I provide a systematic review of trials of school-based smoking prevention programs that had at least 15 sessions, preferably with some in high school, that reported significant short-term effects, and that included long-term follow-up. This is supplemented with a description of some other programs that produce short-term effects that portend large long-term effects. I conclude that school-based programs can have long-term effects of practical importance it they: include 15 or more sessions over multiple years, including some in high school; use the social influence model and interactive delivery methods; include components on norms, commitment not to use, intentions not to use, and training and practice in the use of refusal and other life skills; and use peer leaders in some role. School-based programs of this type can reduce smoking onset by 25–30%, and school plus community programs can reduce smoking onset by 35–40% by the end of high school. Some early childhood programs that do not have smoking prevention as their main aim, including home nursing, the Good Behavior Game, the Positive Action program and others, seem to change the developmental trajectories of children so that they are less likely to engage in multiple problem behaviors, including smoking, as adolescents. This review makes it clear that effective school-based smoking prevention programs exist and can be adopted, adapted and deployed with success – and should be. Researchers and others have developed many school-based tobacco prevention programs over the past 30 years. Over a dozen reviews of approaches to tobacco control or substance abuse prevention published since the early 1990's have included school-based smoking prevention within their realm . Some ofDuring this same period there were many reviews -28 and mHowever, findings in the field are sometimes confusing to practitioners and policy makers because some early or short psychosocial programs reported promising short-term effects that did not last -40. In aIn an accompanying paper  I providMy objective is to determine which kinds of school-based prevention can be effective over the long term and what their costs might be. This required a focus on studies of programs that both were successful in reducing smoking in the short term and also included follow-up data into high school (grades 10–12) or beyond. The general protocol of locating studies was through multiple sources; searches of PsycINFO, MedINFO, the Google Scholar search engine , and word of mouth. The duration of that search was from January, 1970 through July 2008. Any article or report in the English language that included data regarding the evaluation of school-based smoking prevention programs was included. Only studies with a control condition were included.Since the purpose was to determine the size of the effects that could be obtained by the best programs that have been tested, the decision was made, based on the review of past reviews , to limiGiven the small number of Category I studies, evaluations of other programs with the promise of medium- and long-term effectiveness was also conducted. These Category II studies consist of school-based and school+ programs that had large effects that were of a large enough scope and sequence to suggest likely medium- and long-term effects. Ten studies of 8 programs met these criteria.All of the studies reviewed in these two sections are from developed countries . Table post, expressed as a percentage.I use percent relative improvement (RI) as the indicator of effect size in this review for two reasons. First, it is readily available for all programs, whereas the detailed statistics needed to calculate an effect size are sometimes incompletely reported. Second, RI is readily understood and utilized in cost and benefit calculations. For randomized trials, pretest levels of smoking should be the same in both program and control groups, and RI would be the difference between posttest control (C) and program (P) groups divided by the control group level . However, pretest levels were not always the same, and these should be adjusted for; thus, in cases where pretest data were reported, RI is the posttest difference between groups minus the pretest difference between groups, divided by the control group posttest level, that is, (%ρC – %ρP)/%COne may ask how to compare the effect sizes reported in meta-analyses and RIs. An approach commonly used by meta-analysts  is to trOn the assumption that investigators reporting only one outcome may have chosen to report the measure with the largest effect size, the estimates are likely to be biased upward.Another complication in determining effect sizes is that different studies reported different levels of smoking as their outcome variable. For both short- and long-term effects, the most commonly used outcomes were ever (lifetime) use, use in the past month, or use in the past week. When studies reported more than one of these, I report them all in the tables. An additional level of complication is that some researchers report outcomes only for the subset of their sample who were nonsmokers at baseline/pretest. I noted reports on only those students who were nonsmokers at baseline in the text but not the tables. While relatively few studies reported more than one outcome measure, when they did so, the RIs were remarkably consistent across outcomes (with some exceptions). To recap, Category I includes high-quality studies of programs that were both a) successful in reducing smoking in the short term and b) also reported significant follow-up effects into high school grades 10–12) or beyond. For each of these programs, Additional file 0–12 or bReviews of evaluations of three school-based programs are included in this sectionThe Tobacco and Alcohol Prevention Project, developed by Bill Hansen and colleagues at UCLA in the early 1980s  was a 15TAPP was evaluated in two cohorts of seventh grade classes in a nonrandomized study in Los Angeles County. Only cohort 1, conducted in two moderately sized school districts, was followed into grade 10. Health education and social studies teachers received 2 days of training prior to delivering the program. As shown in Additional file This was an early study of the social influences approach, and it demonstrated that the approach can be very effective. The use of peer leaders probably enhanced what program effects would have occurred with teacher-only delivery -53 The wLife Skills Training is one of the most researched school-based substance use (including smoking) prevention programs. Developed by Gil Botvin , originaMultiple studies over 25 years have demonstrated the effectiveness of the program when delivered by different providers, in different kinds of schools, and for different kinds of students  were not statistically significant. However, as shown in Additional file The program was evaluated in 22 schools with ethnically diverse populations in the San Diego, California area, some suburban and some rural. Schools were assigned randomly to program and control conditions after matching on pretest levels of tobacco use. Effects observed at the end of 8The pattern of effects observed suggest that much of the long-term effect was due to personal attention via newsletters and phone calls in grades 9 and 11. Indeed, one has to wonder if the personal attention set up a response bias among respondents such that those who received personalized newsletters and phone calls were motivated to tell the researchers what they wanted to hear; however, lack of a differential response rate to the surveys by condition speaks against this, at least in part. Considerable research suggests that the power of similar-age peers and the power of college-age counselors for high school students should not be underestimated. Although the cost of the intervention as studied was kept down by the use of volunteer students, it is not clear how easily this model can be disseminated. The results also strongly suggest, however, that even a brief intervention during high school was enough to actually increase the effect observed at the end of grade 9.Erkki Vartiainen and colleagues at the National Public Health Institute of Finland -71 testeThe results reported here can only be interpreted as the joint effects of the school-based smoking prevention program and the community-wide heart disease prevention campaign (which had a reduction of smoking as one of its targets). Thus, these results suggest effects that are larger than those of the school-based programs reviewed above. The larger effects obtained by regular teachers suggests that programs might be more effective when delivered by regular classroom teachers than when delivered by visitors to classrooms, possibly because of the ongoing relationships that teachers establish with students. However, at the long-term follow-up, teachers and visitors produced similar effects.This project, conducted by Cheryl Perry and colleagues , then atThe smoking prevention program was evaluated with a design in which students in all of the schools in one community received both the community-wide cardiovascular intervention and the school-based smoking prevention program, and students in all the schools in another community did not. All students in one cohort were surveyed every year from grade 6 to grade 12. As in all school-based studies, attrition occurred continuously over the 6 years and, by grade 12 only 45% of the original participants were surveyed. There were no differences in smoking rates at sixth grade. By the end of seventh grade, after the core smoking prevention content had been delivered, weekly smoking prevalence was about 40% lower in the program condition, and this effect was maintained through twelfth grade , developed by Mary Ann Pentz and colleagues at the University of Southern California, tested a school-plus-community (and mass media) version of the social influences approach in eight communities in the Kansas City metropolitan area. The school-based component consisted of 10 sessions delivered by classroom teachers to sixth or seventh grade students (depending on the year of transition to middle school) and 5 sessions delivered the following year . Of these schools, 8 were assigned randomly to conditions, 24 other schools elected to deliver the program and 18 others elected to wait till after the project. Mass media programming was available to all communities every year. Other community-based programming started in the third year and likewise was available in all communities.At the 2-year follow-up, the RI was 37.5% : an average of a 27.6% (range 18.7–44.1) relative reduction in smoking. The extraordinary effects of Project SHOUT may have been due to the added content on tobacco industry activities, the teaching and encouragement of advocacy skills, and the personal attention. These results need to be replicated. The long-term effects suggest that a minimal personal contact intervention of this kind in high school could increase the effects of any other program delivered in middle school. On the basis of these studies, I conclude that The school+ studies produced short-term RIs of about 40%, significantly higher than the school-only programs, consistent with my prior review . These eThe use of multiple delivery modalities increases effectiveness over those obtained from school-only programs . This isongoing school plus mass media or community programs can produce a long-term RI of between 35 and 40%% or an ES between 1 and 1.3.Thus, we conclude that This section provides a brief review of 10 studies of 8 programs that show exceptional promise or provide other important insights to help estimate the potential and likely relative reduction in smoking onset if prevention programs were widely implemented. These results are summarized in Additional file William Hansen and John Graham of the University of Southern California  tested tAlthough this program focused mostly on alcohol, it also produced effects for cigarette smoking. These effects were not too different in magnitude from those reported earlier from TAPP , although, as might be expected because the program was not focused on smoking, these effects were not maintained as well.Steve Sussman and colleagues, also at the University of Southern California -96, deveInvestigators at the American Health Foundation developed the Know Your Body program in the early 1980s as a comprehensive health education program that included social influences and competence prevention components. It consisted of 384 lessons delivered during grades 4 through 9. In a randomized trial in two New York State communities, Walter and colleagues -102 founIn a second randomized trial with Washington DC African-American students, Bush and colleagues ,104 obtaThese are exceptionally large effects. Without long-term follow-up data we cannot be sure how well they would have been maintained, but these studies show that strong prevention effects can be obtained by comprehensive health education programs that also include proven approaches to prevention.Shepard Kellam and colleagues at The Johns Hopkins University  applied A second trial tested the effects of only one year of the GBG in grade 1 on smoking behavior 6 years later . At the These studies demonstrate that important changes in life course trajectories of behavior brought about early in life can lead to important changes in adolescent behavior, including smoking. Other school-based programs that improve elementary school children's behavior also have this kind of potential, for example, Fast Track , the SeaThe Positive Action (PA) program was developed by Carol Gerber Allred, a teacher and administrator, beginning in 1977 and expanded and revised multiple times since then based on process evaluation data and user feedback. It is a comprehensive character education program designed to improve character, pro-social and other positive behaviors and school performance as well as negative  behaviors, including smoking. The program consists of developmentally-appropriate, 15-minute lessons, delivered by trained teachers on up to four days per week for grades K-12. In addition, the school principal and a PA coordinator/committee implement school-wide climate change activities to encourage and support positive behaviors by students. School counselors can provide additional lessons to high-risk students and parents can conduct weekly lessons with their children that parallel the school lessons. A community involvement component is also available. The program makes students (and their teachers and parents) aware of how they feel good about themselves when they do positive actions, how that good feeling can lead to further positive thoughts and actions, and that there is always a positive way to do everything. Students are taught what actions are positive in the physical , intellectual , emotional  and social  areas of their lives. Students learn the skills they need to perform all of these behaviors through lessons, discussion, role-play and practice. The program is very interactive ,116; intThree quasi-experimental evaluations used archival school-level data from Hawaii, Nevada and a large south-eastern school district in matched controlled designs ,118 to eth graders were 47% and 29% less likely to have started smoking than 5th graders in control schools in Hawaii and Chicago, respectively [Two randomized trials of PA have recently been completed, one in 10 matched pairs of Hawaii elementary schools and one in 7 matched pairs of Chicago elementary schools. After 3 years of PA, 5ectively ,120 , but with large variation (12–50% for short term and 14–81% for medium term). The medium-term effects of these programs were larger than the long-term effects of Category I programs. Given that Category I programs actually had increased effects over time, these results suggest that long-term effects considerably higher than those derived from Category I programs may be possible with more comprehensive or newer school-based programs.The results of the Good Behavior Game and Positive Action programs are particularly intriguing because they demonstrate the power of changing the trajectories of behavior early in life. A prevention program provided to these students in middle and high school might have much larger medium- and long-term effects on smoking and other health-related behaviors – but no evaluations of such combinations are available to date.This review suggests that interactive social influences smoking prevention programs that provide 15 or more lessons, start in upper elementary or middle school, and continue into high school can produce solid long-term effects. Other conditions that appear to improve the effectiveness of school-only programs relate to content , how well they are delivered (related to how well teachers are motivated and trained), and the involvement of older peers. The findings are consistent with the 13 components of effective programs for elaborated by Nan Tobler as a result of her multiple meta-analyses .Results from three social influence and social competence programs with 15 or more sessions over 2–4 years, preferably with some content in high school, had significant short-term effects of about 22% RI in monthly or weekly smoking that increased during high school in two of the studies to an estimated average of 28% RI. Some other programs (Category II) provided further evidence that (1) the social influence approach can affect tobacco use even when alcohol use was the main focus, (2) comprehensive health education programs that include strong social influence content can be effective, possibly even more effective than stand-alone social influence programs, and (3) programs early in life can alter developmental pathways for the better, including less tobacco use in adolescence.the possible long-term (end of high school or age 18) effects of a national program of well-implemented, school-based smoking prevention programs of proven effectiveness could be as high as 25–30%.Based on an average of the long-term effects of Category I studies, and supported by the estimated medium-term effects of Category II studies, I estimate that the possible long-term effects of a national program of well-implemented school- plus-community and/or mass media smoking prevention programs of proven effectiveness could be as high as 35–40%.The four Category I school-plus-community studies produced short-term RIs of about 40%, decaying to long-term effects of about 31%. As noted earlier, program effects were maintained at a higher level  for those programs that included a high school component (North Karelia and Minnesota Class of 89). Thus, Program effects are likely to decay beyond high school. Unfortunately, few studies are available to guide us in how large or small this decay might be. National U.S. data may allow for an estimate, though it is unclear how applicable it will be in other countries. The U.S. National Household survey on Drug Abuse data suggest that about 3.012%  of 18 year-olds who are not smoking daily become daily smokers by the time they are 25 [y are 25 . The MonThere are at least two other factors that could reduce the effects of even the best programs in real-world implementations (1) rate of adoption by schools and communities, and (2) level and quality of implementation or delivery.Clearly, less-than-complete implementation could dramatically reduce the expected effect size of any national effort.Active adoption of a program does not necessarily translate into high implementation of it. For example, among Oregon schools who obtained funding to implement smoking prevention in 2001 (from Master Settlement funds), 37% implemented at a very low level and produced less than 1% RI in student smoking when compared with control schools (who did not obtain smoking prevention funding); another 39% implemented at a medium level, producing an 11.5% RI; and 24% implemented at a high level, producing a 48% RI in student smoking . These fExperience in the USA suggests that getting effective prevention programs adopted (and used) by schools is not easy ,126,127.In actuality, not even all schools entered into studies always carry through with their willingness to implement the program. For example, we noted above how 6 program schools were dropped from the LST analysis because of lack of implementation . In anotA clear mandate to include tobacco prevention in the curriculum together with earmarked funding and monitoring of adoption could help obtain rates of adoption of evidence-based school-based programs of up to 75% or more.In these days of high demands on schools, they are unlikely to address prevention unless they have to, or unless it can be shown to also improve achievement, and they are unlikely to adopt a program unless they have the funding for it. Adoption probably would not be 100% even with a clear mandate and earmarked funding, although it might increase over time, following the standard S-shaped adoption curve as successes are publicized . A clearGetting comprehensive programs implemented fully and with integrity, even when they are adopted with full information and commitments, also is no small task, and the level and quality of implementation are clearly related to program effectiveness . FactorsFor some programs with high levels of monitoring, levels of implementation might be high. For example, the Conduct Problems Prevention Research Group  reportedWithout ongoing monitoring, implementation might be much more uneven. Uneven implementation of a national program could reduce the effect size substantially – but by how much? The effect sizes reported for LST already took incomplete implementation into account. The authors reported that about 76% of the students received 60% or more of the program from trained teachers in schools who had signed onto the study . The 20%under conditions of ongoing monitoring or evaluation a high level of implementation (60% or more) could be achieved.The tobacco industry has sponsored adoption, implementation, and evaluation of LST ,133  campaigns.The final objective of this review is to estimate the costs and cost-effectiveness of school-based smoking prevention. It is difficult to estimate the costs and the value of benefits of successful prevention programs and, therefore the benefit/cost ratio ,135. TheOne analysis estimated the cost of an effective 30-session prevention program in the U.S. at $US150 per student for program materials, training, teacher time and other costs . HoweverThe cost of an effective smoking prevention program in Canada has been estimated at $CAN67 (1996 dollars) . AssuminScholars disagree on whether lives saved actually save money or add costs for society. According to Hodgson , smokerssmoking prevention is more efficient than most health/medical interventions.Another group estimated the costs of Project TNT at $US48 per student and that TNT would cost about $US20,000 per quality-adjusted life-year (QALY) gained . While nThe social benefits of even broader behavior improvement programs could be considerably greater. Steve Aos and his colleagues at the Washington State Institute for Public Policy conducted an analysis of the cost-effectiveness of about 70 different prevention programs . They esClearly, from a societal perspective, the costs of effective prevention are well worth it both to the individual students and to society as a whole, at least in developed countries. In developed countries, cost-benefit estimates ranged from 2 to over 100. It is difficult to determine the likely cost-benefit in developing countries, but the high costs of program delivery in developed countries combined with the high social and health costs of not preventing tobacco use in developing countries, suggest that prevention would also be cost-effective in many developing countries.There are at least 10 limitations to the studies that met the criteria for this review.The most appropriate design for evaluating school-based prevention programs is the school-based randomized trial, where schools are assigned to conditions and data are analyzed taking into account the nesting of students in schools [1. Many prevention studies – including some of those reviewed here – did not use randomization, but instead used matched controls or other designs. Some so-called quasi-experimental designs  may be a schools -148.Replication studies, especially in different cultural groups and countries, are urgently needed.2. Although more than one program has reported significant long-term effects, none of the individual programs has more than two evaluations of long-term effects. Thus, although we can conclude that comprehensive, interactive programs, using the social influences approach, with 15 or more sessions, including in high school, can have long-term effects, we do not yet know whether the long-term effects of any one of the programs meeting these criteria can be replicated. it would be wise to have some studies with different ethnic groups and in developing countries use biochemical verification of self reports of smoking with high school students and young adults.3. There is a reliance on self-report measures of tobacco use. For many years, the validity of self-reports of sensitive behaviors was questioned. After a series of studies of the use of biochemical validation or the collection of biochemical samples for use in a "bogus pipeline" procedure -151. populations are promising. We need many more truly long-term studies of the ongoing effects of smoking prevention programs, preferably up to age 25.5. The last time of data collection in most of these studies was while youth were still in high schools. mediating variables need to be measured in future research, and their mediating effects on tobacco use behavior demonstrated.6. There is great variability in the way researchers and evaluators assess outcomes. Researchers have used ever smoking, smoking in the past month or week, and other indicators of youth smoking. Some researchers included only baseline never smokers in their analyses. Fortunately, there was reasonable consistency in estimates of prevention effectiveness across measures in most of the reviewed studies. Nevertheless, it would help future reviewers if researchers could settle on consistent measures or report results for multiple measures. In addition, however, future research needs to include assessment of multiple short-term effects (or mediating variables) in addition to tobacco use. For example, programs are designed to improve knowledge of the influences on behavior (including tobacco industry promotions); knowledge of the physical, economic, environmental and social consequences of tobacco use; perceptions of risk; normative estimates or beliefs; decision-making, peer pressure resistance, and coping skills; and possibly student's activism against smoking in their environment. All of these try to find which kinds of programs produce significant effects, or the largest effects (as well as for which kinds of people and under what conditions), as Tobler et al. [7. There was large variation across studies in program content, which affects the validity of some prior reviews of this literature. Conducting meta-analyses of these studies seems like comparing apples with oranges, or even with yams . The variation makes it difficult to compare programs. In other disciplines, one would not conduct a meta-analysis or review of such different kinds of programs and draw a conclusion for all programs as a group. One would not, for example, conduct a meta-analysis of all treatments for breast cancer and conclude that breast cancer treatment does not work. Rather, one would attempt to determine which kinds of treatments work the best (and for whom and under what conditions), and then adopt the best treatment as the standard of practice. Unfortunately, some meta-analysts of various smoking prevention programs have treated them as a homogeneous group and concluded that they are not very effective, and that they do not have medium- or long-term effects. It would be more appropriate to r et al. , Hwang er et al. , and thievaluations of general behavior-improvement and youth development programs will contribute to the knowledge base of smoking prevention as well as youth development.8. We still lack consistency and continuity across developmental stages (preschool through college), and this clearly is an area where continued research is desirable. At the preschool and elementary school levels, implementation of more general and promising approaches such as the Good Behavior Game or the Positive Action program should be used to prepare students to adopt tobacco-free lifestyles. Increasing evidence suggests that behavior improvement or positive youth development programs can have pervasive effects on behavior, including reducing tobacco use, and also can improve school performance. However, the lack of replicated findings regarding specific effects on tobacco use to date suggests that they should be accompanied by rigorous evaluations. Such The field is urgently in need of independent replications of the findings summarized in this paper [9. Program developers were involved in all of the evaluations reported. It is quite probable that the effect sizes reported when program developers are involved were larger that those that will be obtained under other conditions. is paper .Much more research on school-based smoking prevention in developing countries is urgently needed.10. There is a relative dearth of rigorous research on school-based smoking prevention in developing countries. However, several countries are making progress with the help of researchers from more developed countries ,156-168.Despite, or maybe because of, the above limitations, there are multiple reasons to suspect that estimates of effect sizes derived from the small number of studies reviewed here (RI of 25–30% for school-only programs or 35–40% for those that add on-going community activities or mass media campaigns) might be conservative (under) estimates of effects that might be observed under ideal conditions. First, some of the effect sizes were derived from studies that already included less than optimal implementation. Second, if a program was implemented nationwide, for multiple years, there might be increasing effects for a while as new generations of students passed through the program. For example, as fewer young adults become smokers, there will be less social support for smoking and fewer adolescents will be tempted to try smoking. Third, the possibility of larger effect sizes were suggested by the larger short-term effects of the Towards No Tobacco and Know Your Body projects, the promising effects of general behavior improvement programs such as the Good Behavior Game and the Positive Action program, and the extraordinarily large effects of Project SHOUT with minimal high school boosters.The current research demonstrates a phenomenon of decreasing effect sizes over time . They should evaluate currently-funded programs to determine if benefits occur and if they exceed costs. They should develop the specialized knowledge needed to keep abreast of the latest research from around the world.It is time for the world to face up to the fact that preventing as many children and youth as possible from starting to smoke cigarettes is feasible and worthwhile, both economically and in terms of improved health of the population.The author is married to the developer and owner of the Positive Action program, one of the programs discussed in this paper.Table S1. Short and Long-term Effects of Seven Selected Social Influence Programs with Follow-up Into High School.Click here for fileTable S2. Other Programs of Interest.Click here for file


A comparative genomic and proteomic study of halophilic and non-halophilic prokaryotes identifies specific genomic and proteomic features typical of halophilic species that are independent from genomic GC-content and taxonomic position. Halophilic prokaryotes are adapted to thrive in extreme conditions of salinity. Identification and analysis of distinct macromolecular characteristics of halophiles provide insight into the factors responsible for their adaptation to high-salt environments. The current report presents an extensive and systematic comparative analysis of genome and proteome composition of halophilic and non-halophilic microorganisms, with a view to identify such macromolecular signatures of haloadaptation.Comparative analysis of the genomes and proteomes of halophiles and non-halophiles reveals some common trends in halophiles that transcend the boundary of phylogenetic relationship and the genomic GC-content of the species. At the protein level, halophilic species are characterized by low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure. At the DNA level, the dinucleotide abundance profiles of halophilic genomes bear some common characteristics, which are quite distinct from those of non-halophiles, and hence may be regarded as specific genomic signatures for salt-adaptation. The synonymous codon usage in halophiles also exhibits similar patterns regardless of their long-term evolutionary history.The generality of molecular signatures for environmental adaptation of extreme salt-loving organisms, demonstrated in the present study, advocates the convergent evolution of halophilic species towards specific genome and amino acid composition, irrespective of their varying GC-bias and widely disparate taxonomic positions. The adapted features of halophiles seem to be related to physical principles governing DNA and protein stability, in response to the extreme environmental conditions under which they thrive. Halophiles are organisms adapted to thrive in extreme conditions of salinity. There is a wide range of halophilic microorganisms belonging to the domains Archaea and Bacteria. The intra-cellular machinery of these prokaryotes has evolved to function at very high salt concentrations -5. A detThe stable and unique native structure of a protein is a basic requirement for its proper functioning -11. To uHaloquadratum walsbyi is so far the only exception, with a remarkably low genomic GC-content of 47.9% [Halobacterium sp. NRC1 [H. walsbyi [Several studies have suggested that high genomic GC-content (well above 60%) is also a common feature of extreme halophiles, presumably to avoid UV induced thymidine dimer formation and possible accumulation of mutations ,19. The of 47.9% . At the sp. NRC1 , but not walsbyi . Thus, aThe current report presents an extensive and systematic analysis of the genome and proteome composition of halophilic organisms, along with a comparative study of non-halophiles, with a view to characterize the molecular signatures of halophilic adaptation. We consider 6 completely sequenced obligatory halophiles and compare them with 24 non-halophiles from various phyla of both Archaea and Bacteria with comparable GC-content to minimize the phylogenetic influence and the effect of mutational bias on their nucleotide/amino acid usage patterns. We examine the preferences, if any, in amino acid replacements from non-halophile to halophile orthologs in an attempt to understand which residues are instrumental for halophilic adaptation. Finally, we show how observed patterns of change in amino acid compositions in response to extreme conditions of the environment are related to physical principles that govern stability of proteins under such conditions. This study examines in detail the genome and proteome-wide adaptations to extreme environments, knowledge of which has important potential applications in various fields, including the engineering of industrial biomolecules.Escherichia coli, while the right panel offers a pictorial representation of relative amino acid usage in the respective organisms. As the relative abundances of the residues increase from 0.35 to 1.80, the color of the respective block changes from red to green, that is, the greener the color, the more abundant is the residue in that organism compared to E. coli. Halophilic organisms show quite distinct usage of amino acid residues compared to non-halophiles, elucidated by the presence of either more red or green blocks in Figure H. walsbyi, probably due to its significantly lower genomic GC-content  of axis 1 with the GC-content of the respective genomes identifies GC-bias as one of the major sources of inter-species variation in global amino acid composition, while the contributions to axis 2 come from hydrophobicity  and the ratio of negatively versus positively charged amino acid residues  of the encoded gene products of the organisms. This indicates, therefore, that the proteins of halophilic organisms are characterized by less hydrophobicity (or higher hydrophilicity) and relatively higher usage of negatively charged amino acids compared to non-halophile proteins. Figure Similar to the cluster analysis, correspondence analysis (COA) on amino acid usage also segregates the halophilic organisms from the non-halophiles along the second principal axis Figure . The firMethanosaeta thermophila, Thermoplasma acidophilum and so on , appears in the same cluster along with the GC-rich halophiles, while the three non-halophilic species with similar GC-content and  cluster with the other non-halophiles, most of which are characterized by much higher GC-content. It is worth mentioning at this point that organisms with high growth temperature also cluster together  share the same node. The distinct branching pattern of three thermophiles with relatively low genomic GC-content  suggests that the overall GC-content also plays a significant role in shaping the amino acid composition of such organisms, as observed previously by Kreil and Ouzounis [All these trends are specifically exhibited by halophiles irrespective of their taxonomic origins and their genomic GC-content  between halophilic and non-halophilic organisms was performed to identify the underlying factors for halophilic adaptation in more detail. Table p < 10-2 for set II; p < 10-3 for set I; and p < 10-6 for sets III and IV). They contribute 56%, 52%, 66% and 63% of the replacements for set I , set II , set III  and set IV , respectively . The top 20 replacements in all these sets suggest that there are two clear trends in amino acid substitution patterns in terms of highest gain as well as highest ratio /2 = 190 possible pairs of replacements) between the orthologous sequences in the direction from non-halophile to halophile proteins . There are 59 , 51 , 81  and 76  pairs of amino acids for sets I, II, III and IV, respectively, that have significant directional replacement bias  compared to C. vibrioforme MDH (48.5% decrease). The comparison of aligned sequences of secondary structure regions using the DSSP program also lends supports to this notion , the cumulative frequency of Asp and Glu is 20.5%, whereas in C. vibrioforme MDH  it is 12.9%.One pair of crystal structures of the protein malate dehydrogenase (MDH) from halophilic The distinct amino acid usage pattern in halophiles might have originated from compositional bias operating at the nucleotide level, or from the preference for, or avoidance of, specific amino acid residues as a tool for halophilic adaptation. With a view to distinguish between these two possibilities, we randomly re-shuffled the nucleotides in the coding sequences of all genomes and calculated the average amino acid composition of the hypothetical protein sequences of halophiles and non-halophiles obtained from the theoretical translation of the reshuffled gene sequences. If the selection had operated at the mono-nucleotide level, proteins translated from such randomly reshuffled hypothetical sequences of halophiles should feature similar trends as depicted by their true proteomes, since the nucleotide bias of the reshuffled sequences would have remained the same as those of the real gene sequences. On the contrary, if the distinct amino acid composition of halophile proteomes had evolved due to environmental adaptation of these extremophiles, the trends in amino acid usage in reshuffled hypothetical sequences would differ from those of actual halophilic proteins. In Figure Thermoplasma acidophilum. However, on the basis of dinucleotide frequencies at the first and second codon positions of genes, these organisms cluster together irrespective of any phylogenetic relationship. In order to figure out the possible impact of the relative abundance of specific dinucleotides on the mechanical properties of halophilic genomes, we calculated the likelihood of their sequences forming a Z-DNA structure, using ZHunt software [2 = 0.54, p < 10-4) between the propensity of DNA to flip from the B-form to the Z-form per kilobase of genome and the relative abundance of the CG dinucleotide.We calculated the dinucleotide abundance of all genomes to find out whether any specific nucleotide composition has significant influence on the genomic signature of obligatory halophiles. Clustering on dinucleotide abundance by city-block (Manhattan) distance clearly segregates the halophilic organisms  from the non-halophiles Figure . The higsoftware . We foun3 values , indicating separation of genomes according to their genomic GC-content.In an attempt to examine whether the pattern of synonymous codon usage in halophiles follows any specific signature, COA was performed on the relative synonymous codon usage (RSCU) of 82,927 predicted open reading frames (ORFs) from 30 microbial genomes  of the COA of RSCU, the variation along the third major axis  separates the halophiles from the non-halophiles. The distribution of codons along axis 3 Figure  depicts The present study discerns the nucleotide and amino acid biases in extreme halophiles and thereby characterizes the genomic/proteomic determinants of halophilic adaptation in prokaryotes. From this study, it appears that specific trends in amino acid usage are required for halophilic adaptation of organisms, irrespective of their genomic GC-content and taxonomic position. Evidence in favor of specific selection on dinucleotide and synonymous codon usage are apparent for halophiles Figures  and 7a. M. thermophila from Methanogens group II and T. acidophilum from Thermoplasmatales - organisms very close to haloarchaea as per the 16s rRNA tree . Among bacterial non-halophiles, we chose members from different bacterial phyla, such as proteobacteria, firmicutes, cyanobacteria, actinobacteria and especially P. luteolum from the bacteroidetes/chlorobi group to which the halophilic bacteria S. ruber belongs . It can be concluded, therefore, that the determinants of genomic/proteomic architecture in halophilic organisms are high salt adaptation specific, and transcend the boundary of phylogenetic relationships and the genomic GC-content of the species.In order to subtract out the phylogenetic influence, we have included both bacterial and archaeal organisms in the dataset Table . The datH. marismortui in our analysis and found that the amino acid usage, dinucleotide relative abundance and synonymous codon usage of chromosome II are quite different from those of chromosome I . A high GC-content in halophilic genomes is thought to help in avoiding UV-induced thymidine dimer formation and the possible accumulation of mutations in their specialized habitat , characterized by high levels of UV irradiation [H. walsbyi, the disadvantage of a low GC-genome is thought to be partly compensated for by the presence of a relatively higher number (four copies) of photolyases [2 = 0.54, p < 10-4) between the propensity of Z-DNA formation per kilobase in genomes with a relatively high abundance of CG dinucleotides supports this notion. We also observed that the pattern of synonymous codon usage in halophiles is significantly different from that in non-halophiles , except for adiation ,19. In Htolyases . Our anatolyases . It is atolyases . Hence, tolyases . A signiThe present study demonstrates the generality of the mechanisms of macromolecular adaptation of extreme salt-loving organisms, irrespective of their genomic GC-content and taxonomic position. At the protein level, these include: convergent evolution towards a specific proteome composition, characterized by low hydrophobicity; over-representation of acidic residues, especially Asp; higher usage of Val and Thr; lower usage of Cys; and a lower propensity for helix formation and a higher propensity for coil structure. Among the signatures of halophilic adaptation at the DNA level, the abundance of GA, AC and GT dinucleotides may partly be coupled with the specific amino acid requirements, while CG dinucleotide abundance may be an additional halophilic signature of DNA stability at high salt concentration. The synonymous codon usage in halophiles also seems to have converged to a single pattern regardless of their long-term evolutionary history.H. walsbyi, all the extreme halophilic organisms are GC-rich, so to minimize the GC-compositional effect on amino acid usage comparison (as well as on codon usage), most of the chosen non-halophilic organisms are similarly GC-rich, while some others have GC-content comparable to that of H. walsbyi.All protein coding sequences of the chromosomes of 6 extreme halophiles  and 24 non-halophiles from Archaea (both euryarchaeota and crenarchaeota) and bacteria  were retrieved from NCBI GenBank version 145.0) and Halolex databases   and standardized amino acid usage values, respectively. The over-representation or under-representation of standardized amino acid usage values of the organisms in the matrix are shown in green or red colored blocks in Figure To find the differences in amino acid usage between extreme halophilic and non-halophilic organisms, the cluster analysis on amino acid composition was carried out using STATISTICA  for all 30 organisms Table . The amiCOA on amino acid usage was performed using the program CODONW 1.4.2  to identIn order to identify any halophile-specific genome signature, dinucleotide abundance values ,43 of ge-10. Hits less than 60% similar and having more than 20% difference in length with the query were removed from the dataset. Putative membrane proteins and proteins likely to be secreted or localized to the cell surface, predicted using TMHMM2.0 [S. ruber and the non-halophile P. luteolum - both belonging to the phylum bacteroidetes/chlorobi. Set II contained 104 orthologous sequences from two species with similar GC-content (Table H. marismortui (Ch-I) and non-halophilic bacteria Pseudomonas putida. Set III contained 584 orthologous proteins from a halophilic and a thermophilic archaeon, namely H. marismortui (Ch-I) and M. thermophila. Set IV incorporated 574 orthologous proteins of the halophilic archaeon Natronomonas pharaonis and uncultured methanogenic archaeon RC -I.Four sets of orthologous sequences between halophiles and non-halophiles were identified  using the BlastP program  using a TMHMM2.0  and SignTMHMM2.0 , were alThe amino acid sequences of these four sets of orthologous genes were aligned using ClustalW  and the -3 to 10-6. For each pair of replacements, the first and second rows of the 2 × 2 contingency table represented the number of replacements from one particular residue  to another  of the pair and the total count of the remaining replacements  from the residue i (where k ≠ j), respectively.While examining the trends in amino acid or secondary structure replacements, the direction of conversion of non-halophile proteins to extreme halophile proteins were taken by convention as the 'forward' direction. Under unbiased conditions, the ratio of forward to reverse replacements was expected to be 1:1 for each pair of replacements. To test this hypothesis, the observed and expected numbers (based on a 1:1 ratio) were recorded for each pair of residues belonging to a particular group. In all cases, the chi-square test was applied to assess the significance of the directional bias, if any, at significance levels of 10Indices like RSCU , GC-contp-value 1e-38) MDHs from H. marismortui (1D3A) [C. vibrioforme (1GV1) [We obtained one pair of protein structures for extreme halophilic and non-halophilic organisms from the Protein Data Bank. The pair contains (Blast i (1D3A)  and C. ve (1GV1) . The sece (1GV1)  and DSSPe (1GV1) .COA, correspondence analysis; MDH, malate dehydrogenase; ORF, open reading frame; RSCU, relative synonymous codon usage.SP and SKB made substantial contributions to the conception of the study, devised the overall strategy, performed genome sequence analysis and drafted the manuscript, developed relevant programs for sequence analysis and performed sequence alignment. SD participated in the initial work, development of the work plan and manuscript preparation. ETH made thoughtful and constructive suggestions during preparation of the manuscript. CD participated in the design and coordination of the study and revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.P. luteolum and S. ruber orthologs. Additional data file P. putida and halophilic H. marismortui chromosome I orthologs. Additional data file M. thermophila and halophilic H. marismortui chromosome I orthologs. Additional data file Methanogenic archaeon and halophilic N. pharaonis orthologs. Additional data file P. luteolum to halophilic S. ruber orthologs. Additional data file P. putida to halophilic H. marismortui chromosome I orthologs. Additional data file M. thermophila to halophilic H. marismortui chromosome I orthologs. Additional data file M. archaeon to halophilic N. pharaonis orthologs. Additional data file The following additional data are available with the online version of this paper. Additional data file Phylogenetic relationship with 16s rRNA using the neighbor joining method with Tamura-Nei model.Click here for fileP. luteolum and halophilic S. ruber orthologs.Trends in amino acid replacements in non-halophilic Click here for fileP. putida and halophilic H. marismortui chromosome I orthologs.Trends in amino acid replacements in non-halophilic Click here for fileM. thermophila and halophilic H. marismortui chromosome I orthologs.Trends in amino acid replacements in non-halophilic Click here for fileM. archaeon and halophilic N. pharaonis orthologs.Trends in amino acid replacements in non-halophilic Click here for fileP. luteolum to halophilic S. ruber orthologs.Number of amino acid replacements from non-halophilic Click here for fileP. putida to halophilic H. marismortui chromosome I orthologs.Number of amino acid replacements from non-halophilic Click here for fileM. thermophila to halophilic H. marismortui chromosome I orthologs.Number of amino acid replacements from non-halophilic Click here for fileM. archaeon to halophilic N. pharaonis orthologs.Number of amino acid replacements from non-halophilic Click here for fileDinucleotide relative abundance for all the organisms under study.Click here for fileDinucleotide frequency at the first and second codon positions for all the organisms under study.Click here for fileRSCU of genes at the positive and negative extremes  of axis 3 of COA on RSCU.Click here for file


The effect of visual arts interventions on development of empathy has not been quantitatively investigated. A study was conducted on the effect of a visual arts-based program on the scores of the Jefferson Scale for Physician Empathy (JSPE).A total of 110 clerks (n = 92) and first-year postgraduate residents (PGY1s) (n = 18) participating in the program were recruited into this study. The 4-hr program covered the subjects of learning to interpret paintings, interpreting paintings relating to medicine, illness and human suffering, the related-topics of humanitarianism and the other humanities fields and values and meaning. The JSPE was completed at the beginning (pretest) and the end (posttest) of the program.p = 0.0358). An increased but not significantly mean posttest JESPE score was noted for the subgroup of PGY1s. Neither the females nor the males had higher posttest JSPE scores than the pretest scores.There was no significant difference between the pretest and posttest JSPE scores. The average of the scores for the pretest was lower in the subgroup of PGY1s than the subgroup of clerks (Although using a structured visual arts-based program as an intervention may be useful to enhance medical students’ empathy, our results failed to show a positive effect on the JSPE Scores for a group of clerks and PGY1s. This suggests that further experimental studies are needed if quantitative evaluation of the effectiveness of visual-arts based programs on empathy is to be investigated. The role of the humanities including visual arts, literature and many other fields has been highlighted in many publications,2. A litJefferson Scale of Physician Empathy (JSPE) is a validated instrument designed to measure empathy specific to health professionals (HP version) and medical students (S version)[The version). In a crversion), althougversion). It is iOur hospital  is a 1,600-bed medical center and is one of the main teaching hospitals in central Taiwan. In this country, there are 7-year (applicants are high school graduates) and 5-year graduate medical programs. Some programs integrate Chinese medicine courses. After 2–4 years of pre-medical and basic sciences, medical students require 2 years of clerkship and 1 year of internship before graduation. Recruitment of participants in this study was through convenience sampling. From 2010 October to 2011 January, all of 98 medical students in clerkship and 22 PGY1s at our hospital participated in a required visual arts-based program. Among them 113 (95 clerks and 18 PGY1s) were recruited into this study. The participants were divided into 12 discussion small groups so that there were less than 12 persons for each group. Those already in the same block rotations were allocated into one small group when possible. The complexity of different rotations and duty loads of clinical care made some participants have to shift to join one of the pretest or posttest sessions in the other groups. There were 3 of the recruited unable to complete the 2-h sessions within 1 week as planned. The data were excluded from analysis. To this end, there was a total of 110 (92 clerks and 18 PGY1s) recruited participants in 10 groups, that completed the study. Ninety of the 92 clerks were the year 6 students of 7-year medical programs. Only 2 of 92 were the year 4 students of 5-year graduate medical programs. All PGY1s were graduates of 7-year program. Data from these 110 students were used for analysis of the effect of the program on the JSPE scores.We implemented a visual arts-based program as an intervention. The program was initially initiated with the goals of giving medical students entering their clinical training in our hospital an appreciation of paintings, helping them use visual arts as a tool to develop competencies as physicians and interesting them in visual arts. It was As the JSPE was developed to specifically measure the empathy in medical professionals and students, the JSPE (student version) was used in this study. In the questionnaire, there were twenty items to be answered with a seven-point Likert scale (1 = strongly agreed to 7 = strongly disagreed). Among them, ten were negatively worded items, which were to be reversely scored. A higher score indicates a higher level of empathy. A sample of positively worded question is: “I believe that empathy is an important therapeutic factor in medical treatment” and a sample of negatively worded question is “It is difficult for a physician to view things from patients’ perspectives.”The JSPE has been translated into about 25 languages. The version used in this study was translated into Chinese and then back-translated into English by two bilingual translators to ensure the accuracy of translation. The psychometric characteristics of the translated version needed to be defined. Data collected from the study were subjected to exploratory factor analysis (maximum likelihood) with Varimax rotation. In order to expand the volume of the data, those JSPE scores collected at the beginning of the first session of the studies from all of 113 recruited were analyzed. Three factors retained using Velicer’s minimum average partial criteria. The firIn this study, the 4-hr program was divided into two 2-hr sessions (each one week apart). Both recruited and not recruited might attend the same group. The (pretest) JSPE with another anonymous questionnaire for program assessment were distributed at the beginning of the first session. No limitation was for the time to complete the JSPE. At the end of the program, the (posttest) JSPE were distributed again with another anonymous questionnaire for post-lecture program assessment. Students recruited were asked to complete both while not recruited to complete at least the program assessment. Those not recruited were informed that they may complete the JSPE but their scores would not be included in the analysis. The pretest and posttest JSPE scores collected were used for analysis.The hospital’s Educational Board approved the program and the Institutional Review Board of the hospital approved the study.Paired t-test was used to test the significant difference of the JSPE scores between the pretest and posttest. Unpaired t-test was used to test the significant difference of the JSPE scores between the subgroups of PGY1s and clerks.p = 0.0358) with a Cohen’s d = 0.553. Significant difference was not noted for the posttest scores between the subgroups of PGY1s and clerks. In terms of gender, either the females or the males did not have a higher posttest JSPE scores than the pretest. No difference between the pretest and posttest for the females or males in the subgroup of clerks, respectively. Neither was it for the subgroup of or PGY1s.One hundred and ten participants  completed both sessions of the program. The distribution of the participants and the results of the pretest and posttest JSPE scores are shown in Table Empathy is a cognitive attribute that involves an understanding of the inner experiences and perspectives of the patient, combined with a capability to communicate this understanding to the patient. It is eFemale medical students were reported to have higher JSPE scores than male in a previous study. It was To our knowledge, no previous research has been reported using a validated measurement tool to quantitatively evaluate the effect of a visual arts program on empathy. Whether enhancement of empathy score using such interventions is possible for the medical students and residents and more remarkable for one training level than the others and for females than males is interesting to know. Further experimental studies with larger sample size are needed to clarify this variance. Intervention of such a program with extension of time or increase of the frequency of interventions may also be worthy of studying.Although using a structured visual arts-based program as an intervention may be useful to enhance medical students’ empathy, our results failed to show a positive effect on the JSPE Scores for a group of clerks and PGY1s. This suggests that further experimental studies are needed if quantitative evaluation of the effectiveness of visual-arts based programs on empathy is to be investigated.The author declares that they have no financial or non-financial competing interests.The author contributed to the conception, design and implementation of the program and drafted the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/13/142/prepub


This paper aims to investigate the effectiveness of the Tier 1 Program of the Project P.A.T.H.S.  based on eight datasets collected between 2005 and 2009. A total of 1,138 students who participated in the program were randomly invited (from the whole grade or in some classes) to write a piece of journal in the form of a weekly diary in order to reveal their perceptions and feelings regarding the program and the perceived benefits of the program. Based on an integration of findings from different databases, results showed that the respondents generally (1) had positive views on the program, (2) had positive views on the instructors, and (3) perceived that they had acquired competencies at the societal, school, familial, interpersonal, and personal levels after joining the program. Acknowledging the limitations of diaries, the present qualitative findings provide support for the effectiveness of the Tier 1 Program of the Project P.A.T.H.S. in Hong Kong. The purpose of this paper is to report evaluation findings of a positive youth development program in Hong Kong  based on weekly diaries written by students participating in the program. The personal diary is an important source of information to understand the social reality. For example, Anne Frank's diary documents the thoughts, feelings, and experiences of a Jewish teenage girl who had fled and hid in Amsterdam with her family when Nazi forces conquered The Netherlands. In view of the invaluable information gathered from people's diaries, diaries for analyzing human behavior, discovering history, or evaluating a program represent an attractive research approach. In the context of social sciences research, diaries are commonly used in nonexperimental research to collect data on human behavior. For example, Leigh  asked paIn the field of education, it is not uncommon to find that a teacher would keep reflective journals as a means to assess personal teaching performance, identify one's pedagogical strengths and weaknesses, and evaluate the effectiveness of the changes implemented in teaching . It is bSimilarly, in contrast to the traditional learning model where students mainly take a passive role in receiving what their teachers' transmit or reciting the information from books, students are perceived as active learners who construct their personal knowledge based on their own experiences in reflective learning models, where they are encouraged to be autonomous learners who reflect on their learning and to have more awareness on their learning processes, competencies, and limitations. Therefore, reflective observation is highlighted to be an indispensable process in translating concrete experiences to concepts and actions in the experiential learning process , in whicIn order to guide students to become autonomous learners, students studying in human services programs were commonly asked to write diaries or reflective journals in different ways. First, some courses simply aimed to encourage students to be critical and reflective learners. For example, in a module on management education, students were asked to write eight diaries with predesigned topics throughout the course . Second,Besides understanding the subjective world views of the informants, a survey of the literature in the field of evaluation shows that there is an increasing number of studies using diaries as a tool for evaluation. Wagner  used stuThe main purpose of this paper is to report evaluation findings of the Project P.A.T.H.S. using student diaries as an evaluation method. The project P.A.T.H.S. is a positive youth development program that attempts to promote holistic development among adolescents in Hong Kong, with an initial earmarked grant of HK$400 million by The Hong Kong Jockey Club Charities Trust. The Project is divided into two tiers of programs. The Tier 1 Program is a universal curriculum-based program for secondary 1 to 3 students in participating schools and the Tier 2 Program is a selective program for students with greater psychosocial needs. In contrast to mainstream approaches that focus on tackling youth developmental problems, the field of positive youth development focuses on enabling the talents, strengths, interests, and future potentials in children and adolescents. Roth et al.  pointed To understand the program effects of the Project P.A.T.H.S., several complementary program evaluation strategies, including both quantitative and qualitative methodologies, have been adopted . These iTo further understand the perceived program effects of the Tier 1 Program, evaluation findings based on weekly diaries written by students are reported in this paper. Actually, there are published papers on the evaluation of the Project P.A.T.H.S. based on weekly diaries , 29. In From 2005 to 2009, a total of 244 secondary schools participated in the P.A.T.H.S. Project. Among all the participating schools, 12 schools that joined the secondary 1 level of the Project, 12 schools that joined the secondary 2 level, and 10 schools that joined the secondary 3 level were randomly selected and invited to take part in eight research studies. In each of the selected schools, the school contact person was asked to randomly invite some students (from the whole grade or in some classes) to write a piece of journal in the form of a weekly diary in order to reveal their perceptions and feelings after joining the Tier 1 Program of the project. The students were informed of the purpose and confidentiality of this study, and their consent for participation was sought prior to data collection. The title of the reflective journal was “Participating in the Tier 1 Program of the Project P.A.T.H.S.: Experiences and Feelings.” The reflection was expected to be not less than 200 words in Chinese, and it should be related to the students' experiences, feelings, and comments in connection with their participation in the Tier 1 Program. The students could either complete it at home or during class time. The total number of students' diaries received was 1,138, comprising 369 written by secondary 1 students, 486 written by secondary 2 students, and 283 written by secondary 3 students see . As the While the basic research design of the studies generating the datasets was primarily qualitative in nature , it is possible to use quantitative methods  to create a descriptive profile of the findings . In the In each of the eight studies, the data were analyzed using general qualitative analyses techniques involving three steps . First, Responses regarding the students' perception of the program and the instructor, as well as the perceived benefits of the program, are presented in this paper. The integrated findings are presented in Tables “This is my first time joining the P.A.T.H.S. Project. I learned about emotion, choice and other knowledge about development from the program … I personally think that this program has great impact on me and my classmates. We could learn via the worksheet activities as well as from the advice given by the classmates. What we learned are closely related to reality which could be applicable to daily life. Therefore, I think the program is very meaningful.”“After completion of the Tier 1 Program in the first semester, I felt I became more mature in interpersonal communication and personal growth. We studied various problems such as teenagers' learning attitudes and hedonism during class. Through games or group discussion, we learned how to analyze and acquire correct information, and how to face difficulties and remain positive under adverse circumstances. I like this program very much because all the program content is closely related to common teenage problems, such as body shaping and substance abuse, which is closely related to our daily life. If we do not have good virtues and healthy mind, we will follow the wrong track easily. Therefore, the program serves as not only a light in the dark but also a teacher who teaches and guides us to the right way.”“Fortunately, I joined the Project P.A.T.H.S. from Secondary 1 to Secondary 3. This is one of my favorite subjects. The atmosphere during the class was relaxing that made the subject become more acceptable. Moreover, I really liked the design of the program. Teachers and social workers used multiple resources for teaching in order to guide the students to think from different angles and make judgment. Problems such as school orientation, conflicts with parents and peer relationship that troubled me a lot were discussed in the class. I could solve these problems by applying the skills I learned from the class. Obviously the project works.”There were 558 responses regarding “views on instructors,” which were categorized into “overall impression,” “teaching performance,” “teaching attitude,” and “others” see . About 9“I remembered when I first joined the Project P.A.T.H.S., I felt very excited … Miss Wong was our instructor. Although the topics of each P.A.T.H.S. session were different, the goal was the same—to help students to develop good interpersonal relationship. The instructor would encourage the students to share their opinions on a topic and then asked other students to discuss or analyze each student's viewpoints. The instructor would also arrange some activities such as group discussion and role-play. These activities promoted the relationship among classmates in only one year's time.”“There was a period of time when I lost self-confidence due to a failure. I kept myself in the corner of my world and could not find a way out until I heard some sharing from the instructor. She said, “Life is like an obstacle race. No matter what obstacles you encounter during the race, once you pass the finishing line, you will have victory.” I suddenly realized that self-pitying did not help anything. Rather, I should regain my strength to face new challenges. The words from the instructor become my motto, which drives me to keep moving forward.”n = 1,936), particularly in the subcategory of “positive self” , followed by “general gains”  and “cherishing life” . In addition, many respondents reported that they acquired interpersonal competence (n = 773), which could be categorized into “general interpersonal competence”  and “specific interpersonal competence” . Some narratives based on the respondents revealed that the program was perceived positively by the program participants.The perceived benefits of the program to the students are shown in “I learned much useful knowledge in this program. Firstly, I learned many interpersonal skills such as how to get along with classmates, teachers, friends, senior people and family members. I also learned to appreciate others and myself. I knew myself much more than before. By realizing my strengths and weaknesses, I was able to improve myself. In addition, I learned to distinguish between what I should do and what I should not. Most important of all, the program enabled me to meet new friends and improve my communication skills. All these skills will be very useful for my future development.”“When I looked back, I really learned a lot of things. I felt I truly grew up. In familial aspect, I understood that family members should mutually help each other and care for one another. I need to help doing the family chores when I have time. As parents have been taking care of us whole-heartedly, we have to repay and be thankful to them. I learned the importance of communication. Now I would spend more time to chat with my parents and share my feelings with them. In doing so, my parents were reassured and became less worried about me … In social aspect, I knew that we should follow social disciplines such as keeping the voice down in public area to avoid interrupting other people … In global aspect, I learned that there were many people in the world living in poverty. We should do whatever we could do to help these people.”“I could still remember when I first participated in the Project P.A.T.H.S., I did not understand why we should sacrifice two sessions of lesson for studying this program. However, I was gradually involved in it. This program provided me an opportunity to know more about my teachers and classmates … Although the three-year program is going to end, we still have a lot to learn throughout our lives. Among the units I have learned, the most impressive topic is “Life and Death”. “Life and Death” is actually not far away from us. Remember the Wenchuan earthquake last year? Life of the students in Sichuan was changed in only one second. As Hong Kong students, we should make deepest reflection—bliss is not taken for granted!”n = 1,138 students) was involved. Second, eight datasets collected across three grades at different points in time were analyzed. Third, this is the first known scientific evaluation study of a positive youth development program utilizing student diaries based on different cohorts in China. Fourth, this is also the first evaluation study utilizing diaries based on such a large sample of participants in the global context. Finally, the data were analyzed in both quantitative and qualitative ways. As far as the design and data collection of the studies are concerned, the study has the characteristics of naturalistic inquiry, inductive analysis, holistic perspective, qualitative data, unique case orientation, and contextual sensitivity. Regarding data analyses, both qualitative  and quantitative (mainly counting) analyses were used.The purpose of this study was to evaluate the Tier 1 Program of the Project P.A.T.H.S. using findings based on weekly diaries collected in the experimental and full implementation phases (2005–2009) of the project. There are several characteristics of this study. First, a large sample of participants ., 31.27, The present findings demonstrate the utility of using student diaries to evaluate positive youth development programs. By asking the program participants to reflect on their experiences, subjective perceptions and perceived benefits of the program can be properly understood in a less mechanical and artificial manner. Such an approach can help to capture the dynamic nature of the perceived qualities and effectiveness of the program. In addition to its holistic emphasis and ease of data collection, Maxwell  commenteShek et al.  proposedNevertheless, several limitations of the study should be noted. First, for students who are sensitive about the issue of “invasion of privacy” , the useFourth, although the present findings are interpreted in terms of the positive program effects and experiences of the program participants, it should be noted that there are several alternative explanations. These alternative explanations should be considered because high proportions of responses were positive across the eight studies. The first alternative explanation is that the students were afraid of being punished by the program implementers if they did not respond in a favorable manner. However, this possibility can be partially dismissed because the teachers did not mark the weekly diaries and the identities of the students were basically anonymous. The second alternative explanation is that the students consciously acted in a “nice” manner to help the workers to illustrate a positive program effect. However, this alternative explanation can be partially dismissed because negative comments were recorded and students are normally encouraged to express their views honestly. The third alternative explanation is that the high proportion of positive responses observed, were in fact, biased responses. However, this alternative explanation can also be dismissed because the schools and students were randomly selected. The fourth alternative explanation is that because there is no control group, natural developmental maturation may account for the positive evaluation findings. That is, through maturation, the participants may see things in a more favorable manner. Furthermore, threats to internal validity in the study should be realized.Finally, although the 11 principles proposed by Shek et al.  were uph


Streptococcus thermophilus is the archetype of lactose-adapted bacterium and so far, its sugar metabolism has been mainly investigated in vitro. The objective of this work was to study the impact of lactose and lactose permease on S. thermophilus physiology in the gastrointestinal tract (GIT) of gnotobiotic rats. We used rats mono-associated with LMD-9 strain and receiving 4.5% lactose. This model allowed the analysis of colonization curves of LMD-9, its metabolic profile, its production of lactate and its interaction with the colon epithelium. Lactose induced a rapid and high level of S. thermophilus in the GIT, where its activity led to 49 mM of intra-luminal L-lactate that was related to the induction of mono-carboxylic transporter mRNAs (SLC16A1 and SLC5A8) and p27Kip1 cell cycle arrest protein in epithelial cells. In the presence of a continuous lactose supply, S. thermophilus recruited proteins involved in glycolysis and induced the metabolism of alternative sugars as sucrose, galactose, and glycogen. Moreover, inactivation of the lactose transporter, LacS, delayed S. thermophilus colonization. Our results show i/that lactose constitutes a limiting factor for colonization of S. thermophilus, ii/that activation of enzymes involved in carbohydrate metabolism constitutes the metabolic signature of S. thermophilus in the GIT, iii/that the production of lactate settles the dialogue with colon epithelium. We propose a metabolic model of management of carbohydrate resources by S. thermophilus in the GIT. Our results are in accord with the rationale that nutritional allegation via consumption of yogurt alleviates the symptoms of lactose intolerance. Streptoccocus thermophilus (S. thermophilus) is a dairy bacterium consumed by Humans for centuries. S. thermophilus is in the European Qualified Presumption of Safety list of food bacteria and is a generally recognized as a safe species (GRAS status); it has a long documented history of safe use in food and its genome is devoided of potential virulence functional genes S. thermophilus is found in numerous cheeses and is one of the two bacteria in yogurt with its obligate partner Lactobacillus delbrueckii sp. bulgaricus (L. bulgaricus). Its high performance in fermenting milk and its resistance to elevated temperatures are two essential properties that accounted for a large utilization of strains of the species S. thermophilus in dairy industry. In addition, S. thermophilus is also recognized as a probiotic and, therefore, used to promote health L. bulgaricus and S. thermophilus that compensate the deficiency of the intestinal enzymes by their own β-galactosidase S. thermophilus in the gastrointestinal tract (GIT) of gnotobiotic rats. Previously, we have shown that S. thermophilus adapts its physiology to GIT by enhancing proteins devoted to carbohydrates metabolism S. thermophilus is boosted in the GIT, thus explaining how the consumption of this viable microorganism may help the digestion of carbohydrates S. thermophilus is higher in the GIT after lactose supplementation Recently, a general claim on yogurt on aiding lactose digestion has been accepted by the European Food Safety Authority. The latter considers that a cause and effect relationship is established between the consumption of live yogurt culture and improvement of lactose digestion in individuals with lactose mal-digestion . In Humans, only a low proportion of population retains the capacity to degrade the lactose into adulthood, because the intestinal enzyme lactase-phlorizin hydrolase  activity commonly starts decreasing after the first months or years of life S. thermophilus can metabolize a number of different sugars , lactose remains its favorite substrate. Lactose transport, metabolism, and regulation have been extensively studied in vitro by using different strains of S. thermophilusS. thermophilus, lactose is transported into the cell via the LacS permease which operates as a lactose/galactose antiporter or as a galactoside/H+ symporter S. thermophilus is the archetype of lactose-adapted bacterium and on the other hand, we have observed that this specialized bacterium revealed a high adaptability of its carbohydrate metabolic pathways to the gut environment S. thermophilus physiology in the GIT of gnotobiotic rats. We studied the sequenced strain LMD-9 for its colonization capacities, lactate production, proteomic profile and the related host responsiveness when animals received 4.5% of lactose that corresponded milk content of lactose. This work sheds new light on the lactose-related metabolism of S. thermophilus in the GIT and the subsequent crosstalk with colonic epithelium. By comparing the wild-type S. thermophilus strain and a mutant strain inactivated in lactose permease LacS, we also studied the involvement of its transporter in the carbohydrate metabolism in vitro and in vivo. Our results led us to propose a metabolic model of management of carbohydrates resources by S. thermophilus in vivo.Although 8 CFU of S. thermophilus LMD-9 and received lactose (4.5% wt/vol) in the drinking water (Ino-LMD9+lac)   . S. therhe colon . The max311+lac) . This ra lactose  and [12]S. thermophilus metabolism - accumulated at a 49.9±5.7 mM concentration in Ino-LMD9+lac caecal content, that represented 20 fold higher L-lactate than in GF+lac (2.5±1.5 mM). D-lactate was not detected in all groups of rats assessed. In parallel to this luminal L-lactate accumulation, a significant (p<0.05) acidification was noted with a luminal pH of 6.5±0.1 in Ino-LMD9+lac when compared to 7.4±0.5 in GF+lac. In Ino-LMG18311+lac, 55.8±4.2 mM L-lactate accumulated in caecal content. Thus, S. thermophilus – either LMD-9 or LMG18311 strain - when colonizing the GIT was metabolically active by producing a high amount of L-lactate.L-lactate - an end product of +lac epithelium was analysed by HES staining , GF+lac (204±17 µm) and GF +lac was characterized by large and slack crypts and by the presence of crypts which seems to be ready to split (crypt fission). This global aspect of epithelium was reminiscent of our previous observations with various mono-associated and germ free rats S. thermophilus stimulates an increases in monocarboxylic acid transporters mRNAs (SLC16A1 and SLC5A8) and a lactate-sensitive protein involved in the cell cycle arrest (p27kip1) in colon +lac (n = 5) relative to GF+lac (n = 3). p27kip1 protein was 1.8 (±0.03) induced in Ino-LMD9+lac in comparison with GF+lac  and after 30 days residence in the GIT (faeces of Ino-LMD9+lac) (We compared the proteomic profiles of MD9+lac) . Sixty-tMD9+lac) .S. thermophilus population was maintained at a high level (109 CFU/g) in the GIT even with a low expression level of these pathways, whereas its growth in milk required a high expression and availability of these enzymes.Down-regulated proteins were mainly involved in transcription, translation, nitrogen metabolism , and purine and pyrimidine metabolisms. Thus, the +lac faeces, compared to milk alone, belong to the carbohydrate metabolism  Primarily listing the proteins differentially expressed in the Ino-LMD (without lactose) digestive tract versus milk and ii) a second listing of proteins differentially expressed in the Ino-LMD9+lac digestive tract versus milk . Thirty four of these intestine-repressed proteins that were both repressed in presence or absence of lactose . The final level of colonization was identical with ΔlacS and LMD-9 strains, with 1.5 109 CFU/g (lacS+lacversus 34.1±7 mM in Ino-LMD9+lac). However, the kinetic of colonisation was altered with ΔlacS strain in the first days after inoculation as the maximal colonization ΔlacS being delayed in comparison with the wild-type strain. In concordance with this delayed colonisation, the L-lactate was undetectable in the first days after inoculation in Ino-ΔlacS+lac faeces, whereas it was already high (41±12 mM) in faeces of Ino-LMD9+lac. These results indicated that LacS permease inactivation delayed the colonisation of S. thermophilus and suggested that an adaptation process occurred during the first 15 days after inoculation.The Δ09 CFU/g . ConcordS. thermophilus leading to higher level of luminal lactate  did not correlate with higher stimulation of epithelial transporters and p27kip1. All these results highlight the homeostatic responsiveness of the epithelium, as we have previously observed after intestinal resection versus 34.1±7 mM in faeces). In such models, the major proportion of lactate was excreted, because of the absence of other bacteria that normally inhabit the gut and metabolize lactate. In healthy human adults, lactate produced by gut microbiota is nearly undetectable in faecal samples since it is absorbed by host and also consumed by lactate-utilizing bacteria to produce mainly butyrate S. thermophilus could contribute to microbial ecosystem by favoring lactate-utilizing enzymes or inhibiting low-pH sensitive bacteria The present work confirmed our previous data stating that lactate produced in Ino-LMD9 by S. thermophilus physiology and demonstrates the flexibility of this bacterium to adapt to digestive environmental constraints and its capacity to diversify the use of alternative sugars. In absence of lactose, S. thermophilus develops a capacity to use alternative sugars in vivo ant this suggests that similar mechanisms occurred in the absence of a functional LacS permease. The presence of lactose enhanced LMD-9 colonization in the GIT, probably by favoring the use of diverse sugars in the GIT via the induction of metabolisms of galactose, sucrose, maltose, and glycogen. Each metabolic pathway feeds glycolysis, suggesting that S. thermophilus metabolism converges to ATP production in the GIT. This also indicates that most of the sugar routes predicted by genome analysis, and so far not yet studied, are probably functional in S. thermophilus LMD-9. ΔlacS strain needed about 15 days to stably and maximally implant and thus possibly to set up a metabolic response by using alternative sugars and/or by favoring the entrance of lactose by other unknown transporters. By studying the physiology of S. thermophilus in the digestive tract, we also propose that the protein MalQ may be involved in glycogen synthesis rather than in maltose metabolism. In absence of lactose, MalQ was induced in gut and we hypothesized that S. thermophilus stocked glycogen in order to face limiting environmental conditions S. thermophilus. A similar simultaneous glycogen synthesis and degradation have been described in Corynebacterium glutamicum where glycogen is constantly recycled malQ and glgP  are an operon in LMD-9. Glycogen could be of prime importance in the colonization, adaptation and survival of S. thermophilus within the GIT, as previously observed for other microorganisms, when present in starvation conditions Our study presents data on S. thermophilus and lactose by revealing that lactose enhanced S. thermophilus kinetics, level of colonization and fermentative activity in the GIT. Our work suggests that a food product containing both a live S. thermophilus and lactose would favor the colonization and fermentative activity of S. thermophilus in vivo. In this context, our results follow the rationale of previous clinical observations and the nutritional assertion that the consumption of yogurt (containing lactose) could alleviate the symptoms of lactose intolerance.Our present work sheds new light on the established association of S. thermophilus LMD-9 , and LMG18311  were used. Stock cultures of S. thermophilus LMD-9 and LMG18311 were prepared in reconstituted 10% (wt/vol) Nilac skim milk  as previously described S. thermophilus monocultures were obtained by inoculating Nilac milk with 106 CFU/ml of stock cultures and incubated at 42°C until pH 5.4–5.5. One ml of culture was used for rat gavage and the remaining aliquots were frozen in liquid nitrogen and stored at −20°C until protein extraction. The cultures were enumerated a posteriori by plating appropriate dilutions on M17 agar lactose (10 g/L) for S. thermophilus. After 16 h (S. thermophilus) incubation at 42°C under anaerobiosis , colonies were counted.The strains S. thermophilus strains were performed in M17 supplemented with 10 g/L lactose, glucose, sucrose or fructose; the apparent growth rate (μmax) was defined as the maximum slope of semi-logarithmic representation of growth curves assessed by O. D.650 nm measurements.Cultures (n = 3) of 5′CCAGCGAACCATTTGA3′) and AphA3-R (5′GTTGCGGATGTACTTCAG3′) primers. The 1489 bp DNA fragments flanking the lacS gene were PCR-amplified using the Phusion DNA polymerase, the LMD-9 DNA as a template, and primers LacS-up (5′TATGTGCCTGCCAGTCCA3′)/Kana-up-R (5′AGGGGTCCCGAGCGCCTACGAGGAATTTGTATCGATGACCTTTAGATTTTTCCAT3′) for the upstream fragment and primers LacS-down (5′AACTGGAACGACTTCAAC3′)/Kana-down (5′CTTACCTATCACCTCAAATGGTTCGCTGGGTTTATCGTAACTAATTCAGAAAAA3′) for the downstream fragment. The 3′ end of the upstream generated fragment contained a sequence complementary to the 5′ end of the kana cassette whereas the 5′ end of the downstream generated fragment contained a sequence complementary to the 3′ end of the cassette. This allowed joining of these three fragments subsequent to Taq Phusion PCR using primers LacS-up and LacS-down. After purification with a QIAquick PCR purification kit, 500 ng of the resulting 3.4 kb fragment was further used to transform LMD-9 natural competent cells as described by Gardan et al. The kanamycin cassette of the plasmid pKa 2000 was PCR-amplified using the Phusion high fidelity DNA polymerase with AphA3-F  or S. thermophilus LMG18311 . 1 ml of a culture of S. thermophilus in Nilac milk (5×108 CFU/ml) was transferred to GF rats by oral gavage. As a control, GF+lac rats were also inoculated with 1 ml of sterile Nilac milk (without bacteria). Following gavage, rats received water enriched with lactose (4.5%wt/vol). GF and mono-associated rats were housed in sterile Plexiglas isolators . All groups of rats received the same standard diet (UAR), which was sterilized by gamma irradiation. Twice a week S. thermophilus was enumerated by plating serial dilutions of the faeces on M17 lactose agar. All rats were euthanized at three months old, 30 days after gavage.All procedures were carried out in accordance with European and French guidelines for the care and use of laboratory animals. Permission 78–123 is a permit number dedicated to M. Thomas. MICALIS (Microbiologie de l'Alimentation au Service de la Santé) review board specifically approved this study. At the age of 2 months, germ-free (GF) rats  were inoculated either with S. thermophilus ΔlacS strain was inoculated in GF rats to obtain Ino-Δ+laclacS rats. The ΔlacS inoculum was grown in M17+glucose and bacteria were enumerated by plating serial dilutions of the faeces on M17 glucose agar supplemented with kanamycine (1 mg/ml). Ino-ΔlacS+lac (n = 5) rats drank 4.5% lactose-enriched water. The presence of kanamycin cassette disrupting the lacS gene was checked by PCR in faeces of Ino-ΔlacS+lac. Ino-ΔlacS+lac rats were euthanized 18 days after gavage.et al.Rats were anesthetized with isoflurane and tissues were recovered. The colon was immediately used, for colonic epithelial cell isolation or for histological procedures as it has been described in Cherbuy kip1 . GAPDH was used as loading control. Signals imprinted on autoradiography films were quantified by scanning densitometry of the autoradiograph using Biovision 1000 and logiciel bio1D .Colonic proteins from isolated epithelial cells were used for Western blot analysis as previously described d- and l-lactates were measured in caecal contents and faeces with the Biosentec d/l lactic acid enzymatic kits according to the manufacturer instructions  as also described in Rul et al. +lac, n = 5; Ino-LMG18311+lac, n = 3).Colon samples were cut into 2 cm sections, fixed in 4% paraformaldehyde , dehydrated and embedded in paraffin according to standard histological protocols. Four micrometer sections were mounted on SuperFrost® Plus slides. Slides were stained with Hematoxylin-Eosin-Safran (HES) for histological analysis. Crypt depths were determined with NDP.view software (Hamamatsu). Only U shaped longitudinally cut crypts with open lumina along the crypt axis were analysed. +lac (n = 3), and Ino-LMD9+lac (n = 5) by the guanidinium thiocyanate method. The slc16a1 and slc5a8 mRNA quantification was performed as we have previously described Total RNAs were extracted from colon epithelial cells of GF+lac rats). A volume of cytosolic fraction corresponding to 250 µg of proteins was treated as previously described et al.http://pappso.inra.fr). The proteins were identified using MS-FIT (http://prospector.ucsf.edu).Cytoplasmic proteins were extracted from bacteria as previously described P value lower than 0.05. Statistical analysis was performed using the JMP® software .Figure S1S. thermophilus LMD-9 between faeces in presence of lactose and late growth phase in milk.Fold changes in protein abundance (2-DE) of (DOCX)Click here for additional data file.