Loyalty programs are a consolidated marketing instrument whose adoption in many sectors has not been associated with appropriate comprehension of either their management elements or their effects. The purpose of this research is to contribute to knowledge about the effect of loyalty programs on repeat purchase behavior. More specifically, it seeks to discover whether joining a program changes the buying behavior of its members, and, if so, to study the profile of those whose behavior changes most. The intention was also to provide new study variables pertaining to multi-vendor loyalty programs, such as where they are joined or purchases in associated outlets as a result of behavioral loyalty. Research was carried out using a sample of 1200 individuals  belonging to a multi-vendor loyalty program. The study period was 13 years, 4 months, and split into two phases: before and after the joining the program. Different methodological approaches, such as the use of transactional databases that included pre-program-enrollment data and of the same sampling units throughout the study, were incorporated into the research with the aim of advancing academic knowledge regarding multi-vendor loyalty programs. Moreover, a type of program and market hardly dealt with in the relevant literature was analyzed. The results showed while the loyalty program had managed to reduce the time between purchases, it had not affected purchase volume or average expenditure. They also demonstrated the existence of a differential profile of customers who had changed their buying behavior to a greater extent. Finally, recency was identified as being the decisive variable in behavioral change. Competition in today's market is intense, which makes acquiring new customers increasingly complicated and less profitable; hence the importance of securing the loyalty of existing customers. In this regard, Alet i Vilaginés and Nueno  point toThe various existing conceptualizations of loyalty mainly revolve around two accepted dimensions . This is because, in practice, only repeat purchase behavior is rewarded, and not attitude, as loyalty schemes are often based on classic promotional techniques, with delayed, or immediate rewards  or relationship marketing techniques , which encourage consumers to purchase more often and remain loyal to the store. We can thus assume, that if a certain number of loyalty cardholders make similar changes in their buying behavior, there will be visible changes in repeat purchase patterns at a store level .Finally, for the purposes of making advances in loyalty program research with adequate scientific rigor, a new methodology was applied, consisting of the use of transactional databases incorporating purchases made prior to when the program was joined and sampling units, that remained constant throughout the study .Authors such as Lewis , Taylor Other authors have also confirmed the influence of loyalty programs on behavioral loyalty, but limiting their effects to the short term, therefore, usually associating them with promotional rather than relationship marketing activities .This can be attributed to the “double jeopardy phenomenon”: that is to say, the greater the market penetration of the brand, the greater the buying frequency and the category quota bought, and vice-versa, so loyalty programs are more effective for market leaders than for small companies  of customers in a multi-vendor loyalty program influences behavioral loyalty toward one of the member establishments.Multi-vendor loyalty programs exhibit differentiating elements, with respect to mono-sponsor ones, which it is necessary to analyze. The possibility of accumulating and redeeming points in the different member establishments of the multi-vendor platform makes a deeper study of this differential aspect necessary. Thus, questions as to whether the establishment in which the program is joined exerts an influence on behavioral loyalty or whether purchasing in other member establishments implies a specific buying behavior arise.If prior related literature is analyzed, according to Meyer-Waarden , the proSo, taking this into account, the following hypothesis regarding the establishment where enrollment takes place was proposed:H4: The establishment where a program is joined has an influence on the change in buying behavior after joining a multi-vendor loyalty program.There have been very few studies on the impact, that the associate companies in a multi-vendor loyalty program have on buying behavior, since the majority of studies have been focused on mono-sponsor programs where relationships with “partners” simply do not exist.Among those who have analyzed multi-vendor programs, Dorotic et al.  comparedGiven the scarcity of bibliographical sources specifically relating to multi-vendor programs, prior literature regarding mono-sponsor programs with associated companies was also analyzed and taken into account, since these are the programs that most resemble multi-vendor ones.In this area, authors such as Lemon and Wangenheim  studied They specifically verified how, as use of and satisfaction with the core service increased, so did cross-purchasing from other services in the program, and vice versa. However, they confirmed that cross-purchasing in one category did not seem to strengthen cross-buying between other categories; that is to say that the relationship was only confirmed when there existed a main product/core service within the program. Moreover, according to these authors, this relationship was influenced by the kind of service that the partners in the program offered; the more, that this fit with and complemented the core service, the greater was the cross-purchasing effect.In contrast with these results, other authors have found, that satisfaction with the core service of an enterprise did not exert a positive influence on the cross-purchase of other services, and also, that this itself was negatively influenced by the number of services joined to it in the recent past Verhoef, . The proThus, it would be interesting to know whether the customers that only shopped at the optical shop (as if the program were mono-sponsor) demonstrated higher behavioral variable values  than the customers that also bought in other member companies . Therefore, the following hypotheses were proposed:H5a: Purchasing in other multi-vendor program member establishments influences behavioral loyalty toward other companies associated with the program .H5b: The number of different sectors in the program in which a customer buys using the multi-vendor program influences behavioral loyalty toward other companies  associated with the program.As explained in the presentation of the dimensions of loyalty, like other studies . The study period was 13 years 4 months, giving it a greater timespan than previous research. The study period was divided into two parts: (a) “before joining the program”  and (b) “after joining the program” . The date the establishment joined the program was April 1, 2002.The sampling frame comprised those customers of the optical shop chain who, being members of the selected loyalty program, had made at least one purchase in each of the two periods under study. The selection procedure for the sampling units within the program used was simple random sampling.A sample of 1,200 individuals who had effected a total of 31,746 purchasing actions in the two periods studied was obtained.Concerning the information about the ethics requirements, this study was made according to criteria of the Ethics Department of the Rey Juan Carlos University and to the ICC/ESOMAR International Code on Social Research. Personal or sensitive data were deleted of the initial database because they did not provide relevant information for the analysis.The study variables were calculated by cross-referencing the electronic transactional data from the multi-vendor program with that of the retailer studied.Visual Basic for Applications were used. Also, specific new variables on the operation of loyalty programs were added based on the recommendations given by industry experts.The initial database was modified with a complex conversion process to suit the objectives of the study, and some existing variables were transformed according to the criteria used in the literature; to calculate these variables, algorithms programmed in Finally, a total of 45 variables grouped into 9 constructs were used for the analysis, related to: (a) purchase behavior, (b) change in buying behavior after joining the program, (c) purchase behavior by category of product purchased, (d) recency, (e) multi-vendor purchase behavior (variables related to purchases made in other establishments), (f) type of consumer, (g) level of accreditation, (h) communication channels used by the multi-vendor program, and (i) redemption of points.T-test for related samples and, later, a Wilcoxon test to confirm the results obtained were carried out , while in the Wilcoxon test they were not (p = 0.06); the significance level was slightly higher than the critical value. It could therefore not be confirmed that joining the program had caused a change in average annual expenditure.The last measurement variable used for the purposes of this research was the average annual expenditure of the customers. As was the case with the average purchase price, there was a decrease in the second period analyzed. However, here, the two tests used differed. In the These results partially confirmed Hypothesis 1. Although a change took place in all the variables used, after joining the program, the differences only turned out to be significant and positive for the establishment in the case of reducing inter-purchase time and increasing the average number of shop visits.Next, in order to analyze the profile of those customers who had most changed their buying behavior after joining the program, the sample was split into two groups comprised of those customers who had greatly changed their behavior compared to those who had done so to a much lesser degree. The difference in averages considered was the difference between the 5% of customers that had most increased their buying behavior and the remaining 95% who had done so to a much lesser extent. Afterwards, a comparison of the differences in behavior for the two sub-samples both “before” and “after” was carried out for each of the buying behavior variables.The results obtained for each one of the buying behavior variables demonstrated, that the change produced was related to several variables from the period prior to when the loyalty program was joined. Thus, the variables, that had most significantly influenced the buying behavior of the customer group analyzed were: recency in the pre-joining period and previous average expenditure on key optical products, lenses and graduated lenses , this greater change was noted for all the buying behavior variables except that of the average number of articles purchased.It is also interesting to note, that the consumers who showed the greatest change in number of store visits (-0.14) were customers whose enrollment had taken place in program member establishments other than the optical shop in question and who bought in other sectors, such as clothing (0.14). Moreover, purchasing in the fuel sector caused a greater increase in the average purchase price for the customers in the analyzed group (0.016). Therefore, on the basis of these results, Hypothesis 2 (H2) can be accepted.p-values confirmed the absence of correlation between the analyzed variables. Therefore, the third hypothesis was rejected.Having determined the influence of recency on buying behavior change, the influence of the point accumulation rate on behavioral loyalty was then verified. In order to do this, the degree of relationship between the average accumulation rate and each one of the buying behavior variables in the post-joining time period was studied  = −2.79, p = 0.01, and W = 162.940, p = 0.02]; and (b) average number of visits, which was higher for those who joined at other establishments. Here it should be pointed out that the results obtained with the T-test  were not significant, whereas with the non-parametric contrast of the Wilcoxon test it was confirmed that they in fact were , although with only the minimum critical level. Therefore, Hypothesis 4 was partially rejected.First, the influence of the “enrollment establishment” on buying behavior change after the multi-vendor program was joined was examined. The results of the tests carried out confirmed, that the differences, that existed on the basis of the enrollment establishment were only significant with regard to the variables  = −2.0, p = 0.05 y W = 103, 437, p = 0.05). Thus, Hypothesis 5a was partially rejected.With respect to the effect on behavioral loyalty of buying at other multi-vendor program member establishments, it was verified, that the customers who purchased at other program establishments, and not only at the optical shop, had greater basket volumes, a higher average purchase price, a lower inter-purchase time, and therefore, a higher average number of visits to the store, and, finally, a greater average annual expenditure. On the other hand, those customers who used the program as if it were mono-sponsor, only using the loyalty program when buying at the optical shop, did so on average with a higher number of articles and categories. However, these differences between the two groups of customers were only found to be significant with respect to the average annual expenditure variable [H-test was used  customers, but not in making them loyal.It was also confirmed that the category of products sold, in this case less frequent, medium-priced consumer goods, obtained worse results than other types of products more typical of research focused on loyalty programs . For this reason, the results of this work have provided important business decision-taking information for the different stakeholders involved—the program management and the member companies. This information concerns the type of company that should join the program and the need to simultaneously coordinate program activities with those specific to each member brand in order to improve the results.An important decision to be taken by companies, with regard to the incorporation of a loyalty program into their strategies, is to choose the type of program to which they adhere. For many companies, the decision to join a particular multi-vendor platform is based on management complexity and the higher costs associated with mono-sponsor programs. However, in view of the results of this work, companies wishing to optimize the benefits offered by these programs should assess their limited effects on behavioral loyalty and consider that their advantages will be conditioned by the type of company that is associated with them, as well as the development of a management style that favors the company's own objectives against those of all the other companies promoting the same multi-vendor program.It is important to point out that the company managing the program should not only include associate companies from different sectors in the program, given that behavior in one sector does not affect behavior in the rest, but it should also, preferably, make sure that the services of these associated establishments are complementary. This would greatly improve effectiveness. Likewise, it has also been shown that the relative importance of each member company within the multi-vendor loyalty program is a determining factor in behavioral loyalty. The driving-force companies of the program will improve the behavioral loyalty of their customers to a greater extent than those companies of lesser importance within the program itself. Thus, the total accumulation rate in the program will only be an interesting segmentation variable for the companies that carry more weight within the program.With regard to the benefits related to loyalty that have been shown, the increased frequency of visits to related businesses has very positive effects for the management of the establishment, to the extent that:greater possibilities for cross-selling are offered, which helps to identify the most suitable products for sale through promotions.personalized communication and service, as well as targeted advertising campaigns, is favored in comparison to communications made through mass media.the chances that visits will be made to local competitors are diminished.The fact that multi-vendor programs represent a very useful marketing tool which permits the management of a heterogeneous client base is also verified. From an operational point of view, the ability to identify the profiles of customers who are more sensitive to program actions subsequent to joining, highlights the need to carry out differentiated relationship activities. However, the process of customer segmentation should be made independently by each of the associated companies and not by the company managing the program. This recommendation, which is derived from the results obtained jointly in the program, does not correspond to the behavior seen in each of the participating establishments as previously explained. In this regard, it is recommended that those responsible for the companies that are associated with a multi-vendor program manage information proactively, adapting it to their needs. This implies a greater commercial training of managers and employees in contact with customers, in addition to building a good relationship with the program promoters, which will help the program to adapt, as well as develop specific targets for each establishment based on their situation and the developments in their respective markets. This has obvious implications for companies that choose to adopt multi-vendor programs in order to reduce their marketing costs. For example, a company associated with these programs does not avoid—if they wish to optimize their benefits—having to realize their own communication actions since the generic communication program integrates all associated brands, making it less effective. Communication activities of the program are designed to benefit the whole program and its objectives do not necessarily match the conditions that each of the companies taking part in the program require. Since communications made by the program are not sufficiently segmented, each associated company must decide what their objectives are and what to communicate. Therefore, if an associated company is participating more actively in the management of the program to suit their own particular needs, they will obtain better results, thus improving customer loyalty.The relationships between loyalty programs and behavioral outcomes are more complex than has been assumed. This empirical investigation had certain limitations: the effect of the loyalty program was only tested with regard to behavioral loyalty. The integration of attitudinal variables to complement the behavioral approach to loyalty would be a promising area of research.It would likewise be interesting to continue to progress with the exploratory models used in this study, with a view to more precisely explaining changes in buying behavior after a customer joins a multi-sponsor loyalty program. Therefore, new variables should be incorporated related to purchases at competing establishments, such as share of wallet, a variable that, as seen in the literature review, has been included in numerous studies on behavioral loyalty. In other words, it should be determined whether the increase in the number of store visits is concentrated in the program and, therefore, decreases the number of visits to the competition or if, on the contrary, the frequency of visits increases under the program but remains unchanged with regard to the rest of the stores. This would mean that, although the program would improve purchasing behavior at the associated establishment, high levels of customer loyalty would not be achieved, as customers would continue to divide their purchases between the different competing brands.Additionally, future studies should address segmentation in loyalty programs based on customer lifetime value. As proposed by Kumar and Shah , it woulFinally, given that the data analyzed correspond to a single multi-sponsor loyalty program , and even though the program's representativeness is justified as there is only one other program with the same characteristics in Spain, it would be advisable to compare the results with those of other international programs.The directors of research have been the professors PR and ER. The three co-authors have participated in all stages of work, including the conception and design of the research, the revision of intellectual content, and drafting the work. The analysis and interpretation of data has been the responsibility of the professor TV.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Summary: JSBML, the official pure Java programming library for the Systems Biology Markup Language (SBML) format, has evolved with the advent of different modeling formalisms in systems biology and their ability to be exchanged and represented via extensions of SBML. JSBML has matured into a major, active open-source project with contributions from a growing, international team of developers who not only maintain compatibility with SBML, but also drive steady improvements to the Java interface and promote ease-of-use with end users.Availability and implementation: Source code, binaries and documentation for JSBML can be freely obtained under the terms of the LGPL 2.1 from the website http://sbml.org/Software/JSBML. More information about JSBML can be found in the user guide at http://sbml.org/Software/JSBML/docs/.Contact:jsbml-development@googlegroups.com or andraeger@eng.ucsd.eduSupplementary information:Supplementary data are available at Bioinformatics online. It enables systems biology information to be expressed in Java data structures patterned after the SBML format for fast access. JSBML 1.0 implements the ability to encode, exchange and use all parts of SBML, up to the current release, SBML Level 3 (L3), including all community-approved  SBML packages for L3, as described later.The Systems Biology Markup Language (SBML) is a widely used format that enables easy distribution of systems biology data, models and diagrams, and it allows the easy exchange of data and models between a variety of software systems . Given tOne important aspect of SBML is its ability to provide additional capabilities to encode specific types of systems biology models. These capabilities are extensions of the core SBML L3 format and are known as ‘packages’. Although many models can be fully represented using only the core set of SBML constructs, these extensions support (i) other model features that cannot be formulated with the SBML core standards and (ii) additional constructs that enable users in specific fields to formulate, interface and use the SBML framework more easily within their modeling approaches. SBML packages enable the ability to build models that encompass several formalisms within one overall framework. Several tools today use JSBML 1.0 in this way and encode multiple types of systems biology models; examples include iBioSim  allows species in a model to have non-quantitative or non-continuous levels  provides a generic framework to encode models as hierarchical entities in SBML (comp implementation provides access to elements within sub-models and interfaces with other models.At the time of writing, all approved SBML L3 extensions are supported by JSBML 1.0. We describe the support below, and provide insights into the mapping between modeling formalisms and the corresponding JSBML data objects. An abbreviated version of the JSBML class hierarchies are presented in nts fbc,  encodes Spatial Processes (spatial, Groups (groups, Arrays (arrays,Required Elements (req, Distributions (distrib, Dynamic Structures (dyn, Rendering (render, layout to provide symbol and style information for diagrams. The last package, Multistate and Multicomponent Species (Draft specifications are available for the remaining SBML packages; they are encoded in JSBML with varying maturity. JSBML fully supports the current specifications of seven packages whose community approvals are pending:  groups,  agglomernts req,  allows adistrib,  encodes  Species  is stillarrays package. Prior to the development of arrays in JSBML, the specification lacked important validation rules that serve as quality controls for math operations done with arrays data structures. JSBML also provided the environment to ensure arrays was compatible with other SBML L3 packages, helping to build a more robust specification. Finally, JSBML development of the arrays package was used to generate simulatable examples for the SBML L3 arrays specification. Therefore, JSBML provides a means to test and implement new package development for future versions of SBML.Some package specifications have been influenced by JSBML development. For example, protocols for validation and flattening of array constructs in the JSBML version 1.0 marks the maturation of this software library as an essential component for any systems biology pipeline that runs in the Java Virtual Machine, and joins other biological exchange format interpreters (Paxtools,


Communication is important for the quality of clinical practice, and programs have been implemented to improve healthcare providers’ communication skills. However, the consistency of programs teaching communication skills has received little attention, and debate exists about the application of acquired skills to real patients. This study inspects whether (1) results from a communication program are replicated with different samples, and (2) results with standardized patients apply to interviews with real patients.A structured, nine-month communication program was applied in two consecutive years to two different samples of healthcare professionals . Results were assessed at four different points in time, each year, regarding participants’ confidence levels (self-rated), basic communication skills in interviews with standardized patients, and basic communication skills in interviews with real patients. Data were analyzed using GLM Repeated-Measures procedures.Improvements were statistically significant in both years in all measures except in simulated patients’ assessment of the 2008 group. Differences between the two samples were non-significant. Differences between interviews with standardized and with real patients were also non-significant.The program’s positive outcomes were replicated in different samples, and acquired skills were successfully applied to real-patient interviews. This reinforces this type of program structure as a valuable training tool, with results translating into real situations. It also adds to the reliability of the assessment instruments employed, though these may need adaptation in the case of real patients. Studies consistently report improvements in communication skills among participants in programs teaching these competencies in healthcare settings -3. The eImprovements in communication skills after programs aimed at these competences are noticeable in participants’ subjective sense of confidence in their clinical practice ,9 and reStudies have shown that improvements in communication skills after a program are observed in encounters with both simulated and real patients ,21,22, bSince effective communication has been shown to be relevant to clinical practice -30, and The program had a duration of nine months and was offered in 2008 and, again, in 2009 as a post-graduate program on communication skills. Each year, the same content was taught by the same faculty members, following the same structure and sequence. The program has been described in detail in a previous work  and is sClasses met twice a week for five hours each day and typically included theoretical presentations of the materials, role-modeling through video viewing and discussion, and role-playing in small groups of peers (six or seven students), with feedback by both peers and faculty. Participants were divided into the smaller groups according to their professional backgrounds, to maximize group heterogeneity and exposure to professional diversity. Two faculty members per group tutored the same students throughout the program, encouraging participation and discussion. The role-playing practice represented a substantial part of the program (about 67 percent of the course load) and used previously written scripts based on cases of patients from the faculty’s and the students’ clinical experiences.In this observational study, participants were evaluated at four moments in time: before the program (T0); three months into the program, at the end of the basic communication skills section (T1); six months into the program, at the end of the advanced skills section (T2); and three months later, at the end of the program (T3). In each moment, participants conducted a 25-minute first interview with SPs. All interviews were videotaped with participants’ consent. Participants were also asked to record a 25-minute first interview with one of their RPs, in a real situation, at T1, T2 and T3.Participants were evaluated on their communication performances in the videotaped interviews with SPs (by two trained faculty members per interview and by the SP after the interview) and with RPs (by one trained faculty member per interview). They were additionally evaluated on their (self-rated) levels of confidence in conducting clinical encounters at T0, T2 and T3. Participants were informed that, with the exception of the interview at T0, all interviews with SPs and with RPs counted towards their grades. Participation was voluntary, and data were used for research purposes with participants’ consent. The study was approved by the Ethics Committee for Health of S. João Hospital/School of Medicine of Porto, and complies with the Declaration of Helsinki.The interviews in 2008 and 2009 used the same five professional actors  from one theatre company who met with the students during these evaluation times exclusively. The actors shared the same ethnic background as the students . Three were in their thirties, one male was in his late twenties and one male was in his fifties. Each actor met with an average of three to eight participants per evaluation time each year.The actors had no previous experience as SPs, though two had majored in psychology and a third had attended several workshops dealing with communication in medical settings. The team of faculty members  coached the actors on the purposes of the interviews, on the scripts of ‘patients’ created for the SPs and on the specific aspects of the encounters, such as the amount and kind of “talking” and “acting” they should do for the purposes of these interviews. Each script was reviewed with each actor, and possible responses and interactions rehearsed. The initial preparation took about two hours with each actor, plus an average of over two hours actors reported spending with each script at home. At each evaluation point, actors were again briefed on the scripts, the acting and the purposes of the program. Further discussions of the cases and the interviews occurred throughout the programs on demand. Additionally, faculty members systematically monitored and gave SPs feedback about their performances throughout the programs in order to maintain acting quality and consistency, and keep it within the evaluation goals.Written by faculty members with clinical experience, the scripts featured situations based on real cases of patients. Tailored to the actors (each actor had his or her own scripts) and adapted to the professional areas of the participants in this study, all scripts followed the same format and contained detailed information on patients’ personal circumstances , sets of symptoms , health history, family health history, life style , and psychosocial aspects . Since the main goal of the study was communication, actual clinical symptoms were consistently kept simple across scripts, namely to ensure equivalent situations across the different professional areas involved.Faculty members assessed participants’ communication skills in interviews with SPs and with RPs using the SEGUE framework . SPs ratThese instruments, as well as the rationale for their choice, are described elsewhere ,15,31-34The ICSC was developed by the eight medical schools in the New York City Consortium for Clinical Competence. It is a 17-item (yes/no) checklist designed to assess interpersonal and communication skills that has shown acceptable psychometric properties .Developed to assess residents’ attitudes towards psychosocial skills used in medical care, Smith et al.’s questionnaire is a 38-item, 7-point Likert scale that evaluates self-efficacy [Twenty-five healthcare professionals participated in the program in 2008 and another 20 enrolled in 2009. The two groups are comparable in several ways: professional background composition , professional experience , and gender .For the SEGUE framework, and for the ICSC, the score was the percentage of items checked ‘yes’ in each interview. The mean of the scores given by the two different SEGUE raters to interviews with SPs was calculated and used. For the measure of confidence, the mean of the ratings  was computed for the used items. Total scores for each measurement time were obtained by calculating the mean of all students’ scores in each instrument.To address the first goal of this study, GLM Repeated-Measures procedures were conducted in PASW  to inspect changes over time in the measures of confidence and of performance (SEGUE and ICSC) with SPs, and to examine differences between samples, following a factorial plan of 2 × 4 – (2008/2009 group) × . Bonferroni correction was used for multiple comparisons .In order to assess whether changes with the program transfer to real situations with RPs , another GLM Repeated-Measures procedure was conducted on the measure of performance (SEGUE) according to a factorial plan of 2×3 – (SPs/RPs) × . Participants’ interviews with RPs were added to their SPs’ interviews, leading to a database of 90 subjects. Because the instruction in both situations asked for a first encounter with a patient, students who conducted other types of encounters with RPs were excluded. Thus procedures were carried out for a total n = 70.Performances follow similar patterns in the 2008 and 2009 groups Figure . The 200F = 24.79, p < 0.01), but not for the 2008 group  = 19.14, p < 0.01), at T2  = 45.75, p < 0.01) and at T3  = 30.90, p < 0.01). By contrast, SPs already rated 2008 group performances at high levels before the beginning of the program, approximating them to their subsequent evaluations at T1, T2 and T3.Effects of taking the course assessed by SPs are statistically significant for the 2009 group  = 17.61, p < 0.01), as well as the 2008 sample  = 6.90, p < 0.01). At the end of the program (T3), participants were significantly more confident in their communication skills in clinical interviews than before taking it  = 10.17, p < 0.01; 2009 group, F = 24.76, p < 0.01), and the increase in confidence was statistically significant also at T2 for the 2009 group  = 11.17, p < 0.01), though it was non-significant for the 2008 sample, reflecting the former group’s greater change with the program, mentioned above. Pairwise comparisons further indicate that, for the 2009 group, the change between T2 and T3 is also statistically significant , though it is non-significant for the 2008 group, with its less pronounced increase.Effects of taking the course on self-confidence are statistically significant for the 2009 group  = 48.69, p < 0.01), as well as the 2008 group  = 40.31, p < 0.01). Comparing with T0, the increase in communication skills was statistically significant for the two samples at T1  = 68.52, p < 0.01; 2009 group, F = 46.18, p < 0.01), T2  = 104.83, p < 0.01; 2009 group, F = 117.85, p < 0.01) and T3  = 44.02, p < 0.01; 2009 group, F = 93.88, p < 0.01) . As mentioned before, in both samples, the change in competence at T3 remains statistically significant, comparing with T0, despite the decline .Results show the numerical similarity between ratings for SPs and for RPs throughout the program Table .ParticipRegarding the first goal of this study, the analyses indicate that the positive outcomes of this structured, comprehensive training program are replicated in different samples in two different years. These positive outcomes are reflected, each year, in statistically significant increases in confidence, self-rated by participants, and in communication skills, assessed by external observers and by SPs, with between-group effects indicating that no differences exist overall between the 2008 and the 2009 groups throughout the program. The slight decay observed for both the 2008 and the 2009 groups at T3 has no statistical significance in either external observers’ or SPs’ assessments. These findings substantiate the robustness of the procedures, validating the program through confirmation of its results with a different sample and in a different year . They also show the stability of the instruments employed, which adds to their potential for comparative use .The fact that SPs’ ratings at T0 are lower in 2009 (close to faculty’s ratings) than in 2008 may partly be an effect of learning. Throughout their first year, SPs were exposed to professionals increasingly more effective at communicating, which may have created new (and higher) standards for them, then applied to the next year’s participants at T0. They may also have become more familiarized with the specificities of the competences taught, moving away from the global ways in which patients tend to evaluate health professionals -36 and pOn the other hand, participants in the 2009 class scored lower at T0 than those of the 2008 group in all measures. This could indicate that the 2009 group began with fewer communication skills than did the 2008 sample . However, the generally smaller standard deviations in 2009 for SPs  support the learning hypothesis  self-assessments with evaluations by external observers and by SPs; including evaluations at different points in time, namely before and after the program; using two different samples of participants; and including SPs in controlled situations, as well as RPs in real situations. Future studies with control groups and participant randomization in a pretest-posttest design can help disentangle the effects of confounding variables, such as participants’ own experience, or the effects of the more advanced classes of the program, on the observed increase in basic communication skills and in self-confidence. Once the outcomes of the overall program are established, its defined components can also be manipulated to assess their specific effects in the results. Qualitative interviews will provide relevant information on participants’ perspectives about what makes the communication training effective and why. Finally, the program’s robustness can further be tested when taught by different instructors in future studies.The consistent positive results in this study indicate that this type of program structure and procedures can be used to improve communication skills among healthcare professionals. The skills learned and practiced within the program in artificial settings are transferrable to controlled situations with simulated patients, and results are replicated in different samples of participants. They also transfer to real-life situations with real patients, the ultimate goal of these courses. Future follow-up studies with these samples will assess the permanence over time of these communication skills in clinical interviews.The authors declare that they have no competing interests.All authors participated in the conception of the study after RMC’s initial proposal, and in the data collection process. IPC, VGP and FRS conducted the literature review and IPC analyzed the data. IPC wrote a first draft of the manuscript, which was read by the other authors. All authors contributed to the paper’s final version. All authors read and approved the final manuscript.Parts of this work were presented at the International Conference on Communication in Healthcare in Verona, Italy, September 2010.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6920/14/92/prepub


Online education programs are becoming a popular means to disseminate knowledge about evidence-based practice (EBP) among healthcare practitioners. This mode of delivery also offers a viable and potentially sustainable solution for teaching consistent EBP content to learners over time and across multiple geographical locations. We conducted a study with 3 main aims: 1) develop an online distance-learning program about the principles of evidence-based practice (EBP) for chiropractic providers; 2) test the effectiveness of the online program on the attitudes, skills, and use of EBP in a sample of chiropractors; and 3) determine the feasibility of expanding the program for broader-scale implementation. This study was conducted from January 2013 to September 2014.This was an exploratory randomized trial in which 293 chiropractors were allocated to either an online EBP education intervention or a waitlist control. The online EBP program consisted of 3 courses and 4 booster lessons, and was developed using educational resources created in previous EBP educational programs at 4 chiropractic institutions. Participants were surveyed using a validated EBP instrument (EBASE) with 3 rescaled (0 to 100) subscores: Attitudes, Skills, and Use of EBP. Multiple regression was used to compare groups, adjusting for personal and practice characteristics. Satisfaction and compliance with the program was evaluated to assess feasibility.p < .001) and skills  subscores, but not the use subscore . The majority of participants agreed that the educational program was ‘relevant to their profession’ (84 %) and ‘was worthwhile’ (82 %). Overall, engagement in the online program was less than optimal, with 48 % of the Training Group, and 42 % of the Waitlist Group completing all 3 of the program courses.The Training Group showed modest improvement compared to the Waitlist Group in attitudes (Δ =6.2, Online EBP training leads to modest improvements in chiropractors’ EBP attitudes and skill, but not their use of EBP. This online program can be delivered to a wide national audience, but requires modification to enable greater individualization and peer-to-peer interaction. Our results indicate that it is feasible to deliver an online EBP education on a broad scale, but that this mode of education alone is not sufficient for making large changes in chiropractors’ use of EBP. Evidence-based practice (EBP) has been widely adopted as a standard by all health disciplines. Defined as a systematic approach for integrating the best research evidence with clinical expertise and patients’ unique values and circumstances , it requInterest in EBP among chiropractors, one of the most frequently-used Complementary and Integrative Health (CIH) disciplines in the US , has shoThere are barriers to chiropractors engaging in EBP, including lack of training, skills and resources, as well as time constraints and concerns regarding the relevance of existing research . These cOnline education programs have been shown to be as effective as in-person instruction for improving attitudes and knowledge of EBP among healthcare practitioners  and thisOur long term goal is to develop a comprehensive EBP literacy campaign on a national scale for chiropractors and other CIH providers. To this end, our study had 3 aims: 1) develop an online distance-learning program on the principles of evidence-based practice (EBP) for chiropractic providers; 2) test the effectiveness of the online program on the attitudes, skills, and use of EBP in a random sample of chiropractors; and 3) determine the feasibility of expanding the program for broader-scale implementation on a national basis.The study adopted an exploratory, multi-phase, multi-method approach. We previously published the results of a national cross-sectional survey of US chiropractors, which comprised the first phase of the study and captured a baseline measure of EBP literacy . The secOur study was designed to explore several questions and “unknowns” about the processes and outcomes of online EBP training. First, we found a paradoxical discordance between good attitudes but poor use of research evidence, which begged the question “why”? Secondly, although online educational programs have been shown to be generally effective, we wanted to know if an online educational program specifically focused on research and EBP would be effective in improving chiropractors’ level of EBP literacy and use. Lastly, we wanted to explore questions about which attributes of our online educational program would private practice chiropractors find useful, as well as the barriers and facilitators to completion of the online program.The study was an open-label, prospective, parallel-groups randomized controlled trial with a delayed waitlist control. Volunteers came from a random sample of chiropractors who completed the first phase of the study. They were randomized using a computer-generated algorithm, prior to which group allocation was concealed from all participants and study personnel. The study was funded by the National Institutes of Health/National Center for Complementary and Integrative Health (R21 AT007547), with Institutional Review Board approval (exempt status) granted by the University of Pittsburgh (PRO12060417) and all other participating institutions. This study was conducted January 2013–September 2014.To participate in this RCT, individuals had to hold a doctor of chiropractic degree, reside in the US, have Internet access, have a valid email address, and have participated in the cross-sectional national survey. There were no participant exclusion criteria.Participants were recruited from an original pool of 1,314 chiropractors who completed a cross-sectional national survey . We sentThe online educational program was developed and adapted from competencies and resources created through previous National Center for Complementary and Integrative Health funded EBP educational grants awarded to four chiropractic institutions –15. PartThe EBP educational program was delivered online over a 7-month period for each group. The overall goal was to provide foundational skills for practitioners to become ‘informed consumers’ of research . ProgramTo incentivize participation, up to 10 h of continuing education credits were available to participants from qualifying states. E-mail reminders were sent 2 to 4 times per month to encourage participation, and phone calls to stimulate interest were made to the subset of participants who registered for the online program, but did not complete any courses. The EBP educational program consisted of two parts: 1) a series of online educational courses and 2) 4 monthly online booster lessons.The first part of the educational intervention consisted of a series of 15 to 40-min modules, divided into 3 general courses. The entire program contained a total of 18 modules and required an estimated total of 10 h to complete. While participants were encouraged to complete the courses at their own pace over 2 months, they were provided program access for the entire 7-month intervention period. The targeted learning objectives for the program focused on the EBP topics summarized in Table Four online booster lessons, consisting of 30-min interactive presentations, provided opportunities for participants to review and practice their EBP skills Table . Three mWe collected demographic and baseline EBP information using online self-report questionnaires during our national cross-sectional survey . Our effFeasibility data collection included course and booster session completion rates. A program evaluation survey was also administered to gain insight as to participants’ satisfaction with the program. The survey included 23 items addressing the educational program overall, and the online course modules and boosters specifically, using a 5 point Likert scale .Power analysis showed that a sample size of n = 125 per group would yield 88 % power to detect a between-groups, standardized effect size of 0.4 on E-BASE subscore changes. We achieved this projected enrollment goal by randomizing a total of N = 293 participants; n = 147 in the Training Group and n = 146 in the Waitlist Group.Descriptive statistics, including measures of central tendency and variability, were generated to examine the demographic characteristics and EBASE scores for the immediate access and waitlist groups. We examined the effectiveness of the educational program by comparing the differences in the between-group changes in EBASE attitudes, skills, and use subscores from baseline to Time 2 (9 months). We used general linear models with robust standard errors and controlled for baseline EBASE subscore value, gender, practice focus, education, number of patients seen daily, and years in practice. These clinically relevant covariates were each correlated with the outcomes (p < 0.05). Prior to these analyses, we rescaled each EBASE subscore to be on a 100 point scale  by normalizing the data and multiplying by 100. This linear transformation was conducted to facilitate ease of interpretation of the results by using the same scale for the 3 subscores. All statistical analyses were performed using SPSS 22  with statistical significance set to .05.Although the primary endpoint for the between-group analysis was Time 2, a third EBASE (Time 3) was administered at 16 months, for two reasons. First, we wanted to explore descriptively the within-group changes in EBASE subscores following the program for the Wait-list Group (Time 3 – Time 2), to see if it was comparable the change following the program in the Training Group (Time 2 – Time 1). Second, we wanted to explore the sustainability of the within-group changes in the Training Group, 7 months after they had completed the educational intervention (Time 3 – Time 2).Feasibility was assessed by calculating frequencies and percentages of the number of courses and booster sessions completed for each group. These descriptive feasibility data were collected at Time 2 for the Training Group, and at Time 3 for the Waitlist Group. We tracked the total number of people in each group who completed each online course and booster lesson, which was reported as the frequency. We then divided the number of completed courses and booster lessons by the total number of people in that group to arrive at the completion rates.To ease interpretation of the program evaluation surveys, we created dichotomous variables by collapsing the response categories ‘strongly agree’ and ‘agree’ into one ‘agree’ variable, and combining ‘neutral’, ‘disagree’, and ‘strongly disagree’ responses into one ‘disagree’ variable. Frequencies and percentages were then calculated using these dichotomous variables for each group.Table Figure In terms of data collection follow up, at Time 2 (primary endpoint), 59 % of the training group completed the second EBASE and the Program Evaluation Survey, compared to 84 % of the Waitlist Group. At Time 3, 61 % of the Training Group and 60 % of the Waitlist Group completed the third EBASE questionnaire.p < .001). There was no statistically significant difference between groups for the Use subscores . Table Table Feasibility was measured by rates of online EBP course completion and the monthly booster session completion rates. Generally the course and booster session completion rates among those randomized was low, with only 71 (48 %) randomized to the Training Group completing all three courses compared to 61 (42 %) in the Waitlist Group. For the boosters, 79 (54 %) of participants randomized to the Training group compared to 59 (41 %) in the waitlist, failed to participate in any booster lessons. Only 23 (16 %) in the Training Group and 35 (24 %) in the Waitlist Group completed all four booster lessons.overall educational program was ‘relevant to their profession’ (84 %) and ‘was worthwhile’ (82 %). Participant views regarding specific aspects of the online course modules and the booster lessons are also illustrated in Table Table ession’ 8 % and ‘wThis is one of the first studies to systematically explore the effectiveness and feasibility of delivering an online EBP educational program for chiropractors, one of the most widely utilized CIH disciplines in the United States .Significant differences between the training and waitlist groups were observed in self-reported EBP attitudes and skills, but there was no meaningful change in EBP use following exposure to the educational program. However, the magnitude of these significant group differences were only modest, and given that no established standard exists for what constitutes a meaningful difference in EBP outcomes, the potential impact of these findings is unknown.The modest improvement in EBP attitudes and skills, and the absence of any change in EBP use were unexpected findings. This is in light of literature showing improvements in EBP knowledge, skills, attitudes, and behaviors following EBP training in other health disciplines, including allied health , nursingFirst, it is possible that the small change in attitudes was due to self-selection bias and an associated ceiling effect. That is, participants were already favorably inclined towards EBP and motivated to take part in the study, resulting in high pre-intervention attitude sub-scores with little room for improvement. These same factors might also explain the modest change in EBP skills. Another explanation for the observed outcomes is that the previously developed online courses were designed as a ‘companion’ intervention to be used with other more interactive educational venues such as classes, workshops, and other formats . While tFurther, if our EBP educational program is considered within the context of relevant behavioral theories, the lack of social support in the program may be a critical and absent component. While the program did provide opportunity to enhance EBP capabilities critical for enacting and enabling EBP behaviors , 34, theOur choice of outcome measure may also have influenced the observed results. Ideally, outcome measures should be both psychometrically sound and mapped specifically to what an intervention aims to achieve . While tGiven the educational program was considered foundational in nature, we did not anticipate large changes in the EBASE ‘use’ domain. In fact, it is unlikely that >any educational program will result in important changes in research use due to the inherent complexity of EBP-related behaviors , 8. InstRegarding our feasibility aims, we were able to successfully develop and host a series of online educational modules and deliver monthly booster lessons to almost 300 chiropractors in different geographic regions of the US. However, we found generally poor compliance with completion of the online courses and booster lessons. The engagement data illustrated that a number of individuals (14–26 %) failed to register for the educational program after randomization. In this study, participants were required to take a number of online steps (clicks) in order to proceed from registration to commencement of the educational program, the purpose of which was to accommodate the processing of continuing education credits. This may have proved un-motivating to some, especially those unaccustomed to online education formats .did register for the program was also somewhat disappointing, especially given efforts encouraging participation via e-mail and telephone reminders. Of those that registered for the program, 55–63 % completed at least one course with 42–48 % completing all three courses. Participation in the booster lessons was far less, with 32–33 % completing at least one lesson, and only 16–24 % completing all four. Indeed, motivating individuals to complete online courses is a well-recognized challenge for education. Others have noted a 10–20 % greater drop-out rate for online courses compared to traditional classroom environments [The engagement rates for those that ronments , and comronments .Noteworthy is that of those in both groups who completed one course in our program (n = 171), the majority went on to complete all three (n = 132). This observation, particularly in light of other online participation rates, suggests our program was successful once individuals overcame the initial obstacles to commencement.A strength of this study is the randomized, waitlist-controlled design with careful attention paid to the feasibility of implementation. Further, given that there has been little research investigating the effectiveness of online learning for improving EBP attitudes, knowledge, and behaviors , our stuThe results of this study have several implications for those embarking on future EBP educational initiatives, particularly those aimed at broader scale implementation regardless of healthcare discipline. Future programs should consider including the use of moderated online discussions and other interactive methods as ways to provide individualized feedback, collaborative learning and instructor-student interaction; all of which are known to facilitate adult learning , 31, 32.This exploratory study found that an EBP educational program can be delivered in an online format; however relatively poor engagement suggests several barriers exist which need to be considered in future research and implementation efforts. Our online educational program resulted in small positive improvements in chiropractors’ attitudes and skills in EBP, but not their level of EBP uptake (use). This suggests that such online programs can provide an opportunity to enhance practitioners’ motivation and capacity for EBP, but that online education alone is not sufficient to make large changes in EBP use behaviors. The feasibility and effectiveness results from this study can be used to design and refine future, and more robust EBP training and implementation initiatives that can have larger impact on a broader scale.http://www.csh.umn.edu/research/foundations-evidence-informed-practice-modules.The online courses described in this manuscript are freely available at


Lymphovascular invasion (LVI) is the primary and essential step in the systemic dissemination of cancer cells. The aim of our study was to assess the independent prognostic role of LVI for pT1 urothelial carcinoma with squamous differentiation in bladder cancer. We retrospectively analyzed the clinical and pathological information of 206 patients diagnosed pT1 urothelial carcinoma with squamous differentiation. Of the 206 patients, LVI was detected in 57 (27.6%) patients. The 5 year cancer specific survival (CSS) rates were 87.2% in LVI (−) and 52.4% in LVI (+) (p < 0.001). According to univariate analysis, tumor multiplicity, tumor size, recurrence and LVI were the prognostic factors associated with CSS. Additionally, tumor size and LVI significantly influenced the CSS in multivariate analysis. TURBT had shorter median CSS than RC in recurred patients with LVI (+). Our study suggested that LVI is an important predictor for survival of pT1 urothelial carcinoma with squamous differentiation. LVI positive status and tumor size ≥3 cm led to a higher risk of death. RC should be routinely performed in recurred LVI (+) bladder cancer patients of pT1 urothelial carcinoma with squamous differentiation. Squamous differentiation is well known to occur in the bladder urothelial carcinoma and represents the most common form of mixed differentiation12367891011We retrospectively analyzed the clinical and pathological information of 206 patients who were diagnosed as pT1 urothelial carcinoma with squamous differentiation from 2003 to 2014 in our institution. Clinical data was collected by a retrospective review of the medical records. We excluded patients with a history of previous urothelial carcinoma and concomitant upper tract urothelial carcinoma. All the patients underwent transurethral resection of bladder tumor (TURBT) and intravesical chemotherapy. All recurrent patients underwent re-TURBT or radical cystectomy (RC) according to individual histological grade. All the patients were divided into 2 groups by LVI or not, and the recurred patients was divided into 2 groups by TURBT and RC. Cystoscopy has been suggested to be given during the postoperative follow-up according to the European and US guidelinesAll surgical specimens were submitted en bloc for pathological evaluation. Sectioning was performed on a case by case basis to provide adequate evaluation of grade and stage. Independent pathologic re-review of three representative slides from each patient was performed by two pathologists on all specimens to confirm reported pathologic findings and to confirm LVI status. The presence of intercellular bridges or keratinization was indicative of squamous differentiation. The presence of LVI in TURBT specimens was assessed using conventional hematoxylin and eosin (H&E) staining and immunohistochemical staining (IHC) markers against the lymphatic (D2-40) and vascular endothelium (CD 31)14t –test were used to evaluate the association between categorical and continuous variables, respectively. The Kaplan-Meier method was used to calculate overall survival trends, and differences were assessed using the log-rank statistic. Univariate and multivariate Cox regression models were used to analyze overall survival after operation. All reported P values were two-sided, and a P value of ≤0.05 was considered to indicate statistical significance. Statistical analysis was performed with SPSS software (Version 22).Cancer specific survival was considered from the day of surgery to the day of bladder cancer specific death. The chi-squared test and Student’s Clinical and pathological characteristics are listed in Fifty-one patients out of 206 (24.3%) died of cancer metastasis during the median follow up of 62.2 months (range 4 to 112 months). The overall 5 year CSS rate was 78.3%. Moreover, the 5 year CSS rates were 87.2% in LVI (−) and 52.4% in LVI (+)  and LVI (−). Patients operated by TURBT had shorter median CSS duration than those operated by RC in LVI (+) et al., who conducted retrospective analyses of 118 patients reported that LVI, as an independent prognostic factor of progression and metastasis in pT1 bladder cancer, was significantly associated with disease recurrenceMcDonald and Thompson reported the value of LVI as a criterion to assess the severity of urothelial bladder tumors for the first timeHowever, there are short of data on the significance of LVI in patients of TURBT with urothelial carcinoma with squamous differentiation in bladder cancer, especially pT1 urothelial carcinoma of bladder. Our study showed further evidence suggesting that LVI was a pathological variable that might play an important role as a prognostic indicator in patients with pT1 urothelial carcinoma with squamous differentiation in bladder cancer. In our study, the presence of LVI was an independent prognostic factor related with disease survival (P < 0.001). In addition, there are hardly any data on the survival of the TURBT or RC in recurred patients in LVI for pT1 urothelial carcinoma with squamous differentiation in bladder cancer. Our finding indicated that recurred patients operated by TURBT had shorter median CSS duration than those operated by RC in LVI (+) (P = 0.025). However, no significant difference was observed between the two groups in LVI (−) (P = 0.466). According to the results, we suggest surgeons should operate RC routinely in recurred patients for pT1 urothelial carcinoma with squamous differentiation with LVI (+) in bladder cancer.et al. conducted a retrospective review of 4257 radical cystectomy specimens and stratified the patients by LVI status and pathological stageet al. demonstrated that ureteral UC had a higher recurrence rate and poorer survival rate than renal pelvic UCShariat The presence of LVI in patients with newly diagnosed T1 urothelial carcinoma in bladder cancer is associated with decreased recurrence-free survival22LVI is an important predictor for survival for pT1 urothelial carcinoma with squamous differentiation in bladder cancer. LVI positive status and tumor size ≥3 cm led to a higher risk of death which led to a higher mortality rate. Surgeons should operate RC routinely in recurred patients for pT1 urothelial carcinoma with squamous differentiation with LVI (+) in bladder cancer.How to cite this article: Li, G. et al. Poor prognostic value of lymphovascular invasion for pT1 urothelial carcinoma with squamous differentiation in bladder cancer. Sci. Rep.6, 27586; doi: 10.1038/srep27586 (2016).


Herniated discs may exhibit calcification, and calcified discs may complicate surgical treatment. However, the osteogenic potential and expression of osteogenic markers in degenerative discs of different degenerative grades are still unclear. Our purposes are to study the differences in calcification rate and osteogenic potential of herniated discs according to different degenerative grades.t tests. Differences in the posterior portion of removed discs of different Pfirrmann grades were analyzed by one-way analysis of variance (ANOVA), and comparisons of data between discs of any two grades were completed with least significant difference (LSD) tests.Fifty-eight lumbar intervertebral discs were removed from 41 patients. After grading according to the Pfirrmann scale, calcification was analyzed by micro computed tomography (μ-CT), and expression of osteogenic markers was analyzed by immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). Data from μ-CT scans were compared with the Kruskal–Wallis test. The Mann–Whitney test was applied to compare data between any two groups. Differences in osteogenic mRNA expression in different regions of the removed discs (posterior vs. anterior) were analyzed by paired Significant differences in calcification according to μ-CT scanning were observed between discs of different degenerative grades. Nearly half of the discs of Pfirrmann grade V showed the highest degree of calcification compared to Pfirrman grade II discs. Bone morphogenetic protein (BMP)-2, Osterix, and Osteocalcin were detected histologically in discs of Pfirrmann grades III–V. Alkaline phosphatase (ALP) expression was observed in discs showing evidence of calcification. The qPCR analysis showed that BMP-2, Osterix, and Osteocalcin were expressed in most degenerated discs. We also observed greater expression of these osteogenic markers in the posterior portion of removed discs than in the anterior portion.The osteogenic potential of degenerated intervertebral discs appears to increase with the severity of degeneration and to be greater in the tissue near the spinal canal than in tissue in the inner portion of the disc. Degenerative intervertebral disc disease leads to low back pain and radiating leg pain that severely diminishes patients’ quality of life . HerniatThe purpose of this study was to clarify the osteogenic potential and expression of osteogenic markers in degenerative discs of different Pfirrmann grades and to investigate the differences in osteogenic marker expression between the portion of the intervertebral disc near the spinal canal and that near the inner region of the disc.Forty-one cases were diagnosed as lumbar disc herniation or lumbar disc herniation combined with spinal stenosis and were treated by discectomy via a posterior approach in Peking University Third Hospital between February 2014 and December 2014. In 24 cases, a single segmental procedure was performed, whereas a two segmental procedure was performed in 17 cases. Fifty-eight discs were removed: 14 discs of L3/4, 31 of L4/5, and 13 of L5/S1. Patients with bone metabolic disease, congenital bone malformation, gout, renal dysfunction, or hypercalcemia were excluded. Our study was approved by the Medical Scientific Research Ethics Committee of Peking University Third Hospital, and the patients provided informed written consent to participation in this study.Total RNA was extracted using Trizol reagent (15596–026) from Invitrogen Life Technologies . Reverse transcription was performed using the RevertAid first strand cDNA synthesis kit (K1633) from ThermoScientific . The real-time quantitative polymerase chain reaction (qPCR) was carried out using Faststart Universal SYBR Green Master from Roche . Primers were synthesized by Invitrogen Biotechnology Co., Ltd., . Anti-BMP2 antibody (ab14933), anti-alkaline phosphatase (ALP) antibody (ab95462), anti-Sp7/Osterix antibody (ab94744), and anti-Osteocalcin antibody (ab93876) were purchased from Abcam . Secondary antibody and a diaminobenzidine (DAB) kit (K5007) were obtained from DakoCytomation .The Pfirrmann grading system based on magnetic resonance imaging (MRI) T2 weighted images was used to classify the degenerative discs according to the severity of degeneration . The opeAfter the lumbar lamina was removed, the nerve root was retracted in the posterior lumbar discectomy procedure, leaving space for the posterior longitudinal ligament and annulus fibrosus of the herniated side to be incised circularly. Then the discs were removed using the nucleus pulposus forceps. We designated the first piece of tissue removed as the specimen. Eighteen specimens  were selected to be fixed in test tubes filled with 10 % neutral buffered formalin with the anterior part of the specimen leaning on the bottom of the tube for 12–24 h. Specimens were then washed and temporarily steeped in 75 % ethanol at 4 °C before μ-CT analysis. The other forty samples  were transferred to a frozen storage tube immediately with the anterior part of the specimen leaning on the bottom of the tube and stored at −80 °C until analysis.All 58 specimens were analyzed using a Siemens Inveon μ-CT scanner  according to the following parameters: X-ray beam voltage, 80 kV; current, 500 μA; and effective resolution, 13.6 μm. According to the semi-quantitative grading system of Rutges et al. , we desiHematoxylin-eosin (HE) staining was used to evaluate tissue organization, Von Kossa staining was used to determine mineralization, and immunohistochemical staining was applied to investigate the expression of BMP-2, Osterix, Osteocalcin, and ALP. After dehydration and paraffin embedding, the specimens were sectioned at 5 μm. The sections were deparaffinized and hydrated, and heat treatment in citrate buffer (pH 6.0) was used for antigen retrieval. After the primary antibodies were added at proper dilutions , the sections were incubated for 2 hours at 37 °C. After addition of the secondary antibody, DAB was used as the chromogen. All sections were counterstained with hematoxylin.After μ-CT analysis, each removed intervertebral disc was divided into posterior and anterior parts for qPCR analysis. After being mixed with Trizol reagent and ground sufficiently, the specimens were centrifuged and reconstituted in Methenyltrichloride and propyl alcohol. The product of centrifugation was total RNA. Reverse transcription for cDNA synthesis was completed following instructions provided by the manufacturer of the kit employed (RevertAid first strand cDNA synthesis kit).β-actin was used as the reference gene. We randomly chose five posterior portions of samples from each grade, and eight samples that had been divided into posterior and anterior parts for qPCR analysis. Because no osteogenic markers were detected in grade II samples in our preliminary experiment, grade II samples were excluded from qPCR analysis. qPCR was completed following the protocol provided by the manufacturer of the product  with the primers listed in Table t tests. Differences in the posterior portion of removed intervertebral discs of different Pfirrmann grades were analyzed by one-way analysis of variance (ANOVA), and comparisons of data between discs of any two grades were completed with least squares difference (LSD) tests. P < 0.05 was considered statistically significant, except in the Mann–Whitney test for which a corrected P < 0.007 as statistically significant. The corrected P value in the Mann–Whitney test was calculated using the following formula:Statistical analysis was completed using SPSS 20.0 for Windows PC version . The kappa value was calculated to determine interobserver agreement regarding the Pfirrmann grade. Data from μ-CT scans were compared with the Kruskal–Wallis test. The Mann–Whitney test was applied to compare data between any two groups. Differences in osteogenic mRNA expression in different regions of the removed discs (posterior vs. anterior) were analyzed by paired P value (P = 0.05), and “n” represents the number of groups, which was 4 in our study. P’ was approximately 0.0071 based on the formula, and thus, we defined P’ < 0.007 as statistically significant.where P’ indicates the corrected P < 0.01). The μ-CT results for intervertebral disc calcification in discs of different Pfirrmann grades indicated that the percentage of discs with a calcification grade of ++ increased from 0 % among the Pfirrman grade II discs to 45.4 % among the Pfirrmann grade V discs , grades II and IV (P < 0.001), and grades II and V (P < 0.001), whereas no significant difference in grades III and IV (P = 0.105), grades III and V (P = 0.174), and grades IV and V (P = 0.805) was observed. Thus, our μ-CT results indicate a significant difference in the extent of degeneration and calcification between discs of Pfirrmann grade II vs. III, IV, or V. In addition, we observed more ossification sites in the posterior portions of removed intervertebral discs than in the anterior portions , Osterix (P < 0.01), and Osteocalcin (P < 0.05) than the anterior portions  and the Osteocalcin group (P < 0.05), but no differences were observed in Osterix expression (P = 0.124). Although LSD tests revealed no statistically significant differences in gene expression between any pair of adjacent Pfirrmann grades, the comparison of gene expression data for Pfirrmann Grade III and Grade V discs indicated that grade V discs expressed higher levels of BMP-2 (P < 0.01), Osterix (P < 0.05), and Osteocalcin (P < 0.01) than the grade III group (Fig. One-way ANOVA comparing osteogenic marker expression among discs of different Pfirrrmann grades also confirmed significant differences in the expression of BMP-2 (p P < 0.0, but no Our μ-CT results indicate a significant increase in the degree of intervertebral disc calcification with increasing severity of degeneration based on the Pfirrmann grading system, and this finding is consistent with the results of a previous study . The lacDystrophic calcification is a passive pathologic process caused by necrosis or malnutrition, but it has been reported that the calcification of intervertebral discs is an active process that occurs along with hypertrophic differentiation . Our obsBMP-2 is a type of multifunctional growth factor involved in the regulation and control of development, differentiation, and apoptosis of osteoblasts, chondroblasts, neurocytes, and epithelial cells . Takae eOur μ-CT and qPCR results both demonstrated that the posterior part of removed intervertebral discs possesses greater osteogenic potential than the anterior part. This phenomenon may be caused by differences in the local environments of these regions of discs. Interestingly, the posterior part of intervertebral discs has also been shown to exhibit more severe inflammation, fibrosis, and angiogenesis than the anterior part . HoweverOur immunohistochemical staining experiments revealed the presence of the osteogenic markers BMP-2, Osterix, Osteocalcin, and especially ALP, which hydrolyzes calcium pyrophosphate  to initiDegenerated intervertebral discs appear to possess osteogenic potential in direct relation to the severity of degeneration. The more severely degenerated discs have greater osteogenic potential than the less severely degenerated discs. Moreover, the osteogenic potential of the intervertebral disc tissue near the spinal canal is greater than that of the tissue in the relative inner portion of the disc.Our study was approved by the Medical Scientific Research Ethics Committee of Peking University Third Hospital, and the patients provided informed written consent to participation in this study.Not applicable.The datasets supporting the conclusions of this article are included within the article.


There are errors in the Funding section. The correct funding information is as follows:This work was supported by the Mid-career Researcher Program Grants  and  through National Research Foundation (NRF) funded by the Ministry of Science, ICT, and Future Planning; the Ministry of Food and Drug Safety Grant  in 2015; and the R&D Convergence Center Support Program of the Ministry of Agriculture, Food and Rural Affairs (to SHC), Republic of Korea. MRP was supported by the National Institutes of Health (R01 AI077628-05A) and YI was a University of Washington-Cystic Fibrosis Foundation Research and Development Program (UW CFF RDP) Fellow. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Chikungunya virus (CHIKV) is present or emerging in dengue virus–endemic areas. Infections caused by these viruses share some common signs/symptoms, but prognosis, patient care, and persistent symptoms differ. Thus, accurate diagnostic methods are essential for differentiating the infections. We evaluated 4 CHIKV serologic diagnostic tests, 2 of which showed poor sensitivity and specificity. Togaviridae family, genus Alphavirus), is clinically characterized by sudden-onset fever and severe arthralgia, which may persist for weeks, months, or years after the acute phase of the infection (Disease caused by chikungunya virus (CHIKV), a mosquitoborne arbovirus , are established in tropical and temperate regions of the world. The vulnerability of Europe to transmission of CHIKV and other arboviruses has been shown: autochthonous cases of CHIKV infection occurred in Italy in 2007  technique. Although IFA tests have good sensitivity and specificity . The 2 other tests were ELISAs for the detection of CHIKV IgM and IgG: Chikungunya IgM μ-capture ELISA and Chikungunya IgG Capture ELISA  and Anti-Chikungunya Virus ELISA IgM test and Anti-Chikungunya Virus ELISA IgG test .We evaluated 4 commercially available serologic tests that are approved for CHIKV testing by the European Commission. Two of the tests were RDTs for CHIKV IgM: SD Bioline Chikungunya IgM  and We obtained 2 sets of serum samples for testing: panel A (23 samples) and panel B (30 samples). The samples had been submitted to the French Armed Forces Biomedical Research Institute  for arbovirus testing during 2005–2014. We chose the serum samples on the basis of their reactivity against CHIKV and other genetically or clinically related arboviruses.Panel A was characterized in the laboratories of 2 National Reference Centers for Arboviruses by using in-house ELISAs as previously described , 2.Panel B was tested by using in-house techniques at IRBA. Because sample volumes were limited, we used panel A to test the commercial kits and used panel B only if the specificity and sensitivity of tests on panel A were >70%. Commercial tests were performed according to manufacturers’ protocols.We used SD Bioline and CTK Biotech RDTs to process panel A samples plus serum samples (1 each) infected with Mayaro virus and o’nyong-nyong virus , 2. NeitWe used chikungunya IgM/IgG ELISAs from Euroimmun and IBL International , 2 to prELISAs from both companies detected o’nyong-nyong virus IgM and IgG. The Euroimmun ELISA detected Mayaro virus IgG but not IgM; the IBL International ELISA did not detect Mayaro virus IgG or IgM. This cross-reactivity highlights the fact that seroneutralization is necessary to differentiate between viruses in the same serogroup. The IBL ELISA had a specificity of 88% (IgM) and 96% (IgG) and a sensitivity of 79% (IgM) and 52% (IgG). For IgM detection, 12% of the IBL ELISA results were false positive and 21% were false negative. The Euroimmun ELISA had a specificity of 82% (IgM) and 95% (IgG) and a sensitivity of 85% (IgM) and 88% (IgG). For IgM detection, 18% of the Euroimmun ELISA results were false positive and 15% were false negative.In our evaluation, the commercial RDTs that we compared with in-house ELISAs from 2 National Reference Centers for Arboviruses performed poorly. A previous evaluation study that used the same RDTs to process serum samples from residents of Indonesia had results in the same range as our results (If the current outbreak of CHIKV infection in the Americas follows the same trend as that seen in the 2005 Réunion Island outbreak, increased circulation of the virus can be expected, and diagnostic laboratories must be prepared. A 2009 international evaluation of the diagnostic quality of 30 expert laboratories showed that most of the laboratories needed more sensitive CHIKV IgM detection assays; results for IgM were correct in only 50.7% of cases (


HvBot1 and HvNIP2;1.Boron (B) is an important micronutrient for plant growth, but is toxic when levels are too high. This commonly occurs in environments with alkaline soils and relatively low rainfall, including many of the cereal growing regions of southern Australia. Four major genetic loci controlling tolerance to high soil B have been identified in the landrace barley, Sahara 3771. Genes underlying two of the loci encode the B transporters HvBot1 and HvNIP2;1 across barley germplasm, and identified five novel coding sequence alleles for HvBot1. Lines were identified containing either single or multiple copies of the Sahara HvBot1 allele. We established that only the tandemly duplicated Sahara allele conferred B tolerance, and this duplicated allele was found only in a set of nine lines accessioned in Australian collections as Sahara 3763–3771. HvNIP2;1 coding sequences were highly conserved across barley germplasm. We identified the likely causative SNP in the 5’UTR of Sahara HvNIP2;1, and propose that the creation of a small upstream open reading frame interferes with HvNIP2;1 translation in Sahara 3771. Similar to HvBot1, the tolerant HvNIP2;1 allele was unique to the Sahara barley accessions. We identified a new source of the 2H B tolerance allele controlling leaf symptom development, in the landrace Ethiopia 756.We investigated sequence and expression level diversity in HvBot1 B tolerance alleles derived from Sahara 3771, may be valuable as alternative parents in breeding programs targeted to high soil B environments. There is significant diversity in B toxicity tolerance among contemporary Australian barley varieties but this is not related to variation at any of the four known B tolerance loci, indicating that novel, as yet undiscovered, sources of tolerance exist.Ethiopia 756, as well as the cultivar Sloop Vic which carries both the 2H and The online version of this article (doi:10.1186/s12870-015-0607-1) contains supplementary material, which is available to authorized users. Hordeum vulgare L.) across southern Australia by up to 17 % .


Evidence, particularly practice-based evidence, is needed to guide public health practice. With the goal of contributing to practice-based evidence, the Division for Heart Disease and Stroke Prevention at the Centers for Disease Control and Prevention combined and streamlined aspects of an evaluability assessment and an effectiveness evaluation to create the Enhanced Evaluability Assessment (EEA). This approach offers a viable and less costly alternative to evaluators and practitioners by quickly identifying and evaluating models with evidence of effectiveness that can be replicated and expanded. The EEA can be applied to a range of public health topics, not just cardiovascular health. This article provides a step-by-step description of the EEA. Public health agencies often face substantial fiscal constraints and challenges to identifying and implementing strategies that have the greatest effect on improving health. Given this reality, there is an increased need for efficient methods to identify programs or interventions with demonstrated evidence of effectiveness  that canWith the goal of contributing to practice-based evidence, the Division for Heart Disease and Stroke Prevention (DHDSP) at the Centers for Disease Control and Prevention (CDC) used a staged evaluation approach — a pre-evaluation assessment to appraise a program’s capacity and readiness for an effectiveness evaluation and then an effectiveness evaluation of the selected site. This staged approach guides subsequent investment of resources in evaluating public health practices and serves as a platform for developing strong evaluation designs for programs in the field, thereby yielding practice-based evidence.We describe a newly created evaluation approach, the Enhanced Evaluability Assessment (EEA), which incorporates key aspects of the staged approach while streamlining and truncating the process to provide a quicker assessment of readiness, an effectiveness evaluation, and dissemination of practice-based evidence. This approach may improve informed decision making and lead to enhanced evaluation and implementation of public health programs. This approach can be applied to all areas of public health inquiry, not simply cardiovascular health. We describe each step of the EEA approach, including considerations and lessons learned.Since 2008, DHDSP has used pre-evaluation assessments — the Systematic Screening and Assessment (SSA) and the Evaluability Assessment (EA) — to determine whether a program demonstrates potential promise to address cardiovascular disease outcomes. The SSA verifies that the intervention is 1) fully implemented, 2) operating as intended, 3) operating consistently over time, 4) capable of collecting and extracting data on outcomes, and 5) achieving its desired effects, according to the data , because the effort relies on telephone calls and sharing existing documents rather than making a site visit.Effectiveness evaluation. The EEA uses a mixed-methods evaluation approach, similar to the previously used method, but relies more on existing data and a shorter timeframe than the 30-month effectiveness evaluation.Expert panel. Under the SSA and EA, expert panels are instrumental in 1) the initial selection of the site to undergo an EA and 2) determining the site’s suitability for evaluation after the EA. Expert panels include people with diverse yet relevant backgrounds and experiences to ensure that multiple perspectives are represented. DHDSP selects subject matter experts from clinical, academic, state, and local practice settings; CDC; and other federal agencies. The EEA uses an internal expert panel at the end of the evaluation period to determine the intervention’s public health impact and the quality of the evidence . Once the list is compiled, prospective programs are contacted by CDC to discern their fit for an effectiveness evaluation. The point of contact and key staff at the potential site are asked to provide relevant program documents and verification of access to data sources needed for the evaluation. It is essential that the selected program have access to at least 6 months of data, that these data are accessible to the program, and that the intervention itself is well-established. The EEA approach can be applied to a range of program interventions. The intervention drives the need for the type of data; for example, the data could be health outcome data or behavior change outcomes.The selected program is involved in the entire EEA process and has regular  meetings or calls with the evaluators to discuss progress and provide input into the project. Having a champion and single point of contact at the site is critical to successfully conducting the EEA and obtaining the requested data in a short timeframe. It is estimated that site staff engagement in the EEA process is approximately 6 hours per week. This level of engagement is slightly less than the anticipated engagement for an effectiveness evaluation, which is approximately 10 hours per week.2. Evaluation questions and evaluation design. The selected site is substantially involved in the evaluation planning and implementation process. Once the site has agreed to participate in the EEA, the next step is to engage the selected site in an in-depth discussion of their evaluation needs and determine a mutually satisfying focus for the EEA. It is important that the evaluation be value-added for the site, because little compensation is being given to the selected site to participate in the evaluation.3. Data collection. Data for the EEA are collected through document review, site visit, interviews, and quantitative data abstraction. The evaluators obtain existing program documents, reports, and intervention descriptions to prepare a comprehensive program description including an accurate logic model. This information is obtained by working with a point of contact at the site of the selected program. After a thorough review of this information, a site visit is scheduled.The purpose of the site visit is to conduct interviews of key personnel and partners involved in program implementation. Interviews and focus groups are conducted as needed, and site visitors are given an opportunity to observe the program firsthand. This is also the time when the evaluators can get clarification on any lingering questions that arise during the earlier review of written materials. In addition to the document review and site visit, a quantitative data abstraction is completed.Evaluators work with the program to abstract relevant data to be used as evidence of promise or effectiveness in the strategies being implemented. In many cases, a modest stipend is given to the selected program to secure the requested health outcome or implementation data. Once the initial program description is completed, based on the document review, site visit, and other data collection, the information is shared with the program to determine whether there are any clarifications or misunderstandings that need to be resolved.4. Data analysis. The data collection for an EEA is a rigorous process that uses both quantitative and qualitative methods. Therefore, the analysis portion of the study includes a review of documents and identification of themes and a statistical analysis of the quantitative data collected. The analyses vary based on the needs of the study but always include triangulating the data and gaining consensus of all reviewers.5. Preliminary results shared with program. A key discussion point occurs with the program after the analysis of process and outcome data. These preliminary findings are presented to the program, usually through a conference call or in webinar format, so that they can be reviewed carefully and discussed as a group. Any potential contradictions or areas that need follow up or clarification are identified through this step before final reports are written and findings are further disseminated. Any changes are then incorporated into the reports.6. Report writing. Two reports are written that summarize the findings of the evaluation. The first report, the program description, is usually written midway through the evaluation project and provides a detailed description of the program and key findings from the qualitative data collection. This report includes the goals and expected outcomes of the program, the core components of the program, factors affecting program implementation, and the logic model of the program. The second report incorporates much of the program description report but focuses on the final results of all data collection and analyses. Specific elements of the report include a brief executive summary that can be used as a stand-alone document, a background section, an overview of the study design and methods, results, and a conclusion with recommendations. The overview of the evaluation design and methods describes the components, design, and evaluation questions; outlines the qualitative and quantitative methods; and addresses study and data limitations. The bulk of this report is the results section. The results begin with information from the program description report, then a discussion of implementation effectiveness, practice and program related outcomes (including program reach), and program effectiveness. Program effectiveness results generally include health system outcomes and clinical and patient-level outcomes of interest in the evaluation. The conclusion provides a discussion of the findings and considerations for replication. The considerations for replication also link to the original selection criteria so that the program can be assessed by an expert panel using those criteria.7. Final briefing with program. A formal final briefing is provided to the program in webinar format. The presentation includes an overview of the evaluation methods, process and outcome evaluation findings, final conclusions, and reflections on the evaluation. Staff from the program who were not directly involved in the evaluation study are invited to participate to share thoughts related to the evaluation methods, findings, and future implications. Clarifications and refinements are made to the final report based on this discussion.8. Expert panel convened. A unique aspect of the EEA is the use of an expert panel at the end of the evaluation to assess the intervention using a CDC-created conceptual framework (ramework . Two comTo prepare the panel for the small-group discussion, a description of the intervention, outcomes, literature related to the intervention, instructions for reviewing materials, and a scoring sheet are emailed in advance for submitting scoring sheets to CDC. Scores are tallied, and overall comments are compiled. Summaries are shared during the small group discussion in PowerPoint presentation format. The goal of convening the small group is to provide panel members the opportunity to discuss assessments, engage in dialogue, and explore content related to the level of evidence based on the public health impact determination and quality of evidence provided. A CDC representative convenes the panel, facilitates the discussion, and clarifies points as needed. Input from the expert panel is incorporated into the final evaluation documents.9. Dissemination. In collaboration with the program, findings from the evaluation are shared internally with the program and CDC and shared externally on the websites of both the program and CDC. Implementation guides, field notes, or other briefing documents may also be created to enable replication by other programs. Whenever possible, the evaluation findings are also disseminated through peer-reviewed journals and professional conferences.To build practice-based evidence, appropriately designed evaluation methods must be implemented with rigor and efficiency. As a new methodology, the EEA incorporates these essential qualities by collecting primary and secondary data while reducing costs and time. With one EEA completed and 2 others under way, early indications suggest that this approach offers a viable and less costly alternative to evaluators and practitioners by quickly identifying and evaluating models with evidence of effectiveness that can be replicated and expanded.As with every methodology, there are some challenges and lessons learned that should be considered when determining whether to implement an EEA versus other evaluation approaches. As noted, the pool of nominations may be smaller than the broader call for nominations used by the SSA, which may inadvertently omit some innovative or promising practices. Also, an approach with a broader call for nominations may be more appropriate when the focus area is new or emerging or when the evaluator does not have access to networks to identify potentially promising programs. It is also essential with the EEA that the evaluators establish a strong relationship with the site at the beginning of the process for the evaluation to be successful. It is important to consider the evaluation needs of the program and to offer feedback and reports that are useful to the program for program improvement, building evaluation capacity, and demonstrating successes in ways that are relevant for their stakeholders. DHDSP has found this to be a key incentive for programs’ participation and ongoing engagement. Another lesson learned is that it is critical during the initial discussions with potential programs to understand what data are collected or are accessible and how these data are managed and extracted. Using the expert panel at the end of the evaluation is a unique aspect of the EEA. Because DHDSP is using criteria from the CDC framework, it is necessary for the summaries of the findings to speak clearly to these criteria or it is difficult for the panel to assess during the scoring process. Depending on the needs of the evaluation and the focus of the program’s intervention, evaluators may wish to explore other frameworks or assessment criteria for the expert panel to use.Keeping these early lessons learned in mind will help evaluators determine whether the EEA is a viable evaluation approach for their work. Highlighting this approach is intended to increase evaluation capacity, support informed decision making, and lead to improved evaluation and implementation of public health programs. Because the EEA is responsive to stakeholder needs and enables the timely dissemination of evaluation findings, the EEA has the potential to have broad application for a range of public health topics.