We have utilized plant-based transient expression to produce tobacco mosaic virus (TMV)-based nano-rods of predetermined lengths. This is achieved by expressing RNAs containing the TMV origin of assembly sequence (OAS) and the sequence of the TMV coat protein either on the same RNA molecule or on two separate constructs. We show that the length of the resulting nano-rods is dependent upon the length of the RNA that possesses the OAS element. By expressing a version of the TMV coat protein that incorporates a metal-binding peptide at its C-terminus in the presence of RNA containing the OAS we have been able to produce nano-rods of predetermined length that are coated with cobalt-platinum. These nano-rods have the properties of defined-length nano-wires that make them ideal for many developing bionanotechnological processes. Particles of Tobacco mosaic virus (TMV) consist of a 6.4 kb molecule of single-stranded RNA encapsidated by approximately 2130 copies of the 17.5 kDa coat protein arranged with helical symmetry. TMV particles are hollow cylinders 300 nm in length with external and internal diameters of 18 and 4 nm, respectively. TMV is probably the highest yielding plant virus and thus its particles have attracted considerable interest for use in bio- and nanotechnology. For example, peptides have been fused to exposed locations of the coat protein for multi-epitope display and particles have been both genetically and chemically modified. Given their rigid rod-shaped morphology, a particular potential use of TMV particles is the production of electrically conductive “nanowires” for the incorporation into nanoscale devices , linked to the OAS sequence , . After digestion with AgeI and XhoI this was ligated into similarly digested pEAQ-HT . pEAQ-HT-TMV-CP(CP9)/OAS was constructed in a similar manner (Gene Art),  but with the addition of a sequence encoding the peptide CNAGDHANC∗ . Two oligonucleotides, KS 109 and KS 108 (Table 1) were synthesized to clone via PCR with Phusion DNA polymerase (New England Biolabs), a TMV coat protein possessing C-terminal six histidine residues. pEAQ-HT-TMV-CP/OAS was used as a DNA template for this PCR reaction. The wild-type coat protein sequence in pEAQ-HT-TMV-CP/OAS was then replaced with the modified coat protein sequence via its AgeI and SalI restriction sites  to yield pEAQ-HT-TMV-CPHis/OAS.A DNA molecule comprising of the TMV  pEAQ-HT  to yieldAGDHANC∗  after thHT-TMV-CP/OAS and pEAQ-HT-TMV-CPHis/OAS were removed by digestion with SalI and XhoI . The resulting DNA was self-ligated to create pEAQ-HT-TMV-CP and pEAQ-HT-TMV-CPHis.The OAS of pEAQ-Table 1). The resulting DNA was cloned via the NgoMIV and SalI restriction sites, (underlined) into BspEI and SalI digested pEAQ-HT-TMV-CP/OAS to yield pEAQ-HT-TMV-CP/VP60/OAS. DNA corresponding to TMV nucleotides 5420–5546 inclusive  VP60 coding region DNA was derived from pBinPS2NT  by PCR aP-EMS-11  were utiHT clones were used to transform Agrobacterium tumefaciens LBA4404 and were subsequently used for plant infiltration experiments . Verified pEAQ-eriments . InfiltrA buffer . Equal vAgrobacterium tumefaciens infiltrated leaf tissue was harvested 8 days post infiltration and homogenized in three volumes of extraction buffer . After squeezing through Miracloth , the extracts were clarified by centrifugation at 13,000 × g for 20 min at 4°C. To improve the quality of the resulting nano-rods, the supernatant was incubated with one quarter volume of CHCl3 and mixed at this stage and an additional centrifugation step of 13,000 × g for 20 min at 4°C was performed. To the resulting supernatant was added one quarter volume of a 20% (w/v) PEG 6000, 1 M NaCl solution and the mixture was stirred overnight in a cold room. Protein was sedimented at 13,000 × g for 20 min at 4°C and the pellet was re-suspended in 10 mM sodium phosphate pH 7.0 and further clarified at 27,000 × g for 20 min at 4°C. Nano-rods in the resulting supernatant were recovered by centrifugation at 118,700 × g for 3 h at 4°C, re-suspended in 10 mM sodium phosphate pH 7.0 and were subjected to centrifugation for 24 h at 209,627 × g (10°C) in a CsCl gradient formed of equal volumes of 42, 49, 57, and 65% (w/v) CsCl in 10 mM sodium phosphate pH 7.0. After centrifugation, the gradients were illuminated with white light from directly above and photographed. Nano-rod-containing bands were removed from the CsCl gradient by side puncture with a syringe and the material was dialysed against H2O overnight in a cold room.Protein samples were denatured in NuPAGE sample buffer (Invitrogen) supplemented with 5% (v/v) 2-mercaptoethanol and by subsequent heating to 95°C for 5 min. Denatured proteins were resolved on 12% (w/v) NuPAGE polyacrylamide gels run with MOPS buffer (Invitrogen). See Blue Plus, protein molecular weight markers (Invitrogen) were run on all NuPAGE gels. Instant Blue stain (Expedeon Ltd.) was used to visualize the protein bands. Western blotting to detect either TMV coat protein with an anti-TMV antibody,  or the six C-terminal histidine residues with a monoclonal antibody prepared to six histidine residues,  was performed as described by 3 with the addition of 0.2 volume 10% (w/v) SDS and 0.1 volume 3 M Na acetate pH 4.8. After mixing and centrifugation at 16000 × g for 10 min, the RNA in the aqueous phase was recovered following overnight precipitation at -20°C with the addition of 2.5 volume of ethanol and by centrifugation at 16,000 × g for 20 min. RNA pellets were washed with 70% (v/v) ethanol and after centrifugation at 16,000 × g for 20 min, the RNA was air-dried and suspended in 20 μL H2O. 5 μL of RNA was denatured by the addition of formaldehyde (0.7 vol) and formamide (2 volume), incubated at 65°C for 5 min and subsequently resolved in 1.2% (w/v) agarose gel containing 2.2 M formaldehyde. The RNA size marker, catalog number 15623-200 (Invitrogen) was run alongside the extracted RNA samples and the RNA detected under UV light. For northern blot analysis, the RNA resolved in an agarose gel was stained by ethidium bromide and subsequently transferred to a positively charged nylon membrane by overnight capillary transfer in 20 × SSC . The RNAs containing the OAS sequence were detected by hybridisation with a complementary 126 nucleotide DIG-labeled RNA probe , the latter possessing the T7 RNA polymerase promoter sequence.Encapsidated RNA was isolated from purified nano-rods by extraction with phenol/CHClNA probe  containiNA probe . This waParticle preparations were spotted onto carbon-coated copper grids and when necessary were negatively stained with 2% (w/v) uranyl acetate. The grids were examined using a Tecnai 20 transmission electron microscope. Nano-rod length of TMV rod-like structures that clearly resembled TMV-like particles, was determined by utilizing AMT camera software version 3.2 (Supplementary Table S1).2, 50 mM H2PtCl6 and 100 mM NaBH4 for 22 or 69 h with constant agitation. Nano-rods were spun-washed at 3,000 × g with 1 mL H2O in Microcon YM-100 MWCO 100 kDa spin filters (Millipore) prior to electron microscopy.After gradient purification, a volume of nano-rods were incubated at room temperature with an equal volume of a 1:1:1: solution of 50 mM CoClin cis approach. This synthetic DNA was cloned into pEAQ-HT , a plant-based expression vector that produces abundant quantities of highly translatable RNA .As a first step to creating defined length nano-rods, we designed a DNA sequence that included the TMV coat protein open reading frame with a TMV OAS element to its 3′ side. This reverses the order of the OAS and coat protein (CP) that is found in TMV U1 genome and was done to ensure that the majority of RNA encapsidation would occur in the fast 3′–5′ direction. Upon transcription, this gene construct would direct the synthesis of TMV CP which will subsequently interact with the OAS element to form nano-rods. As both the CP and OAS are present on the same RNA, we term this the able RNA  to yieldQ vector , approxiNicotiana benthamiana leaves were separately infiltrated and extracts were subjected to the standard TMV purification protocol including precipitation with PEG and ultracentrifugation, followed by isopycnic centrifugation on caesium chloride (CsCl) gradients. Material banding at the same density as an authentic TMV control was collected and the CsCl removed by dialysis. Figure 2A shows the typical position in a CsCl gradient of the nano-rods resulting from the infiltration of plant leaves with pEAQ-HT-TMV CP/OAS. NuPAGE gel electrophoresis and Instant Blue staining revealed the presence of a single band of the size expected for TMV coat protein in the nano-rod-containing fractions of the CsCl gradients in the case of pEAQ-HT-TMV-CP/OAS and pEAQ-HT-TMV-CP/VP60/OAS . The identity of this protein was confirmed by MALDI-TOF analysis of tryptic digests of the material in the bands (data not shown). By contrast, no band corresponding to TMV CP could be seen in extracts prepared from leaves infiltrated with pEAQ-HT-TMV CP, a construct that is designed to produce TMV CP but no OAS-containing transcripts. These results indicate that expression of both the TMV CP and the presence of an RNA containing the OAS is essential for the formation of TMV nano-rods and that the buoyant density of such artificial rods in CsCl is similar to that of native TMV. This indicates that the nano-rods have a similar RNA:protein ratio, and therefore structure, to native TMV. The main difference between the results obtained with pEAQ-HT-TMV-CP/OAS and pEAQ-HT-TMV-CP/VP60/OAS was the level of CP detected in latter which was only about 10% of that for the shorter construct in samples from which equal masses of leaf material was processed.To determine if nano-rods could be formed from the above constructs, Figure 3A, panels 1 and 2). The length of a TMV particle is dependent upon the length of its encapsidated genome with one TMV coat protein subunit interacting with three nucleotides. Therefore, the expected length of each type of nano-rod can be calculated from the number of nucleotides in the encapsidated RNA. The length of a representative number of particles was measured from the micrographs in Figure 3A and there was very good agreement between the calculated and measured lengths for each class of nano-rod , confirming our hypothesis that it should be possible to vary the size of nano-rods by changing the length of the RNA containing the OAS.To confirm that the TMV CP was incorporated into TMV-like rods of defined length the particles were examined by transmission electron microscopy using negative staining. The particles appeared to have identical external and internal diameters to native TMV particles but in both cases were shorter . To examine the ability of these RNAs to assemble into rods, they were independently co-infiltrated with the pEAQ-HT-TMV CP gene construct (lacking the OAS) to create an in trans viral assembly system, . Isolation and examination of the rods formed was carried out as described for the in cis system . Negatively stained images of the particles indicated that the in trans system gave a mean rod length close to the expected length in the case of pBin-KS-mim-OAS-mp del  whereas the rods produced from pBin_CPMV-2_OAS where somewhat shorter than expected . We do not know the reason for this discrepancy but it may reflect the lack of sufficient CP to encapsidate the longer RNA. However, it may also be due to the fact that two gene constructs are needed for nano-rod formation with the in trans system in contrast to in cis nano-rod formation where only a single gene construct has to be present in a transformed cell. As shown previously, co-infiltration is a less efficient process than infiltration with a single construct  in our study , is in keeping with the variation measured and reported by in vitro formation of nano-rods. In this previous study, in vitro synthesized RNA of a known length was incubated with purified TMV CP for up to 48 h to form nano-rods. Similarities in the variation of the lengths of the nano-rods are apparent for both systems. Indeed the in vitro study reports that there is a greater variation in the length of the nano-rods resulting from the incubation of the longest RNAs when presented to CP, unlike the current investigation where a similar degree of variation in the length of the nano-rod is observed irrespective of its predicted length.To investigate the possibility of  deleted  and a fu deleted  to give onstruct . The varHT-TMV-CPHis/OAS . This construct was designed to express TMV CP modified to contain six histidine residues at its C-terminus (see below); this observation suggests that this tagged CP is not as assembly competent as wild-type (WT) CP. In all cases the samples of RNA extracted from partially purified nano-rods also contained substantial quantities of low molecular weight nucleic acid migrating more quickly than the 0.5 kb marker. To determine if this material was encapsidated into nano-rods or represented co-purifying host or Agrobacterium-derived nucleic acid, RNA was extracted from a sample of nano-rods obtained by infiltration pEAQ-HT-TMV-CP/OAS that had been purified from a CsCl gradient . In this case, a single band migrating close to the 1.5 kb marker could be seen – a size consistent with the encapsidation of the OAS-containing transcript from pEAQ-HT-TMV-CP/OAS. Significantly none of the shorter nucleic acids banded at the density of nano-rods suggesting that they are not encapsidated but rather represent nucleic acid fragments that are co-precipitated during the PEG precipitation step.When RNA was extracted from particles purified without the CsCl gradient step, bands corresponding to RNA of the expected length, ranging in size between 1.5 and 3.8 kb could be seen on denaturing agarose gels stained with ethidium bromide apart from the sample produced from plants infiltrated with pEAQ-Figure 4A, lanes 1–5) represented encapsidated RNA containing the TMV OAS, a replica agarose gel was subjected to northern blot analysis using a probe specific for the OAS. The specificity of the probe was confirmed by its ability to readily detect TMV RNA but not commercially available RNA size markers . In all cases, the high molecular weight RNA extracted from nano-rods hybridized with the OAS-specific probe to a degree consistent with the intensity of the ethidium bromide-stained bands; in contrast the lower molecular weight material hybridized less well, thereby suggesting that much of this material was not TMV-specific. In addition a high molecular weight band of hybridizing material could be seen in all samples  which we believe represents plasmid DNA which has co-precipitated with the nano-rods. One significant difference between the results obtained by northern blotting and ethidium bromide staining was that there was evidence from the former that the RNA transcribed from pEAQ-HT-TMV-CPHis/OAS could be incorporated into nano-rods by the coat protein containing a 6 × His sequence at its C-terminus albeit less efficiently that the equivalent WT construct. The results of the study of the RNA encapsidated within the nano-rods suggest rods are likely to be of a more precisely defined size than implied by the TEM analysis of the negatively stained particles. This difference is probably a reflection of the harsh nature of the TEM analysis leading to shearing of some of the particles on the grids.To confirm that the high molecular weight bands seen in the ethidium bromide-stained gel (HT-TMV-CP/OAS to give pEAQ-HT-TMV-CPHis/OAS and pEAQ-HT-TMV-CP(CP9)/OAS . Both constructs were designed to encapsidate an RNA of 1.5 kb producing nano-rods of around 75 nm. pEAQ-HT-TMV-CPHis, similar in design to pEAQ-HT-TMV-CP but possessing six C-terminal histidines, was also constructed to investigate in trans incorporation .In addition to controlling particle length, another potential advantage of using transient expression to produce nano-rods is the possibility of expressing CP variants which may be incompatible with a productive infection. To examine this possibility, two CP variants with additional sequences at the C-terminus were produced, one with the addition of six histidines and the second with the sequence CNAGDHANC. The incorporation of six histidines has been shown to permit the assembly of the modified CP into a number of protein-only structures but the modified protein has been reported to be poorly incorporated into RNA-containing rods . The CNAHT-TMV-CPHis/OAS, the yield of nano-rods obtained after PEG precipitation of extracts from leaves infiltrated with pEAQ-HT-TMV-CPHis/OAS was about 1% of that obtained from leaves infiltrated with pEAQ-HT-TMV-CP/OAS, judged by the detection of CP (arrowed) by western blotting with a polyclonal antibody to TMV . Here equal masses of infiltrated leaf material harvested at 7 days post infiltration from both infiltrations was processed. Instant Blue-stained gels of preparations of nano-rods isolated by PEG precipitation , showed the presence of CP suggesting that the modified CP could be assembled into nano-rods via the in trans reaction,  This substantiated the finding above, concerning the detection of encapsidated RNA in the in cis derived six histidine rods by northern blotting, . This was confirmed by TEM analysis which showed the presence only a few nano-rods of the expected size (data not shown). An attempt to incorporate the six histidine C-terminal CP into nano-rod structures via the in trans reaction upon the co-infiltration of both the WT and the six histidine CP coat protein constructs were fruitless, . This again highlights the difficulty of incorporating this modified CP into RNA containing rods, discussed above. However, the Instant Blue stained gel, , revealed the presence of CP in PEG purified nano-rods (generated via the in trans reaction). Thus, it is likely that the C-terminal six histidines have been removed probably by protease digestion thereby allowing for the formation of WT nano-rods. Taken together, these results show that assembly of the His-tagged protein into RNA-containing nano-rods is inefficient compared to the incorporation of wild-type coat protein. This may be because of the previously noted propensity of the His-tagged coat protein to assemble into protein-only aggregates rather than nucleoprotein rods (In the case of pEAQ-ein rods . Indeed HT-TMV-CP(CP9)/OAS appeared to contain sufficient material to allow further purification. After centrifugation of PEG-precipitated extracts from equal masses of leaves infiltrated with pEAQ-HT-TMV-CP/OAS or pEAQ-HT-TMV-CP(CP9)/OAS through CsCl gradients, the yield of the CP9 nano-rod was estimated to be approximately one third that of the WT nano-rod as judged by Instant Blue staining of coat protein resolved in NuPAGE gels . CP9 nano-rods were morphological indistinguishable from the WT nano-rods when examined by transmission electron microscopy after negative staining with uranyl acetate . Following CoPt deposition, CP9 nano-rods could be visualized without negative staining . In this case, the nano-rods had a dark rod-like appearance but with a hazy outline. Intense darker regions on the CP9 nano-rods can also be seen without staining, especially at higher magnifications . By contrast, nano-rods produced from WT coat protein could not be visualized by transmission electron microscopy unless the samples were subjected to negative staining , even after being subjected to the CoPt deposition reaction. Negative staining of the CP9 nano-rods following CoPt deposition revealed the characteristic nano-rod structure . The build-up of density, on the surface of the CP9 nano-rods is also evident  which correlates with the darker regions observed on the unstained CP9 nano-rods . These darker regions were more evident in the samples which underwent a longer deposition reaction (69 h) compared with those which were reacted for a shorter time, (22 h).By contrast, extracts of leaves infiltrated with pEAQ-in vitro formation of nanoboomerangs and tetrapods, formed upon RNAs with more than one OAS element has been demonstrated (in vitro reassembly experiments. The ability to metallise rods of different sizes, as demonstrated in the case of CoPt, increases the scope for the deployment of TMV-based structures in nanoelectronics.In this paper we have demonstrated that it is possible to synthesize defined length nano-rods by transiently expressing the TMV coat protein and an RNA containing the OAS element. These designer nano-rods have the general morphology of native TMV rods whose length can be controlled. In addition, they will lack the ability to cause an infection since the genomic RNA is not present. The ability to control their length, makes these synthetic nano-rods ideal for many emerging nanobiotechnological applications. In recent times, the concept of using plants as biofactories for the development and production of suitable pharmaceutical proteins has been gaining strength and is now very much a commercial reality . With sunstrated  and we aConceived and designed the experiments: KS and GL. Performed the experiments: KS. Analyzed the data: KS and GL. Wrote the paper: KS and GL.GL declares that he is a named inventor on granted patent WO 29087391 A1 which describes the pEAQ vector system used for the work described in this manuscript. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study examined the effects of an integrated health care program in elementary school students. The integrated program comprised exercises (3–4 times/week) and six sessions on nutritional and psychological education. Anthropometric measurements were recorded before the intervention. Additionally, physical fitness, dietary habits, nutrition knowledge, and psychological changes were assessed before and after the program. In total, 29% of the subjects were overweight and obese before the intervention (32% boys and 26% girls). There was a significant increase in flexibility, endurance, and cardiovascular endurance after the implementation of the program. Additionally, as a result of the program, participants showed improvement in nutrition knowledge and dietary habits. After the training, children tended to exhibit increased self–efficacy and lower stress, but the findings were not statistically significant. Implementation of an integrated health care program for the prevention and treatment of obesity could have a positive impact on children’s health. It is hoped that continued research on the long-term effects of such programs is conducted along with the development of various programs.  Childhood, which encompasses elementary school age, is a critical period for physical and mental growth and development as well as for character building. Further, this is a period in which dietary habits and physical activity habits – the factors that affect current and future health status – are established, based on which children build physical strength that serves as the basis for lifelong health2. In addition, multiple studies have raised concerns regarding children, including reduced physical activity, nutritional imbalance, increased obesity rate, school maladaptation, depression, and school violence, all of which threaten children’s health. According to the Ministry of Education, over the past 15 years (1999–2013), the average height and weight of 10-year-old Korean boys increased by 1.1 cm (from 141.6 cm to 142.7 cm) and 3.7 kg (from 36.5 kg to 40.2 kg), respectively, and those of 10-year-old Korean girls increased by 0.8 cm (from 142.4 cm to 143.2 cm) and 2.3 kg (from 36.1 kg to 38.4 kg), respectively. However, in the same period, physical fitness declined in both groups3. Thus, a balance among adequate physical activities, nutritional balance, and psychological stability by reducing stress must be achieved to maintain health. The World Health Organization (WHO) defines health as a state of complete physical, mental, and social well-being and not merely the absence of disease and infirmity5. Further, only a handful of the programs comprise independent sessions of exercise as well as nutritional and psychological education or combined sessions of exercise and nutritional education7. Therefore, the present study sought to identify the effects of a 10-week, integrated physical activity program, consisting of both exercise and psychological education, on the physical fitness, nutritional knowledge, and dietary habits, as well as on the psychological changes (self-efficacy and stress) in the fifth grade participants. Most health-related programs for children in Korea aim at providing secondary treatment for a subset of health-risk groups, such as reducing obesity for obese children, instead of offering a comprehensive health management for the prevention of obesity and diseasesFifty-two fifth grade students (two classes) from anelementary school in Seongbuk-gu, Seoul, were enrolledin the study after obtaining consent from the school andtheir parents. With cooperation from the school, the programwas administered in the playground, auditorium,and lecture halls during 3–4 hours of physical educationsessions allotted for the study. The integrated program comprised 36 sessions of physical activity and six sessions of nutritional and psychological education, each implemented for 10 weeks, from October to December, 2013. The physical activity portion of the program comprised circulation exercise and new sports exercise, mostly based on aerobic exercise and muscle strengthening exercise, to boost the physical strength of schoolaged children. The initial, development, and maintenance stages involved low-intensity exercise , moderate-intensity exercise , respectively. To keep the children interested, the exercise program consisted of bowling, basketball, badminton-like program, floorball, and T-ball baseball, in addition to muscle-strengthening exercise, ranging from light muscle strengthening to heavier training using equipment. With a focus on achieving a balanced diet through appropriate diet and weight management with lower sodium intake, the nutritional education component of the program comprised lectures on concepts of health , evaluation of nutritional status (survey of dietary habits and nutrition intake), functions and roles of nutrients (explanation of the food cycle), appropriate dietary habits , reading nutrition facts, and reducing salt intake . The psychological education component of the program comprised lectures on the importance of mental health, evaluation of psychological state psychological survey), boosting self-respect methods of communication, improving leadership, stress relief (meditation and five-senses therapy), and depression prevention . Prior to the program, the participants’ anthropometric measurements were taken; height was measured using a height scale, and body weight and body fat percentage were measured using InBody 7.0 . The children were classified into underweight (<5th percentile), normal weight (5th percentile–85th percentile), overweight (85th percentile–95th percentile), and obese (>95th percentile or body mass index [BMI] >25) based on the standard percentages of BMI for age and sex as per the growth chart for children and adolescents developed by the Centers for Disease Control and the Korean Pediatric Society. To assess the effects of the intervention in improving students’ physical fitness, flexibility (sit and reach), cardiovascular endurance (round-trip long run), muscular endurance (sit and rollup), and power (50-m run) were measured twice, once before and once after the intervention. To assess the effects of the intervention on nutritional education, changes in students’ nutritional knowledge and dietary habits were surveyed before and after the intervention. Educational materials that we developed as well as data provided from some nutrition-related websites were used for the nutritional education component, and educational sessions included various activities, including lectures, group discussions, and games to capture students’ attention and promote active participation. ① Nutritional knowledge: Nutritional knowledge was measured with 10 items on health, nutrients, and sodium intake. Students were given a score of 10 for a correct answer and a score of 0 for a wrong answer. The mean score for the 10 items was used. All the questions were based on the contents of the nutritional education. 10. ② Survey of dietary habits: A 20-item scale developed by the Korean Society for Health Promotion and Disease Prevention was used to survey dietary habits. For items on desirable dietary habits, a score of 1, 3, and 5 were given for “0–2 days,” “3–5 days,” and “6–7 days,” respectively; the scores were reversed for items on undesirable dietary habits. The items pertained to skipping breakfast; extent of consumption of fruits, vegetables, fast food, ramen, soft drinks, and milk intake; and appropriate dietary habitsThe psychological changes produced by the intervention were assessed using psychological tests before and after the intervention. Items pertaining to self-efficacy, leadership life skills, and daily stress were included in the test. 12. The scale comprises 17 items pertaining to the perception of individual skills that affect outcomes in a variety of situations, and each item is measured on a five-point scale. ① Self-efficacy: Self-efficacy was measured using the self-efficacy scale originally developed by Sherer et al. (1982) and translated and validated by Kim (1997) for use with Korean adolescents13. This 30-item scale comprises five categories of skills related to interacting with others, learning skills, skills in understanding oneself, skills in working with groups, and decision-making skills, and each item is measured on a five-point scale. ② Leadership life skills: The leadership life skills questionnaire for elementary school children developed by Jin (2006) was used to measure leadership life skills③ Daily stress: The daily stress scale for Korean children developed by Han and Yoo (1995) was used to measure daily stress14. This 42-item scale comprises six categories of stress related to parents, family background, friends, studies, teachers and school, and environment, and each item is measured on a five-point scale. All values were presented as mean ± standard deviation (SD). All statistical analyses were performed using the SAS software program , with a significance level of α < 0.05. Sex-specific differences were analyzed using the Chi-square test and student t-tests, and post-intervention changes were measured using paired t-tests. Fifty-two students were enrolled in this study, of whom 25 were boys (48.1%) and 27 were girls (51.9%). The mean height and weight of the boys and girls was 148.5 cm and 42.4 kg and 146.9 cm and 39.1 kg, respectively, showing no significant differences between the sexes. There were no significant sex differences in BMI, but body fat percentage was higher in girls. Further, there were no significant sex differences in the proportion of obesity, with 17 normal weight (68%), six overweight (24%), and two obese (8%) boys, and 20 normal weight (74.1%), five overweight (18.5%), and two obese (7.4%) girls . Changes in physical fitness after the intervention are illustrated in Post-program changes in dietary habits are presented in Post-program changes in nutritional knowledge are shown in Post-program changes in self-efficacy have been shown in Changes in leadership life skills have been shown in Post-program changes in daily stress are shown in 2. Further, there were no sex differences in BMI, but body fat percentage was significantly higher in girls than that in boys. Proportions of overweight and obese children were higher, though not statistically significantly, in boys. The proportions of obese children in this study  were higher than those reported by Kim et al.15 based on the same obesity criterion . The objective of this study was to examine the effects of an integrated physical activity program comprising nutritional and psychological education on the physical fitness, psychological states, dietary habits, and nutritional knowledge of elementary school children. The mean height and weight for boys was 148.5 cm and 42.4 kg, respectively, and for girls was 146.9 cm and 39.1 kg, respectively; these were above the national average for Korean 10-year-olds reported by the Ministry of Education in 2013 16, reduced physical strength not only signifies physical problems such as increased obesity rate, but also leads to an array of social problems such as bullying, violence, suicide, game addiction, and depression. Further, they stated that regular exercise promotes physical fitness and plays a positive role in relieving physical and mental stress. Prior to the intervention, boys had lower flexibility and muscular endurance than the national average for Korean 10-year-olds, as reported by the 2013 Student Health and Fitness Evaluation by the Ministry of Education, but both values increased to higher levels than the national average after completing the program. Further, the participants had higher power than the national average for 10-yearolds before and after the program. Conversely, despite the fact that boys showed significant improvements in cardiovascular endurance after the program, the value was still lower than the national average cardiovascular endurance of Korean 10-year-olds. For girls, flexibility was lower than the national average before the program, but it increased to higher levels than the national average after the program. Muscular endurance was higher than the national average and it significantly increased after participation in the program. Power was similar to the national average, but decreased after participation in the program. Similar to the trend observed in boys, cardiovascular endurance significantly increased after the program, but it was still lower than the national average. In essence, the integrated program improved flexibility, muscular endurance, and cardiovascular endurance in both boys and girls, while power either remained unchanged or reduced. According to Park et al.17. Dietary habits in childhood may have a lasting impact on a child’s lifelong dietary habits; hence, it is critical to shape appropriate dietary habits in childhood. In our study, participation in the program led to significant improvements in nutritional knowledge, and though not statistically significant, the total dietary habit score also increased after the program. In a study that only performed four sessions of nutritional education over 4 weeks in fifth graders, children showed significant improvements in nutritional knowledge, dietary attitude, and dietary behaviors18. However, long-term systematic nutrition education is required for increased nutritional knowledge to produce changes in dietary attitudes, behaviors, and intake, but such changes are difficult to be delivered through most of the current nutrition education programs for children, as they are generally administered via shortterm programs. Many studies have suggested that nutritional education increases children’s nutritional knowledge and brings about desirable changes in dietary behaviors and dietary intake12. Further, academic stress is known to have a vast impact on self-respect during childhood, the most important period in life20. After participating in our program, the participants’ self-efficacy improved mildly, with an overall reduction in daily stress, but not to significant levels. Therefore, we speculate that our program contributed to the positive alteration of the participants’ psychological states. Self-efficacy refers to the belief in one’s own capacity to perform behaviors that would bring about certain outcomes. Daily stress refers to trivial problems that most people commonly encounter in their daily lives. In Kim’s study on adolescents, self-efficacy was enhanced with advancing stage of exercise, and regular participation in exercise increased the significance of self-efficacy as a psychological variableImplementation of an integrated health management program for the prevention and treatment of obesity in childhood, a critical period that shapes a child’s lifelong health, may have a positive impact on children’s health. Hence, we hope that future studies continue to develop various programs and examine the long-term effects of programs for children.


HealtheSteps™ participants are provided with in-person lifestyle coaching and access to eHealth technologies delivered in community-based primary care clinics and health care organizations.Physical inactivity is one of the leading causes of chronic disease in Canadian adults. With less than 50% of Canadian adults reaching the recommended amount of daily physical activity, there is an urgent need for effective programs targeting this risk factor. Healthesteps™, we will conduct a 6-month pragmatic randomized controlled trial with integrated process and economic evaluations of HealtheSteps™ in 5 clinic settings in Southwestern Ontario. 110 participants will be individually randomized  to either the intervention  or comparator (Wait-list control). There are 3 phases of the HealtheSteps™ program, lasting 6 months each. The active phase consists of bi-monthly in-person coaching with access to a full suite of eHealth technology supports. During the maintenance phase I, the in-person coaching will be removed, but participants will still have access to the full suite of eHealth technology supports. In the final stage, maintenance phase II, access to the full suite of eHealth technology supports is removed and participants only have access to publicly available resources and tools.To determine the effectiveness of HealtheSteps™ program, and the direct cost for each person participating in the program as well as the costs associated with delivering the program at the different community sites. These results will inform future optimization and scaling up of the program into additional community-based primary care sites.This trial aims to determine the effectiveness of the program in increasing physical activity levels and improving other health behaviours and indicators, the acceptability of the HealthNCT02413385 . Date Registered: April 6, 2015. Physical activity (PA) is one of the most modifiable risk factors for preventing chronic disease, yet less than 50% of Canadian adults are considered physically active , 2 and oResearch shows that many chronic diseases can be prevented by decreasing four main behavioural risk factors: physical inactivity, poor nutrition, smoking and alcohol consumption , 10. PubeSteps™ program was developed in response to the literature demonstrating the effectiveness of primary care-based chronic disease prevention programs [eSteps™ program was developed from an extensive research base [The Healthprograms , consultprograms . The Hearch base , 20–27; eSteps™ program provides individuals with a specific plan of action to improve their PA levels, healthy eating habits, and reduce their sedentary behaviour. This is achieved through personalized coaching, grounded in the principles of Motivational Interviewing [eSteps™ is the pragmatic nature of the program and ability to work within the existing workflow of an organization. While certain elements of the program must be conducted exactly as designed to follow the evidence-based protocols, other aspects are flexible. For example, the program may be conducted in-person in small groups or one-on-one; it can be run in a variety of primary care based settings; and it can be integrated with other existing programs.The Healthrviewing  and Co-Arviewing , and supeSteps™ program in helping at-risk individuals increase PA levels , improve eating habits, and improve other health behaviours and health indicators.To conduct an outcome evaluation to determine effectiveness of the HealthTo conduct a process evaluation to determine acceptability of the intervention, to inform program improvement and optimization.eSteps™ program at different community sites, and the average cost per participant involved in the HealtheSteps™ program.To conduct an economic evaluation to determine the direct cost associated with delivering the HealtheSteps™) or the comparator (Wait-list control). Participants in both groups will complete measurement sessions at baseline and 6 months; participants in the intervention group will complete additional measurements at 12 and 18 months. Participants in the comparator group will be given the opportunity to start the HealtheSteps™ program after a 6-month delay. See Fig. A 6-month, two-arm, pilot, pragmatic, randomized controlled trial (RCT) with integrated process and economic evaluations will be conducted within five clinic/health care organization settings in both urban and rural communities in Southwestern Ontario. Participants will be individually randomized  to either the intervention .eSteps™ program. Sites will be recruited opportunistically through connections with the research team. A variety of staff from each site will be recruited for training to be coaches in the program. Meetings with sites will occur with the central research team to ensure the coaches are trained in the program protocol and understand the commitment required . HealtheSteps™ coaches will be trained by the central research team or via eLearning modules available through the HealtheSteps™ website [This trial was designed to recruit primary care sites and/or health service organizations as they provide greater opportunities to recruit participants at risk or diagnosed with a chronic disease. We will solicit interest from primary care community sites for participation in the trial by contacting primary care leads in target communities where they had not been previously exposed to the Health website . In ordeWe set a recruitment target of 110 participants. Baker and colleagues  conducteParticipants will be recruited at the sites through poster advertisements, word of mouth, health care provider referrals, staff e-mails, and in-person recruitment booths. Those interested in participating will be contacted by the central research team and asked to attend an in-person screening session. At this session they will be screened for eligibility and if deemed eligible will take part in baseline measurements. Randomization will occur after baseline assessments. All individuals who contact a study site will be asked to report where they heard about the program. Participants who are ineligible and those who choose not to enroll in the study after initial contact will be tracked, along with reasons for ineligibility/not enrolling. Acceptability of randomization will be estimated from the percentage of participants who adhered to their allocation assignment  and/or attend measurement sessions. Participation in the follow-up measurement sessions will be used to assess retention throughout the trial. Attendance at program sessions will be obtained from coach’s logs and will be used to assess adherence to the program.2; less than 150 min of exercise per week; 3 or more hours sitting per day; consuming less than 8 fruit and vegetable servings per day; diagnosis of metabolic syndrome or type 2 diabetes; and clearance to participate in PA via the Physical Activity Readiness-Questionnaire (PAR-Q) or a health care provider.Individuals 18–85 years of age; one or more self-reported or measured risk factors for chronic disease including: body-mass index (BMI) greater than 25 kg/mIndividuals who are unable to comprehend the letter of information and consent documentation.eSteps™ program) or comparator (Wait-list control). The randomization sequence  will be computer-generated and conducted by a member of the research team who will not be involved in allocation. Following allocation, all participants (intervention and comparator groups) will receive Eating Well with Canada’s Food Guide [Following the baseline measurement, participants at each site will be individually allocated to either the intervention  that occurs while a participant is enrolled in the Healthactive phase that encompasses the first six months, a maintenance phase I for the next six months, and finally a maintenance phase II, which is the last six months of the trial.There are three phases to the intervention: an eSteps™ program within three weeks of completing their baseline measurement session. Participants will receive a handbook that contains documents to help them track their progress throughout the program and will be given a pedometer to use to track their PA. The research team chose to utilize pedometers to measure step counts as they are inexpensive, easy to use, and easily interpreted by the participant and research team [eSteps™ coach who will be trained on how to create an action plan with the participant to help them work towards achieving their lifestyle prescriptions. Strategies for setting lifestyle prescriptions are based on S.M.A.R.T. goal setting principles , as goal setting has been found to produce positive behaviour change in adults [Exercise Prescription: The exercise prescription focuses on making incremental changes in the amount of time the participant engages in moderate to vigorous activity or the amount of time they exercise within their target heart rate zone, up to the recommended 150 min/week. At each program session, participants will complete a sub-maximal fitness test, the Step Test and Exercise Prescription tool (STEP™ Test) [eSteps™ coach will collaborate with the participant to set a weekly target of exercise minutes that includes frequency, duration, and type of exercise to facilitate achievement of their exercise prescription.P™ Test) . Based oP™ Test) . The HeaPhysical Activity Prescription: The PA prescription focuses on making incremental changes to the participants’ daily step count to reach the recommended 10,000 steps per day. This requires setting a baseline step count, which for coaching session 1 will be the participants’ step count recorded during the baseline measurement session. For sessions 2 to 4, the prescription will be based on steps logged in the participants’ handbook completed between sessions. If the participant did not track their step counts between coaching sessions, the coach and participant will work together to develop a reasonable target step count to set a new PA prescription.Healthy Eating Prescription: Using Eating Well with Canada’s Food Guide, participants will work with their coach to determine their current daily intake from each food group, their water intake, and consumption of a healthy balanced breakfast. The healthy eating goal is set through increasing or decreasing a serving in one of the food groups to better meet the recommendations , increasing water intake and increasing the number of days the participant consumes a healthy balanced breakfast.Access to the HealtheSteps™ eHealth Technology Suite: Tyze Personal Networks [maintenance phase I until the 12 month assessments are complete. Tyze Personal Networks is a web-based platform that provides an exclusive network for connecting with central research staff, coaches, and other participants through private chat services and posting information on a CareWall . Sykes provides phone coaching services to support participants in-between coaching sessions and during maintenance phase I. Sykes CareCoaches® are trained in the HealtheSteps™ protocol and act similarly to the HealtheSteps™ coach, working with participants to address any challenges they may be experiencing and providing encouragement for them to meet their goals.Networks  and SykeNetworks  phone coeSteps™ website  for healthy living resources and the HealtheSteps™ and eaTracker smartphone apps. The HealtheSteps™ app (available for free download on Apple and Android devices) has a built-in fitness test and heart rate monitor. Additionally, through the help of a virtual coach, the app provides personalized prescriptions, goals, and tracking options to help participants maintain their goals developed with their in-person coach. The eaTracker allows participants to monitor food and activity choices, analyze recipes, and create meal plans. These publicly available supports are available to participants throughout the active and maintenance phases as well as beyond the trial period.Participants will be encouraged to seek out publicly available healthy living supports, such as the HealthParticipants assigned to the intervention group will start the Healthrch team . In totan adults . The lifeHealth technology support options.Between coaching sessions, participants will be expected to self-direct their healthy living activities, using healthy living resources  and eHealth technology support tools.Participants will no longer have in-person coaching sessions, but will have access to the full suite of eSteps™ website.Participants will no longer have access to the online Tyze Personal Networks, and Sykes phone coaching services, but will still have access to the publicly available resources, including the HealtheSteps™ and eaTracker Smartphone apps, and the HealtheSteps™ program.This group will continue with usual activities without intervention from the study team for the first 6-month period. After the 6 month measurement sessions are completed, participants allocated to this group will be given the opportunity to start the 6-month HealthAverage steps per day, measured using Yamax Digiwalker SW-200 pedometers and self-reported by participants using a 7-day paper log , 42 -minutes/week) measured using the International Physical Activity Questionnaire (IPAQ) Short Form ; and tim2), waist circumference (cm) [Objectively-measured clinical characteristics: weight , Body Mass Index  , and resSelf-reported eating: healthful eating score, measured using Starting the Conversation (STC) questionnaire ; fatty fSelf-rated health measured using the European Quality of Life – 5 Dimensions – 3 Levels (EQ-5D-3 L) visual analog scale (VAS) score , 49.The outcome measures will be taken at baseline and 6 months in both groups, and then again at 12 and 18 months in the intervention group only  feasibility of retaining individuals for an extra 6 and 12 months (to inform a larger study); and ii) within group change to 12 and 18 months to determine the sustainability of any positive changes  observed following the active phase.The primary outcome will be the difference between the intervention and comparator groups in mean steps per day at 6 months. For all secondary outcomes, we will also examine differences between intervention and comparator groups in mean change at 6 months. The additional follow-up data collected from HealthWe will analyze data based on an intent-to-treat approach; thus, we will include all participants with at least valid baseline data according to the randomization scheme. We will analyze data using linear or generalized linear mixed models for repeated measurements and we will retain the baseline outcome as part of the outcome vector and constrain the group means as equal because of randomization . This apTo address participant dropout, we will also compare baseline characteristics of participants who dropped out versus participants who were included in the analysis. Interpretation of study results will primarily be based on estimation and associated 95% confidence intervals . ConfideeSteps™ program, the coaches’ experience delivering the program, and the participants’ experience maintaining their changes. This will inform future optimization of the HealtheSteps™ program.Qualitative data sources will include the Interviewer-Guided Participant Feedback Questionnaires and coach interviews collected at the 6 month assessments and the participant interviews collected at the 12 month assessments. Due to the high volume of participant interviews at 12 months, a representative sample  of interviews from each site will be selected for transcription and analysis; these data sources will be analyzed separately by members of the research team whom will not be involved in direct program delivery. An inductive content analysis will be performed. The research team will read through all of the transcripts and identify common themes and exemplar quotes that represent this theme; following a group discussion, a final list of overarching themes will be created. The findings from the Interviewer-Guided Participant Feedback Questionnaires, and coach and participant interviews will be triangulated to produce a detailed description of the participants’ experience with the HealtheSteps™ program. Total costs will be calculated by summing all item costs, regardless of site, study group, payer perspective, component, and time period. Total cost will then be broken down for each study group and site. Secondary analysis involves calculating the average cost per person of the HealtheSteps™ program. The average cost per person will be calculated by dividing the total cost of the HealtheSteps™ program by the number of participants at each site. Given that participants differ by site, per person cost will be presented by site and by study group (intervention vs. comparator). Accordingly, total cost of the program will be broken down for each study group and site, and subsequently by component, payer perspective, and time period. The same process will be used to calculate the average cost of the HealtheSteps™ program per participant. The final analysis stage will estimate the total and average per person cost to implement the HealtheSteps™ program at a new site. Total costs will be calculated by summing all item costs that contributed to the implementation of the HealtheSteps™ program at a new site. The average cost per person will be calculated by dividing the total cost of the HealtheSteps™ program by the number of assumed participants and coaches (a participant to coach ratio of 15:1) at this hypothetical site. Both the total and average per person cost will also be categorized by component costs .The economic evaluation will be conducted by the Centre for exceLlence in Economic Analysis Research (CLEAR) Team. The primary analysis will consist of calculating the total cost of the HealthLiving a physically active lifestyle along with consuming a healthier diet and reducing tobacco intake can prevent 80% of chronic diseases, such as heart disease, stroke, and type 2 diabetes, and 40% of cancer cases . The majeSteps™ is a lifestyle program developed from an extensive research base and designed to provide participants with action specific goals and strategies to increase their PA and exercise levels, and encourage healthy eating habits, within the confines of their primary health care space. HealtheSteps™ engages participants through multiple avenues, including using technology and phone supports to provide participants with the tools needed to make and maintain a healthy lifestyle. In this study we aim to determine the effectiveness of the HealtheSteps™ program in helping at-risk individuals increase PA and exercise levels, and improve eating habits. This study also aims to determine the acceptability of the program to inform future optimization. Lastly, this study aims to determine the direct cost per participant involved in the program, and the cost to the community or site running the program. If the results are promising, our next step will be to further scale-up the program into rural and remote communities focusing on delivery in primary care, workplace wellness, and community-based facilities.Health


Introduction. Lung adenocarcinoma (LAC) is the most frequent type of lung cancer and has a high metastatic rate at an early stage. This study is aimed at identifying LAC-associated genes. Materials and Methods. GSE62950 downloaded from Gene Expression Omnibus included a DNA methylation dataset and an mRNA expression profiles dataset, both of which included 28 LAC tissue samples and 28 adjacent normal tissue samples. The differentially expressed genes (DEGs) were screened by Limma package in R, and their functions were predicted by enrichment analysis using TargetMine online tool. Then, protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. Finally, LAC-associated methylation sites were identified by CpGassoc package in R and mapped to the DEGs to obtain LAC-associated DEGs. Results. Total 913 DEGs were identified in LAC tissues. In the PPI networks, MAD2L1, AURKB, CCNB2, CDC20, and WNT3A had higher degrees, and the first four genes might be involved in LAC through interaction. Total 8856 LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs . Conclusions. These key genes might play a role in pathogenesis of LAC.  As the most common type of lung cancer and a non-small cell lung carcinoma (NSCLC) , lung ad HOXB9 and LEF1 which mediates chemotactic invasion and colony outgrowth [ Oct4 and Nanog, which are homeobox transcription factors important for self-renewal of stem cells, commands epithelial-mesenchymal transdifferentiation, mediates tumor-initiating ability, and contributes to metastasis of LAC [ MALAT-1 enhances motility of LAC cells via regulating motility-associated gene expression in transcriptional and posttranscriptional levels [ BRAF mutation is frequently detected in human LAC, indicating that BRAF may serve as a therapeutic target for a subset of patients with the disease [ caveolin-1 is essential for mediating filopodia formation, which may promote invasion of LAC cells [The WNT/TCF signaling can promote osseous and brain metastasis of LAC cells through targetingutgrowth . Coexpres of LAC . As a nol levels , 9. BRAF disease , 11. OveAC cells . In spitRecently, along with the development of chip technology, microarray data have been obtained and uploaded to Gene Expression Omnibus (GEO) for us to study . Using mhttp://www.ncbi.nlm.nih.gov/geo/), which included a DNA methylation dataset and an mRNA expression profiles dataset. The DNA methylation dataset and the mRNA expression profiles dataset separately were based on the platform of GPL8432 Illumina HumanRef-8 WG-DASL v3.0 and GPL8490 Illumina HumanMethylation27 BeadChip (HumanMethylation27_270596_v.1.2), and both of them included 28 LAC tissue samples and 28 adjacent normal tissue samples.Microarray data GSE62950 was downloaded from the database of GEO  method [p value < 0.05 and |log2fold-change(FC) | ≥1 were taken as the thresholds.Using Limma package  in R, th) method . The adjp values of enriched terms were corrected by Holm-Bonferroni [p value < 0.05 was used as the cut-off criterion.GO is utilized for predicting potential functions of gene products in three categories  . The KEGnferroni . The adjhttp://www.cytoscape.org/) [The STRING  databasepe.org/) . Subsequpe.org/)  in R wasFurthermore, MCODE plugin  in Cytosp value < 0.05 was used as the cut-off criterion. Then, the methylation sites located in sex chromosomes were removed. Finally, the associations between the screened methylation sites and LAC were analyzed by CpGassoc package [p value < 0.05 was taken as the threshold.Using genefilter package  in R, th package  in R. ThAccording to the annotation information of the DNA methylation profiles, the nearest genes to the LAC-associated methylation sites were obtained and then mapped to the DEGs. At last, LAC-associated methylation sites of the DEGs were gained.http://cancergenome.nih.gov/) database, which included 515 LAC tissue samples and 59 adjacent normal tissue samples. Using Limma package [p value < 0.05 and |log2FC| ≥ 1 were also screened. Meanwhile, the methylation data of LAC were also downloaded from TCGA database, which included 459 LAC tissue samples and 32 adjacent normal tissue samples. Moreover, LAC-associated methylation sites were also identified and mapped to the DEGs using the same methods as the above.The RNASeqV2 data of LAC were downloaded from The Cancer Genome Atlas  in LAC tissue samples. In the heat map of the DEGs, LAC tissues could be definitely separated from adjacent normal tissues by the DEGs .p value = 8.59E − 07) in BP category, as well as extracellular region  and extracellular space  in CC category  and cell cycle  , germ cell cancer , embryoma , and embryonal cancer  ( MAD2L1 and aurora B kinase (AURKB). The enriched GO functions for downregulated genes were listed in p value = 5.73E − 05) and single-multicellular organism process  in BP category, as well as cell periphery in CC category . In addition, there were nonsignificant KEGG pathways and DO terms enriched for the downregulated genes.Using TargetMine online tool, functions of the DEGs were predicted by GO, KEGG, and DO enrichment analyses. For the upregulated genes, the enriched GO functions included multicellular organismal catabolic process (category . The enr MAD2L1) . The enr.009481) . And all MAD2L1 (degree = 36), AURKB (degree = 38), CCNB2 (degree = 40), and CDC20 (degree = 42) had higher degrees, and they can interact with each other in the PPI network. Several modules were screened from the PPI network for upregulated genes, and the largest module (module 1) is also showed in PPI networks for upregulated and downregulated genes were constructed, respectively. The PPI network for upregulated genes had 229 nodes and 725 interactions . ParticuPRKG2, degree = 11), VE-cadherin , WNT3A (degree = 15), adenylyl cyclase 8 , and adenylyl cyclase 4  were the top 5 nodes which had higher degrees. What is more, nonsignificant modules were screened from the PPI network for downregulated genes.The PPI network for downregulated genes had 233 nodes and 368 interactions . Connectp value < 0.05. As a result, 230 LAC-associated methylation sites were identified.After screening, total 8856 methylation sites were obtained. Then, the associations between the screened methylation sites and LAC were analyzed under the threshold of  SH3GL2; brain-specific angiogenesis inhibitor 3, BAI3; H-cadherin, CDH13; junctional adhesion molecule 2, JAM2; metallothionein 1A, MT1A; LIM-homeobox containing 6, LHX6; and insulin-like growth factor binding protein-3, IGFBP3)  . And all WNT3A, MAD2L1, AURKB, CCNB2, CDC20, SH3GL2, BAI3, CDH13, JAM2, MT1A, and IGFBP3. In addition, the 29 LAC-associated methylation sites identified in the microarray data of GSE62950 were also detected in the methylation data downloaded from TCGA database.Total 4893 DEGs (including 2191 upregulated genes and 2702 downregulated genes) were identified in the LAC sample data downloaded from TCGA database. There were 691 common DEGs (including 310 upregulated genes and 381 downregulated genes) between the 4893 DEGs identified in the LAC sample data downloaded from TCGA database and the 913 DEGs identified in the microarray data of GSE62950. The common DEGs included genes such as WNT3A, MAD2L1, AURKB, CCNB2, CDC20, SH3GL2, BAI3, CDH13, JAM2, MT1A, and IGFBP3) between the 913 DEGs identified in the microarray data of GSE62950 and the 4893 DEGs identified in the LAC sample data downloaded from TCGA database. In addition, the 29 LAC-associated methylation sites identified in the microarray data of GSE62950 were also detected in the methylation data downloaded from TCGA database. Functional enrichment indicated that WNT3A was involved in single-multicellular organism process and cell periphery. Overexpression of Wnt5a, which belongs to Wnt family that encodes signaling glycoproteins, promotes invasion of NSCLC during tumor progression [ Wnt-7a and Fzd-9 signaling plays role in inducing the receptor tyrosine kinase inhibitor Sprouty-4 and cadherin proteins and is essential for maintaining epithelial differentiation and inhibiting transformed cell growth in some NSCLC patients [In this study, 913 DEGs including 409 upregulated and 504 downregulated genes were identified in LAC tissue samples compared with adjacent normal tissue samples. Total 230 LAC-associated methylation sites were identified, among which 29 LAC-associated methylation sites were located in 27 DEGs. Afterwards, the RNASeqV2 data and methylation data of LAC were downloaded from TCGA database to validate the obtained methylation sites and DEGs. There were 691 common DEGs  and AURKB can result in bad outcome of lung cancer patients; thus, suppression of the CDCA8-AURKB pathway is a potential therapeutic strategy for lung cancer [MAD2) is overexpressed in a high percentage of lung cancers, and multivariate analysis suggests that high-level MAD2 can be a prognostic marker independently [ MAD2L1, AURKB, CCNB2, and CDC20 had higher degrees, and they can interact with each other. Therefore, MAD2L1, AURKB, CCNB2, and CDC20 might be implicated in LAC by interacting with each other.Besides,arcinoma . Overexparcinoma . The 5-ywith LAC , 35. Immrcinomas , 37. Immg cancer . Semiquaendently . In the  SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3). Loss of SH3GL2 is frequently detected in NSCLC and SH3GL2 can mediate cellular growth and invasion through interacting with EGFR [ CDX2, VIL1, and BAI3 levels have significant differences in SCLC and large-cell neuroendocrine lung carcinoma (LCNEC); therefore, they can be diagnostic markers of these tumor types [ CDH13, located on chromosome 16q24.2–3, is downregulated in lung cancer and its aberrant methylation may be a potential marker for cancer detection [β1 integrin subunit and ERK activation in human dermal lymphatic endothelial cells (HDLEC), junctional adhesion molecule-C (JAM-C) contributes to lymphangiogenesis and nodal metastasis, suggesting that JAM-C may be a target for treating lymphatic metastases in NSCLC [MT) can be used as an independent predictor of short-term survival in SCLC patients enduring chemotherapy [ LHX6 is a candidate tumor suppressor gene that has epigenetic silencing in patients with lung cancer [ IGFBP-3 before cisplatin therapy seems to be a biomarker of prognosis, helping to select appropriate therapeutic method for patients [ SH3GL2, BAI3, CDH13, JAM2, MT1A, LHX6, and IGFBP3 might relate to LAC.Additionally, LAC-associated methylation sites were identified and mapped to the DEGs. And there were 29 LAC-associated methylation sites located in 27 DEGs . However, these bioinformatic findings need to be validated by further researches.In conclusion, we carried out a comprehensive bioinformatics analysis to screen LAC-associated genes. We identified 913 DEGs and 8856 methylation sites in LAC tissue samples. Besides, LAC might correlate with several key genes (e.g.,


The way a postgraduate medical training program is organized and the capacity of faculty members to function as tutors and to organize effective professional experiences are among the elements that affect the quality of training. An evaluation system designed to target these elements has been implemented within the framework of the Pediatric Residency Program of the University of Padua . The aim of this report is to describe some aspects of the experience gained in the first 3 years of implementation of the system (2013–2015). Data were collected using four validated questionnaires: the “Resident Assessment Questionnaire”, the “Tutor-Assessment Questionnaire”, the “Rotation-Assessment Questionnaire”, and the “Resident Affairs Committee-Assessment Questionnaire”. The response rate was 72% for the “Resident Assessment Questionnaires”; 78% for the “Tutor-/Rotation-Assessment Questionnaires” and 84% for the “Resident Affair Committee-Assessment Questionnaires”. The scores collected were validated by psychometric tests.Conclusion: The high rates of completed questionnaires returned and the psychometric validation of the results collected indicate that the evaluation system reported herein can be effectively implemented. Efforts should be made to refine this system and, more importantly, to document its impact in improving the Pediatric Residency Program. Postgraduate medical training programs should be continuously monitored to guarantee the quality of the education curriculum . The wayThe Pediatric Residency Program of the University of Padua is a Ministerial accredited and ISO-9001 certified 5-year program that offers a Diploma of Specialization in Pediatrics [To monitor the program, the Resident Affairs Committee of the Pediatric Residency Program has developed an evaluation system according to which Division Chiefs (tutors) are requested to evaluate residents, and residents are requested to evaluate the quality of the training provided by each Division Chief, the professional experience acquired during the various rotations, and the functioning of the Resident Affairs Committee.This evaluation system is based on four validated questionnaires: the “Residents Assessment Questionnaire (AQ)”, the “Tutor-AQ”, the “Rotation-AQ” and the “Resident Affairs Committee-AQ” , 3 that An internet-based system has been developed to handle the entire evaluation process. The web-based system provides the Resident Affairs Committee with a dashboard command to monitor the implementation and the correct use of the evaluation system. On the whole, questionnaires can be completed in less than 10 min. Tutorials to instruct new residents and faculty members on how to complete the questionnaires are organized annually. The psychometric properties of the questionnaires, although previously validated, are regularly monitored to verify (a) the internal consistency of all the questionnaires, namely, to verify whether differences among the scores are true and not due to measurement errors ; (b) theThe number of questionnaires expected and collected in the study period is reported in Table Tutors returned 447 “Resident-AQs”, which represented 72% of the total ones which were supposed to be returned; 55% were returned within the 15-days requested. The number of “Resident-AQs” received per resident in a year varied from one (for fourth year residents) to four (for the ones attending the first 3 years).Four-hundred and eighty-five “Tutor-AQs” and “Rotation-AQs” were returned  of which 65% were returned with the 15-day deadline.Two-hundred and nine “Residents Affairs Committee-AQs” were returned (i.e., 84% of those expected; 75% of them were returned within 15 days. Ninety-nine residents evaluated the Resident Affairs Committee from one to three times.Tutors tended to be generous in assessing the performance of residents Fig. a, while Figure The psychometric properties of the questionnaires (as outlined in the “Feedbacks on the system were received from 76% of residents and 58% of tutors. Interestingly, 80% of the tutors but only 60% of the residents felt that the purpose of the evaluation system was clear, that the meaning of the different items was easy to understand and that the entire system would help to improve the performance of residents and the quality of training. Most residents and tutors thought that the internet-based system was easy to use.The relatively high rates of questionnaires completed and returned in time indicate that the evaluation system adopted within the framework of the University of Padua’s Pediatric Residency Program was successfully implemented. In fact, the analysis of the validity of the various responses provided, as assessed by conventional psychometric tests, and the fact that most tutors and residents returned the questionnaires within the time set, indicate that the task of completing the questionnaires was taken seriously by all.The aim of the evaluation system was to provide residents, tutors and the Resident Affairs Committee with feedback regarding their performance, based on objective data, and thus to provide a base on which to continuously improve the program. We do not yet have objective data of the impact of this evaluation system on the quality of the Pediatric Residency Program. However, the presentation, during the yearly faculty meetings, of the scores obtained by each tutor and rotation has stimulated efforts to improve. Indeed, the scores assigned to tutors and rotations improved over the years. Furthermore, the fact that all the Division Chiefs were interested in having private discussions on the scores received is a sign that they took seriously the judgments received. The same applies to the residents, who were eager to receive, privately, feedback on their performance. Fortunately, no conflicts seemed to have emerged between residents and tutors and no one was offended by negative judgments. We believe that this evaluation system set a positive circle, considering the improvement of many scores of “Tutor-AQs” and “Rotation-AQs” documented over the years and the impression we had had that everybody took quite seriously the judgments received.We acknowledge some intrinsic limitations of the system, namely, the fact that the “Tutor-AQ” was meant to target only the performance of the Division Chief, thus neglecting the contribution of all the other staff members. More should be done in order to refine it and, more importantly, to document its impact in improving the program.Finally, we are aware that comprehensive evaluation systems require common guidelines and rules, well-equipped infrastructures and financial resources. However, we believe that good organization, strong motivation, and a culture of educational commitment can result in an excellent residency program, even with limited financial resources. This is the ultimate reason why we thought appropriate to share this limited experience.


When tested in a randomized controlled trial (RCT) of 31,411 patients, the nurse-led 6-PACK falls prevention program did not reduce falls. Poor implementation fidelity  may explain this result. Despite repeated calls for the examination of implementation fidelity as an essential component of evaluating interventions designed to improve the delivery of care, it has been neglected in prior falls prevention studies. This study examined implementation fidelity of the 6-PACK program during a large multi-site RCT.Based on the 6-PACK implementation framework and intervention description, implementation fidelity was examined by quantifying adherence to program components and organizational support. Adherence indicators were: 1) falls-risk tool completion; and for patients classified as high-risk, provision of 2) a ‘Falls alert’ sign; and 3) at least one additional 6-PACK intervention. Organizational support indicators were: 1) provision of resources ; 2) implementation activities ; and 3) program acceptability. Data were collected from daily bedside observation, medical records, resource utilization diaries and nurse surveys.All seven intervention components were delivered on the 12 intervention wards. Program adherence data were collected from 103,398 observations and medical record audits. The falls-risk tool was completed each day for 75% of patients. Of the 38% of patients classified as high-risk, 79% had a ‘Falls alert’ sign and 63% were provided with at least one additional 6-PACK intervention, as recommended. All hospitals provided the recommended resources and undertook the nine outlined program implementation activities. Most of the nurses surveyed considered program components important for falls prevention.While implementation fidelity was variable across wards, overall it was found to be acceptable during the RCT. Implementation failure is unlikely to be a key factor for the observed lack of program effectiveness in the 6-PACK trial.ACTRN12611000332921 (29 March 2011).The 6-PACK cluster RCT is registered with the Australian New Zealand Clinical Trials Registry, number The online version of this article (doi:10.1186/s12913-017-2315-z) contains supplementary material, which is available to authorized users. In-hospital falls continue to be a major clinical and economic problem for hospitals . They arFalls are a complex problem , occurriDespite repeated calls for the examination of implementation fidelity as an essential component of undertaking trials to evaluate interventions designed to improve the delivery of care , it has “the bottom-line measurement of implementation fidelity” [Examining implementation fidelity is particularly relevant for complex interventions in multi-center trials, where the same intervention may be implemented and received in different ways across sites , 25. It idelity”  incorporn = 31,411 patients), the 6-PACK program did not reduce falls  or fall injuries  [This study concerns the implementation fidelity of the 6-PACK program, a targeted nurse-led multi-component falls prevention program designed specifically for acute hospital wards . It incon = 3), general surgical (n = 3), general medical short stay (n = 1), specialist medical (n = 4) and specialist surgical (n = 1). All patients admitted to participating wards were included in this study.The methodology and findings of the RCT, conducted between January 2012 and April 2013, are described elsewhere , 31. IntThe RCT was a pragmatic trial with the 6-PACK program implemented by ward nursing staff as part of usual care practice. Data from a sample of intervention ward nurses and senior managers from each hospital were used in this evaluation. Sampling procedures are described in the data collection section below.The 6-PACK program Fig.  was implThe implementation of the 6-PACK program was managed at a central level (by the research team) and tailored locally by participating hospitals. It was informed by two mixed-methods studies: 1) perceived acceptability of the 6-PACK program   and 2) pSite clinical leaders were provided with a comprehensive 6-PACK implementation, training and monitoring guide. Throughout the trial, support was also provided by the research team  on a needs basis via telephone, email and site visits. Need was determined by the site clinical leader or the research team and based primarily on ward program adherence. This included review of complex fall cases, additional training of site clinical leaders on the program and implementation strategies such as audit and feedback and trouble-shooting problems arising with equipment use. Further detail on the 6-PACK program and its implementation can be found elsewhere , 31.An adapted version of the framework developed by Carroll and colleagues was used to assess implementation fidelity. Outcome indicators and data collection sources are summarized in Table Nursing practice adherence indicators (program dose) were: 1) falls-risk tool completion each day of admission; and for patients classified as high-risk provision of 2) a ‘Falls alert’ sign; and 3) a sign and at least one additional 6-PACK intervention. As this was a ward-based intervention and all patients admitted to participating wards were recruited as part of this study, program coverage was not specifically examined. Organizational support indicators were: 1) provision of hospital and ward resources ; 2) implementation activities ; and 3) program acceptability among nursing staff.Data were prospectively collected. Data on program adherence were collected through daily audit of patient care plans and structured bedside observation. Data collection was undertaken by members of the research team (site data collectors) using a standardized tool. Budget constraints meant these data were only able to be collected from the commencement of the cluster RCT until December 2013. Weekly site clinical leader resource utilization diaries detailed time spent on program implementation activities. A member of the research team recorded attendance to site clinical leader training sessions and meetings and kept a log on the compliance of hospitals and wards to resource commitment, care-plan integration and data provision. Nurse perceptions were explored as part of a staff knowledge, attitudes and practices survey, developed by the research team. Nineteen items in the staff survey related to program acceptability scored on a 5-point Likert scale . The survey was administered prior to program implementation. The researcher attended handover sessions or designated ward meetings to distribute the survey. Nurses that had worked on an intervention ward for at least 7.5 h per week in the 2 months prior were invited to complete the survey. Completed surveys were placed in sealed boxes collected by the researcher at the end of the 2-week dissemination period. To avoid hierarchical coercion, Nurse Unit Managers were not involved in the distribution of the surveys. The number of surveys distributed to, and returned from, each ward was recorded.Descriptive statistics were used to profile the study sample, program adherence and staff perceptions. Adherence to program content was categorized into three levels: adherent, partially adherent and non-adherent. Program dose was defined as a proportion. Data on hospital and ward resources and implementation activities were measured against expectations and/or recommendations and categorized into three level of adherence: adherent, partially adherent and non-adherent. Items were only coded as adherent if the recommendation was completely met. Analyses were undertaken using Stata MP v13 statistical software. Data visualizations of staff survey item responses were created using Tableau Desktop v9.0.During the RCT there were 22,670 admissions to the intervention wards representing 17,698 individual participants, as some patients were admitted more than once.Data on program use were collected from 103,398 daily observations and medical record audits . All wards implemented low-low beds that lowered to 250 mm from floor level . However, only two of the six hospitals implemented beds that lowered to 100 mm from the floor as recommended.All hospitals appointed an executive sponsor to support program implementation during the RCT. There was general compliance with the appointment of a site clinical leader. However, three of the six hospitals had less than the recommended 0.1 full time equivalent (FTE) per intervention ward. Hospital 4 provided the greatest site clinical leader resources (0.2 FTE per intervention ward) and Hospital 3 supported the least (0.03 FTE per intervention ward). Hospital 4 experienced a change in their site clinical leader 4 months into program implementation. All hospital wards nominated the recommended number of ward champions, although Hospital 2 experienced a number of staff changes that meant the role was vacant for many months.All hospitals integrated 6-PACK documentation into the patient care-plan. Each site clinical leader attended two formal face-face training sessions and were provided with the 6-PACK implementation guide. All site clinical leaders were adherent with implementation activities Table , with vaA total of 208 intervention ward nurses (66% response rate) completed the survey. The majority of respondents were qualified registered nurses (85%) and had worked on the participating intervention wards for more than 12 months (68%) with at least five shifts per week (51%). Results have been summarized in Fig. Nurse responses toward the prevention of falls were less positive. Almost one third agreed that falls cannot be avoided in older people . A degree of uncertainty amongst nurses for some items were noted, for example, more than one third neither agreed nor disagreed that there was strong leadership for falls prevention on their ward . Moderate variation in staff responsiveness on some survey items between hospitals were observed  . Nearly When interpreting these results, the following methodological limitations should be considered. The provision of 6-PACK interventions was reliant on the daily completion of the falls-risk tool and accurate classification of patients at high-risk. In addition, it does not necessarily reflect optimum practice  or allow for the examination of the appropriate use of strategies. The authors acknowledge that qualitative data would provide additional insights into the factors influencing implementation fidelity, yet it was beyond the scope of this study. Considerable but different barriers to implementation appeared to be present in some hospitals/wards during the 6-PACK trial. Further exploration of organizational characteristics, existing infrastructure and surrounding social structures, ward location, the availability, skills and leadership qualities of the local implementation team and other competing priorities experienced by staff should be considered.In-hospital falls represent a complex problem that occurs within the complex environment of a hospital ward, often to individuals with complex care needs. Therefore, it is inevitable that examining implementation fidelity is also challenging. Currently there is limited consensus on how best to define and measure implementation fidelity . The fraIn this study, while program adherence was not optimal at some participating hospitals, we considered overall implementation fidelity sufficient in the 6-PACK trial. Therefore, implementation failure is unlikely to be a key factor for the observed lack of program effectiveness on fall and fall injuries. This study provides valuable learnings for the development and implementation of future falls prevention programs or other nurse-led safety programs. Given the challenges and costs involved with the implementation of complex health interventions, such as falls prevention programs, examination of implementation fidelity should be an integral part of any evaluation of an intervention’s effectiveness.Additional file 1:Program adherence of each individual ward by month (DOCX 43 kb)Additional file 2:Time allocation by site clinical leader to 6-PACK program implementation activities (DOCX 15 kb)Additional file 3:Influencing factors to implementation of the 6-PACK program: staff acceptability by hospital (PDF 136 kb)