unique feature of bacteria or viruses. Toll-like receptors have two parts that perform two different functions: when one part binds the relevant microbial molecules, the other part sends a signal that results in the production of effector proteins. These proteins include interleukin-1β, which helps to fight infection by causing the inflammation of tissue. To date, 12 different types of toll-like receptors have been found in mice, including three—known as TLR11, TLR12 and TLR13—that are not present in humans. Very little is known about the functions of TLR12 and TLR13. Humans, on the other hand, possess 10 different TLRs, only one of which, TLR10, is not found in mice.Li and Chen have now discovered that TLR13 is responsible for detecting a certain type of ribosomal RNA called 23S ribosomal RNA that are present in bacteria but not in eukaryotic cells. Moreover, they have shown that a short sequence of 13 residues within the 23S ribosomal RNA triggers this pathway and leads to the production of interleukin-1β. The sequence of 13 residues is located at an active site in the RNA that catalyzes the synthesis of peptide bonds, and changing just one of these residues stops the production of interleukin-1β. Other forms of ribosomal RNA are unable to trigger the production of interleukin-1β. These results show that TLR13 differs from all other pattern recognition receptors because it is able to recognize a specific RNA sequence. Li and Chen went on to generate mice lacking TLR13 and showed that immune cells isolated from these mice failed to respond to bacterial RNA. These mice can be used to investigate the role of TLR13 in immune responses to bacterial infections in vivo.DOI:http://dx.doi.org/10.7554/eLife.00102.002 Toxoplasma gondii and an unknown ligand from uropathogenic E. coli. The ligands and roles of TLR12 and TLR13 are unknown. To date, 10 TLRs (TLR1–10) have been identified in humans and 12  in mice.Toll-like receptors are evolutionarily conserved transmembrane proteins that detect microbial components on the cell surface or within the endosomes . All TLRIn addition to TLRs, the innate immune system in vertebrate animals consists of other microbial pattern recognition receptors, including RIG-I like receptors (RLRs), NOD-like receptors (NLRs) and C-type lectin receptors (CLRs). RLRs, which include RIG-I, MDA5 and LGP2, detect viral double-stranded RNA in the cytoplasm and activate a signaling cascade that leads to the production of type-I interferons and other antiviral molecules . RecentlIn the course of investigating the mechanism of MyD88-dependennt induction of IL-1β by bacterial RNA, we found that TLR13 was responsible for detecting the 23S ribosomal RNA of both gram-negative and gram-positive bacteria. Remarkably, a short sequence of 13 residues within domain V of 23S rRNA, which is known to be the catalytic center of peptide bond formation , is bothLactobacillus salivarius (LAB), a gram-positive commensal bacterium commonly found in the gastrointestinal tract, strongly induced IL-1β in mouse bone marrow derived macrophages (BMDM) and Raw264.7, a mouse macrophage cell line  did not impair the ability of the RNA to induce IL-1β, but further deletion of another 490 nt (1–1894) did, suggesting that a sequence located between nt 1894 and 2384 is important (Immune Stimulatory RNA from 23S rRNA).Using in vitro transcription by T7 RNA polymerase, we carried out a systemic deletion analysis of 23S rRNA . Deletiomportant . Furthermportant , which wmportant . Furthermportant . Titratimportant . For simTo determine the sequence specificity of ISR23, we introduced point mutations in the sequence 2054–2068. Point mutations at each residue from position 2055 to 2064 abolished the activity of the RNA, whereas an A>G mutation at position 2054 was tolerable . NotablyE. coli, Salmonella, Listeria monocytogenes and LAB  extracts and culture supernatants using an antibody against IL-1β. Stimulation of WT BMDC with the 23S rRNA sequence led to accumulation of pro-IL-1β, but not mature IL-1β (Tlr13 KO cells stimulated by the 23S rRNA sequence (lanes 11 and 12). In contrast, LPS and poly[dA:dT] treatment induced pro-IL-1β and mature IL-1β even in the absence of TLR13 (lanes 9 and 10). These results indicated that TLR13 was essential for the induction of pro-IL-1β by bacterial ribosomal RNA but this receptor alone was insufficient to trigger inflammasome activation. TLR13-deficient macrophages were also completely defective in inducing other cytokines, including IL6, IL10, TNFα and MCP1, in response to the 23S rRNA sequence (We generated TLR13 knockout mice using a targeted ES cell line generated by the knockout mouse project (KOMP), which deleted almost the entire open reading frame in exon 2 and exon 3 of the mouse romosome . PCR of out mice . The Tlrand R848 . The delre IL-1β , lane 5.re IL-1β , maturedsequence . SimilarE. coli sequence) is hydrogen bonded to A2451, the catalytic residue of peptide bond synthesis (6 methylation at A2085 of S. aureus (corresponding to A2058 of E. coli 23S rRNA), which conferred antibiotic resistance, abolished TLR13 stimulation. Complementing these results, we showed that point mutations of the TLR13 recognition sequence in full-length 23S rRNA, including those expected to impair peptide bond synthesis, destroyed its ability to induce IL-1β . RIG-I detects viral and bacterial RNA in the cytosol through the recognition of dsRNA bearing 5′-triphosphate. MDA5 detects viral dsRNA and perhaps some other unknown features. AIM2 binds dsDNA in the cytoplasm to trigger inflammasome activation. None of these cytosolic nucleic acid sensors display significant sequence specificity.H. marismortui , they could mutate or modify other residues in the ISR23 sequences to render the bacterium invisible to TLR13, as shown for ismortui . Second,Toxoplasma gondii and is important for the production of IL-12 in dendritic cells in mice  on the cell surface and traffic to the endosome where it launches the signaling cascades. The TLR13 signaling cascade clearly engage MyD88 and Unc93b1, but the details of the signaling pathway requires further dissection. Finally, although ISR23 may not be an adjuvant for the development of human vaccines, its potent activity in stimulating cytokine production may be employed to boost the production of antibodies in vertebrate animals that possess the TLR13 pathway.http://www.velocigene.com/komp/detail/10438). Two independently targeted ES cell clones were injected into Albino B6 blastocysts to produce chimeric mice, which were bred with Albino mice to obtain germline transmission. The heterozygous F1 progenies were intercrossed to obtain TLR13 knockout mice. For genotyping TLR13 knockout mice, tail genomic DNA was isolated from 7- to 10-day-old pups and then amplified by PCR using primers described in TLR13 knockout mice were generated using ES cells produced by the KOMP Repository (UC Davis) , TLR2−/−TLR4−/− and TLR7−/− mice were bred and maintained under specific pathogen-free conditions in the animal care facility of University of Texas Southwestern Medical Center at Dallas. These strains were maintained on C57BL/6J background. All mice were engineered, housed and used according to the experimental protocols approved by the Institutional Animal Care and Use Committee.Wild type (WT), MavsBone marrow cells were collected from femurs and tibiae of mice. Cells were cultured in DMEM containing 10% fetal bovine serum (FBS), antibiotics, and conditional media from either L929 cell culture or GM-CSF producing cells. 24 hr later, non-adherent cells were transferred to a new plate and fresh conditional medium were added every other day up to the seventh day. Mature macrophages or DC were harvested and transferred to new plates for further experiments.Murine macrophage cell line Raw264.7 was grown in DMEM supplemented with 10% FBS and antibiotics including penicillin, streptomycin and Normocin . Stable TLR13 knockdown cells were generated using lentiviruses expressing shRNA against TLR13 and selected by puromycin (2 μg/ml). The efficiency of TLR13 knockdown or rescue in these stable cell lines was confirmed by either qRT-PCR or immunoblotting using FLAG (M2) antibody . In all experiments except indicated otherwise, 1 or 3 µg bacterial RNA was added to the culture medium and incubated for 6–8 hr before cells were harvested for analyses.Lactobacillus salivarius (LAB) (ATCC 11741) and DH5α were grown in Difco Lactobacilli MRS Broth or Luria Broth (LB) under either anaerobic chamber  without shaking or normal aerobic with shaking at 250 rpm, respectively. After 16–18 hr, bacterial cells were collected into lysing Matrix B tubes  and rapidly frozen down with liquid nitrogen. To isolate the total RNA, TriZol  was added and the tubes were vortexed at high speed with FastPrep  at 4°C. Crude RNA was further purified with RNeasy Mini Kit . RNA was treated with DNase I   to remove potential DNA contamination.Total RNA isolated from either LAB or DH5α was separated by native or denatured 1% agarose gel electrophoresis. Each band containing ribosomal RNA was excised and soaked in RNase-free water overnight. On the following day, 5-butanol was used to remove extra water and then RNA was precipitated with 100% ethanol.MEGAscript T7 kit  was used for in vitro transcription according to manufacturer's instruction. DNA encoding full length bacterial 23S or 16S rRNA was obtained by PCR amplification from DH5α genomic DNA using Phusion DNA polymerase  and subsequently cloned in pGEM-T vector . DNA templates for in vitro transcription were amplified by PCR using primers listed in Mouse TLR13 cDNA was purchased from InvivoGen and subcloned into a pTY lentiviral vector in which TLR13 was fused in frame with a C-terminal Flag tag. The primers for amplification of TLR13 is shown in Transfection of RNA into Raw264.7 or BMDM was carried out using FuGENE (Roche) or Lipofectamine 2000 (Invitrogen). For enzymatic treatments of nucleic acids, 1.0 μg of nucleic acids was treated with RNase III, RNase T1 or RNase V1 (Ambion) at 37°C for 1 hr. Enzyme-treated RNAs were purified with RNeasy Mini Kit (Qiagen) before adding to cell culture.To extract RNA, cells were first lysed in 1.0 ml of TRIzol (Invitrogen). Lysate was mixed with chloroform, and the aqueous phase was applied to RNeasy columns to obtain total RNA (Qiagen). The iScript cDNA synthesis kit  was used to create cDNA from 0.15 μg of RNA. Quantitative RT-PCR was performed in Applied Biosystem Vii7 using SYBR Green and primers described in  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent ; Osamu Takeuchi (peer reviewer).Thank you for choosing to send your work entitled “Sequence Specific Detection of Bacterial 23S Ribosomal RNA by TLR13” for consideration at The Reviewing Editor and the other reviewer discussed their comments before we reached this decision, and the Reviewing Editor has assembled the following comments based on the reviewers' reports. Our goal is to provide the essential revision requirements as a single set of instructions, so that you have a clear view of the revisions that are necessary for us to publish your work.This is a very interesting paper that describes the sequence-specific recognition of bacterial 23S ribosomal RNA by a Toll-like receptor 11 (TLR11) family, TLR13. Although the basic principle for recognition of "non-self" by innate receptors has been for pattern recognition, as opposed to molecular recognition, of microbe-derived molecules, TLR13 is apparently different. TLR13 instead recognizes specific, conserved sequences of the 23S RNA that are essential for the catalytic center of peptide bond formation in gram-negative and -positive bacteria. Therefore, this provides a new view of innate receptor recognition.We will be happy to accept this manuscript, provided that the authors make a single, quick round of revision by taking the following issues raised by the referees.Science Express (19 July 2012 / http://www.sciencemag.org/content/337/6098/1111.abstract).(1) The authors should mention, succinctly, the similarity and novelty of their current findings in respect to the paper that recently appeared in (2) Throughout the study, the only output examined for the TLR13-MyD88 signaling by 23S rRNA or ISR23 is the induction of IL-1β mRNA. It is most likely that this signal induces other genes such as those for TNF-α, IL6 and type I IFNs. It would be preferable that they show mRNA induction for at least one or two of these cytokines, so that the readers will not misinterpret that the TLR13-MyD88 pathway is specifically targeted to IL-1β gene in lieu of a broader response, assuming this is the case.(3) Is there any possibility that TLR13 signals in conjunction with other TLRs? It would be interesting to discuss this issue. http://www.sciencemag.org/content/337/6098/1111.abstract)(1) The authors should mention, succinctly, the similarity and novelty of their current findings in respect to the paper that recently appeared in Science Express  in bacterial 23S rRNA as a ligand for TLR13. Through a completely independent approach, we arrived at a similar conclusion. However, we found that the optimal sequence that activates 23S rRNA contains 13 nucleotides (ACGGAAAGACCCC), which we referred to as ISR23. We also provided direct evidence that point mutations in the ISR23 sequence in the context of full-length 23S rRNA abolished its activity . Furthermore, we now provide genetic evidence that the knockout of TLR13 in mouse macrophages prevented the induction of IL-1β and other cytokines by the 23S rRNA sequence . We have added a paragraph in the paper to note the similar finding by Oldenberg et al.(2) Throughout the study, the only output examined for the TLR13-MyD88 signaling by 23S rRNA or ISR23 is the induction of IL-1β mRNA. It is most likely that this signal induces other genes such as those for TNF-α, IL6 and type I IFNs. It would be preferable that they show mRNA induction for at least one or two of these cytokines, so that the readers will not misinterpret that the TLR13-MyD88 pathway is specifically targeted to IL-1β gene in lieu of a broader response, assuming this is the case.We thank the reviewer for this suggestion and have performed the experiments with results now shown in Figure 6. Specifically, we showed that stimulation of macrophages by the 23S rRNA sequence induced multiple cytokines, including TNFα, IL-6, IL-10, MCP1 as well as IL-1β. Importantly, the induction of these cytokines was completely abolished by the TLR13 knockout, providing the definitive genetic evidence for the essential role of TLR13 in detecting bacterial 23S rRNA.(3) Is there any possibility that TLR13 signals in conjunction with other TLRs? It would be interesting to discuss this issue.We have added the following paragraph to the Discussion:“Our data obtained from the TLR13 knockout macrophages show that TLR13 is essential for cytokine induction by the bacterial 23S rRNA, a phenotype that has not been observed with other TLR mutant mice, including those lacking TLR2, 4, 7 or 11. However, this does not exclude the possibility that TLR13 may cooperate with other TLRs to detect bacterial infections. In fact, in the context of bacterial infections, multiple TLRs are likely to be activated by distinct ligands associated with bacteria, including peptidoglycans, flagellin, lipopolysaccharides, DNA and RNA. Thus, the relative contribution of TLR13 to immune response against pathogenic and commensal bacteria in vivo requires further investigation, which will now be facilitated by the availability of the TLR13 knockout mice.”


Alcohol drinking is a risk factor for harm and disease. A low level of drinking among non-Western immigrants may lead to less alcohol-related harm and disease. The first aim of this study was to describe frequency of drinking in two generations of immigrants in Oslo, contrasting the result to drinking frequency among ethnic Norwegians. The second aim was to study how frequency of drinking among adult immigrants was associated with social interaction with their own countrymen and ethnic Norwegians, acculturation, age, gender, socioeconomic factors and the Muslim faith.The Oslo Health Study (HUBRO) was conducted during the period 2000 to 2002 and consisted of three separate surveys: a youth study ; adult cohorts from 30 to 75 years old ; the five largest immigrant groups in Oslo . Based on these three surveys, studies of frequency of drinking in the previous year (four categories) were conducted among 15-16-year-olds and their parents’ generation, 30-60-year-old Iranians, Pakistanis, Turks and ethnic Norwegians. A structural equation model with drinking frequency as outcome was established for the adult immigrants.Adults and youth of ethnic Norwegian background reported more frequent alcohol use than immigrants with backgrounds from Iran, Turkey and Pakistan. Iranians reported a higher drinking frequency than Turks and Pakistanis. In the structural equation model high drinking frequency was associated with high host culture competence and social interaction, while high own culture competence was associated with low drinking frequency. Adult first-generation immigrants with a longer stay in Norway, those of a higher age, and females drank alcohol less frequently, while those with a higher level of education and work participation drank more frequently. Muslim immigrants reported a significantly lower drinking frequency than non-Muslims, although this did not apply to Iranians.The existence and growth in Western societies of immigrant groups with low-level alcohol consumption contributed to a lower level of consumption at the population level. This may imply reduced drinking and alcohol-related harm and disease even among ethnic Norwegians. Alcohol use has been ranked high as a risk factor for death and loss of healthy life years, as well as being a source of personal and social harm -3. Policth graders, the largest immigrant group (Turks) reported less than half the level of binge drinking in the previous four weeks than those of German descent. The drinking behaviour of the second largest group of immigrants in Germany (from the former Soviet Union) was similar to those of German descent. Otherwise adolescents from Islamic countries living in Germany had a lower lifetime prevalence of drinking than German and Western European adolescents, with the exception of students from Iran [Studies in Europe have generally found that immigrants from non-Western countries tend to drink less or less often than the host population. In the Netherlands, Turkish and Moroccan immigrants reported less alcohol use than the Dutch population in both the first and second generation, and their alcohol consumption did not converge towards the higher rates in the host population ,6. In anrom Iran . In a strom Iran . Albaniarom Iran . Also, irom Iran .In the United States (US) adolescents of Chinese origin were less likely to be drinkers then Chinese adolescents in Hong Kong and American adolescents in the United States . Among MThus, alcohol use among immigrants to Western countries tended to be less prevalent among non-Western immigrants, but more prevalent and more frequent among those residing in the host country for a longer period, i.e., among second-generation immigrants (as compared to first-generation). In addition those immigrants who spoke the language of the receiving country drank more often ,15,20-22Drinking is strongly influenced by other drinkers in a person’s personal network. It has been argued that society at large, or at least large segments of society, behave collectively regarding drinking . A higheThe pattern of drinking in Norway has been characterised by relatively infrequent consumption, but with a high level of consumption and drinking into intoxication, especially on festive occasions . AlcoholThe aim of the Norwegian government’s alcohol policies has for many years been to reduce both individual and societal costs. The measures introduced have included imposing a monopoly for the import and sale of wine and stronger alcohol, limiting access to beer sold in shops , imposing high taxes on all sales and disseminating information on the consequences of alcohol use. Such a system has not been given priority in the EU or in single member countries (with the exception of Finland before entering the EU and Sweden) . NorwegiImmigration to Norway started in the latter part of the 1960s. In 1970 1.5% of the population was registered as immigrants (born outside Norway of non-Norwegian parents or born in Norway with two non-Norwegian parents). Only 0.1% came from non-Western countries at that time . In 2001, at the time of the study referred to here, comparable figures for immigrants were 6.6% in total and 3.4% non-Western at the national level and 19% in total and 13% non-Western in Oslo . The thiThe availability of three health surveys in 2000–2002 covering adolescents, adults, and adults in the largest immigrant groups in Oslo, Norway, enabled us to study alcohol drinking among ethnic Norwegians and three of the five largest non-Western immigrant groups with Islam as the predominant religion in their home country: Iranians, Pakistanis and Turks. The first aim of this study was to describe frequency of drinking in two generations of immigrants in Oslo, comparing the result to drinking among ethnic Norwegians. The second aim was to study how frequency of drinking among adult immigrants was associated with social interaction with their own countrymen and ethnic Norwegians, acculturation factors, age, gender, socioeconomic factors and the Muslim religion.This analysis was based on three sample surveys included in the Oslo Health Study (HUBRO), which was conducted in joint collaboration with the Oslo City Council, the University of Oslo and the Norwegian Institute of Public Health. The study protocol was approved by the Norwegian Data Inspectorate and the Regional Committee for Medical Research Ethics.HUBRO was carried out in the city of Oslo from May 2000 to September 2001 . An inviIn addition, all 15-16-year-olds on class lists in Oslo in the period of January to April in both 2000 and 2001, as well as those who entered the school later, were invited to participate in a school-based survey. A total of 8435 students were registered in or entered schools, including those in special schools for the disabled. Thirty-one were unable to respond to the questionnaire due to impairment and 88 had moved or quit school before the survey was carried out. Thus 8316 students were eligible, among whom 7343 responded to at least one question (response rate 88%). This survey consisted of a questionnaire only. Response rates for Iranians were 82%, for Pakistanis 83% and for Turks 79%. More details on the sampling procedure can be found elsewhere . MissingA third HUBRO study, the Oslo Immigrant Health Study, was conducted among the five largest immigrant groups in Oslo in 2002 . All indItem missing was at 2.2% or lower for gender and country of birth/ethnicity in the three surveys.Three datasets were established for this study, including ethnic Norwegians and/or Iranians, Turks and Pakistanis who had reported frequency of alcohol drinking:1. The 15-16-year-olds in 2000 in HUBRO: 5381 youth with ethnic Norwegian background, 93 Iranians, 559 Pakistanis and 105 Turks. This sample was called the Oslo youth population sample.2. The 30-60-year-olds in 2000/2001 in a merged dataset of HUBRO and the Oslo Immigrant Health Study: 12,259 with ethnic Norwegian background and 604 born in Iran, 607 born in Pakistan, and 436 born in Turkey. Age and gender weights were used to estimate the population distribution. This dataset was called the Oslo adult population sample, constituting the parent generation of the 15-16-year-olds plus those adults who socialised with them.3. A selection from the Oslo adult population sample called the adult immigrant sample: immigrants who had attended the physical examination and delivered the second questionnaire. This sample included 389 born in Iran, 323 in Pakistan, and 270 in Turkey who also reported acculturation variables and other relevant variables. Item non-response for acculturation variables etc. was between 1.2 and 12.9%.Alcohol use among adults varies by gender, age, ethnic or religious groups and socioeconomic status ,34. AnalThe responses were divided into four categories: weekly/monthly/less than monthly/never drank alcohol.Included gender and age, workforce participation , education (number of years of education), and being a Muslim.The place of birth was recorded as it appeared in the Norwegian Population Register. The parents’ place of birth for the 15-16-year-olds was reported by the youth in the questionnaire. The categories by country background were based on the mother’s country of birth, while the father could be from any country other than Norway. The parents of all the Iranians were from Iran, while among Pakistanis 98% were mono-ethnic, as were 97% of the Turks.Various modes of the use of language and of socialisation/affiliation are frequently used to establish measures of competence and identification regarding both one’s own and the host culture -38. In tHow often have you in the course of the past year:· read a newspaper in your own language?· read a newspaper in Norwegian?· had a visit from a Norwegian?· received help/support from a Norwegian?· taken part in meetings arranged by your own countrymen?In your opinion, how good is your knowledge of Norwegian? The first five measurements had four response categories: daily/weekly/less often/never, while the sixth had five response categories: very good/good/average/rather poor/poor.The variable ‘Number of years since moving to Norway’ was also included in the analysis as an exogenous variable to measure the length of the acculturation process.Immigrants have tended to settle in Norway's capital, Oslo, where they represented almost one-fifth of the population in 2001 . In the On the host country national level, the immigrant population studied varied with respect to the status of immigration (migrant workers vs. refugees), main settlement period (indicating differences in the length of the acculturation period), tendency to marry one’s own culture members, number of offspring, Norwegian citizenship, participation in voting, participation in upper secondary school level of education and workforce participation . Such differences among immigrants are also found in the sample  and RMSEA (Root Mean Square Error of Approximation). An acceptable fit can be based on a value of CFI higher than 0.90 and a good fit higher than 0.95 (possible range zero to one), while a value of RMSEA lower than 0.05 represents a good fit and lower than 0.08 an acceptable fit  [A structural equation modelling (SEM) approach was used to model alcohol drinking frequency for adult immigrants. First a factor analysis approach was used to reduce the dimensions of the six acculturation variables and establish the measurement part of the model. The factors found were used as intermediate latent endogenous variables. Exogenous variables were gender, age, Muslim faith, years of education, participation in the workforce and length of residence in Norway. The structural model was developed in AMOS  version 17.0 run within the IBM SPSS Statistics version 19.0, using the modification indices approach . Three ge value) . The accIn the final, most parsimonious model with best fit, associations between the exogenous variables and the outcome alcohol frequency can be both direct and indirect through one or more latent variables. The indirect effects are calculated as the product of any direct effect of an exogenous variable on the latent variables, multiplied by the direct effect of the latent variables on alcohol frequency. The total effect will be the sum of the direct and the indirect effects.A significance level of 5% was used for all hypothesis tests. Few p-values for tests are reported in the text, but statements of differences are all based on tests using the 5% level.Frequency of drinking among adults was significantly different between ethnic Norwegians and all three immigrant groups, both for males and females. Among the immigrant groups, Iranians reported the highest frequency of drinking, Turks were at a middle level and Pakistanis reported the lowest frequency of drinking both for males and females :1. Host culture competence (high level of Norwegian language skills/often read Norwegian newspaper/often had a Norwegian visitor/often had support from a Norwegian)2. Own culture competence (often read newspaper in own language/often participated in meetings arranged by own countrymen)3. Social interaction (often had a Norwegian visitor/often had support from a Norwegian/often participated in meetings arranged by own countrymen)Rotation techniques did not improve or change the interpretation of the factors.The structural equation model with drinking frequency as outcome (four categories) thus included six exogenous variables  and the three endogenous variables identified above. In the baseline model all exogenous variables could be correlated, as could the three endogenous variables. In addition all direct and indirect effects were included. Correlation between the exogenous variables in the regression on the latent variables in the structural model was highest between age and time in Norway (0.40) for the total sample. A somewhat higher value was found for this correlation for Pakistanis (0.47), while all other correlations were lower. Thus colinearity was not an issue in the regression part of the structural equation model.Using the modification indices approach to remove all non-significant correlations and effects, an acceptable fit was obtained for the model shown in the lower section of Figure Being male and having attended school for many years were positively associated with drinking frequency, while being a Muslim and having lived in Norway for a long time were negatively associated with this outcome. Those having lived in Norway longest were mainly Pakistanis with low alcohol consumption. No direct association was found between age and drinking frequency, or between work participation and drinking frequency. Indirectly, however, there were associations between those variables and alcohol frequency through host culture competence. Age was negatively associated with host culture competence (−0.030 (se 0.003)), which was positively associated with alcohol frequency (0.293 (se 0.061)) and thus a total negative association was established between age and drinking frequency. Work participation was in the same way positively associated with alcohol frequency.In addition to the direct negative association between the Muslim faith and drinking frequency, an additional negative association was mediated through a higher level of own culture competence. In addition, the positive association between the number years attending school and frequency of drinking was strengthened by the mediation through own and host culture competence. Regarding gender and years in Norway, the indirect association mediated by host and own culture competence implied a reduction of the direct effects.2 = 25.7 for weights with 18 degrees of freedom, p = 0.11 and Chi2 = 19.9 for means with 14 degrees of freedom, p = 0.13 in a nested model).When country-specific analyses were run, the CFIs were somewhat reduced, while the RMSEAs were somewhat increased for each country – indicating a poorer, but acceptable fit for each country. The same structural model was applied for the three countries, i.e. the non-significant parameters were not removed. Fewer significant results were present in the country-specific analyses, as seen by the lower number of significant coefficients in columns 2–4 in Table 2 = 372.2 with 26 degrees of freedom, p < 0.00). Differences in parameters between countries were identified for the association between alcohol frequency and the three variables gender , being a Muslim  and years in school . For gender the association (direct and indirect) was strongest for Turks and weakest for Pakistanis  and age on host culture competence  were lowest for Iranians. Gender on own culture competence  was highest for Turks. In addition, the correlation between host and own culture competence was significantly different . This correlation was lowest for Iranians.Differences in the structural model were also identified for the regression weights for the exogenous variables on the latent variables. Years in school on host culture competence , but a higher level of drinking than Pakistanis and Turks. They were closest to the population characteristics of the Norwegian population  and the low level of education may have strengthened the Muslim imperative to abstain from alcohol.Turks were a group in the middle position for almost all measurements: population characteristics Table , sample Young immigrants of the first and second generations from Iran, Pakistan, and Turkey drank less than ethnic Norwegians, as did adults from the same countries. This is in line with earlier studies among Turks, Moroccans, Antilleans, Surinamese, Asian Muslims, Hindus, Sikhs, Vietnamese in Europe -12, thosPakistani and Turkish youth adhered to the low drinking frequency of the first generation of adults. In an earlier study in Oslo, young immigrants from Morocco, Pakistan, Turkey and Vietnam reported higher frequency of use in the second generation than in the first, by country and gender, although this was not significant . In the Lower consumption of alcohol among females than males is a worldwide finding. The exception for Norwegian adolescents seen here has been recognised in other studies . The posTime in the host country has been used as an indicator of integration or assimilation for immigrants. In this study drinking level was negatively associated with both mean stay in Norway and age. This reflects the fact that those with the longest mean stay were older, low-consuming Pakistanis and Turks. Country-wise analyses showed a positive association between drinking and mean stay only among Iranians. Thus the use of length of stay as an indicator of immigrant behaviour becoming closer to the host behaviour was group-dependent and not dominant.The latent variable called ‘host culture competence’ loading on the four variables 1) Norwegian language skills, 2) reading of Norwegian newspapers, 3) had Norwegian visitors and 4) had support from Norwegians, also had a social interaction component: knowledge of the host language was necessary for reading and for socialising with the host community. The variable ‘own culture competence’ loaded on two variables: 1) reading newspaper in own language and 2) participating in meetings arranged by own countrymen. Thus this variable also included a social component. The variables were positively correlated, indicating that the same person tended to score high (or low) on both variables. Thus host and own culture competence could not be measured as opposite values along one dimension only. The direction of the association with drinking was as predicted for these factors: high host culture competence was associated with a higher frequency of drinking, while high own culture competence was associated with a lower frequency. The third latent variable, social interaction, loaded on the social interaction variables mentioned above , while they were much lower for the adult cohorts (46%) and the immigrant survey (40%). These were response rates based on the whole population of Iranians and Turks, not response rates for a drawn sample from those populations. In the immigrant survey, a 30% sample was first drawn of Pakistanis. Self-selection may be very important, however. Self-selection according to socio-demographic variables was shown to have little impact on several prevalence estimates in the adult survey and social inequality in health by different socio-demographic variables seemed unbiased . A similCross-sectional studies have limitations and shortcomings in the study of longitudinal social processes. The result of the acculturation process on drinking was measured at the same time as the factors assumed to influence the drinking. This is not a major problem if socialisation patterns are stable, but it is relevant to assume that such patterns develop during the acculturation process. It is a huge challenge to establish prospective studies in immigrant samples, however. It is also seldom that a cross-sectional sample as large as the one used here can be established among immigrants. Even in this study, however, some country-specific results were in line with results for the total sample, but not significantly so. This may partly be a result of less power due to a lower number of observations for each country’s background.The three surveys employed here were conducted in 2000–2002, after 30 years of steady immigration from non-Western countries. During the 10–12 years since then, there has been a further increase in immigration from non-Western countries, and the country background has been extended to Somalia, Iraq and others. Thus the research questions addressed here have not become less topical.The existence of large immigrant groups with a low frequency of alcohol drinking in Oslo contributed to a lower level of consumption at the population level. Social integration and own and host competence were associated with drinking, as were being a Muslim, years in school and gender. Results were specific to the particular country backgrounds, however. Based on the lasting low level of drinking among non-Western immigrants, the established associations between own and host culture competence and social interaction, and also theoretical effects of social interaction on drinking, we can expect to observe reduced drinking also among ethnic Norwegians in the future. This may imply reduced alcohol-related harm and disease.There are no competing interests. The study was financed by the Norwegian Institute for Alcohol and Drug Research.The sole author conceived the idea, carried out the analyses and wrote this manuscript.The author has worked with the topic of substance use among immigrants for several years and has published nationally and internationally plus reviewed articles on the subject for peer-reviewed journals.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/535/prepub