P = 0.019), reduced screen time by 44 minutes (P < 0.001), and improved key dietary behaviors. Overall, FNPA scores increased by 9% (P < 0.001) and 69% of patients with “high risk” FNPA scores at baseline dropped below the “high risk” range by followup. Patients also lowered BMIs (P = 0.011) and age- and sex-adjusted BMI z-scores (P < 0.001) after completing the 7-week program. We hope this report will be useful to medical and public health professionals seeking to develop stage 2 pediatric obesity programs.This paper describes FitKids360, a stage 2 pediatric weight management program. FitKids360 is a physician-referred, multicomponent, low-cost healthy lifestyle program for overweight and obese youth 5–16 years of age and their families. FitKids360 provides an evidence-based approach to the treatment of pediatric overweight by targeting patients' physical activity, screen time, and dietary behaviors using a family-centered approach. The intervention begins with a two-hour orientation and assessment period followed by six weekly sessions. Assessments include lifestyle behaviors, anthropometry, and the Family Nutrition and Physical Activity (FNPA) survey, which screens for obesogenic risk factors in the home environment. Outcomes are presented from 258 patients who completed one of 33 FitKids360 classes. After completing FitKids360, patients increased moderate to vigorous physical activity by 14 minutes ( The epidemiologic evidence of the prevalence and consequences of pediatric obesity is well documented . CurrentStage 1 is referred to as “prevention plus” and generally takes place in the primary care office setting. Key lifestyle behaviors are promoted and if, after 3 to 6 months, the child has not made appropriate improvement the provider can offer the next level of obesity care. Stage 2 entails structured weight management and often includes a multidisciplinary team of health care providers. This level of obesity treatment is distinguished from prevention plus less by differences in the targeted health behaviors and more by the support and structure that is provided to the child in achieving health behaviors. Stages 3 and 4 occur within specialty clinics and include a multidisciplinary team where the intensity of behavior changes and frequency of visits increases. In stage 4 obesity care, medications, very low calorie diets, or bariatric surgery are prescribed.This paper describes the methodology of FitKids360, a stage 2 pediatric weight management program, and provides outcomes in patients who have completed one of 33 FitKids360 classes conducted between 2010 and 2013.FitKids360 is a multicomponent, family-based, low-cost healthy lifestyle program targeting overweight and obese children and adolescents of 5–16 years of age and their families. FitKids360 was conceived and designed by a multidisciplinary team of medical professionals . The overall goal of FitKids360 is to provide an evidence-based approach to the treatment of pediatric overweight and obesity by achieving the following primary aims: (1) improving the patients' physical activity, screen time, and dietary behaviors and (2) improving the family's “obesogenic” risk score.The “360” portion of the program name was suggested by the marketing agency that designed the FitKids360 material. It stems from the logo being 360 degrees and is intended to depict people coming full circle, being well rounded, and the program being part of everything in their life. In addition, it was to represent that the program was of full service to encompass all aspects of obesity, not just the physical component, and to support our belief that a 360-degree perspective is required to address the complex issue of pediatric obesity.The involvement of the family and assisting participants by creating a supportive environment for healthy behaviors is a major component of FitKids360. Indeed, the family component is essential in the development of an environment that supports children's efforts to implement changes in nutrition, physical activity, and screen time , 5. FurtFor background purposes, it is important to note that this program was developed based on a collaborative design; there was no grant funding or hired staff. It was a true community collaborative at its best. Beginning in 2009, key players were actively recruited, including researchers, marketing experts, nonprofits, insurance company representatives, fitness centers, and primary care physicians. As the concept was designed, volunteers filled the needs in the constructing of the curriculum, program development, brand design, and so forth. The original objectives of the program were to be (1) evidence-based, (2) free of charge, and (3) widely available at multiple sites and times within the community (up to 20 or more sessions per year).Patients must be 5 to 16 years of age and have a body mass index (BMI) ≥ 85th percentile  according to the Centers for Disease Control growth chart . In addiAll patients must be referred by a physician or health care provider to the FitKids360 program coordinator. Self-referrals must go through a physician or health care provider. In addition, patients and their parents or support partners are screened for readiness to change . The proFitKids360 is conducted by a multidisciplinary team of experts in their respective discipline and in pediatric obesity. The intervention begins with a two-hour orientation and assessment period followed by six weekly sessions. Each weekly session is two hours in duration and consists of physical activity and nutrition education and behavioral counseling for the patients and their social support person(s). In addition, physical activity is performed intermittently throughout the session for a total of 30–60 minutes. Between weekly sessions, the patients log habitual physical activity, screen time, and dietary behaviors.The orientation consists of an introduction of the FitKids360 staff, a description of the purpose of the program, program expectations, class structure, and a review of the weekly log sheets and other paperworks. In addition, preintervention assessments are conducted (see below for details).What do you think this group will be about and why do you think you were asked to participate?What are your perceptions of the group? What things do you think you have in common?What needs to happen for your family to be successful?Besides general introductions of all participants, we also conduct “getting to know you/icebreaker” activitiesto create a connection to the group. For example, if your name were given as the description for any one word in the dictionary, what word would that be? And why? We also try to provide a positive perception of pediatric obesity treatment by starting the discussion with the following questions.8 to 11 hours of sleep every night;7 breakfasts every week;6 home-cooked meals around the table every week;5 servings of fruit and vegetables every day;4 positive self-messages per day;3 servings of low-fat dairy per day;2 hours or less of screen time per day;1 hour or more of physical activity per day;0 sugar-sweetened beverages per day.The curriculum was developed based on an evidence-based approach to key features related to pediatric obesity. “Healthy counts” (8-7-6-5-4-3-2-1-0) are the cornerstone of the FitKids360 curriculum and more broadly our primary care and public health education campaign:More specifically, the cornerstone lifestyle behaviors of physical activity/sedentary behavior and nutrition were coupled with behavioral strategies and issues. The FitKids360 development team relied upon the expert committee recommendations  to develFamilies monitor their progress towards their goals via tracking logs for diet , physical activity, and screen time. Patients and their families set weekly goals with support from the clinical staff. Besides monitoring goals, the tracking logs are also part of the incentive point system . Weekly To date, 258 of 418  patients who started the program have completed it. In comparison, a recent review reported attrition rates ranging from 27% to 73% (with most on higher end) . Aside fWhile the weekly curriculum provides patients and their families with the foundation for a healthy lifestyle, the Buddy Program presents the opportunity for enhanced and extended support especially since compliance and adherence are major barriers in the treatment of childhood obesity. The Buddy Program serves as a transition program that helps children and their families translate knowledge from weekly sessions into lasting behavior changes.A “Buddy” is a case manager and mentor. The mentoring approach used here is similar to that used in public schools for a variety of goals  , 11. A rThe first phase of the Buddy Program, which occurs concurrently with the FitKids360 weekly sessions, matches participating families with student mentors (Buddies) for accountability, goal setting, motivation, and weekly planning. The Buddies are first and second year students at the Michigan State University College of Human Medicine. Each medical student is assigned one or two families during the first week of class. The Buddies engage patients and their families in weekly communication via phone calls. The minimum requirements for each contact include (1) asking about progress towards individual and family goals, (2) remembering to complete physical activity and screen time logs, and (3) addressing any remaining concerns.The second phase of the Buddy Program occurs during the 6 months following completion of the FitKids360 weekly sessions. This phase builds on the relationships formed during the FitKids360 class session and includes continuation of phone calls or email “checkups” to facilitate structured goal setting and monthly family events to review themes from the FitKids360 classes and demonstrate healthy lifestyle choices .This opportunity for medical students to engage in a pediatric obesity treatment program is unique, since the medical school curriculum does not typically cover details of nutrition, physical activity, healthy lifestyle, or obesity prevention and treatment. This is important since residency and physician training after residency graduation consist of individuals choosing courses on their own to meet continuing medical education requirements without mandatory update training in any specific areas of medicine. Furthermore, training of future physicians in obesity prevention and treatment is critical regardless of their future career paths. Whether they choose primary care or subspecialty paths, obesity does not spare any particular underlying disease process and its presence will complicate the disease processes of children with special health care needs.Assessments are conducted at the beginning and conclusion of the program. The assessment team has consisted of site staff, nurses, medical students, and undergraduate and graduate health science students. These individuals are trained by one of the principal investigators (JMT). The assessment battery includes lifestyle behaviors  as well as physical measures .Physical activity, screen time , and diet are determined by self-report. The self-report question for physical activity is the same question used in the Youth Risk Behavior Survey, asking how many days per week children are engaged in moderate-to-vigorous physical activity (MVPA). Total screen time is determined from self-reported television viewing, computer use, and video games on weekdays and weekends. Dietary consumption of 100% fruit juices, fruit, vegetables, whole grains, sugar-sweetened beverages, sweets/desserts, and dairy was reported as times consumed per day or week.Since the family environment is instrumental to the physical activity, screen time, and nutritional behaviors of the patient, the impact of the program on the family “obesogenic” environment is evaluated using the Family Nutrition and Physical Activity (FNPA) screening tool. The FNPA screening tool was developed by Ihmels and colleagues  through 2) is then calculated from measured stature and body mass. Age- and sex-specific percentiles for height, weight, and BMI are determined using the CDC SAS growth software (http://www.cdc.gov/nccdphp/dnpao/growthcharts/resources/sas.htm). Waist circumference is measured as a proxy for abdominal or visceral adiposity using a Gullick tape to the nearest 0.1 cm at the superior border of the iliac crest.Stature is measured without shoes to the nearest 0.1 cm using a ShorrBoard stadiometer . Body mass is measured to the nearest 0.1 kg and body fatness (%BF) is estimated using a foot-to-foot bioelectric impedance scale . Body mass index . A separate report is sent to the referring physician and also includes educational insight into the key lifestyle behaviors and obesity assessment. In addition, the physician is reminded of the recommended visit schedule for stage 2 obesity treatment (monthly).FitKids360 is offered free of charge to those who qualify. The program is of relatively low cost—about $196 per child at a participation rate of 20 children per seven-week session. This calculation is based on the budget shown in To increase engagement amongst those interested in FitKids360, quarterly workgroup meetings are held and attended by key personnel, staff from existing FitKids360 sites, and any other interested persons or parties. These 1-2 hour meetings include presentations and discussion on topics such as class updates, recent results, funding opportunities, procedural changes, brainstorming sessions, and alerts to related opportunities in the community . As needed, subcommittees are formed and meet periodically. The entire program is overseen by a steering committee comprised of six individuals including two pediatricians, an exercise physiologist, a registered dietician, a researcher, and the program coordinator. The steering committee meets monthly and addresses all pertinent issues to the FitKids360 program.All “teams” interested in conducting a FitKids360 class are required to participate in our 1-day training program. Training sessions are held on a quarterly basis, depending on interest, and are provided by the program coordinator and several members of the FitKids360 steering committee, including the dietician, exercise physiologist, and researcher. The training session begins with an overview and introduction of the FitKids360 program (60 minutes). Given the ethnic and socioeconomic variation in our patients and their perceptions and beliefs about a healthy lifestyle, nutrition, food preparation, and so forth, we provide training on cultural competency and sensitivity (60 minutes) led by a community health educator with experience on this topic. Goal setting and motivational interviewing are taught and time is allowed to practice motivational interviewing techniques (50 minutes). Following the lunch break, an overview of assessments and measurements is provided (60 minutes), and then breakout groups are formed based on area of expertise . This portion of the training lasts about 2 hours to allow an in-depth training on each week of the curriculum. While lesson plans have been developed to engage the entire family, there are cognitive and developmental differences inherent in the wide age range of our patients. Therefore, these breakout sessions include training for adapting the curriculum to different age groups and provide examples of accommodations for younger and older participants. In addition, the FitKids360 “Buddy program” (described previously) also plays a major role in providing individualized support to both older and younger students. The training concludes with a question and answer session (30–45 minutes).P < 0.05.Over the past 4 years a total of 33 FitKids360 classes have been administered at over dozen sites throughout the state of Michigan. Patient outcomes from these classes are presented below. Specifically, baseline characteristics were calculated as means ± standard deviations (SD), and changes in primary and secondary outcomes over the 7-week program were evaluated using within-subjects repeated measures analysis of variance. Before analyses, data were screened for outliers as identified by values >3 SD from the mean. All analyses were performed using PC-SAS (version 9.3) and alpha was set at A total of 418 overweight or obese (BMI ≥ 85th centile) patients aged 5–16 years old were enrolled in one of 33 FitKids360 classes. 258 patients completed the program and the follow-up evaluation (62% retention). Patients were 10.5 ± 2.9 years old at baseline and 59% were female. Upon enrollment, 365 patients (87.3%) were obese (BMI ≥ 95th centile) and 173 (41.4%) of the total sample were severely obese (BMI ≥ 99th centile).P = 0.019). Among those not meeting recommended activity levels at baseline, MVPA increased by 31 min/d and 56% reported MVPA levels sufficient to meet the 60 min/d guideline by follow-up. On average, patients reduced screen time by 44 min/d (0.7 hr/d) (P < 0.001) due to significant decreases in TV viewing (P < 0.001) and video game playing (P = 0.027). Among the 70% reporting >2 hr/d of TV viewing at baseline, 42% reduced to <2 hr/d after completing FitKids360.Mean changes in health behaviors are presented in P < 0.001) and sugar-sweetened soft drinks (P = 0.016) and an increase in the frequency of consumed whole grains (P < 0.001) and fruits and vegetables (P = 0.017) (P < 0.001), and scores improved by 8.5 points (16%) among families identified as having high-risk family environments and behaviors according to baseline FNPA levels [Significant changes in reported nutrition behaviors included a reduction in the frequency of consumed sweets/desserts = 0.017) . In addi . In addP < 0.001) and weight (P = 0.030) over the 7-week program resulting in a small but significant reduction in BMI (P = 0.011). Similarly, a significant decrease was measured in age- and sex-adjusted BMI z-scores (P < 0.001). Waist circumference and body composition estimates remained relatively unchanged after the program.Anthropometric changes are displayed in Based on the results, it appears that FitKids360 may be an effective program for initiating positive health behavior changes and supporting weight management efforts, at least in the short term. Patients who completed FitKids360 made meaningful improvements to their physical activity and sedentary behaviors and reported significant progress in their nutrition habits, including consuming whole grains, fruits, and vegetables more frequently and sweets/desserts and sugar-sweetened soft drinks less frequently.Perhaps most significantly, participants (and their families) improved overall health behaviors  as indicated by the FNPA survey. Ihmels et al. have shown that youth in the lowest tertile for FNPA scores had 1.7 times higher odds of being overweight or obese (BMI ≥ 85th percentile) when compared to youth in the highest tertile . In the z-scores over the 7-week program. This is a notable result since typical BMI trajectories for growing children in this age range are positive. Future research is needed to assess the long-term sustainability and health impact of the health behavior and anthropometric changes that took place during FitKids360 classes.Due to the short duration of the FitKids360 curriculum, substantial changes in body composition were not expected. Yet, patients who completed the program did experience small, but significant improvements in both BMI and BMI One limitation of this study is the lack of a control/comparison group, which introduces several potential threats to internal validity, including confounding, regression toward the mean, and mortality. In addition, several of the lifestyle behaviors evaluated in this study were self- or parent-reported, which introduces potential social desirability bias. However, the self-report tools used in FitKids360 consist of surveys and questions which have been previously validated. In addition, the results presented here are fairly robust, as they include a relatively large sample from over 30 different FitKids360 interventions. Moreover, the FitKids360 program was designed as an evidence-based, yet feasible and practical pediatric weight management program rather than a randomized clinical trial. To this end, the data presented here provide a “real-world” indication of short-term outcomes after participating in the FitKids360 program.The FitKids360 team is currently working to develop a standard follow-up procedure to allow long-term tracking of patients. We have initiated the process by sending a report to the patients' physicians asking them to continue long-term follow-up. Based on the expert recommendations , monthlyIn summary, we have described the program design and intervention and measurement components of a low-cost stage 2 pediatric obesity program called FitKids360, which has been implemented in several locations throughout Michigan and has now expanded outside of the state as well. In addition, we have reported outcomes on over 250 youth who have completed the FitKids360 program, including improvements in physical activity, nutrition, and sedentary behaviors, as well as BMI. We hope that this report will be useful to researchers, public health professionals, and medical professionals seeking to develop similar stage 2 pediatric obesity programs.


BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of β-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.The down-modulation of the β-catenin antagonist Chibby 1 (CBY1) associated with the  BCR-ABL1 rearranged gene encodes a p210-kDa chimeric protein where the ABL tyrosine kinase (TK) is converted into a constitutively activated isoform by fusion with the BCR amino acids 1 to 63 . It. ItBCR-A(p<0.05)  9]. Fur. FurBCR-(p<0.05) . Unlike (p<0.05)  [9]. TheC22orf2 K562 cell line   . βcatenictively) . These rBCR-ABL1 TK by IM is an early event, followed by its retrograde activation by mTOR [CBY1 binding with 14-3-3 proteins σ and ξ is mediated by AKT phosphorylation at serine 20, located within the mode II 14-3-3-binding motif . Notably by mTOR ,24. The C22orf2 K562 cell line was preliminary assayed , an mTOR inhibitor promoting persistent AKT inactivation, was used to investigate the impact of AKT inhibition on CBY1 and βcatenin subcellular localization and expression in relation to their interaction with 14-3-3σ . The abs62 cells . CBY1 re62 cells . The inc-ABL1 TK .C22orf2 K562 (<0.01) and left steady CBY1/14-3-3σ binding in the nuclear compartment (p<0.1) (C22orf2 K562 cells (p<0.01)   by c-Jun N-terminal kinase (JNK) promotes the dissociation of client proteins . The imp (p<0.1) . Further(p<0.01) . These fctively) . These fell line .BCR-ABL1+ cells compared to the HD pool (p<0.01 or less) . Clinical characteristics of CML patients included in our study are illustrated in the or less) . CBY1 reor less) . These rBCR-ABL1 [C22orf2 K562, CBY1 exhibited a significant increment in response to bortezomib (5 nM for 24 h) (p<.001)  or preventing (SP600125) CBY1 release from 14-3-3σ were probed with anti-SUMO antibodies. CBY1 increment following its release from 14-3-3σ in response to IM, BV02 and RAD001 was associated with a significant reduction in the signal intensities with anti-SUMO antibody (p<0.01) (Selective degradation of the great majority of intracellular proteins is executed by the ubiquitin proteasome system (UPS) –31. BortBCR-ABL1 . In C22o(p<.001) . The fin(p<0.01) . Convers(p<0.01) . The finTo confirm that SUMOylation is a component of CBY1 down-modulation in CML, we compared signal intensities of CBY1 IP products from MCF of CML patients and HD probed with anti-SUMO antibody. In all CML patients but one (patient #1) CBY1 reduced expression was associated with a significant increment in its SUMOylation compared to the HD pool (p<0.05 or less) . DensitoBCR-ABL1 TK activity and by CBY1 interaction with 14-3-3σ. 14-3-3 proteins are ubiquitous adapter proteins whose physiological effects on cell cycle, signal transduction, protein trafficking and apoptosis are contingent upon binding with client proteins at a so-called amphipathic groove, which accommodates the mostly phosphorylated interaction motif of their partner proteins [BCR-ABL1 TK signaling pathways to CML phenotype [BCR-ABL1+ cells . The central role of BCR-ABL1 TK activity in CBY1 binding with 14-3-3σ is promoted by reduced CBY1 interaction with the scaffolding protein in response to IM , further supporting the impact of 14-3-3σ binding on CBY1 stability  was associated with CBY1 enhanced interaction with 14-3-3σ and reduced phosphorylation of JNK and 14-3-3σ at residues involved in the complex persistence in response to SP600125 . In C22orf2 K562 cells CBY1 increment in response to drugs  was associated with CBY1 reduced SUMOylation, while CBY1 reduction in response to SP600125 (which let the persistence of CBY1 interaction with 14-3-3σ) was associated with CBY1 upraised SUMOylation , JNK-induced reduction of oxidant-enhanced SUMOylation of ataxin-1, SUMOylation impact on JNK activation under oxidative stress conditions and JNK-dependent SUMOylation of retinoid X receptor α in hepatocellular carcinoma –46. Our  and JNK ,47,48.BCR-ABL1 TK downstream target AKT. The interaction with 14-3-3σ reduces CBY1 stability in BCR-ABL1+ cells through events encompassing UPS and, in particular, protein SUMOylation. Our results open new routes in the search for molecular pathways promoting the proliferative advantage of leukemic hematopoiesis, particularly in the LSC compartment which is resistant to TK inhibitors.Results shown here suggest that CBY1 interaction with 14-3-3σ is promoted by CBY1 post-transcriptional modification by the S1 Fig(PDF)Click here for additional data file.S2 Figst up to 5th h of IM treatment. B-5mC and DNMT1 recruitment at C22orf2 promoter were concurrently reduced in response to IM.A-Both CBY1 transcript isoforms (340 bp and 200 bp) were raised since 1(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


The Jackson Heart Study (JHS) is committed to providing opportunities for expanding the understanding of the epidemiology, diagnosis, prevention, and treatment of cardiovascular diseases. The JHS Graduate Training and Education Center (GTEC) has initiated the Daniel Hale Williams Scholar (DHWS) program where students are afforded the opportunity to interact with epidemiologists and other biomedical scientists to learn to identify, predict, and prevent cardiovascular disease using the Jackson Heart Study data. This study describes the structured programs developed by JHS GTEC seeking to alleviate the shortage of trained professionals in cardiovascular epidemiology by training graduate students while they complete their academic degrees. The DHWS program provides: (1) an enrichment curriculum; (2) a learning community; (3) quarterly seminars; and (4) a Summer Institute. Students attend enrichment activities comprising: (1) Applied Biostatistics; (2) Cardiovascular Disease Epidemiology; (3) Social Epidemiology; (4) Emerging Topics; and (5) Research Writing. Training focuses on developing proficiency in cardiovascular health knowledge. The DHWS program is a unique strategy for incorporating rigorous academic and career-focused training to graduate students and has enabled the acquisition of competencies needed to impact cardiovascular disease management programs. Accornd below . These snd below . Treatmend below . Reductind below  to advanThe JHS began in 2000 as a partnership among three institutions, Jackson State University, University of Mississippi Medical Center, and Tougaloo College. Jackson State University developed and managed the Coordinating Center, University of Mississippi Medical Center managed the Examination Center, and Tougaloo College had the responsibility of operating the Undergraduate Training Center. Three exams have been completed so far; Exam 1 was conducted during 2000–2004, and 5301 participants were recruited; Exam 2 was conducted from 2005–2008; and Exam 3 extended from 2009–2012. Data collection continues through annual follow-up activities. A substantial number of publications is available presenting research conducted by JHS investigators including staff at the three partner institutions, as well as external investigators who operate through various Working Groups and Vanguard Centers. In 2013, the structure of the JHS was changed from three centers to five centers with Jackson State University contracted to operate a new Graduate Training and Education Center (GTEC) and a new Community Outreach Center (CORC), University of Mississippi Medical Center contracted to operate the Coordinating Center and the Field Center, and Tougaloo College contracted to manage the Undergraduate Training and Education Center. The primary responsibility of GTEC is to train graduate students in cardiovascular epidemiology through the DHWS Program. Additional initiatives are needed to address the paucity of African-American public health and biomedical scientists in Mississippi, as well as to increase the number of students who pursue graduate education in the health sciences in Mississippi. In order to improve disease detection, treatment, and prevention in the African-American community that comprises about 80% of the population in Mississippi and are disproportionately affected by CVD, there is a need for an innovative approach to encourage and train African-American scholars to aspire to become health professionals so that they can become involved in doing research, treating patients, and developing prevention strategies for the population at risk where health disparities persist . TraininIn August 2013, Jackson State University was awarded a contract from the National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH) to implement a JHS GTEC. GTEC is designed to implement a robust education training program by applying evidence-based approaches to train graduate students in the social, behavioral, and medical sciences. The JHS has an objective to build capacity among its communities, and one focus is on developing core competencies from which students in the biomedical and public health disciplines can develop the knowledge, skills, abilities, and other professional attributes that are required to perform successful service in cardiovascular disease management.The mission of the JHS GTEC at Jackson State University is to provide opportunities in public health and biomedical research techniques for scholars to improve their capacity in community-based research and practice. To achieve the goals, GTEC initiated the DHWS program to prepare graduate students at the masters’ and doctoral levels utilizing mentoring and training in public health with a focus on the underserved and at- risk populations in Mississippi. The program promotes public health and biomedical science education and provides enrichment opportunities to equip GTEC scholars with the capacity to contribute to the prevention of CVD disease and disability. As a service goal, GTEC’s operations promote the development of technical expertise, advocacy skills, and translational capabilities in the scholars to support the mission of its internal stakeholders  and external stakeholders  .For many minority students who specialize in public health and biomedical sciences, the current university graduate curriculum does not prepare them to immediately assume a position/career in their chosen field upon graduation. Some educators have expressed alarm that after the long process on the way to graduating with a professional degree, students still may not have acquired skills that are comprehensive enough to make them competitive, or enable them to immediately impact the workplace with a high degree of efficiency where they can put into practice all of the theory they have learned in the classroom and the expertise they have acquired. Providing avenues for participation in an enrichment graduate education program is essential and critical for maintaining healthy communities ,13. It iRecently, there has been support for a paradigm shift towards utilization of learning communities to provide accelerated professional development of public health scholars ,15. LearThis study reports on the implementation of the Jackson Heart Study GTEC DHWS program that serves as a singular, comprehensive medium to facilitate the development of the identified core competencies. This study describes the methods of the Jackson Heart Study GTEC for delivering this effective, unique, comprehensive training program to achieve stated goals. We provide a description of how the Jackson Heart Study is organizationally structured for realizing our objectives.The Jackson Heart Study has developed and is conducting a two-year graduate training program for African American graduate and health professional students through GTEC, under the auspices of Jackson State University, Jackson, Mississippi. A key component of GTEC is the installation of the DHWS program that utilizes the learning community (LC) concept to provide expert training to the scholars who are selected to participate. The organizational structure of GTEC comprises a staff serving in a variety of capacities. They are described in the To complete the learning community structure, GTEC enlists the services of four learning community advisors and four instructors. This team makes up the core Daniel Hale Williams Scholar Program learning community (LC). Additional expertise is acquired periodically as needed from expert investigators from around the United States as the program progresses through its phases. The LC Advisors are selected from the faculty/investigators from the two JHS institutions involved in graduate training, JSU and UMMC.The day-to-day management of GTEC and the DHWS program is undertaken by its administrators through the organization of a Working Group/Committee structure. Five working groups have been established with distinct functions to ensure that all of the requirements for the GTEC and the DHWSP are effectively developed, implemented, managed, monitored, and evaluated. The GTEC Working Groups are presented in The overall goals of the GTEC are to: (1) enrich the educational experiences and skills of African American graduate students to prepare them for future careers in academia, industry, and government research settings; (2) enhance the professional skills of graduate students to increase the likelihood of success and completion of graduate school; and (3) increase the interest in, and likelihood of, African American graduate students entering careers in biomedical sciences following degree and program completion. The GTEC is dedicated to achieving these goals by engaging the affiliated faculty, staff, mentors, and institutional leaders. These leaders are a diverse group of multidisciplinary faculty with the depth of experience, dedication, and commitment to the provision of education and research training for graduate students to advance cardiovascular research and improve the quality of health care delivery for underserved and vulnerable populations.With the administrative staff and Working Groups installed, the first objective of GTEC was to establish a graduate-level training program focused on cardiovascular epidemiology research; we achieved that objective by establishing the DHWS program. The DHWS program utilizes an empirically-tested learning model to develop, implement, and evaluate a two-year graduate program in cardiovascular epidemiology, with an emphasis on social epidemiology and health disparities. In developing the DHWS program, we examined the recommendations from the Sullivan Commission as the primary guide to identify, select, implement, evaluate, and maintain the health professional educational training ground and mentorship programs that are critical to improve the racial diversity of the healthcare workforce ,18. GTECThis innovative training program recruits full-time students in good standing who are enrolled in Psychology, Sociology, and Public Health graduate programs at Jackson State University and students enrolled in medicine, nursing, pharmacy, and dentistry schools at the University of Mississippi Medical Center. This program is designed to provide a strong foundation in epidemiology, cardiovascular disease, health disparities, and professional development to increase the likelihood of African American graduate students entering careers in biomedical sciences. All course offerings are held in the Jackson Medical Mall, where the School of Public Health of Jackson State University and the Jackson Heart Study are located.Recruitment of students is done using several media. Announcements are posted on billboards that are strategically located in relation to the targeted campuses . Announcements are posted on the websites of the institutions, as well as on bulletin boards in the departments of the students who are targeted. In addition, announcements are made at all JHS public events, as well as the Universities’ events, the JHS Newsletter, and local news media. The deans, faculty, and staff of the targeted departments also serve as spirited recruiters for the program.All classes/activities in the DHWS program are considered enrichment activities that are offered to supplement the students’ full-time class load in their graduate programs. Students are recruited and selected with the understanding that they must have two years to devote to the program, they must maintain good academic standing required by their institution, and they must meet all the criteria established by their institution in order to maintain their scholarship.The first cohort of Daniel Hale Williams Scholars selected for the 2013–2014 academic year comprised three students, the second cohort for the 2014–2015 academic year comprised four students. The third cohort for the 2015–2016 academic year comprised four students. It is expected that by the end of the current contract period in 2018, GTEC will have recruited and trained a total of 17 scholars. Scientists from public health departments and other institutions interact with students and provide training during the orientation and the one week training camp. In addition, the scholars participate in summer training institutes where they engage in research training activities from nationally-known scientists from around the country. The LC advisors are matched with the students based on their familiarity with the JHS data and their research interests. Throughout the two years of scholars’ eligibility, the LC advisors guide the scholars through the manuscript proposal development process, the research process, and the final manuscript development process. The evaluation component of the program ensures that GTEC is able to monitor progress, address areas of concern, make necessary adjustments, and measure success of the program. The evaluation comprises a student evaluation of the instructors and the LC advisors, the instructors’ and LC advisors’ evaluation of the students’ progress, as well as an evaluation of all of the DHWSP activities. We have developed a tracking system that will track our scholars to document milestones such as graduation rates and professional employment. Our tracking system will measure long-term success of the scholars and the program. Shorter term outcomes that will be measured include the scholars’ completion of research projects, presentations of research at conferences, completion of proposals using JHS data, submission of the proposals to the JHS P and P for approval, completion of a manuscript, and subsequently publication of a manuscript.The JHS Graduate Training and Education Center utilizes four key components of core competencies which serve as the basis of the DHWS program: (1) the enrichment curriculum; (2) learning community; (3) quarterly seminar; and (4) Summer Institute. These activities enable GTEC to provide quality cardiovascular epidemiology training to the graduate scholars enrolled in the program. The DHWS program curriculum was developed to train students to design, analyze, and evaluate epidemiologic and related biomedical research; demonstrate an understanding of and ability to, apply biostatistics and other appropriate quantitative research and evaluation methods; and apply current research methods in cardiovascular disease epidemiology. The first year of the program consists of interactive delivery of courses on the design, analysis, and evaluation of epidemiology research, with an emphasis on cardiovascular disease. During the first year, scholars also attend a Summer Research Training Program. The minimal goals for the scholars for Year 1 include: at least 85% attendance at DHWSP activities, timely submission of DHWS program reports, abstract submission, and presentation on Research Day at the Annual Conference on Eliminating Health Disparities Sponsored by the Center for Excellence in Minority Health and Health Disparities at Jackson State University.During Year 2, the emphasis is on finalizing and submitting a manuscript for publication. The second year is characterized by hands-on opportunities to apply acquired research knowledge using a data set derived from the Jackson Heart Study data. Upon completion of this program, students will receive a certificate of completion. The Quarterly Seminar Series focuses on social epidemiology, examining differential exposures and health outcomes among populations. During this series, seminars also include presentations on identifying social interactions, while addressing experimental social epidemiology. Speakers consist of national and local experts, including those from the Jackson State University (JSU) and the University of Mississippi Medical Center (UMMC).The Research Camp is a one week intensive program, focusing on increasing students’ overall quality and knowledge of research, and improving their oral and written communication skills. It utilizes didactic and interactive approaches. Lectures and exercises address strategies for reading and critiquing the scientific literature; scientific writing; enhancing skills in evaluation and statistics; designing epidemiological studies; and career navigation. Mentoring is a critical element for the advancement and success in academic careers. At GTEC, our scholars are paired with a learning community advisor whose research expertise mirrors their research interest. The learning community advisors guide them through the research process.Scholars participate in an intensive didactic and interactive summer research program focused on social epidemiology, cardiovascular disease, cardiovascular epidemiology, health disparities/health equity research, and the JHS. Researchers with substantial expertise in these areas deliver the curriculum via in-person, didactic sessions, as well as participate in career mentoring sessions. Faculty from other institutions travel to Jackson to provide training for the scholars during the orientation and the one week long Research Camp, as well as during the Quarterly Seminars. The scholars also travel to participate in Summer Institutes and conferences. Summer training is conducted in collaboration with institutions around the nation, including the University of Michigan, Ann Arbor and Drexel University in Pennsylvania.The DHWS program began in 2014, and has enrolled three cohorts of scholars to date for a total of 11 students. We anticipate training 17 students by the end of the school year in 2018. The scholars’ success is measured by their completion of the academic training activities, successful interaction with the learning community advisor, completion of 85% of prescribed DHWS program activities, See  as well We began operation of the DHWS program with the expectation that a well-organized learning community is associated with a strong graduate student collective efficacy, hence making our scholars competent for entry in the health care profession. This design is an emerging trend in higher education and professional development, and has been touted as a strategy to increase student success and retention ,20. For The main strength of the GTEC DHWS program is that the scholars have an opportunity to access data from the largest epidemiological study ever conducted in an African American cohort. The scholars also have the opportunity to interact with Jackson Heart Study investigators on site who are intimately familiar with the data and can assist them with their research development. Through the GTEC DHWS program, they have an opportunity to gain an understanding of emerging risk factors for CVD and the relation of variables that contribute to the prevalence and incidence of those diseases in an African American population. Students also have the opportunity to publish manuscripts and attend and present at major conferences. A limitation of the program is the process for navigating the Jackson Heart Study bureaucratic framework for acquiring data to conduct research. This process is challenging for scholars who have a limited amount of time to complete their assigned tasks, and delays in the approval process for manuscript proposals and acquisition of data could severely limit their ability to meet all of their objectives.The Jackson Heart Study GTEC DHWS program has a rigorous academic and career-focused curriculum, and its core competencies offer a comprehensive approach to identifying, recruiting, and providing training to graduate students as they further develop the competencies needed to impact cardiovascular disease management programs. As mentioned earlier, there is no other institution in the United States that offers a graduate program in cardiovascular epidemiology leading to a certificate of completion. In the State of Mississippi where cardiovascular disease is most rampant, particularly among African Americans, the opportunities provided by the initiation of the DHWS program have the potential to increase the pool of public health and biomedical scientists who can provide services within the Mississippi communities to help alleviate the impact of these diseases and reduce the prevalence of risk factors that eventually lead to the development of CVD. The DHWS program goes beyond the foundational and disciplinary-specific material typically presented in these courses in the regular academic curriculum. The enrichment activities are complementary to the core curriculum and are typically designed to further develop research skill sets. GTEC aims to contribute to building evidence-based approaches to training minority graduate students in the social, behavioral, and medical sciences through research by maintaining a network of partners who can implement robust education training programs uniquely tailored to minority students addressing the historic and current challenges experienced by this group. The entire program is facilitated by the GTEC investigators’ deep familiarity and engagement with the JHS and the existence of state-of-the-art research resources and infrastructure for leading the GTEC. The GTEC has enjoyed strong successes in the two years of operation and the program has resulted in positive student results. The students enter this formal program with a common purpose and become empowered through their social interaction to complete their courses and other activities as a cohort, while, at the same time, pursuing individual and group learning opportunities. As mentioned by Thompson , the schThe GTEC in the School of Public Health at Jackson State University is a very new program. Future plans are to compare milestones, such as graduation rates of the GTEC scholars and the entry of the scholars into professional employment. We have already compiled a list of presentations completed by the scholars from the first three cohorts at conferences, as well as publications that they have completed. Plans are in place to compare the scholars’ scientific productivity, as well.


Recommended screening and brief intervention (SBI) for alcohol use during pregnancy is impeded by high patient loads and limited resources in public health settings. We evaluated the feasibility, acceptability and validity of a new self-administered, single-session, bilingual, computerized Screening and Brief Intervention (SBI) program for alcohol and sugar sweetened beverage (SSB) use in pregnancy.We developed and tested the computerized SBI program at a public health clinic with 290 pregnant women. Feasibility, acceptability, and validity measures were included in the program which had several modules, including those on demographics, health and beverage use. Time to complete the program and user experience items were used to determine program feasibility and acceptability. Validity analyses compared proportions of prenatal alcohol use identified by the program versus in-person screening by clinic staff.Most program users  completed the entire program; 91% (n = 263) completed the key screening and brief intervention modules. Most users also completed the program in ten to fifteen minutes. Program users reported that the program was easy to use (97%), they learned something new (88%), and that they would share what they learned with others (83%) and with their doctors or clinic staff (76%). Program acceptability did not differ by age, education, or type of beverage intervention received. The program identified alcohol use in pregnancy among 21% of users, a higher rate than the 13% (p < .01) found via screening by clinic staff.Computerized Screening and Brief Intervention for alcohol and SSB use in public health clinics is feasible and acceptable to English and Spanish speaking pregnant women and can efficiently identify prenatal alcohol use. Competing priorities of high patient loads, multiple patient health issues, and resource limitations in public health clinics impede implementation of evidence-based screening and brief interventions (SBI) for alcohol use during pregnancy . ComputeIn 2000, Kaskutas and colleagues developed Early Start Plus (ESP), a novel computerized intervention for pregnant women. The ESP intervention incorporated detailed drink size assessment and feedback into Early Start (ES), a prevention program for pregnant women enrolled in a health maintenance organization ,7. A uniRandomized control trial data showed that ES and ESP recipients had better neonatal and maternal outcomes, i.e., higher birth weight and lower rates of preterm labor respectively, than controls . HoweverMore recently, Tenkku and colleagues  developeThus, existing computerized SBI programs have limited appeal as they require clinical staff ,7, multiAnother innovation of our new computerized SBI is the inclusion of sugar sweetened beverages (SSB). While reducing prenatal alcohol use has been a longstanding priority , SSB conThe objective of our study was to examine the feasibility, acceptability and validity of our self-administered, single-session, bilingual (Spanish and English) computerized SBI for alcohol and SSB use during pregnancy as implemented in a public health clinic.th grade readability of all program materials. The computerized SBI program was pilot tested at a local public health clinic. Implementation was preceded by 4 weeks of intensive beta testing, specifically testing for software programming errors or failures, by the first author and 2 research assistants, and administration to 30 randomly selected women in the clinic.Our study involved a test implementation of the computerized SBI program and therefore used an observational design. Expert review was used to ensure relevance and 5All study protocol and procedures were reviewed and approved by the Institutional Review Boards of the Public Health Institute (PHI) and the Contra Costa Medical Research Center.The SBI program was administered using an ATM-like kiosk with a 19-inch touch-screen monitor. The kiosk was placed in the waiting area of the clinic in order to increase its visibility and facilitate use. All pregnant women attending the clinic for the first time were provided a brightly coloured handout about the study with the clinic paperwork. Posters about the study were also posted conspicuously on the clinic walls. No other active recruitment was used for the study, i.e., no research personnel were present at the clinic to recruit participants and no incentives were provided for participation. Participants provided consent electronically by selecting “I agree” on the pertinent screen of the computerized program. Consent screens included contact information for the principal investigator (the first author) and the PHI IRB chair. Printed copies of this contact information were also visibly placed near the kiosk and available with clinic staff.Participants were recruited at a Women Infant and Children’s (WIC) clinic in northern California from June 2012 through January 2013. The federally funded WIC program serves a large portion of childbearing age women across the United States, mostly low-income and of minority status  SBI. The screening assessed alcohol and sugar sweetened beverages (SSB) consumption and provided a brief intervention for women reporting consumption of these beverages  drinking.Women reporting alcohol use in the past month or prior to pregnancy were asked about frequency of alcohol use, types of alcoholic beverages consumed, and the two most frequently consumed beverages in each relevant time period. These details about drinking were assessed in two separate modules based on the time frame of most recent use. Thus, women who reported recent (past month) alcohol use were asked about their drinking in the past 30 days. Those not reporting current use and reporting drinking in the 12 months prior to their pregnancy were asked about their drinking in the year prior to their pregnancy. Women who reported drinking in a time frame that coincided with their pregnancy but denied both past month use and use in the year prior to their pregnancy (n = 4) were precluded from the detailed alcohol use assessment due to additional costs to program separate modules and the limited resources we had for software programming.This module examined user’s beverage-specific drink (pour) sizes for the two most frequently consumed beverages by determining: a) the drink container or vessel that the participant usually drank from, and b) how much they would pour into their chosen vessel. Actual drinking vessels (9 glasses) were displayed in a box placed above the kiosk. If one of the 9 glasses was not the chosen drink container, photographs of other drink containers  were presented on the computer screens to assess beverage-specific drink sizes. Women indicated their pour size in the container they selected by using letter markings on their chosen vessel. More details on the drink size assessment are available elsewhere .This module comprised two key components: feedback on drink size and a personalized plan for reducing consumption. The module began with individualized feedback on the woman’s drink sizes, reviewed discrepancies between her drink size and the standard size for each beverage, including that for usual and maximum consumption, and highlighted where pertinent, that she may be drinking more than she thinks she does. The module ended with a personalized plan for reducing consumption which included goal setting, an analysis of high risk situations for drinking alcohol, and suggested coping strategies.Women who did not select complete abstinence (stop drinking) as a goal were encouraged to reduce consumption and to set a goal for reduced drinking. They were then guided through ways of doing so, such as to replace half the drinks per week with non-alcoholic drinks, drink one less each drink each week, or drink one less day each week. For women reporting current (past month) alcohol use, the intervention also provided information on the harmful effects of drinking during pregnancy. For women who did not report current use but use in the 12 months prior to pregnancy, information and feedback focused on reducing harms related to drinking alcohol in the future, should they choose to resume drinking.SSB screening included items on consumption of four types of sugar sweetened beverages in the past 30 days, including energy drinks, aqua fresca or horchata, regular soda or pop, and sweetened fruit drinks. Sugar-free or diet drinks were not included. Items used were from prior national surveys ,20 with These modules were completed by all women who screened positive for SSB use by reporting consumption of any of the four SSB types in the past month. The modules were identical in format to that for alcoholic beverages described previously and focused on the two most frequently consumed sugar sweetened beverages.All women signing in for their first appointment at the clinic were provided with a handout describing the study along with other paperwork routinely given to women while checking in for their appointment. Women approached the computer kiosk voluntarily, selected the language they wanted to use and “continue” on the bilingual introductory screen to begin the program. To maximize participation in this new program, no identifying information, such as date of birth, medical record number, or phone number was obtained from program users.This was measured by the time it took each user to complete the program. The program recorded the time, in minutes and seconds, at which the woman touched the “continue” button on the first screen to start the program and the time at which the last screen was reached. For those who did not complete the program, this last screen appeared upon their pressing “stop/discontinue”.Program acceptability was assessed through items on user experience which included “Is this program a good idea for asking women about their drinking?”, “Was the computer program easy to use?”, “Did you learn anything new?”, “Will you talk about what you learned from the computer program with other people?”, “Will you talk with your doctor or clinic staff about anything you learned?”, “Did you feel you had enough privacy to answer the questions in this program honestly?”, and “Was it difficult to understand the questions or program materials?”.To assess validity of the assessment of prenatal alcohol use by the program, we compared overall rates of alcohol use among pregnant women attending the WIC clinic with that based on responses to the alcohol use screening item in the computerized program. In order to determine whether a woman drank at a time when she was pregnant, WIC compares drinking data recorded by nutritionists in terms of calendar month and year with the number of months of pregnancy computed from the estimated delivery date in the medical record. We estimated alcohol use during pregnancy by cross-tabulating the woman’s response to the item on most recent alcohol use  by the number of months of pregnancy she reported in the program’s demographics module.To assess representativeness of our sample, we compared program user demographics with those of WIC clinic attendees during the months of data collection for the present study (June 2012 to January 2013). WIC maintains self-reported data obtained at the first clinic visit for all women enrolled in the program at the end of each month. Monthly data are not mutually exclusive and include several of the same women across months. To estimate demographic distributions for WIC attendees for the 7 months of our data collection, we averaged percentages for demographic groups in the WIC data (numbers per month ranged from 765 to 840) and compared these percentages to those for our sample using estimations of effect sizes to compare proportions .Analyses assessing feasibility and acceptability examined time to complete the program and overall proportions of women endorsing different response options to the user experience items, respectively. Differences in feasibility and acceptability by user demographics and type of beverage  intervention received were also examined using T-tests (time to complete program) and chi-square tests (user-experience). Analyses assessing program validity used chi-square tests to compare the overall proportion of women identified as using alcohol during pregnancy by the computerized SBI program with monthly estimates in WIC data obtained via in-person screening by WIC nutritionists. We used the Statistical Package for Social Sciences (SPSS) version 15 for all analyses.All study protocol and procedures were reviewed and approved by the Institutional Review Boards of the Public Health Institute (IRB# I11-014) and the Contra Costa Medical Research Center .A total of 450 women began the program and completed initial screens up to informed consent. Data from the informed consent screens indicated that 69% 310 of 450) of the women agreed to study participation , roughly a quarter were in their second (n = 77) and third (n = 67) trimester, respectively. Three-quarters of the women (72%) reported they received nutritional counseling at WIC, and 60% were receiving prenatal care outside the health center that the clinic was a part of. Twelve percent of women reported ever being told that they had diabetes. Of these women, 65% reported diabetes only during this pregnancy, 16% reported diabetes in both a prior and the current pregnancy.Small effect sizes .10 or less) obtained indicated that our sample was representative in terms of age, language, marital status, and ethnicity. However, our sample had more women with more than a high school education  and fewer women in their first trimester  were first-time visitors: 215 (82%) reported attending the clinic for the first time. Thus, we estimated that the reach of our SBI program for pregnant women visiting the clinic for the first time was 22% (215/980).Roughly twenty one percent (n = 60) of all women who completed the alcohol screening reported that they last used alcohol during a time period that coincided with their pregnancy . Compared to those who did not drink during pregnancy (see Table A large majority (87%) of women reported SSB use but did not differ in demographics from women who did not use SSB or from the general WIC population of pregnant women . When those completing the relevant assessment and intervention modules but not all program modules were included in program time estimates, 75% of alcohol users took 11 minutes or less, 80% of sweetened beverage users took 16 minutes or less.Table Overall (results not shown in Table), large majorities reported that the program was easy to use (97%), they learned something new (88%), thought the program was a good idea , would share what they learned from the program with others (83%) and with their doctors or clinic staff (76%), and did not have any difficulty with the program materials (87%). Women receiving the SSB versus alcohol intervention were more likely to say that they learned something new and that they would talk to the doctor or WIC staff about what they learned. Acceptability did not differ between women reporting drinking only sips of alcohol and other alcohol users.Because our sample was comprised of a significant proportion of women with more than a high school education, we also examined if education was associated with program acceptability  or “not sure” (n = 8) to “Did you feel you had enough privacy to answer the questions honestly?’. Therefore we also examined program acceptability by reporting of privacy (results not shown in tables). These 32 women were less likely to report that they learned something new or share program information with others or with their doctor, more likely to prefer a person asking about their drinking, and completed the program in less time than those who reported adequate privacy.2 = 6.78, p < .01) and the average rate of 13.3%  in the WIC data for the same time period.The prenatal alcohol use rate obtained by our program was 20.6% (60 of 290). This rate was significantly higher than the highest rate of 15.2%  program for alcohol and sugar sweetened beverage (SSB) consumption among pregnant women in a public health clinic. Almost all women completed the program in 15 minutes or less and none required staff assistance. Regardless of the type and language of intervention received, women also responded very positively to the program and found the program information novel and worth sharing with others, including their doctor and health care professionals. Finally, our study documented higher identification rates for prenatal alcohol use than that obtained by routine clinic screening. Although the higher rates of alcohol use may be due to our sample having women with more education and educated women being more likely to drink alcohol, we note that the clinic data was missing information on education for roughly one in four women. Thus any biases in our findings due to our specific sample may be artificially inflated due to missing data in the clinic records.While the 67% study participation rate may appear relatively low, it is at best a conservative estimate of future use of computerized SBI given the lack of active recruitment and incentives in our study. Since the kiosk was a stand-alone, un-monitored tool, ineligible users  may have declined participation and, thus, inflated the study refusal rate. We estimated the reach of our program for pregnant women visiting the clinic for the first time to be 22%. It is noteworthy that 18% of our program users were non first time visitors who did not receive information about the kiosk program when they checked in at the clinic. This suggests that these women found the kiosk and program appealing and that future studies can enhance program reach by including all pregnant women attending the clinic and not just first time visitors. Future use of the program could also be increased by its integration into routine care, e.g., by having reception staff point out the kiosk to pregnant women checking in to the clinic, explain its short duration and ease of use, and clarify that women can complete the program while waiting or after meeting with clinic staff.Our SBI includes several improvements over other SBIs. First, it extends SBI to another health risk behavior for weight gain and diabetes related obstetric complications , SSB conDrinking prior to pregnancy recognition is often missed when past month use alone is assessed ,23. Our A key innovation of our computerized SBI is that it is bilingual. As the fastest growing ethnic group in the United States  with theEmbarrassment and fear of judgment can impede identification of prenatal alcohol use via face-to-face screening . WIC staDue to lack of data in WIC records on the frequency and quantity of alcohol consumption, particularly at the level of detail as in our program, we could not assess validity of alcohol use patterns. However, we expect that women will be more willing to more honestly report details about their alcohol use in a computer program than to a staff person. We also did not specifically assess validity for SSB use as it is not currently assessed by clinic staff but computerized programs may also minimize barriers to disclosure of SSB use. Overall, though, the higher identification rates obtained by our program for alcohol use support the use of computerized SBI in prenatal clinics.Because the computerized SBI was completely anonymous, we lacked identifying information needed to compare alcohol use in SBI versus WIC staff reports at an individual level. Future studies should link data obtained by computer versus clinic staff to assess program validity.Placing the computer kiosk in the clinic waiting area maximized its visibility and facilitated use by women but also reduced privacy. Women reporting lack of adequate privacy completed the program more quickly, and were less satisfied with it than those reporting adequate privacy. Use of a privacy screen for the monitor can interfere with use of the touch screen. However, future implementation can increase privacy with the use of a barrier, e.g., a wall, enclosure, or booth.The self-selected nature of our program users and possible biases due to their higher education may also limit generalizability of our study findings. Finally, our study demonstrated feasibility and acceptability but not efficacy of computerized SBI because of lack of follow up outcome data. Examining program efficacy in reducing alcohol and SSB consumption and improving maternal and child outcomes with controlled studies is a key area of focus for our planned research.Our findings suggest that computerized SBI provides a low resource, time-efficient, acceptable and valid tool to address gaps in healthcare for women aimed at preventing poor birth and fetal outcomes. Low-income women are less likely to receive appropriate counseling regarding alcohol use during pregnancy ,31 and t


RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation  Post-transcriptional control of RNA is important in health and disease. Here, the authors show that the RNA-binding protein Quaking guides pre-mRNA splicing and transcript abundance during monocyte to macrophage differentiation, and that Quaking depletion impairs pro-atherogenic foam cell formation. Monocytes serve as danger sensors within the circulation. The activation of blood-borne monocytes by inflammatory stimuli triggers their adhesion and homing to sites of tissue injury, where they differentiate into macrophages and collectively aid in the resolution of damage156Dynamic changes in gene expression are associated with monocyte to macrophage differentiation, where PU.1 ref. , Signal 111516In contrast to miRNAs, RBPs mediate both quantitative and qualitative changes to the transcriptome, interacting with pre-mRNAs to influence  splicing, transcript stability, editing, subcellular localization and translational activation or repression18192123262930313234Here we show that the RBP QKI plays a critical role in regulating the conversion of monocytes into macrophages in, for example, atherosclerotic lesions. Our studies pinpoint QKI as a dynamic regulator of pre-mRNA splicing and expression profiles that drive monocyte activation, adhesion and differentiation into macrophages, and facilitates their conversion into foam cells.+ macrophages from early and advanced atherosclerotic lesions of human carotid arteries. QKI mRNA was 4.2-fold enriched in macrophages derived from advanced as compared with early atherosclerotic lesions  micewt) littermate controls (LM) into atherogenic LDLR−/− mice. Although qk knockout mice die as embryos, the qkv mouse harbours a spontaneous ∼1 Mb deletion in the QK promoter region that leads to reduced levels of QKI mRNA and proteinqkv BM-derived monocytes to macrophages showed subtly reduced QKI-5 mRNA and protein levels, and almost a complete ablation of QKI-6 and -7 protein  from QKI viable , intermediate (CD14++/CD16+) and non-classical (CD14+/CD16+) monocytes derived from human peripheral blood (PB)Having identified high QKI expression in macrophages in atherosclerotic lesions, we first explored whether QKI mRNA expression levels differ in macrophage precursors, namely classical  and M-CSF, to stimulate their conversion to pro-inflammatory and anti-inflammatory macrophages, respectively . We obseQKI+/−) and her sister control (Sib-QKI+/+)QKI alleles specifically reduces QKI expression by 50% in both QKI mRNAQKI+/+; To further assess the role of QKI in monocyte and macrophage biology, we undertook an in-depth analysis of a unique, QKI haploinsufficient individual  analysis. Although monocyte subset ratios were not different , the expb-QKI+/+ . As CSF1QKI+/− and Sib-QKI+/+ monocytes from venous blood and treated the cells for 7 days in the presence of either GM-CSF or M-CSF. Similar to the results in QKI+/+ monocytes possessed the capacity to adopt the characteristic pro-inflammatory macrophage morphology, whereas monocytes from Pat-QKI+/− generally retained a monocytic morphology . In contrast, the expression levels of several key pro- and anti-inflammatory markers indicated an anti-atherogenic shift in Pat-QKI+/− macrophages . At the genome-wide level, QKI haploinsufficiency altered the abundance of 2,433 and 1,306 mRNA species in monocytes and macrophages  against the transcript Log2FC (x axis: Pat-QKI+/−/Sib-QKI+/+) and stratified for either putative QKI targets (with QRE) or non-targets (no QRE). In PB monocytes, a reduction in QKI was associated with significantly lowered target mRNA expression  relative to background exons, as well as an increase in ACUAA motif frequency downstream of exons with increased skipping (QKI activated) relative to background. This suggested that similar to C2C12 myoblastsGiven previous reports that QKI is involved in splicing of pre-mRNAs23394041 changes . This ann events . PreviouADD3), alternative 5′-splice sites (PARP2), alternative 3′-splice sites (M6PR) and intron retention , thereby expanding the detection of veritable QKI-regulated events beyond cassette exons (splice event ‘se' location defined by brackets). Importantly, strong correlations were observed between QKI expression levels and the magnitude of the splicing event, be it between the patient and sibling control, or between monocytes and macrophages , using primers in flanking exons . Collect+ cells; ADD3 and FcγR-IIb (FCGR2B) (n=3 donors). It should be noted that the inability to remarkably reduce the expression of the nuclear QKI isoform, namely QKI-5, could be responsible for the discrepancy between the striking shift in splicing observed in the QKI haploinsufficinet patient as compared with the GapmeR-mediated abrogation of QKI expression. Taken together, these studies clearly pinpoint QKI as a regulator of pre-mRNA splicing during monocyte-to-macrophage differentiation and implicate QKI gene dosage as a determinant of splicing event magnitude.To extend results obtained with the QKI haploinsufficient patient, we abrogated QKI expression in naive primary human monocytes harvested from freshly drawn venous blood of healthy controls. We designed GapmeR antisense oligonucleotides that either targeted QKI for degradation (QKI-Gap), or are scrambled as a control (Scr-Gap), coupled with a 5′-FAM label to track their cellular uptake. The QKI-Gap and Scr-Gap compounds were administered to the freshly isolated monocytes, concomitant with GM-CSF for 96 h, to drive the differentiation to pro-inflammatory macrophages. In contrast to our attempts to reduce QKI mRNA levels using other well-established approaches, we observed virtually no signs of cytotoxicity or apoptosis following GapmeR treatment. Furthermore, the treatment did not hamper the capacity of monocytes to differentiate into macrophages  top, whiQKI+/− versus Sib-QKI+/+ monocytes, sh-QKI THP-1 ‘monocytes' displayed an inability to adopt the ‘macrophage' morphology following stimulation with PMA as compared with sh-Cont THP-1 ‘monocytes' . Similar to QKI haploinsufficient macrophages, several markers in sh-QKI THP-1 ‘macrophages' displayed an anti-atherogenic phenotypic shift  as compared with sh-Cont THP-1 ‘macrophages' -induced transition of THP-1 ‘monocytes' to ‘macrophages' was associated with the following: (1) significantly increased expression of all QKI mRNA transcripts ; (2) barnocytes'  arrows. nocytes' . Next, aophages' .At the genome-wide level, the reduction of QKI significantly altered the abundance of 359 and 573 mRNAs in THP-1 ‘monocytes' and ‘macrophages', respectively  bottom. n=3). Detected splicing events are illustrated in y axis and x axis, respectively) of transcript-specific hybridization probes directed to either the constitutive or alternatively spliced exons are plotted. The separation score, obtained by determining slope differences, indicates the magnitude of the splicing event. Similar to the motif enrichment analyses performed for the RNA-seq of PB monocytes and macrophages, these studies confirmed that exon skipping frequency was significantly correlated with alternative exons that had an ACUAA motif in the upstream intron  enrichment of coordinately regulated pathways during monocyte-to-macrophage differentiation. As shown in Our experimentally determined changes in (pre)-mRNA splicing and expression, as well as bioinformatically predicted changes in biological processes, prompted us to evaluate whether these QKI-induced posttranscriptional modifications could affect monocyte and macrophage function. To test this, we first assessed whether cell survival is affected by a reduction of QKI expression in THP-1 ‘monocytes'. Importantly, the cumulative population doublings and apoptotic rates were not affected by decreased QKI levels . Next, win vitro by seeding sh-QKI or sh-Cont THP-1 ‘monocytes' into transwell migration chambers and assessed their ability to migrate towards the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Indeed, depletion of QKI in monocytes inhibited migration  and PPARG (PPARγ) expression, as well as cholesterol uptake (CD36 and LDLR) and efflux (ABCG1) receptors, as compared with WT monocytes. These effects were diminished on conversion to macrophages , along with significant increases in cholesterol efflux genes (ABCA1 and ABCG1). Taken together, these studies suggested that changes in QKI expression could have an impact on the net balance of genes that control lipid metabolism and homeostasis.Having identified that changes in QKI expression levels had an impact on lipid-associated gene expression, we investigated whether lipid loading affected QKI expression levels. Indeed, treatment with either acetylated low-density lipoprotein (acLDL) or β-very low-density lipoprotein (β-VLDL) led to significant increases in QKI-5 mRNA levels, while QKI-6 and QKI-7 levels also increased, albeit not significantly . In contQKI+/− macrophages we observed significantly less lipid staining after β-VLDL treatment , FCGR2B and VLDLR are unknown. However, given that phosphorylation of the γ-adducin C terminus triggers dissociation from spectrin and cortical actin lossFCGR2B could potentially have an impact on the inhibitory role of this protein in monocyte- and macrophage-mediated phagocytosis at sites of vascular injuryOur findings also suggest that the abundant expression of QKI mRNAs in naive monocytes could serve to prime these cells with the capacity to rapidly respond to pro-inflammatory triggers at sites of injury. Although we did not assess the translational kinetics of QKI mRNA into protein, our investigation of monocyte activation, adhesion and differentiation strongly suggests that the determination of pre-mRNA fate by QKI critically has an impact on the capacity of the monocyte to aid in response to vascular injury. To date, the genome-wide  splicing patterns during monocyte-to-macrophage differentiation had not been described. However, the consequences of many splicing events herein described, such as γ-adducin . Scoring of plaque stage, based on characteristics such as thin cap fibroatheroma (vulnerable or ruptured plaques), vascularization, IPH and/or thrombi/fibrin deposits, were scored by a trained pathologist.−1, clone N147/6; UC Davis/NIH NeuroMab Facility, Davis, CA, USA), mouse anti-human QKI-5 ; mouse anti-human QKI-6  or mouse anti-human QKI-7  diluted in Tris-buffered saline containing 1% BSA and 0.1% Tween 20. Subsequently, sections were washed in Tris-buffered saline and incubated with a secondary biotinylated sheep-anti-mouse antibody . Next, the sections were incubated with streptavidin ABC-alkaline phosphatase  and colour was developed using the Vector Red staining kit (Vector Laboratories), followed by haematoxylin counterstaining. No primary antibody was used for the negative control. QKI/CD68 co-localization immunostaining was achieved using CD68  and pan-QKI antibodies, with CD68 and QKI being counterstained with Vector Blue and Vector Red, respectively. For quantification, slides were analysed in a blinded manner using a Leica DM3000 light microscope (Leica Microsystems) coupled to computerized morphometry (Leica Qwin 3.5.1).Paraffin tissue sections from human carotid arteries were deparaffinized and rehydrated. After pre-treatment with TE buffer (pH 9 for QKI-5 and QKI-7) or citrate buffer (pH 6 for QKI 6) for antigen retrieval, sections were incubated overnight at 4 °C with primary mouse-anti-human pan-QKI . Drinking water was infused with antibiotics (83 mg l−1 ciprofloxacin and 67 mg l−1 polymyxin B sulfate) and 6.5 g l−1 sugar and was provided ad libitum. BM transplantation studies were performed as previously described with minor modifications−1, 200 kV, 4 mA) total body irradiation, using an Andrex Smart 225 Röntgen source  with a 6-mm aluminium filter, 1 day before transplantation. After 24 h, BM cell suspensions were prepared by flushing the femurs of ∼12-week-old qkv mice or age-matched LM controls  with PBS, after which 5 × 106 cells were injected into the tail vein of recipient mice. After 8 weeks of recovery, the mice were placed on a Western-type diet, containing 0.25% cholesterol and 15% cacao butter  for 8 weeks (n=12 per group). Immunohistochemical analysis and quantification of the aortic root using anti-Monocyte and Macrophage-2 (MOMA2) antibody (Sigma-Aldrich) for MOMA2-positive cell area , VSMC content (smooth muscle α-actin-positive cells) and collagen (picosirius red staining) was performed in a blinded manner. Haematologic chimerism of the transplanted LDLR−/− mice was validated using the following primers: qk forward primer 5′-TGTGACTTGGGGACTGTCAA-3′; qk reverse primer 5′-AAAGGGAAAATTTAGCAACAA-3′.Male WT and qkv monocytes were isolated using CD115+ antibody coupled to magnetic beads  and differentiated for 7 days to macrophages using mouse recombinant M-CSF  in RPMI 1640 medium  containing 10% FCS  and 0.01 μg ml−1 glutamine, 50 units per ml penicillin and 50 μg ml−1 streptomycin.BM-derived −1 puromycin (Sigma-Aldrich) for 72 h.Human THP-1 ‘monocytes'  were transduced with lentiviral particles encoding either sh-QKI or sh-Cont . Stable transductants were selected using 3 μg mlA single-stranded RNA–DNA hybrid antisense oligonucleotide (GapmeR) was designed to target exon 2 of QKI , along with a scrambled GapmeR control. Both the GapmeR and scrambled control were 22 nucleotides in length and consisted of RNA  and DNA  in a 6-10-6 manner, with a phosphorothioate backbone (*) and a 6-FAM label on the 5′-end. GapmeR sequences were as follows:5′-A*C*A*U*G*U*dC*dT*dT*dT*dC*dC*dG*dT*dA*dC*U*C*U*G*C*U-3′ for the QKI-targeting GapmeR and 5′-U*G*C*C*U*C*dT*dC*dT*dC*dG*dT*dA*dC*dC *dG*U*A*U*U*U*A-3′ for the scrambled control.−1 CD14-FITC and 1 μg ml−1 CD16-Pc5  for 30 min at 4 °C and FACS sorted using a FACSCalibur . RNA was isolated from the subpopulations using Trizol reagent .Human monocytes were derived from healthy donor buffy coats  following Ficoll density gradient centrifugation and isolated from the PB mononuclear cell fraction using a negative selection cocktail to isolate unlabelled monocytes . Purified monocytes were subsequently incubated with 1 μg ml−1 glutamine at 37 °C and 5% CO2. Differentiation of primary CD14+ monocytes to pro-inflammatory macrophages was achieved by stimulating with 5 ng ml−1 GM-CSF (Thermo Fisher Scientific) or 5 ng ml−1 M-CSF (Miltenyi Biotech). Human THP-1 ‘monocytes' were cultured in RPMI media supplemented with 10% FCS and 0.01 μg ml−1 glutamine at 37 °C and 5% CO2, with differentiation into macrophages being induced by treating with 100 nM PMA+/+ and Pat-QKI−+/ macrophages for 24 h with either 25 μg ml−1 β-VLDL or 50 μg ml−1 acLDL, or 10 μg ml−1 oxLDL, after which the cells were fixed with 10% Formafix, and Oil-Red-O stained and haematoxylin counterstained. Oil-Red-O area per field of view was divided by the number of cells using ImageJ software.Human monocytes were isolated from buffy coats of healthy donors with an antibody to CD14, conjugated to magnetic beads to allow for MACS sorting (Miltenyi Biotech). Cells were cultured in RPMI media supplemented with 10% FCS and 0.01 μg ml−1 HEPES, 132 mmol l−1 NaCl, 6 mmol l− KCl, 1 mmol l−1 MgSO4, 1.2 mmol l−1 KH2PO4, 5 mmol l−1 glucose, 1.0 mmol l−1 CaCl2 and 0.5% BSA) for 2 min. Sh-Cont and sh-QKI THP-1 were resuspended in flow buffer at a concentration of 4 × 106 ml−1, after which the cells were perfused over the substrate for 5 min at 1 dyne cm−2. Cellular adhesion was tracked visually using a Leica DMI5000 microscope. The flow rate was subsequently increased to 2 dynes cm2 for 2 min, followed by the visual assessment of firm adhesion of perfused monocytes for a duration of 3 min, after which photomicrographs of ten random fields of view in the fluidic chamber were taken and quantified.Glass coverslips were coated with type I collagen, after which the system was perfused with platelet-rich plasma for 10 min. Next, the system was flushed with flow buffer  or 10 ng ml−1 macrophage chemoattractant protein 1  for human primary monocytes were performed as previously described5 cells) and incubated overnight at 37 °C. The following day, cell migration (adherent cells) was quantified by manual counting or ImageJ.Transwell cellular migration studies of sh-Cont and sh-QkI THP-1 ‘monocytes' towards 1 nM RNA was isolated from unstimulated sh-Cont and sh-QkI THP-1 ‘monocytes' (day 0), and from sh-Cont and sh-QkI THP-1 cells stimulated with 100 nM PMA for 3 days using Trizol (Thermo Fisher Scientific) and the RNeasy Mini Kit  according to the manufacturer's instructions. RNA quantity and quality was measured using a NanoDrop spectrophotometer  and an Agilent 2100 bioanalyser . Samples meeting a RNA integrity number critieria of >8 were used for further analysis.P-value that a particular probe's intensity arose from the background of all probes. Probes were stratified for GC content . For each probe set, the median P-value of the set of individual probes in the probe set was used as the P-value for that probe set. Before assessing for alternatively spliced transcripts from a particular locus, we first determined whether the gene was expressed. Next, if the expression criteria was met, we determined whether RNA containing two or more alternative splice junctions was detectable using the isoform-specific probes. In situations where the probe sets for two or more alternative isoforms were ‘present' in any sample of the data, an alternative splicing event was scored. For these events, the junction probe sets that hybridized to individual isoforms were identified and the probes they contained were used for the Kruskal–Wallis test. Next, we normalized individual probe intensities and grouped the replicate values. Subsequently, using R software, the kruskal.test function was used to test the hypothesis that the probe intensities come from identical populations. If the resulting P-value was small enough, the null hypothesis was rejected and the alternative hypothesis that the probes were differentially expressed was accepted. To determine an appropriate value for the 0.01 significance level, 12,740 Kruskal–Wallis tests on randomly selected probe sets were performed, yielding an α-value that associated with the 1% quantile of randomly selected probes (1.975486 × 10−3). To account for multiple testing, a Bonferroni-corrected α-value of 1.975486 × 10−3/1.2740 × 104=1.550617 × 10−7 was used as a P-value cutoff for significance. The Sepscore is log2 (Include/Skip ratio) of the experimental sample over the reference sample. Exon inclusion generates a positive Sepscore, whereas exon exclusion generates a negative Sepscore.Microarray data is deposited in GEO under the accession number GSE74887. Targets were prepared from three replicate cultures for each sample. Complementary DNA synthesis and amplification was performed using the WT Expression Kit . Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit . Labelled target was hybridized to the HJAY Chip (Affymetrix 540091). Chips were washed and scanned using the Fluidics Station 450 and Affymetrix Gene ChIP Scanner 3000 7G (Affymetrix). Data were analysed as previously describedP-value that the motif frequency at each point within the peak is greater than background. As the points were not independent, we estimated a q-value for each peak by finding the most significant P-value for any point in the peak and applying Bonferroni correction for the number of points within the peak. A second Bonferroni correction controlled for the number of possible positions at which a peak might occur, yielding the final q-values. To explain further; the effect of QKI depletion on every assessed exon was calculated from the RNA-seq or the splicing-sensitive microarray: that is, whether a reduction in QKI causes inclusion or exclusion from the final mature mRNA transcript.Counts of all 5-mers (ACUAA) in the selected region of an exon set are compared with their counts from a background set of sequences using Fisher's exact test, with multiple testing correction. For motif mapping, we plotted the frequency of specific motifs in 50-nucleotide windows slid along the intron sequences upstream and downstream of each selected exon set with 5-nucleotide sampling intervals. At each interval, we computed the average number of motifs in exons activated or repressed in shRNA-treated cells, and background exons in the same experiment with no splicing change. For the background set, we empirically estimated the 95% confidence interval of motif frequency (error bars). For peaks of consecutive points outside the 95% interval, we applied the Mann–Whitney–Wilcoxon test to estimate a Next, the intronic regions around differentially spliced exons were analysed for the presence of ACUAA motifs (5-mers). By doing this for every alternatively spliced exon, we could detect an enrichment of ACUAA motifs in the upstream introns of the exons that were included in a ‘QKI-deficient' cell: QKI is not binding upstream; thus, the exon is included, as measured by RNA-seq or splicing-sensitive microarray.In contrast, we could detect an enrichment of ACUAA motifs in the downstream introns of the exons that are included on QKI reduction. QKI binding in the downstream intron would normally give inclusion, but now QKI cannot bind downstream; thus, exclusion is now favoured as assessed by the RNA-seq or splicing-sensitive microarray.RNA-seq data are deposited in GEO under the accession number GSE74979. For each sample, the non-ribosomal fraction of 3 μg of total RNA was isolated using a Ribo-Zero rRNA removal Kit . Ribo-Zero-treated RNAs were used to generate barcoded cDNA libraries using the TruSeq RNA Sample Preparation kit (Illumina), with the following additions. Size selections were performed before and after cDNA amplification on an E-gel Safe Imager (Invitrogen) using 2% E-gel SizeSelect gels (Invitrogen). The cDNA fraction of 300 bp in size (including adapters) was isolated and purified. Indexed libraries were pooled and sequenced (paired-end 50 or 100 bp reads) on the Illumina HiSEQ platform to a depth of 41–70 million reads per sample (QB3 Vincent J. Coates Genomics Sequencing Laboratory). After removal of PCR duplicates and repeats, there were 21–26 million uniquely mapping paired-end reads (37–61%).All samples were mapped using Tophat2 refs , 64 withhttps://github.com/STAR-Fusion/. Non-splice junction reads from the macrophage samples that mapped from the QKI gene on chr6 to any location on chr5 were inspected and several were found, which mapped from QKI intron 4 to a site that is strongly transcribed from chr5 in the patient but not at all in her sibling.For mapping of reads that are chimeric to the reference genomeq-value cutoff of q≤0.05.Human mature mRNA sequences were downloaded from UCSC RefSeq database (hg19). Computational screening was performed for the QRE motif, UACUAAY N1-20 UAAY, and counts calculated for the longest annotated transcript. After cross-referencing these transcripts with either the transcripts annotated on the microarray or the RNA-seq, we annotated the number or QREs in the https://github.com/wyleung/rnaveer).Mixture of isoforms (MISO) analysis was used to assess, quantify and visualize alternative transcripts based on RNA-seq data. Sequences obtained from RNA-seq were aligned using TopHat2 to the human genome v19 transcriptome . Using the alignment files (BAM files), MISO analyses was performed on our RNA-seq paired-end sequencing data to identify alternative splicing events, as previously describedPolyacrylamide gel electrophoresis was used to resolve proteins from cellular lysates harvested in RIPA buffer. Protein determinations (BCA) were performed to ensure equal loading of protein on a per-sample basis. QKI-5, -6 and -7 were detected using primary mouse monoclonal antibodies that target pan-QKI , QKI-5 , QKI-6  or QKI-7 , or polyclonal antibodies targeting QKI-5 , QKI-6  and QKI-7 . For loading references, rabbit polyclonal antibodies were used to detect β-actin or Histone H3 . All gels were run and blotted with Bio-Rad TGX pre-cast gels and blotted on nitrocellulose 0.2 μm using the Bio-Rad TurboBlot system (Bio-Rad Laboratories). Full blots are shown in RNA was harvested from monocytes and macrophages using Trizol reagent (Thermo Fisher Scientific). Standard mouse and human cDNA was made using oligo-dT primers (Invitrogen), whereas cDNA for alternative splicing studies was synthesized using random primers (Invitrogen). Primer sets designed for specific pre-mRNA amplification by quantitative RT–PCR analysis are provided in N defined the number of biological replicates. All in vitro and in vivo results were analysed using GraphPad software with either a Student's t-test or analysis of variance (with a Bonferonni post test being used). All results are expressed as mean±s.e.m. Differences in P-values<0.05 or <0.01 were considered significant and indicated as follows: *P<0.05 or **P<0.01, respectively.For all experiments, Informed consent was obtained for all patient-derived samples. Approval for these studies was provided by the relevant medical ethics committees, namely Maastricht University Medical Center, The Netherlands (Professor Dr E.A.L. Biessen), for immunohistochemical studies, and Leuven University Hospital, Belgium (Professor Dr H. Van Esch), for the QKI haploinsufficient and control materials. All mouse experiments were approved by the regulatory authorities of the Leiden University and were in compliance with the Dutch Government Guidelines.Accession code: RNA sequencing and microarray data were deposited in NCBI's Gene Expression Omnibus (GEO) under accession codes GSE74979 (for RNA-seq data) and GSE74887 (for the splicing-sensitive microarray data).How to cite this article: de Bruin, R. G. et al. Quaking promotes monocyte differentiation into pro-atherogenic macrophages by controlling pre-mRNA splicing and gene expression. Nat. Commun. 7:10846 doi: 10.1038/ncomms10846 (2016).Supplementary Figures 1-10, Supplementary Table 1 and Supplementary Referencein vitro cell perfusion system. Glass coverslips were coated with type I collagen, after which the system was perfused with platelet-rich plasma for 10 minutes, leading to the deposition of effector molecules to which the monocyte can adhere. This movie displays the perfusion of sh-Cont THP-1 ‘monocytes' over this bio-active substrate, leading to their attachment to the surface. Total cellular perfusion time was 5 minutes with a flow rate of 1 dyne cm-2. The movie is representative of at least three perfusions.Initial adhesion of sh-Cont THP-1 ‘monocytes' in an in vitro cell perfusion system. Glass coverslips were coated with type I collagen, after which the system was perfused with platelet-rich plasma for 10 minutes, leading to the deposition of effector molecules to which the monocyte can adhere. This movie displays the perfusion of sh-QKI THP-1 ‘monocytes' over this bio-active substrate, leading to a reduction in their attachment to the surface as compared to that seen for sh-Cont THP-1 ‘monocytes' in Initial adhesion of sh-QKI THP-1 ‘monocytes' in an Hematologic profile of whole blood harvested from LDLR-/- mice 16 weeks after transplantation with bone marrow from C57Bl6 control (WT littermates) and quaking viable (qkv) mice (8 week recovery and 8 weeks high-fat diet).RNA-seq derived mRNA abundance as CPM after quantile normalization in Sib-QKI+/+ and Pat-QKI+/- PB monocytes and macrophages.RNA-seq profiling of alternative splicing events in Sib-QKI+/+ and Pat-QKI+/- PB monocytes and macrophages.ACUAA motif enrichment analysis based on the splicing-sensitive microarray and RNA-seq data.Microarray profiling of mRNA abundance in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'.Splicing-sensitive microarray analysis of sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages' and RNA motif analysis for alternative splicing events observed in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'.Ingenuity(r) Pathway Analysis (IPA) of THP-1 and PB monocytes and macrophage datasets.