PD) in an earlier randomized controlled trial. This study aimed to describe the adaptation process and feasibility of implementing the HiBalance program for PD within primary healthcare settings.Translating evidence into practice requires adaptation to facilitate the implementation of efficacious interventions. A novel highly challenging balance training program  was found to improve gait, balance, and physical activity in persons with Parkinson’s disease  received HiBalance training three times weekly for 10 weeks while another (n = 4) trained twice weekly plus a once weekly home exercise program (HEP). Balance performance was the primary outcome, while secondary outcomes  were also evaluated.Feasibility was assessed in terms of study processes and scientific evaluation. Nine persons with mild–moderate PD.Regarding process feasibility, attendance was high (approximately 90%) in both groups, and experiences of the group and home training were positive. Newly selected outcome measures were feasible. The scientific evaluation revealed few adverse events and no serious injuries occurred. Concerning outcomes per group, the average change in balance performance and gait speed was equal to, or exceeded, the minimally worthwhile treatment effect commonly used in The findings support the feasibility, in terms of process and scientific evaluation, of the adapted HiBalance program for implementation within clinical settings. A sufficiently powered study is required to ascertain whether the newly proposed program offers similar short and long‐term effects as the original program. Results from the process and scientific feasibility will be used to inform the larger implementation trial.2.3HiBalance program have previously been described in detail  was proposed and developed over several months by the same expert group during a workshop and thereafter circulated for comments, where progression and variation aspects of exercises were added. Patients attending rehabilitation clinics in Sweden are always given home exercise programs on top of their rehabilitation interventions to aid self‐management and promote overall cardiovascular fitness. This adjunct program mainly focuses on aerobic capacity, strengthening of lower extremity and core muscles—components that can be performed unsupervised with minimal risk of falls, and which have been shown to improve balance control and gait in PD , was used as in the efficacy trial. Conversely, gait speed was manually assessed with the timed 10‐m walk test, as opposed to the electronic walkway. The 6‐min walk test was added to evaluate exercise endurance. Lastly, accelerometery  measured free‐living physical activity by aggregating step counts. Self‐reported measures included the Falls Efficacy Scale–International (FES‐I) for evaluating concerns of falling, the Walk‐12  evaluated self‐perceived limitations in walking ability, and the EQ‐5D assessed health‐related quality of life. Time taken to complete testing was recorded for all individuals. Upon program completion, participants reported their perception of the balance training and HEP using a questionnaire.For the assessment of process feasibility, attendance/adherence rates, eligibility criteria, the appropriateness of data collection tools/outcome measures, time taken to complete all measurements, and participants’ experiences of the program were investigated. 2.4.2Safety of the intervention was assessed by recording the nature and frequency of adverse events which included falls, injuries, fatigue, and pain. To assess the effectiveness (responsiveness) of the newly proposed dose, participants were randomized to (1) receive training three times weekly (Group 3x), (2) receive group training twice weekly and perform the HEP once a week (Group 2x + HEP).2.5Data were analyzed descriptively, using the proportion of individuals per group who changed their outcome status at post‐testing. This study was not sufficiently powered to test between‐group differences and report on the statistical superiority of one training dose over the other. However, we discussed the observed changes in relation to their clinically meaningful important differences.33.1Group 3x attended 137 of 150 sessions (91%) and Group 2x + HEP, 74 of 80 therapist‐led sessions (93%) and 32 of 40 (80%) home training sessions. All data collection procedures, that is, self‐reported outcomes and clinical tests, were completed with ease by participants, indicating the clinical feasibility of selected measures. Measurements took between 90 and 120 min to complete. Concerning recruitment, a number of patients under 60 years, but who fulfill all other criteria, was excluded, which suggested broadening of the inclusion criteria. Both groups reported therapist‐led sessions as balance challenging and exercise difficulty as progressive in nature, especially with the introduction of dual‐tasking. All participants, including the subject who had missed some official HEP sessions, in the Group 2x + HEP, expressed their willingness to continue with the HEP after the intervention period.3.2Concerning adverse events during therapist‐led sessions, three participants fell without causing injury; all stumbled over artificial hindrances that were created to challenge balance. Another participant reported pain associated with the training which lasted more than 2 days, and another felt dizzy during the session but recovered after a resting period. No participant reported negative events during the HEP.Four of five individuals in Group 3x improved their balance performance and two of four in Group 2x + HEP, while no one reduced their balance function Figure . The ave4Results of this study reveal that the adapted HiBalance program was feasible in mild–moderate PD within a primary healthcare setting, in that attendance rates were high, adverse events few, and the effects on balance performance and secondary outcomes were detectable and favorable. The results support initial feasibility of the intervention which was adapted to suit the healthcare context. However, a larger and more rigorously designed multisite study is required to establish whether the newly adapted program offers similar short‐ and long‐term effects as the original program.According to the feasibility typology, assessment of the process revealed good adherence as approximately 90% of the total sessions were attended by participants. The eligibility criteria were similar to that used in the RCT (Conradsson et al., As part of the scientific evaluation, adverse events were few during the therapist‐led training program and HEP. Three falls were recorded during the training sessions, with all of them occurring during the advance stages of exercise progression. All falls occurred on a soft surface and no injuries were reported. Risk of falls, however, cannot be removed during training sessions due to the highly challenging nature of the program. To minimize falls, challenging balance exercises were not included in the HEP, while PT’s remained near participants during training sessions.Training resulted in improved balance performance. Despite the small sample size, both groups improved their balance performance by at least one point on the Mini‐BESTest, which is also the minimal detectable change in mild–moderate PD (Löfgren, Lenholm, Conradsson, Ståhle, & Franzén, This study presented with several limitations. Firstly, the sample size was too small to sufficiently power outcomes and to adequately evaluate adverse events. Also, the lack of a control group undermined true treatment effects. Lastly, the lack of assessor blinding may have introduced measurement bias. Taken together, future larger and more rigorously designed studies are warranted to provide evidence on the cost‐effectiveness of the adapted HiBalance training program within clinical settings. Translating this training program into practice may offer clinicians an effective option to retrain and maintain balance and gait in persons with PD, an area that lacked proven interventions (Conradsson, Leavy, Hagströmer, Nilsson, & Franzén, In conclusion, this study demonstrates that the adapted HiBalance program is feasible from both a process and scientific/effectiveness perspective. A larger multisite study is needed to test the effectiveness of the adapted program on balance and gait outcomes as well as to inform future widespread implementation.All authors have no conflict of interest to declare.


Zimbabwe adopted voluntary medical male circumcision (VMMC) as a priority HIV prevention strategy in 2007 and began implementation in 2009. We evaluated the costs and impact of this VMMC program to date and in future.Three mathematical models describing Zimbabwe’s HIV epidemic and program evolution were calibrated to household survey data on prevalence and risk behaviors, with circumcision coverage calibrated to program-reported VMMCs. We compared trends in new infections and costs to a counterfactual without VMMC. Input assumptions were agreed in workshops with national stakeholders in 2015 and 2017.The VMMC program averted 2,600–12,200 infections (among men and women combined) by the end of 2016. This impact will grow as circumcised men are protected lifelong, and onward dynamic transmission effects, which protect women via reduced incidence and prevalence in their male partners, increase over time. If other prevention interventions remain at 2016 coverages, the VMMCs already performed will avert 24,400–69,800 infections  through 2030. If coverage targets are achieved by 2021 and maintained, the program will avert 108,000–171,000 infections  by 2030, costing $2,100–3,250 per infection averted relative to no VMMC. Annual savings from averted treatment needs will outweigh VMMC maintenance costs once coverage targets are reached. If Zimbabwe also achieves ambitious UNAIDS targets for scaling up treatment and prevention efforts, VMMC will reduce the HIV incidence remaining at 2030 by one-third, critically contributing to the UNAIDS goal of 90% incidence reduction.VMMC can substantially impact Zimbabwe’s HIV epidemic in the coming years; this investment will save costs in the longer term. Zimbabwe has mounted a strong response to its generalized HIV epidemic –3. AveraVoluntary medical male circumcision (VMMC) has been established as an effective and efficient intervention against the spread of HIV; three randomized clinical trials in the early 2000s showed that it reduces the risk of female-to-male HIV transmission by 60% –10, and Zimbabwe adopted VMMC as a priority HIV prevention strategy in 2007 and began service delivery in 2009. Its initial plan was to reach 80% coverage among young men (ages 15–29) by 2017. Pilot studies and modeling of expected impact and cost –18 inforIn 2016 the original targets were revised using a simple linear projection model, the Decision Makers' Program Planning Tool (DMPPT) . The proAvenir Health applied the Spectrum Goals model , which hAll models independently projected the historical, ongoing, and future HIV epidemic in Zimbabwe: the Goals and ICL models for each of Zimbabwe’s ten provinces, and EMOD for the country as a whole but additionally fitting age-specific behavioral and epidemic outcomes  was determined from self-reported MC rates in Demographic and Health Surveys (DHS) from 2005/6  and 2010Numbers of VMMCs performed by age stratum were taken from client intake records at facilities for 2009–2014 and from the National District Health Information System  for 2015Although it may be beneficial to include HIV-positive males in VMMC programs , ZimbabwOur counterfactual throughout is a 'no VMMC ever' scenario; that is, we assume that there was/is never a VMMC program in Zimbabwe, and MC coverage instead remains constant at the 2008 level in each province .Our first scale-up scenario considers the program up to 2016 only; that is, the models capture the VMMCs performed over 2009–2016 (described above), but assume that no further VMMCs would be performed after 2016.Our second scale-up scenario supposes that the VMMC program targets—scale-up to 80% coverage among 15–29 year old men and 30% among 10–14 year-olds—are achieved by 2021 and maintained thereafter. We evaluate this scenario in two possible settings, which capture developments with other HIV interventions in Zimbabwe over the same time period. In a 'status quo' setting, the VMMC program could be accompanied by continuations of recent trends in antiretroviral therapy (ART) scale-up, with constant coverage of prevention interventions and constant rates of risk and (non-VMMC) protective behaviors. For the Goals model, this translates to extrapolating the trend in the proportion of people on ART from 2014–2016  through For an additional "pessimistic" model scenario, we assume that the funders of Zimbabwe’s existing program—PEPFAR, the Global Fund to fight AIDS, Tuberculosis and Malaria, and the Bill & Melinda Gates Foundation—discontinue district-level programs when their current commitments end in 2018/2019.In the Goals model, ART reduces the HIV transmission probability per sex act by 80% in the 'status quo' scenarios , increasing to 90% in the Fast Track scenario in all provinces. In the ICL model, ART reduces the per-partnership transmission probability by 85% in all scenarios. EMOD simulates viral suppression from ART and assumes suppressed individuals are non-infectious.Results for each model and scenario were evaluated and compared for: new HIV infections, numbers of VMMC conducted, cost of VMMC and cost of ART, and the resulting number of VMMCs or cost per infection averted, relative to the counterfactual scenario without the VMMC program. We did this in turn for multiple timeframes, looking either historically (over 2009–2016) or into the future (projecting forward from 2017 through 2030). In the Results section, the presented ranges reflect differences in parameters and structure between the three models  continues to increase over 2022–2030.In our scenarios in which no future VMMCs are performed (i.e. the program ended in 2016) or the program is halted in 2018/19 as current funder commitments expire, coverage in the 15–49 year old cohort falls gradually back to the baseline level of 9.2% as uncircumcised 14-year-old boys age into the cohort and circumcised 49-year-old men age out.Across the three models, the VMMC program has averted an estimated 2,600–12,200 HIV infections over 2009–2016. This has required 69–335 circumcisions or a cost of $7,600-$36,500 to avert one new infection .Against a 'status quo' backdrop of continued ART scale-up, we evaluated the impact of VMMC over the longer timeframe of 2009–2030 . The VMMAchieving the VMMC targets by 2021, with maintenance thereafter, will avert 108,000–171,000 infections  over 2009–2030. VMMC is an efficient intervention, requiring only 19–30 circumcisions per infection averted. Sustaining the accelerated VMMC program will require continued funding and support. Indeed, the US President's Emergency Plan for AIDS Relief (PEPFAR), one of the main funders, plans to continue supporting VMMC programs in 14 countries and aims to achieve saturation by 2020 in priority districts . HoweverWhile additional funds will be required to meet the VMMC target by 2021, after the target is reached, fewer annual resources will be required to maintain VMMC coverage. Over 2009–2030, the cost to avert one HIV infection with VMMC will be only $2,100–3,250. The models agree that once the target VMMC coverage levels have been reached, the net annual cost of maintaining treatment provision and the VMMC program together will be lower than what it would cost to maintain treatment in the absence of the VMMC program, because the aversion of new infections by VMMC will reduce the long-term treatment need. The 'break-even point' occurs in 2022 in the Goals projections, 2030 in the ICL projections, and 2035 in the EMOD projections . The redIn the Fast Track scenario with accelerated scale-up of ART and additional prevention interventions alongside VMMC  MC coverage taken from the 2015 DHS and 2016 PHIA surveys rather than program data  in the number of infections averted and slightly improved cost per infection averted over 2009–2016, but a small reduction (7.5%) in infections averted and cost-effectiveness over 2009–2030 . These eProjections by our three independent models show that the Zimbabwe VMMC program will substantially impact the country’s HIV epidemic in the coming years. Cost per infection averted is low compared to most other HIV prevention interventions, as well as in comparison to key global non-HIV public health/infectious disease interventions  stem from the ICL model considering only ages 15–49  and depicting a lower proportion of total incidence occurring among men than women, though both models are consistent with historical data. Additional uncertainty comes from the age distribution of incidence relative to the age distribution of new VMMCs. The age-structured EMOD model may, in this respect, have provided the best impact estimate, although data limitations make it difficult to fully validate modeled age structure in sexual networks. Nevertheless, our results are in line with earlier evaluations of VMMC cost-effectiveness in other sub-Saharan African settings; for example, VMMC was estimated to cost between $131 and $3,160 per infection averted across three regions in Mozambique , and $4,Compared to projections used to inform Zimbabwe’s first VMMC program plan (ASCOP) in 2014 , the curOur current analysis—and past cost-effectiveness analyses , 40, 41—Preliminary outputs from the 2016 PHIA  estimateOur ingredients-based unit cost for VMMC included national-level costs for demand creation but excluded the costs of monitoring and evaluation, provincial and district management and coordination, resource mobilization, and partner-level overheads . This maOur consensus modeling results, together with identified uncertainties and data limitations, suggest actions that may help Zimbabwe's VMMC program to maximize its impact and cost-effectiveness, and strengthen future evaluations. In particular, uncertainty in the coverage of male circumcision achieved by 2016 .(DOCX)Click here for additional data file.S2 FileTable A. Self-reported Male Circumcision status, in national household surveys.Table B. Program-reported numbers of Voluntary Medical Male Circumcisions in Zimbabwe, by age group, province and year, 2009–2016; and resulting estimated proportion of men circumcised at 2016.(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(XLSX)Click here for additional data file.S5 File(XLSX)Click here for additional data file.


My Journey, a culturally attuned curriculum for 6–8th graders. My Journey is grounded in traditional values and teachings to promote self-efficacy in sexual health decision-making and engagement in prosocial behaviors. We conducted a pilot study with 6–8th grade students (n = 45), aged 11–14 years . Pilot study findings confirm program feasibility and acceptability. The process evaluation revealed that teachers liked the curriculum, particularly its adaptability of cultural components and ease of student engagement. The outcome evaluation demonstrated that My Journey provided an avenue for NA youth to increase their sex refusal self-efficacy. Application of the culture cube framework revealed My Journey has made a meaningful practice-based evidence contribution as a community-defined, culturally integrated curriculum that is effective. Future directions include broader implementation of My Journey, including adaption for additional populations.A clear need exists for teen pregnancy prevention programs that are responsive to the specific needs and cultural contexts of Native American communities. Recent data indicates that the birth rate for Native teens is nearly two and a half times the rate for White teens (32.9 versus 13.2). To address this disparity, we conducted formative research with Northern Plains Native American community members, resulting in  The teen birth rate  has steadily declined over the past decade, with a decrease of 55% from 2007 41.5) to 2017 (18.8), a historic low for the United States (U.S.) . Althoug1.5 to 20Previous research suggests several reasons why teen pregnancy rates among Native American adolescents are higher. Youth Risk Behavior Survey (YRBS) results reveal that, compared to their peers, a greater proportion of Native youth reported a cluster of related risk behaviors, including early sexual debut, sex with multiple lifetime partners, dating violence, forced sex, early alcohol use, and early drug initiation and lifetime drug use ,7. DespiOur previous research with Native American communities about birth control and sex helps elucidate why Native teens may be less likely to use contraception. For instance, Northern Plains Native youth stated that many Native teens believe birth control is a woman’s responsibility, not a man’s, because women, it is supposed, are “more aware” and “more responsible” . AdditioThe unique cultural context within which such beliefs about sex and contraception develop emphasizes the need for innovative, culturally-specific sexual health curriculums for Native youth. Although a number of efforts have built on the assets and strengths of NA cultures to improve the reproductive health of Native youth , there remains a dearth of culturally attuned programs for this at-risk population . EvidencOur work utilizes a practice-based evidence (PBE) framework, “a range of treatment approaches and supports that are derived from, and supportive of, the positive cultural attributes of the local society and traditions,”Isaacs et al. . Much ofFor researchers who work with Native American communities and other non-majority populations, the evidence is ideally derived from the community itself, beginning with the formative research, when researchers ask community members and stakeholders to identify community needs and interventions that would likely yield the most positive results. Indeed, community-based participatory research (CBPR) goes hand-in-hand with PBE, as many researchers have acknowledged ,25, and The inclusion of culture is also critical in the development of PBE intervention programs, and requires the full participation of community members and stakeholders in order for a program to be both accepted and effective. As Walsh-Buhi  titled hMy Journey, the culture cube serves as the empirical framework from which we were able to articulate how culture informed the development of this program, how culture was infused into the development of our evaluation tools and methods, and how local Indigenous ways of knowing and being are integrated into the conceptual underpinnings of My Journey and serve as the community-defined evidence for determining the effectiveness of the program  and elders, as well as 20 interviews with local health care providers and school personnel. Questions focused on facilitators and barriers to teen pregnancy prevention, reasons youth have/do not have sex and use/do not use contraception, attitudes toward teen pregnancy, and recommendations for the content and implementation of a teen pregnancy prevention program. A detailed description of the recommendations for program content and activities is published elsewhere . The topm see in  and utilm see in . My Journey is to equip students with information, tools, and skills that enable them to take charge of their decision-making. Through My Journey, youth consider how culture and environment play important roles in a person’s fundamental beliefs and values. My Journey has youth identify these factors and explore the effects their decisions have on their personal lives, futures, and goals as well as the effects their decisions have on the people in their lives and places in which they live. By equipping students with skills and self-efficacy to make healthy decisions and increasing their positive ethnic identity, we aim to foster their attainment of positive life goals through reducing risk behaviors that lead to unintended teen pregnancy. For more details on the curriculum content, including lesson objectives and activities, see the Curriculum development and pilot testing took place over 3.5 years from start to professional printing. The aim of My Journey is the incorporation of elements of Indigenous cultures throughout the curriculum, which is important to PBE to base programs on local Indigenous worldviews and ways of knowing and being. Culture was an important element uncovered in the needs assessment, which underscores the importance of using a framework such as the culture cube assess if culture is accounted for appropriately. As summed up by this urban female youth (non-parent), “……I think if people were more educated about their culture and like traditions, and how things used to be, and what should be carried on today, maybe they would focus more on that. And like, and maybe that would become more time-consuming and more of like a value too than, rather than choosing to be sexually active” . . . . They also like talking about personal experiences with people they know or what they have read in the media. . . . The students said they learned a lot about pregnancy today.”Many of the observer’s comments focused on the students’ acceptability of the curriculum, namely the engagement and enjoyment of the lesson activities. For example, observers noted, “The kids really liked the Hoop Pass game. They seem like they are beginning to understand the Chain Reaction Model”. Aspects of the activities students appeared to particularly enjoy were the traditional games and STI transmission activity, as seen in the following example quotes:My Journey also noted many strengths of the program, particularly in terms of its acceptability. For example, facilitators observed high levels of student investment and engagement in the course material, attributing this, in part, to the cultural aspects of the program and kinesthetic teaching methods, such as traditional games. Additionally, process evaluation findings provide evidence that My Journey effectively worked towards achieving the outcomes of increasing ethnic identity and pride and sexual health knowledge and responsibility. Observations of My Journey was implemented, program fidelity was highest in the second semester (spring of 2016). This may be due in part to this being the second semester the facilitator was teaching this class, with greater experience, familiarity, and comfort with the curriculum. Fidelity was lowest the third semester . This may reflect the fact that this was the first semester My Journey was taught at this particular school, and this facilitator’s first semester with the curriculum. However, had evaluation of the program continued, we would have likely observed fidelity increasing over time.Of the three semesters M = 3.69) compared to pre-intervention (M =3.47). Although not significant, the decision-making skills and reasons for not having sex also increased while reasons for having sex decreased. Somewhat unexpectedly, we did not find a significant increase in ethnic identity or its subscales of affirmation/belonging and exploration. Similarly, there was no change found on prosocial connectedness.My Journey with the 6 aspects of the culture cube , we were able to determine that My Journey is a community-defined, effective, culturally integrated prevention curriculum for Native youth. My Journey as aligned with the visible and invisible elements of the cube, which was helpful for reflecting on the effectiveness of the cultural elements of the program, such as the Chain Reaction Model, the application of the medicine wheel, and the various activities that draw upon Indigenous cultures and values in order to encourage positive sexual health decision-making. By aligning the elements of My Journey, a culturally attuned sexual health curriculum for Native youth, to address the need for teen pregnancy prevention programs for this population. The results of our pilot study indicate that, not only are the facilitators pleased with the curriculum and its application within tribal youth classrooms (grades 6–8), but My Journey has a positive effect on the self-efficacy of Native teens. Decision-making skills and a respect for one’s self and others are encouraged through the emphasis My Journey places on Indigenous cultures and values, a major component of the curriculum. The lack of significant changes in the study’s quantitative measure of ethnic identity may be due to a ceiling effect, as the students reported high levels of this measure of ethnic identity at the pre-test assessment. Support for this interpretation has been found in a previous study which a youth program failed to create a strong Native American identity, likely because it was attracting Native youth with an already high ethnic identity [My Journey.We created identity . The preMy Journey draws on the rich history and depth of Indigenous cultures to engage and inform Native youth, and as such, culture is one of the strongest, most innovative aspects of the My Journey curriculum. After the application of the culture cube, our confidence in the cultural underpinnings of My Journey, and the community-defined evidence for the curriculum has been bolstered. Developed through CBPR methods and analyzed with a PBE cultural lens, My Journey was developed for a specific population and piloted with a small number of students within that group, it was expected that the numbers might not immediately align with Western definitions of significance. But, as a growing number of researchers have argued, in order to develop any culturally attuned, innovative program for minority populations, we must seriously consider strategies and evidence that may or may not align with Western scientific methods [My Journey should be changed [Increasingly, researchers are beginning to acknowledge the need for innovative program design, implementation, and evaluation when working with culturally and ethnically diverse populations. Many have called for a reevaluation of what constitutes “evidence” when serving non-majority populations, since “evidence” often indicates the use of randomized controlled trials, which require higher numbers of participants than certain communities can provide, as well as a “controlled” environment, which negates the rich, cultural and extemporaneous nuances of the human experience. Because  methods ,46. Some changed  to utiliMy Journey. Our hope is to expand, sustain, and adapt My Journey by securing funding that will allow us to implement the program in other communities and schools, including elementary schools, as requested by the community. Expanding the program beyond Northern Plains tribes would require considerable time, effort, and resources, which may not be readily available. This is especially true for many rural, reservation schools with limited resources whose students and communities continue to face the long-term effects of historical trauma. In future iterations of the program, community input will continue to be critical in order to adapt and create culturally aligned evaluation methods and tools and assess the community’s awareness and satisfaction with the program, how it is being implemented, and its’ impact. We encourage researchers to expand upon these efforts by developing more diverse research teams, including local educators, students, elders, curriculum writers, and researchers in planning and implementation. This will be particularly important when My Journey is adapted and implemented in communities outside of the ones in which it was developed, as this would require adjustments to the PBE used to establish the efficacy of the program. Each tribal community and school is unique and requires thoughtful attention and respect in order to develop culturally attuned PBE interventions. The utilization of the culture cube is one way researchers can develop and/or evaluate PBEs, hopefully leading to a future in which the creation of innovative, culturally attuned PBEs for diverse, non-majority communities is accepted in the broader scientific community.We are encouraged by positive facilitator insights and student growth due to


Bacillus subtilis WB800.Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in P43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive PP43 or PHpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL.Six recombinant strains harboring different plasmids with a single promoter  contains supplementary material, which is available to authorized users. Bacillus subtilis natto (B. subtilis natto) [Nattokinase NK, E.C. 3.4.21.62) was first identified by Sumi et al. from “Natto”, which is a popular traditional Japanese soybean food .4.21.62 . The NK s natto) .B. subtilis is a good host strain for the industrial production of the NK enzyme, as NK was isolated from B. subtilis natto. B. subtilis is a gram-positive bacterium and is a well-studied host for the expression of heterologous proteins because of its many attractive features [B. subtilis is widely used in laboratory studies because it is easy to culture and has a high-level secretory system. In addition, B. subtilis is a food-grade safety strain and presents no safety concerns, as reviewed by the U.S. FDA Center. Some efficient expression systems have been constructed to promote the production of homologous and heterologous proteins in B. subtilis, because of its well-characterized physiological and biochemical properties and nonpathogenicity [B. subtilis strains has been engineered as extracellular-protease deficient strains for the overexpression of subtilisin and β-lactamase in B. subtilis WB600 [B. subtilis WB700 [B. subtilis WB800 [B. subtilis strains [The species features . As a mogenicity –15. B. sis WB600 , 17, theis WB700 , 19, andis WB800 . In addi strains , 22.B. subtilis [B. subtilis and found that the expression level of the gene could increase by using expression systems that contain two or three tandem promoters in contrast to a single promoter. The study demonstrated that the expression of aprL achieved a high level by combining the mutant amyQ promoter with the promoter of the cry3A gene [Thermus scotoductus was overexpressed in B. subtilis, and its productivity was elevated by more than ten-fold when promoted by a dual-promoter system, compared to that of the single HpaII promoter system [B. subtilis. In addition, the dual-promoter PgsiB–PHpaII gave the best performance, which was much higher than PHpaII and PgsiB [HpaII-PamyQ′ was found to sustain superior expression of β-cyclodextrin glycosyltransferase in a B. subtilis strain (CCTCC M 2016536) [Escherichia coli [As is well known, the promoter-regulated gene transcription is usually located upstream of the gene. There are two kinds of promoters: the constitutive promoter that is active in all circumstances and the regulated promoter that become active only in response to specific stimulation in the cell. Because the promoter is a crucial aspect of the expression system, many strong promoters have been screened and characterized in subtilis –26. Recesubtilis , 28. Widy3A gene . The ther system . Researcnd PgsiB . The sys2016536) . Okegawahia coli .B. subtilis WB800 by mediating the gene expression promotion by tandem promoters. Six constitutive promoters, PHpaII, PP43, PBcaprE, PluxS, PgsiB and PyxiE, were selected, and a series of expression cassettes containing single promoters, dual-promoters and triple-promoters was achieved by arranging promoters in different orders. The efficacies of these multiple tandem promoters for controlling the expression of NK are presented.In this study, we aimed to increase the secretory expression of NK in HpaII, PP43, PBcaprE, PluxS, PgsiB and PyxiE, were selected as targets for enhancing the production of NK, and their origins and characteristics are listed in Additional file -PHpaII was constructed in our previous study [-pro-NK with no promoter was constructed first, and five promoters were employed to construct the plasmids pSG-PP43, pSG-PBcaprE, pSG-PluxS, pSG-PgsiB and pSG-PyxiE following the MEGAWHOP method . These six promoters were further inserted into the downstream region of different promoters to result in fourteen kinds of plasmids in which the NK was controlled by dual-promoters. Based on the NK expression level of recombinant strains under the control of dual-promoters, promoter P43 and HpaII were combined in the pattern of three and four promoters in tandem, and ten different kinds of promoters were successfully obtained.As shown in Fig. X) was constructed, as shown in Fig. In addition, another type of tandem promoter  and PluxS (99.2 ± 3.8 FU/ml) were similar, second only to the expression under the control of PHpaII. The promoter PyxiE (20.2 ± 2.0 FU/ml) exhibited the lowest expression level of NK among the six promoters, and its promoter strength was only 14% of PP43. The results of SDS-PAGE and the fibrin plate assay supported the above fibrinolytic analysis results  compared with PP43 (140.5 ± 3.9 FU/ml), PHpaII-PHpaII (199.4 ± 4.8 FU/ml) compared with PHpaII (110.8 ± 3.6 FU/ml), PBcaprE-PBcaprE (120.3 ± 2.4 FU/ml) compared with PBcaprE (103.5 ± 4.2 FU/ml), and PgsiB-PgsiB (48.0 ± 2.2 FU/ml) compared with PgsiB (44.6 ± 2.9 FU/ml). These results showed that the experiments involving PgsiB in tandem or separately did not exhibit an efficient expression of NK.To investigate whether two of these promoters in tandem could enhance NK production, fourteen types of dual-promoters were constructed. The effects of these dual-promoter systems on the expression of recombinant NK were compared by SDS-PAGE and by measuring the fibrinolytic activity Fig. . The NK HpaII-PyxiE was approximately 166.7 ± 2.5 FU/ml, but the production under the control of PyxiE-PHpaII displayed an obviously opposite effect, in which the expression of NK was undetected (0 FU/ml). Similar results were observed in strains harboring pSG-PgsiB-PHpaII (164.9 ± 3.0 FU/ml) and pSG-PHpaII-PgsiB (0 FU/ml), pSG-PBcaprE-PHpaII (175.5 ± 5.0 FU/ml) and pSG-PHpaII-PBcaprE (0 FU/ml), and pSG-PluxS-PHpaII (77.5 ± 4.0 FU/ml) and pSG-PHpaII-PluxS (0 FU/ml).Intriguingly, the dual-promoter system containing different promoters showed that the order of two promoters has an important effect on the expression of NK. The NK activity under the control of PHpaII and PP43 were arranged in tandem, NK was expressed at a high level in the recombinant strain B. subtilis WB800. The NK yield mediated by pSG-PP43-PHpaII reached the highest value (231.7 ± 6.0 FU/ml), which increased by 109% when compared with PHpaII and 64.9% when compared with PP43. The strain harboring pSG-PHpaII-PP43 exhibited the second highest expression of 210.6 ± 5.2 FU/ml. The result of the SDS-PAGE analysis  and PP43-PHpaII-PP43 (182.3 ± 5.6 FU/ml) showed similar promoter strengths, and the production that was promoted by both improved considerably compared with the production of PHpaII and PP43. In contrast, PP43-PHpaII-PHpaII (47.5 ± 3.1 FU/ml) did not exhibit an efficient expression of NK, and PHpaII-PP43-PP43 (0 FU/ml) exhibited no expression of NK. These results indicated that the arrangement of the promoters in tandem has intrinsic effects on the expression level of the target protein.Analysis of NK production showed that promoters PHpaII-PHpaII-PHpaII (213.3 ± 5.1 FU/ml) was increased by 92.2% compared with that under the control of pSG-PHpaII, and by 7% compared with that under the control of pSG-PHpaII-PHpaII. Furthermore, pSG-PP43-PP43-PP43 (219.2 ± 7.7 FU/ml) enhanced the NK production by 55.9% compared with pSG-PP43, and 39.4% compared with pSG-PP43-PP43. The above results of the fibrinolytic activity assays were consistent with those of SDS-PAGE analysis  were constructed, as shown in Fig. X was determined by the fibrinolytic activity and SDS-PAGE analysis  was increased by 81.2% compared with pSG-PHpaII. However, the NK production promoted by 3CPHpaII (138.3 ± 3.8 FU/ml) decreased by 31.1% compared with that promoted by 2CPHpaII. It could be seen that the NK expression that was mediated by pSG-5CPP43 (254.2 ± 5.1 FU/ml) was 80.9% higher than that mediated by pSG-PP43. The expression level of NK increased with the increase in the number of core regions of PP43 up to five. The SDS-PAGE analysis showed that the NK expressive quantity in the supernatant produced by pSG-nCPHpaII , the scale-up of recombinant NK production was completed in a 5-L fermenter using the strain harboring pSG-PHpaII-PHpaII-PP43. The process for the cultivation in the fermenter is shown in Fig. 600 value of 33.0 ± 0.4 at 20 h. Similar to the cell growth, NK production was significantly increased and reached the highest value of 816.7 ± 30.0 FU/ml at 20 h, which was the highest value ever reported. NK production was about two-fold higher in the 5-L fermenter compared to that of the shaking flask experiments. These results indicated that the strain harboring pSG-PHpaII-PHpaII-PP43 had great potential for the industrial production of NK.Our results indicated that the highest overexpression level of NK was produced by the triple-promoter PB. subtilis WB800, and the overexpression of NK mediated by those single promoter systems exhibited significantly different levels. In our study, the highest expression level of NK driven by a single promoter was 140.5 ± 3.9 FU/ml as induced by PP43. The order of the strength of the six single promoters mediating NK expression in B. subtilis was PP43 > PHpaII > PBcaprE > PluxS > PgsiB > PyxiE. However, Guan et al. reported that the activity of the single promoter PP43 was lower than that of PluxS and PyxiE for aminopeptidase expression in B. subtilis [yxiE exhibited higher expression strengths than PP43, both in B. subtilis and E. coli [Six promoters having high expression strength were selected to overexpress the NK enzyme in subtilis . In addi E. coli . The exp E. coli . The groP43-PP43 reached 157.2 ± 3.0 FU/ml, which was 10% higher than that induced by the single promoter PP43. Similar results were observed between the dual-promoter PBcaprE-PBcaprE and the single promoter PBcaprE and the dual-promoter PgsiB-PgsiB and the single promoter PgsiB. However, the strength of the dual-promoter containing two PHpaII was 1.8-fold higher than that of the single promoter. The two promoters PP43 and PHpaII exhibited higher promoter activity for NK expression than that of the other promoters. We further carried out the experiments of arranging PHpaII and PP43 by combining three or four promoters in tandem. As shown in Table P43-PP43 was increased by 11.9% compared with that promoted by PP43, and the NK production mediated by PP43-PP43-PP43 increased by 39.4% compared with that promoted by PP43-PP43. However, the NK expression level increased by only 1.7% when promoted by PP43-PP43-PP43-PP43. Furthermore, NK production decreased under the control of four PHpaII in tandem compared with the expression controlled by three tandem promoters. The results were in agreement with studies that suggested that the length of the promoter affects its expression activity [The NK expression level driven by a dual-promoter Pactivity . AlthougP43 or PHpaII in tandem on the expression level of NK, a series of promoters with core-region repeats (nCPP43 and nCPHpaII) were constructed. The whole sequence of PHpaII is 284 bps; however, the core-region sequence of PHpaII is only 31 bps. The NK production mediated by 2PHpaII and 2CPHpaII almost reached the same level, which suggested that the core region of PHpaII could efficiently initiate the NK overexpression. However, it was unexpected that the strength of 3CPHpaII for NK expression was 35.2% lower than that of 3PHpaII. Further studies will be needed to explore the difference between the whole sequence and the core regions of PHpaII for the level of gene expression. In addition, the whole sequence of PP43 is 300 bps, and the core region of PP43 is 29 bps. The NK expression mediated by the whole sequence of PP43 in tandem increased to that mediated by 4PP43. The NK production that was initiated by core promoters of PP43 in tandem gradually increased as the number of core regions increased. It was found that both strong promoters, PP43 and PHpaII, have distinct characterization and differential expressions of NK. The analysis of the expression level of NK induced by more core regions of PP43 in tandem will be carried out.The core region of a promoter plays an important role in regulating transcription initiation and is the minimal portion of a promoter that is required to properly initiate transcription . To undeB. subtilis. It has been reported that σA- and σB-promoters can function cooperatively. The promoter synergism resulting from the double promoters was found only when the σB-promoter was located upstream of the σA-promoter, and the expression level of reporter gene was severely reduced by switching the locations of the σA- and σB-promoters [gsiB is σB-dependent  compared with that by PHpaII (110.8 ± 3.6 FU/ml) and that by PHpaII-PgsiB (0 FU/ml), suggested that PHpaII might be a σB-dependent promoter. Similar phenomena were observed in the results of NK expression mediated by PBcaprE-PHpaII (175.5 ± 5.0 FU/ml) and PHpaII-PBcaprE (0 FU/ml), by PluxS-PHpaII (77.5 ± 4.0 FU/ml) and PHpaII-PluxS (0 FU/ml), predicting that PluxS and PBcaprE are σB-dependent promoters. Whereas PyxiE is σA-dependent , compared with that by PyxiE-PHpaII (0 FU/ml) and that by PyxiE (20.2 ± 2.0 FU/ml), suggested that PHpaII might also be recognized by σA RNA polymerase. Therefore, promoters PHpaII and PP43 might be recognized by both σA and σB RNA polymerases. Our results showed that the NK expression that was promoted by the dual-promoter system makes a large difference, which could be due to the synergistic effect of the double promoters.Obviously different effects on NK production are caused by different arrangements in the dual-promoter system. The promoter is recognized by the σ factor of RNA polymerase to initiate gene transcription. Several σ factors have been defined in romoters . Since PHpaII and PP43 were generated, from which the NK production showed different levels. Among these 8 strains, one strain harboring the plasmid pSG-PHpaII-PP43-PP43 lost the ability to express NK, and one strain harboring the plasmid pSG-PP43-PHpaII-PHpaII exhibited low activity of NK expression (47.5 ± 3.1 FU/ml). The other six strains harboring the plasmid containing a triple-promoter exhibited relatively high production of the secreted NK, and the NK expression of four strains were higher than 200 FU/ml. The growth curves of strains containing triple-promoters were approximately the same  containing the promoter (PP43) was stored in our laboratory. Transformants were selected on LB agar , supplemented with 100 μg/mL ampicillin for E. coli JM109 or 50 μg/mL kanamycin for B. subtilis WB800. E. coli JM109 was cultivated in LB medium supplemented with 100 μg/mL ampicillin. B. subtilis WB800 was incubated in TB medium  additionally containing 0.02% CaCl2 and 50 μg/mL kanamycin. All of the strains were cultivated at 37 °C under shaking conditions at 200 rpm. Cell densities were measured using a UV-1800/PC spectrophotometer . Strains and plasmids used in this study are summarized in Table The plasmid pMA0911-HpaII from the plasmid pMA0911-pro-NK was carried out following megaprimer PCR of the entire plasmid (MEGAWHOP) [DpnI, and the resulting plasmid was transformed into JM109 to yield plasmid pSG-pro-NK without a promoter  , 42 usinB. subtilis 168 with primers P1 and P2. The amplified product was cloned into pSG-pro-NK by the MEGAWHOP protocol, yielding plasmid pSG-PP43. The other single promoters  were synthesized by Shanghai Sangon Biotech Co., Ltd. and were employed to construct the plasmids pSG-PBcaprE, pSG-PgsiB, pSG-PyxiE and pSG-PluxS, respectively, using the same procedures as for pSG-PP43.The promoter P43 gene was cloned from the genomic DNA of P43-PHpaII was constructed by two steps. The fragment of P43 was amplified from pSG-PP43 using primers P1 and P11, and then the PCR product was inserted upstream of the promoter HpaII in pMA0911-pro-NK following the MEGAWHOP protocol, thereby yielding pSG-PP43-PHpaII. The same procedures were used to construct the other dual-promoter plasmids.The six single promoters were further employed to construct 14 kinds of expression cassettes under the control of two promoters in tandem. The plasmid pSG-PHpaII-PHpaII-PP43, the fragment of HpaII was amplified from pMA0911-pro-NK with primers P12 and P13 and then was inserted into the front of the promoter PHpaII-PP43 in pSG-PHpaII-PP43 following the MEGAWHOP protocol. The other triple-promoter plasmids and two quad-promoter plasmids (pSG-4PHpaII and pSG-4PP43) were obtained after being treated in the same manner as for pSG-PHpaII-PHpaII-PP43.To construct the triple-promoter plasmid pSG-PX harboring the multiple tandem core promoter regions were synthesized by Shanghai RuiDi Biological Technology Co., Ltd. All plasmids were constructed and cloned in E. coli JM109 and were sequenced by Shanghai RuiDi Biological Technology Co., Ltd.The plasmids pSG-nCP600 value of 0.2(v/v), and then was cultivated at 37 °C for 84 h under a shaking condition at 200 rpm for the expression of NK. The supernatant was collected for the following research by centrifugation  at 4 °C.Plasmid transformation was carried out according to the method as previously reported , 44. A s2PO4, 0.2% Na2HPO4, 0.02% CaCl2, and 0.05% MgSO4) containing 50 μg/ml kanamycin. The pre-inoculum culture was 50 mL TB medium including 50 μg/mL kanamycin, which was incubated at 37 °C under a shaking condition at 200 rpm. After 12 h, the culture was inoculated into the 5-L fermenter. The inoculation volume was 8%. The cultivated condition was maintained at 37 °C, and the dissolved oxygen (DO) was performed above 30% under the control of the inlet air and the exponential feeding of glycerol and soybean peptone. During the cultivation process, the pH was controlled at 7.0 through the automatic addition of 50% ammonium solution. Samples were taken every 2 h.Fed-batch cultivations were carried out in a 5-L bioreactor, and the initial medium was 2 L . In brief, 1.4 mL Tris-HCl  and 0.4 mL fibrinogen solution (0.72%) were pre-incubated in a 37 °C water bath for 5 min. Thereafter, 0.1 mL thrombin solution was added, followed by the addition of 0.1 mL diluted sample after 10 min. The mixture was incubated at 37 °C for an hour. Finally, trichloroacetic acid solution (0.2 M) was added and incubated at 37 °C for 20 min to stop the reaction. The supernatant was transferred into a microtest tube after centrifugation , and the absorbance of the supernatant at 275 nm was read and recorded. One unit (1 FU) was defined as the amount of the enzyme that increased the absorbance of the filtrate at 275 nm by 0.01 per minute. The analysis of fibrinolytic activity was independently carried out in triplicate, and the data are presented as the mean ± s.d.The fibrinolytic activity was determined using the method described by the Japan Nattokinase Association (Samples were incubated at room temperature for 30 min with 5 × SDS-PAGE loading buffer and protease inhibitor PMSF (phenylmethane sulphonyl fluoride). Then, the samples were heated at 100 °C for 5 min and were applied into 12% SDS-PAGE with 5% stacking gels. Finally, the gels were stained by Coomassie Blue R-250.Additional file 1:Table S1 Characterization of single promoters used for the NK production. Figure S1 The growth curves of recombinant strains harboring different plasmids with a single promoter. Figure S2 The growth curves of recombinant strains containing a triple-promoter. (DOCX 297 kb)


The ability to predict the eventual size of an earthquake during its early growth stage is a crucial component of earthquake early warning systems. Recent studies have revealed that the onsets of small and large earthquakes are variable but statistically indistinguishable. However, it is unknown whether small and large earthquakes can share the same processes at the same location. Here we show clear evidence of almost identical growth processes shared by repeating earthquakes of various sizes that have occurred in the Naka region, eastern Japan. Our results indicate that a large earthquake is a failure with a large characteristic spatial scale that is initially triggered by a failure with a small characteristic scale, which may also occur independently controlled by subtle differences in the physical conditions, suggesting the existence of a hierarchical structure on the plate interface. Earthquakes are random, but they may also be controlled by such structures. The degree to which small and large earthquakes share the same rupture processes remains unknown. Here, the authors reveal earthquakes of magnitude 3–5 share almost identical growth processes shared, but while they are controlled by some characteristic structures, their final size remains unpredictable. The nucleation model, which is proposed primarily from observations in laboratory experiments3, requires a precursory slow slip that is related to the final earthquake size. The observation of foreshocks is considered a by-product of the slow nucleation process in this model10. However, various types of scale independence12 and the power-law nature13 of natural earthquakes also suggest a large degree of self-similarity, and this forms the basis of the cascade model, in which the earthquake rupture grows as a cascading failure of hierarchical characteristic structures over a range of scales and without any distinguishable nucleation process4.Do small and large earthquakes undergo different nucleation mechanisms? While capturing their initial rupture processes is a non-trivial task, these details are crucial in predicting the event sizes and improving earthquake early warning systems. Inference on the early evolution of a given earthquake lies between the two end-members of the conceptual earthquake rupture model: the nucleation model and cascade model20, the conclusion seems to be that nucleation and rupture growth are statistically common for all earthquakes up to a magnitude limit that is determined by the tectonic environment8. Although this observation favours the cascade model, the applicability of the model, especially the existence of a hierarchical characteristic structure, is still unclear, because previous investigations of the initial nucleation of rupture in seismic waveforms employed either many seismograms from a broad spatiotemporal distribution of earthquakes, or just a few records from a series of closely located events. A more detailed analysis that employs many station records from a series of closely located earthquakes is therefore required to confirm the existence of a hierarchical characteristic structure.The applicability of these two end-member models has been investigated by direct observations of the very beginning of seismic waveforms from earthquakes of various sizes. After more than two decades of debateM ~ 4.8 earthquake sequence has been observed since 2003, consisting of five events to date. The recurrence interval was about 4 years for the first three events, and showed a slight decrease after the 2011 Tohoku-Oki earthquake (M9.0), probably due to the afterslip of the Tohoku-Oki earthquake  that represent the ~M4.8, ~M4, ~M3, and ~M2 events, respectively. Given the relative locations, expected source sizes, and possible errors, it is likely that the rupture areas of the group M events overlap with each other, as for groups A, B, and C. Furthermore, the rupture areas of the group M events include the rupture areas of the events in groups A, B, and C, which suggest a hierarchical structure that is embedded on the plate interface.Some of these earthquakes have been recognised as repeating earthquakes that rupture almost the same area each timeP-wave onsets indicate that the rupture of M2015 and the group B events originated from approximately the same location. Their waveforms are also indistinguishable during the first 0.07 s of the selected events, which is comparable to the typical rupture duration of a M3 earthquake. Furthermore, M2015 nearly stopped growing at ~0.07 s, i.e., at the same time that marks the end of the rupture growth of the smaller events. Although, the Fig. Some of these closely located earthquakes share quite similar initial waveforms. Hereafter, for the investigation of initial waveforms, we return to look at the original seismograms without any filtering. Figure M ~ 3). This small difference indicates that all of these processes occurred within the typical area of a M3 earthquake, which is ~150 m for a stress drop of 38 MPa.We measured the similarity of waveforms using the mean of cross correlation (MCC) between seismograms from 0.1 s before to 0.07 s after the onset, calculated for every station. The stations used here for MCC calculation were the seven stations shown in Fig. Figure However, at this moment, we cannot conclude that all group M events share common initial waveforms with some smaller events. Figure The behaviour of group M events is various since ~0.1 s after the onset. M2015 grew rapidly from 0.07 s, while M2007, M2011, and M2013 had a longer (~0.15 s) period with relatively small amplitude before their rapid growth . Thus, the cascade-up probability from the rupture of the small patch (~M3) to the rupture of the mainshock patch (M4.8) can be estimated to be 40%.It is therefore important to estimate the probability of rupture from a smaller patch growing into the rupture of a larger patch, although our sample domain is currently limited to only five larger earthquakes. Our results indicate that the M2013 and M2015 events started from the rupture of the same small patch, which also occurred as independent group B earthquakes (~M5 Naka earthquake sequence, as well as those of smaller earthquakes, which tend to behave like foreshocks in a broad sense. This represents the first clear observations of such hierarchical rupture growth. Our results suggest the existence of a time-independent characteristic structure that is represented by a hierarchical patch distribution on the plate interface9. A large earthquake is the failure of a large patch, which is the consequence of successive cascade-up processes from a small patch. The small patch may also host an independent smaller earthquake. Whether a small patch rupture grows into a large-scale rupture or not depends on subtle differences in the physical conditions, which we cannot observe here, but may be able to evaluate in a probabilistic sense. Earthquakes are random processes, but they may not be completely random, as the pre-existing characteristics of the seismic region may control the rupture process to an extent. Evaluating the cascade-up probabilities would thus improve the probabilistic forecasting of earthquakes.In summary, the raw seismograms from many stations show minimal differences in the seismic waveforms during the first 0.07–0.13 s of the repeating ~24, with further information available in ref. 26. We used a 4-s window, starting 1 s before the onset of the P-wave and shear-wave arrivals. A 1–10 Hz band-pass filter was applied to the data prior to analysis. The seismic stations that acquired that data analysed here are equipped with 1-Hz 3-component seismometers, and the velocity seismograms are recorded at a sampling rate of 100 Hz.We relocated events using relative arrival times determined by waveform cross-correlation. This method consists of two main steps. First, we estimated the relative hypocentre location for each pair of events by maximising the summation of the waveform cross-correlation for all stations and components. We then determined the set of centroids that was consistent with the relative locations by solving a least-squares problem. This approach is described in ref. For the seismogram comparison in Figs. Supplementary InformationPeer Review File


Erratum: BMC Musculoskeletal Disordershttps://doi.org/10.1186/s12891-018-2048-0weight loss advice and support paragraph of the manuscript is outlined below. All changes to the protocol were made and approved before starting the trial and were prospectively changed on our trial registration (ACTRN12617001595303).After the publication of this protocol , our colAmended weight loss advice and support paragraph: If the patient has a BMI ≥27 kg/m2, the patient will be offered the option of participating in the remotely-delivered weight loss program. The Australian Commonwealth Scientific and Industrial Research Organisation’s (CSIRO) “Total Wellbeing Diet” is based on an evidence-based weight management strategy that utilises a structured, nutritionally balanced eating plan designed to be incorporated into a balanced lifestyle program [http://www.sphealth.com/) on behalf of the CSIRO. After completion of the 12-week program, patients may elect to continue the basic program for an additional 12-weeks. Patients who elect to undertake the online weight-loss program will continue to be supported by the PARTNER Care Support Team throughout their time on the weight-loss program. This program will be undertaken in conjunction with the PARTNER exercise program and educational resources on healthy lifestyle change. program . The pro


A recent study demonstrated the effectiveness of the New Hampshire Colorectal Cancer Screening Program’s (NHCRCSP) patient navigation (PN) program. The PN intervention was delivered by telephone with navigators following a rigorous, six-topic protocol to support low-income patients to complete colonoscopy screening. We applied the RE-AIM  framework to examine implementation processes and consider potential scalability of this intervention.N = 443) were analyzed. Researchers were provided in-depth call details for 50 patients randomly selected from the group of 443. The case study included review of program documents, observations of navigators, and interviews with 27 individuals including staff, patients, and other stakeholders.A mixed-methods evaluation study was conducted including 1) a quasi-experimental, retrospective, comparison group study examining program effectiveness, 2) secondary analysis of NHCRCSP program data, and 3) a case study. Data for all navigated patients scheduled and notified of their colonoscopy test date between July 1, 2012 and September 30, 2013 , and 0.7% late cancellations. Trained and supervised nurse navigators spent an average of 124.3 min delivering the six-topic PN protocol to patients. Navigators benefited from a real-time data system that allowed for patient tracking, communication across team members, and documentation of service delivery. Evaluators identified several factors supporting program maintenance including consistent funding support from CDC, a strong program infrastructure, and partnerships.Factors supporting implementation included funding for colonoscopies, use of registered nurses, a clinical champion, strong partnerships with primary care and endoscopy sites, fidelity to the PN protocol, significant intervention dose, and a real-time data system. Further study is needed to assess scalability to other locations. The effectiveness of patient navigation (PN) in increasing breast, cervical, and colorectal cancer (CRC) screening among disparate populations has been empirically studied since the mid-1990s –5.p < 0.001) [p < 0.115), and more adequate bowel preparation compared to the usual care group  [The New Hampshire Colorectal Cancer Screening Program’s (NHCRCSP) was funded by the Centers for Disease Control and Prevention (CDC) from 2009 to 2015 to provide CRC screening and related support services, including PN. The NHCRCSP served a low income and ethnically diverse population. A recently completed effectiveness study of the NHCRCSP PN program demonstrated a significantly higher rate of colonoscopy completion among navigated patients compared to a similar group receiving usual care  a quasi-experimental, retrospective, comparison group study examining program effectiveness, 2) secondary analysis of NHCRCSP program data, and 3) a case study. This paper presents the methods and results for the second and third evaluation components only; the comparison group study methods and results are presented elsewhere . This stThe NHCRCSP staff team included a program director, a medical director , two navigators, a secretary responsible for assisting in enrolling patients, and a data manager tasked with collecting and reporting program data to CDC. Two registered nurse navigators, one full-time and one quarter-time, delivered PN services telephonically to several hundred patients across the state annually whose screening costs were compensated by NHCRCSP with CDC funding. Patients were New Hampshire (NH) residents at average or increased risk for CRC, ages 50–64, < 250% federal poverty level, and uninsured or underinsured. NHCRCSP patients received their colonoscopy at one of 12 endoscopy sites throughout the state with which NHCRCSP contracted. Given limited resources, the most northern area of NH was not targeted given its small population. The CDC grant supported payment of relevant clinical costs, including colonoscopy. Navigators followed a prescribed protocol addressing six specified topics: (1) patient engagement, CRC screening education, and barrier assessment ; (2) bowel preparation (prep) education and barrier resolution; (3) prep review and readdressing barriers; (4) assessment of prep and confirmation of test day details; (5) day of colonoscopy check-in; and (6) follow-up and assurance of patients’ understanding of results and recommended follow-up . The navData used in the evaluation included a de-identified, patient-level analytic dataset provided by the NHCRCSP and navigator call notes on a subset of patients. Case study data included program documents, results of a patient satisfaction survey, navigator observations, and interviews. All data sources and related analysis are described in detail below.N = 443). To assess colonoscopy completion and other outcomes , data were assessed for a 12-month period from the notification date. Data variables included, among others: patient demographics, residence zip code, comorbidities and CRC personal and family history, number and length of calls (in five minute increments) per navigation topic, appointment no-shows and cancellations, bowel preparation quality, and colonoscopy completion. Basic descriptive statistical analysis of data for the 443 NHCRCSP patients navigated during the defined time period was conducted using SPSS Version 20 to assess patient demographics and outcomes. To determine program reach for this time period, patient zip codes were mapped using geographic software.The analytic dataset included data for all NHCRCSP patients scheduled and notified of their colonoscopy test date between July 1, 2012 and September 30, 2013 . For analysis, documents were reviewed carefully and used to develop a logic model depicting the relationship between programmatic activities and outcomes and a patient flow diagram representing steps to completing colonoscopy including areas of interface with program staff and partners . The logCDC researchers were provided a summary report of satisfaction surveys completed by patients served by NHCRCSP from July 1, 2012 to June 30, 2013. A total of 405 patients were mailed a brief survey following program participation and were encouraged to voluntarily complete and return it. The survey was anonymous and completed by 235 patients (58% response rate). The patient satisfaction survey results were reviewed by the research team.Four CDC researchers each conducted 4 h (16 person-hours) of in-person observations of the navigators on May 13, 2014. Two researchers observed one navigator and two other researchers observed the second navigator. During the observation period, the navigators provided telephonic navigation to patients and made calls needed to facilitate service delivery . Researchers used a standardized, pilot-tested template  to recorTo analyze observation data, field notes were more fully transcribed by observers into electronic format. The team of observers met to review field notes and the call matrices developed during the observations. Researchers focused on defining navigator characteristics  and identifying techniques .n = 6), program stakeholders (n = 9), gastroenterologists (n = 2), practice administrators at endoscopy sites (n = 5), and primary care providers (n = 5). The average length of the interviews was 45 min.Purposeful sampling  was usedTo conduct the interviews, the four CDC researchers split into two teams, each with a trained qualitative researcher with extensive interviewing experience. Within each team, one researcher led the interview and the second served as note taker. Interviewers followed open-ended interview guides specific to the interviewee role. Questions addressed the following topics: aspects of the PN model, experiences related to program implementation including reach, perceived challenges and facilitators to implementation, navigator characteristics and skills, perceptions of program effectiveness, partnerships, sustainability, and patient experiences.CDC researchers conducted additional interviews by telephone with nine NHCRCSP patients from September 12–25, 2014. Patients were asked about their experience with the program and the navigator and interviews lasted, on average, 11 min. Across all interviews, saturation was achieved suggesting an adequate number of interview participants.For analysis of interviews, a professional transcription service transcribed verbatim the digitally audio-recorded data. Researchers verified the accuracy of the transcription by re-listening to the interview audio and making needed corrections in a revised, final transcript. Immediately following vetting, the team created an analytic summary of each interview. Analysis of transcripts involved several iterative steps, consistent with inductive analytic techniques and constant comparative methods. Evaluators conducted initial, focused, and theoretical coding , in progThe RE-AIM framework has five dimensions -- reach, efficacy, adoption, implementation, and maintenance , 11. ReaTwo navigators (1.25 FTE) delivered the PN intervention to 443 patients during the 14-month study period. By delivering PN via telephone, navigators were able to reach patients across the state, aside from a small geographic area representing the least populated area in the state. Patients had access to a cell phone or landline. Patients were offered call cards with pre-paid minutes for their cell phones if needed. Patients received colonoscopy at one of 12 contracted endoscopy sites across the state.Patients were recruited through provider referral (91.2%), self-referral (4.7%), and referral through another CDC-funded screening program, the National Breast and Cervical Cancer Early Detection Program (4.1%). Patients who self-referred included those who heard about the program through an outside source . NHCRCSP engaged federally qualified health clinics (FQHCs) as an important referral source given many FQHC patients are un- or under-insured.The program successfully recruited patients from the intended priority population who met the established age, income, and insurance eligibility guidelines Table . Among tResults of a comparison group study examining outcomes among NHCRCSP patients and a control group of similar patients at one of the 12 endoscopy sites have been reported elsewhere . AnalysiThe medical director, a well-respected gastroenterologist in New Hampshire, acted as the program champion, promoting the program among fellow endoscopists, and was a key component to this successful program. The medical director was viewed as a strong advocate for increasing high quality colonoscopy screening and had credibility with colleagues throughout the state. As a result, twelve endoscopy sites, geographically dispersed across the state, adopted the program and were contracted by NHCRCSP. Navigators also recruited local champions working within endoscopic and primary care practices who adopted the program as their own and promoted it within their settings.Partnerships were critical to program adoption and the medical and program directors had extensive relationships in the cancer community, drawing on them to engage primary care practices and other stakeholders such as the American Cancer Society. In addition, a pharmaceutical chain with locations throughout the state and accessible to NHCRCSP patients was enlisted to provide patients bowel prep materials that were reimbursed by NHCRCSP. A potential challenge for adoption was the lack of resources to pay for cancer treatment among persons diagnosed through NHCRCSP, given the population was uninsured and CDC resources could not be used to pay for cancer treatment. However, prior to implementation, program staff obtained commitments from directors of health systems associated with all the endoscopy sites for free cancer care for the few colorectal cancers anticipated to be diagnosed through the screening program. This commitment was readily given and adhered to for patients requiring cancer care. While the issue of the cost of cancer care was mentioned in interviews, interviewees may have been unaware of commitments made by their leadership, and researchers did not find strong evidence suggesting it was a significant barrier. Table Navigators spent an average of 124.3 min delivering the six-topic PN protocol to patients (range: 40–240 min). Protocol fidelity with four of the six topics was 95% or greater adherence , and partnerships . Staff turnover was also low. The medical director and program director were constant for the full CDC grant period. The data manager and secretary exhibited deep understanding and passion for the program. Navigators originally hired for NHCRCSP were replaced with different nurse navigators prior to our study. Those two navigators remained with the program through the study’s end Table .This study applied the RE-AIM framework to examine implementation processes of a PN intervention found effective in improving outcomes, including colonoscopy completion.Results identified multiple implementation factors that likely contributed to intervention effectiveness including using telephonic navigation to extend reach and providing payment for colonoscopy in order to serve a low-income population. A gastroenterologist serving as a program champion, strong partnerships with primary care clinics and endoscopic sites, and a commitment of charity care for those diagnosed with cancer all supported program adoption. Implementation was aided by fidelity to a rigorous protocol, significant intervention dose, use of well-trained registered nurses with regular clinical supervision, and a real time data system for patient tracking and program monitoring. Maintenance of the intervention was possible given consistent program resources, a strong infrastructure, and low staff turnover.We also found that applying the RE-AIM framework allowed examination of dimensions of implementation often ignored but essential to inform potential transferability and scalability. While calls for comprehensive evaluations addressing dimensions beyond effectiveness and efficacy have increased , 17, comImportant considerations of reach for adopting this intervention in new settings include the ability to recruit the identified priority population and contact them by phone, as well as the availability of a payment source for colonoscopy and, if necessary, cancer treatment. For programs without a ready source of patients, partnerships with health systems serving the priority population will be needed. Also, in this study, while NH is largely rural, limited resources led NHCRCSP to prioritize more populated areas and, therefore, the program was unable to include those in the least populated parts of the state. Others have identified challenges in recruiting rural populations for cancer screening programs and recommend targeted outreach strategies , 19. NexAlong with results of the comparison study previously reported , data frWhile a few stakeholders suggested NHCRCSP patients may have been especially motivated to complete colonoscopy, data assessing patient motivation were not collected, and, therefore, this hypothesis is speculative. In our study, colonoscopy costs were covered for both the NHCRCSP patients and the comparison patients, eliminating cost as a distinguishing factor. Results might be different if patients for whom the cost of colonoscopy is not covered are compared to patients for whom it is; it is reasonable to assume the latter group might be more motivated. At the time of enrollment, patients did agree to participate in a colonoscopy, suggesting that they were amenable to the procedure. Given the complexity of colonoscopy , programAdoption of the NHCRCSP PN intervention was facilitated by program champions and multiple, strong partnerships. There is evidence that champions contribute to the effectiveness of public health programs . The priA potential barrier to adoption if serving an under- or uninsured population is the availability of treatment resources for patients diagnosed with cancer. Other CDC-funded cancer screening programs have identified this as a significant challenge . In suchIn regard to implementation, results suggest that the PN intervention was delivered with fidelity to the PN protocol and with significant dose . These factors have been shown to contribute to positive health outcomes . Use of https://rtips.cancer.gov/rtips/index.do). The manual details all facets of the intervention including the 6-topic protocol. Appendices include sample job descriptions for the navigators, the training curriculum, referral forms, and other needed resources to adopt the intervention. This resource is intended to aid future adoption and maintenance of the intervention. The sustainability of the intervention was not evaluated, and, therefore, it is not clear whether the intervention could be maintained without CDC or other resources.Maintenance of the PN intervention was enhanced through availability of consistent program resources, a strong infrastructure, and limited staff turnover. This suggests that replication of the intervention will benefit from well documented program materials and a carefully vetted and selected staff. CDC, in collaboration with NHCRCSP, has recently produced a replication manual of the PN intervention . The NatOverall, evaluation results, based on the RE-AIM framework, are positive and support further study of the NHCRCSP PN intervention in new settings. Given that this was a relatively small program in a single state (the program served about 2000 patients throughout the state over 5–6 years), additional research is needed to examine scalability and explore generalizability. Scalability is defined as “the ability of a health intervention shown to be efficacious on a small scale and or under controlled conditions to be expanded under real world conditions to reach a greater proportion of the eligible population, while retaining effectiveness” . Future Limitations of the current study should be noted. First, the retrospective design of the effectiveness evaluation render results that are not generalizable. Second, evaluators had limited data  for calculating some process measures, including time spent delivering the intervention and fidelity with the six-topic PN protocol. Third, patient satisfaction surveys were not completed by all participants and it may be that those patients more satisfied chose to complete and return them. However, nearly 60% of patients completed the survey, which is a relatively high response rate.This evaluation of NHCRCSP PN intervention provides robust evidence supporting further investigation to explore scalability. Factors supporting implementation included funding for colonoscopies, use of registered nurses, a clinical champion, strong partnerships with primary care and endoscopy sites, fidelity to the PN protocol, significant intervention dose, and a real-time data system. For public health practitioners considering use of the NHCRCSP PN model, results presented here offer rich information to help assess whether the intervention is a good fit for their unique context and whether they have the capacity to adopt and adapt it. A replication manual for the intervention has been developed and evaluations are underway to assess the intervention in new settings to examine adaptation.Additional file 1. Interview guide used with NHCRCSP non-navigator staff.Additional file 2. Interview guide used with NHCRCSP navigators.Additional file 3. Interview guide used with endoscopists involved in the NHCRCSP.Additional file 4. Interview guide used with practice administrators at endoscopy sites involved in the NHCRCSP.Additional file 5. Interview guides used with primary care providers involved in the NHCRCSP.Additional file 6. Interview guides used with partner organization staff involved in the NHCRCSP.


The factors influencing pubertal timing have gained much attention due to a secular trend toward earlier pubertal onset in many countries. However, no studies have investigated the association between the Great earthquake and early puberty. We aimed to assess whether the Wenchuan earthquake is associated with early puberty, in both boys and girls.We used data from two circles of a survey on reproductive health in China to explore the impact of the Wenchuan earthquake on early puberty , and a total of 9,785 adolescents  aged 12–20 years from 29 schools in eight provinces were recruited. Wenchuan earthquake exposure was defined as those Sichuan students who had not experienced oigarche/menarche before May 12, 2008. Early puberty was identified as a reported onset of oigarche/menarche at 11 years or earlier. We tested the association between the Wenchuan earthquake and early puberty in boys and girls. Then, subgroup analysis stratified by the age at earthquake exposure also was performed.In total, 8,883 adolescents  with a mean (SD) age of 15.13 (1.81) were included in the final sample. In general, children exposed to the earthquake had three times greater risk of early puberty . Subgroup analysis showed that the adjusted RR was 1.90 [1.19–3.03] for boys and 2.22 [1.75–2.80] for girls. Earthquake exposure predicted almost a fourfold (RR [95%CI] = 3.91 [1.31–11.72]) increased risk of early puberty in preschool girls, whereas the increase was about twofold (RR [95%CI] = 2.09 [1.65–2.64]) in schoolgirls. Among boys, only older age at earthquake exposure was linked to early puberty (RR [95%CI] = 1.93 [1.18–3.16]).Wenchuan earthquake exposure increased the risk of early puberty in boys and girls, and preschoolers were more at risk than schoolchildren. The implications are relevant to support policies for those survivors, especially children, to better rebuild after disasters. Pubertal timing is of great interest to both health providers and the public. There’s evidence that early puberty is associated with a broad range of short- and long-term adverse health consequences, especially for adolescent girls . The phyIn addition to chemical exposures and obesity, emotional stress is another culprit that could jump-start puberty earlier . AccordiWar is another harsh environmental stressor that could influence pubertal timing . The 193The 8.0-magnitude Wenchuan earthquake that occurred on May 12, 2008, in Sichuan Province was the biggest and deadliest earthquake since the 1950 Tibet earthquake and 1976 Tangshan earthquake in China. This disaster impacted over 15 million people, rendered at least 4.8 million homeless, and left 69,197 people dead, 374,176 injured and 18,222 listed as missing . The disThe data in the current study are drawn from 2013 and 2015 cycles of a multisite survey on the reproductive health among Chinese middle school students aged 12-20 years from 29 schools in eight provinces. The 2013 survey included Sichuan, Hebei, Shaanxi, Shandong, Heilongjiang, and Guangxi, while the 2015 survey sampled Sichuan, Anhui and Inner Mongolia. The data was collected electronically in student computer labs , and incThe institutional review board of Shanghai Institute of Planned Parenthood Research reviewed and approved this study (2012–01). Written consents were obtained from both parent and child before the survey.For the present study, which was not originally designed to estimate the individual-level exposure to Wenchuan earthquake, we labeled the students who lived in Sichuan as a nature indicator of the Wenchuan earthquake exposure, and their peers in other provinces as a non-exposure group. The Sichuan students were sampled from Wenchuan country, Mianzhu city, Deyang City, Luojiang country, and Zhongjiang country. The distance from the quake’s epicenter (kilometers) is 9.60, 94.23, 100.94, 130.76 and 147.76 respectively.We measured pubertal timing by age at first ejaculation (oigarche) for boys or period (menarche) for girls and defined early puberty as a reported onset of oigarche/menarche at 11 years or earlier . OigarchWe identified potential confounders by drawing causal acyclic graphs (DAGs) based on prior knowledge in the exposure-outcome association . In thisUnivariate statistics were used to describe the mean age, the frequencies of the individual level and macro level characteristics, and the rate of early puberty. We examined the association of Wenchuan earthquake exposure with early oigarche and early menarche and calculated the strength of associations that measured by rate ratio (RR) with 95% confidential interval (CI) . We alsoWe estimated the strength of association of earthquake exposure with RR because this natural experiment can be understood as a retrospective cohort study that earthquake exposure was identified from the recorded information. Statistical analysis used Stata/SE, version 15.1 .A total of 8,883 adolescents from 29 schools in eight provinces were included in the analysis, of which 4,543 were boys (51.14%), and 4,340 were girls (48.86%) . The meap < 0.001]. Similarly, p < 0.001).The rate of early puberty was 2.55% for boys and 10.14% for girls . As illuP < 0.050; girls: 2.22 [1.75–2.80], P < 0.050). As shown in p = 0.003; schoolgirls: RR = 2.09 [1.65–2.64], p < 0.001). The association was non-significant among adolescent boys who were exposed to the Wenchuan earthquake before age seven years , but significant among the surviving schoolboys We conducted subgroup analysis for males and females respectively, given that the children’s age at the time of the earthquake may serve as an effect-measure modifier on the association between earthquake exposure and early puberty . The earTo our best knowledge, this is the first study to investigate the potential impacts of the Great earthquake on pubertal timing. The 2008 Wenchuan earthquake offers a unique opportunity to explore the effects of the earthquake on the quake survivors’ later lives. In this natural experiment, exposure to the Wenchuan earthquake doubled the risk ratio of early puberty . What’s more, there may be effect modification by children’s age at earthquake exposure. Earthquake exposure predicted almost a fourfold increased risk ratio of early puberty in preschool girls, whereas the increase was about twofold in schoolgirls. Similarly, earthquake exposure doubled the risk ratio in schoolboys. However, the association of earthquake exposure with early puberty was not statistically significant in preschool boys. The main reason for the unanticipated result was the sample was too small (Unexposed = 50[Cases = 2]).Factors that influence pubertal maturation have gained much attention recently because of the secular trend to earlier puberty . There aIn the opinion of evolutionary-mind thinkers, accelerated somatic development was adaptive under dangerous or unpredictable environments because this tactic increased the chances of dispersing genes across generations before dying . The adaConsiderable research on adverse health consequences of disaster events including earthquakes and hurricanes has focused on mental health . Our finThe underlying biological mechanisms that earthquakes might influence the early puberty are still barely understood. The stressors, including early childhood adversity and earthquake exposure, can affect not only the hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes individually, but the cross-talk between the two axes, possibly mediating HPA-HPG coupling . The mecThe primary strength of this study is the clear temporal relationship between Wenchuan earthquake exposure and early puberty, which improved our ability to make a valid causal inference. To avoid temporal ambiguity, we excluded individuals who had experienced oigarche/menarche before May 12, 2008. Also, this study took advantage of the natural experiment generated by the Wenchuan earthquake to investigate the long-term effect of the Great earthquake on early puberty.Our study has several limitations worth noting. First, the earthquake exposure was not measured at an individual level that could have allowed the higher accuracy of exposure estimates. There was not any information on death nor illness caused by the earthquake in their family members in the present study. Second, we failed to investigate the association of severity of exposure with earlier puberty due to the the small sample size in the most damaged areas (237 in Wenchuan and 303 in Mianzhu). Third, the data on oigarche/menarche were collected by self-report and thus may be prone to recall bias. However, there is much evidence to show a high degree of validity in recalled age at menarche, especially among adolescents . Fourth,Exposure to the Wenchuan earthquake elevated the risk of early puberty in both boys and girls, and preschoolers are more vulnerable to earthquakes compared with schoolchildren. The findings suggest that the health of children, especially younger children, must remain a focus in the recovery from disasters.10.7717/peerj.5085/supp-1Supplemental Information 1Click here for additional data file.