Health-related quality of life (HRQoL) was found to improve in participants of weight management interventions. However, information on moderately overweight youth as well as on maintaining HRQoL improvements following treatment is sparse. We studied the HRQoL of 74 overweight, but not obese participants  of a comprehensive and effective six-month outpatient training at four time-points up to 12 months after end of treatment.HRQoL was measured by self-report and proxy-report versions of the generic German KINDL-R, including six sub domains, and an obesity-specific additional module. Changes in original and z-standardized scores were analyzed by (2×4) doubly multivariate analysis of variance. This was done separately for self- and proxy-reported HRQoL, taking into account further socio-demographic background variables and social desirability. Additionally, correlations between changes in HRQoL scores and changes in zBMI were examined.2 = 0.14-0.19). Generic HRQoL further increased after end of treatment. The largest effects were found on the dimension self-esteem . Correlations with changes in weight were gender-specific, and weight reduction was only associated with HRQoL improvements in girls.There were significant multivariate time effects for self-reported and proxy-reported HRQoL and a significant time-gender interaction in self-reports revealed (p < .05). Improvements in weight-specific HRQoL were evident during treatment  children and adolescents could be demonstrated. Improvements in HRQoL were not consistently bound to weight reduction. While changes in weight-specific HRQoL were more immediate, generic HRQoL further increased after treatment ended. An extended follow-up may therefore be needed to scrutinize HRQoL improvements due to weight management.clinicaltrials.gov NCT00422916 Overweight and obesity pose an increasing health problem in most industrialized countries ,2. The GHealth-related quality of life (HRQoL) relates to the self-perception of health and consists of ratings of well-being and functionality in important life areas, including physical functioning, bodily pain/symptoms, emotional well-being, self-esteem, social functioning and family relations ,9. WhileResearch shows that in overweight or obese youth weight-specific as well as generic HRQoL is likely to be impaired ,10-12, eCorrespondingly, HRQoL was found to improve in participants of weight management interventions -13,15-20The aim of the present paper was to analyze changes in HRQoL among overweight children and adolescents participating in a six-month outpatient training programme for weight reduction with a follow-up period of 12 months and to relate changes in weight to changes in HRQoL. We expected a positive impact for weight loss on HRQoL.The study was designed to be a randomized controlled trial (RCT) with a waiting list control group (participation in the intervention after six months without any intervention) to assess the effects of a six-month outpatient training on weight reduction and different secondary outcomes, including HRQoL. Details and results of the main study are described elsewhere ,22.th percentile and ≤97th percentile, according to German percentiles [Families were invited for participation in the study mainly by media  and paediatricians. To be included in the study, children had to be 8 to 16 years old, overweight, apparently healthy and not be taking any medication. Overweight was defined as a BMI ≥90centiles . Obese cParticipants differed in the time period that elapsed between study enrolment and start of intervention. Children of the pilot study and those from the RCT control group (n = 32) had to wait six months before entering the treatment, while children of the RCT intervention group (n = 34) and those enrolled after end of recruitment for the RCT were assigned to the next available training course. Participants with longer waiting periods did not differ in any of the compiled characteristics from those who started the intervention directly at beginning of the treatment, and participants of the RCT did not differ from those not randomly allocated. Furthermore, length of the waiting period before intervention did not affect the results described. The groups were therefore combined for the presented analysis. The final sample, after exclusion of 30 dropouts before beginning of training, 10 cases ineligible at start of training , and 4 cases with missing questionnaires at second follow-up, consisted of 74 children.HRQoL and weight status of all participants were measured at beginning (pre-treatment) and end (post) of the six-month training, as well as six (follow-up 1) and twelve months (follow-up 2) after end of intervention. In order to follow an intention-to-treat approach, missing values due to drop-out during intervention (n = 2) or lost to follow-up after end of intervention (n = 1) were set back to baseline values and included in the analyses. Excluded cases were compared with the analyzed sample and no significant differences were found regarding age, weight status, gender, foreign background, socio-economic or family status, social desirability or baseline HRQoL scores (p > .05).Written informed consent was obtained from all children and their parents prior to study start. The study was approved by the local ethics committee of the University of Bremen.The six-month outpatient training “Obeldicks light” was offered in two cities in north-west Germany. The intervention has been described in detail . BrieflyPreviously published analyses showed that the intervention was successful in significantly reducing several parameters such as body weight, body fat percentage, waist circumference, and blood pressure as compared to the control group at the end of treatment. 94% of children reduced their body mass index z-score (zBMI) during the training and 24% reached normal weight . Follow-Health-related quality of life was measured by German age-specific self-report versions and parent-proxy versions of the KINDL-R questionnaire . KINDL-RThe KINDL-R was chosen because of its sensitivity to change ,27 and bHRQoL measures were z-standardized using German norms from a recent representative sample  to allowTo calculate children’s BMI, height was measured to the nearest centimetre using a rigid stadiometer. Weight was measured in underwear to the nearest 0.1 kg using a calibrated balance scale. The degree of overweight was quantified using Cole’s least mean square method expressing BMI as a standard deviation score (zBMI)  using reBackground variables such as socio-economic status (SES), ethnicity and family status were assessed by parent questionnaires ,32. SES Data were screened for missing values and outliers. As missing values on single occasions and subscales were common and Little’s MCAR test indicated missingness at random, individual missing values were estimated by a formula based on the individual mean throughout measurements and the group mean at the measurement in question . In geneNot all scales showed normal distribution. Since distributions differed between subscales and time points, no uniform normalizing transformation was possible. However, sample size was large enough to assume robustness of MANOVAs .HRQoL data were tested for univariate and multivariate outliers by inspection of boxplots and Cook’s distance. There was one case with a high Cook’s distance (1.85). However, as MANOVA results were very similar with and without this case, we included it unchanged in the analyses.Descriptive statistics were computed for boys and girls and the total sample and compared using independent t-tests for continuous measures and chi-square-tests for categorical variables. For the main analyses doubly multivariate analyses of (co-)variance were computed separately for child and parent-proxy HRQoL original and z-scores to compare the four measurements from pre to follow-up 2 using GLM procedures of IBM SPSS Statistics 20.0 . In subsequent univariate analyses successive HRQoL scores were contrasted with pre-treatment scores.Given that weight-specific HRQoL and weight complaints were only reported by adolescents (n = 25), these two variables were examined univariately only and not included in the multivariate child models. Aside from that, there were no z-scores for weight-specific HRQoL and weight complaints, so MANOVAs on z-scores were constrained to generic HRQoL.In all models gender and SES were included as potential between-subjects factors, testing the main effects and interactions with time. Because of resulting small group sizes, these factors were only included in the final models, if there was a significant interaction with time. Furthermore, duration of the waiting period between study enrolment and start of the training, baseline age, zBMI at baseline, and social desirability were tested as potential covariates and included in the final model, when there was a significant interaction with time.Spearman’s rank order correlations between changes in zBMI over the course of the treatment (short-term from pre to post) as well as from start of training until the 12-month follow-up (long-term from pre to follow-up 2) and short- and long-term changes in HRQoL scores were analyzed. Since we expected weight reduction to correlate with HRQoL improvements, significances of the correlations are given for one-tailed tests.Unless otherwise specified, p-values ≤ 0.05 were considered significant.Power analysis revealed that with a sample size of 74 and correlations between repeated measures of r = 0.5, an effect of medium size would be detected with a power > 0.95, and the power for univariate contrasts exceeded 0.80 after adjusting alpha-level for multiple tests. For self-reported weight-specific HRQoL in adolescents (n = 25), however, a power > 0.80 was given for large effects only .Table Table Overweight according to zBMI was significantly reduced during treatment and remained relatively stable until follow-up 2 .Neither age, length of the waiting period, baseline zBMI, nor social desirability as covariates showed significant main effects or interactions with time. There was also no significant main effect or interaction of SES or gender. For the raw scores there was a significant multivariate effect of time on HRQoL (see Table For the z-scores the time effect was likewise significant Table . Univari2 = 0.10).For children’s self-reported raw scores we found a significant effect for social desirability, where children with higher scores had significantly higher HRQoL scores, but there was no interaction with time, so social desirability was not included in the final model. All other tested covariates and SES showed neither main effects nor interactions with time. The main effec of time only approached significance (p < .10), but there was a significant time-gender interaction in the multivariate test Table . Univari2 = 0.07).In terms of the z-scores, results for the covariates resembled those of the raw scores. There was a significant time effect, as well as a nearly significant time-gender interaction. Univariate contrasts showed that only self-esteem at follow-up 2 remained significant after Bonferroni adjustment. The unadjusted interaction contrast for gender × time was p < .05 for school at follow-up 2  and long-term (follow-up 2 minus pre) changes and Spearman’s rank order correlations between HRQoL and zBMI changes were computed for the entire sample as well as for girls and boys separately . Inspection of gender-specific correlations showed, however, that the described patterns were primarily true for girls.In terms of generic HRQoL, short-term weight reduction was significantly correlated with improved physical well-being during treatment and long-term improvements in school-related HRQoL (self-reported). Self-reported short- and long-term improvements in school-functioning as well as short-term improvements in self-esteem were significantly associated with long-term weight reduction. All these relations, however, again were only present in girls. Girls’ self-reported changes in emotional well-being during treatment also correlated significantly with long-term weight changes.Correlations contrary to the expected direction (namely that a reduction in zBMI would be associated with improved HRQoL) were found on some HRQoL subscales in boys, primarily for self-reported HRQoL. Some parent-reported HRQoL scores also declined while zBMI was reduced during treatment, revealing associations opposite to those in girls, but these correlations did not reach significance.Significant associations were found between boys’ short-term changes in social well-being (friends) and weight complaints and long-term changes in zBMI, which indicate that improved well-being was rather associated with a weight gain or that reduced well-being in these domains preceded long-term weight reduction. Long-term weight reduction was furthermore significantly associated with reduced self-reported HRQoL total score, emotional, familial and social well-being as well as parent-reported physical well-being in the long run. Associations between boys’ weight reduction and decreased parent-reported weight-specific HRQoL showed the same trend, although not statistically significant.The magnitude of correlations was low to medium in general, except for self-reported HRQoL in girls.The main aim of the present study was to analyze changes in HRQoL during and after participation in an outpatient training programme for weight reduction in overweight children and adolescents. Furthermore, changes in HRQoL were related to weight changes.Results from children’s self-report and parent proxy-report showed significant improvements in several HRQoL dimensions. All subscales reached or outreached population means at follow-up 2. The largest increases were found for weight-specific HRQoL during treatment. By contrast, subscales of generic HRQoL often continued to increase after end of treatment and were mostly not significantly higher than pre-treatment until follow-up 2 with the exception of parent-reported self-esteem. In general, parents reported more marked changes and changes were more obvious with age- and gender-specific norm scores than with raw scores. Correlations in the expected direction, namely associations of weight reduction with HRQoL improvements, were found primarily in girls.Our results on HRQoL-improvements in participants of a successful weight management intervention are in line with the results of other studies that analyzed mainly obese children and adolescents ,16,18,20Compared to age- and gender-specific population norms , parent Concerning changes in HRQoL dimensions, we found the largest increases in weight-specific HRQoL during treatment. Because disease-specific instruments should be more sensitive to changes during treatment , this reIn terms of generic HRQoL we found the most notable increases in self-reported as well as parent-reported self-esteem equivalent to a moderate effect-size, although not all contrasts reached significance. While self-esteem is not included in most HRQoL-instruments and was therefore not covered by all studies, some studies also demonstrated significant increases ,16,20,40With respect to other HRQoL dimensions the literature reveals inconsistent results which may be due to different instruments, self-report versus parent proxy-report versions and differences between samples and treatments. However, studies with obese participants found significant HRQoL increases on at least some HRQoL subscales during treatment . GriffitAccording to the literature ,42,43, iFrom our results in relation to other studies it can be supposed that the expectable magnitude of positive HRQoL changes during and after weight management training in general varies with the degree of impairment before treatment . Larger Effects on generic HRQoL were more pronounced when looking at norm scores than on original scores. In general, HRQoL decreases with age during adolescence , so thatThe observed changes of HRQoL during our study have important implications for future studies on weight management: As improvements continued after end of treatment on most HRQoL dimensions and most scores were not significantly different post-treatment, longer follow-up periods and larger study populations seem necessary to verify psychosocial improvements at least in only moderately overweight children and adolescents. In addition, it may be that in some preceding studies on obese youth more improvements would have been revealed with longer follow-up measurements. This is in line with Tsiros et al. , who conDirect correlations between weight reduction and HRQoL changes were low in our study for the overall sample and mainly significant for weight-specific HRQoL. Other studies confirm these low associations. Studies by Yackobovitch-Gavan and colleagues ,19, for However, unlike other studies we additionally looked at gender-specific associations and found clear gender differences in results, where weight reduction of girls was more favourably correlated with HRQoL changes, while in boys some correlations opposite to the expected direction were found. These gender-specific effects partially averaged out to low correlations for the overall sample. Gender-specific associations were particularly pronounced in terms of long-term changes. None of the previous studies reported associations with long-term changes. Short-term HRQoL changes in our study were often associated with long-term weight changes and pointed to improvements in emotional well-being, self-esteem, school, and weight complaints preceding long-term weight maintenance in girls, while in boys long-term associations were rather unfavourable for generic HRQoL. Possible negative effects as well as gender-specific results should be further monitored, although the gender effects observed in our study may well be sample-specific, for our male subsample was quite small (n = 24). Furthermore, HRQoL scores increased in both boys and girls during and after treatment, so that there is no indication that treatment-induced weight reduction overall showed detrimental effects.Our results point to HRQoL changes precdicting long-term weight changes in girls rather than vice versa, since on some scales short-term HRQoL changes were associated with long-term weight changes, while no direct correlations were found between long-term changes in HRQoL and long-term weight development. Although no causal effect can be proved by this study, an effect of weight change that occurs later in time on previous HRQoL changes can be ruled out. A possible interpretation, therefore, is that in girls an improved HRQoL, especially in terms of self-esteem and emotional well-being, helped in sustaining a reduced weight, while in boys no such effect was revealed.In general, weight reduction seems not to be the only critical factor for HRQoL changes during or subsequent to weight management treatment, since it was not consistently associated with HRQoL changes. Further, associations with weight reduction may differ between boys and girls. Because increases in generic HRQoL were not directly related to weight reduction in most cases, improvements may depend primarily on specific content of programme  more than on diet changes that directly lead to weight reduction, or improvements may not parallel weight changes temporally. This has important implications for practice. While long-term weight reduction may be difficult to achieve for many overweight youth, interventions that lead to HRQoL improvements may increase psychosocial health and well-being even in the absence of weight loss. Hence, they may be an alternative to help those who are not able to achieve a healthy weight in coping with their condition. Future studies should therefore clarify which specific components of the intervention result in HRQoL improvements in girls and boys.As far as we know, our study is the first to demonstrate HRQoL-changes during and after treatment in moderately overweight children and adolescents. It has the strengths to track changes on generic and weight-specific HRQoL dimensions over multiple occasions until one year after end of treatment from the perspective of the children themselves as well as from parents’ point of view and relate these changes to weight reduction.However, there are some limitations to be considered, when interpreting our results. At first, reliability of some HRQoL subscales was unsatisfactory, especially for friends, self-esteem and school; although internal consistency was only slightly lower than in other samples ,45. ThisParticipation in a six-month outpatient training programme for weight reduction had favourable effects on HRQoL of moderately overweight children and adolescents. Improvements in HRQoL were most notable one year after end of treatment in generic HRQoL and near-term rather in weight-specific HRQoL. These HRQoL improvements were hardly related to weight reduction in the entire sample, but associations were visible in girls. Positive effects on HRQoL were demonstrated for an overall successful treatment, though, and therefore may not hold true for programmes that do not result in weight reduction.Given that HRQoL improvements continued after treatment, future studies should include longer follow-up periods to document HRQoL development in the long run. An issue that should be further monitored is the potentially negative impact weight reduction may have on some HRQoL-dimensions in boys.HRQoL: Health-related quality of life; zBMI: z-score of body mass index calculated using Coles LMS method.The authors declare that they have no competing interests.EF participated in the acquisition of data, conceptualized and performed the statistical analyses and drafted the manuscript. PK and TR conceived of the study and its design and helped in interpreting the results. AS, KW, and TR participated in acquisition of the data and were involved in implementing the intervention. All authors critically revised the manuscript and read and approved the final manuscript.


In current study, we report an adult patient with the tumor. The patient presented similar morphologic, immunophenotypic and genotypic changes of the disease with that occurred in children, whereas clinically, he showed a prolonged clinical course without hepatosplenomegaly or generalized lymphadenopathy. Whether there are some differences in biologic behavior between children and adults still remains unknown and it is necessary to collect more data to observe and to investigate in the future. In current study, an adult patient with hydroa vacciniforme-like lymphoma was reported with review of the literature.Hydroa vacciniforme-like lymphoma (HVLL) is a cutaneous cytotoxic T-cell or natural killer (NK) cell originated lymphoma occurring in children and adolescents, but not in adults, mainly reported from Asia, Central and South America. In the updated WHO Classification of tumor of Haematopoietic and lymphoid tissues (2008), hydroa vacciniforme-like lymphoma has been recognized as one of the Epstein-Barr virus (EBV)-positive lymphoproliferative disorders of childhood . In curr9/L, white blood cell (WBC) 4.10×109/L, hemoglobin 124 g/L; Lactate dehydrogenase (LDH) 461 IU/L, CD3 subpopulation 83.20%. EBV-DNA level was 1.33E+04 copies/mL detected by real-time quantitative Polymerase Chain Reaction (RT-qPCR). EBV-VCA-IgA was positive, while EBV-EAD-IgG was negative. Type-B ultrasonic examination showed portal vein thickening and splenomegaly. Sputum culture found Pseudomonas aeruginosa. Cervical lymph node biopsy was performed and systemic EBV-positive T-cell lymphoproliferative disease was diagnosed. Clinically, respiratory tract infection was also considered. The protocol of this study was approved by the Institutional Review Board for ethical committee of West China Hospital of Sichuan University, and the study protocol adhered to guidelines by Helsinki Convention.A 19-year-old Asian male, complained of repeatedly hyperpyrexia for two years, multiple papules of the head for more than one year, papules and blisters of the face and trunk for eight months  include extranodal NK/T-cell lymphoma nasal type, aggressive NK-cell leukemia, systemic EBV-positive T-cell lymphoproliferative disorder of childhood, and hydroa vacciniforme-like T-cell lymphoma. All occur more frequently in Asians, and in the Native Americans from Central and South America and Mexico [008 incluHydroa vacciniforme-like lymphoma (HVLL) has also been called angiocentric cutaneous T-cell lymphoma of childhood , and hydHVLL is characterized by recurrent papulovesicles which mainly presented in the sun-exposed area, and often accompanied by fever, lymadenopathy, hepatosplenomegaly and increased liver enzyme, and the two year survival rate is 36% . In currHydroa vacciniforme-like lymphoma should be distinguished from a group of T-cell and NK cell lymphoproliferative disorders presenting in the skin. The first is Hydroa vacciniforme (HV). Typical HV is a self-limited skin disease characterized by vesiculopapular eruptions on the sun-exposed regions with vacciniforme scarring. Systemic involvement is absent. Patients with HV-like lymphoproliferative disorders may show some clinical features mimicking typical HV at the onset of the disease . In addiWith regard to the treatment of the tumor, Interferon-α (IFN-α) is the most commonly selected whose major side effects are fever and cytopenia. Corticosteroid is usually used to control the symptoms, but it is not suitable to be used persistently. Chemotherapy is uncommonly used, because only 30% of patients may have partial response. Xu reported six Chinese cases of childhood, in which, four patients received IFN-α, and two of them also had chemotherapy (the regimen was unknown) . In currWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MW, SW and QPY conducted data analysis and helped to draft the manuscript. YML, LMG and HS carried out the immunohistochemical staining and molecular genetic studies. WPL wrote the manuscript and supervised throughout the study. All authors read and approved the final manuscript.


Whether healthy older people can benefit from cognitive training (CogTr) remains controversial. This study explored the benefits of CogTr in community dwelling, healthy, older adults and compared the effects of single-domain with multi-domain CogTr interventions.A randomized, controlled, 3-month trial of CogTr with double-blind assessments at baseline and immediate, 6-month and 12-month follow-up after training completion was conducted. A total of 270 healthy Chinese older people, 65 to 75 years old, were recruited from the Ganquan-area community in Shanghai. Participants were randomly assigned to three groups: multi-domain CogTr, single-domain CogTr, and a wait-list control group. Twenty-four sessions of CogTr were administrated to the intervention groups over a three-month period. Six months later, three booster training sessions were offered to 60% of the initial training participants. The Repeatable Battery for the Assessment of Neuropsychological Status , the Color Word Stroop test (CWST), the Visual Reasoning test and the Trail Making test (TMT) were used to assess cognitive function.P < 0.05). At the 12-month posttest, the multi-domain CogTr showed training effects on RBANS, delayed memory and visual reasoning, while single-domain CogTr only showed effects on word interference. Booster training resulted in effects on RBANS, visual reasoning, time of trail making test, and visuospatial/constructional index score.Multi-domain CogTr produced statistically significant training effects on RBANS, visual reasoning, and immediate and delayed memory, while single-domain CogTr showed training effects on RBANS, visual reasoning, word interference, and visuospatial/constructional score (all Cognitive training can improve memory, visual reasoning, visuospatial construction, attention and neuropsychological status in community-living older people and can help maintain their functioning over time. Multi-domain CogTr enhanced memory proficiency, while single-domain CogTr augmented visuospatial/constructional and attention abilities. Multi-domain CogTr had more advantages in training effect maintenance.Chinese Clinical Trial Registry. Registration number: ChiCTR-TRC-09000732. Declining cognition in older people accounts for a major portion of increased health care costs . As adulHigh levels of mental activity can potentially decrease an individual's risk for developing dementia by approximately 50%  and can et al. [et al. [Several studies have explored the effects of CogTr interventions in healthy older individuals . Howeveret al.  focused  [et al.  found no [et al. ,9-14, re [et al. ,15-17, p [et al. ,17-21, r [et al. , solving [et al. , or stra [et al. . For exa [et al. . However [et al. .et al. [Previously, few studies evaluated the effects of multi-domain CogTr interventions in healthy older adults. Recently, Oswald et al.  demonstret al.  conducteThe first aim of the present study was to explore whether community dwelling, healthy, older adults could benefit from CogTr. The second aim was to distinguish the different effects of single-domain and multi-domain CogTr interventions in healthy older adults. Specifically, we used a block-randomized, controlled, single blind trial to answer the following questions: Can healthy older adults benefit from CogTr? Does multi-domain CogTr improve and maintain cognitive abilities more than single domain CogTr? Can single-domain CogTr enhance untargeted cognitive functions in healthy older adults? Does booster training either augment or help maintain previous training effects? Which cognitive domain benefits most from CogTr?This study was approved by the Human Research Ethics Board of Tongji Hospital in Shanghai, China and all participants gave written informed consent before being enrolled in the study (LL(H)-09-04).All participants were community dwelling, older adults with good functional capacity and no evidence of significant cognitive impairment, living independently at the time of enrollment. The participants were recruited from three community centers around Tongji Hospital in Shanghai via a dispatched notice and broadcasting by the local institute of community service from March 2008 to April 2008.All participants were interviewed in person by professional interviewers before being admitted to the study. To be eligible, subjects could not exhibit hearing, vision, or communication difficulties that would prevent completion of cognitive training. Vision, hearing and communication status were evaluated during the eligibility screening by a trained interviewer. Other eligibility criteria included age (65 ≤ age ≤ 75 years) at screening, and educational level (≥ 1 year). The Chinese version of the Mini-Mental State Examination score of 19 or above was required for enrollment . The nor2×(Ζα/2+Ζβ)2, α = 0.05, Ζα/2 = 1.96, β = 0.1, Ζβ = 1.65, the values of σ and Δ were determined on the basis of our previous studies. Combined with our previous drop-out rate, ninety individuals were assigned to each group. The statistical power level was set to 0.80, and the statistical significance level was set at 0.05. The non-training control group served to match for the social contact associated with CogTr and to determine the net effects on cognitive function specific to our CogTr intervention. Each intervention group received 24 sessions of CogTr over a three month period, conducted by qualified trainers. Three booster-training sessions were offered to 60% of the initial training sample six months after completion of the first 24 sessions of CogTr. All participants, including the control group, attended a lecture on different aspects of healthy living every two months. Cognitive assessors were blinded to intervention assignment. Training exposure and social contact were equal across interventions. Measurement outcomes were collected at baseline, immediately after CogTr completion, and during a 6-month and 12-month post training follow-up interview.A block-randomized, controlled design was used to test whether community dwelling elderly could benefit from CogTr. Using the Statistics Analysis System (SAS) software, the randomization table was generated with a block randomization procedure (randomization occurred within three blocks) provided by an independent statistician who had no information about the study subjects. Each subject received a number within a concealed envelope indicating his/her randomization assignment. The study employed a three-group design, including one wait-list control group and two intervention groups, one receiving single-domain CogTr and one receiving multi-domain CogTr. According to the sample size equation n = (σ/Δ)CogTr employs standardized exercises to provide structured practice of tasks relevant to specific aspects of cognition with the intention of specifically addressing cognitive functioning and/or cognitive impairment . Most deCogTr intervention took place in small group settings, with an average class size of 15 individuals. The training occurred at a frequency of twice a week over a 12 week period for 24 sessions from June to August 2009. The multi-domain CogTr targeted on memory, reasoning, problem solving strategies, visuospatial map reading skill development, handcraft making, and health and physical exercise. The single-domain CogTr focused specifically on reasoning training, including the towers of Hanoi, numerical reasoning, Raven Progressive Matrices, and verbal reasoning.Each session lasted 60 minutes. A lecture was presented during the first 15 minutes of each hour, focusing on education about the diseases common in older persons. Then, all participants were trained in one specific cognitive technique during the second 30 minutes of each session. The trainer taught the participants about a certain strategy or technique. All participants received instructions about the rule, including its methods and function and how to use it in daily life, such as Loci memory training, face/name memory training, and so on. Sometimes, the participants needed to be tested on the content to assess their learning success. The last 15 minutes were used to consolidate the newly practiced skills by solving some real-life problems. Participants received structured training in topics delivered via PowerPoint presentations. The instructor followed a manual of structured curricular format for training, but allowed discussion based on the needs and interests of participants. Participants were assigned homework once a week including cognitive tasks relating to the content of the latest session, health knowledge reading, Chinese calligraphy, and simple graph drawing. The homework was reviewed every week.At the end of each session, participants evaluated the type of intervention used for that session. Subjects were asked to provide feedback on performance achievement, to describe their opinion of or to provide suggestions for each intervention, and to document the self-training practice they performed outside the training sessions during their daily life in a booklet which they handed in to the study personnel after completing all 24 training sessions. A study observer monitored each individual's performance and assessed the quality of the skill-related practice during the intervention. The observers provided useful feedback to the instructor with the aim of preventing participants from developing poor practice habits and precluding the inadvertent reinforcement of detrimental training.Six months after completion of the initial 24 cognitive training sessions, booster training was provided to a randomly selected 60% of subjects in each intervention group. Booster training, which comprised one additional 60-minute training session every month for the next three months from April to June 2010, reinforced the initial training by reviewing previously practiced content.Composite outcome measures were created to represent multiple cognitive domains that matched the content of each intervention. All measures were administered at baseline and three times post-test after the intervention. Each of the composite measures was designed to assess ability rather than performance on a single test. The outcome measures included the Repeatable Battery for the Assessment of Neuropsychological Status  which has good reliability and validity in a Chinese community-living older sample ,30, the The RBANS consists of six scores: Total Score and five Index Scores. The latter include Immediate Memory, Visuospatial/Constructional, Language, Attention, and Delayed Memory. With the exception of the delayed memory index which is based on four subtests, each of the other four Index Scores is based on two subtests. The RBANS was designed for evaluation of disease progression and intervention efficacy . All parThe Color Word Stroop Test (CWST) was administered according to standard techniques . The mosThe visual reasoning test is a part of the visual gnostic function tests included in the the World Health Organization Neuropsychological Battery of Cognitive Assessment Instruments for the elderly (WHO-BCAI). It consists of nine items. The test involves tasks requiring identification of patterns in graphic series problems. The visual reasoning test is correlated with cognitive functions of reasoning, summarizing, and analytical ability .The Trail Making Test (TMT) is one of the most frequently used neuropsychological tests in clinical practice, largely due to its good reliability, validity, and sensitivity to cognitive impairment and brain damage . The TMTTo evaluate the effects of initial CogTr and booster training over one year, a general linear model (GLM) - repeated measures was used. The within-subject factor was defined as time, and the between-subject factor was defined as group. The within-subject dependent variables were outcome measures at four time points: baseline, immediately after intervention completion, 6 months post-test and 12 months post-test. The outcome measures at baseline were defined as covariates to control for the slight, but not statistically significant, between group imbalance prior to intervention. Main effects for group and time were determined. The time*group interaction determined the net effect of CogTr intervention, whereas the time*booster*training interaction assessed the training-specific effects of each booster intervention . The intP < 0.05 as the significance level.The net effect size (NES) was used to compare outcome scores  to baseline scores and to control group scores. The NES of training at a specific timepoint was defined as: ((trained mean - control mean at timepoint n) - (trained mean - control mean at baseline))/intra-subject SD. Similarly, the NES of each booster training was defined as: ((booster mean - non-boosted mean at timepoint n) - (booster mean - non-booster mean at baseline))/I ntra-subject SD [From the 320 eligible individuals who were contacted for participation, 270 agreed to attend our study while 50 subjects refused to participate prior to randomization from March 2008 to April 2008. Reasons for refusal included lack of enthusiasm, too busy to participate, and unwillingness to attend the training sessions. Ninety individuals were randomly assigned to each group. One hundred and ninety three individuals completed cognitive assessments at baseline from November to December in 2008, while 173 subjects finished the immediate follow-up after CogTr from September to October in 2009, 166 subjects finished the six-month follow-up after CogTr from March to April in 2010 and 165 subjects finished the one-year follow-up after CogTr from October to November in 2010, respectively. The flow of participants through the entire study is illustrated in Figure The intervention groups were comparable with the control group with regard to age, education and sex composition at each assessment. Subject characteristics of the study groups are summarized in Table P > 0.05)). Attendance-rates per session for the two intervention groups were also not statistically different ). Attendance data for each session is detailed in Figure Intention-to-treat analyses were used. Sixty individuals attended at least one session of multi-domain CogTr, while 59 individuals completed a single-domain CogTr session , indicating that cognitive abilities across study groups were largely comparable before training. The Students-Newman-Keuls test was used to draw a parallel between groups. We found that although the two intervention groups showed no significant between group baseline differences, the intervention groups (M = 0.02) together were significantly different from the control group (M = 0.11) at baseline. The control group showed a little more approximate error number.Cognitive performance outcome measures at baseline are summarized in Table The results of the GLM-repeated measures estimations are summarized in Table P values < 0.01). The NES for each intervention group showed a significantly improved effect. The immediate posttest NES showed no significant difference between multi-domain training and single-domain training . Single-domain CogTr showed a better effect at the 6-month posttest, while multi-domain CogTr resulted in a better effect at the 12-month posttest. The multi-domain CogTr group presented a stable increase from the pretest to each posttest evaluation , whereas it remained stable at 12-month posttest (d time 4-2 = 2.680). In contrast, the performance of the single-domain CogTr group decreased from 6-month posttest to 12-month posttest (d time 4-3 = -1.981) , without a significant difference between groups .RBANS total score revealed a significant main effect of time, group, and significant interaction between time and group  Figure . The maiP < 0.001). The effect of booster training showed no significant difference among groups .The Mini-mental State Examination (MMSE) score revealed no significance of either a group main effect or time*group interaction, whereas the main effect of time was significant . Then NES for each intervention group showed a significantly improved effect after training, especially at the immediate posttest. There was no significant difference in the NES between multi-domain and single-domain CogTr at the immediate  or six-month posttest . The multi-domain CogTr group gradually increased from pretest to each posttest evaluation , whereas it remained stable and at a good effect level at the 12-month posttest. In contrast, the performance of the single-domain CogTr group decreased from 6-month posttest to 12-month posttest (d time 4-3 = -0.538) and led to a small effect at 12-month posttest . Single-domain booster training  showed a better booster effect than multi-domain booster training .The visual reasoning test score revealed a significant main effect of time, group, and interaction between time and group .RBANS immediate memory index score revealed a non-significant main effect of group but significant main effect of time and a significant interaction between time and group. The NES for multi-domain CogTr showed a significantly improved immediate effect after intervention, which is better than the single-domain CogTr performance. The NES revealed scores increased from the pretest to each posttest evaluation Figure . BoosterP values < 0.01). The NES for multi-domain CogTr showed a significantly improved immediate effect after training, a stronger effect than single-domain CogTr performance. Furthermore, the multi-domain CogTr effect remained stable at the 12-month posttest. The temporal trend showed the multi-domain CogTr group score as a steadily increasing line from the pretest to each posttest evaluation. In contrast, the performance of the single-domain CogTr group decreased from the 6-month posttest to the 12-month posttest (d time 4-3 = -1.717) .RBANS delayed memory index score revealed a significant main effect of time, group, and interaction between time and group  Figure . BoosterP = 0.036) compared to the participants who did not attend a booster session .The RBANS visuospatial/constructional index score revealed significant main effects of time and group, whereas their interaction showed a non-significant effect. The NES for single-domain CogTr showed significant improvement after intervention, which showed a better effect than the multi-domain CogTr group at all posttest performance. Temporal trends of intervention groups showed a similar performance, a gradual increase after training and a decrease from the 6-month to 12-month posttest Figure . The pertime4- 1 = 3.740, P = 0.008), while single-domain CogTr increased significantly at immediate  and 12-month posttest . Booster training showed no significant booster effect .The RBANS language index score revealed a significant main effect of time and interaction between time and group but a non-significant main effect of group. Time differences showed that multi-domain CogTr resulted in a significant increase at 12-month posttest (dP = 0.123).The RBANS attention index score only showed a significant main effect of time but no effect of group or interaction. Temporal trends indicated that the single-domain CogTr group improved more than the multi-domain CogTr group after intervention Figure . BoosterThe CWST interference score and number of naming errors showed a significant effect of time but a non-significant effect of group and interaction. The word interference score of the single-domain CogTr group showed a significant training effect at the 12-month posttest. Booster training had no significant effect on the CWST interference score or the number of naming errors.trails A = 6.289, P = 0.013, F trails B = 7.352, P = 0.007).The completion time of Trails A and B, and the approximate error number of Trail A of the TMT showed a significant effect of time but no effect of group or interaction. Booster training had a significant effect on completion time (F P < 0.05); the completed training group outperformed the partial training group. Detailed data are shown in Table The attendance-rates per session for the two intervention groups were not statistically different. To explore the impact of completed training sessions on cognition, we divided the training subjects into two groups according to whether they had completed 80% of the training sessions (≥ 19 sessions): completed training group and partial training group. Controlling the baseline assessment as a covariate, we compared group differences at the three time-points of every cognitive test. There were ten posttest assessments that showed group differences P < 0.0; the comTo the best of our knowledge, this is the first randomized, controlled study to compare multi-domain with single-domain CogTr intervention in community-living healthy older people. Immediate and long-term follow-up provided powerful proof for the improvement and maintenance of training-related effects on cognitive function.The data presented here demonstrated that: (1) cognitive function varied along with time. RBANS total score, index scores and the visual reasoning test showed that the change in cognitive outcomes varied with time due to different group design and that each group benefitted differently after CogTr. CogTr was effective in improving test-related cognitive functions. (2) Multi-domain CogTr produced a significant effect on the RBANS total score, the visual reasoning test and immediate and delayed memory indices, while single-domain CogTr showed improvement in RBANS total score, the visual reasoning test, delayed memory and visuospatial/constructional index score, and CWST word interference. These results demonstrated that both multi-domain and single-domain CogTr resulted in training-related effects in cognitive improvement. Multi-domain CogTr resulted in better improvement in memory ability, while single-domain training gave better results for visuospatial/constructional and attention ability. (3) Besides the visual reasoning test, single-domain CogTr revealed effective improvement on the RBANS total score, delayed memory, visuospatial/constructional abilities, language, and CWST word interference. These results showed the generalization of benefit to non-trained cognitive functions, and such benefits persist. (4) It is notable that the RBANS total score, delayed memory and the visual reasoning test showed significant training effects in the multi-domain CogTr group at the 12-month posttest, while the single-domain CogTr only showed training effect on CWST word interfere at the 12-month posttest. The multi-domain CogTr effect showed better performance than single-domain CogTr at long-term follow-up and resulted in a steady gradual increase after intervention in most measure outcomes. These results proved that multi-domain CogTr had a better training effect on maintenance. (5) Booster training had a significant effect on the RBANS total score, the visual reasoning test, the completion time of TMT, and the visuospatial/constructional index score. These results prove that booster training was effective and enhanced the initial training effect on reasoning, and executive and visuospatial/constructional ability. (6) Reasoning ability showed a good training effect in both intervention groups and had no significant group differences at immediate and 6-month follow-up. Meanwhile, the multi-domain group showed better performance at the 12-month follow-up, which was consistent with the training effect maintenance on other cognitive tests. Furthermore, booster training also had a significant effect on the visual reasoning test. These results indicate that reasoning ability may be the domain most sensitive to CogTr (7). The completed training group outperformed the partial training group on visuospatial/constructional, reasoning, attention and processing speed abilities. These results also proved that cognitive training was effective.Compared to previous studies on CogTr in healthy older adults ,25,39-42Some important findings are described in this study for the first time. First of all, the comparison between multi-domain and single-domain CogTr with healthy older adults found that certain domains benefit differently from CogTr. Memory ability benefits more from multi-domain CogTr, while visuospatial/constructional and attention abilities benefit more from single-domain CogTr. Furthermore, although the CogTr impact on cognitive function decreased over time, it remained statistically significant, attesting to the durability of the intervention effects. Multi-domain CogTr has more advantages for effect maintenance. Secondly, the largest effect was observed in reasoning ability. The NES of initial training and booster training were larger than the values showed in the ACTIVE study . The difIt is interesting that the single domain training, which focused on reasoning, outperformed multi-domain in other cognitive domains but not in the reasoning domain. Why did single-domain training outperform multi-domain training in other cognitive domains? The reason might be the generalization of the training effect. Several studies have proved that non-trained cognitive domains could also be improved after single-domain training ,44. The . Thus, CogTr not only can improve cognition but may also have positive effects on daily functioning. CogTr, which produces no toxic effects, deserves to be further studied for its possible preventive and palliative therapeutic value. With acceptable compliance and training benefits and reasonable cost of implementation, our study may encourage future use of these CogTr methods to improve or maintain cognitive and daily function in cognitively impaired older adults.One limitation of this study is that 53 subjects drop out before completing the baseline cognitive testing due to the following reasons. First, there was a long time lag between randomization and baseline assessment, therefore, some older adults had totally forgotten our study or changed their contact information. Second, the baseline assessment was conducted in summer. Some older adults refused to attend the assessment due to the hot weather. Third, all subjects in our study came from three communities in Shanghai, which could not be fully representative of the entire population of non-demented older people in China. This may cause bias in our study and limit the generalization of our results. We may need to select multiple cities for recruitment in the future to avoid this bias. Whether our conclusions could be applied to other populations deserves to be further studied. Although the limitations exist, this study represents an important extension of previous knowledge of the effects of CogTr on brain aging. Several cognitive domains, such as memory, visual reasoning, visuospatial/constructional and attention improved significantly after CogTr. More importantly, such benefits were maintained over time. The clinical role of CogTr with healthy individuals was primary prevention to reduce disease incidence . DeclineOverall, cognitive training can improve memory, visual reasoning, visuospatial/construction, attention and neuropsychological status in community-living older persons and can help maintain their functioning over time. Multi-domain CogTr enhanced memory proficiency and single-domain CogTr augmented visuospatial/constructional and attention abilities, while multi-domain CogTr had more advantage in training effect maintenance. Booster training measurably enhances the training efficacy.ACTIVE: Advanced Cognitive Training for Independent and Vital Elderly; AD: Alzheimer's Disease; CogTR: cognitive training; CWST: Color Word Stroop Test; ES: effective size; GLM: general linear model; ITT: intention to treat; MMSE: mini-mental state examination; NES: net effect size; RBANS: Repeatable Battery for the Assessment of Neuropsychological Status; TMT: trail making test.The authors declare that they have no competing interests.Y C carried out the cognitive training and booster training, drafted the manuscript and participated in the training design. WF, YC and YS contributed to the participants' enrollment and cognitive assessment. JQW helped carry out the training. QWL and XZ contributed to the data collection and performed the preliminary data preparation. CBL conceived of and designed the study and performed the randomization procedure, and reviewed the manuscript. WYW conceived of the study, and participated in its design and coordination and helped review the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/30/prepub


Aortic dissection (AD) is associated with high morbidity and mortality; mortality rates increase by 1-2% per hour1, therefore timely diagnosis is pivotal. Point-of-care ultrasound (PoCUS) narrows the number of differentials in patients with shock, chest pain and shortness of breath2; however, reports addressing its routine use in the ER are scarce.Retrospective assessment of factors influencing use of PoCUS and its impact on time-to-diagnosis in patients with AD.We reviewed medical records and charts of patients with confirmed diagnosis of acute AD between May 2010 to May 2012.Twenty-seven patients (out of 45.630 presenting to the ER) with AD were identified . Diagnosis was confirmed with contrast enhanced CT in 25 patients, and with PoCUS (during CPR) and autopsy in two. 14 (52%) had prior PoCUS . PoCUS did not affect time-to-discharge from the ER significantly . PoCUS was performed more often in unstable patients  and in patients with equivocal clinical presentation . On a multivariate model, atypical clinical presentation emerged as an indenpedent predictor of PoCUS use after adjustment for age, gender, and hemodynamic stability (p=0.047).Our findings suggest that point-of-care ultrasound is increasingly used in the initial ER management of patients with AD, especially in hemodynamically unstable patients and in patients with atypical clinical presentation.


Non-necrotizing epithelioid granulomas have been described in association with many primary tumors. In such cases, they are designated as sarcoid-like reaction. Although it is more seen in carcinomas than in sarcomas, it is very rarely reported in renal carcinoma. Here, we describe a rare association of prominent peritumoral sarcoid-like reaction without intratumoral granulomas and conventional clear cell renal carcinoma in a 62-year-old-male, without clinical or laboratory finding of sarcoidosis. At 30 months follow-up, he had no recurrence.http://www.diagnosticpathology.diagnomx.eu/vs/4054525336657922The virtual slide(s) for this article can be found here:  Non-necrotizing granulomas have been described in association with several primary cancers. Histologically, this sarcoid-like reaction is undistinguishable from granulomas found in systemic sarcoidosis. It is composed of a focal accumulation of epitheloid cells and multinucleated giant cells . Few casA 62-year-old caucasian male known for dyslipidemia and scalp psoriasis was admitted to the emergency room for right renal colic. The patients had no history of constitutional symptoms, gross hematuria or abdominal pain. Laboratory findings were unremarkable. A computerized tomography (CT) was performed, which showed a 3.3 cm heterogeneous enhancing lesion in the upper pole of the right kidney consistent with a renal carcinoma Figure . PatientMacroscopically, the tumor lesion revealed a 3.5 cm encapsulated yellowish mass with bosselated surface with small foci of hemorrhage and necrosis.Histological examination showed a conventional clear cell type renal carcinoma of Fuhrman nuclear grade III, without sarcomatoid features Figure . There wThe frequency of sarcoid like reactions in certain tumor types and in different locations varies from 4% in carcinoma, to 20% in lymphoma . Non-casAntigens expressed by the neoplastic cell or soluble tumor antigens trigger an immune response which leads to the formation of non-caseating granulomas. Such reaction, locally mediated by T-cell, can be found in involved or uninvolved remote site, in regional lymph nodes and less frequently in tumoral areas -11.th case of clear cell renal cell carcinoma associated with sarcoidosis, this association is very rare.A certain number of cases in the literature reports sarcoid-like reaction associated with renal cell carcinoma in patient with sarcoidosis -10. RenaFew cases reported sarcoid-like reaction associated with renal cell carcinoma in patients without sarcoidoisis -6. We prInterestingly, in our case we did not find granulomatous reaction within the tumor as described in some carcinoma, including breast , renal -5 and heWe report a rare association between conventional clear cell type renal carcinoma and peritumoral sarcoid-like granulomatous reaction in a patient without clinical, radiologic or laboratory finding of sarcoidosis. Due to the low number of published cases, prognostic value of peritumoral non-necrotizing epithelioid granulomas has yet to be determined. Further cases are needed to provide information on the mechanism and prognostic value of peritumoral granuloma reaction in renal cell carcinoma.Written informed consent was obtained from the patient for publication of this Case Report and any accompanying images. A copy of written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.SO drafted the manuscript and provided clinical information. RA carried out the histological evaluation and helped drafted this manuscript. RS was the surgeon, supervised and helped drafted this manuscript. All authors read and approved the final manuscript.


The use of microfluidic systems for screening of aptamers and their biomedical applications are reviewed in this paper. Aptamers with different nucleic acid sequences have been extensively studied and the results demonstrated a strong binding affinity to target molecules such that they can be used as promising candidate biomarkers for diagnosis and therapeutics. Recently, the aptamer screening protocol has been conducted with microfluidic-based devices. Furthermore, aptamer affinity screening by a microfluidic-based method has demonstrated remarkable advantages over competing traditional methods. In this paper, we first reviewed microfluidic systems which demonstrated efficient and rapid screening of a specific aptamer. Then, the clinical applications of screened aptamers, also performed by microfluidic systems, are further reviewed. These automated microfluidic systems can provide advantages over their conventional counterparts including more compactness, faster analysis, less sample/reagent consumption and automation. An aptamer-based compact microfluidic system for diagnosis may even lead to a point-of-care device. The use of microfluidic systems for aptamer screening and diagnosis is expected to continue growing in the near future and may make a substantial impact on biomedical applications. The ligto 1016) –5. The lto 1016) . Aptamerto 1016) .Since the discovery of aptamers, many researchers have used the SELEX process to select aptamers with high affinities and specificities for their targets –15. Many2.Typically, the SELEX method is an iterative process of incubation, separation, and nucleic acid amplification. Multiple rounds of selection are generally necessary to screen aptamers with a sufficient specificity and a high binding affinity, which requires more sample/reagent consumption and time . In orde2.1.Recently, some research groups have demonstrated CE microfluidic chips as an efficient SELEX selection method (CE-SELEX) see  22,25–2–225–27. In particular, the CE-SELEX method has shown excellent selection efficiencies for protein targets that exhibit significant shifts in the electrophoretic mobility; protein-binding aptamers in the low-nanomolar range have been isolated after a small number of selection rounds ,25. Howe2.2.Sol-gel entrapment of biomolecules, such as proteins, has been used as a promising selection method of considerable interest to researchers . For ins2.3.Recently, some research groups reported the application of microfluidic technologies for SELEX utilizing magnetic separation in a continuous-flow, magnetically-activated, chip-based separation device ,20,21,24Recently, integrated microfluidic systems using magnetic beads to provide an automated approach to rapidly screen aptamers has been reported ,32. FurtUsing a multi-function microfluidic chip, the aptamer specific to alpha-fetoprotein (AFP) and C-reactive protein (CRP) were successfully screened. Using a multi-function microfluidic chip, the aptamer specific to alpha-fetoprotein (AFP) and C-reactive protein (CRP) were successfully screened. It is envisioned that the fully-automated SELEX process on an integrated microfluidic system may provide a user-friendly, efficient and rapid platform for screening aptamers for a variety of biomedical applications.Another issue involved in the aptamer screening process is that it requires a time-consuming competitive assay to select ssDNA with a high affinity and specificity for subsequent sequencing and synthesis ,33. This3.A modification of the traditional SELEX process that uses cells as targets has been named Cell-SELEX –36. Cell4.Another interesting area of aptamer research is their applications in clinical diagnostics and biosensing. Their high affinity, high specificity and ease of modification make aptamers excellent diagnostic or biosensing tools. For aptamer-based diagnostics or biosensors, binding of an aptamer to its target molecule have been extensively explored –42. DeteFor example, a microfluidic-based, aptamer-sandwich-type assay using electrochemical detection of human thrombin has been reported . ThrombiAnother excellent work using a highly specific and sensitive DNA aptamer, instead of using antibodies, for the brain natriuretic peptide (BNP) assay has been reported by using Agilent lab-on-a-chip  technology . BNP is Alternatively, SPR is an optical technique that detects refractive index changes and has been extensively applied to bio-analysis . SPR allAnother aptamer-based assay reported a microfluidic device capable of aptamer–protein specific binding for the determination of CRP by using an optical detection scheme . Herein,Aptamers have a better applicability than antibodies in microfluidic systems because they are more easily labeled with fluorescent tags and more stable for longer periods of time under various conditions. Recently, aptamers have been used as excellent affinity probes in microfluidic chips using different methods. For instance, electrophoretic separation and detection of aptamer-protein complexes for the analysis of thrombin in plasma has been reported . An aptaA microchip-based, aptamer-sandwich-type sensor probe has the potential for screening different levels of biomarkers in a complex sample. One of the most challenging targets in aptamer-based assays is cancer cells. Aptamer-coated microchannels for detecting cancer cells have been recently demonstrated . A microFinally, the selection of aptamers for specific subtypes of circulating tumor cells and stem cells (or cancer stem cells) using microfluidic systems that can perform extraction, enrichment, multiplexing, sorting, and separation poses great promise. Thus, counting rare cancer cells can be used to diagnose metastatic relapse, monitor response to drugs and therapies, and to track tumor progression. Cell-SELEX on microfluidic systems may play an important role in the development of these promising detection platforms. Different microfluidic systems for the SELEX technology have been applied for different kinds of clinical targets. Inorganic and small organic molecules, proteins, as well as complex targets like target mixtures or whole cells were used for SELEX. In this review, several publications summarized the various target molecules according to different microfluidic chips. Human immunoglobulin E , protein5.In this review article, recent works on automating aptamer selection using microfluidic devices have been extensively reviewed. Microfluidic systems for sample pretreatment to purify the specific samples in an aptamer-based assay have also been reviewed and discussed. Even with the advances in these microfluidic systems, there are several issues that were identified and need to be resolved before they can be used for clinical applications. However, based on the selected aptamers, applications including clinical diagnostics and biosensing for the identification of various target molecules can be realized. For each of these applications, the initial choice of enabling technology and detection strategy is important to select an aptamer and/or target molecule with optimal binding properties. Moreover, the microfluidic technology has many advantages over conventional counterparts including less sample/reagent consumption, lower power consumption, less contamination, and automation. Microfluidic devices and systems have extensively used aptamers for a variety of biomedical applications such as cell-based assays, molecular diagnosis, immunoassays, and biochemical assays. It is expected that these microfluidic devices and systems may soon provide a fast and accurate clinical analysis with an aptamer-based sensor. Most importantly, point-of-care devices and analysis systems may eventually be feasible in the near future.


Ruegeria pomeroyi, a member of the Roseobacter clade, an abundant group of marine bacteria, as a seed for the annotation of the whole clade.The structural and functional annotation of genomes is now heavily based on data obtained using automated pipeline systems. The key for an accurate structural annotation consists of blending similarities between closely related genomes with biochemical evidence of the genome interpretation. In this work we applied high-throughput proteogenomics to R. pomeroyi was obtained after searching over 1.1 million MS/MS spectra against a six-frame translated genome database. We identified 2006 polypeptides, of which thirty-four were encoded by open reading frames (ORFs) that had not previously been annotated. From the pool of 'one-hit-wonders', i.e. those ORFs specified by only one peptide detected by tandem mass spectrometry, we could confirm the probable existence of five additional new genes after proving that the corresponding RNAs were transcribed. We also identified the most-N-terminal peptide of 486 polypeptides, of which sixty-four had originally been wrongly annotated.A large dataset of peptides from Roseobacter isolates sequenced to date (twenty different genera), we propose the correction of the assigned start codons of 1082 homologous genes in the clade. In addition, we also report the presence of novel genes within operons encoding determinants of the important tricarboxylic acid cycle, a feature that seems to be characteristic of some Roseobacter genomes. The detection of their corresponding products in large amounts raises the question of their function. Their discoveries point to a possible theory for protein evolution that will rely on high expression of orphans in bacteria: their putative poor efficiency could be counterbalanced by a higher level of expression. Our proteogenomic analysis will increase the reliability of the future annotation of marine bacterial genomes.By extending these re-annotations to the other thirty-six  Haemophilus influenza [in-silico genome analysis estimated almost 60% erroneous start codon prediction in some prokaryotic genomes [The first complete bacterial genome to be sequenced was that of nfluenza . Seventenfluenza . Genome nfluenza ,4. An in genomes . The genCaenorhabditis elegans [Vitis vinifera [Mycoplasma mobile [Deinococcus deserti [Thermococcus gammatolerans [Pristionchus pacificus [Geobacter sulfurreducens [i.e. those with large genomes [New strategies have been proposed to better annotate genomes with the integration of experimental data collected at the transcriptome or proteome levels . The ex elegans  and Vitivinifera , producivinifera ,11. The vinifera  and has vinifera . The resvinifera . The mapa mobile , Deinoco deserti , and Thetolerans . Integraacificus  and Geobreducens . The mai genomes .et al. [Mycobacterium genus, was tentatively called ortho-proteogenomics. A similar study has also been performed on Yersinia pestis KIM and extended, respectively, to the twenty-one closest Yersinia species sequenced [On the basis of evolutionary constraints, re-annotations obtained by proteogenomics for one organism can be extended to all orthologous genes present in all phylogenetically related species. This approach, first proposed by Gallien et al.  for the equenced . The intRoseobacter clade is a group of bacteria that can represent up to 20% of bacterioplankton communities in coastal environments and more than 10% in the open ocean [Roseobacter genome to be sequenced was that of Ruegeria pomeroyi DSS-3. This genome consists of a 4.11 Mb chromosome and a 0.49 Mb megaplasmid, named pDSS-3 [Roseobacter isolates have since been sequenced and many more are in progress in order to explore this important clade and to understand how ocean ecosystems function.The en ocean . The fird pDSS-3 . A high R. pomeroyi genome using experimental data obtained from a large proteomic survey recorded for this micro-organism. This ortho-proteogenomic study is the first to propose the extension of proteogenomic corrections to a large bacterial clade. Moreover, we have detected highly abundant orphan proteins and discuss their significance.In the present study, we propose a re-annotation of the R. pomeroyi, cultivated in various conditions, resulted in a large dataset of MS/MS spectra . To assign these spectra to peptide sequences, we created a database comprising all translated stop-to-stop amino acid sequences of more than forty residues, taking into account the six-frame translation of the genome of R. pomeroyi. Thus, this database comprises all the coding domain sequences (CDS) of the bacterium mixed with a large number of aberrant polypeptide sequences. All of the MS/MS spectra were searched against this database using the Mascot engine, resulting in the identification of a restricted set of 4425 probable ORFs. A second search against this restricted database led to the assignment of 594 902 spectra corresponding to 22 805 non-redundant tryptic peptides  and showing no overlap with other proteomic-detected genes. Applying these criteria resulted in the selection of seven targets. To give higher confidence to the validation of these 'one-hit-wonders', we checked whether these genes were transcribed. We extracted total RNA from bacteria grown in the physiological conditions in which each polypeptide had been most abundantly detected during the proteomic survey. We performed RT-PCR amplification for five of these targets  whereas the mean length of polypeptides encoded in the genome is greater than 320 amino acids. Curiously, the well conserved RNA helicase, SPO_PG024, was not previously annotated, due to the report of a putative translational frameshift in the sequence [Roseobacter members, being a possible hotspot for the accumulation of mutations and the reason why a putative frameshift was at first predicted.As shown in Table sequence . We defiRoseobacter clade  and sequence, with no inter-species similarity. The existence of the previously annotated SPO0504 gene is undoubtedly false. It was assigned to the other DNA strand and the corresponding short polypeptide product (forty-one residues) did not show any similar protein by PSI-BLAST. SPO_PG028 is also highly detected by MS/MS as 1395 spectra were assigned to this polypeptide . The C-terminal region of this newly detected gene overlaps the N-terminal region of the stop-to-stop SPO0877 ORF. A PSI-BLAST search with the annotated sequence of SPO0877 clearly indicates, by comparison with its closest homologues, that this conserved esterase lipase in R. pomeroyi should be 127 amino acids shorter. In such a case, the start codon of SPO0877 would be in position 935 284 on the chromosome, being consistent with the existence of SPO_PG034.Interestingly, the stop-to-stop ORF comprising SPO_PG031 overlapped the stop-to-stop ORF comprising an already annotated gene SPO2724) detected during the proteomic survey. SPO2724 was validated with twenty non-redundant peptides  before overlapping SPO3316 , as shown in Figure Roseobacter clade. However, the first residues were found to be very dissimilar in comparison with its closest homologues. We sequenced the locus and found a sequence error with an extra G between positions 2 029 022 and 2 029 023. This would be the reason why a shorter sequence was at first assigned to this gene. The corrected N-terminus of the polypeptide sequence is now similar to its closest homologues.The search for peptides located further upstream than the previously annotated start codons  led to tThe search for semi-tryptic peptides led to the discovery of the N-terminal peptides of 486 proteins , meaning that these are no longer unique to R. pomeroyi.An ortho-proteogenomic analysis was carried out in order to extend the genomic re-annotations proposed for es Table  among thRoseobacter strains had been wrongly annotated. For this, a local tBLASTn analysis with the 486 protein sequences was performed for all the annotated CDSs of each of the thirty-six sequenced Roseobacters. A total of 9887 polypeptides sharing high similarities with these R. pomeroyi polypeptides were found and are listed in Additional file R. pomeroyi protein and its respective homologue in other Roseobacter members was observed. Nevertheless, in 1082 cases the position of the most-N-terminal sequence annotated was not in agreement with the N-terminus of the R. pomeroyi protein. For all of these, we propose the correction of the annotation after manual inspection. We checked whether the new gene starts corresponded to a canonical initiation codon  and, in the case of a sequence extension at the N-terminus, if there was evidence of sequence similarities with the R. pomeroyi polypeptide. The rate of erroneously identified start codons among the Roseobacter genomes was 11% (1082 of 9887). Of note, this value may be an underestimate because i) we discarded many doubtful corrections during manual inspection, and ii) most of the BLAST results mainly matched highly conserved genes that are generally better annotated by comparative genomics. The rate decreased to 6.8% when considering only those curated genomes .The confidently detected N-terminal sequences of the 486 CDSs listed in Additional file Panel B shows the locus where the two novel genes, SPO_PG09 and SPO_PG027, are encoded. As these two genes are equally oriented as the rpsE and rpmD genes, both encoding conserved ribosomal proteins, we checked whether an operonic structure could be identified. We purified RNA from cells where the two proteins had been detected, and amplified specific cDNA fragments by RT-PCR . We found that the two genes are co-transcribed, as we obtained RT-PCR amplification of the SPO_PG027-SPO_PG09 region. Despite this, no amplification could be obtained between this operon and its downstream rplO or upstream rpmD genes. The latter result was expected because of a putative Rho-independent transcriptional terminator identified by the TransTerm software .We found that some loci comprise several annotation errors. For example, Figure 2The neighbouring genes in the operon structures of the newly annotated CDSs may infer plausible hints about the functions of the novel detected genes. This is the case for SPO_PG026, which we have shown here to be co-transcribed with the SPO_3673 gene. We have previously shown that the latter encodes an RTX-like toxin which is abundantly secreted in the culture medium . RTX opeRoseobacter members . The loci encoding these far-homologues are also located close to genes with functions related to branched-chain amino acid metabolism. However, the genomic context is not always similar to that found in R. pomeroyi. On the other hand, we found that SPO_PG022 is co-transcribed with its downstream gene, SPO1095, and upstream gene, SPO1094, a putative propionyl-CoA carboxylase. SPO1094 converts propionyl-CoA (a derivative product from branched-chain amino acid catabolism) to methylmalonyl-CoA . Strikingly, SPO_PG022 shares similarity with one other annotated gene, in the Roseobacter sp MED193, which also shows the same putative operon structure.RT-PCR amplification was also performed for the SPO_PG023 and SPO_1339 couple, possibly with a linked function. SPO_1339, a signal-recognition, particle-docking protein, is located just upstream of SPO_PG023. Another operon structure detected was that of SPO_PG010 with the upstream SPO_2211-SPO_2212-SPO_2213 genes. These three genes encode the determinants of branched-chain amino acid catabolism . SPO_PG010 shows similarities with only a few proteins found in other R. pomeroyi. Interestingly, SPO_PG006 and SPO_PG016 were shown to be co-transcribed with the genes encoding these enzymes. SPO_PG005, which is co-transcribed with the SPO_0345 gene (whose product has no assigned function), is independently transcribed from its flanking operons, sucCD and sucAB . In all Roseobacter species that were compared, we observed a variable pattern of novel genes inserted within the sucCD operon and in the intergenic region between sucAB and sucCD. These novel genes are poorly conserved between the different Roseobacter members, as shown in Figure sdhCDAB. We demonstrated its co-transcription with its flanking genes, SPO0360 and SPO0361, by a specific RT-PCR amplification. The presence of non-conserved genes included in the sdhCDAB operon is also common amongst Roseobacter members, but not in other bacteria.SPO_PG005 and SPO_PG006, shown in Figure R. pomeroyi cultivated in various conditions, we identified thirty-nine newly annotated genes and nine wrongly described ORFs. We also corrected seventy-four start codons and described five sequencing errors  that consequently modified the characteristics of the genes encoded at these loci. Because of its environmental relevance, the Roseobacter clade is currently subject to intense sequencing efforts [From the large dataset of MS/MS spectra (1 117 372) recorded for proteome samples of  efforts -31. HoweR. pomeroyi genome annotation. This is similar to previous proteogenomic reports for Shewanella or Mycobacterium bacteria that resulted in thirty-eight and twenty-nine new annotations, respectively [Yersinia pestis identified only four novel genes [Enterobacteriaceae family have now been sequenced and annotated, it is reasonable to consider that their genomes are amongst the best for accuracy and reliability. This is in full agreement with the proteogenomic data presented by Payne et al. [R. pomeroyi. The majority of annotation problems come from the identification of CDSs exclusive to a small number of organisms, as comparative genomics is not useful in such a case in confirming the ORF prediction. Their validation requires additional experimental evidence, such as described here. Blending data from complementary approaches, such as protein characterization by tandem mass spectrometry and transcriptomic evidence, is time consuming but results in stronger evidence for small genes. In terms of mass spectrometry, 'one-hit-wonders' are proteins identified with only one, non-redundant peptide tag. They are usually proteins with low molecular weight that are able to generate only a few tryptic peptides. Depending on the score of MS/MS spectrum assignment, these hits may be difficult to ascertain confidently and require manual validation. Gupta et al [We noted that the number of annotation inaccuracies, in terms of structural annotation, is rather large for ectively ,32. In cel genes . As entee et al. . Here weta et al  proposedR. pomeroyi genome sequence also supports the current idea of re-sequencing genomes which were established a decade ago [Another frequent problem encountered during genome annotation is the identification of a CDS located in two different reading frames that clearly encodes a unique, conserved protein. This can be either a real frameshift process occurring for the regulation of protein synthesis, an artefact resulting from a sequencing error, or a pseudogene that has been recently inactivated. As we identified in the present study peptides in different reading frames at the same loci (e.g. SPO_PG036 and SPO_PG037), we confirmed the production of the polypeptides encoded and discounted the existence of pseudogenes. We checked systematically the sequences of the five loci and found in all cases that the plausible frameshifts resulted from sequencing errors. The insertion of an extra nucleotide in the sequence produced a slippage of the coding region to another reading frame in the five cases. This was expected as frameshifts are rare processes of regulation and usually down-regulate the protein synthesis in bacteria, while they are frequent in Archaea or viruses ,35. The cade ago . Here weMycobacterium [Yersinia [Roseobacter clade are distantly related, they all form a robust cluster with a high rate of similarities at the 16S RNA nucleotide sequence level [R. pomeroyi to 9887 homologous genes in the thirty-six sequenced Roseobacter isolates, corresponding to nineteen distinct genera. In this way, 1082 genes that were wrongly annotated were confidently corrected. This represents 11% of the total number of ORFs considered. To highlight the importance of manual curation of genome annotations, the rate of erroneous N-terminal identifications decreased to 6.8% when considering only the four complete Roseobacter genomes included in this study. These error rates are probably slightly underestimated as we only considered the conserved and obvious corrections. It is important to note that the full rate of badly annotated N-terminal genes established on the well-annotated genome of R. pomeroyi was 12.8%. A more comprehensive annotation of the clade could only be accomplished by integrating a comparative proteogenomic analysis of various Roseobacter strains, as previously carried out with the genus Shewanella .It is worth noting that ortho-proteogenomic extension of the corrected annotations to phylogenetically-related microorganisms reinforces the interest of proteogenomic studies for poorly studied bacterial phyla. Ortho-proteogenomic analyses have, to date, been limited to only two genera, acterium  and YersYersinia , and havce level . We haveRoseobacter members, but their presence is a common topic restricted to members of this clade. Whether these genes encode proteins that enhance this central metabolic cycle in these bacteria or are opportunistic genes that specifically appear in this operon because of the advantage of their high expression is an open question. The presence of the novel proteins found in the citric acid cycle operon in R. pomeroyi could represent snapshots of how novel proteins with novel specific functions arise during evolution.Finally, the identification of operon structures by RT-PCR has given insights of the plausible function of the novel proteins identified in the present study. Bacterial genomes are usually well structured and regulated in the form of operons. Remarkably, we found that most of the novel, proteogenomic-detected CDSs were identified in operons encoding catabolic functions for amino acid degradation, RTX-like secreted proteins or central citric cycle metabolism. Because most of the biological conditions were carried out in a peptide broth , this caRoseobacter clade, are necessary to improve genome and even metagenome annotations. Ortho-proteogenomic annotation extension to a whole bacterial clade has proven here to be highly valuable. Such extension could also be applied to metagenome data, taking into account higher constraints.Systematic listing of CDSs in numerous microorganisms, with the help of proteogenomic evidence, should increase the accuracy of annotation software. As demonstrated here, proteogenomic evidences from bacteria belonging to orders that have, thus far, been poorly characterized, such as the R. pomeroyi consisting in 136 nanoLC-MS/MS runs acquired on proteomes and subproteomes from cells grown under 30 different culture conditions [An extensive proteomic analysis was applied to nditions ,24. Brienditions ,39. MS/Mnditions .R. pomeroyi . This Osoftware . A totalsoftware  and ii) software . The resR. pomeroyi cells was performed using the DNeasy Blood and Tissue Kit (Qiagen) following manufacturer instructions for gram-negative bacteria. DNA PCR amplification was carried out with standard conditions using Taq DNA polymerase (Roche). All primers used for specific PCR amplification of the different targets are listed in Supplementary Additional file Genomic DNA extraction of Roseobacters for genes specifying similar proteins to those detected by proteogenomics in R. pomeroyi. These strains were: Citriecella sp. SE45, Dinoroseobacter shibae DFL 12, Jannaschia sp. CCS1, Loktanella vestfoldensis SKA53, Maritimibacter alkaliphilus HTCC2654, Oceanibulbus indolifex HEL-45, Oceanicola batsensis HTCC2597, Oceanicola granulosus HTCC2516, Octadecabacter antarcticus 238, Octadecabacter antarcticus 307, Pelagibaca bermudensis HTCC2601, Phaeobacter gallaeciensis 2.10, Phaeobacter gallaeciensis BS107, Phaeobacter sp. Y4I, Rhodobacteraceae KLH11, Rhodobacterales bacterium HTCC2150, Rhodobacterales bacterium HTCC2255, Rhodobacterales HTCC2083, Roseobacter denitrificans OCh 114, Roseobacter litoralis Och 149, Roseobacter sp. AzwK-3b, Roseobacter sp. CCS2, Roseobacter sp. GAI101, Roseobacter sp. MED193, Roseobacter sp. SK209-2-6, Roseovarius nubinhibens ISM, Roseovarius sp. 217, Roseovarius sp. TM1035, Ruegeria lacuscaerulensis ITI-1157, Ruegeria sp. R11, Ruegeria sp. TM1040, RuegeriaTrich CH4B, Sagittula stellata E-37, Sulfitobacter sp. EE-36, Sulfitobacter sp. NAS-14.1 and Thalassiobium R2A62. The E-value cutoff was set at < 10-20. We also searched protein homologues in the other 36 Roseobacter proteomes with a local BLASTP tool  in order to check specifically their most probable N-termini.Visualization of genome sequences and gene positioning was done with the Artemis browser . The TrFR852579 (SPO_PG036) FR852580 (SPO_PG037), FR852581 (SPO_PG038), FR852582 (SPO_PG039), and FR852583 (SPO1905).The novel Ruegeria pomeroyi DSS-3 nucleotide sequences reported in this paper have been deposited in the EMBL/GenBank Nucleotide Sequence Database under the accession numbers ORFs: Open Reading Frames; CDS: coding domain sequences; MS/MS: tandem mass spectrometry.JC-O designed and performed the experiments of microbiology and proteomics, and analyzed all the data. GC contributed to mRNA analysis and sequence verification. JA conceived, coordinated the study and participated in the analysis of the proteomic data. JC-O and JA wrote the manuscript. All authors read and approved the final manuscript.Non-redundant peptide list detected by MS/MS spectral attributions (p < 0.005). The excel table presents the characteristics of all the non-redundant peptides detected (22805 items) and their corresponding proteins.Click here for fileStop-to-stop polypeptides detected by MS/MS. The excel table presents the whole list (2547 items) of ORFs detected by MS/MS with their annotated function and sequence, as well as the number of non-redundant peptides assigned to each of these ORFs.Click here for fileR. pomeroyi detected by proteogenomics in this studyList of novel genes encoded in the genome of . The excel table presents the 39 new genes found in the present study and their characteristics. The corresponding ORF, the number of non-redundant peptides, the number of MS/MS spectra assigned, the plausible protein sequence, and the conditions where each product has been detected are listed for the 39 genes.Click here for fileAnnotated CDS demonstrated to be longer due to the detection of tryptic peptides situated further N-ter than previously annotated. The excel table presents the evidences for 10 annotated CDS that should be revised in terms of N-terminus. The corresponding ORF label, the number of non-redundant peptides, the new proteomic-corrected CDS sequence and the corresponding peptide evidences  are indicated for each of these 10 CDS.Click here for fileSemi-tryptic peptides representing the most N-ter sequence of the stop-to-stop CDS. The excel table presents the characteristics of all the semi-tryptic peptides detected which correspond to the most N-terminal sequence of the stop-to-stop ORF (22805 items) and their corresponding proteins. We distinguished the peptides starting with a methionine residue encoded by an ATG start codon (lines 5-136), those starting from the residue just after a removed methionine encoded by an ATG start codon (lines 138-463), the peptides starting with a methionine residue encoded by an GTG start codon (lines 465-471), those starting from the residue just after a removed methionine encoded by an GTG start codon (lines 473-489), and those starting from the residue just after a removed methionine encoded by an TTG start codon (lines 491-494).Click here for fileR. pomeroyi to 36 other isolates of the Roseobacter cladeOrtho-proteogenomic based analysis extending the N-ter corrections done on . The excel table presents the extension to 36 isolates for each of R. pomeroyi re-annotated genes . The e-value obtained by BLAST is indicated for each homologue, as well as its N-terminus (60 first residues), the new CDS length, and its location on the corresponding genome.Click here for filePrimers used throughout the study for PCR amplifications. The excel table presents the 37 primers used in the study for PCR amplification of specific genomic loci and their characteristics .Click here for file