Adherence to Internet-delivered lifestyle interventions using multiple tailoring is suboptimal. Therefore, it is essential to invest in proactive strategies, such as periodic email prompts, to boost re-use of the intervention.This study investigated the influence of content and timing of a single email prompt on re-use of an Internet-delivered computer-tailored (CT) lifestyle program.A sample of municipality employees was invited to participate in the program. All participants who decided to use the program received an email prompting them to revisit the program. A 2×3 (content × timing) design was used to test manipulations of prompt content and timing. Depending on the study group participants were randomly assigned to, they received either a prompt containing standard content (an invitation to revisit the program), or standard content plus a preview of new content placed on the program website. Participants received this prompt after 2, 4, or 6 weeks. In addition to these 6 experimental conditions, a control condition was included consisting of participants who did not receive an additional email prompt. Clicks on the uniform resource locator (URL) provided in the prompt and log-ins to the CT program were objectively monitored. Logistic regression analyses were conducted to determine whether prompt content and/or prompt timing predicted clicking on the URL and logging in to the CT program.P=.005) and after 6 weeks . Furthermore, participants who received an email prompt 2 weeks after their first visit logged in to the program significantly more often compared to participants receiving the prompt after 6 weeks . A trend was observed with regard to prompt content. Participants receiving a prompt with additional content were more likely to log in to the program compared to participants who received a standard prompt. However, this result was not statistically significant .Of all program users (N=240), 206 participants received a subsequent email prompting them to revisit the program. A total of 53 participants (25.7%) who received a prompt reacted to this prompt by clicking on the URL, and 25 participants (12.1%) actually logged in to the program. There was a main effect of prompt timing; participants receiving an email prompt 2 weeks after their first visit clicked on the URL significantly more often compared with participants that received the prompt after 4 weeks (odds ratio [OR] 3.069, 95% CI 1.392-6.765, The key findings suggest that boosting revisits to a CT program benefits most from relatively short prompt timing. Furthermore, a preview of new website content may be added to a standard prompt to further increase its effectiveness in persuading people to log in to the program. Internet-delivered lifestyle interventions applying computer-tailoring techniques -3 have rResearch on non-usage attrition has demonstrated an initial rapid decline in program use over the first few weeks ,21. DuriA substantial amount of Internet-delivered interventions use reactive strategies to achieve re-use, implying that a passive approach is used in which users themselves must undertake action to repeatedly benefit from the intervention content . HoweverMore specific evidence on the positive effects of the use of email to promote direct action stems from the field of e-marketing ,35. WithThis study aimed to assess the added value of periodic email prompts to boost revisits to an Internet-delivered CT lifestyle program. We aimed to answer 2 questions: (1) which prompt timing interval is most effective in boosting re-use of the Internet-delivered CT lifestyle program and (2) which prompt content is most effective in boosting re-use of the program? We addressed these questions among participants in an Internet-delivered CT intervention aimed at multiple health behaviors: increasing physical activity, increasing fruit and vegetable intake, smoking cessation, and decreasing alcohol consumption.This study was conducted in close collaboration with the Regional Public Health Service (RPHS) in the northern part of the Dutch region, Limburg. All people employed by the municipalities in this region were invited by the RPHS to participate in an Internet-delivered CT lifestyle program developed by our research group ,42. InviParticipants who logged in to the program to obtain CT advice were sent an email prompting them to re-use the program. Re-use was encouraged to allow participants to monitor their behavior change and to obtain iterative CT advice aimed at behavior change and relapse prevention. Furthermore, participants were offered an opportunity to obtain CT advice regarding an additional health behavior. Finally, re-use was stimulated to keep participants informed about new content added to the program website.A 2×3 design was used to test the effect of 2 factors: prompt content (standard and standard+) and prompt timing . The standard prompt (SP) contained a message reminding people about their previous visit to the program and invited them to re-use the program to monitor their progress and obtain additional feedback. The second version of the prompt (SP+) contained standard content complemented with a message alerting people to new content added to the intervention website. Participants received an email prompt after 2, 4, or 6 weeks. In addition to these 6 experimental conditions, a control condition was included, no prompt (NP). People allocated to the control condition did not receive an additional prompt and were only encouraged at baseline to re-use the program. To participate in the program, participants had to register by using a personal log-in code and password. Immediately after registration for the program, participants were randomly allocated to 1 of the 7 study conditions. Randomization occurred at the respondent level by means of a computer software randomization device. Data for the present study were collected from March to July 2011.People who were allocated to the experimental conditions of the study received an email prompting them to re-use the program. Periodic prompts should be distinguished from reminders, which are also often used in Internet-delivered interventions. Sending reminders is a technique that is used to increase response rates in online data collection by proactively stimulating participation among nonresponders and is used to prevent drop-out attrition ,43. PeriThe CT program integrated established CT programs tested and proven to be effective in randomized controlled trials for increasing smoking cessation, promoting the intake of fruit and vegetables, increasing the level of physical activity, and reducing the consumption of alcohol -48. The The program was embedded in a website designed for the current study. This website entailed general information considering a healthy lifestyle and the selected health behaviors. Furthermore, the website provided specific information regarding the project, contained a direct link to the CT program, and provided background information regarding the study and the research team. During the study, new information  was added to the website.Participants in the experimental conditions (SP and SP+) received an email prompting them to re-use the intervention. Participants had to take 2 steps to re-use the program in order to self-monitor their level of behavior change and obtain additional CT advice. The first was clicking on the URL provided in the email prompt. Clicking on the URL was tracked by referral ID codes that were integrated in the URL. The second step was logging in to the CT program after arriving on the program website. Log-ins were objectively monitored during a 2- week period and log-in dates were compared to baseline dates to determine revisits.To describe characteristics of program visitors, age, gender, and educational level of participants was assessed . Health behavior status consisted of information regarding the 5 key behaviors. Physical activity was measured by the Short Questionnaire to Assess Health-enhancing physical activity (SQUASH)  and guidGeneral descriptive statistics were calculated to describe characteristics of program visitors, as well as main findings concerning adherence to the public health guidelines. Baseline differences between the intervention groups were calculated using the Chi-square test for dichotomous and categorical variables and 1-way analysis of variance (ANOVA) for continuous variables. Finally, logistic regression analyses were conducted. There were 2 dependent variables: whether participants clicked on the URL  and whether participants logged in to the CT program . Prompt content  and prompt timing (dummy coded with 2 weeks as the reference category and 4 weeks as the reference category) and the interactions between these variables were used as predictors in the initial model of each dependent variable. Furthermore, because women and people with a higher educational level and age are more likely to use Internet-delivered lifestyle interventions ,58, age,26= 9.1, P=.17). Furthermore, there were no significant differences between the groups regarding educational level  and age . Overall visitors had a mean age of 50 years (SD 14.99) and most were medium  to highly educated . Regarding the 5 health behaviors included in the program, 12.9% (31/240) did not comply with the Dutch guidelines of at least 30 minutes of moderately intensive physical activity on at least 5 days of the week. With regard to fruit and vegetable intake, 50.8% (122/240) and 60.0% (144/240) did not adhere to the Dutch guidelines of at least 2 pieces of fruit and at least 200 gram of vegetables each day, respectively. Approximately 1 out of 10 participants indicated that they smoked , and 22.1% (53/240) did not comply with the Dutch guidelines for alcohol intake.In total, 240 participants visited the program, of which 73.3% (176/240) were female. Participants were randomly allocated to each of the 7 study conditions and a randomization check revealed that females were equally distributed  reacted to this email by clicking on the URL (step 1), whereas 25 participants (12.1%) actually logged in to the program (step 2). All results concerning clicking on the URL and logging in to the program are described per study group in There was no significant interaction between prompt content and prompt timing  with reg22 = 15.2, P = <.001). Participants who received an email prompt 2 weeks after their first visit, clicked on the URL significantly more often compared with participants that received the prompt after 4 weeks  and after 6 weeks . There was no significant difference in reaction to the email prompt between participants receiving the prompts after 4 weeks, compared with participants receiving the prompts after 6 weeks. Also, no main effects of prompt content could be detected . Participants receiving an email prompt 2 weeks after their first visit, logged in to the program significantly more often compared to participants receiving the prompt after 6 weeks . There was, however, no significant difference between participants receiving the prompts after 4 weeks compared with participants receiving the prompts after 2 or 6 weeks. With regard to prompt content, a trend was observed. Participants receiving a prompt with additional content were more likely to log in to the program compared to participants who received a prompt with standard content. However, this result was not statistically significant .With regard to logging in to the program, analyses of main effects indicated that there was a significant effect of prompt timing (χP<.001).Finally, all experimental groups were compared with the control condition (NP) that did not receive an additional email prompt to boost revisits to the intervention. Results from this analysis revealed that participants who received an email prompt containing additional content after 2 weeks were significantly more likely to log in to the program , but also to the proportion of log-ins to the program (step 2). Secondly, a trend was observed indicating that adding a preview of new website content to a standard prompt increased its effectiveness in persuading people to log in to the program. Finally, sending a prompt with additional content after a 2-week period significantly increased program log-ins compared to using a reactive approach in which no additional prompts were used.The effectiveness of using a relatively brief interval for sending a prompt may indicate that people tend to forget about the program. Therefore, the interval between engaging in a first visit and receiving an email prompt to boost re-use should be kept relatively short. Since the costs of sending a large number of email messages are relatively low , this stResults from the current study also show a trend toward the efficacy of adding additional content to a standard prompt. However, this trend was only present for logging in to the program (step 2) and not for clicking on the URL (step 1). A possible explanation for this effect on log-ins might be the repeated exposure to new website content. People who received an email prompt containing a preview of new website content were exposed to this website content twice: within the email prompt and on the program website. According to ease of processing theory , repeateopt-in email is used when receivers have agreed to the receipt of an email [With regard to clicking on the URL (step 1), significant effects of adding a peripheral cue to a standard prompt failed to occur. People were significantly more likely to respond to emails that were sent at a relatively short interval, irrespective of the email content. A possible explanation for this finding might be that participants consented to participate in the present study after being informed about the possibility of receiving an additional email to boost program use. Within the field of e-marketing, the term an email ,61. Resean email ,61. TherThe current study primarily focused on the effectiveness of using a single email prompt with regard to clicking on the URL and logging in to the program. Combined with results from our previous study , importaWe found that using relatively short prompt timing (2 weeks) resulted in more positive effects compared to using a longer time period. This effect not only referred to clicking on the URL, but also to the proportion of log-ins to the program. Although a trend was observed about the effectiveness of adding a preview of new website content to a standard prompt, this only referred to persuading people to log in to the program. The findings of this study underline the importance of sending an email prompt relatively shortly after a first visit to the program. Furthermore, it is important to further focus on prompt content because the addition of a peripheral cue may add to the prompt’s effectiveness.


Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving  Much of the difference between individual humans is in the number of copies of genes and lengths of genome. The mechanisms by which copy number variation arises are not well understood. We sought information on copy number change mechanisms by extensive use of array comparative genomic hybridization of whole genomes in bacteria selected for amplification of part of the genome. We report that about 10% of amplified isolates carried other chromosomal structural changes associated with the amplification, a result comparable to that seen in human copy number variants. Importantly, we found a significant occurrence of structural changes that were not involved in the amplification event. These were not seen in a control sample of stressed cells not carrying amplification. This establishes that chromosomal structural change happens in a subpopulation of cells apparently licensed to undergo these changes. Because the changes occur under the stress of starvation and require two of the cells' stress-response systems, we propose that licensing for cell-wide structural change in this subpopulation is a component of response to stress. This idea has implications for the mechanisms of evolution and cancer progression, suggesting that changes occur in a shower of events rather than as isolated random events. CNVs constitute the most ubiquitous differences between individual or personal human genomes lac in the Lac assay system on an F′-plasmid in E. coli requires DNA polymerase I (Pol I) but not excision repair , placing the event at replication forks E. coli amplification is enhanced by 3′ single-stranded DNA ends, suggesting priming of DNA synthesis oriT, and this requirement is suppressed by double-strand cutting near lac on the F′-plasmid Amplification at E. coli− cells carrying a +1 frameshift mutation are spread on lactose minimal medium. Lac+ colonies arise over days from the starving cells. The colonies carry either amplified arrays of the leaky lac allele or a compensating frame-shift mutation (point mutants) + colonies are found to carry secondary unselected mutations at a high frequency (up to 10−2 for some loci) + carry a much lower frequency of unselected mutations + colonies arise from a hypermutating subpopulation (HMS) while the majority of the starved cells do not take part in hypermutability. The HMS is defined by the stress responses that are activated in given cell + colonies arise from a chromosomally unstable subpopulation, though it has been shown that they do not arise from the HMS In the Lac assay in lac. Evidence of secondary chromosomal structural change in amplified isolates that is not seen in controls constitutes evidence of a physiological difference that affects genome stability between cells undergoing amplification and those that do not. We found a significantly higher occurrence of unselected events that would not have bestowed a growth advantage among amplified isolates compared with stressed Lac− control cells. This demonstrates that amplification is happening in a differentiated subpopulation undergoing general chromosomal structural change, suggesting that this differentiation might be mediated by stress responses. Second, we sought further evidence that amplification in E. coli shows similar complexity to human non-recurrent CNV events. We found complexity in the amplification events in over 7% of amplified isolates, mostly in the form of inverted duplications within the amplicons (units of amplification), confirming that there is a tendency for events that mediate chromosomal structural change to be complex.This study reports the use of array comparative genomic hybridization (aCGH) to analyze genome-wide changes in copy number. We sought, first, evidence of secondary unselected cell-wide chromosomal structural instability in those cells that carry amplification at + phenotype, in comparison with a reference DNA sample from the parental strain FC40 + isolates consisted of 284 new isolates from day 7 of adaptive mutation experiments and 16 isolates that were reported before − cells taken from lactose minimal medium starvation plates on day 5 . We also analyzed 60 day seven Lac+ point mutant colonies, and include these with the Lac− isolates from starvation plates as a control of 240 stressed isolates that do not carry lac-amplification. In addition, we studied 60 single cell Lac− colonies that had not been stressed. No change in copy number was found in any of 300 non-amplified samples.Using Oxford Gene Technologies 44K aCGH arrays, giving a resolution of about 100 base-pairs (bp), we analyzed 300 isolates that had an unstable Lac+ isolates were found to carry amplification at lac. The mean copy number at lac was 69.3+/−22.9 (mean +/− SD). The mean length of 298 amplicons was 22.7 kilobase pairs (kb). These same isolates, when grown in lactose minimal medium (to maintain selection for amplification) were found to have about twice the amount of F′-borne chromosomal sequence than sequences that were only present on the chromosome (1.88+/−0.30-fold). There was no increase in copy number of F′ sequences in previously stressed Lac− cells grown in glycerol minimal medium or in previously stressed Lac+ point mutant cells grown in lactose minimal medium (0.99+/−0.10-fold). We sequenced the amplification junctions of 40 amplicons. We found that all had microhomology at the junction sequence. Sixteen of the 40 were located in REP sequences All 300 unstable Laclac-amplified isolates, we identified 28 events that changed chromosomal structure in addition to the amplification at lac (9.3%). The positions of some of these changes on the standard map of E. coli are shown in lac, and might therefore have played a role in lac-amplification. Other events do not appear to offer a growth advantage to Lac− cells on lactose minimal medium, and therefore represent other events occurring in the same cells as lac-amplification. We tested whether one inversion affected the rate of amplification by measuring amplification in a derivative that had lost amplification. In 300 Salmonella entericalac region was included in the embedded duplication. Detailed study of these events showed that the embedded inverted duplications vary in size from 5.2 to 42.6 kb. Two novel junctions were found in each case. The junctions showed microhomology of 3 to 30 bp   is shown in Another event of the same type, PJH2058, that did not involve inversion or duplication of lac, and part of the chromosome was duplicated  was found in an isolate (PJH1475) in which the F′-factor was integrated into the chromosome, so that part of the F′ including plicated . We havebination . Similarlac and had one or both ends outside the chromosomal sequence on the F′. The junctions were not found in the IS3 elements that span chromosomal sequence on the F′-plasmid as has been observed previously Two other large duplications, PJH1477 and PJH1487, were found that included These data establish that, like in human, a significant proportion of events of chromosomal structural change that generate amplification are complex in that more than one structural change occurred, apparently within the same event. This applies to 19 of 300 events resolved by our approach .In the same sample of 300 amplified isolates, we also found six that included a chromosomal structural change that was not apparently directly involved in the amplification. None was seen in the 240 stressed control isolates. The null hypothesis that the amount of that unrelated chromosomal structural change does not differ between amplified and stressed non-amplified isolates, can be rejected : two on the F′-plasmid and two on the chromosome. One of the deletions (PJH1474) was flanked by non-identical IS elements, and so might have occurred by homeologous recombination or alternatively might have utilized the shorter homology stretches to mediate a template switch. The other three show microhomology junctions (1 to 4 bp), and so probably happened by events similar to those generating amplification. The chromosomal deletions were 0.8 and 1.6 kb long, and are situated at about 1.4 and 1.6 megabases on the standard reference E. coli map (PJH2116 and PJH1482 respectively). Deletions of 0.2 and 7.5 kb long (PJH2030 and PJH1482 respectively) were found on the F′ at about 44 kb and 50 kb from lac respectively  , PJH1479onal PCR . When PCSalmonella enterica, have proposed that amplification at lac is not induced by the stress of starvation, but is a product of selection for more β-galactosidase expression with parameters within those established for chromosomal structural changes in growing cells of E. coli. We regard these amplification events as stress-induced because it was not pre-existing Kugelberg et al S. enterica. We report that a significant number of amplification events are complex in that they show more than one novel junction, indicating more than one non-homologous recombination event. The case that amplification events in the Lac assay reflect template switches during replication has been made in detail elsewhere + clone under the conditions of these experiments unless there is a second inverted template switch. This is because a single inverted switch will generate an incomplete F′-plasmid. However, this requirement for a second inversion cannot be the explanation for all complexity because, as shown in If an inverted template switch occurs, the product will not be a viable Laclac. Taking the four deletions and the two inversions that did not share a junction with amplification as events secondary to amplification, we see a significant occurrence of secondary events in cells that underwent amplification compared with starved cells not showing amplification . This is clearly an under-estimate of structural changes because duplications would be expected to be unstable, so it is not surprising that we saw none that did not duplicate lac. Those duplications that include lac presumably provide selection for maintenance of the duplication lac because we did not look elsewhere. Using aCGH, we would detect only those unrelated inversions that overlapped the amplicon, and we found one of these. We also looked for inversion by unidirectional PCR, but only in the 40 kb surrounding the lac locus.To determine whether a secondary structural change might play a role in the amplification process, we screened for loss of amplification in one strain carrying a secondary inversion. When this strain was used in a starvation-induced mutation experiment, the rate of amplification was unchanged from the control strain FC40  suggestilac+ point mutation is correlated with an elevated frequency of other unselected secondary point mutations lac locus.The meaning of the finding that the sample of amplified isolates differs in the frequency of secondary events from non-amplified cells from the same plate is important. First, it means that amplifying cells differ from other cells in their propensity to undergo chromosomal structural change. Second, it shows that this happened in a subpopulation of starved cells rather than in the whole population of starved cells, because starved cells that did not undergo amplification provide the basis of comparison. The identification of chromosomal structural mutations that are secondary to the selected event is analogous to the finding in the Lac assay that The existence of this chromosomally unstable subpopulation is compatible with the concept of stress-induced differentiation to a condition permissive for chromosomal structural change and genomic instability, and is incompatible with models that seek to explain these events as normal change and selection in slowly growing cells. We suggest that this subpopulation is differentiated to a physiological condition that allows chromosomal structural change. This is suggested by the finding that some of the secondary events occurred on the chromosome, indicating that a diffusible factor is involved. Further, we suggest that this differentiation was induced by the stress of starvation.We made the unexpected finding that some inversions involve almost, but not quite, reciprocal non-homologous recombination so that the junctions show insertions or deletions of a few tens of base-pairs. We suggest that this might occur as follows: If a template switch occurred because a replication fork was stalled by secondary structure forming in template DNA, then the complementary sequence on the other template would also be capable of forming a similar secondary structure. If two such sites occurred in a short interval, within the dimensions of a single replication fork, then the series of template switches portrayed in Alu repetitive sequences in the human genome lac that is involved in 20% of the junctions listed in REP is a pseudopalindromic sequence of about 38 bp that occurs in clusters in intergenic regions in vivo strongly suggests that stalling is mediated by hairpin formation on the lagging-strand template at replication forks Study of the positions of novel junctions of amplification show a preference for the stem of potential stem-loop structures. For 40 amplification junctions that we sequenced , 16 are E. coli. For this reason, we suggested previously that template switches occur between different replication forks: the long distance template-switch model lac, we later suggested that the mechanism was a modification of break-induced replication (BIR) at collapsed replication forks, namely that in place of RecA-mediated strand invasion, the broken end annealed by microhomology to nearby single-stranded DNA (MMBIR) SceI endonuclease to make double-strand cuts near to lac that double-strand breaks increase amplification at lacoriT by TraI provides a discontinuity in the DNA template that leads to replication fork collapse We have suggested above that some inversions are formed by template switching within a replication fork. Template switching is much more difficult to apply to other events reported here because most switches cover tens of kb, well beyond the 1.5 kb dimensions of a replication fork in We present evidence that, among stressed cells, a small proportion enters a state of heightened genomic instability during which multiple chromosomal structural changes might occur anywhere in the genome. Many such changes would be expected to be disadvantageous, but rarely a change occurs that allows escape from the stress. Because the events that we studied here in a bacterial model system are similar to those described for copy number changes in human, this conclusion might apply generally throughout biology. This view suggests that genome evolution might occur in bursts of multiple simultaneous chromosomal changes induced by stress. This view also has implications for understanding cancer progression in the stressful tumor microenvironment and the stresses imposed by chemotherapy, both of which might induce showers of chromosomal structural changes.Escherichia coli cells of strain SMR4562 128 with a leaky lac +1 frameshift mutation were initially grown to stationary phase for 3 days at 32° + colonies arise over several days, and are marked daily. Amplification was distinguished from point mutation by its instability as seen by blue and white sectoring of colonies grown on rich medium with X-gal. 284 Lac+ colonies from day 7 together with 16 previously published amplified strains + colonies arising on day 7 that carried point mutations reverting the lac mutation. 180 Lac− stressed FC40 control cells were collected by taking plugs from the same lactose plate on day 5. Sixty colonies derived from unstressed control cells were taken from the initial stationary phase culture.These 584 new isolates are identified by strain numbers PJH1458–PJH1642 and PJH2025–PJH2425. Those described previously + isolates or M9 glycerol medium for Lac− isolates by using the QIAGEN DNA Purification kit. E.coli custom high-resolution genomic microarray (4×44K) containing 44,000 unique sequence oligonucleotides spaced at about 100-bp intervals were obtained from Oxford Gene Technology (OGT). Probe labeling and hybridization were performed following the manufacturer's protocol . Slides were scanned on a GenePix 4000B Microarray Scanner (Axon Instruments). Data extraction, normalization and visualization were achieved by using Agilent Feature Extraction Software A.7.5.1. Extraction data were analyzed for copy number differences by using Microsoft Excel software. All occurrences of two or more adjacent probes showing 2-fold or more increase or decrease in copy number relative to the reference FC40 DNA were investigated further, except those that mapped to repetitive elements or prophages.Total genomic DNA was extracted from exponential culture in M9 lactose medium for LacEscherichia coli K-12 substr. MG1655. Long-range PCR was performed using LongAmp™ Taq Master Mix (New England Biolabs). The PCR products were purified with either a QIAquick PCR Purification Kit (QIAGEN) or a QIAEX II Gel Extraction Kit (QIAGEN) following the manufacturer's instructions, and sequenced by Lone Star Labs . DNA sequences were analyzed by comparison to reference sequences with the use of BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Possible secondary structures in DNA were found by use of DNAMAN version 6 (Lynnon Biosoft).All deletion, inversion and duplication junctions were further validated by PCR and sequencing. Inward-facing primers for deletions and inversions and outward-facing primers for tandem duplication were designed based on sequence from National Center for Biotechnology Information (NCBI) Deamplified lines were derived from amplified isolates by screening for sectors in colonies of amplified strains that showed a low level of β-galactosidase as seen on medium containing X-gal, but yet retained the ability to grow without proline.Figure S1mhpE and mhpT. Amplification junctions that were identified in this region are shown in red.Potential second structure of an array of REP sequences between (DOC)Click here for additional data file.Figure S2Inversion has no effect on amplification. A deamplified derivative strain of PJH1465 shows the same rate of amplification as the wild-type FC40. Mean with SEM of four cultures. Diamonds: FC40. Squares: deamplified PJH1465.(DOC)Click here for additional data file.Figure S3Array scans for PJH1475, PJH1477 and PJH1487.(PPT)Click here for additional data file.Table S1Sequences of 40 amplicon junctions.(DOC)Click here for additional data file.


During the Severe Acute Respiratory Syndrome (SARS) outbreak, high compliance in healthcare workers to hand hygiene was primarily driven by fear. However, the post-SARS period confirmed that this practice was not sustainable. At the Singapore General Hospital, a 1,600-bedded acute tertiary care hospital, the hand hygiene program was revised in early 2007 following Singapore's signing of the pledge to the World Health Organization (WHO) "Clean Care is Safer Care" program.A multi-prong approach was used in designing the hand hygiene program. This included system change; training and education; evaluation and feedback; reminders in the workplace; and institutional safety climate. Hand hygiene compliance rate improved from 20% (in January 2007) to 61% (2010). Improvement was also seen annually in the compliance to each of the 5 moments as well as in all staff categories. Healthcare-associated MRSA infections were reduced from 0.6 (2007) to 0.3 (2010) per 1000 patient-days.Leadership's support of the program evidenced through visible leadership presence, messaging and release of resources is the key factor in helping to make the program a true success. The hospital was recognised as a Global Hand Hygiene Expert Centre in January 2011. The WHO multi-prong interventions work in improving compliance and reducing healthcare associated infections. The WHO Multimodal Hand Hygiene Improvement Strategy comprising a Guide to Implementation and a range of tools constructed to facilitate implementation of each component was used in a 1600-bedded acute tertiary care general hospital in Singapore [The hand hygiene program was revised in early 2007 following Singapore's signing of the pledge to the World Health Organization (WHO) "Clean Care is Safer Care" program.The multi-prong approach used includes:1. System change:a. Alcohol based hand rub was promoted for routine use of hand hygiene instead of the previous 4% chlorhexidine handwash. During SARS, alcohol hand rub bottles were installed at the foot of every patient bed and all lift lobbies in an attempt to enable easy access by healthcare workers to hand hygiene products. From 2007, more alcohol hand rub bottles were installed along ward corridors and near food establishments in the hospital ground.b. Following the review of the WHO Hand Hygiene Manual (Advanced Draft 2006) . Hand mo2. Training & education:a. WHO training DVDs were used to illustrate clinical scenarios of hand hygiene opportunities to all healthcare workers. Although these were in French, they provided clear teaching on the 5 moments to the staffs.b. Creative educational tools were also used to teach healthcare attendants and junior nurses. Teddy bears dusted with Glo-germ were used as patient models in teaching healthcare workers the importance of hand hygiene as well as the WHO 5 moments Figure .c. Powerpoint slides with detailed explanation of the 5 moments were created for doctors, nurses and allied health. These were uploaded on the hospital intranet for easy access by the staffs.3. Evaluation and feedback: Feedback surveys are conducted annually amongst staffs to gather feedback and comments on products used or issues faced during practice. The last survey done in November 2010 confirmed that the use of posters have helped to remind staffs and public on the messages of hand hygiene.4. Reminders in the workplace: More than 150 posters were designed from March 2007 for display at lifts and walkways Figures . Giant p5. Institutional safety climate: Leadership's support and commitment was clearly visible at events and meetings. The Chief Executive Officer (CEO) led the hospitals staffs in November 2009 in a pledge of commitment to the Hand Hygiene Program  to 61% 2010)  in 2006 in their "Protecting 5 Million Lives from Harm" campaign. It comprised the following elements :1. Hand Hygiene2. Decontamination of the environment/equipment3. Active surveillance cultures4. Contact precautions for infected and colonized patients5. Device bundles Most of the elements were routine practices in the hospital except for active surveillance cultures, which were implemented hospital-wide for high risk patient groups from 2008. Healthcare-associated MRSA infections were noted to reduce from 0.6 (2007) to 0.3 (2010) per 1000 patient-days Figure .Leadership's support of the program evidenced through visible leadership presence, messaging and release of resources is the key factor in helping to make the program a true success. The hospital was recognised as a Global Hand Hygiene Expert Centre in January 2011 Figure . The useWe have received support of our hand hygiene program from the following companies:1. B. Braun2. Schülke3. Mölnlycke Health CareMLL was the advisor to the hand hygiene program. KBH was the coordinator in the hand hygiene program. All authors have read and approved this manuscript for publication.


C. difficile colitis. Guidelines recommend development of antimicrobial stewardship programs for these facilities to promote optimal antimicrobial use. However, the effectiveness of these programs or the contribution of any specific program component is not known. For this review, publications describing evaluation of antimicrobial stewardship programs for long term care facilities were identified through a systematic literature search. Interventions included education, guidelines development, feedback to practitioners, and infectious disease consultation. The studies reviewed varied in types of facilities, interventions used, implementation, and evaluation. Comprehensive programs addressing all infections were reported to have improved antimicrobial use for at least some outcomes. Targeted programs for treatment of pneumonia were minimally effective, and only for indicators of uncertain relevance for stewardship. Programs focusing on specific aspects of treatment of urinary infection – limiting treatment of asymptomatic bacteriuria or prophylaxis of urinary infection – were reported to be effective. There were no reports of cost-effectiveness, and the sustainability of most of the programs is unclear. There is a need for further evaluation to characterize effective antimicrobial stewardship for long term care facilities.Intense antimicrobial use in long term care facilities promotes the emergence and persistence of antimicrobial resistant organisms and leads to adverse effects such as  Clostridium difficile colitis . T-9. T6-9]C. difficile colitis cases on the long term care wards was also observed.The interventions described were relatively limited for two of the reports. One trial randomized 8 long term care facilities to intervention or control . The 36 The two other studies describing comprehensive programs implemented multimodal stewardship activities Table . SchwartTwo reports describe programs addressing treatment of pneumonia in residents of long term care facilities Table . NaughtoThree studies describe stewardship programs which addressed specific aspects of management of urinary tract infection Table . A clustThis review identified a limited number of studies which evaluate the implementation and effectiveness of antimicrobial stewardship programs for long term care residents. The programs described in these reports are all unique. There is no standardization of program components, implementation strategies, or evaluation. Thus, the generalizability of any of the programs for more widespread use in other facilities is uncertain. Most of the studies included simultaneous implementation of several different program components as well as input from practitioners to tailor specific program components for local implementation. Several distinct activities were usually implemented simultaneously, so the efficacy and relative importance of any single program component is unknown. While it seems appropriate to customize some program content for specific facility or regional needs, there should also be a core set of elements identified which are recommended for all antimicrobial stewardship programs. It is also desireable to standardize how outcomes are measured and reported, to allow comparisons among different programs.Most of the studies reviewed report at least some improvements in antimicrobial use following introduction of the stewardship interventions. However, the improvements in some outcomes seem relatively limited given the intensity of the programs described ,10-12. LC. difficile colitis. The stewardship programs which addressed more limited goals relevant to urinary infection – treatment of asymptomatic bacteriuria or antimicrobial use for prophylaxis for urinary infection – were both reported to be effective [The studies restricted to pneumonia both reported no impact of comprehensive programs including education, guidelines and algorithms on overall antimicrobial use for this indication ,11. The ffective ,14. Howeffective . Thus, fNone of these reports describe the impact of stewardship on the incidence or prevalence of antimicrobial resistance, an outcome of major current concern. In addition, for most of the studies reported, outcomes were evaluated only while the program remained active. Only one report described sustainability of changes in practice beyond the period of the intervention . FinallyC. difficile colitis is also needed.Current evidence is insufficient to support recommendations for a specific program, or any specific program components. Further evaluation of antimicrobial stewardship programs in long term care facilities, including the impacts of specific program components, is needed. Provision of on-site infectious diseases specialty consultation may be an effective intervention, but this is likely not realistic for most facilities. It seems reasonable for programs to have flexibility for customization to address local considerations. However, it should also be possible to standardize stewardship activities recommended for all facilities by identifying a library of effective program elements. Evaluation of the impact of stewardship programs on the relevant adverse outcomes of antimicrobial resistance and The author declares that she has no competing interests.The author is solely responsible for the inception, review, content, and writing of the manuscript.


To have public health impact, evidence-based interventions (EBIs) must be implemented appropriately at meaningful scale. The Center for Disease Control and Prevention’s Replicating Effective Programs and Diffusion of Effective Behavioral Interventions programs disseminate select EBIs by providing program materials and training health providers on their appropriate use and implementation. Sociometrics’ HIV/AIDS Prevention Program Archive (HAPPA) and Program Archive for Sexuality, Health, and Adolescents (PASHA) are likewise the largest EBI collections targeting sexual risk behaviors in the private sector. This study examined the extent to which organizations that obtain EBIs from HAPPA and PASHA implement, adapt and evaluate them and factors associated with program implementation.Survey data were collected from 123 organizations that acquired, and had been in possession for a minimum of six months, at least one EBI from HAPPA or PASHA between January 2009 and June 2011. Data regarding program characteristics and date of acquisition were obtained from Sociometrics’ sales and marketing databases. Logistic regression was used to assess barriers to program implementation.Among organizations that obtained an EBI from Sociometrics intending to implement it, 53% had implemented the program at least once or were in the process of implementing the program for the first time; another 22% were preparing for implementation. Over the three-year time period assessed, over 11,381 individuals participated in these interventions. Almost two-thirds (65%) of implementers made changes to the original program. Common adaptations included: editing content to be more current and of local relevance (81%); adding, deleting or modifying incentives for participation (50%); changing the location in which the program takes place (44%); and/or changing the number, length and/or frequency of program sessions (42%). In total, 80% of implementers monitored program delivery. Participant outcomes were tracked by 78%; 28% of which used evaluation designs that included a control or comparison group. Lack of adequate resources was significantly associated with decreased likelihood of program implementation .Findings provide greater understanding of implementation processes, barriers and facilitators that may be used to develop strategies to increase the appropriate use of EBIs. Since the beginning of the epidemic, considerable resources have been spent developing and rigorously evaluating HIV prevention programs. A wide variety of evidence-based interventions (EBIs) have been shown to reduce HIV-related sex and drug risk behaviors among a range of high risk populations. Consequently, over the past several years, government and other funding agencies have expressed a preference for making use of existing, well-tested HIV prevention interventions and approaches ‘that work,’ rather than spending resources on innovation -3. The CSeveral strategies have been developed to promote the translation of efficacious HIV prevention programs into real-world settings. For example, the CDC’s Replicating Effective Programs (REP) and Diffusion of Effective Behavioral Interventions (DEBI) projects provide materials, training, and technical assistance to support implementation of EBIs ,7. AlthoThe availability of replication kits for effective HIV prevention programs facilitates, but does not guarantee, their appropriate use by practitioners . HarshbaA growing body of literature has identified a wide variety of reasons why organizations may have difficulty implementing EBIs -19. AmonThe overall objective of this study was to examine how EBIs for HIV prevention disseminated via Sociometrics’ HAPPA and PASHA are used in real world settings. Specifically, we assessed: the extent to which these EBIs are implemented; the extent to which the EBIs that are implemented are modified, evaluated and/or sustained over time; and organizational characteristics associated with implementation. This is the first implementation study of EBIs for HIV prevention to assess a private-sector alternative to the CDC’s REP and DEBI dissemination programs.We employed a universal sampling scheme to reach all organizations that had obtained at least one EBI via HAPPA or PASHA in the recent past (between January 2009 and June 2011). During this time, a total of 187 organizations obtained 381 program packages for 50 unique programs. Reasons an agency may order multiple program packages include, but are not limited to, that they plan to implement a particular program in multiple sites or that they would like to closely examine materials for a variety of EBIs before selecting which, if any, to implement. A total of 16 (9%) organizations were unable to be contacted, 48 (26%) were successfully contacted but did not participate in the survey because an individual responsible for the program(s) obtained could not be identified or declined to participate, and 123 (66%) completed the survey. Individuals who completed the survey had worked an average of 9.8 years (standard deviation [SD] = 7.5 years) in HIV prevention and included executive directors, program directors, managers, coordinators, and facilitators, health educators, and other prevention specialists. In total, 107 (87%) were female. A total of 86 (70%) self-identified as being White, 25 (20%) as Black, 10 (8%) as Hispanic, 1 (1%) as Asian, and 1 (1%) as mixed race/ethnicity.A minimum of five phone call attempts were made and two emails sent to reach an eligible representative from each organization. Eligibility requirements included being 18 years of age or older and responsibility for the selection and/or implementation the HIV prevention program package(s) obtained from Sociometrics. All organizations had been in possession of their selected EBI(s) for six months to almost three years prior to being contacted.i.e., number, age, sex, race/ethnicity, sexual orientation, HIV status, and substance use behaviors of persons enrolled in the program); type of adaptations made ; reasons for program adaptations made ; process evaluation activities conducted ; outcome evaluation activities conducted ; and training and technical assistance received . Data regarding characteristics of the EBIs such as type , duration , original implementation setting , program package format , and number of months organizations were in possession of the program prior to completing the survey, were obtained from Sociometrics’ sales and marketing databases.Survey data were collected from September to December 2011 via SurveyMonkey. Items assessed included: organizational characteristics such as type and main activities of the organization, type of area served, extent to which HIV prevention is a priority, number of paid full-time equivalent staff, number of paid full-time equivalent staff who work specifically in HIV prevention, and implementation of other EBIs; reasons for EBI acquisition; extent to which program implementation was achieved; and level of agreement (1 = strongly disagree to 5 = strongly agree) regarding six factors posited to influence program implementation, including lack of program alignment with the needs of local population and/or setting, poor program fit with other agency programs or goals, lack of availability of funding and other resources, absence of a ‘champion’ committed to program implementation, lack of organizational support for the program, and lack of support from local stakeholders. For each factor, responses were dichotomized as ‘yes’ or ‘no’ that it was a barrier to program implementation. Participants representing organizations that had implemented or were currently implementing the program were additionally asked a series of yes or no questions regarding: characteristics of the target population reached (Respondents completed the survey for only one program obtained per organization. For organizations that had obtained multiple programs (as opposed to multiple copies of the same program), study staff randomly selected the program about which the participant completed the survey. The surveys lasted approximately 10 minutes to 30 minutes, depending on whether the organization had ever implemented the program obtained. Survey participants were compensated $25 for their time, either as a personal check or donation to their organization. All of the procedures were approved by Sociometrics’ Institutional Review Board.All analyses were conducted using SPSS 17.0 . Initial descriptive analyses were performed to examine the extent to which program implementation was achieved as well as reasons for not implementing the EBI obtained. Statistical analyses were performed in a step-wise fashion using logistic regression with 95% confidence intervals for each odds ratio. First, we tested whether various organizational and program characteristics were associated with whether program implementation had occurred. All of the variables that were significantly associated with program implementation were then entered in a single block. All analyses controlled for length of time an organization had been in possession of the program prior to completing the survey. Statistical significance was defined as p <0.05.Table The majority of EBIs obtained by organizations included in the study were small-group (76%), multi-session (93%) programs, originally implemented in community-based (40%) or clinic-based (39%) settings. A total of 57% of the program packages for these EBIs were obtained from Sociometrics in a downloadable, versus hard-copy, format. On average, organizations had been in possession of their EBIs for 16.4 (SD = 8.1) months.A total of 45 (37%) organizations had neither implemented their selected EBI nor were planning to do so. Of these, 19 (42%) had not ever intended to implement the program. Rather, commonly cited reasons for EBI acquisition included: to obtain information about the program for a grant application or research proposal (11%); to use activities from the intervention as part of another program offered (5%); to help inform the development of a new program (11%); and/or to provide training or technical assistance to others implementing the program (42%).Among organizations that obtained their selected EBI with the intent to implement it (N = 104), 55 (53%) had implemented the program at least once or were implementing it for the first time , and another 23 (22%) were in the process of preparing for implementation. Among those that had implemented their selected EBI at least once (N = 31), 68% had done so more than once, and 48% were still offering the program.EBIs obtained by organizations surveyed for this study had been implemented with more than 11,381 individuals. Table e.g., updating information presented to be more current and of local relevance; adding, deleting, or modifying incentives for participation; changing the location in which the program takes place) and more substantial  changes to the EBIs’ original content and designs. A total of 78% of organizations that made such changes did so to increase the relevance, acceptability, or appeal of the program to the target population (78%), followed by 47%, 22%, and 19% that did so to increase the feasibility to implement it given resource constraints, relevance and compatibility of the program with their mission, and/or ‘buy-in’ from other local stakeholders, respectively.Among organizations that implemented the EBI they obtained (N = 55), 36 (65%) reported having made changes to the original content and design. Table Among organizations that implemented the EBI they obtained (N = 55), 44 (80%) had conducted or were conducting one or more of the following process evaluation activities: tracking program participation (84%); assessing participant satisfaction with the program (86%); assessing staff attitudes toward the program (66%); and assessing fidelity of program implementation (64%). In total, 43 (78%) had assessed and/or were tracking participant outcomes such as changes in participant knowledge, attitudes or behaviors. Study designs for these assessments included: post-test survey only (5%); pre-test and post-test surveys (53%); and pre-test, post-test, and follow-up surveys (42%). A total of 28% included a control or comparison group. Among organizations that reported having made adaptations to the EBI they implemented or were currently implementing (N = 36), 89% and 75% assessed or were monitoring program delivery and/or assessing participant outcomes, respectively.None of the organizational or program characteristics listed in Table In bivariate analysis, program implementation having not occurred was associated with a lack program alignment with the needs of local population and/or setting; lack of adequate funding, staff, and other resources; and lack of the presence of a ‘champion’ committed to program implementation. In multivariable analysis, only lack of adequate funding, staff, and other resources remained independently associated with decreased likelihood of organizations having implemented the EBI they obtained .Among program implementers (N = 55), 85% reported having received a grant or donation to fund the program. Only 5% reported that no additional funds were needed for program implementation; rather, the EBI had been integrated into other ongoing initiatives, programs or activities. The remainder did not specify how program implementation had been or was being supported.Among organizations that implemented the EBI they obtained (N = 55), 76% reported having received training or technical assistance with program implementation and/or evaluation activities including: training on program implementation in general (45%); training on how to implement or help implement a specific program (47%); training on how to adapt EBIs in general (47%); training on how to adapt or help adapt a specific program (33%); training on program evaluation in general (45%); and/or training on how to evaluate or help evaluate a specific program (31%). Likewise, among organizations that were planning for but had not yet begun program implementation (N = 23), 65% reported that staff had received or would receive training and/or technical assistance in one or more of the areas described above.i.e., preparing for implementation). Thus, a total of 75% of organizations were actively working with their respective programs within a minimum of six months of having obtained them. Moreover, many organizations that obtained EBIs via HAPPA and PASHA have successfully been able to sustain program implementation over time.In order to maximize their public health impact and successfully change behavior among diverse populations, EBIs must be widely disseminated, appropriately implemented, and sustained over time. Our findings indicate that organizations may obtain materials for evidence-based HIV prevention programs for a variety of reasons, including: to implement the program, to obtain information for a grant application or research proposal, and/or to provide training or technical assistance to others implementing the program. Among those who obtained EBIs with the intent to implement them, a majority (53%) had implemented the program at least once or were in the middle of implementing it for the first time. Almost another quarter (22%) were in the preimplementation phase  of program implementers had made changes to the original content and/or design is consistent with previous implementation studies of DEBI programs, where adaptations were found to be commonplace ,20,22,23The most common type of changes reported by study participants were editing content to be more current and of local relevance, and adding, deleting or modifying incentives for participation. These are relatively minor, and generally considered allowable, adaptations. Conversely, changing the number, length and/or frequency of program sessions, substituting activities, and/or deleting activities  are much more likely to threaten program fidelity and, in turn, potentially negatively affect desired outcomes. The ADAPT and ADAPT-ITT frameworks are logic models that detail the steps involved in organizations’ adoption, adaptation and implementation of EBIs ,24. TherIn recent decades, the nonprofit sector has faced increasing pressures of accountability for program success, with an emphasis on measuring outcomes and impacts rather than just activities and inputs . Many fue.g., culture, climate, work attitudes) and program implementation should be the focus of future research [Although use of EBIs is considered to be a more cost-effective approach to HIV prevention than developing new programs, of the organizational characteristics assessed in this study, availability of funding, staff, and other resources was nonetheless the only factor found to be independently significantly associated with whether program implementation took place. For most (85%) of the organizations that had achieved program implementation, this work was supported by an outside grant or donation. Given funding to implement an EBI, two other important factors associated with program implementation are the goodness of fit between the intervention and needs of the local context, and the presence of a program champion. The relationship between other factors associated with organizational context not assessed in this study . Although the survey asked about attempts at evaluation, we do not know what measures were assessed or how many of these efforts were able to document significant changes. Owing to the cross-sectional nature of this research, we also cannot make causal interpretations regarding factors that influence program implementation. The study procedures controlled for the influence of agencies that purchased more than one type of program package. It is a good strategy for organizations to consider multiple options and choose that which is the best fit for their contexts. By randomly selecting the program about which representative from these agencies completed the survey, in some cases, it might appear that an agency had failed to implement an EBI when in fact stakeholders may have made a thoughtful determination to implement an alternative one.There were several limitations to this study. Participants represented only 66% of eligible organizations; thus, caution should be exercised when generalizing the findings. Selection bias among the respondents may have led to a skewed picture of EBI implementation. This study also relied on self-reported, rather than objective, measures of program implementation, adaptation, and evaluation activities, thus may be subject to recall and/or social desirability biases. We attempted to mitigate the former by limiting our sample to include only organizations that had obtained their respective EBIs in the past three years. Likewise, the selection criteria for respondents included that they have been involved in the review, selection, implementation, and evaluation of evidence-based programs at their respective organizations and were familiar with the program package that was obtained from Sociometrics. It is possible, however, that staff turnover may have affected the reliability of some respondents’ answers (The decision to acquire a program is distinct from the decision to implement it and how it gets implemented. When translating EBIs to real world settings, challenges are to be expected. Despite its limitations, this study demonstrates the success of Sociometrics’ program archives to promote the adoption, implementation and evaluation of a wide variety of evidence-based HIV prevention programs among a diverse pool of practitioners nationwide. Moreover, its findings provide greater understanding of implementation processes, barriers and facilitators that may be used to develop strategies to increase the use of EBIs.Both authors were employed by Sociometrics Corporation at the time the research was conducted.SDC conceptualized the research design, conducted the interviews and analyses, and served as the lead writer. JJC supervised all aspects of the study and assisted with writing. Both authors have read and approved the final manuscript.


The Great East Japan (Tōhoku/Kanto) earthquake of March 2011was followed by a major tsunami and nuclear incident. Several previous studies have suggested a number of psychological responses to such disasters. However, few previous studies have modelled individual differences in the risk perceptions of major events, or the implications of these perceptions for relevant behaviours. We conducted a survey specifically examining responses to the Great Japan earthquake and nuclear incident, with data collected 11–13 weeks following these events. 844 young respondents completed a questionnaire in three regions of Japan; Miyagi (close to the earthquake and leaking nuclear plants), Tokyo/Chiba (approximately 220 km from the nuclear plants), and Western Japan . Results indicated significant regional differences in risk perception, with greater concern over earthquake risks in Tokyo than in Miyagi or Western Japan. Structural equation analyses showed that shared normative concerns about earthquake and nuclear risks, conservation values, lack of trust in governmental advice about the nuclear hazard, and poor personal control over the nuclear incident were positively correlated with perceived earthquake and nuclear risks. These risk perceptions further predicted specific outcomes . The strength and significance of these pathways varied by region. Mental health and practical implications of these findings are discussed in the light of the continuing uncertainties in Japan following the March 2011 events. Drawing on transaction appraisal In this paper we predict that earthquake and nuclear risk perceptions will correlate with two sets of outcomes. Earthquake risk perception will positively correlate with those activities most likely to ameliorate the consequences of an earthquake, namely keeping an earthquake kit and making modifications to the home. Similarly, nuclear risk should positively correlate with particularly precautionary activities; namely avoidance of particular foods, avoiding going outside, and wearing a face mask to avoid radiation. Stocking up on food, and contemplating leaving Japan are relevant to both risk events, and will be positively correlated with both risk assessments (earthquake and nuclear risks). In line with previous research into risks th earthquake and nuclear incident.In this paper we collected data from young people two to three months after the Great East Japan earthquake. Data was collected on individual values, the anxieties of friends and family, perceived control over risk, trust in government advice, perceived risk, and actions taken as a result of the earthquake and nuclear incidents. We collected this data three different regions of Japan: Miyagi, close to the epicentre of the earthquake N = 235; Tokyo and Chiba, N = 247; Western Japan (Yamaguchi and Nagasaki), N = 362). All data was collected over 12 days between May 30th and June 11th 2011 (i.e. 11–13 weeks after the earthquake). Data collection dates did not differ significantly across sites: site collection dates were 30th May –7th June (Western Japan); 7th June –9th June (Tokyo) and 9th June –11th June (Miyagi).Respondents were 844 university students attending seven Universities located in the three areas listed above  M = 2.70, SD = .84), the second normative concerns about the nuclear incident . Our third predictor (perceived control over safety) similarly included two items . while our fourth predictor assessed trust in government’s advice about the radiation risks . Perceived risk itself was measured by two items, one assessing perceived risk from a further earthquake, a second risk from a future nuclear incident. Behavioural responses to the earthquake or nuclear incident were assessed by three sets of items, the first set specific to earthquake risk (two questions), the second specific to nuclear risk (three questions), and the third applicable to both risks (two questions).Details of the questionnaire items are provided in 2x = 1441.598, df = 66, p<.001, GFI = .757, CFI = .286, RMSEA = .157, SRMR = .145) r = .31). The last cluster consisted of a single item, “considered leaving Japan.” We utilized cluster scores by averaging items within each cluster to model the relationships among variables. After fitting the model to the pooled data first, we moved to comparing the model fit across three different sites, using a step-by-step multiple-group comparison procedure Following descriptive analysis of respondents across sites, we examine significant differences by region in both risk perception and behavioural responses using analysis of variance, controlling for sex and age. We then perform a structural equation analysis using AMOS (version 18.0) to examine predictors and outcomes of both earthquake and nuclear risk perception. First, data distribution were examined and multivariate normality evidenced in light of Mardia’s coefficient x2 (2) = 23.97, p<. 01; 59% of respondents were male in Miyagi compared to 41% in Western Japan and 39% in Tokyo/Chiba). Our Tokyo/Chiba respondents were also significantly older  = 69.78, p<.001: M age = 23 for Tokyo/Chiba, M age = 19 in each of the other two data sites).Respondent characteristics from the three regions were first analysed across age and sex. There were significant effects for age and sex across the samples. There were more male respondents in our data set from Miyagi  participants in general perceived ‘some’ risk of a future earthquake or nuclear incident seriously impacting on their safety  and 2.75 (.73) on a 4-point scale, high scores indicating higher risk: see Given a four point scale  = 9.59, p<.001), although it was those in Miyagi that were most likely to report having someone close to them killed or seriously injured in an earthquake  = 53.72, p<.001). Respondents in Western Japan were significantly less likely to perceive a risk from a nuclear incident than those in the other two areas  = 8.66, p<.001). Respondents in Western Japan were less likely to report avoiding foods or drink  = 29.39, p<.001) or going outside  = 28.74, p<.001), and were less likely to consider leaving Japan  = 6. 98, p<.001). Those in Western Japan were also less likely to report keeping an earthquake kit (2x (2) = 138.58, p<.001), making changes to the house (2x (2) = 120.49, p<.001) or stocking up on food since the earthquake and nuclear incident (2x (2) = 168.52, p<.001).Analysis by region indicated significant differences in both risk perception and behavioural responses see . Control2x = 415.11, df = 25, p<.001, GFI = .913, CFI = .610, RMSEA = .136, SRMR = .101)  , we first tried to fit a fully-mediated baseline model with parceled items, which showed a poor fit ( = .101) . After f = .046) . In this2x = 145.34, df = 81, p<.001, GFI = .969, CFI = .930, RMSEA = .031, SRMR = .052). The resultant fit indices suggested that the same structural configurations can be fitted to the three different samples 2x = 192.21, df = 105, p<.001, GFI = .958, CFI = .905, RMSEA = .031, SRMR = .066). Further comparative tests however indicated that not all the structural coefficients were uniform across the three sites see .As can be seen from the figures, normative indicators of earthquake or nuclear risk were significant positive predictors of earthquake and nuclear risk across all the sites, with regression weights ranging from.16 (Western Japan) to.21 (Miyagi) for the relationship between earthquake norms and earthquake risks, and from.14 (Western Japan) to.32 (Miyagi) for nuclear norms and nuclear risks. Normative nuclear risks also had a direct positive relationship with nuclear related actions across sites (regression weights ranging from.10 (Western Japan) to.27 (Tokyo)). In addition, across all sites, perception of control over a nuclear risk was significantly negatively related to both anxiety about a nuclear risk (weights ranging from −.11 (Western Japan) to −.14 (Migayi)), and positively related to nuclear actions (from.11 (Tokyo) to.15 (Western Japan)). However, trust in governmental advice was significantly (negatively) related to nuclear risk perception in only Miyagi (−.19) and Tokyo (−.24), while conservation values were only significantly (positively) related to either earthquake or nuclear risk perceptions in Tokyo . Earthquake risk was significantly positively related to earthquake acts in Miyagi (regression weight = .14) and Western Japan (.12); nuclear risk was significantly positively related to nuclear acts in Tokyo (.26) and Western Japan (.20). Finally, nuclear risk was positively related to contemplating leaving Japan in Miyagi (.13) and Tokyo (.18), but not Western Japan (.10). We discuss these variations in our conclusions below. Although included in our overall model , earthquThe great East Japan earthquake, the subsequent tsunami, and the continuing uncertainty about nuclear leakages provide serious challenges to the citizens of that country. Consistent with our expectations, we found significant relationships between four antecedent variables  and their perceptions of risk. These risk perceptions in turn predicted changes in preventive actions  and avoidance behaviours . Our results, however, indicated significant differences in regional responses to the threat, with greater anticipated risk of a future earthquake in Tokyo than Miyagi or Western Japan, while behavioural changes were larger in areas most affected by the March 2011 events. Our findings also indicated significant regional variations in the relationship between values, trust in government and risk perceptions, and between risk perceptions and behavioural outcomes. We discuss each of these in more detail below.First, let us consider our findings from across the samples. Consistent with previous work on the psychological predictors of anxiety and risk perception Shouganai (“it cannot be helped”). Controllability therefore may be less of a significant predictor of anxiety or avoidant activities when faced with such naturally occuring threats.As anticipated, normative perceptions of threats correlated significantly with perceived risks: during widespread threat, individuals seek to reduce their anxieties by sharing their concerns with others Our findings also suggest that anxiety about future earthquakes and anxiety about nuclear threat may lead to different behavioural outcomes. Although relatively few houses collapsed as result of the 2011 Great East Japan earthquake Our analyses suggested significant regional differences in both levels of risk perception and behavioural responses, and the relationship between the variables in our structural model . We consth earthquake and the following aftershocks. This may be due to several reasons. Those who live or study in ‘high risk’ areas may find themselves in a ‘dissonant state’, where their desire for safety may clash with their potential ‘high risk’ habitat hibakusha) was reported in earlier generations, amongst those exposed to the A-bomb In our data, it was the residents of Tokyo and nearby Chiba that were most anxious about further earthquakes, not those nearer the epicenter of the March 11Turning to regional variations in our model , individWhat are the mental health implications of the 2011 earthquake? Despite the stress –related responses that often follow earthquakes and other natural disasters, resilience to such disasters has been reported in several studies in Japan For all this, particular groups are likely to remain vulnerable, with earthquakes liable to trigger multiple negative life events The media are likely to play an important role in risk perception ‘making sense’ of traumatic events, informing the public about necessary reactions, as well as providing information about the continuing threat to the wider public Our study had several limitations. Students may not represent the wider Japanese community, who may be differentially impacted by the earthquake. Young mothers, for example, may be a particular risk group following the Fukushima nuclear incident, with large numbers of such mothers leaving areas such as Tokyo and Chiba th earthquake and tsunami (estimated to number around a third of a million persons) Further outcomes can also profitably be explored, particularly given the continuing challenges faced by the huge number of refugees created by the March 11Figure S1Original conceptual model. Note: For the poor-fit original model, we tested all the regression paths from predictors to mediators, then from mediators to outcomes variables. However this fully mediated model does not fit well (see text above). For parsimony purpose, we did not draw all the regression paths but instead use two large arrows to illustrate the structural relationships.(TIF)Click here for additional data file.Figure S2Baseline model for all respondents. Note: *p<.05, **p<.01, ***p<.001. D0– D4 denote disturbance terms. D0 and D1 are correlated, whereas D2, D3 and D4 are correlated with each other. E. Norm = earthquake normative concern, Conserv = conservation scores, N. Norm = nuclear normative concern, N. Trust = trust in governmental nuclear advice, N. Contr. = control over safety, E. Risk and N. Risk = risk of an earthquake (or nuclear event) affecting safety. Nuclear acts are “avoid certain foods and drinks,” “avoid going outside,” and “wear masks.” Quake acts’ are “kept an emergency kit’ and “modifying houses.” Leave is “considered leaving Japan.”(TIF)Click here for additional data file.Table S1Residual matrices for the three study sites.(DOCX)Click here for additional data file.


For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors.Biosensors have attracted increasing attention as reliable analytical instruments in mapp) was calculated to be 3.78 μM.A novel disposable biosensor was constructed by immobilizing laccase (Lac) with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs)-doped screen-printed electrode (SPE). Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA) as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 μM with a detection limit of 0.42 μM (S/N = 3), and the Michaelis-Menten constant (KThe immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE). This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds. Laccase (Lac) has been widely used to construct electrochemical biosensors for phenolic and their derivatives, because it can catalyze the oxidation of phenolic compounds accompanied by the reduction of oxygen ,2. The hThere is of intense interest in the construction of Lac-based biosensors using nanomaterials, due to their unique and particular properties . Silica Multi-walled carbon nanotubes (MWCNTs), with high surface area and excellent biocompatibility, are also a promising candidate as the matrix material to incorporate enzyme and construct enzyme-based biosensors ,12. Impoin-situ analysis [Screen-printed electrode (SPE) is a kind of planar sensor device with various substrates that are coated with layers of electroconductive and insulating inks at controlled thickness . Severalanalysis -22. It ianalysis ,24. Howeanalysis . While panalysis , ionic lanalysis  and enzyanalysis ,27-dopedHerein, our goal is to construct a disposable electrochemical biosensor by immobilizing Lac on the MWCNTs-doped SPE using silica spheres as immobilization matrix (Lac/Si/MWCNTs/SPE) without cross-linking reagents. The morphology and the electrochemical properties of the constructed biosensor were characterized. Moreover, its electrochemical sensing properties were evaluated by selective measurements of dopamine (DA). Figure Trametes versicolor , dopamine (DA), ascorbic acid (AA) and tetraethyl orthosilicate (TEOS) were purchased from Sigma-Aldrich . All other reagents were of analytical reagent grade purity and all solutions were prepared using deionized water obtained with a Mili-Q system . MWCNTs were supplied by Shenzhen Nanotech Port Co. Ltd.  with a typical diameter of 10–30 nm and length of 5–15 μm, and their purity was 95-98%. Besides, silver paste, carbon paste, silver/silver chloride paste , and insulating paste  were used to fabricate SPEs.Laccase from Scanning electron microscopy (SEM) results were obtained by using a Zeiss utra 55 field-emission SEM instrument . All the electrochemical measurements were performed with a CHI-1211A portable electrochemical workstation . The measurements were performed at room temperature (~15°C).2 working area for each were fabricated according to the process described by our previous work [As the base electrodes for the printing process, SPEs with a standard three-electrode system and a 3.1 mmous work ,29 with Silica spheres were synthesized according to the Stöber’s method . Typical-1, prepared in 0.10 M pH 5.0 PBS) stock solution thoroughly in a volume ratio 2:3 for 24 hours. Then, 2.5 μL mixed solution of silica-Lac was coated onto the surface of the MWCNTs/SPE to form Lac/Si/MWCNTs/SPE as the disposable biosensor. After the solvent evaporated, the constructed biosensor was washed with deionized water to remove excess Lac. For comparison, Lac modified SPE (Lac/SPE), Lac modified MWCNTs/SPE (Lac/MWCNTs/SPE), and silica spheres modified MWCNTs/SPE (Si/MWCNTs/SPE) were fabricated with the similar steps. All of the modified electrodes were stored at 4°C.Before modification, the bare SPEs were pretreated in pH 7.0 potassium phosphate buffer solution (PBS) by applying an anodic potential of 2.00 V for 300 s. The synthesized silica spheres colloidal suspension was mixed with Lac  are usually electrochemically active and subsequently re-reduced on the surface of the electrode at the appropriate potentials. DA, as a typical model of  are usuap) are 0.051 V and 0.044 V, characteristic of a two-electron and two-proton quasi-reversible redox process of DA at both SPEs [The results of Figure oth SPEs . No cathoth SPEs . Furtheroth SPEs .Since the proton participates in the electrochemical reaction, the pH value of the supporting electrolyte is considered to be an important parameter affecting the electrochemical behavior of the biosensor . The curAnother important parameter affecting the responses of the target analyte at the disposable biosensor is the loading amount of MWCNTs. SPEs doped with different mass proportions of carbon paste and MWCNTs were investigated in order to choose an optimum loading amount of MWCNTs. As shown in Figure DA (μM) – 2.091, R = 0.9908)  of the disposable biosensor were evaluated , which shows a better resolution and a higher signal-to-noise ratio comparing with CV . As dispequation :s refers to the steady-state catalytic current, Imax is the maximum current measured under saturated conditions, and C is the concentration of DA. The Kmapp value was estimated to be 3.78 μM. This small Kmapp means that Lac immobilized with silica spheres on the MWCNTs/SPE possesses very high enzymatic activity for the determination of DA. These results indicate that the disposable biosensor has a good analytical performance for DA.where IThe selectivity of the disposable biosensor to DA determination was also investigated. AA was added into the solution which contained 85.5 μM DA  containing of 60.0 μM DA. It was found that the current responses stayed at the same level after 20 sweep segments, indicating that Lac was immobilized stably on the SPE. The results further proved that the proposed immobilization method was effective and it was not necessary to use any other cross-linking reagents. The possible reason may be due to the large loading area, good biocompatibility and the stabilizing property provided by silica spheres and MWCNTs, attributing to their porous and three-dimensional architecture. The nanosized pores on silica spheres and MWCNTs could act as small cages surrounding the Lac, consequently offering a protective chemical microenvironment which is similar to the microenvironment near enzyme in biological cells. Furthermore, the interconnected pores and a well-defined three-dimensional network of the proposed immobilization matrix can prevent Lac from leaching into the solution while allow free diffusion between the matrix and product molecules from/to the catalytic active sites. Therefore, even if in the absence of cross-linking reagents, the developed biosensor can still show good stability during the detection ,36.To verify the reproducibility of the disposable biosensor, five different Lac/Si/MWCNTs/SPEs fabricated by same steps independently were chosen randomly from 50 pieces of store SPEs. The R.S.D. for the cathodic peak current responses to 60.0 μM DA was 6.5%, meaning that the constructing procedures were reliable and the modified SPEs had a good reproducibility. In addition, the same Lac/Si/MWCNTs/SPE was used to detect DA for five times successively. As a result, the R.S.D. value was 4.7%, showing a good repeatability.The storage stability of the disposable biosensor was also investigated. After 10 and 30 days saving at 4°C, the current response of Lac/Si/MWCNTs/SPE reached to 91.0% and 86.0% of the initial response respectively in the 60.0 μM DA solution. The good stability may ascribe to the effective protection of the bioactivity of Lac due to the consistent stability of silica spheres and the biocompatible microenvironment provided by silica spheres and MWCNTs.in situ determination of DA with good sensitivity, selectivity and reproducibility. In summary, the proposed approach of enzyme immobilization shows a great potential for the construction of biosensors without using cross-linking reagents, and the constructed biosensor displays an excellent analytical performance for phenolic compounds in a rapid and cost-effective way.A novel disposable biosensor has been successfully constructed on MWCNTs/SPE by immobilizing Lac with silica spheres. Due to the large specific surface area and excellent biocompatibility of MWCNTs and silica spheres, the biosensor can effectively provide a suitable microenvironment for the immobilization of Lac and exhibit a good electrocatalytic performance for DA. In addition, based on a non-oxidative electrochemical mechanism, the biosensor enables the The authors declare that they have no competing interests.YL carried out the experimental work, participated in data collection and analysis, involved in drafting and revising the manuscript. ML participated in fabricating SPEs and data analysis. LZ, DL, ZP revised it critically for important intellectual content, and gave final approval of the version to be published. All authors read and approved the final manuscript.


We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons.Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (K Ras is a member of a large family of small GTPase proteins that bind to and hydrolyze guanosine triphosphate (GTP) into guanosine diphosphate (GDP) In order to image intracellular signaling activity, many sensors based on FRET have been developed Recently, a more sensitive imaging method based on FLIM in combination with a FRET-based Ras sensor optimized for FLIM, FRas, has been developed R59A-mRFP) accumulates in the nucleus, limiting the sensitivity of the Ras sensor. In this paper, we identify the sequence in RBD that causes nuclear localization and introduce a point mutation to generate a new sensor, FRas2-F, which overcomes this problem. In addition, we develop a FRas2 variant, FRas2-M, with slightly increased RBD affinity. The new sensors show significantly improved sensitivity over previous sensors in both 293T cells and neurons.While it has been demonstrated that the sensitivity of FRas-F is sufficiently high for imaging Ras signaling in small subcellular compartments such as dendritic spines of pyramidal neurons R59A-mRFP were previously described R59A-mRFP or pCI-mRFP-RBD-mRFP to produce pCI-mRFP-RBDR59A,K108A-mRFP and pCI-mRFP-RBDK65E,K108A-mRFP, respectively.The constructs pCI-mEGFP-HRas, pCI-mRFP-RBD-mRFP and pCI-mRFP-RBD6)-superfolder GFP (sfGFP)-HRas without the C-terminal CAAX membrane targeting sequence 6-mRFP–RBD mutants were cloned into the pET bacterial expression vector. Proteins were overexpressed in Escherichia coli (BL21(DE3)pLysS), purified with Ni+-nitrilotriacetate (NTA) column  and desalted on a desalting column  equilibrated with 50 mM Tris-HCl (pH 8), 100 mM NaCl, 3 mM MgCl2 and 1 mM dithiothreitol (DTT). The concentration of the purified protein was measured by the absorbance of the fluorophore p) and GDP by incubating in the presence of 15 mM ethylenediaminetetraacetic acid (EDTA) for 30 min at 37°C. Then, 10 mM MgCl2 was added to the reaction Purified sfGFP-HRas (without the CAAX membrane targeting sequence) was loaded with 25-fold molar excess of 2′,3′-O-The dissociation constant . Approximately 16–18 hours after transfection, the medium was replaced with DMEM with low FBS (0.5%) for 8 hours, and subjected to imaging in a solution containing 30 mM HEPES (pH 7.3), 130 mM NaCl, 2.5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 2 mM NaHCO3, 1.25 mM NaH2PO4, and 25 mM glucose 293T cells (ATCC #: CRL-11268) were cultured in Dubelco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% COin vitro (DIV) 2 to inhibit the proliferation of non-neuronal cells. Cells were transfected at DIV 3 using Lipofectamine 2000 (Invitrogen) as described previously Cortical neurons were prepared from newborn Sprague Dawley rats at postnatal day 0 as described previously A custom-built 2-photon microscope equipped with a Ti:sapphire pulsed laser  tuned at 920 nm and a mode-locked Ytterbium-doped laser  were used for imaging mEGFP and mRFP distribution, respectively, in 293T cells. The intensity of each laser beam was independently controlled with electro-optical modulators . The two laser beams were combined using a beam-splitting cube and passed through the same set of galvano-scanning mirrors and objective . Imaging of dissociated cultures of cortical neurons were performed on a different custom-built two-photon microscope with a Ti:sapphire pulsed laser  tuned to 920 nm for imaging of mEGFP- and mRFP-tagged constructs. Fluorescence was divided with a dichroic mirror  and detected by photomultiplier tubes  for green, R3896 (Hamamtsu) for red) after wavelength filters  To generate the fluorescence lifetime image, we calculated the mean photon arrival time, To quantify Ras activation, the fraction of mEGFP-Ras bound to mRFP-RBD-mRFP was calculated by fitting the fluorescence decay curve summed over the whole image with R59A-mRFP) was concentrated in the nucleus, with relatively low expression in the cytosol  show cytosolic localization   .R59A: 2.0 µM; RBDR59A,R108A: 1.7 µM). The affinity of FRas2-M  was ∼0.5 µM, in between FRas  and FRas-F, as expected To further characterize the effect of the K108A mutation on the affinity between Ras and RBD, we measured the affinity between purified sfGFP-HRas without the CAAX membrane targeting sequence and mRFP-RBD or its mutations in the presence of either non-hydrolyzable GTP analog (Gpp(NH)p) or GDP using fluorescence lifetime measurements in a cuvette ; Table 1To test the sensitivity of FRas2 variants (FRas2-F and FRas2-M) compared to that of FRas and FRas-F, we transfected these Ras FRET sensors in 293T cells, and imaged them with 2-photon fluorescence lifetime imaging microscopy (2pFLIM) . To actiR59A-mRFP) was strongly accumulated in the nucleus ; and fast sensor with low affinity and lower sensitivity (FRas2-F). To quantify the spatiotemporal dynamics of Ras using these sensors, one must measure the dependency of sensor kinetics on RBD concentration and extrapolate to zero expression level Previously, it has been reported that the inactivation of FRas-F is much faster than FRas due to its lower affinity FRas-F has been shown to be useful for imaging Ras activity in single dendritic spines undergoing synaptic plasticity