Aboriginal and Torres Strait Islander women experience higher rates of obesity, chronic disease, and are less active than non-Indigenous Australian women. Lifestyle programs designed to increase physical activity and encourage healthy eating are needed to ameliorate this disparity. The aim of this study was to identify participants’ perceived barriers and enablers to attend group exercise classes as part of a 12-week fitness program.To understand the factors that influence attendance, a mixed method process evaluation was undertaken in which a quantitative measure of attendance in the group exercise classes was used to identify cases for further qualitative investigation. Aboriginal and/or Torres Strait Islander women aged 18 to 64 years were recruited to a research trial of a fitness program. The 12-week program included two 60-minute group exercise classes per week, and four nutrition education workshops. Semi-structured interviews were conducted at program completion. Participants were stratified by attendance, and interviews from the highest and lowest 25 percentiles analysed. Rigour was strengthened through use of multiple data analysts, member checking and prolonged engagement in the field.Analyses of the post-program interviews revealed that participants enrolled in the program primarily for the perceived health benefits and all (with one exception) found the program met their needs and expectations. The atmosphere of classes was positive and comfortable and they reported developing good relationships with their fellow participants and program staff. Low attendees described more barriers to attendance, such as illness and competing work and family obligations, and were more likely to report logistical issues, such as inconvenient venue or class times.Attendance to the ‘Aboriginal and Torres Strait Islander Women’s Fitness Program’ was primarily influenced by the participant’s personal health, logistics and competing obligations. Low attendees reported more barriers during the 12-week period and identified fewer enabling factors than high attendees.Australian New Zealand Clinical Trials Registry ACTRN12610000224022 Compared to non-Indigenous Australian women, Aboriginal and Torres Strait Islander women are more likely to be obese, have lower levels of physical activity and higher rates of type 2 diabetes mellitus (T2DM). It is cThere is conclusive evidence that regular physical activity is an effective primary and secondary preventive measure against chronic diseases such as T2DM, and cliSeveral lifestyle programs to prevent and/or manage T2DM among Indigenous adults, or behavioural risk factors for the development of chronic disease in Indigenous populations have been implemented-24. MostImplementation assessment has been long recognised as a critical element of program evaluation, as it can strengthen the basis for understanding differences in results and explaining how breakdowns in implementation processes can impact hypothesised causal pathways-28. As sIndigenous people across a number of health programs have reportedly low attendance rates. Clifford et al. completed a review on lifestyle interventions that targeted Indigenous Australians. Of the four included physical activity and nutrition programs, all reported less than 80 per cent of participants completing the study. AttendaThe ‘Aboriginal and Torres Strait Islander Women’s Fitness Program’ was a structured, progressive 12-week group program aimed at reducing waist circumference and improving metabolic health. The program is described in further detail elsewhere. LogistiThe study aim was to identify the perceived barriers and facilitators to attendance of Aboriginal and Torres Strait Islander women registered in a 12-week structured physical activity and nutrition program.Aboriginal and Torres Strait Islander women aged 18 to 64 years residing in Adelaide, South Australia, were invited to participate in the research project. The inclusion criteria is described elsewhere. ParticiTo understand the factors that influence attendance, a mixed method process evaluation was undertaken in which a quantitative measure of attendance in the group exercise classes was used to identify cases for further qualitative investigation. High and low attendees were identified objectively from attendance records across the 12-week program. These cases were used to understand the factors underpinning variation in attendance.The study was informed by Chen’s program planning framework which identifies program and participant-related factors as influencing aspects related to program implementation. Of inteEach participant was encouraged to attend two classes per week during the 12-week program. The days and times of the classes were organised around the preferences of each group. A staff member recorded participant attendance to classes. Attendance was noted as a participant being present for the session. There was no measure of effort or exertion. A continuous measure of participation was computed and constituted the quantitative component of the mixed method process evaluation.For these analyses participants were purposively sampled based on their attendance to exercise classes. Attendance was stratified and only participants located in the top 25th percentile and bottom 25th percentile were analysed. The bottom 25th percentile included eight participants who had attended eight or less classes, these are referred to as the ‘low attendees’. The top 25th percentile, ‘high attendees’, included eight participants, each of whom had attended 18 or more classes. These two extreme groups were chosen for analysis to compare and their perceived barriers and enablers to attendance to the exercise sessions.Participants were invited to take part in a one-on-one semi-structured interview following completion of the 12-week program. The interviewer was a local Aboriginal woman with extensive experience in conducting research interviews. Interviews took place in workplaces, homes, at the university and, if a face-to-face interview was not possible, over the telephone.The interview guide was designed to capture individual, interpersonal and environmental factors influencing participant engagement in the program in relation to their family, work, study and community responsibilities and obligations. The interviewer used exploratory, amplificatory and explanatory probes to clarify and better understand the individual, family, social and work-related factors that acted as barriers or facilitators to attendance.Interviews were digitally recorded and professionally transcribed. Following each interview, the interviewer completed a contact summary sheet detailing the quality of responses, the main issues or themes that emerged and any changes in body language throughout the interview. The conDemographic data was collected at baseline via questionnaire. This questionnaire captured personal information and socio-demographic information, including home address, date of birth, employment status, and highest educational attainment. Household information was also included, such as household income and the number of adults and children in the home. Even though all forms were received, not all questions were completed.Objective assessments of metabolic health were taken including; basic anthropometrics  and blood pathology ). Further details of these assessment protocols, including the equipment and assays used, are described elsewhere. AssessmThe demographic and metabolic health data of the high and low attendees were compared using mean values for continuous data and numbers of occurrence for categorical variables. Means were calculated using Stata 11.To ensure that interview data were coded in the context of each case, audio files were listened to, the transcripts read and re-read. Each interview was analysed independently by two researchers. Thematic network analysis was applied to identify basic themes, organising themes and higher-order global themes. SegmentThe socio-demographic factors and baseline health assessments of the low and high attendees were compared. Low attendees were more likely to be employed (7 low attendees compared to 5 high attendees), were less likely to have dependants under the age of 18 (4 low attendees compared to 2 high attendees), and on average lived 2.6 km further from where classes were held. Compared to high attendees, low attendees were on average nearly 4 years younger and 7.6 kg heavier, with a waist circumferences 9.5 cm larger, and a fasting insulin 5.6 mmol/L higher. There was no difference between groups for educational attainment, annual household income, fasting glucose, HbA1c or lipid profile. These differences were noted, but due to the small number of participants no statistical analysis was conducted and no firm conclusions can be drawn about their significance.On average, attendance at the group exercise classes was low. Participants were encouraged to attend a total of 24 classes; two classes per week for twelve weeks; although group A was offered 5 classes a week to meet the needs of the participants. Of the 51 participants the average number of classes attended was 9.5 . Seven participants did not attend any exercise classes, six of whom withdrew from the program. Of the participants who completed post-program interviews, the eight highest attendees attended an average of 20.4 classes (range 18 – 30 classes) and the eight lowest attendees attended an average of 6.5 classes (range 2 – 8 classes).Interview length averaged 40 minutes. Interview quality was reported by the interviewer providing an overall subjective rating based on the richness of responses, the openness of the participant to share their story and any issues that arose . There was little difference in interview quality between the high and low attendees overall. The atmosphere created by the interviewer contributed to most of the women feeling comfortable to speak freely and share their story.The factors that influenced class attendance are synthesised and displayed in FigureThe global theme of program motivation was comprised of participant’s initial motivation to enrol, and their motivation to attend the fitness program following enrolment. As depicted in Figure“I just thought it might be a good way to kick start my health again.” For others, their health concern was linked to improving a specific health-related outcome, for example, “to hopefully get fit and lose a bit of weight” or improve an existing medical condition, “I’ve got high blood pressure and I want to try and get off as much tablets as I can.” Only one participant did not mention personal health as a motivation for enrolling, however she was a low attendee who provided a short phone interview which had gaps in the transcript due to telephone line interference and background noise.The motivations driving participants to enrol were similar regardless of attendance level. All participants, with the exception of one, identified a desire to improve their personal health and wellbeing in general or to mitigate an existing health issue as the impetus for registering. For example, “I just thought I'd just try it and see what it was like” and because there was no financial cost they felt they had nothing to lose, “any exercise you can do for free, why not!” Other participants were attracted by its social aspect to either “meet new people”, to network with other Aboriginal and Torres Strait Islander women, or thought it was a great opportunity to “get together as a group of Aboriginal and Torres Strait Islander people …and do it.” Finally, a secondary motive for joining the program related to participants supporting the research, “…and also to support the program because I think it’s a really good program.”Some participants were curious about the program and thought they would see what it had to offer, From the women’s stories we identified five sources of motivation related to their ongoing attendance, specifically, their original motivation to enrol in the program, their self-motivation to be active, the motivation they received from family and friends, motivation gained from experience they had in the program, and their personal circumstances.“I just made that a priority for me and then I just worked around that.” Participants were also motivated and encouraged to attend regularly by their family and friends:Self-motivation was realised through participants prioritising their needs and/or a strong commitment to completing the program. “… my family and friends – they knew I was participating in the program and they thought it was really good and especially my Mum – she reminded – “Oh, you’ve got that thing tonight at six – remember that.”“I loved it; I enjoyed going there, that’s why I went there all the time.” When asked if there was anything about the program they found particularly motivating, a participant explained:Overall, a participant’s program experience seemed to have either a positive or a negative impact on their motivation for ongoing attendance. “I think just the atmosphere of everyone was willing to give it a go, that was motivating and the fact that they were also supportive, just (staff members) – they were like really supporting. They were like “C’mon …, you can do another one of those.” And plus all the ladies – I found them really supporting because I was the youngest one and they were all just encouraging me little by little you know. So it was just the atmosphere really that was motivating me to keep going.”The personal circumstances of each participant also impacted on their motivation to attend classes. Some of these factors are described below and even though most were not specifically linked by some participants to their motivation others noted that factors such as logistical issues could make them feel ‘reluctant’ to attend.A participant's personal circumstances influenced their ability to engage in the program regardless of their motivation to enrol. Personal health issues, ‘logistics’, competing priorities, and instrumental support acted to either constrain or enable their participation.Personal health issues surfaced as a strong barrier to class attendance. Low attendees were more likely to report long-term injuries or illnesses during the 12-week program:“Well for me I know that I missed the first couple of weeks because I was actually injured from netball.”High attendees, on the other hand, only experienced mild or short-term illnesses or injuries.For high attendees logistical factors were not identified as barriers to attendance although, for some, parking was noted as a nuisance. Most high attendees found the class times and location convenient, as illustrated in the examples below:“I found it good, like the location, because it was in close proximity to my work”,“The timing was really good because they do it at 6-7[pm] … Tuesday and a Thursday so yeah if it was any earlier then maybe I wouldn’t have been able to.”Whereas, for some low attendees, the location was a barrier to attendance,“Yeah I did half the program. I stopped because it got too far away for me.Even though some logistical issues were more ‘inconveniences’ rather than barriers, participants noted the effect on their motivation to attend:“Sometimes it made me reluctant. Do I feel like sitting through traffic?”Crèche was often mentioned by the women who used the service; however, it is unknown if its absence would have been a limiting factor for these participants or just an added convenience. The fact that the program was ‘free’ was regularly mentioned by interviewees. Some participants also discussed the cost of gyms and other activities:…a lot of people can't afford something like that (gym membership) because most people in (suburb) are on the dole or on a pension (government welfare).”““going away on block study caused a bit of an interruption.” High attendees reported less competing obligations, while low attendees either reported having several obligations, or they experienced major life events that completely hindered attendance for a significant block of time, such as death in the family.Competing obligations were barriers to class attendance and included family obligations, work, study and/or other commitments. The impact of competing obligations varied significantly; some were one-off events, such as a family member’s birthday, while others were short-term priorities, such as being asked to work back late for several days, or study commitments, “I was able to keep up with it for the first month, then after the first six weeks I started decreasing because I had other  issues and other (study and work) interruptions.”“…but then unfortunately during the last few weeks I had a death in the family so I had to go out of town so that didn’t really help because I was out – I was completely out in the middle of nowhere.”Instrumental supports emerged as tangible or practical supports normally offered by family members or friends. In our analysis, we found that instrumental supports modified a competing obligation or logistical issue. For example a participant who was normally responsible for getting her children ready in the morning and taking them to childcare and kindergarten received instrumental support from her husband so that she could attend morning exercise classes:“So obviously my husband is quite supportive in terms of like getting the kids up … because I would leave home by 6:30 am.”“I used her (mother’s) car for most of the time … so I wouldn’t be catching the buses that late.” However, having support did not prove essential to attendance. Participants who did not identify any barriers or competing priorities did not require support to enable their attendance.Another participant who attended evening classes was assisted by her mother who loaned her a car, “Well I didn’t have really, any family commitments, because my daughter’s away, and really, not that much work really, it’s just your 9 to 5 during the week, so I didn’t have really, no problem about attending any sessions or exercise classes and things like that, so, no that was pretty easy for me.”All participants, with the exception of one, reported their program experiences to be all positive. The program experience reflected the program suitability, professionalism of the program, class encouragement (from participants and staff), program atmosphere, and personal reinforcing factors.Participants reported that the program was a good fit for themselves and other participants. It met their needs and expectations. They particularly liked the fact that the activities accommodated participant’s different fitness levels:“(they) catered for all different levels, you know what I mean, like say if you were just a beginner or something … you could work at that level or you could be higher fitness level, whatever and it's a, it accommodates to that as well.”“I was really enjoying the classes so that just kept me coming back for more.”Participants enjoyed the exercises, and this impacted their motivation to attend, Unfortunately one participant, a low attendee, found that the program was not what she expected. She also felt the program was not suited to her fitness level, consequently she felt “uncomfortable.”“Well at first I thought the circuit class was going to be good but I found some of the exercise was a little bit hard for me … so yes I did an adaptation of some of those exercises that the instructor would say if you can’t do that one well you do it this way and she’d show you a different way and that was good but I still felt a bit uncomfortable … maybe if it was like one group was over one side of the room and the other group was on the other side so that we were able to go at a slower pace…”“easy and not a chore.”The professionalism of the program contributed to the overall program experience. Most women reported that they felt confident that the staff were knowledgeable and professional, reliable and had good communication skills. They also found the program well organised which made the program “I thought that it was really good and run really well, kept up to date with everything and good communication, people were friendly.”During the interviews many women discussed the encouragement they received from program staff and their fellow participants. Participants indicated that they found the encouragement during class motivated them to put their full efforts into the activities, thereby influencing their motivation to attend the classes.“When the instructors sort of pushed you, and challenged you to try the new things actually it was surprising to think, oh my goodness I can actually do that exercise…”“I think that was just the environment and the people that were there, and a lot goes to the staff that were doing it. Like you know they were very encouraging whether it was (staff member) or (researcher) or any of the instructors that were there, everyone was encouraging and yeah.”a group of women and they were going to be with other Nungas or other Aboriginal, Torres Strait Islander women” or that the participants shared goals or were on a similar journey or that they “felt comfortable.”The atmosphere of classes had a significant impact on the participants’ program experience. This included the relationships that the women formed with fellow participants and program staff. When participants were asked what they found particularly motivating about the class most talked about the group environment, a positive atmosphere and feeling comfortable. All participants made reference to liking the fact that the program was “‘I think it was supportive like everyone was really encouraging … being all Aboriginal women was good, you didn’t feel out of place … It was a program that specialised to make their environment as comfortable as it could be for participation … it just helped with the participation or your ability to do some of the exercises.’There was a sense of women sharing a journey together and in a culturally safe environment in which they felt ‘no shame’. These factors contributed to their program experience and ongoing motivation to attend the classes.All participants reported perceived improvements on one or more aspects of their physical or emotional well-being. The most common reinforcing factors were participants perceiving physical improvements such as feeling the exercises becoming ‘easier’ or feeling ‘fitter’ during the 12-week program.“I could feel the difference in the fitness level of myself. I guess and I was able to do some of the exercises a lot more easier than when I first started.”These physical improvements extended to women talking about losing weight, sleeping better, having improved blood pressure, as well as experiencing reduced pain in their joints and improved mobility.“…like I said, I feel fitter, healthier, more energy”,The women also reported having ‘more energy’ Beyond improvements in energy, women also shared stories about perceived improvements to their mental well-being, such as feeling less stressed or feeling better about themselves.“…since I started the program and exercising and stuff it just lifted me and helped me to feel better about myself. I wasn’t depressed or anything, but I just didn’t have any motivation to do anything, whereas now I know with exercise and eating well you feel good and you know, it’s a benefit.”‘felt good’ after the exercise sessions. Additionally high attendees reported a sense of achievement or accomplishment:Participants also commonly reported that they “…it was hard work, and we felt really good after it, a real high after it. And it took away all that stress and you felt really happy.”As discussed earlier and depicted in FigureThis is one of the first mixed method process evaluations embedded with a pragmatic controlled fitness trials in an Indigenous population to examine the barriers to participation of high and low attendees. Despite sharing positive motivations and positive program experiences with high attendees, those who struggled to attend classes either experienced several competing obligations and logistical issues that made attending very difficult in the absence of instrumental support, or they had a major event occur, such as a death in the family. In particular, low attendees experienced more competing family obligations and work obligations.There were similarities in the stories shared by the women in the positivity of their program experience and program motivations. The motivations of the women to enrol in the program, and to continue attending the program, were also similar and there did not appear to be a discrepancy between the two groups. Participants reported enrolling in the program primarily for the perceived health benefits. Participants perceived the program to be of high quality and all but one only had positive things to say about their program experience. Participants felt comfortable and reported a positive program atmosphere. The fact that the program was designed by an Indigenous woman, for Indigenous women, and was implemented in a culturally safe environment contributed to the women’s positive program experience.A review of the literature did not reveal any qualitative studies on the barriers to participation of Indigenous women engaged in equivalent structured group-based lifestyle intervention. However, previous studies on at-risk minority populations report similar findings. The community health assessment and promotion project (CHAPP) was a ten-week group-based exercise and nutrition intervention in an urban setting, which provided crèche and transportation. All parIn another study, focus groups amongst African-American women on the perceptions of physical activity and personal barriers and enablers found that lack of time, lack of self-motivation and competing priorities (putting their family first) as the most frequently cited perceived barriers. A qualiThe role of competing family obligations cannot be under-estimated. With 40 per cent of Aboriginal and Torres Strait Islander Australians under the age of 15 and with a higher number of children per household, compared with non-Indigenous women, AborigiAn older, comprehensive, review of physical activity determinants found that self-motivation, support from a spouse and reinforcement from program staff and or activity partner influenced adherence. Self-moThe collective process of data analysis and interpretation challenged the researchers to reflect critically on their personal biases and how these biases were reflected in their interpretations of the data.The firsThe findings of this study have implications for the design and delivery of group-based exercise programs targeting Indigenous women living in urban areas. Based on our findings, there may be some benefit to implementing pre-program workshops with participants to identify potential barriers and solutions to their program attendance. We found, for example, that it was important to provide instrumental supports like crèche and transport to enable some women to attend the exercise classes. Having a process that identifies and addresses the barriers emerged is important. It is additionally important that the program invest resources to develop a professional program that offers consistent training, technical support and feedback to staff responsible for implementing the program. Offering a well-organised program, run by qualified, supportive and motivating staff contributes to a positive and engaging atmosphere for participants. We also found that running the program only for Aboriginal and Torres Strait women, in a non-gym setting, created a comfortable place for women to exercise and positively influenced their motivation to attend.The study findings should be interpreted in light of the following two limitations. First, the study is based on a small number of interviews from participants who completed the program and consented to an interview. Interviews from participants who withdrew from the program or were lost to follow-up would likely have provided greater insight into the factors influencing participation. Consequently, the circumstances and barriers affecting the participants that were not interviewed are unknown. Second, although the semi-structured interview was designed to capture participant’s barriers and enablers to attendance in relation to their life circumstances, how they came to enrol in the program and what motivated them to continue attending, the interview guide probed into other issues as well, and this may have detracted from the richness of the women’s stories about their engagement.s between high and low attendees of the Aboriginal and Torres Strait Islander Women’s Fitness Program, giving some insight into the factors that influenced their attendance. There were five key sources of motivation to attend classes; their motivation to enrol, their self-motivation for exercise, motivation from family and friends, their program experience and their circumstances. Most women talked extensively about their experience with the program, including the relationships they developed with the staff and other participants, enjoying the classes and the exercises, feeling safe and ‘comfortable’ and perceived improvements in their own health and well-being. High and low attendees differed in their circumstances during the program with low attendees experiencing more barriers, particularly logistics and competing obligations in the absence of instrumental support, and fewer enabling factors. Future programs should focus on reducing barriers to attendance. Barriers that are unavoidable and unpredictable will always be present; however, some can be modified by instrumental support. Pre-program workshops could prove useful to help identify potential instrumental supports that service providers could offer and/or assist participants to identify sources of instrumental support from their family and friends.This mixed method study uncovered some key similarities and differenceThe project gained ethics approval from the University of South Australia’s Human Research Ethics Committee and the Aboriginal Health Council of South Australia’s Human Research Ethics Committee.The project sought the guidance, advice and support of their collaborating partners, Nunkuwarrin Yunti of South Australia Inc. and the Aboriginal Sobriety Group Inc. in Adelaide and their specially formed advisory committee of local women.The authors declare that they have no competing interests.KJC developed the 12-week program, contributed to the study design and was involved in the data collection and delivery of the project. RAM developed the study design and led the NHMRC Project Grant. BS and KJC coded and conducted the analysis under the supervision of MC. BS was also involved in the data collection and delivery of the project. MC also contributed to the study design. KJC was primarily responsible for drafting the manuscript. All authors approved the final manuscript.


Responsible beverage service (RBS) training designed to improve the appropriate service of alcohol in commercial establishments is typically delivered in workshops. Recently, Web-based RBS training programs have emerged. This report describes the formative development and subsequent design of an innovative Web-delivered RBS program, and evaluation of the impact of the program on servers’ knowledge, attitudes, and self-efficacy.Formative procedures using focus groups and usability testing were used to develop a Web-based RBS training program. Professional alcohol servers (N = 112) who worked as servers and/or mangers in alcohol service settings were recruited to participate. A pre-post assessment design was used to assess changes associated with using the program.Participants who used the program showed significant improvements in their RBS knowledge, attitudes, and self-efficacy.Although the current study did not directly observe and determine impact of the intervention on server behaviors, it demonstrated that the development process incorporating input from a multidisciplinary team in conjunction with feedback from end-users resulted in creation of a Web-based RBS program that was well-received by servers and that changed relevant knowledge, attitudes, and self-efficacy. The results also help to establish a needed evidence base in support of the use of online RBS training, which has been afforded little research attention. Drunk driving crashes are responsible for nearly 11,000 fatalities and one-third of all car accident fatalities in the United States each year. MoreoveAs of 2011, 17 states in the U.S. had enacted mandatory RBS laws that required training and certification of RBS. A total of 15 of these states – including Oregon – mandate RBS training for managers and 13 states require RBS training for servers. RBS cerThis report describes the formative development, design, and preliminary test of a Web-based RBS training program designed to encourage responsible alcoholic beverage service by servers and managers of commercial alcohol service establishments.Initial project development meetings were convened for a multidisciplinary team composed of individuals from Deschutes Research, Inc. (a company focused on the dissemination of evidence-based programs), InterVision Media (a Oregon-based technology media and Web intervention development programs), and Oregon Research Institute  to generate ideas regarding the design, development, and evaluation of a Web-based RBS training program. Team members had experience and expertise in the development and evaluation of alcohol treatment programs, health behavior change programs, Web-based behavioral interventions, and the programming of complex interactive websites. In addition, the team had been previously involved in a Phase I SBIR research program that developed and tested a portion of the full RBS Web-based program that is described in this report. The team followed an incremental and iterative formative development process spanningchunks that worked best for Web delivery, ways that encouraged interaction and provided user with opportunities for self-evaluation.The first step in the process involved development of written program specifications that included the design of each program module, its functionality as a component within the website, the information architecture and instructional design that would best allow the content to engage the user and facilitate the learning and mastery of the material. ProgramFocus group procedures were informed by the team’s experience and by published guidelines. These falpha version of the Web-RBS program. Participants were encouraged to discuss their feedback with the group regarding the overall "look and feel" of the program, its navigation, graphics, interactive features, and the amount, arrangement, and type of information presented. Participants were also asked about any information they felt was missing in the program and how the website compared to what they had experienced in the current workshop-based RBS training mandated by the Oregon Liquor Control Commission (OLCC). Sessions were audio taped and transcribed, which permitted us to identify themes and highlight possible changes to the program.Focus group sessions for the current project involved nine experienced alcohol servers, bartenders, and managers. Four of these participants were female and most were Caucasian, with one participant indicating more than one race. During the 1.5-hour sessions, focus group participants received a guided tour of the features of a preliminary or hands-on approach as he/she accomplishes certain assigned tasks while receiving minimal direction from the facilitator. During this time each tester is asked to describe their thoughts using a think-aloud technique derived from cognitive science[For optimal adoption and accurate use, Web-based tools like the RBS program should embody established usability standards,14. A re science that has science,19. As n science, the thi science. At the Two rounds of usability tests were conducted. The first round involved seven alcohol servers and managers  of whom six described themselves as Caucasian, one as African American, and one as Hispanic/Latino . The second usability test consisted of 3 female alcohol servers and managers who described themselves as Caucasian and non-Hispanic/Latino. Usability testers were observed as they completed a series of tasks on the website including user registration, reviewing program information, completing section quizzes, and completing the online assessments.The formative research steps described in this report directly informed the design of the resulting RBS Web-based training program for alcohol servers, managers and licensees. Other sources that shaped program design included the field experience of team members, feedback received from expert consultants , and the requirements of the OLCC. As depihouse policies for risk reduction, self-protection, customer cooperation, training methods and the server-customer relationship, support and supervision through communications skills, and encouraging teamwork.The six separate server modules included: (1) Introduction to RBS training, (2) Effects of Alcohol, (3) Who Not to Serve, (4) Alcohol Problem Prevention, (5) Skills and Resources, and (6) an online Knowledge (certification) Test. Effective alcohol server responses to challenging situations were modeled in Modules 3–5 assisted by online role playing video vignettes. For example, videos demonstrated how to card (check the identification) of customers, ways to refuse service to an intoxicated customer, and how to discourage an intoxicated customer from driving. Each server module concluded with an interactive quiz that asked questions derived from the information covered in that module. A successful score on the Knowledge Test earned participants an informal certificate of program completion. The module for managers focused on their specific knowledge, roles, and responsibilities, including the State of Oregon system regulations, the We conducted a preliminary acceptability and feasibility study that assessed the extent to which participants using the RBS training program displayed improvements in their knowledge about alcohol service, attitudes about RBS service, self-efficacy, and intention to change their behavior in their establishments following the training experience. The Web-RBS program was made available to all staff who worked in an alcohol establishment, including servers, bartenders, and owners. Screening, informed consent, and study assessments were all completed online. Both Baseline and Post-test assessed alcohol server attitudes, knowledge, and self-efficacy. The Post-test also measured consumer satisfaction with the website.All data associated with the study were maintained in a confidential manner. All transmission of data over the Internet was protected by our use of Secure Sockets Layer (SSL) – an industry standard encryption protocol. Study data were stored on secure servers located within a locked computer room that could only be accessed by approved study staff. In addition, each study participant was assigned a unique username and password which provided secure access to the program website. All study results were described only as aggregate data and never in a manner that could be used to identify any participant’s identity. Participants agreed to an online informed consent before they were able to interact with the Web-based intervention. The research protocol was approved by the Institutional Review Board of Oregon Research Institute.Study participants were recruited using informational announcements sent to local alcohol service establishments listed in the OLCC licensee database. EstabliAn introductory recruitment meeting was held at each establishment attended by servers and establishment management. During this meeting, a research staff member provided an overview of the project. Topics included the study design, details about logging onto the RBS website to complete the screening, and the importance of completing the online informed consent. Printed materials were also left for any prospective participants who could not attend the meeting. Each study participant was given a card with a website address (url) and their unique registration information. Study participants had unique usernames and passwords, and they used this information to complete an online informed consent. Establishment owners/managers received $150 for supporting their staff to participate in the program. Study participants who completed Baseline and Post-test assessments received $50.Twenty-two establishments with 112 participants were recruited for the feasibility and acceptability study of the Web-RBS intervention. More than 60% of participating establishments were tavern/bars. The typical participant was a 35 year-old female bartender or server with some college education, 10 years of hospitality service , and currently certified to sell alcoholic beverages in Oregon. More precise descriptions of participant characteristics are shown in Table A set of 15 multiple choice questions was included on both the Baseline and Post-test assessments to measure knowledge of RBS content required by the State of Oregon and covered in our online RBS training program. For example, one item asked: "The brain is vulnerable to alcohol damage through the approximate age of 15, 20, or 25." Another item asked: "A server under 21 with a minor service permit in Oregon may: a) not mix drinks but may serve alcohol in a bar or lounge, b) only serve alcohol in areas where minors are permitted such as a restaurant, or c) serve alcohol from a kitchen but may not enter a restaurant bar or lounge)."The Baseline and Post-test assessments used a set of 40 items to test RBS attitudes related to content covered in our online RBS training. For example, one item asked respondents to rate the severity of the problem of fetal alcohol spectrum disorders and another item asked for a rating of confidence (self-efficacy) in dealing with types of challenging situations.The Post-test included six Likert-scaled items that asked about whether the program provided new information useful for work, whether the information was personally interesting, whether they would recommend the program to other alcohol servers or owners/manager, how well organized the program was, and how they would compare the online RBS training program with live RBS training.Unobtrusive measures of participant exposure to the RBS training were collected unobtrusively by the program, including the frequency and duration of each participant session and more detailed data on the viewing of individual program web pages.t-tests. Individual item analysis was accomplished using classification table tests.Each participant was assigned a score defined as the sum of knowledge items answered correctly (possible range = 0–15). Analysis of knowledge change from Baseline to Post-test was examined using paired a) which resulted in dropping 2 scales thus yielding 5 subscales: Usefulness at Work , Self-efficacy , Risks , Being Prepared , and Establishment Policies . A composite score for each subscale was assigned to each participant defined as the mean of ratings of subscale items. Baseline to Post-Test comparisons of attitudes used t-tests for independent samples for each subscale. Analysis of changes in attitudes from Baseline to Post-test was examined using paired t-tests.The set of 40 attitude items was initially grouped into 7 subscales according to theme and similarity of rating scales. All items were scaled in a manner such that a higher rating reflected a more desirable or positive attitude. Subscales were further refined by examining their internal reliability  was the number of visits and overall time spent using the program.A total of 84.8% (95/112) of study participants completed both the Baseline and the Post-test. The mean elapsed time between completion of the Baseline and the Post-test was 5.4 days .t-test results revealed that this improvement was statistically significant . Analysis of individual items using classification tables tests revealed significant improvement on 73% (11/15) of knowledge items.Improvement in RBS knowledge was observed among 95 study participants by comparing the mean number of correct items out of the 15 item assessment at Baseline  and the Post-test . Paired t-tests for change from Baseline and Post-test. In each instance, statistically significant improvements in mean scale ratings were observed , that it was useful for their work (41.9% indicated "a lot" and 48.4% indicated "some"), and that the information was personally interesting (32.3% indicated "a lot" and 53.8% indicated "some"). Similarly, 79.6% of participants reported that they found the program to be very well-organized and 82.8% reported that the program contained "about the right amount of information." They also reported that they would recommend the program to other alcohol servers or owners/managers (38.7% answering "very strongly recommend" and 39.8% "might recommend"). The majority of participants reported that they liked the online RBS program "much better" (32.3%) or "better" (26.9%) than live RBS trainings.Similarly very positive results were obtained regarding participant ratings of usefulness of the 10 program sections Table . Each ofMost study participants visited the Web-based program on the first day it was made available for their use and participants visited an average 2.9 times . They viewed an average of 133.4 program webpages , and they spent an average of 113.2 min viewing the program .The development of an innovative Web-based RBS training program was informed by formative research procedures and the contribution of a multidisciplinary design team. An acceptability and feasibility study of a completed version of this program was conducted with servers recruited from alcohol service establishments. Almost all participants completed the program, they improved their RBS knowledge, attitudes and self-efficacy, and they reported that they found the program to be useful, engaging, personally interesting and well-organized. The magnitude of these effects using Cohen’s d was encoThis study has some limitations. It is important to acknowledge that Baseline to Post-test changes may not indicate that participants had a deeper understanding of RBS content nor do they provide a test of long-term retention of relevant RBS content. The preIt is also important to acknowledge that the efficacy of any brief RBS training intervention is likely to be enhanced were it to be used within the broader context of a community effort to institutionalize programs to prevent alcohol problems-36. For Finally, it remains for further research to examine other questions that could have important implications for RBS practices. For example, one research direction might examine the extent to which combining RBS classroom training with a Web-based program adjunct might prove more beneficial than providing training by either modality on its own. Additional research might also include direct behavioral interventions in alcohol service settings. Moreover, future research might examine the use of pseudo patrons and observational assessments with longer follow-up intervals to assess for meaningful longer-term improvements in RBS practices. It is clear from the significant number of people killed, injured, and affected by drunk driving in the United States that there is a public health need for ongoing research on evidence-based, effective RBS practices to reduce these staggering statistics. The current study has added to this body of research in an important way.OLCC: Oregon Liquor Control Commission; RBS, Responsible Beverage Service: The appropriate sale and service of alcohol in commercial establishments.The authors declare that they have no competing interests.BD participated in the design of the study, performed the statistical analysis, and helped draft the manuscript. JD conceived of the study and participated in the study design. TS participated in the study design, study coordination, and helped draft the manuscript. HS participated in the study design and helped draft the manuscript. MT participated in the study design, study coordination, and helped draft the manuscript. EM participated in study coordination and helped draft the manuscript. SC participated in the design and development of the website and the online evaluation of the intervention program. All authors read and approved the final manuscript.


An effective method for obtaining resistant transgenic plants is to induce RNA silencing by expressing virus-derived dsRNA in plants and this method has been successfully implemented for the generation of different plant lines resistant to many plant viruses.Tobacco mosaic virus (TMV) movement protein (MP) gene and the partial Cucumber mosaic virus (CMV) replication protein (Rep) gene were introduced into the plant expression vector and the recombinant plasmids were transformed into Agrobacterium tumefaciens. Agrobacterium-mediated transformation was carried out and three transgenic tobacco lines  immune to TMV infection and three transgenic tobacco lines  immune to CMV infection were obtained. Virus inoculation assays showed that the resistance of these transgenic plants could inherit and keep stable in T4 progeny. The low temperature (15℃) did not influence the resistance of transgenic plants. There was no significant correlation between the resistance and the copy number of the transgene. CMV infection could not break the resistance to TMV in the transgenic tobacco plants expressing TMV hairpin MP RNA.Inverted repeats of the partial We have demonstrated that transgenic tobacco plants expressed partial TMV movement gene and partial CMV replicase gene in the form of an intermolecular intron-hairpin RNA exhibited complete resistance to TMV or CMV infection. Tobacco mosaic virus (TMV) or Cucumber mosaic virus (CMV) is found worldwide. The two viruses are known to infect more than 150 species of herbaceous, dicotyledonous plants including many vegetables, flowers, and weeds. TMV and CMV cause serious losses on several crops including tobacco, tomato, cucumber, pepper and many ornamentals. During the last decade, several laboratories have tried to introduce resistance to TMV or CMV by genetic engineering. Virus resistance in plants containing virus-derived transgene, usually by the expression of functional or dysfunctional coat protein, movement protein or polymerase gene, has been widely reported. The TMV coat protein gene was used in the first demonstration of virus-derived, protein-mediated resistance in transgenic plants dCTP-labelled TMV MP or CMV Rep gene as probe prepared by random primer procedure according to the Prime-a-Gene Labeling System .The recombinant vector pBIN-TMVΔg method  and tranescribed . Rooted B method , and anar TMV MP  and primTransgenic plants and wild plants were grown in greenhouse condition for 5 weeks before virus inoculation. Plants were mock-inoculated with phosphate buffer or inoculated with leaves sap extracts [diluted in 0.02 M phosphate buffer (pH 7.2)] from plants infected with TMV, CMV or both TMV and CMV (TMV+CMV). The inoculated plants were observed for virus symptoms after virus inoculation.Leaf tissues (0.1 g) from new emergent leaves of each plant infected with TMV, CMV, TMV+CMV or inoculated with buffer were collected at 15, 25, 45 dpi. The virus concentration in the inoculated plants was detected by triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) as described . The abs® Gold nucleic acid gel stain (Invitrogen). Membranes were hybridized as described [α-32P]dCTP-labelled MP or Rep gene as probe prepared by random primer procedure according to the Prime-a-Gene Labeling System (Promega) overnight at 40℃ in 50% formamide buffer. 10-min three time post-hybridization washes were performed sequentially at 40℃ with 1× sodium chloride-sodium citrate buffer (SSC) supplemented with 0.1% SDS. Hybridization signals were detected by phosphorimaging using a Typhoon 9200 imager .Plants tissues were ground to a fine powder in liquid nitrogen and RNAs were extracted with TRIzol , according to the manufacturer's instructions. The same RNA extract was separated to high- and low-molecular-mass RNAs using 30% PEG  and 3 M NaCl as described . The higescribed  with [α-The authors declare that they have no competing interests.QH, YN, KZ, YL performed the experiments. QH, XZ analyzed the data and drafted the manuscript. XZ provided overall direction and conducted experimental design, data analysis and wrote the manuscript. All authors read and approved the final manuscript.


Successfully completing an internal medicine residency is a great accomplishment. With the great need for primary care physicians, many residents are considering entering the field of general medicine where one can care for the whole patient. However, for those who have a drive to become an expert in one particular organ system, the path to the completion of training is only half over. Though you may think you have figured out the ‘application process’ after successfully gaining admission to college, medical school, and residency, there is one more hurdle that stands between you and your ultimate goal of sub-specialization – the fellowship application process. Unfortunately, this last application may be the most difficult of all. As a former cardiology program director, I have trained over 100 fellows and reviewed over 3,000 applications. Here is some practical advice from the ‘inside’ on how to be successful at this process. Choosing a specialty is perhaps the most important choice of your young medical career. It is what you will most likely be doing on a daily basis even 30 or 40 years from now. Before choosing a specialty, try to get some experience during residency or medical school in that specialty. Don't just spend time in the hospital with a specialist. Go to his or her office and get a good feeling for what they do all day. Is it something that will interest you and keep you stimulated 20 years from now or will it be a boring grind? Consider your options carefully before you take the plunge.If you have an academic career interest, get involved in research in the field. Meaningful research will likely result in an abstract or manuscript at the conclusion of the research project, and these publications will strengthen your application.Spending a year as a chief resident is also very worthwhile, as it is a sign of both seasoning and maturity and is likely to be viewed favorably for any fellowship candidate. The fellows I have trained who have done a chief residency are invariably some of the most mature and successful trainees with whom I have worked.On the flip side, spending a year or two as a hospitalist or moonlighter to work off some debt may not be optimal. Be aware that the longer you are away from training, the less desirable candidate you may become. A maximum of a year or two doing this type of work is a realistic limit before it hurts your chances. Also, try to find a research project in your area of true interest. Nothing is worse in an interview as a candidate who has little interest in their research and has merely done it to ‘buff up’ their application.Study the websites of the various training programs, ask colleagues and mentors about programs they recommend, and contact people from your residency program that are already training at the institutions where you have interest. You should come up with a reasonable list but unless you have a strong aversion, always apply to your home program as well. Your chances at your own program are likely better than elsewhere, especially if you have been a ‘star’ in your residency program.Make sure you understand all the requirements of the application process at a particular institution including visa status. Don't bother to apply if your visa status is not accepted at the institution (many academic centers do not accept H1B visas). I could never even consider these applications and applying was really a complete waste of everyone's time.you. Though most programs have a deadline, many will start looking at applications as they come in. You stand a better chance for your application to get a better look before the program director or admissions committee has seen over 400 of them. I always worried about the time management skills of people who submitted applications on the last day by FedEx.Carefully plan out what you want to say in the application. Since these are now done electronically, you only need submit one so put some time and effort into it. Get started early. Note that the specialty match timing has changed (in my opinion for the better) for candidates who wish to start fellowships in July 2013. The match will open in August of your third year of residency (August 2012), the interview process will probably occur through the late summer and fall and match day will be in early December 2012. This will allow you more time to decide on the right specialty, spend more time on electives, do research, and develop relationships with mentors and specialists who may provide recommendations for Remember to be completely truthful. If you did some significant research, state it, but don't overplay your involvement. You will be asked about it. If you have a limited and superficial knowledge of the subject, it will be very clear to the interviewer. Remember, you are often talking to an expert in the field. I remember one conversation with an applicant where he wasn't even pronouncing the name of the disease correctly that he supposedly had been doing research on for the past 2 years. Clearly, he had little or no real involvement in the project.Also, it pays to note your outside interests aside from medicine. I was always interested in learning whether we were considering a normal, well-rounded individual. Things like hobbies, sports, music, and other interests and special life experiences often showed me whether this would be a nice person to have on our team. The purpose of the essay is to tell a bit about you. Lecturing me on the future of cardiology or the ‘great advances’ that have come over the past few years gets very boring after the 400th essay. Keep it personal, interesting, and short. Think of it like a TV commercial. You only have a few moments to capture the audience's attention.Dear Program Director, Resident X came to teaching rounds twice with me and answered one of my silly questions correctly. I have no real idea who he is, but I am a big name in the field, and he begged me to write him a letter’ (or something to that effect). I have gotten too many of those types of letters in the past. Avoid the temptation unless they really know you well. It is much better to have a letter from a lesser known specialist or your internal medicine program director that says, ‘I worked with Resident X extensively on the wards, in the ICU, at night, and on call. He is fantastic and is someone who I would not hesitate to have care for any one of my family members. He is hard working, reliable, always upbeat and cheerful, and a great team player. We will be recruiting him heavily to remain here in our fellowship program’.References should be from people who know you well, not just a ‘big name’ specialist with whom you had little real contact. Those letters usually read something like ‘You can call once to make sure your application is complete, but do not call more than that. Don't ask if you are being considered for an interview. The secretary won't know and you will be contacted by the program if they want to meet with you. Don't be a pest; it can actually work against you. When contacted for an interview, call back quickly and pick one of the dates being offered. If you have to change the date or cancel, do so as far in advance as possible. Canceling an interview shortly before it is scheduled is not fair to the program or to the other applicants. It is too late to fill one of these coveted interview spots and often the program coordinator has done a lot of work scheduling your interviews with the faculty. Even if you don't think you will attend that institution for fellowship, the academic world is small and one day you may be asking that same program director for a job. They may remember if you did something rude and thoughtless when applying for fellowship.Plan your trip to arrive the night before, so there is no problem with travel delays. Sleep well the night before and show up on time. Being late for the interview day is disruptive and speaks poorly to your reliability. Dress professionally and be courteous to everyone. The program director knows his coordinator and secretaries a lot longer than he has known you, so if you are discourteous, arrogant, or condescending to the administrative staff it will definitely get back to the program director! The reaction of the administrative staff can be ‘the kiss of death’ for your application if you make a negative impression. My program coordinator always told me who was nasty, condescending, or arrogant when I was out of earshot and that told me volumes about their real character. So even if you are nasty, condescending, or arrogant, hide it well from everyone that day!Sit up straight, make eye contact with the interviewer, and be prepared and enthusiastic. I have had interviewees who literally looked at their shoes the entire time and never looked me in the eye or would slump in their chairs and asked me things like ‘so why should I come here?’ I smiled but was secretly thinking ‘don't worry, no chance of that happening!’Think ahead about an interesting case in the field as you may be asked to present something you have seen. I have had people who have been through 4 years of medical school and 2 years of residency who couldn't think of a single interesting case to tell me about! If you did research, state what you did in the project clearly and succinctly. Understand the background and details of the research. As I mentioned, you may be talking to an expert and you can't ‘wing it’ as they will find you out. Have some pertinent questions about the program ready and know clearly why you are applying there. You may ask a few logistical questions about things like on call schedules, but don't dwell too much on the work aspects of the fellowship. It may send a red flag to both the faculty (and the current fellows) that you don't want to work hard and might be a complainer. Of all the headaches for a fellowship director, the chronic complainers are by far the worst. Obviously this is not the impression you want to make.Writing thank you notes afterward is a nice touch but be sure to personalize them. I have gotten so many ‘form’ thank you notes that most just go right in the trash. Sometimes I have even gotten ones addressed to other program directors by mistake!Think carefully about where you want to train. Take family considerations into account as both you and your family must be reasonably content for the duration of your fellowship. Family strife, an unhappy spouse and kids, or long distance separations can make concentrating on your training nearly impossible. However, make sure there is some permanence to the relationship before making a major career decision. I have known residents who picked a program they didn't particularly like in a certain city just because their girlfriend/boyfriend or fiancée was there and then they broke up before even starting their fellowship! You are then left with an undesirable program and a broken personal life. Not a great way to start a new chapter in your life. Definitely take into account where you will live, but remember trivial things like the weather, city amenities, and so on shouldn't greatly decide where you train. You will only be there for 2 to 3 years, and where you train has a tremendous effect on your future career for the next 40!Some programs continue to accept some candidates outside the match. If you are in a very competitive field (like cardiology) you may want to consider taking a spot if it is outside the match and offered to you. This is an individual decision that needs to be weighed based on the competitiveness of the field, the program's desirability, and the strength of your application.Rank your programs based on their desirability, not where you think you will get in. The match process will take care of that. I also never thought there was a great need to call the program director to express interest. The match actually discourages this practice. There is no need to tell the program director that they are your ‘top choice’. If you do, call only the program you are most interested in and don't have anyone else call for you  unless that truly is your top choice. I received a call once from a program director just 2 days before the match list went in, who told me in the clearest possible terms how we were the applicant's number one choice and how much she really wanted to come to our program. She was a great candidate and I had already ranked her high enough to match with us. When she didn't match, I called the program director. He felt very silly for being misled by one of his residents for whom he had done a favor. Remember, medicine is a small world and these lapses in truthfulness could eventually come back to hurt you someday.Hopefully these inside tips will be useful as you start the fellowship application process. Certainly put your best foot forward, but always be yourself and be truthful. After many years of ‘the game’, most program directors have a sense for the ‘good ones’ out there.


Tobacco mosaic virus (TMV) causes significant losses in many economically important crops. Contaminated soils may play roles as reservoirs and sources of transmission for TMV. In this study we report the development of an immunocapture real-time RT-PCR  assay for direct detection of TMV in soils without RNA isolation. A series of TMV infected leaf sap dilutions of 1:101, 1:102, 1:103, 1:104, 1:105 and 1:106  were added to one gram of soil. The reactivity of DAS-ELISA and conventional RT-PCR was in the range of 1:102 and 1:103 dilution in TMV-infested soils, respectively. Meanwhile, the detection limit of IC-real-time RT-PCR sensitivity was up to 1:106 dilution. However, in plant sap infected by TMV, both IC-real-time RT-PCR and real-time RT-PCR were up to 1:106 dilution, DAS-ELISA could detect at least 1:103 dilution. IC-real-time RT-PCR method can use either plant sample extracts or cultivated soils, and show higher sensitivity than RT-PCR and DAS-ELISA for detection of TMV in soils. Therefore, the proposed IC-real-time RT-PCR assay provides an alternative for quick and very sensitive detection of TMV in soils, with the advantage of not requiring a concentration or RNA purification steps while still allowing detection of TMV for disease control. Since its description in the late 19th century, diseases caused by TMV continue to cause heavy agricultural losses worldwide [orldwide . As one orldwide –5. CropsNicotiana tabacum Xanthi-NC by necrotic lesions [Currently, a variety of techniques have been established for the detection of TMV, including biological assay on  lesions , ELISA [ lesions , and RT- lesions . General lesions .In this study, three different methods were assessed for their capacities for detecting TMV from soil. Additionally, the method of immunocapture real-time quantitative RT-PCR from soil for TMV detection was established, and immunocapture sample preparation was incorporated to permit the entire test, from sample preparation to amplicon detection, to be completed in a single tube.2.2.1.Nicotina tabacum cv. K326 in the authors’ laboratory. TMV particles were purified according to the method of Gooding and Herbert [Tomato mosaic virus (ToMV) provided by professor Jiang Guoyong of Qingdao Agriculture University was used as negative control.The assays were developed and assessed with 68 soil samples collected from nine Chinese provinces where tobacco and tomato that were infected by TMV was planted. TMV was characterized and maintained in  Herbert . Tomato 2.2.1, 1:102, 1:103, 1:104, 1:105 and 1:106 , respectively. One gram of air-dried TMV-contaminated or uncontaminated soil samples were suspended in 1 mL extraction buffer (0.01 M phosphate buffer pH 7.0) in Eppendorf tubes. The suspensions were agitated on a vortex mixer for 1 min, shaken on a rotary shaker (200 rpm) for 30 min at room temperature, incubated at 4 °C overnight, and then centrifuged at 10,000×g for 2 min. The supernatants were used for DAS-ELISA and IC-real-time RT-PCR. Based on Horm’s study [To confirm the sensitivity of ELISA, RT-PCR and IC-real-time RT-PCR for detecting TMV in plant and soil, one gram aliquots of soil were treated with a series of TMV infected leaf sap dilutions of 1:10’s study , direct 2.3.TMV antibody and its enzyme conjugate were supplied by ADGEN . Double antibody sandwich ELISA was carried out following the instruction of the manufacture. Leaf tissue or soil mentioned in Section 2.2 were prepared. Absorbance at 405 nm was measured with an ELISA reader, SpectraMax Plus 384 . A sample was considered positive if its absorbance reading was at least two times higher than that of the non-treated control sample (plant and soil).2.4.™ RT reagent kit  according to the manufacturer’s instructions. The forward primer (5′-ATTAGACCCGCTAGTCACAGCAC-3′) and the reverse primer (5′-GTGGGGT TCGCCTGATTTT-3′) were designed using the PerlPrimer software based on the conserved nucleotide sequences of TMV CP in GenBank [CP RNA sequences using the DNASTAR package . The conventional PCR was performed using the above synthesized cDNA. The amplication was carried out in a 25 μL total reaction volume by using rTaq polymerase (TaKaRa) with 25 pmol of forward and reverse primers and 0.5 μL cDNA according to the manufacturer’s protocol. The thermal profile of PCR was the same as used for real-time PCR (see below), performed in a T3 Thermocycler . The primers 5′-CAAGGAAATCACCGCTTTGG-3′ (forward) and 5′-AAGGGATGCGAGGATGGA-3′ (reverse) for the Actin gene of tobacco were used as internal controls.First strand cDNA were synthesized by reverse transcribing total RNA of leaf tissue or soil using an Exscript GenBank , which w2.5.2O. The thermal cycling process and fluorescence signal detection were carried out with the Applied Biosystems 7500 Real-Time PCR System . The cycling parameters were: reverse transcription for 5 min at 42 °C, denaturation for 10 s at 95 °C, followed by 40 cycles of 10 s at 95 °C for denaturation and 36 s at 62 °C for annealing and extension. To generate a standard curve, three replicates of a series of 5-fold dilutions of purified virus  after immunocapture were amplified using One Step SYBR® PrimeScript® PLUS RT-PCR Kit. The standard curve was constructed using the cycle threshold values obtained against the known concentrations of serially diluted virus. The identities of the amplicons and the specificity of the reaction were verified by agarose gel electrophoresis and melting curve analysis, respectively. The relative RNA levels of TMV in different virus samples were computed with respect to the standard curve.Immunocapture was conducted following the method described by Jacobi . Briefly2.6.t-test by parametric one-way analysis of variance (ANOVA), and those with p-values less than 0.05 were considered significant.Results were tested for significance using the Student’s 3.3.1.1 to 106 TMV-infected leaf sap  were added to one gram TMV-free soil, and those soils and above leaf saps were determined by DAS-ELISA, respectively. The results of three repeated tests indicated that the limit of DAS-ELISA to detect TMV in soil was diluted at 1:102    . The resv, g/mL) . DAS-ELI3.2.6 dilution. However, when a 10-fold dilution series of the same TMV-infected leaf sap were added to 1g air-dry soil, from which total RNA was extracted as described in Section 2.2, the sensitivity of conventional RT-PCR for detection in soil was only 1:103 . Tobacco leaf saps with the same dilution were also used to IC-real-time RT-PCR analysis. In this experiment, IC-real-time RT-PCR was capable of detecting TMV in all dilutions in three replicated tests of soil or plant  and TMV Fumeng isolate (GenBank accession: HE818416) isolate that represent two subpopulations of nature TMV populations in China, demonstrated a high degree of specificity for TMV by amplifying TMV only. The method yielded negative results on other viruses and virus-free soil tested  tested positive by DAS-ELISA. Direct RNA extraction was performed on 68 samples with the QIAamp kit, and TMV viral RNA was detected in 16 samples (23.5%) by conventional RT-PCR. The IC-real-time RT-PCR was used to detect TMV from 68 samples without RNA extraction, and 27 samples (39.7%) tested positive .4.Diagnosis of the pathogen plays an important role in crop improvement and disease management strategies. TMV was detected at 0–60 cm depths of clay textured soil 19 months after tobacco waste application [3- and 102- times higher in plant materials and soils, respectively. When IC was used to prior to single-step real-time RT-PCR to detect TMV in plant saps and soils the sensitivity were 104 and 103 times higher relative to ELISA, respectively.The ELISA method was used to detect TMV in clay soil . Due to 2-times higher than that of RT-PCR to detect TMV in soil, however, the sensitivity of both methods were similar to detect TMV in plant materials. TMV and ToMV are closely related, and both infect pepper, potato, tomato and several other hosts. Applying the IC-real-time RT-PCR could detect TMV in soil infected by TMV [RT-PCR technology is widely used to detect viruses with RNA genomes, and has gained importance in recent years. Many new PCR methods have been developed, and have become routine for virus detection in the laboratory. Amplification in real-time PCR reactions is often affected by inhibitors present naturally in different matrices, the effect is often revealed by reduced amplification efficiency . The effd by TMV . No posi5.In conclusion, a IC-real-time RT-PCR assay was used to detect TMV in soil. It was more rapid, sensitive, and specific than either the RT-PCR or DAS-ELISA assays. This assay method could help accurately determine the TMV content of soil. Based on the accurate determination of virus occurrence in soil, crop rotations could be utilized or resistant varieties planted, in order to control TMV and curtail viral disease outbreaks in the crop.


Eucalyptus is an important hardwood tree genus used in the pulp and paper industry and has potential as biofuel feedstock. Xylan removal is expensive and uses environmentally harsh chemical treatments [Eucalyptusgrandis genome  provides a unique opportunity to analyze the native endo-(1-4)-beta-xylanase proteins involved in xylan modification in eucalypt fibre cell walls. Detailed knowledge of endogenous xylanolytic enzymes from Eucalyptus could facilitate the development of strategies to enhance the processing of woody biomass for cellulose and biofuel production. The aims of this study are to identify xylem secondary cell wall-related endo-(1-4)-β-xylanase genes in the E. grandis genome and to perform a comparative analysis of the Eucalyptus xylanasepeptide sequences with those of previously studied Arabidopsis orthologs to provide a framework for assigning function to the Eucalyptus enzymes.The hemicellulose xylan constitutes the major non-cellulosic component of plant secondary cell walls. It has been shown that xylan adsorbs to cellulose fibres and also covalently binds a carbon moiety of lignin ,2. Eucaleatments . Previoueatments . The recE. grandis genome sequence on Phytozome v7.0 (http://www.phytozome.net) for putative endo-(1-4)-β-xylanase genes resulted in the identification of 18 putative GH10 family members. The expression profile of each family member was assessed  to identify members with putative roles in xylem secondary cell wall metabolism. Egrandis_v1_0.001952m (designated EgrXYN1) showed the highest xylem to phloem and xylem to leaf expression ratios of the expressed E. grandis GH10 genes [A. thaliana genome for putative orthologs to EgrXYN1 and co-phylogenetic analysis of all 18 E. grandis enzymes with the putative A. thaliana xylanases revealed that AtXYN1 (At1g58370) [EgrXYN1  revealed that EgrXYN1 contains three successive N-terminal β-sandwich carbohydrate binding modules IV  which were also observed in AtXYN1. A protein motif search (http://motif.genome.jp) revealed that EgrXYN1 also contained a conserved and identical C-terminal GH10 active site sequence “GLPIWFTELDV” at amino acid position 802-812. Finally, de novo motif search of both AtXYN1 and EgrXYN1 using MEME revealed the presence of three additional novel C-terminal motifs present within both enzymes . One of these, EgrXYN1 is highly preferentially expressed in Eucalyptus xylem tissues and shows highest similarity to AtXYN1. The similarities between AtXYN1and EgrXYN1 suggest similar biochemical properties and biological functions. Previous studies showed that AtXYN1::eGFP localized to the cell wall providing support for its function in cell wall modification. AtXYN1prom::GUS constructs expressed predominately in the vascular bundles suggesting that AtXYN1 (and therefore putatively EgrXYN1) is involved in secondary cell wall modification [The fication ,7. Futur


Decoy receptor 3 (DcR3) is a soluble secreted protein that belongs to the tumor necrosis factor receptor (TNFR) superfamily. DcR3 inhibits the Fas ligand (FasL)/Fas apoptotic pathway by binding to FasL, competitively with Fas receptor. Previous studies have reported that overexpression of DcR3 has been detected in various human malignancies and that DcR3 functions as a ‘decoy’ for FasL to inhibit FasL-induced apoptosis. In addition, recent studies have revealed that DcR3 has ‘non-decoy’ functions to promote tumor cell migration and invasion, suggesting that DcR3 may play important roles in tumor progression by decoy and non-decoy functions. We have previously reported that overexpression of DcR3 was observed in human malignant fibrous histiocytoma (MFH), however, the roles of DcR3 in MFH have not been studied. In the present study, to elucidate the roles of DcR3 in tumor progression of MFH, we examined the effects of DcR3 inhibition on cell apoptosis, migration and invasion in human MFH cells. siRNA knockdown of DcR3 enhanced the FasL-induced apoptotic activity and significantly decreased cell migration and invasion with a decrease in the activation of phosphatidylinositol 3 kinase (PI3K)/Akt and matrix metalloproteinase (MMP)-2. The findings in this study strongly suggest that DcR3 plays important roles in tumor progression of human MFH by decoy as well as non-decoy functions and that DcR3 may serve as a potent therapeutic target for human MFH. Malignant fibrous histiocytoma (MFH), which has recently been classified as undifferentiated pleomorphic sarcoma (UPS), is the most common high-grade soft tissue sarcoma that occurs in late adult life ,2. AdvanDecoy receptor 3 (DcR3) is a newly identified member of the tumor necrosis factor receptor (TNFR) superfamily. DcR3 lacks a transmembrane domain and is thought to be a soluble secreted protein. DcR3 is known to act as a decoy receptor for Fas ligand (FasL) , LIGHT  and TL1ARecent studies have demonstrated that DcR3 also functions as an effector molecule independently of the FasL/Fas apoptotic pathway. DcR3 overexpression promotes migration and invasion of nasopharyngeal carcinoma cells . In breaOverexpression of DcR3 has been reported in various malignancies such as lung and colon cancers , EBV or Based on previous studies, DcR3 may contribute to tumor progression by not only neutralizing FasL-induced apoptosis but also by promoting tumor migration and invasion, which are important for the development of metastasis. We hypothesized that DcR3 may play important roles in tumor progression and metastatic potential in human MFH through the inhibition of the apoptotic pathway and the signaling pathway that is related to migration and invasion. In the present study, we evaluated the effects of DcR3 inhibition on cell apoptosis, migration and invasion using human MFH cell lines to elucidate the roles of DcR3 in human MFH.2 atmosphere. For all experiments, we used the DMEM containing 10% FBS without the antibiotic solution.Two human MFH cell lines, TNMY1 and Nara-H, were used in this study. TNMY1 was previously established in our laboratory  and NaraTo evaluate the effect of DcR3 knockdown, we transfected MFH cell lines with DcR3-specific small interfering RNA (siRNA). Briefly, 1 day before transfection, cells were seeded in 6-well culture plates in growth medium. Then, cells were transfected with 60 nmol of either a specific siRNA against human DcR3 (DcR3-si)  or a negative control siRNA (Ctrl-si) (Invitrogen) using Lipofectamine 2000 transfection reagent according to the manufacturer’s protocol (Invitrogen).After siRNA transfection, DcR3-si transfected cells were incubated in medium with 0 (DcR3-si cells) or 3 μg/ml of recombinant DcR3-Fc , or 3 μg/ml of recombinant IgG-Fc  for 24 h. Ctrl-si transfected cells were incubated without any recombinant Fc proteins (Ctrl-si cells).To induce apoptosis, cells were treated with FasL  for 6 h after siRNA transfection and Fc treatment.To evaluate kinase activities, cells were pretreated with 0 or 20 μM of LY294002  in DMSO  for 2 h followed by a 1-h treatment with DcR3-Fc or IgG-Fc.DcR3, MMP-2 and human β-actin (control) were synthesized by Hokkaido System Science . Primer used were: DcR3: 5′-TCAATGTGCCAGGCTCTTC-3′ and 5′-AGCCACAAAG TCGATGACG-3′; MMP-2: 5′-ACAGCAGGTCTCAGCC TCAT-3′ and 5′-TGCCTCTGGACAACACAGAC-3′; β-actin: 5′-GATGAGATTGGCATGGCTTT-3′ and 5′-CACCTTCA CCGTTCCAGTTT-3′. The values were normalized with those for β-actin and relative expression was analyzed using the ΔΔCt method.We isolated total RNAs from cell lines using an RNeasy Mini kit according to the manufacturer’s protocol  and first strand cDNAs were transcribed. Quantitative real-time PCR (qRT-PCR) was performed in a 20-μl reaction mixture using the Power SYBR Green Master Mix reagent  on an ABI PRISM 7500 sequence detection system (Applied Biosystems). The PCR conditions were as follows: 1 cycle at 95°C for 10 min followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Primers for human Lysates were extracted from cells using a whole cell lysis buffer  supplemented with a protease and phosphatase inhibitor mix . The protein content of lysates was then quantified using BCA Protein Assay reagent . Samples containing equal amounts of protein were electrophoresed through 12% polyacrylamide gels and transferred onto PVDF membranes. After blocking membranes were incubated overnight at 4°C with the following antibodies in CanGet Signal Solution 1 : anti-human DcR3 , anti-human Fas , anti-human PARP , anti-human cleaved PARP , anti-human caspase-3 , anti-human cleaved caspase-3 (1:500), anti-human MMP-2 , anti-human Akt , anti-human phosho-Akt , anti-human ERK1/2 , anti-human phospho-ERK1/2 , anti-human JNK , anti-human phospho-JNK , anti-human p38 , anti-human phospho-p38 . All antibodies were purchased from Cell Signaling Technology. Following washes, membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase and were exposed with ECL Plus western blot detection system reagent . Antibody binding was detected by Chemilumino analyzer LAS-3000 mini . Membranes were reprobed with anti-human α-tubulin antibody (Sigma-Aldrich) to confirm equal protein loading.3 cells/well in 100 μl culture medium. After siRNA transfection and Fc treatment, cells were treated with or without FasL to induce apoptosis. At the indicated incubation times , 10 μl of the CCK-8 solution was added into each well and incubated for 1 h. Optical density was measured at a wavelength of 450 nm using a Model 680 Microplate Reader . The relative number of viable cells in each well was calculated.To evaluate the involvement of DcR3 in MFH cell proliferation, we performed WST-8 cell proliferation assay using Cell Counting Kit-8 . Cells were seeded in 96-well culture plates at a density of 5×10in vitro scratch wound healing assays as previously described  and the lower wells were filled with complete growth medium. The chambers were incubated for 30 h to allow cells to invade from the upper wells towards the lower wells. After incubation, non-invading cells on the upper surface of membranes were removed by scrubbing and invading cells on the lower surface of the membranes were fixed, inspected with a microscope and imaged. The number of invading cells was counted in three random fields.The effect of DcR3 on cell invasion was assessed by Transwell chamber invasion assays, as previously described . After sTo evaluate the enzyme activity of MMP-2, we performed gelatin zymography as previously described . The celEach experiment was performed independently at least three times and data are presented as the mean ± standard deviation (SD). The statistical significance of the differences among means was evaluated by ANOVA with post hoc test. Results were considered significant at P<0.05.*P<0.05, We performed transfection of DcR3-siRNA and DcR3-Fc treatment to evaluate the effects of DcR3 in MFH cells. In both MFH cell lines, DcR3-siRNA transfection strongly suppressed DcR3 mRNA *P<0.05,  and protImmunoblot analysis revealed that DcR3 knockdown or DcR3-Fc treatment without FasL treatment did not affect the expressions of caspase-3, PARP and their cleaved forms in either cell line . FasL tr*P<0.05, A previous report demonstrated that DcR3 knockdown with FasL treatment significantly suppressed cell proliferation in human pancreatic adenocarcinoma cells , therefoin vitro scratch wound healing assays, DcR3 knockdown significantly decreased cell migration in both MFH cell lines at 12 and 24 h after wounding and DcR3-Fc treatment significantly restored the migration ability compared with control cells, respectively . In addition, DcR3-Fc treatment after DcR3 knockdown significantly restored the invasiveness in both cell lines (*P<0.05, Next, we performed the transwell chamber invasion assays to evaluate the role of DcR3 in MFH cell invasion. In both cell lines, the number of invading cells in DcR3-siRNA transfected cells was decreased to 61% TNMY1,  and 64% *P<0.05, .Because DcR3 has been reported to be involved in the activation of various kinases –17, we eMMP-2 mRNA expression was significantly suppressed by DcR3 knockdown and was restored by DcR3-Fc treatment in both MFH cell lines (*P<0.05, As previous reports suggest that DcR3 may affect MMP-2 regulation via the PI3K/Akt pathway ,17,39, w*P<0.05, . Immunob*P<0.05, . Consist*P<0.05, , both th*P<0.05, .DcR3 has been identified as a decoy receptor for FasL in lung and colon cancers  and receet al reported that siRNA knockdown of DcR3 increased FasL-induced apoptosis in human pancreatic adenocarcinoma cells, which highly express DcR3 (Because resistance to apoptosis is a characteristic property of malignant tumor cells to escape from immune attack by host immune systems , we firsess DcR3 . In thisMoreover, several studies have demonstrated that DcR3 functions as an effector molecule in various cells, independently of the FasL/Fas pathway, by regulating migration and invasion abilities ,11, ERK Previous studies have also suggested the involvement of DcR3 in kinase phosphorylation –17. TherIn conclusion, we demonstrated that in human MFH cells DcR3 may increase tumor progression as a decoy, promoting cell proliferation via inhibition of FasL-induced apoptosis and as a non-decoy, regulating cell migration and invasion by MMP-2 activation via the PI3K/Akt pathway . Althoug