PLoS Biology, Toh B et al (2011) Mesenchymal transition and dissemination of cancer cells is driven by myeloid-derived suppressor cells infiltrating the primary tumorPLoS Biol9 e1001162, we show that a subset of granulocytic cells already known to suppress antitumour immune responses also promotes cancer cell proliferation and metastasis by inducing epithelial to mesenchymal transition. This subset plays a key role in early dissemination of cancer cells throughout the body and may therefore represent a novel target for therapeutic intervention.Most studies aimed at understanding the link between inflammation and cancer progression have focused on macrophages. In a recent study published in  Indeed, metastases are usually the cause of death in patients with cancer, not the primary tumor. The prevailing model of metastasis stipulates that cancer cells disseminate to distant organs during the latter stages of disease progression. Over time, cancer cells gain sufficient mutations to allow them to overcome putative barriers to metastasis . However T cells . Given tSBiology , we now +Gr1hiF4/80− as opposed to M-MDSC which are CD11b+Gr1loF4/80+. Besides these morphological and phenotypic differences, the mediators that determine the immunosuppressive abilities of MDSC also differ between the two subsets. The G-MDSC subset has been shown to be greatly increased in the spleen and blood of tumor-bearing animals and have been shown to be up-regulated in human cancers as well [MDSC are immature myeloid cells defined by their ability to suppress T cell function. MDSC are broadly made up of two subsets – G-MDSC and monocytic (M)-MDSC, which morphologically resemble granulocytes and monocytes respectively. In addition, G-MDSC are CD11b as well . G-MDSC, as well .Rfp-ret oncogene in melanocytes. We have previously shown that primary tumors develop in the choroid layer of the eye in RETAAD mice, followed by metastasis to distant organs including lungs, lymph nodes, bone marrow and the reproductive tract. Disseminated cancer cells can be detected as early as 3 weeks of age in these animals. This spontaneous tumor model, unlike many transplanted tumor models [This work was carried out in RETAAD mice which spontaneously develop melanoma due to over-expression of the r models , allows The accumulation of G-MDSC was due to the expression of CXCR2 ligands, CXCL1, CXCL2 and CXCL5, in the primary tumors but not in the metastases. Indeed, adoptive transfer of either wild-type or CXCR2-knockout bone marrow into RETAAD mice revealed that only wild-type G-MDSC were capable of migrating to the primary tumor, demonstrating an absolute requirement for CXCR2 in mediating G-MDSC accumulation in primary tumors. Furthermore, high expression of CXCL5 by the tumor cells leads us to believe that this is the main factor attracting G-MDSC to the primary tumor. Once present at the tumor site, it is likely that G-MDSC secrete CXCL1 and CXCL2 and attract additional G-MDSC, thus forming a positive feedback loop .in vitro by co-culturing cancer cells with G-MDSC. Although the mechanism of this increased proliferation is still unknown, we determined that secreted factors were critical, because separation of G-MDSC from the cancer cells with a 0.3 micron membrane still resulted in increased proliferation.To further investigate the role that G-MDSC play in the primary tumor, we developed two schemes to deplete G-MDSC in RETAAD mice; 1) depletion before the onset of the primary tumor at one week of age, and 2) depletion after development of the primary tumor . Interestingly, we found that depletion of G-MDSC resulted in smaller primary tumors with a lower mitotic index. No difference in tumor size could be seen in the metastases, consistent with observed lack of G-MDSC infiltration in these satellite tumors. Increased proliferation of cancer cells could be recapitulated Another interesting outcome of depleting G-MDSC was the reduced number of metastases in the skin, lymph nodes and lungs. However, this difference could only be seen if the G-MDSC were depleted before clinical detection of the primary tumor. Depletion of G-MDSC after the age of 5 weeks did not alter the number of metastases. We therefore concluded that G-MDSC were important for cancer cell dissemination, and that cancer cells disseminating before 5 weeks were the main source of metastases. Indeed, primary tumors usually display a multi-nodular morphology, but the frequency of nodules in primary tumors was diminished in the absence of G-MDSC. This finding is also consistent with the observation that metastatic tumors that normally lack G-MDSC do not exhibit a multi-nodular structure. These data led us to hypothesize that G-MDSC are involved in the induction of a motile phenotype in cancer cells that affects both the local site (within the primary tumor) as well as distant tissues (organs outside the eye) by supporting dissemination at an early stage.in vitro, we showed that G-MDSC induced a mesenchymal phenotype that resulted in increased motility of NBT-II cells. This increase was accompanied by down-regulation of E-cadherin on the surface of the NBT-II cells, which is indicative of a mesenchymal-like phenotype. Co-culturing G-MDSC with cancer cells purified from RETAAD tumors, or with a human melanoma cell line, similarly resulted in the down-regulation of E-cadherin in vitro. This mesenchymal-like phenotype was also observed in vivo in RETAAD tumors, and mesenchymal markers S100A4 and Vimentin were down-regulated in primary tumors depleted of G-MDSC. In vitro inhibition of transforming growth factor-β, hepatocyte growth factor and epidermal growth factor, either individually or in combination, was able to partially or completely block the mesenchymal transition. These data indicate that the induction of mesenchymal transition in cancer cells occurs via multiple pathways, although the key factor which initiates this transition process currently remains unknown.Using NBT-II cells  as reporter cells in vivo. Thus the locations of the different immune cells throughout the tumor microenvironment might determine the exact nature of the tumor cells in selected areas within the tumor.G-MDSC stimulated both cancer cell proliferation and mesenchymal transition. However, histopathological examination of the primary tumors revealed that proliferating cancer cells were spread throughout the entire tumor, while those that exhibited a mesenchymal phenotype were located at the edge of the tumor. This distinct distribution might be explained by the fact that G-MDSC induce proliferation through production of soluble factors, while mesenchymal transition seems to require close proximity or even direct contact with cancer cells. Alternatively, we do not rule out the possibility that other immune cells, such as macrophages, may promote mesenchymal transition as well The ability of immune cells such as G-MDSC to regulate both the proliferative and metastatic phenotypes of cancer cells compels us to re-visit our current views of cancer progression. With the accumulation of new evidence that supports early cancer cell dissemination, the model of a slowly evolving tumor might not be relevant in this context. The cancer stem cell hypothesis postulates the existence of a defined subset of cancer cells that are able to initiate metastatic tumors. However, could it be that certain cancer cells, due to their location at the edge of the tumor, are simply more susceptible to the effects of the microenvironment, including the secretions of G-MDSC? Could external stimuli such as immune mediators contribute to the ‘stemness’ of these cells? With the emergence of better cancer models such as the RETAAD mouse, it is now possible to observe the tumor as a complex system made up of both cancer cells and tumor stroma. Cancer cells are not isolated but act in concert with various cells in the microenvironment. Understanding these interactions will be key to unlocking this disease.


In this study, Würtz and colleagues conducted high-throughput profiling of blood specimens in two large population-based cohorts in order to identify biomarkers for all-cause mortality and enhance risk prediction. The authors found that biomarker profiling improved prediction of the short-term risk of death from all causes above established risk factors. However, further investigations are needed to clarify the biological mechanisms and the utility of these biomarkers to guide screening and prevention.Please see later in the article for the Editors' Summary Early identification of ambulatory persons at high short-term risk of death could benefit targeted prevention. To identify biomarkers for all-cause mortality and enhance risk prediction, we conducted high-throughput profiling of blood specimens in two large population-based cohorts.n = 9,842; age range 18–103 y; 508 deaths during a median of 5.4 y of follow-up). Biomarkers for all-cause mortality were examined using stepwise proportional hazards models. Significant biomarkers were validated and incremental predictive utility assessed in a population-based cohort from Finland . Four circulating biomarkers predicted the risk of all-cause mortality among participants from the Estonian Biobank after adjusting for conventional risk factors: alpha-1-acid glycoprotein , albumin , very-low-density lipoprotein particle size , and citrate . All four biomarkers were predictive of cardiovascular mortality, as well as death from cancer and other nonvascular diseases. One in five participants in the Estonian Biobank cohort with a biomarker summary score within the highest percentile died during the first year of follow-up, indicating prominent systemic reflections of frailty. The biomarker associations all replicated in the Finnish validation cohort. Including the four biomarkers in a risk prediction score improved risk assessment for 5-y mortality .106 candidate biomarkers were quantified by nuclear magnetic resonance spectroscopy of non-fasting plasma samples from a random subset of the Estonian Biobank  spectroscopy and mass spectrometry. These tools have the potential to be used to screen the general population for a range of different biomarkers.Identifying new biomarkers that provide insight into the risk of death from all causes could be an important step in linking different diseases and assessing patient risk. The authors in this study screened patient samples using NMR spectroscopy for biomarkers that accurately predict the risk of death particularly amongst the general population, rather than amongst people already known to be ill.The researchers studied two large groups of people, one in Estonia and one in Finland. Both countries have set up health registries that collect and store blood samples and health records over many years. The registries include large numbers of people who are representative of the wider population.The researchers first tested blood samples from a representative subset of the Estonian group, testing 9,842 samples in total. They looked at 106 different biomarkers in each sample using NMR spectroscopy. They also looked at the health records of this group and found that 508 people died during the follow-up period after the blood sample was taken, the majority from heart disease, cancer, and other diseases. Using statistical analysis, they looked for any links between the levels of different biomarkers in the blood and people's short-term risk of dying. They found that the levels of four biomarkers—plasma albumin, alpha-1-acid glycoprotein, very-low-density lipoprotein (VLDL) particle size, and citrate—appeared to accurately predict short-term risk of death. They repeated this study with the Finnish group, this time with 7,503 individuals (176 of whom died during the five-year follow-up period after giving a blood sample) and found similar results.The researchers carried out further statistical analyses to take into account other known factors that might have contributed to the risk of life-threatening illness. These included factors such as age, weight, tobacco and alcohol use, cholesterol levels, and pre-existing illness, such as diabetes and cancer. The association between the four biomarkers and short-term risk of death remained the same even when controlling for these other factors.The analysis also showed that combining the test results for all four biomarkers, to produce a biomarker score, provided a more accurate measure of risk than any of the biomarkers individually. This biomarker score also proved to be the strongest predictor of short-term risk of dying in the Estonian group. Individuals with a biomarker score in the top 20% had a risk of dying within five years that was 19 times greater than that of individuals with a score in the bottom 20% (288 versus 15 deaths).This study suggests that there are four biomarkers in the blood—alpha-1-acid glycoprotein, albumin, VLDL particle size, and citrate—that can be measured by NMR spectroscopy to assess whether otherwise healthy people are at short-term risk of dying from heart disease, cancer, and other illnesses. However, further validation of these findings is still required, and additional studies should examine the biomarker specificity and associations in settings closer to clinical practice. The combined biomarker score appears to be a more accurate predictor of risk than tests for more commonly known risk factors. Identifying individuals who are at high risk using these biomarkers might help to target preventative medical treatments to those with the greatest need.However, there are several limitations to this study. As an observational study, it provides evidence of only a correlation between a biomarker score and ill health. It does not identify any underlying causes. Other factors, not detectable by NMR spectroscopy, might be the true cause of serious health problems and would provide a more accurate assessment of risk. Nor does this study identify what kinds of treatment might prove successful in reducing the risks. Therefore, more research is needed to determine whether testing for these biomarkers would provide any clinical benefit.There were also some technical limitations to the study. NMR spectroscopy does not detect as many biomarkers as mass spectrometry, which might therefore identify further biomarkers for a more accurate risk assessment. In addition, because both study groups were northern European, it is not yet known whether the results would be the same in other ethnic groups or populations with different lifestyles.In spite of these limitations, the fact that the same four biomarkers are associated with a short-term risk of death from a variety of diseases does suggest that similar underlying mechanisms are taking place. This observation points to some potentially valuable areas of research to understand precisely what's contributing to the increased risk.http://dx.doi.org/10.1371/journal.pmed.1001606Please access these websites via the online version of this summary at biomarkersThe US National Institute of Environmental Health Sciences has information on Biomarker Qualification Program to help researchers in identifying and evaluating new biomarkersThe US Food and Drug Administration has a Estonian Biobank is availableFurther information on the high-throughput biomarker profiling by NMR spectroscopyThe Computational Medicine Research Team of the University of Oulu and the University of Bristol have a webpage that provides further information on  Concentrations of metabolites and proteins in the circulation can be indicative of future disease outcomes. The existing molecular biomarkers for all-cause mortality, however, display modest predictive power and risk discrimination In this observational study, two population-based cohorts in Estonia and Finland were followed for all-cause mortality via population registries. All participants provided written informed consent. The Ethics Committee of Human Studies, University of Tartu, Estonia, and the ethical committee of the National Public Health Institute, Finland, approved the studies. An overview of the study design is illustrated in p>0.05 for characteristics in Biomarker profiling was conducted by NMR spectroscopy of non-fasting plasma samples for a random subset of 9,842 individuals (pregnant women excluded). Clinical and demographic characteristics of the subset population did not differ from those of the entire cohort  and serum (FINRISK study) samples was used to quantify the concentrations of 106 circulating lipids, proteins, and metabolites. These candidate biomarkers include 85 lipoprotein lipid measures, four abundant proteins, and 17 low-molecular-weight metabolites, including amino acids, glycolysis precursors, and other small molecules . The canp-value in the Cox model adjusted for age and sex only was included as a predictor. Subsequently, the biomarker leading to the smallest p-value in the multivariate model adjusted for age, sex, and the first biomarker was included in the prediction model. The process was repeated until no additional biomarkers were significant at the Bonferroni-corrected threshold of p<0.0005, accounting for testing of 106 candidate biomarkers.All biomarker concentrations were scaled to standard deviation (SD) units. Cox proportional hazards models were used to assess the association of each candidate biomarker with the risk of all-cause mortality. Age at blood sampling was used as time scale—this effectively corresponds to adjusting for age The hazard ratios (HRs) of the four identified biomarkers for all-cause mortality were subsequently examined in a multivariate model adjusted for age, sex, and conventional risk factors that were significant predictors of mortality in the Estonian Biobank cohort: high-density lipoprotein (HDL) cholesterol, current smoking, prevalent diabetes, prevalent cardiovascular disease, and prevalent cancer (Model A). The biomarker associations were further assessed with additional adjustment for body mass index, systolic blood pressure, total cholesterol, triglycerides, creatinine, cigarettes smoked per day, years smoked, and alcohol consumption (Model B). Proportional hazards assumptions of the regression models were confirmed by Schoenfeld's test. Sub-analyses of the four biomarkers were also conducted for cause-specific mortality. Here, analysis of cardiovascular mortality was adjusted for age, sex, blood pressure, antihypertensive treatment, current smoking, total cholesterol, HDL cholesterol, prevalent diabetes, and prevalent cardiovascular disease http://www.r-project.org/).Biomarker associations with all-cause mortality in the Estonian Biobank were replicated in the FINRISK validation cohort. Cox regression models were evaluated during the first 5 y of follow-up in the FINRISK study in order to match the follow-up time in the Estonian Biobank cohort. The same set of adjustment factors was used as for the discovery cohort (see above). The incremental predictive value of the four circulating biomarkers was tested in the FINRISK validation cohort by comparing a risk prediction score composed of conventional risk factors (Model B) to a risk prediction score extended with the four biomarkers. The risk prediction scores for 5-y mortality in the FINRISK study were calculated based on the regression coefficients derived from the Estonian Biobank cohort in the age range 25–74 y . DiscrimThe discovery analyses of biomarkers predictive of the risk for all-cause mortality comprised 9,842 individuals from the Estonian Biobank cohort with NMR-based circulating biomarker profiles. The findings were validated in a cohort of 7,503 individuals from the FINRISK study. Baseline characteristics of the study populations are shown in The associations of the 106 candidate biomarkers with all-cause mortality are listed in r = 0.53) and triglyceride levels (r = 0.82). The multivariate effect observed for alpha-1-acid glycoprotein and VLDL particle size, with the two biomarkers being more strongly associated with the risk of death when both measures were included in the model, is further illustrated in The four circulating biomarkers were associated with all-cause mortality to a similar extent when adjusted for conventional risk factors that were significant predictors of mortality in the Estonian Biobank cohort : alpha-1-acid glycoprotein , albumin , VLDL particle size , and citrate . All four biomarkers were also associated with all-cause mortality during 5 y of follow-up in the FINRISK validation cohort, with consistent HRs . The resr = 0.38), yet extreme biomarker score values were seen across all age groups. Excess mortality within 5 y of follow-up was observed for higher age, but in particular in combination with an elevated biomarker score (p = 0.48 for interaction with age). To illustrate the strong association of the biomarker summary score in the Estonian Biobank cohort, the cumulative probability of death was derived across quintiles of the biomarker score  and the IDI . The discrimination curves are illustrated in p = 0.001) was achieved when incorporating the four biomarkers into the risk prediction score. Specifically, 81 out of the 157 persons who died during the 5-y follow-up were shifted towards higher risk estimates, while 76 were shifted downwards in risk (net 3.1%); among the 6,953 individuals who did not die, 4,283 persons were shifted towards lower risk estimates and 2,670 were shifted upwards in risk (net 23.2%). The category-based NRI was 9.2% (p = 0.08) when persons were assigned to one of four groups  according to their 5-y risk of death. The category-based reclassification was driven by down-classification of risk among persons who did not die during the 5-y follow-up , as detailed in p>0.01, To illustrate the potential of the circulating biomarkers to improve risk discrimination for all-cause mortality in an independent cohort, risk prediction scores for all-cause mortality with and without the biomarkers were derived in the Estonian Biobank cohort and evaluated in the FINRISK validation cohort. The regression coefficients used for calculating the two risk scores are listed in p>0.05). To examine the biomarker associations with all-cause mortality among apparently healthy persons, we conducted analyses excluding persons with prevalent diabetes, cardiovascular disease, and cancer in both cohorts. Here, all four circulating biomarkers remained predictive of the risk of death with essentially unaltered HRs  is elevated in response to infection and inflammation Plasma albumin, as available from a routine blood test, is a marker of liver and kidney function, nutritional status, and inflammation Triglyceride-mediated lipoprotein metabolism is recognized as a risk factor for cardiovascular disease, particularly in the non-fasting state Citrate is an intermediate in the Krebs cycle and thus central to energy metabolism. Circulating citrate levels are tightly regulated, since citrate acts as a chelator to modulate calcium, magnesium, and zinc ion concentrations, and thereby exhibits anticoagulating properties Out of all available risk factors, the biomarker summary score was the strongest predictor of all-cause mortality in the Estonian Biobank cohort. The biomarker score stratified the short-term risk of death: persons with a very high biomarker score were associated with substantially higher mortality rates than those with average levels of the biomarker score, indicating prominent reflections of frailty in the systemic biomarker profile . ImportaAlthough the associations of the four biomarkers were largely unaffected by potential confounders  and S5, Some limitations of our study should be considered. The molecular coverage available from NMR spectroscopy is limited compared to that afforded by mass spectrometry, which holds further promise for risk assessment and elucidation of disease pathways In summary, high-throughput molecular profiling by NMR spectroscopy highlighted four circulating biomarkers—alpha-1-acid glycoprotein, albumin, VLDL particle size, and citrate—predictive of the short-term risk of death from all causes. The biomarker associations were replicated in an independent population and were consistent when limiting analyses to persons free of apparent disease. All four biomarkers were predictive of death from cancer and nonvascular causes in addition to cardiovascular mortality, and may therefore indicate novel relationships between systemic biomarkers and diverse morbidities. Incorporating the biomarkers into risk prediction scores led to improved discrimination and reclassification of 5-y mortality in the validation cohort. Further investigations are required to clarify the utility of these circulating biomarkers for guiding screening and targeted prevention based on the molecular profile of an individual.Figure S1Correlations between biomarkers for mortality and metabolic risk factors.(PDF)Click here for additional data file.Figure S2Scatter plot of very-low-density lipoprotein particle size versus alpha-1-acid glycoprotein and observed mortality in the Estonian Biobank cohort.(PDF)Click here for additional data file.Figure S3Calibration of risk prediction scores for 5-y all-cause mortality in the FINRISK cohort.(PDF)Click here for additional data file.Figure S4Hazard ratios for all-cause mortality among individuals free of apparent disease at baseline.(PDF)Click here for additional data file.Figure S5Hazard ratios for all-cause mortality upon adjustment for potential confounders in the FINRISK cohort.(PDF)Click here for additional data file.Table S1Circulating candidate biomarkers quantified by high-throughput NMR profiling.(PDF)Click here for additional data file.Table S2Reclassification tables of individuals who died and who did not die during 5 y of follow up in the FINRISK cohort.(PDF)Click here for additional data file.Table S3Hazard ratios for all-cause mortality in the Estonian Biobank cohort stratified by gender.(PDF)Click here for additional data file.


Frailty in later life is viewed as a state of heightened vulnerability to poor outcomes. The utility of frailty as a measure of vulnerability in the assisted living (AL) population remains unexplored. We examined the feasibility and predictive accuracy of two different interpretations of the Cardiovascular Health Study (CHS) frailty criteria in a population-based sample of AL residents.CHS frailty criteria were operationalized using two different approaches in 928 AL residents from the Alberta Continuing Care Epidemiological Studies (ACCES). Risks of one-year mortality and hospitalization were estimated for those categorized as frail or pre-frail (compared with non-frail). The prognostic significance of individual criteria was explored, and the area under the ROC curve (AUC) was calculated for select models to assess the utility of frailty in predicting one-year outcomes.Regarding feasibility, complete CHS criteria could not be assessed for 40% of the initial 1,067 residents. Consideration of supplementary items for select criteria reduced this to 12%. Using absolute (CHS-specified) cut-points, 48% of residents were categorized as frail and were at greater risk for death  and hospitalization . Pre-frail residents defined by absolute cut-points (48.6%) showed no increased risk for mortality or hospitalization compared with non-frail residents. Using relative cut-points (derived from AL sample), 19% were defined as frail and 55% as pre-frail and the associated risks for mortality and hospitalization varied by sex. Frail (but not pre-frail) women were more likely to die (RR 1.58 95% CI 1.02-2.44) and be hospitalized (RR 1.53 95% CI 1.25-1.87). Frail and pre-frail men showed an increased mortality risk  while only pre-frail men had an increased risk of hospitalization (RR 1.58 95% CI 1.15-2.17). Although incorporating either frailty measure improved the performance of predictive models, the best AUCs were 0.702 for mortality and 0.633 for hospitalization.Application of the CHS criteria for frailty was problematic and only marginally improved the prediction of select adverse outcomes in AL residents. Development and validation of alternative approaches for detecting frailty in this population, including consideration of female/male differences, is warranted. Residents of assisted living (AL) facilities represent a unique population in relation to frailty research. AL is an increasingly important residential care option for older adults in North America ,2. AL reAlthough there is no consensus on how best to identify frailty in an older person, criteria proposed by Fried and colleagues using data from the Cardiovascular Health Study (CHS) have attracted the most interest ,5,6. Devabsolute cut-points) for determining select criteria while the second was norm-referenced and identified the poorest performers for that criterion within the AL population (relative cut-points). We hypothesized that the absolute cut-points, developed in a community-based cohort, would be too inclusive and would limit the ability of frailty to discriminate AL residents at higher risk for adverse outcomes. Specific objectives were to: i) determine the feasibility of assessing frailty in AL residents using the CHS criteria; ii) estimate the prevalence of frailty based on CHS criteria and determine associated risks for all-cause mortality and hospitalizations in AL residents; iii) compare the predictive accuracy of the criteria using differing interpretations; and, iv) assess how well models with and without frailty predict mortality and hospitalization.This is the first paper to examine the operationalization, utility and predictive accuracy of the CHS frailty criteria in an AL population. We compared two different interpretations of these criteria. One used CHS-specified cut-points  cohort, a provincial study of the health and quality of care issues in AL and nursing home facilities. The ACCES-AL cohort included residents of designated (publicly-funded) assisted living and supportive housing facilities (DAL) in five former health regions  in the province of Alberta, Canada.These facilities offer a wide array of services in diverse settings and often provide a combination of housing, personal support and health care while promoting autonomy, privacy and independence . A facilAll eligible DAL residents within these facilities were approached for participation. Residents were excluded if they were less than 65 years of age, recently admitted < 21 days), or receiving palliative care . A total of 1,089 participants were enrolled and assessed with 1,067 providing consent for linkage with administrative data  and frailty measures at baseline (2006-2007) and at 1-year. The interRAI-AL tool is a comprehensive, standardized assessment of residents' sociodemographic characteristics, physical and cognitive status, health conditions, behavioural problems, and use of medications and services [Trained research nurses (RNs) administered the Resident Assessment Instrument for Assisted Living (interservices . Residenabsolute cut-points) for determining select criteria  while the second was norm-referenced and identified the poorest performers for that criterion within the AL population (relative cut-points). Also, as described in Table Slow gait speed, muscle weakness (grip strength), low activity level, self-reported weight loss and exhaustion were assessed at baseline with residents categorized as "normal" or "impaired" using the methods described in Table Primary outcomes were one-year mortality  and hospitalizations (determined via linkage with the Alberta Inpatient Discharge Abstract database).RAI-AL tool. Other baseline characteristics included three validated scales derived from items on the interRAI-AL tool: the Cognitive Performance Scale (CPS) [A baseline co-morbidity score was calculated based on the Charlson index  and a vale (CPS) , Depressle (CPS) , and Actle (CPS) .RAI-AL tool were used to assign values in the following manner.Of the 1,067 residents consenting to data linkage, it was possible to assess all five CHS frailty criteria for 648 (60.7%). File comments by the RNs indicated that missing values usually occurred when residents refused or failed to comprehend what was requested. Over half (58%) of the cohort had a diagnosis of dementia. Consequently, additional (observed) functional and health items assessed as part of the interRAI-AL items as assessed by RNs. A positive response to the item, "Subject has had weight loss of 5% or more in last 30 days, or 10% or more in last 180 days" was used for weight loss while a positive response to the item "Due to diminished energy, is unable to finish normal day-to-day activities, or start some or any normal day-to-day activities" was used for exhaustion. This reduced missing values to two for weight loss, and zero for exhaustion.Gait speed was missing for 287 (26%) residents. If the RN documented that at least "limited assistance" was required with "walking between locations on the same floor indoors" and that "in the last 3 days the longest distance walked without sitting down was less than 5 meters", the resident was coded as impaired. This reduced missing gait speed values to 97 . Muscle weakness was missing for 105 residents (10%). Impairment was determined as present if RNs stated the resident was physically unable to perform the grip test. This reduced missing muscle weakness values to 74 (7%). Missing data on weight loss and exhaustion occurred in 116 (11%) and 149 (14%) of residents respectively. Assignment for these variables was based on yes/no responses to interafter value assignment); two were lost to follow-up, and one moved out of province after baseline . The final analysis sample was 928 - see Figure Of the enrolled cohort of 1,089, 22 residents refused consent for administrative data linkage (or no linkage was available); 136 had one or more missing frailty value(s)  were assessed in multivariable models. The models considered potential effect modification and confounding by age, sex and co-morbidity. Predictive accuracy was assessed by comparing the area under the receiver operating characteristic curve (AUC) of a baseline model with only age and sex with that of models with age/sex/co-morbidity, age/sex/frailty, and age/sex/frailty/co-morbidity respectively. To facilitate model comparisons, 95% confidence intervals for differences in AUC estimates  were calculated as has been reported by others .M-1)r, where M is the mean cluster size and r is the estimated intraclass correlation coefficient. The mean cluster size was 15.7 residents among the 59 facilities and the estimated intraclass correlation coefficient was 0.0012, resulting in a design effect of 1.018. As the magnitude of the design effect was small , we have presented the results of our main analyses without adjustment for clustering - which permits the use of simpler statistical approaches without the loss of information. SAS Version 9.2  was used for analyses.The level of clustering of residents within facilities was quantified by calculating the design effect as 1+ with an average age of 84.9 years. For 364 of the 421 eligible residents not enrolled, age and sex were available and showed a similar distribution  to the enrolled cohort.Most baseline characteristics of the analysis sample did not differ significantly from the enrolled cohort Table , althougThe CHS absolute cut-point for gait speed categorized 69% of AL residents as impaired Table . AlthougThe proportion of residents categorized as frail was 48% (445) using the CHS absolute cut-points and 19.2% (178) using the relative cut-points. The proportions defined as not frail (robust) or pre-frail were 3.4% (32) and 48.6% (451) using absolute cut-points and 25.6% (238) and 55.2% (512) with relative cut-points, respectively. During follow-up, there were 142 deaths , and 375 residents had at least one hospitalization . Of the individual frailty criteria, the best predictors of death in a model adjusting for age, sex, co-morbidity index and other frailty criteria were the relative definition of slow gait  1.01-1.83), either definition of low physical activity , and exhaustion   and to be hospitalized  within one year after adjusting for age, sex and co-morbidity  of 0.648 . The addition of co-morbidity resulted in an AUC of 0.600 - slightly higher than the models with frailty . Including both co-morbidity and frailty led to an AUC of 0.631 and 0.633 for absolute and relative frailty cut-points respectively.For mortality and hospitalization, the fully adjusted models with the absolute (Model 4) or relative (Model 6) frailty measures did not differ significantly from each other but both were associated with significantly higher AUCs (predictive accuracy) compared with Model 2 .RAI-AL tool.The above findings were essentially unchanged in models incorporating a co-morbidity count derived from diagnoses recorded on the interThe AL population is often described as 'frail', yet surprisingly it represents a care setting little studied in frailty research. The CHS frailty criteria have been widely examined in community-dwelling older populations. They have been investigated within diverse settings and cultural groups -22 and aAlthough both frailty measures produced adjusted models with comparable predictive accuracy, the relative measure illustrated potentially interesting sex-differences with regard to the differential detection of risk across levels of vulnerability among AL residents. Further, the use of this relative frailty measure demonstrated the importance of considering possible competing risks when examining the prognostic significance of selected frailty measures for particular outcomes of interest . Specifically, the failure to observe a significantly increased hospitalization risk for frail men in the present study was a consequence of their particularly high mortality risk within the one year follow-up.RAI-AL tool) greatly improved the performance of our mortality models in terms of increasing the AUCs; whereas the addition of co-morbidity did result in models with improved predictive accuracy for hospitalization.Of the five CHS criteria, slow gait and low physical activity most consistently predicted death and hospitalization. There is growing evidence of the significance of slow gait speed as an independent predictor of health outcomes, in particular all-cause mortality, among older community-dwelling adults ,32. ConsThe feasibility and predictive accuracy concerns raised by our findings likely reflect, in part, the inherent differences between our AL population and the community-based population used to derive the CHS criteria and frailty 'phenotype' . NotableA single standard frailty measure used in all settings would allow comparisons of vulnerable populations across care settings and the tracking of individuals as they transition from one setting to the next. Such a single measure, though, may be problematic depending on its composition and definition. Some measures may not be feasible or may have limited discriminatory ability  in more disabled populations. For gait speed, different cut-points have been suggested for less and more disabled populations . This maSome limitations of our study warrant consideration. Over four hundred eligible residents did not enroll in the study. Although the age and sex distribution of this sample was comparable to our enrolled cohort, this may raise concerns regarding the generalizability of our findings to the larger AL population. We restricted eligibility to residents of publicly-subsidized (or designated) AL facilities in Alberta. Our findings may not apply to residents in private AL or other AL-type facilities across Canada. Hospitalizations were captured using provincial data and may have missed the rare event that occurred outside Alberta. Our assessments for low physical activity, unintentional weight loss and exhaustion varied slightly from the specific measures used in the CHS . While wThis study represents the first attempt to examine the utility and prognostic accuracy of frailty among older AL residents. The rapid expansion of AL across North America and the significant heterogeneity in functional, psychosocial and health needs of residents in this care setting makes it an important target for frailty research. Our findings suggest that the CHS definition of frailty, based on physical performance measures and self-reported data, may have limited practical and prognostic value among residents of AL facilities. At the same time, our results highlight the need for further research on frailty in the AL setting including consideration of cognitive and social determinants ,39,44 ofThe authors declare that they have no competing interests.EF was responsible for the main analysis and interpretation of the data and drafted the initial manuscript. CM and LS were responsible for the conception and design of the study, directed the acquisition of data and made substantial contributions to the analysis and interpretation of data. DH, SP and HS provided substantial input regarding clinical content and analysis/interpretation of the data. ME made substantial contributions to the study design and analysis of data. All authors were involved in revising the manuscript critically for important intellectual content and have given final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2318/11/23/prepub


Several lines of evidence suggest a possible functional role of Matrix metalloproteinase -2 (MMP-2) in obesity. The aim of this study was to evaluate the role of MMP-2 promoter polymorphisms in percentage body fat (PBF) as a measure of childhood obesity in a New Zealand population.546 samples from the Auckland Birthweight Collaborative (ABC) study were genotyped for the three MMP-2 promoter SNPs -1306 C/T (rs243865), -1575G/A (rs243866) and -790 T/G (rs243864) using the Sequenom genotyping platform. The results demonstrated that an MMP-2 promoter haplotype is associated with PBF in New Zealand 7 year old children.We have previously determined that environmental factors are associated with differences in PBF in this study group, and now we have demonstrated a possible genetic contribution. Obesity is reaching epidemic proportions worldwide and is now occurring at younger ages. In New Zealand, one in twelve youngsters aged 2 to 14 years are considered to be obese and one in five are classified as overweight .Obese children are at increased risk of a number of health problems including diabetes, sleep problems, joint problems, early puberty or menarche, asthma and other respiratory problems . ObesityBody mass index (BMI) is the most common method used to determine obesity. However, although BMI may facilitate an adult obesity diagnosis, using this method to diagnose obesity in children may yield more ambiguous results, as BMI is a marker of relative weight and doesn't directly measure body fat . MeasuriChildhood obesity is thought to be the result of an interplay between many genetic and environmental factors. We have previously investigated environmental factors in the Auckland Birthweight Collaborative (ABC) study, and found maternal overweight/obesity, maternal age, female gender, sedentary activity time and hours of television viewing to be independently associated with PBF in 7 year old children . We are Several lines of evidence suggest a possible functional role of Matrix metalloproteinase -2 (MMP-2) in obesity. The main role of MMP-2 is in the degradation of type IV collagen, the major structural component of basement membranes. However, the enzyme also has activity toward a spectrum of functional molecules including growth factor-binding proteins and growth factor receptors, which are known to be involved in obesity. For example, MMP-2 can cleave insulin-like growth factor-binding proteins and release insulin-like growth factors . It has We undertook an association analysis of 3 functional polymorphisms in the promoter of the MMP-2 gene , to investigate the possible role for MMP-2 as a genetic risk factor for obesity (in terms of increased PBF) in the ABC study.The -1306 C/T (rs243865) polymorphism is located in the CCACC box of the Sp1-binding site and displays a strikingly lower promoter activity with the T allele . The -15The ABC study was designed as a case-control study to determine risk factors for small for gestational age (SGA) infants and has been described in detail previously . Data haPercentage body fat (PBF) at age 7 years was the outcome measure analysed in this paper. This was measured using bioelectrical impedance analysis (BIA). This allows calculation of fat free mass which was calculated according to the formula of Schaeffer et al  and consAt 11 years 546 participants consented to collection of peripheral blood (n = 397) or a buccal swab (n = 149) for DNA extraction and genotyping.http://www.hapmap.org.DNA was extracted from the blood/buccal samples using Qiagen's DNA extraction kit and following the manufacturer's instructions. Genotyping was performed with the MassARRAY and iPlex systems of the Sequenom genotyping platform , which uses the MALDI-TOF primer extension assay ,18, accoSNPs were tested for deviation from Hardy-Weinberg equilibrium (HWE) using a chi-square goodness-of-fit test.A Generalised linear model (GLM) was used to test the linearity of genotype-phenotype relationship for quantitative traits. For linearity of the genotype-phenotype relationship for quantitative traits, each SNP was coded 0, 1, and 2 for each tested allele ,20. For To determine LD (linkage disequilibrium) between the SNPs we uploaded our data into haploview . HaplotyStatistical analyses were carried out using R  and SAS The study received ethical approval from the Northern Regional Ethics Committee. Signed consent for the study and extraction of DNA was given by the parents of the children and assent also given by the child.The genotype counts (frequencies) for the three genotyped SNPs are shown in table The 3 MMP-2 promoter SNPs are in high LD with each other Figure , so we uThis paper reports for the first time the role of MMP-2 promoter polymorphisms in childhood obesity as measured using PBF. Although we were unable to demonstrate an association for the 3 SNPs individually after correcting for confounding factors, the GTA  haplotype was statistically significant. It has previously been suggested that studying haplotypes could be more informative than the study of individual SNPs .Genetic variation in MMP-2 has been previously investigated for association with obesity, in adults from Korea . WhereasThe genetic association we report in the MMP-2 promoter may result from altered MMP expression, as the 3 SNPs investigated are all functional SNPs capable of affecting transcriptional activity. Indeed, associations with MMP-2 circulating levels in mouse models and obese children and adults have been previously reported. High expression of MMP-2 has been demonstrated in adipose tissue of mice with nutritionally induced obesity, as well as in genetically obese mice ,10,27. BAdditional studies are now required to further investigate MMP-2 involvement in the development of obesity. It may be also be worth considering other matrix metalloproteinases.In conclusion the results presented here demonstrate that an MMP-2 promoter haplotype is associated with PBF in New Zealand 7 year old children. We have previously determined that environmental factors are associated with differences in PBF in the ABC study, and now we have demonstrated a possible genetic contribution.The authors declare that they have no competing interests.ARM made substantial contributions to this study. She designed the study, carried out the genotyping experiments and was primary author of the manuscript. DYH was responsible for the statistical analysis. JMDT helped in establishing the ABC cohort and its data collection and contributed to editing of the manuscript. EAM established the ABC cohort and its data collection and contributed to editing of the manuscript. LRF participated in editing of the manuscript. All authors read and approved the final manuscript.


Thiol self-assembled monolayers (SAMs) are widely used in many nano- and bio-technology applications. We report a new approach to create and characterize a thiol SAMs micropattern with alternating charges on a flat gold-coated substrate using atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). We produced SAMs-patterns made of alternating positively charged, negatively charged, and hydrophobic-terminated thiols by an automated AFM-assisted manipulation, or nanografting. We show that these thiol patterns possess only small topographical differences as revealed by AFM, and distinguished differences in surface potential (20-50 mV), revealed by KPFM. The pattern can be helpful in the development of biosensor technologies, specifically for selective binding of biomolecules based on charge and hydrophobicity, and serve as a model for creating surfaces with quantified alternating surface potential distribution. Thiol self-assembled monolayers (SAMs) are promising for many nano- and bio-technology applications as they offer a reliable method to produce surfaces with desirable properties. These properties can be used for specific and non-specific binding of biomolecules and nanoparticles and, therefore, can serve as useful templates for nano- and micro-fabrication. The first systematic study of thiol chemicals was reported by Zisman and co-authors , and hasWith the invention of scanning probe microscopy and other nanoscale characterization techniques, much interest has been created in nanoscale fabrication for nanoelectronics and biosensing. The advantage of sensing on the nanoscale using miniaturized devices created a demand in producing thiol SAMs of a repeated pattern, which can be used as biosensing platforms. Many nanopatterning techniques require electron-beam or photo-lithography in vacuum environments , using a2 squares at least 512 lines were required. The scratched squares produced by the AFM probe were immediately filled by thiol 2 present in solution due to the higher concentration of this thiol. The second thiol must have a higher affinity for the metallic surface to replace the first thiol removed from the surface by AFM probe. This process makes it possible to create an alternating charge pattern, composed of two different thiols. AFM and KPFM were used to characterize the pattern in terms of topography and surface potential.Uniform surface coverage with one thiol was created by incubating a gold surface for 24-72 h in this thiol solution. After incubation, the sample was exposed to a second thiol solution in an AFM liquid cell. The AFM probe, with a spring constant of 5 N/m, was inserted into the liquid cell and was used to scratch squares of defined dimension, varying from 10 by 10 nm to 10 by 10 μm in contact mode. The force applied was just high enough to remove thiol molecules from the surface (approximately between 10 and 50 nN), thus leaving the gold undamaged. We have performed scratching at high speeds (10 lines/s) in order to reduce thermal drift and decrease the time required to create a pattern. The number of lines per square depends on the tip geometry, but we found for 10 μm3-terminated thiol molecules on gold-coated glass for 24 h. Figure 3-terminated) displacement [We first incubated a solution of CHlacement . During lacement .3-terminated thiol) with thiol 2 (COOH-terminated thiol). Topography does not show much contrast as the two thiols do not differ significantly in height. The cross-section plot for topography image shows flat profile, with exception of few impurities. Figure Figure In summary, we showed that thiol SAM pattern with chemical functionality and desired surface potential differences can be created using AFM-based nanografting method. In addition, we demonstrated for the first time that small differences in surface potential maps associated with organic thiol patterns can be resolved by KPFM in mV range. Such patterns with controlled differences in surface potential can be useful in nano- and bio-technology applications and to study interactions of charged species, such as nanoparticles and macromolecular ions with non-uniformly charges surfaces.3, NH2, and COOH-terminated surfaces, respectively. All chemicals were used as received with no further purification. Thiols were dissolved in ethanol at 5 mM concentration.Decanethiol, cysteamine hydrochloride, 3-mercaptopropionic acid, and HPLC grade ethanol were purchased from Sigma-Aldrich Chemical Co. . These chemicals produce CHGold-coated mica slides were purchased from Agilent Technologies, Inc. . Before use, these gold surfaces were glued to clean glass cover slips using Epo-Tek 377 glue from Epo-Tek, Inc. , which was cured at 150°C for 1 h . The mic2 to few μm2.AFM uses a sharp probe over a sample surface and allows for imaging the topographical features at the nanoscale. Two common modes of operation are contact mode and intermittent contact mode. Thiol-modified surfaces were imaged in intermittent contact mode with a JPK Nanowizard II atomic force microscope. In intermittent contact mode, the tip is oscillated at the resonant frequency of the cantilever, and a feedback loop maintains constant amplitude over the entire image to insure the gentle imaging conditions. The probes used were Nanoworld NCH tips with a resonant frequency of approximately 338 kHz and 42 N/m spring constant. In contact mode of imaging, the tip usually lightly touches the surface and is moved up and down with the topographical features of the sample. With increased force the probe can interact strongly with the surfaces and remove soft matter from the surface. This approach was used for nanografting. Automated patterning was achieved by programming the JPK AFM imaging software to scratch a square of defined size and then move to the next defined location. Alternating this process produces the pattern with the size of few nmKPFM is an extension of AFM that provides the ability to map the surface potential in addition to imaging sample topography -17. KPFMSurface potential images were recorded in air using Nanoworld NCH cantilevers with a JPK Nanowizard II AFM in a hover mode KPFM. The gold substrates were grounded to eliminate sample charging.AFM: atomic force microscopy; KPFM: Kelvin probe force microscopy; thiols: SAMs (self-assembled monolayers).The authors declare that they have no competing interests.BM and JS carried out the thiol SAM preparation and nanografting experiments, participated in the manuscript draft preparation. BM carried out KPFM imaging. SX participated in nanografting experiments and participated in the manuscript draft preparation. ZL conceived of the study, participated in its design and coordination and finished the final draft of the manuscript. All authors read and approved the final manuscript.


People can inhibit an action because of an instruction by an external stimulus, or because of their own internal decision. The similarities and differences between these two forms of inhibition are not well understood. Therefore, in the present study the neural correlates of intentional and stimulus-driven inhibition were tested in the same subjects. Participants performed two inhibition tasks while lying in the scanner: the marble task in which they had to choose for themselves between intentionally acting on, or inhibiting a prepotent response to measure intentional inhibition, and the classical stop signal task in which an external signal triggered the inhibition process. Results showed that intentional inhibition decision processes rely on a neural network that has been documented extensively for stimulus-driven inhibition, including bilateral parietal and lateral prefrontal cortex and pre-supplementary motor area. We also found activation in dorsal frontomedian cortex and left inferior frontal gyrus during intentional inhibition that depended on the history of previous choices. Together, these results indicate that intentional inhibition and stimulus-driven inhibition engage a common inhibition network, but intentional inhibition is also characterized by additional context-dependent neural activation in medial prefrontal cortex. In daily life, most people experience and exercise a degree of voluntary control over their actions. The concept of intentional action is well recognized in the neuroscience literature. Several studies have focused on the voluntary choice between alternative actions , and the voluntary choice of when to initiate action (the “when–component”) . Within neuroscience research stimulus-driven inhibition has been extensively studied using different paradigms, such as go/nogo tasks  also involved in intentional inhibition, or is a different neural network involved in intentional inhibition (including dFMC)? Additionally, is intentional inhibition dependent on preceding context, as has been previously observed for stimulus-driven inhibition  participated in the experiment. All participants had normal or corrected-to-normal vision, and no neurological or psychiatric impairments according to self-report. Before participating in the experiment, all participants signed informed consent. In accordance with guidelines of the Leiden University Medical Center, all anatomical scans were reviewed by a radiologist. No anomalous findings were reported. To obtain an estimate of cognitive functioning participants completed two subtests of the Wechsler Adult Intelligence Scale (WAIS) .Twenty-four healthy right-handed adults between 18 and 26 years of age  and instructed (green) trials discouraged participants from strategically pre-deciding a pattern of intentional action, such as act-inhibit-act-inhibit etc.The stop-signal task  a stop-signal was presented. In this case the arrow suddenly changed color to red. This color change indicated that participants had to inhibit responding to the direction of the arrow. Stop-signal delay (SSD) was adjusted using a staircase-tracking procedure to guarantee a 50% inhibition rate . The first 2 volumes of each run were discarded in order to allow for equilibration of T1 saturation effects . After the functional runs a high-resolution 3D T1-FFE scan for anatomical reference was obtained . Head motion was restricted by using foam inserts between the head and the head coil. Visual stimuli were projected onto a screen in the magnet bore that could be viewed through a mirror attached to the head coil.Scanning was performed with a standard whole-head coil on a 3.0 Tesla Philips scanner at the Leiden University Medical Center. The marble task consisted of 3 event-related runs, each lasting approximately 6 min, and the stop-signal task consisted of 2 event-related runs, both lasting approximately 5 min. Functional data were acquired using T2For the marble task, repeated measures analyses of variance were performed to examine the effect of preceding context on intentional inhibition. Planned comparisons were performed between the different numbers of preceding green trials, to examine which conditions differed from each other.The use of response selection strategies on the marble task was evaluated by computing the Random Number Generation 2 (RNG2) index using the program RgCalc  was calculated according to the horse-race model of stopping . Images were corrected for rigid-body motion. Structural and functional volumes were spatially normalized to T1 templates. The normalization algorithm used a 12-parameter affine nonlinear transformation involving cosine basis functions, and then resampled the volumes to 3-mm cubic voxels. Translational movement parameters never exceeded 1 voxel (<3 mm) in any direction for any subject or scan. Templates were based on the MNI305 stereotaxic space .Region of interest (ROI) analyses were performed to further characterize the involvement of brain regions in intentional inhibition. ROI analyses were performed with the MarsBaR toolbox in SPM8  = 18.09, p < 0.001  and when there was 1 compared to 3 preceding green trials (p < 0.05). The level of inhibition was comparable for the conditions where there were 1 or 2, and 2 or 3 preceding green trials .Participants successfully responded to the green marble on 63.22% of the trials. Participants intentionally inhibited responding to the white marble on 53.17% of the trials. Participants more often decided to inhibit responding to the white marble when there were fewer preceding green trials, SD = 0.012) was observed. To examine the randomness the participants' RNG2 index was compared with a RNG2 index computed over a set of randomly generated sequences of go- and nogo-responses. For the randomly generated set of go- and nogo-response sequences a mean RNG2 index of 0.801 (SD = 0.002) was observed. Although the RNG2 index for the randomly generated sequences was marginally but significantly smaller compared to the participants' RNG2 index, F = 5.71, p < 0.05, these results indicate that the participants' behavior was close to being random and not driven by simple alternation strategies.To examine the randomness of response selection the RNG2 index was computed. A mean RNG2 index of 0.807  compared to the white marble trials , F = 32.55, p < 0.001, indicating that the decision process in the white marble trials took more time. However, longer reaction times on the white marble trials might also be partially related to the slower marble speed on those trials. Reaction times on the white marble trials were faster when there were more preceding green trials, F = 5.52, p < 0.01 . Reaction times did not differ between the other conditions of preceding green trials .Reaction times were shorter for the green marble trials (SD = 133). Participants successfully inhibited responding to the direction of the arrow on 46.03% of the stop-trials. SSRT was 281 ms (SD = 45).Participants successfully responded to the direction of the arrow on 96.46% of the go-trials. The average reaction time on the successful go-trials was 519 ms  was not correlated with stimulus-driven inhibition as measured by the stop-signal task (SSRT), First, to identify the brain regions underlying the intentional inhibition decision process the contrast intentional inhibition > stimulus-driven action (White NoGo > Green Go) was computed. This analysis revealed activation in a widespread neural network . This analysis revealed activation in bilateral IPL, left IFG, left MFG, right medial temporal gyrus (MTG), and occipital lobe  . For left IFG pars orbitalis and pars triangularis contrast values were significantly different from zero when there were 0 preceding green trials .In order to examine the effect of the number of preceding green trials on intentional inhibition decision outcomes, a parametric analysis of the number of preceding green trials was performed on the contrast intentional inhibition > intentional action (White NoGo > White Go). This analysis revealed stronger activation in dFMC, left IFG pars orbitalis, left IFG pars triangularis, and right SFG when there were fewer preceding green trials  both intentional action and intentional inhibition decisions resulted in a large network of activation including the lateral prefrontal cortex, parietal cortex, and preSMA, regions previously referred to as the intentionality network  prefrontal cortex. Indeed, the contrast intentional action vs. stimulus-driven action (white vs. green marble Go responses) confirmed that this network was largely engaged, consistent with prior studies in the literature on intention action (Lau et al., per se (Karch et al., Secondly, we tested whether there were brain regions uniquely related to the intentional inhibition decision outcome by contrasting intentional inhibition with intentional action. A network of brain regions was active for intentional inhibition compared to intentional action including bilateral IPL and left IFG, suggesting that the inhibition process cannot be reduced to intentionality Contrary to prior findings by Kühn et al. , we obseoptions (Cisek, The results of the parametric analysis shed important light on the role of dFMC in intentional inhibition, and on the significance of intentional inhibition more generally. Briefly, we found dFMC activation was reduced when previous trials had created a prepotent urge to act. Our design differs from the original free-choice whether decision of Brass and Haggard , by incls Cisek, .This reciprocal antagonism corresponds to the common intuition that inhibition of action is harder when the drive to act is strong—for example in cases such as craving and addiction. Interestingly, these are exactly the circumstances when intentional inhibition may also be most necessary. It may also explain why we did not find dFMC activation in our main contrast, while previous studies that did not use instructed action trials to enforce a prepotent urge to act did (Brass and Haggard, side-by-side comparison between the intentional and the stimulus driven tasks was made. Stimulus driven inhibition resulted in the expected network of activation, including the right IFG and pre-SMA (Aron et al., Third, a Some limitations of the present study deserve mention. First, the fact that two different tasks were used to measure intentional and stimulus-driven inhibition pre-empted the possibility to compute a direct contrast between intentional and stimulus-driven inhibition. Future research might benefit from using one single task to measure both forms of inhibition, to allow for such a direct contrast. Second, for the marble task we cannot completely rule out the possibility that participants have pre-decided not to initiate an action on the intentional inhibition trials, instead of deciding in the instant to inhibit an already initiated action. The observed pattern of results showing that intentional inhibition was less likely following a run of instructed action trials, suggests that this was not the case. However, future research could shed more light on this issue, for instance by including electromyography measures to ascertain that the initial action initiation is also present in intentional inhibition trials.per se (Karch et al., Taken together, this study was the first to test the neural correlates of intentional and stimulus-driven inhibition within the same subjects. The results confirmed the hypothesis that these two types of inhibition rely on the same neural network including lateral PFC and preSMA, regions previously associated with intentionality (Lau et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique heterodimeric DNA-topoisomerase IB (LdTopIB). LdTopIB is a heterodimer made up of a large subunit and a small subunit that must interact with each other to build an active enzyme able to solve the topological tensions on the DNA. As LdTopIB is located within the nucleus, one or more nuclear localization signals (NLS) should exist to ensure its nuclear translocation. In this report three novel NLS have been identified through a sequential deletion study of the genes encoding of both subunits fused to that encoding the green fluorescent protein (GFP). NLS1 is a highly basic sequence of 43 amino acids in the C-terminal extension of the large protomer. We found two well-defined sequences in the small protomer: NLS2 is a 10-amino acid motif located in the N-terminal extension of the protein; NLS3 consists of a complex region of 28 amino acids placed in the vicinity of the catalytic Tyr-222 included at the conserved SKINY signature within the C-terminal. Furthermore, by means of yeast cell viability assays, conducted with several LdTopIB chimeras lacking any of the NLS motives, we have revealed that both subunits are transported independently to the nucleus. There was no evidence of LdTopIB accumulation in mitochondria or association to the kinetoplast DNA network. The results rule out the former hypothesis, which attributes nucleocytoplasmic transport of LdTopIB entirely to the large subunit. The LdTopIB is localized to the nucleus only. Leishmania donovani, has several evolutionary peculiarities that hinder the fight against this disease. These include, the plasticity of its genome  for cytotoxic assays . As control, assay was performed with the pESC-URA expressing vector without any insert (“mock”) under same conditions as the wt-transformed vector. All plates contained 0.4% DMSO.To analyse the  purpose . The bicLeishmania expression vector pXG-GFP+2 . This fusion chimera (TopL1), which involves the entire subunit (aa 1-635), was cloned and transfected into L. donovani promastigotes. As expected, TopL1 targeted the nucleus with no cytoplasmic compartmentalization , included the short and non-conserved N-terminal extension, as well as the first 125 amino acids of the core domain. Since NLS of the human TopIB are placed at its N-terminal domain, we expected to find a similar distribution in LdTopIL. Surprisingly, this chimera behaved differently from TopL1, with the protein fluorescence scattering throughout the cytoplasm. As no NLS was found in this region, we prepared TopL3 and TopL4 that ranged from residues 171 to 337 and 338 to 548, respectively. The main feature of TopL3 construct is the presence of the catalytic Arg-314 residue. Meanwhile, TopL4 represents the most conserved region of the protein, harboring the other three residues involved in the catalytic activity; Lys-353, Arg-410 and His-453. The subcellular localization of TopL3 and TopL4 was examined by direct fluorescence of transfected cells. eus only .Once the region responsible for the nuclear translocation of LdTopIL was roughly determined, we proceeded to define the NLS with more accuracy through the analysis of the constructs shown in At this end we have successfully identified a highly basic region of 44 amino acids (aa 549-592) able to target the whole subunit to the nucleus. By analyzing these constructions all together we can state that Lys-enriched stretches involving residues 548-566 and 578-592 could constitute a putative NLS. However, the role played by the amino acids between these two regions remained unclear. In order to identify this role, we performed two serial internal deletions removing these nuclear-targeting regions. In the first instance the sequence between residues 548 to 566 was deleted (TopL10), which was used as template for the second internal deletion process removing amino acids 578 to 592 yielding the TopL11 construct. Confocal analysis of this last chimera revealed a clear nuclear localization of green fluorescence, thus suggesting that this 14-amino acid sequence (aa 562-575) acts as a potential NLS. Consequently, one would think that LdTopIL contains three NLS regions. Nonetheless, the sequence continuity as well as the high density of basic residues makes us think that it may be a NLS complex , in which several stretches can assume the role of NLS when any of them is removed. This makes it impossible to further delineate a smaller sequence responsible for this nuclear-transport process.As no NLS motifs were expected to be present in LdTopIS it was amazing to find out how the entire small subunit fused to the GFP (TopS 1 aa 1-262), targeting the nucleus with no cytoplasmatic compartmentalization . This ouLdTopIS N-terminal extension (aa 1-210) contains a long non-conserved motif (aa 85-136) rich in serine residues as well as an undecapeptide that is highly conserved in trypanosomatids (aa 171-183), which in turn, confers sensitivity to CPT [11]. Finally, the C-terminal end aa 211-262) is phylogenetically conserved and contains the active site "SKxxY" in which Tyr-222 fulfils the function of cleaving the DNA substrate. We have also identified the similarity between the acidic-NLS DEDDAD motif (aa 117-146) described in the hTopIB [2 is phylIn the first extent four constructs were designed and fused to a GFP encoding gene in the pXG-GFP+2 at the end of the GFP ORF : TopS2 (TopS2 chimera amplified a fragment ending just before the start of the APPPKVRRVE sequence (aa 86-96). This fragment displayed a clear accumulation of green fluorescence in the cytoplasm  demonstrTopS6 construct extends from the initial methionine to residue 109, being the sum of TopS2 chimera, the PPPKVRRVE motif and the entire serine cluster. In order to narrow the sequence constituting NLS2, a construct ending just before the serine cluster (TopS9) was cloned and put under study. As shown in Despite having identified a NLS signature within this region, it still remained unsure the possible role of DDDSSSDD sequence (aa 110-117), which shares a high resemblance to the acid signal described for hTopIB (aa 117-146) . To thisOnce NLS2 within the TopS3 sequence was defined, it was time to study the NLS identified region at the C-terminal end of the subunit. For this purpose we generated a TopS12 construct , which eFinally, we wanted to verify if the whole subunit could be transported to the nucleus in the absence of NLS3. TopS18 chimera along with TopS13 confirms that both NLS are able to drive LdTopIS to the cell nucleus independently.Once NLS sequences were delineated in both LdTopIB subunits , we deciBecause we have used a TopIB-deficient strain, the heterologous co-expression of both LdTopIB subunits would make MBY3 cells sensitive to CPT, thus inducing yeast death. As expected, all the constructs perfectly grew in the presence of glucose, because LdTopIB is only expressed in the presence of galactose . In order to assess whether enzyme expression is deleterious or not by itself, cultures were grown in SC-Ura plates containing 2% (w/v) galactose and 0.5% (v/v) DMSO (CPT solvent). As shown in the second column of in vivo.When NLS1 complex was removed from the large subunit (L-ΔNLS1 + S-wt) the culture displayed no sensitivity to any CPT concentration. As a result that no activity related residues were altered or removed , this inL. donovani promastigotes, DNA was stained by incubation with a DAPI solution (see section 2.6.). As shown in all the figures in the present report, neither LdTopIL, nor LdTopIS fused chimeras were driven to the mitochondria and/or kDNA. These results demonstrate that LdTopIB lacks signature motives responsible for kinetoplast signaling or mitochondrial accumulation, which have been described in higher organisms.To assess the subcellular localization of all the studied GFP-fusion proteins in To catalyze changes in DNA superhelicity, TopIB should be located inside the nucleus, where it produces temporary single DNA-chain scissions, which allows relaxation before the encoded information is accessible for transcription, reparation or replication enzymes .Proteins operating in the nucleus are synthesized in cytosol and therefore, they must be driven to the nucleus by a specific group of karyopherins called importins. Importins translocate those proteins that contain recognizable NLS motifs in their sequence –31. NLS L. major KIN13-1 kinesin, a 60-residues-length non-canonical NLS placed at the C-terminal end of the protein [L. donovani Tdp1 [L. donovani Top II fused to GFP delimited another region of around 60 amino acids at the C-terminal end, which contains potential NLS motifs responsible for the translocation of this enzyme to the cell nucleus [Leishmania [Both its nature and length are very heterogeneous factors in trypanosomatids NLS. While Kumar et al.  found a  protein . Anotherani Tdp1 . A delet nucleus . These rshmania ,21.NLS2 is a ten-residues-length motif placed at the N-terminal end of LdTopIS, heading a large cluster of serine residues. This fact is relevant, as the PPPKVRRVES peptide requires the presence of at least one Ser residue to fully complete a functional NLS. A plausible explanation could be that at least one Ser residue must be phosphorylated for transportation to take place, as previously described for other NLS motifs within different proteins . In the in vivo.Some research groups have hypothesized that LdTopIB must be necessarily assembled in the cytoplasm to be translocated to the cell nucleus. Furthermore, LdTopIL should be acting as a carrier of the proenzyme . HoweverT. brucei, both associated with DNA minicircles and therefore, they are operative to solve any topological problem within the organelles during cell replication. However, Das and coworkers [There are several studies that show that eukaryotic TopIB is dually accumulated in both nucleus and mitochondria . Trypanooworkers  indicateThree putative NLS sequences have been identified in LdTopIB. LdTopIL subunit contains the NLS1 motif formed by 44 amino acids located in its C-terminal end. This is a large region within which small groups of adjacent basic amino acids can assume the role of driving the protein to the cell nucleus. LdTopIS subunit contains two NLS. NLS2 sequence is comprised of a PPPKVRRVES motif located between positions 88 and 97, whereas NLS3 involves 28 amino acids (residues 219-246) at the C-terminal end including the catalytic Tyr-222. Finally, neither of the entire subunits, nor any of the constructs fused to GFP were driven to the kinetoplast.


Dental caries is unevenly distributed within populations with a higher burden in low socio-economy groups. Several attempts have been made to allocate resources to those that need them the most; there is a need for convenient approaches to population-based monitoring of caries risk over time. The aim of this study was to develop the geo-map concept, addressing time trends in caries risk, and demonstrate the novel approach by analyzing epidemiological data from preschool residents in the region of Halland, Sweden.The study population consisted of 9,973 (2006) and 10,927 (2010) children between 3 to 6years of age (~77% of the eligible population) from whom caries data were obtained. Reported dmfs>0 for a child was considered as the primary caries outcome. Each study individual was geo-coded with respect to his/her residence parish (66 parishes in the region). Smoothed caries risk geo-maps, along with corresponding statistical certainty geo-maps, were produced by using the free software Rapid Inquiry Facility and the ESRI ArcGIS system. Parish-level socioeconomic data were available.The overall proportion of caries-free (dmfs=0) children improved from 84.0% in 2006 to 88.6% in 2010. The ratio of maximum and minimum (parish-level) smoothed relative risks (SmRRs) increased from 1.76/0.44=4.0 in 2006 to 2.37/0.33=7.2 in 2010, which indicated an increased geographical polarization of early childhood caries in the population. Eight parishes showed evidential, positional changes in caries risk between 2006 and 2010; their corresponding SmRRs and statistical certainty ranks changed markedly. No considerable parallel changes in parish-level socioeconomic characteristics were seen during the same time period.Geo-maps based on caries risk can be used to monitor changes in caries risk over time. Thus, geo-mapping offers a convenient tool for evaluating the effectiveness of tailored health promotion and preventive care in child populations. The prevalence of dental caries has declined globally over the past decades but all children have not benefited from the improved oral health. Widening inequalities between social classes and certain minority ethnic groups are evident -5. CarieIn a recent paper we suggested the use of geo-maps for presenting epidemiological data based on childhood caries risk in a Swedish population and we advocated this novel approach for allocation of preventive care . In ordeThe vast majority of all children (around 90%) in the region are listed as regular patients at the Public Dental Service that provides free dental care between 1 and 19years with recall intervals varying from 3 to 24months depending on the individual need. Data on the experience of manifest (dentin) caries is registered according to the WHO-criteria  and annuThe present study included 9,973 children between 3-6years of age for whom caries data were reported in 2006 and 10,927 children with corresponding data from 2010. The age distributions of the children examined in 2006 and 2010, respectively, did only differ marginally . The overall coverage of the total 3-6-year population of the Halland region was 76% in 2006 and 77% in 2010. The remaining children were not recalled for a regular check-up that particular year or visited a private dentist at a clinic not among the in-reporting ones or located outside the region. The study was approved by the Halland Hospital Ethical committee as well as The Swedish Data Inspection Board.The Halland region consists of six municipalities that are subdivided into 66 parishes. Geo-maps were produced by using the ESRI ArcGIS system . Each child was geo-coded with respect to his/her residence area (parish).Statistics Sweden provided parish-level data from years 2006 and 2010, respectively, on three socio-economic indicators: (i) the proportion with post-secondary education (any schooling beyond the high-school level) among all residents; (ii) the proportion of immigrants  among all residents; and (iii) the proportion of families with low purchasing power  among all residing families with at least one child .smoothed RRs (SmRR) for each parish were obtained by running a Bayesian hierarchical mapping model, which allow parish-specific RRs to be smoothed towards global and local average risk levels across the study region [red in the certainty geo-map. By contrast, a parish with evidently lowered caries risk, Pr(RR<1|data)=1 - Pr(RR>1|data)>0.95, was colored green. The remaining parishes were colored yellow, which indicates a weaker statistical evidence for an elevated/lowered relative risk.Reported dmfs>0 for a child was considered as the primary caries outcome. The method for estimating caries risks at a given year of reporting has been explained previously . Brieflyy region . We undeEvidential, positional changes in the caries risk estimated for the children living in a specific parish in 2006 and 2010, respectively, were assessed by comparing the certainty geo-maps along with the posterior probabilities, Pr(RR>1|data): if the certainty color changed and Pr(RR>1|data) differed at least 25% for a specific parish, when comparing the results for the study years 2006 and 2010, the positional change was considered evidential. Hence, the rationale for classifying a positional change as evidential is based on the change over time in the statistical evidence for an elevated (or lowered) relative risk. Clearly, it seems extraordinary to observe a parish with a positional change from an evidently elevated caries risk (red) in 2006 to an evidently lowered caries risk (green) in 2010, or vice versa. Nevertheless, there were more modest, yet substantial, positional changes  that we considered evidential. Also notice that a parish could change certainty color without being indentified to have an evidential positional change (provided Pr(RR>1|data) differed less than 25%).The statistical analyses were performed using the free software Rapid Inquiry Facility , which pThe geo-maps of early childhood caries risk in 2006 and 2010, respectively, are presented in Figure For the eight parishes showing evidential positional changes in caries risk, no notable parallel changes in the parish-level socioeconomic characteristics were seen Table . AlthougWe demonstrated that geo-maps based on caries risk can be used to monitor changes in caries risk over time. An important finding from the present study was that the geo-map concept was sensible enough for disclosing changes in geographical caries risk patterns among preschool children over a 4-year period, in spite of minor changes in absolute caries risk and stable contextual socioeconomic characteristics. We also underline that the Bayesian smoothing of the relative risks yielded essentially conservative estimates, meaning moderate sensitivity but high specificity . The regThe geo-mapping methodology proved to be rapid and robust albeit, as always, depending on the quality of the input data. The high coverage of data (>75%) indicates that the findings are valid for the entire population. There is always a risk that non-examined children or children of families avoiding dental visits have an impaired oral health but that risk was low in the present study groups. In fact, the dominating reason for not being examined was a prolonged recall interval (up to 18months) due to an estimated low caries risk .The finding of an increased polarization of caries risk in spite of a generally improved dental health over the 4-year period was unwanted but not unexpected in the light of current papers on caries trends -20. The Our purpose was to disclose changes geographical patterns in childhood caries risk over time, without adjusting the parish-level relative risks (SmRRs) for contextual socioeconomic characteristics . If a pWe are fully aware that there is limited evidence for methods to bridge inequalities in early childhood caries and that compliance for both self-applied and professional measures is a paramount concern . HoweverGeo-maps based on caries risk can be used to monitor changes in caries risk over time. Thus, geo-mapping offers a convenient tool for evaluating effectiveness of tailored health promotion and preventive care in child populations.The authors declare that they have no competing interests.All of the listed authors contributed to the conduct of the study. US, AH and ST analyzed and interpreted the data. US and ST drafted the manuscript. KE provided technical and administrative support. All authors approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/12/9/prepub


Epicardial fat was closely related to atrial fibrillation (AF). Transthoracic echocardiography (TTE) has been proposed to be a convenient imaging tool in assessing epicardial adipose tissue (EAT). The goal of the present study was to investigate whether the EAT thickness measured on TTE was a useful parameter in predicting procedural outcomes of AF ablations.2 score, left atrial diameter and EAT thickness were independent predictors of recurrence after catheter ablations. At a cutoff value of 6 mm for PAF and 6.9 mm for non-PAF, the measurement of EAT thickness could help us to identify patients at risk of recurrences.A total of 227 paroxysmal AF (PAF) and 56 non-paroxysmal AF (non-PAF) patients receiving catheter ablations from 2008-2010 were enrolled. Echocardiography-derived regional EAT thickness from parasternal long-axis view was quantified for each patient. Free of recurrence was defined as the absence of atrial arrhythmias without using antiarrhythmic agents after ablations. The mean EAT thickness of the study population was 6.1 ± 0.8 mm. Non-PAF patients had a thicker EAT than that of PAF patients . During the follow-up of 16 ± 9 months, there were 95 patients (33.6%) suffering from recurrences of atrial arrhythmias. Non-PAF, chadsEAT thickness may serve as a useful parameter in predicting recurrences after AF ablations. Compared to other imaging modalities, TTE can be an alternative choice with less cost and time in assessing the effects of EAT on ablation outcomes. Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and has been associated with a marked morbidity, mortality, and socioeconomic burden ,2. As tePatients with symptomatic drug refractory AF who received radiofrequency catheter ablation for the first time under the guidance of a NavX mapping system  from 2008-2010 were studied. Those who didn’t receive regular follow up for at least 1 year after ablations were excluded. Finally, there were 227 paroxysmal AF (PAF) and 56 non-paroxysmal AF (non-PAF) patients enrolled in the present study. This study complies with the Declaration of Helsinki, and the study protocol was approved by the Institutional Review Board at Mackay Memorial Hospital, Taipei, Taiwan.Before catheter ablation, each subject underwent two-dimensional and M-mode transthoracic echocardiography using a Hewlett-Packard Sonos 5500 equipped with a 2.5-MHz to 4.5-MHz transducer. Epicardial fat is identified as the echo-free space between the myocardium and pericardium. At the parasternal long-axis view, EAT thickness was measured at the right ventricular free wall on M-mode cuts generated by placing the ultrasound beam perpendicular to the aortic annulus as proposed by Iacobellis et al.  11]. Th. Th11]. In order to confirm the reproducibility of the measurement, the EAT thicknesses of 120 study subjects (95 had PAF and 25 had non-PAF) were repeatedly measured by two physicians independently. The intraobserver and interobserver intraclass correlation coefficients for EAT thickness were 0.984 and 0.964, respectively . Using the Bland-Altman method [For the same 25 patients mentioned above, the volumes of total and peri-atrial EATs were measured by contrast-enhanced CT. The details about the measurements have been described in our previous publication . The TTEEach patient underwent an electrophysiological study and catheter ablation in the fasting, non-sedative state after written informed consent was obtained. The details have been described previously -17. In bIf non-PAF persisted after PV isolations and linear ablations, an additional complex fractionated electrographically guided substrate ablation was performed sequentially based on complex fractionated electrographic maps after PVI. Complex fractionated electrographic ablation was confined to the continuous complex fractionated electrograms (>5 seconds) in the left atrium (LA) and proximal coronary sinus ,20. The After sinus rhythm was restored from AF by procedural AF termination or electric cardioversion, mapping and ablation were applied only to spontaneously initiating focal atrial tachycardias and non-PV ectopy that initiated AF. If any non-PV ectopy initiating AF from the superior vena cava was identified, isolation of the superior vena cava was guided by circular catheter recordings from the superior vena cava–atrial junction.After the catheter ablation, all patients received anti-arrhythmic drugs for 8 weeks to prevent any early recurrence of AF. Patients underwent regular follow-up  at our cardiology clinic or with the referring physicians. During the follow-up, 24-hour Holter monitoring and/or cardiac event recording with a recording duration of one week were performed. The recurrence was defined as an episode of atrial arrhythmias lasting more than 30 seconds and confirmed by electrocardiograms 3 months after the ablation (blanking period). Freedom of recurrence was defined as the absence of atrial arrhythmias without using any antiarrhythmic agents after the catheter ablation. The long-term efficacy was assessed clinically on the basis of the clinical symptoms, resting surface 12-lead electrocardiogram, 24-hour Holter monitoring and/or 1 week cardiac event recordings.t test for normally distributed continuous variables, the Mann-Whitney test for skewed variables, and the chi-square test for nominal variables. A Cox regression analysis was used to identify the factors associated with recurrence. The optimal cut-off value of the EAT thickness in the prediction of recurrence was identified using the receiver operating characteristic (ROC) curve. The recurrence – free survival curve was plotted via the Kaplan-Meier method with the statistical significance examined by the log-rank test. All statistical significances were set at p value < 0.05 and all statistical analyses were carried out by SPSS 17.0 (SPSS Inc. USA).Differences between continuous values were assessed using an unpaired 2-tailed The baseline characteristics of the study population were shown in 2 score, LA diameter and EAT thickness were identified to be independent predictors of recurrence after catheter ablation  suffering from recurrences of atrial arrhythmias. The recurrence rates were 28.2% and 55.4% for PAF and non-PAF patients, respectively . The factors associated with recurrences in the univariate and multivariate Cox regression analysis were shown in ablation .In the subgroup analysis for PAF and non-PAF patients, EAT was consistently thicker in patients with recurrences than those without  . Figure The ROC curve based on EAT thickness in predicting recurrences after catheter ablation of non-PAF was shown in In this study, we investigated the associations between the TTE-derived EAT thickness and recurrences after AF ablations. The main findings were as follows: (1) EAT thickness assessed by TTE was significantly correlated with the volumes of total and peri-atrial EATs measured by cardiac CT. (2) EAT was thicker in non-PAF than PAF patients, which suggested that EAT was associated with AF chronicity and may play a role in AF progression(3). EAT thickness was a convenient parameter to predict recurrences after catheter ablation of AF.Obesity was recognized as a significant risk factor for AF ,22, and Increased regional EAT thickness measured by TTE was reported to be associated with increased left ventricular mass, metabolic syndrome, endothelial dysfunction, carotid atherosclerosis and coronary artery disease -29. In tAlthough increasing volume of literature has showed that EAT is significantly increased in AF patients ,5,23,24,To the best of our knowledge, the present study was the first one to demonstrate that the TTE-derived regional EAT thickness may be helpful in identifying patients at risk of recurrences after AF ablations. For patients with a great EAT thickness and received catheter ablation, follow-up should be performed more closely to detect subtle recurrences. Besides, it deserves a further study to investigate whether these patients could obtain benefits from decreasing EAT amount through body weight reduction, regular exercises or the avoidance of high-fat diets.There were several limitations of the present study. First, we hypothesized that inflammation could be the possible mechanism for the association of the increased EAT thickness and higher recurrence rate after catheter ablations. However, the speculation was based on the results of previous investigations, and inflammatory markers, such as CRP and interleukin-6, were not available in the present study. Second, other components of body fat, such as visceral fat of abdomen, and waist circumference were not measured. These measurements may provide additional information on the effects of local versus systemic adiposity. Third, the cutoff values of EAT thickness in predicting recurrences were derived and tested in the same study population and it may exaggerate the effectiveness of the EAT thickness in predicting ablation outcomes. Lastly, the accuracy of the TTE-based measurement of EAT in predicting recurrences after AF ablations was not compared to that of CT or MRI-based measurements. However, the main purpose of the present study was to provide a convenient alternative method in evaluating the effects of EAT on ablation outcomes, rather than claiming that the EAT thickness assessed by TTE was better than that derived from other imaging tools.EAT in our study was shown to be a significant predictor of recurrences of atrial arrhythmias after catheter ablations of AF. Compraed with CT or MRI imaging, echocardiography is a reliable alternative imaging tool that requires less cost and time and may help us to identify patients who are at high risk of recurrences after ablations.