Consistently in wild-type cells, pravastatin, an HMGR inhibitor decreased ergosterol and squalene, and the effect was more pronounced on squalene. In hmg1-1 mutant and in wild-type cells treated with pravastatin, the decrease in the levels of farnesyl pyrophosphate and geranylgeranyl pyrophosphate respectively was larger than that of ergosterol but was smaller than that of squalene. In Δerg6 or Δsts1 cells, mutants of the genes involved in the last step of the pathway, ergosterol was not detected, and the changes of intermediate product levels were distinct from that of hmg1-1 mutant. Notably, in wild-type cells miconazole and terbinafine only slightly decreased ergosterol level. Altogether, these studies suggest that the pleiotropic phenotypes caused by the hmg1-1 mutation and pravastatin might be due to decreased levels of isoprenoid pyrophosphates or other isoprenoid pathway intermediate products rather than due to a decreased ergosterol level.Currently, statins are the only drugs acting on the mammalian isoprenoid pathway. The mammalian genes in this pathway are not easily amenable to genetic manipulation. Thus, it is difficult to study the effects of the inhibition of various enzymes on the intermediate and final products in the isoprenoid pathway. In fission yeast, antifungal compounds such as azoles and terbinafine are available as inhibitors of the pathway in addition to statins, and various isoprenoid pathway mutants are also available. Here in these mutants, treated with statins or antifungals, we quantified the final and intermediate products of the fission yeast isoprenoid pathway using liquid chromatography-mass spectrometry/mass spectrometry. In  The isoprenoid pathway is essential for all organisms. Regulation of the isoprenoid pathway has been extensively studied in mammals for many years, because this pathway produces such critical end-products as steroid hormones, cholesterol and bile acids Statins are selective inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which inhibit the biosynthesis of cholesterol and thereby reduce serum cholesterol levels in humans. In addition to the inhibition of cholesterol synthesis, statins have been shown to possess anti-inflammatory and immune-modulatory pleiotropic effects, even in patients with normal cholesterol levels Schizosaccharomyces pombe (S. pombe), the primary role of the isoprenoid pathway is the synthesis of the essential sterol and nonsterol isoprenoids. Nonsterol isoprenoids are same as that of mammals, but the essential sterol is ergosterol, the main sterol of most higher fungi erg1+ product squalene epoxidase erg11+ product lanosterol demethylase that is inhibited by azoles S. pombe) provides a useful model system to study the regulation mechanism of isoprenoid pathway. We previously isolated an allele of the essential hmg1+ gene encoding HMGR, hmg1-1, as a mutant that showed hypersensitivities to high temperature and to FK506, a calcineurin inhibitor hmg1-1 allele contained an opal nonsense mutation in its N-terminal transmembrane domain, yet in spite of the mutation a full-length protein was produced. We also showed that the amount of the mutated gene tagged with GFP protein was lower (approximately 30–50%) than the wild-type protein expressed in wild-type cells by immunoblot analysis hmg1-1 mutant showed hypersensitivity to pravastatin, an HMGR inhibitor, suggesting it has defective HMGR activity. In particular, the mutant showed defects in cell wall integrity and exhibited different phenotypes from those of the disruption mutants of ergosterol biosynthesis genes, and it showed normal filipin staining as well as normal subcellular localization of small GTPases. These data suggest that the pleiotropic phenotypes reflect the integrated effects of the reduced availability of ergosterol as well as various intermediates of the isoprenoid pathway In unicellular eukaryotes such as Here, we quantified the final product (ergosterol) and the pathway intermediates  in various isoprenoid pathway mutants, treated with statins or antifungals using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). The results showed that compounds such as pravastatin, allylamine terbinafine, and miconazole inhibit Hmg1, squalene epoxidase (Erg1), and lanosterol demethylase (Erg11), respectively, and the inhibition was associated with significant changes in the levels of the pathway products and intermediates. Notably, the ergosterol level showed substantial changes but the changes were smaller in magnitude when compared with FPP and GGPP in response to these drugs.r) higher than 0.990.Selected reaction monitoring (SRM) chromatograms of squalene, lanosterol, ergosterol, pyrene , FPP, and GGPP in the standard solution are shown in The SRM chromatograms of ergosterol and pyrene in the blank sample, the zero sample, and the lower limit of quantification (LLOQ) sample are shown in sts1+ gene encodes C-24 (28) sterol reductase that catalyzes the final step in ergosterol biosynthesis sts1 cells. Therefore absolute ergosterol was quantified by constructing a calibration curve using chloroform/methanol  extract from Δsts1 cells as a blank sample. The concentration of ergosterol in wild-type cells was determined to be 23.0±2.6 nmol/mg protein (n = 5 experiments).The sts1 cells treated with terbinafine or miconazole are shown in sts1 cells compared with that in wild-type cells. Furthermore, the squalene levels in Δsts1 drastically increased up to 40-fold and 300-fold by addition of miconazole and terbinafine to the medium, respectively. These results are in good agreement with the fission yeast isoprenoid pathway shown in The quantification results of squalene and ergosterol in wild-type cells and Δspo9+ gene encodes geranylgeranyl pyrophosphate synthetase that is a key enzyme in isoprenoid biosynthesis spo9 deletion on the levels of the intermediates  and the final sterol product (ergosterol) in the isoprenoid pathway. spo9 cells was almost equal to that in wild-type cells, however, GGPP was not detected in Δspo9 cells , Novartis Pharma K.K. , Mochida Pharmaceutical Co., Ltd. , respectively. Squalene, lanosterol, and ergosterol (85.0+% (HPLC)) were purchased from Wako Pure Chemical Industries . Farnesyl pyrophosphate ammonium salt (≥95% (TLC)), and geranylgeranyl pyrophosphate ammonium salt (≥95% (TLC)) were purchased from Sigma-Aldrich, St. Louis, MO, USA. All other reagents were from commercial sources.Schizosaccharomyces pombe strains used in this study are listed in S. pombe genetics were performed as described previously sts1). Proteins are denoted by roman letters and only the first letter is capitalized .Extraction of squalene, lanosterol, and ergosterol from cells was performed essentially as previously described Extraction of FPP and GGPP from cells was performed essentially as previously described + ion was selected as a precursor ion to detect squalene. The [M+H-H2O]+ ion was selected as a precursor ion to detect ergosterol and lanosterol.Squalene, lanosterol, and ergosterol were measured using LC-MS/MS under previously described conditions − ion was selected as a precursor ion to detect FPP and GGPP.FPP and GGPP were measured using LC-MS/MS under previously described conditions The peak area ratios of the compounds squalene, lanosterol, and ergosterol to pyrene in mutants or knockout cells were compared to those in wild-type cells. The peak area values of FPP and GGPP in mutants or knockout cells were compared to those in wild-type cells. The level of the respective compounds in wild-type cells was taken as 100%, and the level of the corresponding compounds in mutants or knockout cells was calculated as a percentage of that of the wild-type cells.The calibration curve of each standard was constructed to confirm that LC-MS/MS system used in this study gave correct quantitative data. Each standard solution to construct a calibration curve was prepared as follows. Squalene and lanosterol were dissolved in methanol. The concentrations of the standard solutions of squalene were 1, 5, 10, 50, 100, and 500 µmol/l. The concentrations of the standard solutions of lanosterol were 0.1, 0.2, 0.5, 1, 2, 5, and 10 µmol/l. Pyrene was used as an internal standard. The concentration of the standard solution of pyrene was 90 µmol/l.FPP and GGPP were dissolved in water/methanol . The concentrations of the standard solutions of both FPP and GGPP were 10, 20, 40, 100, 200, and 400 nmol/l. An internal standard for the quantification of FPP and GGPP was not used.sts1 cell extract ) to serve as blank sample because ergosterol was not detected in Δsts1 cells as shown in sts1 cell extract. The zero reading sample to serve as an internal standard for ergosterol was prepared by adding the pyrene standard solution to the Δsts1 cell extract. The samples for constructing the calibration curve were prepared by adding the ergosterol standard solution and the pyrene standard solution to the Δsts1 cell extract. The concentrations of ergosterol in the calibration curve samples were 1.9, 3.8, 19, 38, 190, and 380 nmol/mg protein. The concentration of pyrene in the calibration curve samples was 340 nmol/mg protein. The protein concentration of the cell extract was quantified using the Bio-Rad protein assay kit  by Bradford method Ergosterol was quantified absolutely by constructing a calibration curve of ΔFigure S1LC-MS/MS analysis of isoprenoids. A) SRM chromatograms of squalene, lanosterol, ergosterol, and pyrene in the standard solution. B) SRM chromatograms of FPP and GGPP in the standard solution.(TIF)Click here for additional data file.Figure S2r = 0.9997, A), lanosterol , FPP , and GGPP .Calibration curves of squalene ((TIF)Click here for additional data file.Figure S3LC-MS/MS analysis of ergosterol in fission yeast extracts. SRM chromatograms of ergosterol (Left) and pyrene (Right) in the blank sample (A), the zero sample (B), and the LLOQ sample (C).(TIF)Click here for additional data file.Figure S4r = 0.9967).Calibration curve of ergosterol ((TIF)Click here for additional data file.


Paired sample t-tests revealed significant differences between the pretest  and posttest  scores . A Pearson correlation revealed no significant relationship between individual step count and the difference between PACER pre- and posttest . The program improved aerobic capacity, but an increase in pedometer-calculated step count was not a predictor.Childhood obesity affects 1 of every 6 youth in the United States. One contributing factor to this statistic is a lack of physical activity (PA). Demands related to accountability which are placed on educators to demonstrate academic achievement often result in resistance to allocating time during the school day for PA. One possible solution is to consider utilizing time after school to integrate PA programs. The purpose of this study was to assess the impact of a 12-week after-school pedometer-focused PA program on aerobic capacity and to examine the relationship between step count and aerobic capacity in elementary school aged children. A group of elementary students ( Obesity-related risk factors and diseases are becoming increasingly prevalent in pediatric populations. Over the past thirty years, the obesity rate in the United States has escalated, creating an epidemic  [Research suggests that regular participation in PA plays an important role in sustaining good health and has been a topic of investigation for several decades . The CenThe need for enhancing appropriate PA levels in order to reverse increasing trends of obesity and the prevalence of other health-related diseases associated with physical inactivity is at an all-time high –8. PreviIn addition to the health benefits, academic achievement has also been linked to PA levels. Research indicates that daily PA improves concentration and academic achievement and can enhance test scores in math, reading, and writing , 18. ForAlthough the research suggests that the recommended “dose” of PA increases health and academic performance, only 8% of elementary schools, 6.4% of middle schools, and 5.8% of high schools provide daily physical education to all of their students . AdditioLimited research has been conducted on the effects of after-school programs on increasing PA levels of the pediatric participants involved . With a In order to further investigate the benefits of after-school PA programs, it is important to use a validated measurement system. The use of pedometers to chart PA also continues to grow in popularity. Pedometers have gained widespread acceptance among PA researchers over the past decade . A litern = 24), 9.5 ± 0.9 years (range 8 to 12)—the ratio between males and females was equally distributed—participated in and completed a pedometer-focused 12-week after-school PA program called Step up for Health (SUH). The SUH staff conducted outreach and registration activities with the aim of maximizing students' and parents'/guardians' knowledge of the program and increasing the convenience of the registration process. Prior to the beginning of testing, all procedures were approved by a local university's International Review Board (IRB). Once approved, parental consent forms were sent home with all potential participants (students' grades 3–6). Written and oral explanations  regarding procedures and potential risks to the participants were provided to parents from whom parental consent had been obtained. After a two-week period, the procedure was repeated to recruit more participants for the program. After obtaining parental permission and written or oral assent from each participant, baseline measurements were taken. A total of 34 students enrolled in the program but 10 dropped out for various reasons and did not complete the final assessment.A group of elementary students , followed by group stretching. After the warm-up the participants engaged in a group discussion involving a “running tip”  and then broke into preestablished teams and completed the prescribed running workout . After the workout the participants completed a group activity  and a cool-down and recorded their pedometer steps. In addition, home workouts were provided to encourage participants to engage in PA at home and to promote exercise as a family activity.Each participant was assigned a pedometer to wear in order to track his or her steps taken during each session of the program. A daily step goal was provided, and each participant was instructed to make daily pedometer step goals. The pedometer step counts were then calculated and converted to miles, allowing the participants to have a better understanding of the accomplishments. The supervisor of the program provided the participants with rules and guidelines on using their pedometer to ensure that the pedometer was being used appropriately. Additionally, personal journals were assigned to each participant in order to record step totals and document his/her thoughts and reflect on the activity session.A licensed physical education teacher underwent a one-week training, conducted by an experienced university faculty in physical education, prior to the start of the program. The licensed physical education teacher with the support of university faculty supervised all sessions.The Cooper Institute created Fitnessgram in 1982 in order to provide physical education teachers with a convenient way to evaluate fitness and report fitness levels to students and parents. In 2012, the President's Council on Fitness, Sports and Nutrition (PCFSN) adopted the Fitnessgram as the new school-based PA and health promotion program. The assessments were created to evaluate the five areas of health-related fitness: cardiovascular fitness (aerobic capacity), muscular strength, muscular endurance, flexibility, and body composition .One of the assessments, the progressive aerobic cardiovascular endurance run (PACER), also known as the multistage fitness test, was used to assess cardiovascular fitness. The PACER test has been shown to be a valid and reliable assessment related to a participant's VO2 max or aerobic capacity. The participants must jog/run 20 meters each time a signal sounds using the Fitnessgram Testing Administration CD. With the passing of each minute, the running time is reduced by one half second, encouraging the participants to increase their running speed and pace themselves with the length of time allowed . Each paDuring this test, each participant's score was recorded as the total number of laps. This assessment was conducted during week one and week twelve of the program to track improvements across the program. Prior to the beginning of the program, the licensed physical education teacher, who acted as the supervisor to the program, underwent a one-hour training to ensure that she was familiar with administering the test. The participants were briefed on best practices on calculating and recording PACER results. Each participant was paired with a peer during the test, and the participants were divided into smaller groups of 6–8 participants to ensure appropriate counting. Scoring of the test was performed with the assistance of undergraduate students enrolled in a physical education course of study. The student received training on the PACER test prior to being recruited to assist with the project.P > 0.05). Subsequently, paired samples t-test was employed in order to assess differences in pre- and postworkout data. A Pearson bivariate correlation was utilized to determine the relationship between individual step count and the difference between PACER pre- and posttest data. Significance was set a priori to alpha <0.05 for all analyses, and all statistical analyses were performed using SPSS version 20.0.Data was first analyzed for normality via Shapiro-Wilk procedures and was not found to be significantly deviant from being normally distributed . A paired sample t-test revealed significant differences between the PACER pretest   scores  .As a group, the elementary student participants totalled 1,388,155 steps for the project; individual steps ranged from 27,419 to 62,158 steps for the duration of the program (D = 9.9)  and post≤ 0.001) . These rThis study evaluated the impact of a pedometer-focused 12-week after-school PA program on aerobic capacity as well as the relationship between step count and aerobic capacity in elementary school aged children. The following discussion addresses three topics: program effectiveness in terms of aerobic capacity, incorporating pedometers, and after-school programming as a solution to physical education decline while the obesity epidemic is on the rise.t-tests revealed significant differences between the pretest  and posttest  on the PACER test.Limited research has been conducted on the effects of after-school PA programs.Pate and O'Neill  reviewedThe results of this study demonstrate the positive impact of the after-school PA program on the aerobic capacity of the participants; however, step count did not predict improvement in aerobic capacity. This may be largely due to the variation in starting levels of fitness of the participants and the data collection of the pedometer being limited to only a volume of training assessment. Pedometers lack the ability to track intensity of movement which is critical to determining zones  of training. The results revealing that pedometer step count and aerobic capacity were not linked may seem contradictory to the overwhelming support for the use of pedometers as a tool to increase PA in adults . HoweverBravata et al.  acknowleBecause pedometers have gained widespread acceptance, their accuracy in step count has also been a source of research study. Schneider et al.  tested aSchools have access to nearly all of a nation's youngsters. Developing strong partnerships with our schools may provide the framework needed to focus on the prevention through increased activity and education. Physical inactivity in American youth is an ongoing concern. It has been suggested that school-based programs be promoted to increase the opportunities for pediatric populations to engage in the recommended daily “dose” of PA. Although initiatives have been implemented and preestablished structures  provide an opportunity for pediatric populations to be active during the school day, students are still not meeting the recommendations. It seems that, with academic rigor being emphasized in schools, there is little time for PA. An attractive and fairly unexplored method to promote increased daily PA is structured after-school programming.Although this study helped to confirm results of previous studies which indicate that after-school running programs have the potential to increase aerobic capacity and PA, after-school programs geared for twice a week in this study and three times a week for others fall very short of the CDC guidelines which recommend 60 minutes of exercise a day for all students attending the school . HoweverThe after-school programming to meet CDC requirements may not provide a total solution to address obesity and meet the needs of students, but certainly it would play an important role in meeting those needs. However, the culture of inactivity is more complicated than a simple solution of increased PA after-school can solve. Addressing the underlying behavioral and psychological mechanisms leading to the rising obesity trajectories is also an important step to solving the problem . FurtherThe initial success of the SUH after-school running program as a service-learning project provides a working model for implementation of similar projects in the future. Although this project was focused upon implementation in an elementary school setting, application of these concepts by other academic units with similar goals of positively impacting their community through PA is recommended. According to the findings in this project, students' fitness levels can be improved through a program such as SUH that utilized pedometers as motivation to succeed. In this current study, the aerobic capacity of the students was improved even though pedometer step count was not a predictor. Future studies may wish to consider expanding to include all K-12 students and examine all aspects of fitness to better understand and promote healthy lifestyles in pediatric populations.


Crystal structures of the tobacco mosaic virus (TMV) coat protein (CP) in its helical and disk conformations have previously been determined at the atomic level. For the helical structure, interactions of proteins and nucleic acids in the main chains were clearly observed; however, the conformation of residues at the C-terminus was flexible and disordered. For the four-layer aggregate disk structure, interactions of the main chain residues could only be observed through water–mediated hydrogen bonding with protein residues. In this study, the effects of the C-terminal peptides on the interactions of TMV CP were investigated by crystal structure determination.19). Overall, N-His-TMV CP19 protein self-assembled into the four-layer aggregate form. The conformations of residues Gln36, Thr59, Asp115 and Arg134 were carefully analyzed in the high radius and low radius regions of N-His-TMV CP19, which were found to be significantly different from those observed previously for the helical and four-layer aggregate forms. In addition, the aggregation of the N-His-TMV CP19 layers was found to primarily be mediated through direct hydrogen-bonding. Notably, this engineered protein also can package RNA effectively and assemble into an infectious virus particle.The crystal structure of a genetically engineered TMV CP was resolved at 3.06 Å. For the genetically engineered TMV CP, a six-histidine (His) tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated (N-His-TMV CPThe terminal sequence of amino acids influences the conformation and interactions of the four-layer aggregate. Direct protein–protein interactions are observed in the major overlap region when residues Gly155 to Thr158 at the C-terminus are truncated. This engineered TMV CP is reassembled by direct protein–protein interaction and maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA. The tobacco mosaic virus (TMV) coat protein (CP) assembly system is composed of inlaid hydrophobic and hydrophilic patches with TMV RNA associated via multiple aromatic interactions between neighboring subunits to stabilize a helical virus structure 19 protein was crystallized using hanging-drop vapor diffusion and the micro-seeding method. The crystal structure of the four-layer aggregate of N-His-TMV CP19 was determined at 3.06 Å. The major differences between N-His-TMV CP19 and the previously reported TMV CP structures are the manner of subunit assembly and the protein-protein interactions in the major overlap region. It should be noted that this engineered truncation mutant still maintains the normal function of the four-layer aggregate of TMV CP in the presence of RNA.Here, we identify the effect of the C-terminal peptides on the molecular packaging and interactions of subunits within TMV CP aggregates. In order to purify and crystallize TMV CP, we constructed a TMV CP variant with a His-tag at the N-terminus and a truncation of residues Gly155 to Thr158 at the C-terminus. The engineered N-His-TMV CP19 for crystallization, four amino acids  were truncated at the C-terminus, and a 6-His-tag was incorporated at the N-terminus. Freshly purified protein was examined in solution using size exclusion chromatography (SEC). In 20 mM sodium phosphate and 100 mM NaCl (pH 8.0) , the retention time of the N-His-TMV CP19 protein revealed its oligomeric state as a dimer and monomer. After equilibration in a buffer containing 300 mM ammonium sulfate and 100 mM Tris-HCl (pH 8.0), the oligomeric state of N-His-TMV CP19 was converted to a 20S structure. These results show that the assembly of N-His-TMV CP19 is influenced by the environmental conditions.To prepare N-His-TMV CP19 using hanging-drop vapor diffusion and a micro-seeding method. The micro-seeding method facilitated efficient and reproducible crystal growth of N-His-TMV CP1919 with a typical maximal dimension of 0.2 mm ×0.3 mm within several days Several TMV CPs have previously been crystallized by the micro-dialysis method against a buffer containing 300 mM ammonium sulfate and 100 mM Tris-HCl (pH 8.0) 19 crystals belong to an orthorhombic space group P21212, with unit cell dimensions a = 173 Å, b = 222 Å, and c = 226 Å . The structure was solved by molecular replacement and refined to a resolution of 3.06 Å (PDB code 4GQH). The local noncrystallographic symmetry (NCS) axis was refined with a grid search wherein we varied the orientation or translation of the asymmetrical unit using XPLOR rigid body dynamics Figure S2A, Figure S2B and Figure S2C). The well-ordered portions of the virus subunit Figure S2D). The ordered segments of the a- and b-chains can be superimposed on each other by a rigid body translation with a RMSD of 0.2 Å for the carbon-alpha atoms . The present atomic model also details the locations and orientations of the side chains involved in the inter- and intra-subunit interactions based on the error estimated by Luzzati statistics Table S1. The HR and LR residue densities corresponding to fitted coordinates are supplied in Figure S5. It is clear that the density for the large hydrophobic residues, some of the small hydrophobic residues and the basic side chains are in agreement with the fitted coordinates. The N-His-TMV CP19 can form disks and infectious viruses . The density corresponding to the side chains of N-His-TMV CP19 is mapped in Figure S9. The 2Fo-Fc electron density maps have been contoured at 1.0 σ level, and the residues have been shown according to structure sequence. Similar to previous studies, one asymmetric unit was observed containing four aggregate disks, with every disk containing 17-fold monomers 19, which contains a hexahistidine tag at the N-terminus to facilitate purification 19 four-layer aggregate was about ∼176 Å in diameter . In the LR region, a poor-quality electron density corresponding to the protein was mapped . Therefore, residues 91–113 are absent in the b-ring atomic model, and residues 92–106 are absent in the a-ring atomic model , and native-PAGE and TEM, after equilibration with an ammonium sulfate solution as above . N-His-TMV CP19 refolding and further self-assembly was carried out at a protein concentration of 1.8 mg/mL. When the N-His-TMV CP19 was incubated at a concentration of 1.8 mg/mL at 22°C for 5 hr, the N-His-TMV CP19 disk or helical structures were formed as observed using TEM in solution . Thus, the N-His-TMV CP19 four-layer aggregate can effectively package and protect the nucleic acid under conditions of 20 mM PB, 100 mM NaCl and pH 7.2. For infection of tobacco leaves, the TMV CP disks undergo disassembly and reassembly mediated by RNA, and the reconstituted virus-like particles are made up of the helical form as determined by TEM . In other words, the RNA can stabilize the disordered residues in the LR region.The N-His-TMV CPFigure S10, The inter-molecular hydrogen bonds between residues are indicated by magenta dashed lines, The residues are shown according to N-His-TMV CP19 sequence. The a-ring subunits in the LR region loop residues are of higher order than the b-ring subunits with observable inter-chain interactions between Asn25-Ser15, Tyr72-Thr28, Phe35-Asp88, Gln36-Asp88 and Arg122-Asp88  tag was introduced at the N-terminus, and the C-terminal residues 155 to 158 were truncated; direct inter-chain interactions between Asn25-Ser15, Tyr72-Thr28, Phe35-Asp88, Gln36-Asp88 and Arg122-Asp88 were observed in N-His-TMV CP19 . Because the oligomer terminal residues were changed, the conformations of residues Gln36, Thr59, Asp115 and Arg134 in the HR and LR regions of N-His-TMV CP19 were found to be significantly different from those observed in the helical structure (PDB code: 1EI7)  and in the four-layer aggregate disk structure of TMV CP (PDB code: 3JO6) . Protein–protein interactions on the salt bridges between Asp19 and Arg134, and between Asp66 and Arg134 were observed in the dihedral-related ba-chain pair. In the dihedral related aa-chain pair, the protein–protein interactions on the salt bridges between Lys53 and Glu22 and the hydrogen-bonding interactions between Thr59 and Thr59 were observed in the middle between trans-layers. All interactions between the four-layer aggregate disk had similar bond lengths (∼3.5 Å)  are depicted in Figure S13B. For the previously reported TMV CP disk structure, protein–protein interactions between Asp19-Arg134 and Asp66-Arg134 for the b-pairs and a-pairs were mediated by water. The electron density maps of protein–protein interactions involving salt-bridges between Lys53 and Glu22 are depicted in Figure S13C. The electron density maps of hydrogen-bonding interactions between Thr59 and Thr59 were observed in the middle trans-layers . As shown in Figure S11 and  a close-up electron density map and fitted coordinates show the inter-chain residues of N-His-TMV CP19 and the previously reported TMV CP (PDB code: 1EI7) subunits. Compared to the previously reported structures, these direct hydrogen-bonding interactions between the protein–protein trans-layers were not observed. In the previous structure (PDB code: 1EI7), the protein–protein interactions involved salt-bridges mediated by water. For the N-His-TMV CP19 structure, the conformations of Asp19, Arg134, Asp66, Lys53, Glu22 and Thr59 were different from the previously reported structure .In the atomic model, the b-ring and a-ring in the HR and MR region are packed in order with observable inter-chain interactions between Asn25-Ser15, Tyr72-Thr28, Phe35-Asp88, Gln36-Asp88 and Arg122-Asp88 . Compared with the previously reported disk structure (PDB code: 1EI7) and the helical structure (PDB code: 3JO6), the major differences are the manner of subunit assembly and protein-protein interactions in the major overlap region. In the N-His-TMV CP19 structure, most protein–protein interactions were observed in the N-His-TMV CP19 inter-chains within 3.5 Å, including salt bridges between Asp19-Arg134 (∼3.12 Å), Asp66-Arg134 (∼3.29 Å), Lys53-Glu22 (∼3.38 Å), Arg122-Asp88 (∼2.43 Å), Arg113-Asp115 (∼3.24 Å) and hydrogen bonding of Asn25-Ser15 (∼2.73 Å), Tyr72-Thr28 (∼2.76 ), Phe35-Asp88 (∼3.03 Å), Gln36-Asp88 (∼3.50 Å) and Thr59-Thr59 (∼3.28 Å). In the previously reported disk structure (PDB code: 1EI7), the charged groups of the interactions were more than 4.0 Å , and most side-chains of the residues interacted via salt bridges and hydrogen-bonding through a network mediated by water 19 b-ring residues 91–113 (a-ring residues 92–106) in the low-radius inner loop region were missing, which is dissimilar to the previously reported TMV CP disk and helical structures. In the TMV CP disk structure (PDB code: 1EI7), the a-ring and b-ring residues 90–110 in the low-radius inner loop region were uniformly disrupted The amino acid sequence of N-His-TMV CPFigure S4). In the atomic model, the region between 0 and 30 Å in radius, corresponding to the central 90-Å-diameter hole in the macromolecular assembly, was completely filled with solvent. In the region between 30-Å and 40-Å in radius, the atomic chain was poorly ordered with no observable inter-chain or intra-chain interactions. In the initial study done by Bhyravbhatla et al. 19 b-ring residues 91–113 (a-ring residues 92–106) are absent in the atomic model. Collecting good low-resolution data and using 17-fold redundancy allowed us to map out the backbone of the polypeptide chain in this region. The side chains are well defined in the electron density map in Figure S5.The structures of the side chains differed: a figure of the electron density map is depicted with an average resolution of 3 Å , indicating a poorly ordered structure in the LR region; the N-His-TMV CP19 had a lower atomic B factor in the central helix  and the C-terminal loop region (residues 148–154), indicating a more tightly packed, well-ordered structure in these regions  and helical structure (PDB code: 3JO6), and these interactions could be interpreted as conformational changes affected by Thr59 and Arg134, as shown in and19 and the previously reported TMV CP disk (PDB code 1EI7) are provided in Figure S15, the N-His-TMV CP19 residues  of the HR and the LR loop regions are different from those residues in the previous structure. To summarize, the main differences between N-His-TMV CP19 and TMV CP disks involve trans-layers proteins.Atoms of the inner loop region (residues 90–110) and the last four residues (154–158) had high B factors both in the previously reported disk structure (PDB code: 1EI7) and helical structure (PDB code: 3JO6), implying that these regions were disordered compared with the average structure 19 is easily overexpressed and purified to large scale; however, it is difficult to crystallize. After truncating four residues at the N-His-TMV CP19 C-terminus, high-quality crystals of N-His-TMV CP19 were easily obtained by hanging-drop vapor diffusion and micro-seeding methods. This phenomenon shows the conformational flexibility of the four residues at the C-terminus, which affects the crystal packing. The structure of N-His-TMV CP19 showed that the HR loop region (residues148–154) at the C-terminus had a low atomic B factor, which indicates a well-ordered structure in the HR loop region. Compared with the helical and disk structures of the previously reported TMV CP, the N-His-TMV CP19 subunits displayed different conformations in the LR and HR regions, and its four-layer aggregate disk was mainly retained by protein–protein salt-bridges and hydrogen-bonding networks between the trans-layers. In addition, the N-His-TMV CP19 could package RNA effectively and could assemble into the infectious virus particle with RNA. Thus, the N-His-TMV CP19 protein can form a reassembled disk or helical structure, and this structure exists in an RNA base-binding pocket. This pocket can be stabilized by TMV RNA and reassembled into a complete virus, and thus can serve as a potential molecular target of anti-plant virus drugs. These principles can also be applied in biology and structure-based drug design.Full-length N-His-TMV CPNicotiana tabacum K326 leaves cultivated in a greenhouse at the Center for Research and Development of Fine Chemicals of Guizhou University. The virus was purified using the method described by Gooding CATATGTCTTACAGTATCACTACTCC-3′5′-GGAATTC, the restriction site is underlined) in 50 mM Tris-HCl (pH 8.0), 8.0 mM magnesium chloride, 75 mM potassium chloride, 10 mM DL-dithiothreitol, 1.0 mM dNTPs, 0.5 unit/μL, AMV reverse transcriptase (TaKaRa), and 1.0 unit/μL RNase inhibitor (TaKaRa) for 1.5 hr at 85°C. The N-His-TMV CP19 gene was amplified by PCR using viral cDNA as template, with primer 1 and primer 9 .TMV (common strain) was isolated from infected 19 protein was constructed by cloning common strains of the TMV CP gene into the Nde I and Xho I sites of pET28a vector (Novagen) and was expressed in E. coli BL21 (DE3) RIL cells (Novagen). The cells were grown in Luria-Bertani medium supplemented with 50 μg/mL kanamycin at 37°C to an OD600 of 0.65, shaking at 220 rpm. Then, the expression of protein was induced with 0.1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) at 16°C overnight. The cells were harvested by centrifugation and then stored at −80°C. Cell pellets were resuspended in lysis buffer  and then lysed at 4°C by sonication. After centrifugation at 15,000 g for 30 min to remove any insoluble material, the supernatant was loaded onto a 5 mL HisTrap High-Performance column , and the protein was eluted with a single gradient of 50 mM to 400 mM imidazole (pH 8.0). The C-terminally truncated protein was further purified by gel filtration (SEC) using a superdex 200 column  in a buffer containing 20 mM PB (pH 8.0) and 100 mM NaCl. Gel filtration was performed at 4°C using a calibrated superdex 200 10/300 GL column  attached to an AKTA purifier fast protein liquid chromatography system . The column was equilibrated with a buffer containing 10 mM PB (pH 7.2) or 100 mM NaCl depending on the TMV CP disk buffer β-lactoglobulin (35 kDa), ribonuclease A (13.7 kDa), cytochrome (13.6 kDa), aprotinin (6.51 kDa), and vitamin B12 (1.36 kDa). The proteins were monitored by absorbance at a wavelength of 280 nm. Native Polyacrylamide Gel Electrophoresis (PAGE) was performed on ice with the N-His-TMV CP19 samples, which were equilibrated overnight in a buffer containing 10 mM PB (pH 7.2) and 100 mM NaCl. A 20 µL sample was treated with 20 µL 2× loading buffer , and then, 8 μL of sample and 4 μL of protein marker were loaded onto a native-PAGE gel (4% stacking and 17% separating gel). Electrophoresis was run in 1× native page buffer  at 0°C for about 1 hr A six-His tag was introduced at the N-terminus to enable protein purification 19 disk (1.8 mg/mL), incubated in 10 mM PB, 100 mM NaCl, pH 7.2 solution, was mixed with 0.2 mL purified TMV RNA (0.5 mg/mL), and then the mixture was incubated at 22°C for 20 h. Suspensions were centrifuged at 5,000 rpm for 1 min, and then the reconstituted virus was obtained 2O. The grid was then stained with 20 μL of 2% aqueous solution of tungstophosphoric acid for 90 s as a negative stain N. glutinosa were mechanically inoculated with reconstructed virus or the WT virus as a control. The local lesion numbers were recorded 3−4 days after inoculation.One milliliter of purified self-assembled N-His-TMV CP19 protein was concentrated to 14.0 mg/mL in a buffer containing 20 mM PB, pH 8.0, 100 mM NaCl, and 1 mM DTT, and then crystallization conditions were carefully screened 19 were mounted and flash-frozen in liquid nitrogen following cryo-protection with reservoir solution containing an additional 30% (v/v) glycerol Crystallization was performed at 22°C by using the hanging-drop vapor diffusion method. N-His-TMV CP1212. The structure was solved by molecular replacement in Phaser 19 protein model was manually built in COOT Diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline 17U. All X-ray data were processed using the HKL2000 program Figure S119Ramachandran Plot of the N-His-TMV CPmonomer.(TIF)Click here for additional data file.Figure S2Comparison of the structure of the truncated TMV CP and previous structures by pyMOL. The following structural comparisons are shown: (A) The N-His-TMV CP19 a-chain and the TMV CP (PDB code: 1EI7) a-chain; (B) The N-His-TMV CP19 a-chain and the TMV CP (PDB code: 1EI7) b-chain; (C) The N-His-TMV CP19 a-chain and the TMV CP (PDB code: 3JO6) a-chain; (D) The N-His-TMV CP19 a- and b-chains and TMV CP (PDB code: 1EI7) a- and b-chains.(TIF)Click here for additional data file.Figure S319 a- and b-chains by pyMOL.Superimposition of the N-His-TMV CP (A) Carbon-alpha trace showing a superimposition of the N-His-TMV CP19 a- and b-chains; (B) Bonds showing the superimposition of the N-His-TMV CP19 a- and b-chains.(TIF)Click here for additional data file.Figure S419 by Coot.Electron density map of the N-His-TMV CP (A) The electron density map of N-His-TMV CP19viewed looking perpendicular to the crystallographic twofold axis, (B) An overview of the electron density map of N-His-TMV CP19 is provided at different angles.(TIF)Click here for additional data file.Figure S519 monomer by Coot.A well-defined electron density map of the N-His-TMV CP (A) Carbon-alpha trace showing a well-defined electron density map of the N-His-TMV CP19 monomer; (B) Bonds showing a well-defined electron density map of the N-His-TMV CP19 monomer.(TIF)Click here for additional data file.Figure S6Electron microscopic analysis of tobacco leaves infected with reconstituted particles. (A) Original image of the tobacco leaves infected with reconstituted particles. (B) Magnified view of the tobacco leaves infected with reconstituted particles. N-His-TMV CP19 discs are enclosed in red boxes.(TIF)Click here for additional data file.Figure S719 and wild type TMV CP as assessed by native-PAGE and SEC.Assembly of the four-layer aggregate disks of N-His-TMV CP (A) Disks of N-His-TMV CP19 eluted at 7.9 mL on SEC. (B) Disks of N-His-TMV CP19 were analyzed by native-PAGE. (C) Disks of wild TMV CP eluted at 7.9 mL on SEC. (D) Disks of wild TMV CP were analyzed by native-PAGE.(TIF)Click here for additional data file.Figure S819.X-ray density map and fitted coordinates of a cross-section corresponding to the HR residues and LR loop residues of N-His-TMV CP (A) Cross-section of an X-ray map and fitted coordinates at the HR residues of N-His-TMV CP19. (B) Cross-section of an X-ray map and fitted coordinates at the LR loop residues of N-His-TMV CP19.(TIF)Click here for additional data file.Figure S919.Side chain density map and fitted coordinates corresponding to the side chain residues of N-His-TMV CP(TIF)Click here for additional data file.Figure S1019 b-ring subunit.Electron density map and fitted coordinates corresponding to the inter-chain interactions within the N-His-TMV CP The electron density map and fitted coordinates corresponding to the inter-chain interactions between the following residues in the N-His-TMV CP19 disk of the b-ring are shown: (A) Phe35-Asp88 and Gln36-Asp88. (B) Tyr72-Thr28. (C) Arg113-Asp115. (D) Asn25-Ser15.(TIF)Click here for additional data file.Figure S1119 and the reported TMV CP (PDB code 1EI7) subunits from Close-up electron density map and fitted coordinates showing the side chain residues of N-His-TMV CP.(TIF)Click here for additional data file.Figure S1219 and the reported TMV CP (PDB code 3JO6) subunits from Close-up electron density map and fitted coordinates showing the side chain residues of N-His-TMV CP.(TIF)Click here for additional data file.Figure S1319 trans-layer.Electron density map and fitted coordinates corresponding to the protein–protein interactions of the N-His-TMV CP The electron density map and fitted coordinates between the following N-His-TMV CP19 protein–protein interactions are shown: (A) The Asp19-Arg134 and Asp66-Arg134 between the b-pair and a-pair. (B) The Asp19-Arg134 and Asp66-Arg134 between the b-pair and a-pair and the Asp19-Arg134 and Asp66-Arg134 between the previously reported TMV CP disk structures (PDB code 1EI7). In the previously reported TMV CP disk structure, the protein–protein interactions involved in Asp19-Arg134 and Asp66-Arg134 between the b-pairs and a-pairs were mediated by water. (C) The Thr59-Thr59 between the a-pair and a-pair. (D) The Lys53-Glu22 between the a-pair and a-pair.(TIF)Click here for additional data file.Figure S1419 and the previously reported TMV CP (PDB code 1EI7) subunits.Close-up electron density map and fitted coordinates showing the inter-chain residues of N-His-TMV CP(TIF)Click here for additional data file.Figure S1519 and the previously reported TMV CP disk (PDB code 1EI7).Cross-section of the X-ray map and fitted coordinates in the HR residues and LR loop residues of N-His-TMV CP Cross-section of the X-ray map and fitted coordinates of N-His-TMV CP19 and the TMV CP disk structure (PDB code 1EI7) are shown for the region of: (A) the HR residues; and (B) the LR loop residues.(TIF)Click here for additional data file.Table S1Data collection and refinement statistics.(DOCX)Click here for additional data file.


Frequently, social interventions produce less for the intended beneficiaries than was initially planned. One possible reason is that ideas embodied in interventions are not self-executing and require careful and systematic translation to put into practice. The capacity of implementers to deliver interventions is thus paramount. Purveyor organizations provide external support to implementers to develop that capacity and to encourage high-fidelity implementation behavior. Literature on the theory underlying this type of program is not plentiful. Research shows that detailed, explicit, and agreed-upon program theory contributes to and encourages high-fidelity implementation behavior. The process of developing and depicting program theory is flexible and leaves the researcher with what might be seen as an overwhelming number of options.This study was designed to develop and depict the program theory underlying the support services delivered by a South African purveyor. The purveyor supports seventeen local organizations in delivering a peer education program to young people as an HIV/AIDS prevention intervention. Purposive sampling was employed to identify and select study participants. An iterative process that involved site visits, a desktop review of program documentation, one-on-one unstructured interviews, and a subsequent verification process, was used to develop a comprehensive program logic model.The study resulted in a formalized logic model of how the specific purveyor is supposed to function; that model was accepted by all study participants.The study serves as an example of how program theory of a ‘real life’ program can be developed and depicted. It highlights the strengths and weakness of this evaluation approach, and provides direction and recommendations for future research on programs that employ the purveyor method to disseminate interventions. In many resource-poor countries, governments and non-governmental organizations struggle with what is generally referred to as ‘lack of capacity.’ This is typically seen as a basic human resource issue: there are inadequate skills and expertise in an organization to perform the required tasks, and in addition, the organization has inadequate governance and management structures to support its personnel ,2. A var’ refers to the support services delivered by purveyors.Purveyors are individuals or organizations that operate as outside experts representing a particular program; they support organizations, systems, and practitioners in striving to adopt and implement that program with fidelity . They ara. A group known as the Practice Group for Purveyors and Intermediary Organizations [Purveyors and intermediary organizations are receiving increasing attention in implementation science. In August 2011, the first Global Implementation Conference  was heldizations  grew outet al. Research has tentatively identified some core factors deemed necessary to ensure high fidelity implementation of interventions [et al. singled out inconsistent implementation strategies and procedures employed by purveyors as a characteristic of the field at present [Authors such as Elliot and Mihalic , Fixsen,ventions . These aventions ,8. The wventions ,12. Fixs present . One sigThese caveats notwithstanding, the purveyor method, in its various shapes and forms, has been applied to a wide variety of human service fields to disseminate interventions: these fields include education , juvenilThree years ago, we became involved in the work of a South African non-governmental organization (NGO) that acts as a purveyor to seventeen local organizations that deliver a peer education intervention to young people as an HIV prevention intervention. It was not easy to classify this NGO as a purveyor or intermediary organization, in the light of the definitions given above. It contains elements of both types of organization, but eventually a decision was taken to classify it as a purveyor, because it provides external expertise, and works with one particular type of program (peer education).It is widely known that South Africa faces a serious HIV/AIDS problem: in 2009, HIV prevalence among adults aged 15 to 49 was calculated at 17.8% of this population, with the number of adults and children aged 0 to 49 living with HIV estimated at 5,600,000 -23. It iThe seventeen provider organizations work in 102 schools in South Africa and Botswana, and have trained more than 5,000 peer educators. At the time of the study, the purveyor (from here on the agency) had been in existence for three years, with a growing demand for its services. Currently, it has an international office in the Western Cape and four provincial offices. The international office supports the provincial offices in their efforts to provide ongoing technical assistance and support to the provider organizations and implementers in the respective provinces. The director is based at the international office, and each province has a provincial manager in charge of its activities. The agency’s purveyor program is divided into the following six functional areas, each with a manager in charge to ensure that services are delivered consistently to all provider organizations and implementers:1. Advocacy and visibility—develops and distributes resources, knowledge, and skill to promote the peer education intervention.2. Quality assurance—provides knowledge and skill to monitor delivery of the peer education intervention.3. Research and development—develops and distributes all resources required to implement the peer education intervention.4. Resource mobilization—develops and distributes resources, knowledge, and skill to help provider organizations to obtain resources independently.5. Stakeholder management—ensures that an increasing number of organizations deliver the peer education intervention. It also ensures that current provider organizations and implementers have opportunities to interact to ensure that they do not feel isolated.6. Training and support— provides implementers with the knowledge and skill needed to deliver the peer education intervention with fidelity.Although the original evaluation approach was formulated in terms of an implementation evaluation of the purveyor program, our initial contact with the agency revealed a problem that needed addressing before we could embark on an implementation study. In program evaluation terms, the agency had no explicit and agreed-upon program theory. To put it differently, it became apparent to us that it was not clear to the stakeholders how the purveyor program should work.Program theory-based evaluation is a well-known evaluation approach , and doeFunnel and Rogers  argue thFunnel and Rogers  identifiA decision was therefore taken, in close consultation with program management, to articulate the ‘theory’ underlying the purveyor program; in other words, the understanding of the way it is supposed to work, both in terms of process and outcome. This was thought to be an important exercise, because it was expected that the agency’s purveyor program would be extended to other provider organizations in the local context.Thus, the primary focus of the study was to unfold the theory of change underlying the agency’s purveyor program. On a practical level, it was argued that this would result in a better understanding of the purveyor program among the agency staff members, and would consequently encourage the strength and fidelity of implementation of the purveyor program to current and future provider organizations and implementers. Less modestly, we also aimed to furnish information that could contribute towards a better understanding of purveyor programs in general. This study also serves as an example of how a detailed, explicit, and agreed-upon representation of program theory could be developed and depicted. In other words, it is a relatively small contribution to the burgeoning field of implementation science and practice.The information used to construct program theory can be obtained from multiple sources, including the following: a review of program documentation; interviews with those closest to the program; observation of the program; prior theory and research in the specific program domain; or exploratory research that tests the critical assumptions of the program .The first decision to be taken was who should be involved in developing the program theory of the purveyor program . The metThere are three main approaches to developing program theory: deductive; inductive; and user-focused . The dedDue to the scarcity of literature and general lack of knowledge about the program theory underlying purveyor programs, the authors opted to combine the inductive and user-focused approaches. The first author took the lead role, with the second author acting as evaluation consultant, which implied that it fitted an approach where evaluators facilitate a collaborative process of developing the program theory . Given tThe development of logic models usually involves the evaluator constructing a preliminary draft based on available program documentation, which is then presented to program staff members for validation. This results in an iterative process of moving back and forth between the development of the logic model and receiving feedback from staff members, which will continue until all staff members agree that the model is an accurate and detailed description of the program as it was originally intended ,39.Numerous site visits to the international office served to develop familiarity with the agency, to establish contact with program staff, to observe the program as it is being delivered, and to identify useful documentation that should be included in the review. Relevant documents were identified and studied, including operational plans, the implementation manual, peer educator portfolios, annual reports, quality assurance mid-year assessments and the like. These documents were analyzed and data sorted into coding categories according to standard logic model components. As a result, we ended up with a preliminary working understanding that covered the following categories: inputs; activities; outputs; pivotal proximal and intermediate outcomes; and distal outcomes.This preliminary logic model was then submitted via a series of relatively unstructured interviews to the individuals selected to participate in the exercise. Two main topics structured these interviews: first, what the goals and objectives were for the specific program areas that these individuals managed; and second, how these aims contributed to the overall effectiveness of the program. Thus, an iterative process was started between our observations, the program documentation, and data resulting from the interviews and the subsequent verification process; this ultimately culminated in a final program logic model. Each iteration resulted in a more refined and accurate model of the program. It took four months to move from initial data gathering to reach the point at which all the participants were satisfied that the model accurately and comprehensively represented how the agency aims to affect change within the provider organizations and implementers.In summary: we developed the program theory of the purveyor program from implicit theories of those closest to the program, observations of the program, and program documentation.The study was approved by an Ethics Review Committee of the Faculty of Humanities at the University of Cape Town. The director of the agency supported the study and communicated its purpose and usefulness to the various evaluation stakeholders and urged them to cooperate. No one refused to participate in the study.Through an iterative process of logic model development, refinement via interviews, and further presentations of the revised logic model, we arrived at the major result of this study: a formalized logic model of the purveyor program’s theory which was accepted by all concerned as an accurate reflection of the work done by the agency. The challenge however is to present such a complex program visually, in a way that is useful to the stakeholders, comprehensible and engaging to those less familiar with it, communicates effectively, and simplifies without trivializing . We decii.e., the reduction of HIV infections among young people. Thus, the purveyor may be improving the capacity of implementing organizations to deliver the peer education intervention as intended, but it remains an open question whether the latter actually do implement peer education with fidelity and strength. At the time of our involvement, the agency had focused its monitoring and evaluation efforts on how provider organizations and implementers deliver the peer education intervention, with little attention given to monitoring the agency’s supportive activities. Although this is understandable, it fails to recognize the extent to which subsequent steps  depend on getting fidelity and strength in the agency’s own activities.Note, in particular, that the present exercise refers only to the two boxes on the left of Figure The next step was to elaborate on these two boxes that represent the purveyor program. One way to deal with this complexity is to use sub-pages, as is the case in visual outcomes, model- building software such as DoView . In otheOf course, this model is still fairly general, because each functional area has a distinctive service delivery plan that includes various activities deemed necessary to result in the function’s expected outcomes. In other words, another sub-page that depicts these aspects is required. We went one step further for the agency, and developed the program theory underlying the activities of each functional area. These six separate logic models are not presented here, as we are more interested in the general principles rather than in the specific details of the purveyor program.The theory flows from left to right, starting with the six program functions. The activities of each function are described below. These activities are presumed to lead to short-term, early changes, which in turn set in motion changes in the medium term, which are expected to result in a distal outcome as indicated.The advocacy and visibility function includes an annual activity to promote the agency’s work, but invests most of its time and resources in developing and distributing marketing resources. Provider organizations receive the marketing material from the agency, and this is intended to assist them in promoting the peer education intervention in the relevant communities.The quality assurance function offers provider organizations a standard monitoring and evaluation system to monitor delivery of the peer education services. The system is based on a Logical Framework Analysis that provides standards and guidelines for implementation practices. Implementers have the skills and knowledge needed to use the system, and they gather information to send to the agency’s quality assurance coordinator at the international office. There, the incoming data is analyzed to identify patterns of delivery to provide systematic feedback on the provider organizations’ performance in comparison to quarterly targets.In terms of research and development, training resources such as the training sessions and workshops are examined and modified if necessary. Research also is conducted with stakeholders to update, for example, the implementation guide. Thus, implementers have easy access to resources that are continuously examined for its relevance and usefulness in those communities where the provider organizations deliver the peer education intervention.The resource mobilization function offers funding to provider organizations and connects them with reliable and sustainable financial partners. In addition, it spends a significant amount of its time and resources on activities to equip provider organizations with the skills and tools needed to obtain their own resources, independently from the agency. These activities include the development and distribution of an information pack, the delivery of a workshop, and the development and distribution of a list of potential donors.The stakeholder management function has the role its title suggests: it identifies and recruits new provider organizations, and maintains the collaboration of current provider organizations with the agency. Particular care is taken to increase the supportiveness of the environment in which the peer education intervention is delivered, via efforts such as peer education forums and peer education workshops.The training and support services offered to provider organizations include training sessions, workshops, mentoring, and coaching for the implementers. The purpose is typical of such interventions: to equip implementers with the skills and support deemed necessary to ensure effective delivery of the peer education intervention. The training function is responsible for delivering eleven training sessions and workshops, and it conducts bi-annual on-site visits and offers continuous telephone and email support to all the implementers.This study was launched by the realization that the agency lacked an explicit model or theory of how the purveyor program was supposed to work, and what it was supposed to achieve. This was even though it had been running for three years, and with a growing demand to extend its reach to include more provider organizations. In practical terms, our first objective was to develop such a theory for the purveyor program and its staff. This was regarded as an important precursor to further evaluation studies and in particular, a possible process and impact evaluation.As indicated earlier, we also had a somewhat more ambitious objective in mind: to use this particular instance of program theory development as a tentative indication of how one could depict purveyor programs. These programs are complex, because they involve multiple implementation sites, and many provider organizations: in this study, 17 provider organizations, with the possibility of expanding this number. Purveyor programs of this kind present an interesting challenge to evaluation, and to program theory-building in particular. It was exactly this realization that diverted our attention from an implementation assessment to a theory-building exercise in the present study.In terms of the actual supportive activities carried out by the agency, it is a relatively simple program: all the functions and their associated activities are well-defined and understood, and delivered in a reasonably consistent and even manualized manner by the agency. Nevertheless, these are all delivered at very different sites, and to provider organizations with very different governances. This presents a challenge in terms of understanding the purveyor program, what the expectancies of various shareholders are, what local variations can be tolerated, and so on. For present purposes, we thought an understanding of the causal path underlying the intervention was particularly important.Thus, the present study focused on those activities and outcomes involving the agency, as depicted in Figure If, however, the agency wishes to understand and/or investigate what happens in the provider organizations themselves, after their involvement with the agency ; Practice and Research Together, of Toronto, Canada (http://www.partontario.org/); The Evidence-based Prevention and Intervention Support Center (EPISCenter) of Pennsylvania State University (USA) (http://www.episcenter.psu.edu/); and the Connecticut Center for Effective Practice, based in New England, USA (http://www.chdi.org/ccep-initiatives.php). The in-text origin of this endnote is in the third paragraph under the Background section.The authors declare that they have no competing interests.CO and JL conceived the study together. CO conducted the fieldwork, and JL provided input on the interpretation of the results. JL prepared the first draft of this manuscript, and both authors provided initial and final refinements. Both CO and JL read and approved the final manuscript.


Dissemination is a critical facet of implementing quality improvement in organizations. As a field, addiction treatment has produced effective interventions but disseminated them slowly and reached only a fraction of people needing treatment. This study investigates four methods of disseminating quality improvement (QI) to addiction treatment programs in the U.S. It is, to our knowledge, the largest study of organizational change ever conducted in healthcare. The trial seeks to determine the most cost-effective method of disseminating quality improvement in addiction treatment.The study is evaluating the costs and effectiveness of different QI approaches by randomizing 201 addiction-treatment programs to four interventions. Each intervention used a web-based learning kit plus monthly phone calls, coaching, face-to-face meetings, or the combination of all three. Effectiveness is defined as reducing waiting time (days between first contact and treatment), increasing program admissions, and increasing continuation in treatment. Opportunity costs will be estimated for the resources associated with providing the services.The study has three primary outcomes: waiting time, annual program admissions, and continuation in treatment. Secondary outcomes include: voluntary employee turnover, treatment completion, and operating margin. We are also seeking to understand the role of mediators, moderators, and other factors related to an organization's success in making changes.We are fitting a mixed-effect regression model to each program's average monthly waiting time and continuation rates (based on aggregated client records), including terms to isolate state and intervention effects. Admissions to treatment are aggregated to a yearly level to compensate for seasonality. We will order the interventions by cost to compare them pair-wise to the lowest cost intervention . All randomized sites with outcome data will be included in the analysis, following the intent-to-treat principle. Organizational covariates in the analysis include program size, management score, and state.The study offers seven recommendations for conducting a large-scale cluster-randomized trial: provide valuable services, have aims that are clear and important, seek powerful allies, understand the recruiting challenge, cultivate commitment, address turnover, and encourage rigor and flexibility.govNCT00934141ClinicalTrials.  National Survey on Drug Use and Health estimates that 23.5 million Americans age 12 and older needed treatment and 2.6 million received treatment from addiction treatment facilities [The field of addiction treatment serves only a small proportion of people who need its help. The 2009 Several barriers stand in the way of people getting treatment. Individuals who call a program for help often encounter problems such as complicated admission systems, poorly designed phone systems, and a rude reception . As a reJust as several barriers impede access to treatment, so do various factors reduce retention. Clients most often leave treatment because of inconvenient treatment services , dissatiProgram leaders set organizational policies and processes that affect all of their clients, and they have the ability to change them. Given that the literature provides solutions for the access and retention problems in addiction treatment, what is the best way to disseminate improvements? Here, the literature provides surprisingly scant information . One way1. Sample size is a frequent limitation. Because randomization and analysis usually happens at the organizational rather than individual level, randomized trials of organizational change require a large number of organizations. Compared to individuals in clinical trials, it can be difficult to get organizations to participate, stay involved, and pursue the same aims.2. Organizations differ and interventions, if they are to succeed, need to be tailored to the organizations. Differences in organizations make it hard to specify treatment as usual at the start of a study, so the researcher needs another baseline.3. Participating organizations may be only those that volunteer and therefore not be representative of organizations in the field.4. The intervention is hard to hold constant. In drug trials, the same pill is given to every subject in the intervention. But if, for instance, the intervention includes consulting, it is hard to hold consultation characteristics constant.5. The environment is also hard to hold constant in studies that take months or years to complete.The complexities of organizational change explain, in part, two criticisms often made of QI studies: the majority of QI efforts involve single organizations and a simple 'before and after' analysis, and QI efforts are multifaceted and studies of QI do not determine the active ingredients . The scaImprovement collaboratives are one tool commonly used to deliver QI to organizations. Improvement collaboratives involve multiple organizations working together  to make The study described here uses improvement collaboratives along with previously described principles and techniques developed by NIATx, formerly the Network for the Improvement of Addiction Treatment ,39. LikeThe primary research question of NIATx 200 is to determine which of four collaborative service combinations produces the greatest improvement in waiting time (days to treatment from first contact), rates of admissions to treatment, and continuation in treatment. The secondary research question is: What is the impact of the study interventions on treatment completion rates, the level of adoption and sustainability of the recommended practices, organizational readiness to adopt and sustain the new practices, voluntary employee turnover, and program margin? Interventions were delivered to and outcome analyses are performed at the program level.We are also examining the cost of delivering each combination of services. The intent of the cost analysis is to assess the cost of disseminating different QI methods to state agencies that regulate substance abuse treatment. Our budget did not allow us to assess the cost to the treatment programs of implementing the services.NIATx 200 is a cluster-randomized trial with four interventions: interest circle calls, coaching, learning sessions, and the combination of interest circle calls, coaching, and learning sessions. Figure Interest circle calls are regularly scheduled (monthly in our case) teleconferences in which change team members from different programs discuss issues and progress and get advice from experts and one another. Interest circle calls are inexpensive and provide a regular meeting time for members to continue collaborating. If interest circle calls produce QIs, then change can be made widely at relatively low cost. But the quality of interest circle calls may vary by facilitator, and because the teleconferences are scheduled at particular times, they sometimes compete with other priorities, limiting participation. Although participants can listen to recordings of calls they miss, they lose the chance to interact.Coaching assigns a process improvement expert to work with program leaders and change teams to help them make and sustain process improvements. In our study, coaching involved one site visit, monthly phone conferences, and e-mail correspondence. Coaches give a program ongoing access to an expert who tailors advice to the program and makes contacts with other experts and programs that have already addressed the same issue. However, coaching is expensive, and the match between program and coach may not be ideal. Program staff may also miss the camaraderie that comes from learning sessions and interest circle calls. Just as facilitator quality in interest circle calls may vary, so may the quality of coaching.e.g., creating business cases for improvements). Learning sessions raise interest in making changes and provide opportunities for program staff to share plans and progress. The sessions promote peer learning, increase accountability and competition, and give program staff the time to focus and plan as a team without distraction. Learning sessions also are costly, and the knowledge and excitement they produce can fade.Learning sessions occur periodically-in our case, twice a year. These face-to-face multi-day conferences brought together program change teams to learn and gather support from outside experts and one another. Participants learn what changes to make and develop skills in QI , ways to measure change, case studies, and other features. What varied were the methods of instruction and support that were wrapped around the learning kit: interest circle calls, coaching, learning sessions, or the combination of all three.The protocol called for all four interventions to have the same goals during the three six-month intervention periods. For example, each program concentrated on reducing waiting time during months 1-6 of their involvement. During each intervention period, programs chose which practices to implement from those shown in Table The study received approval from the institutional review boards at the University of Wisconsin-Madison and Oregon Health and Science University and is registered at ClinicalTrials.gov (NCT00996645).The Center for Health Enhancement Systems Studies (CHESS), located at the University of Wisconsin-Madison, is a multidisciplinary team addressing organizational change to improve healthcare. NIATx is part of CHESS. The Center for Substance Abuse Research and Policy  works at the nexus of policy, practice, and research for the treatment of alcohol and drug dependence to improve evidence-based addiction treatment. The Health Economics Research Group (HERG) at the University of Miami conducts research on the economics of substance abuse treatment and prevention, HIV/AIDS, criminal justice programs, and health system changes. This core research team worked with five states through each state's single state agency (SSA) (the authority for substance-abuse treatment at the state level).SSA administrative systems identified eligible programs. To be eligible to participate in NIATx 200, programs had to have at least 60 admissions per year to outpatient or intensive outpatient levels of care as defined by the American Society of Addiction Medicine (ASAM) and have received some public funding in the past year. Programs that had worked with NIATx in the past were excluded from participating.  All clients treated within an eligible program were deemed eligible and included in the analysis. SSAs and state provider associations promoted the study and helped recruit participants. Before randomization, NIATx assessed programs' readiness for change and management strength, and asked about which other treatment programs most influenced their own operations. Then programs were randomized to study interventions.NIATx 200 has three primary outcomes: waiting time (days from first contact to first treatment), annual program admissions, and continuation in treatment through the first four treatment sessions. Data for the waiting time and continuation outcomes come from patient information collected, aggregated, and sent in by the SSAs at approximately 9, 18, and 27 months after the start of the intervention. Annual program admissions and other secondary outcomes  were collected through surveys of executive directors conducted at baseline, mid-intervention, and project completion. The research team also surveyed staff members at the treatment programs and is using other measures, described below, to understand the role of mediators, moderators, and other factors that contribute to an organization's success in making changes. As others have demonstrated, QI efforts are much more likely to improve quality if they take place in a supportive context ,41.The Organizational Change Manager (OCM) measures an organization's readiness for change. Staff members at treatment programs completed the OCM. The OCM had good inter-rater reliability among respondents in field tests .Organizations adopt many changes, but sustain few . The 10-The management survey measured 14 management practices at the beginning of the study. The survey was based on the instrument developed by Bloom and Van Reenan . The pubThe Drug Abuse Treatment Cost Analysis Program (DATCAP) is a data-collection instrument and interview guide that measures both direct expenses and opportunity costs. Although DATCAP was initially used in the field of drug abuse treatment, the instrument is now used in treatment programs in many social-service settings. DATCAP was modified for this study to capture the economic costs to an SSA of developing and providing services .Power calculations were predicated on the idea that the unit of analysis is the program rather than the client. Power was calculated for various sample sizes, with consideration given to anticipated recruitment levels in each state. An attrition rate of 20% was assumed in the sample size calculations. It was determined that a sample size of 200 programs would provide 80% power to detect a difference of 3.2 days in waiting time, 7.5% difference in continuation, and 14.2% difference in the log of admissions with alpha = 0.05. These levels of improvement for each outcome were deemed to be clinically or organizationally meaningful.The study design calls for nesting of agencies within states; as such, randomization of programs took place state by state. Though recruitment took place over a period of several months, all programs were randomized at a single point in time at the end of the recruitment period in each state. The randomization was stratified by program size and a quality-of-management score generated during a baseline interview with program leaders . The proThe five states participating in NIATx 200 were divided into two cohorts. Cohort one had three states; cohort two had two states. For each cohort, randomization took place in the two to three weeks before the first six-month intervention began. See Table The state authorities recruited programs to participate in NIATx 200. They promoted the study at meetings and by word of mouth, and wrote letters to the CEOs of programs. They also conducted meetings so the leaders of eligible programs could learn more about the study. At these meetings, the research team and SSA directors explained that programs would use one of four methods to improve processes that affect access and retention. Peer programs with NIATx experience explained the improvements they had made using the same methods and showed changes in data that resulted from the improvements. SSA directors outlined the benefits and responsibilities of programs in the study. Programs would gather pretest data, be randomly assigned to one of four study interventions, and receive 18 months of support. During months 1 to 6, they would focus on reducing waiting time; in months 7 to 12, increasing continuation rates; and in months 13 to 18, increasing admissions. Afterward, the program could join state-led activities to sustain changes. The SSA would send client data to the researchers about waiting time, admissions, continuation, and treatment completion. The program CEO would name from the staff an influential change leader. The leader and staff at each program would complete surveys during the pretest period and at 9, 18, and 27 months. These surveys addressed employee turnover, new practices initiated, number of employees, revenue, and operating margin. Staff would complete surveys about how the program makes and sustains changes. The program would receive minimal compensation for reporting these data.The treatment program comprises both the unit of randomization and the unit of analysis in the study. For the primary analysis, the protocol calls for us to aggregate client records to compute monthly averages for each program's waiting time and continuation rates. The unit of analysis will be a vector of program-month results based on these aggregated values. We will fit a mixed-effect regression model to these monthly observations, including terms to isolate state and intervention effects. We are aggregating admissions to a yearly level to compensate for seasonality. We will use random effects to model the correlation among outcomes from the same program. Organization-level random effects will be included to model the correlation among programs within the same organization. Interventions will be ordered by the cost of implementation and compared in a pair-wise fashion to the lowest cost intervention . All randomized programs with available outcome data will be included in the analysis according to the intent-to-treat principle. The analysis will be conducted by originally assigned intervention regardless of how much programs participated in the learning services. Organizational covariates accounted for in the analysis will include program size, management score, and state affiliation.The following scenario shows what a program assigned to the coaching intervention might have experienced. Assignment to the coaching intervention meant that the program had a process improvement coach visit at the beginning of the study, call every month for the next 18 months, and communicate via e-mail.A program began its work once the CEO named a change leader and change team. This group learned from the research team in the first week of their participation how to do a walk-through, which is a tool that shows staff members what it is like to experience dealing with the program as a client does. The program also had a coach assigned to it by the research team. The coach phoned the CEO, change leader, and change team members to introduce herself and set an agenda for a site visit. The coach also reviewed with the change team the results of the walk-through, an experience that revealed many issues to the change team, including long waiting times. The coach also encouraged change team members to examine case studies on the study website.The site visit allowed the coach and change team members to get to know each other. The coach explained evidence-based improvement principles to the change team and different ways of reducing waiting time, the goal of the study in the first six months. The change team had to decide on one change to make first to reach this goal. In this example, the program decided to adopt a change they learned about from a case study on the study website: Eliminate appointments and instead invite callers to walk in the next morning, complete intake and assessment, and start treatment by noon. Among other things, the change team learned that making this change had resulted in the outpatient program's significantly increasing its revenue. Using information from the case study, the coach helped the change team review in detail how the program made this change-what data they collected, what steps they took, and what protocols they used to train staff for handling the high volume of walk-ins. Finally, the coach helped the change team figure out how to collect pretest data and start the rapid-cycle change process. In two weeks, the program would have enough pretest data (on about 25 clients) to start the change process.Once pretest data were collected, the first rapid-cycle change (or plan-do-study-act cycle) began. The first three callers on Monday were invited to come anytime the next morning to be seen right away. Two callers jumped at the chance. One had to work but offered to come after work. The change leader  did the intake and assessment with one client to experience the new process. The clinician did the same with the other client. At the end of the day, the medical director and clinician modified the change to allow walk-ins at the end of the day too. The next version of the change started Thursday and involved the first five people who called and two clinicians. After this change, the staff identified additional concerns and made other adaptations. Throughout the rapid-cycle changes, the change leader worked side-by-side with clinical staff (a key part of the strategy) to understand problems and make modifications. After staff discussions, the group decided on one last change. Now the new process would be tested with anyone who called for the next week . In the space of three weeks, the overall goal of taking walk-ins was achieved by making and adapting small changes several times, until the process worked well. As a result, the program began serving more clients without adding staff.During the remainder of the six months when the goal was to reduce time to treatment, the program continued to introduce and refine other changes  to have enough power to produce reliable results. This determination gave us a sense of the scale and cost of NIATx 200. We recognize that such a study is costly to implement and evaluate, and it will be hard to replicate at a similar scale. It will, however, provide extensive data for the analysis of organizational change for years to come.i.e., access and retention). Second, the sheer scope of the study (201 treatment programs nested within five states) is unprecedented in QI research, and will provide insights on the effectiveness as well as the cost of delivering an organizational change intervention across multiple organizations. Third, in contrast to research that has examined the effects of a single tool or strategy (such as a checklist), NIATx 200 will advance the field by identifying the active ingredients in a multi-factorial QI strategy. Distance learning, coaching, and learning sessions have been key elements in many QI initiatives. Despite their widespread use, few rigorous studies have estimated their effectiveness. NIATx 200 provides a unique and specific test of their relative contributions to improvement.NIATx 200 advances organizational change and QI in several important ways. First, the findings will provide quantitative evidence of whether QI can improve fundamentally important elements of addiction treatment , the study will provide insights about how states manage data systems and how they are prepared to address information system-related issues in healthcare reform. The study also collected information on which treatment programs exert influence over others, making it possible to examine influence characteristics in addiction treatment and how these may affect the dissemination of change.The study examines methods for moving process improvement from a small number of organizations led by a national office to a state-run initiative involving many programs. Qualitative data will show the client experience in seeking care. Because states had to develop new data systems (It may also be possible to identify practices of organizations that are willing to make changes. The study will describe these practices in detail, assess the impact of the practices on clients, and give practical advice about how to implement them. The measures of management quality, readiness for organizational change, and the potential for sustainability allow validation of those instruments and further understanding of the conditions that contribute to lasting organizational change.The data also will allow for detailed studies of moderation. What type of programs benefit from which QI approach? What type of coaching works best with which types of programs? Under what conditions do specific promising practices have the greatest success? One key product of the study will be the resources developed to teach QIs. For example, in preparing for the study, many promising practices were described in 'how to' manuals. These manuals are available to organizations interested in learning about and using these QI techniques. The diverse, real-world results of the study should provide direction for state leaders, heads of programs, and others about how best to improve access to and retention in treatment.Finally, NIATx 200 shows that rigorous randomized trials of organizational change can be designed and conducted. The information produced will expand our understanding of treatment organizations and provide stakeholders in healthcare delivery a body of research they need to move our healthcare systems forward.The authors declare that they have no competing interests.DG and DM designed the study. AQ, JF, and AP contributed to the design and managed the acquisition of data and the implementation of the project. MF and KM led the economic design. AQ drafted the original manuscript. All authors read and approved the final manuscript.


Hillslope instability has been thought to be one of the most important factors for landslide susceptibility. In this study, we apply geomorphic analysis using multi-temporal DEM data and shake intensity analysis to evaluate the topographic characteristics of the landslide areas. There are many geomorphologic analysis methods such as roughness, slope aspect, which are also as useful as slope analysis. The analyses indicate that most of the co-seismic landslides occurred in regions with roughness, hillslope and slope aspect of >1.2, >30, and between 90 and 270, respectively. However, the intersection regions from the above three methods are more accurate than that derived by applying single topographic analysis method. The ground motion data indicates that the co-seismic landslides mainly occurred on the hanging wall side of Longmen Shan Thrust Belt within the up-down and horizontal peak ground acceleration (PGA) contour of 150 PGA and 200 gal, respectively. The comparisons of pre- and post-earthquake DEM data indicate that the medium roughness and slope increased, the roughest and steepest regions decreased after the Wenchuan earthquake. However, slope aspects did not even change. Our results indicate that co-seismic landslides mainly occurred at specific regions of high roughness, southward and steep sloping areas under strong ground motion. Co-seismic landslides significantly modified the local topography, especially the hillslope and roughness. The roughest relief and steepest slope are significantly smoothed; however, the medium relief and slope become rougher and steeper, respectively.The online version of this article (doi:10.1186/2193-1801-2-544) contains supplementary material, which is available to authorized users. It has been commonly accepted that steep topography are of high landslide frequency in active orogenic region. The co-seismic landslides usually occurred in active orogenic regions, which are one of the major secondary nature hazards related to strong earthquakes  data derived from 1:50,000 topographic maps. The analyses indicate that the co-seismic landslides are closely correlated to the topographic conditions as well as the shaking intensity. The topographic effects of the Wenchuan earthquake are also analyzed based on post-earthquake DEM data derived from stereo pair of IRS-P5 (Indian Remote Sensing Satellite) remote sensing images by comparing with pre-earthquake DEM.The Longmen Shan region is one of the steepest margins of the Tibetan Plateau accompany with almost 5000 m altitude decrease across the margin within 50 km distance  contours distribute in the region peripheral to the co-seismic surface rupture with the maximum value of 957.7 gal (Li et al. Co-seismic landslide is a kind of special landslide directly triggered by strong earthquake (Harp and Jibson Landslide occurrence shows clearly correlation with the topographic conditions. At all the three sites, over 50% landslides occurred in the derived roughness, aspect and hillslope areas over the threshold values, respectively Table . In ordePost-earthquake DEM analysis indicates the medium roughness and hillslope regions are becoming rougher and steeper after the Wenchuan earthquake. The roughest relief and steepest slopes are smoothed by the Wenchuan earthquake Figure . This inCollaborative topographic analyses are efficient in landslide susceptibility evaluation. The co-seismic landslides are related to the topographic roughness, hillslope and slope aspect. The topographic thresholds are >1.2, between 90 and 270, and 30, respectively. The co-seismic landslides are also directly related to PGA values, which is usually occurred on hanging wall within the up-down and horizontal PGA contour of 150 and 200 gal, respectively.Topographic conditions such as roughness and hillslope are controlled by tectonic event like the Wenchuan earthquake, however, slope aspect is not modified by such event.The roughest relief and steepest slope regions were smoothed after the Wenchuan earthquake, however the medium roughness and slope regions became rougher and steeper, respectively.Based on the data and analyses of the present study, we arrived at the following conclusions.