Aims. To examine one-year changes in oxidative damage and inflammation level in type 2 diabetic patients undergoing behavioral treatment for subsyndromal depression. Materials and Methods. A randomized controlled comparison of psychoeducation (A), physical exercise (B), and enhanced treatment as usual (C) was performed in 209 eligible subjects in a tertiary diabetes care setting. Depressive symptoms (primary outcome) and selected biomarkers of oxidative damage and inflammation (secondary outcomes) were assessed at baseline and six- and twelve-month follow-up. Results. Out of the 74, 67, and 68 patients randomised into groups A, B, and C, respectively, 201 completed the interventions, and 179 were analysed. Participants in all three groups equally improved in depressive symptoms from baseline to one-year follow-up . Urinary 8-oxo-deoxyguanosine (u-8-oxodG) decreased , as did sialic acid and leukocytes , while uric acid increased  in all subjects during one year. Improvement of depressive symptoms at 6 months significantly predicted one-year reduction in u-8-oxodG . Conclusion. Simple behavioral interventions are capable not only of alleviating depressive symptoms, but also of reducing the intensity of damaging oxidative/inflammatory processes in type 2 diabetic patients with subsyndromal depression. This trial is registered with ISRCTN05673017. Increased prevalence of elevated depressive symptoms in patients suffering from diabetes is well documented , as are  per se promotes excessive production of ROS and depletion of antioxidant defence capacity, resulting in oxidative damage, that is, at least in part, presumably responsible for development of micro- and macrovascular diabetic complications [Recently, research in this area has been focused on biochemical markers reflecting common pathophysiological processes of insulin resistance, inflammation, and oxidative damage assumed to be intertwined in both diabetes and depression. Psychosocial stressors, inappropriate diet, sedentary lifestyle, obesity, and increased intestinal permeability have been proposed as worthwhile intervention targets for treatment or prevention of both diseases –12. Inflications .In spite of the growing evidence that inflammation is associated with type 2 diabetes and depressive symptoms, research on the interventions focusing on the interaction between depressive symptoms and inflammatory response is still rare. Findings obtained in general population have shown that inflammatory markers were higher in patients suffering major depressive disorders than in those without depression and that they are significantly reduced during cognitive-behavioral therapy . WhetherIn this study, being a part of a broader RCT comparing the effects of treating subsyndromal depression, we aimed to examine one-year changes in selected biomarkers of oxidative stress and inflammation in type 2 diabetic patients treated for elevated depressive symptoms that do not reach criteria for clinical depression. Depressive symptoms were primary, while biomarkers of inflammation , oxidative damage (urinary 8-oxo-deoxyguanosine), and endogenous antioxidant capacity (uric acid) were secondary study outcomes.n = 4858) were identified in the patient registry based on inclusion criteria of having type 2 diabetes for at least one year, living in Zagreb, and attending at least one medical check-up during the previous year. Out of 1740 responders (36%), 763 did not report depressive symptoms and 205 expressed no need for receiving professional help in reported mood-related difficulties. After excluding patients with clinical depression and/or psychiatric treatment (n = 134), those with severe physical limitations (n = 94), and those with both physical and psychiatric limitations/illnesses (n = 55), or other obstacles for participation (n = 124), 365 patients were found to be eligible. Eligibility was determined in a phone interview inquiring about personal, disease-related, and sociodemographic data. A presence or absence of clinical depression was determined by phone-administered structured clinical interview for the DSM-IV Axis I disorders. Out of the 365 eligible patients, 265 agreed to participate; among the remaining 100, 58 refused to join the groups and 42 were unreachable after the first contact. Two hundred and nine patients, who gave their informed consent, were randomised to study groups and completed baseline assessments. A computer-generated algorithm stratified by gender was used to allocate patients to the groups and to harmonise the number of female and male participants in each group [A postal screening for depressive symptoms relying on the short form Patient Health Questionnaire-Depression (PHQ-2)  was usedch group . ResearcPatients' flow is shown in N = 59 per group were shown to be needed to have 80% power in demonstrating statistically significant differences in depressive symptoms. A more detailed study procedure was described elsewhere [Primary study outcomes were depressive symptoms and secondary outcomes were endogenous pro- and anti-inflammatory markers , serum uric acid as an indicator of antioxidant capacity, and an overall measure of oxidative damage to DNA (urinary 8-oxo-deoxyguanosine). Sample size calculation was based on the absolute change in depressive symptoms as measured by the CES-D questionnaire. An improvement of 0.5 standard deviations was considered clinically relevant . With allsewhere .The study was carried out at the Vuk Vrhovac University Clinic for Diabetes—Reference Centre for Diabetes Mellitus in Zagreb, Croatia. Laboratory analyses were performed at the Department of Laboratory Medicine, Merkur University Hospital, accredited according to the ISO 15189:2012 “medical laboratories—requirements for quality and competence” standard.Two intervention programs—psychoeducation and physical exercise—and enhanced treatment as usual consisting of brief diabetes reeducation were offered to patients reporting elevated depressive symptoms and a need for receiving professional help in mood-related difficulties. The intervention programs were delivered in small groups attending six 90-minute weekly sessions. One 90-minute intervention session—enhanced treatment as usual—was offered to the control study participants because leaving these patients without any treatment was considered ethically unacceptable.The interventions were considered to have a very low potential for causing harm. Nevertheless, patient information, being a part of the written consent, clearly stated that the participants can discontinue intervention at any time, particularly if they would perceive that their symptoms have been worsened. Qualified professionals involved in treatment carefully monitored patient's condition and routine laboratory results in order to provide a timely referral to physician's care, if needed. In addition, patients allocated to physical exercise underwent ECG and, if indicated, an ergometric test before the study. Exercise intensity was measured by a heart rate monitor and maintained in a light-to-medium intensity range, whereas blood glucose and blood pressure were measured before and after each session.A summary of the programs' objectives, methods of delivery, and specific features are presented in Patient demographic variables included age, sex, education, family status, employment, and economic status. Diabetes-related variables included years of diabetes duration and diabetes treatment with or without insulin.Screening for mood difficulties was carried out using the adapted Patient Health Questionnaire-2 . An addiThe presence or absence of clinical depression was determined by phone-administered structured clinical interview for the DSM-IV Axis 1 disorders, as this method has been shown to be comparable to face-to-face interview . Center for Epidemiological Studies Depression (CES-D) Scale, a 20-item self-report instrument [Depressive symptoms were measured by thestrument . The tot1c was measured by an NGSP-certified automated immunoturbidimetric method  with a total imprecision (expressed as CV) <1.5% and dual reporting of the results .HbALeukocyte count was determined in K3EDTA-whole blood samples with ADVIA120 blood cell counter .Serum uric acid and hs-CRP concentrations were measured by the automated enzymatic and immunoturbidimetric assays, respectively .μg/L.Total serum sialic acid (TSA) was determined by a colorimetric procedure, as described previously . Serum aμg/L and improved specificity [μg/mmol).Urinary 8-oxo-deoxyguanosine (u-8-oxodG) was measured by the competitive ELISA method , with declared assay-range of 0.5–200 cificity . On the 1c, leukocytes, serum hs-CRP, and uric acid were assayed in fresh samples obtained in the morning after an overnight fast. Samples for sialic acid, adiponectin, and u-8-oxodG measurements were frozen (−70°C) until analyzed. The samples from each patient were analyzed with ELISAs at the same microplate in order to avoid possible between-series variability.Variables indicating a level of depressive symptoms and biomarkers of oxidative damage and inflammation were measured at baseline and after six- and twelve-month follow-up periods. HbAData are presented as means (SD), unless otherwise indicated. Due to the nonparametric distribution hs-CRP results were log-transformed prior to the analysis. One-way ANOVA and chi squares were used to determine baseline differences across the three treatment arms and examine differences between continuing participants and dropouts.Data were analyzed per protocol after exclusion of participants who withdrew, did not attend all three follow-up meetings, initiated psychopharmacological treatment, or died during the study period; these results are presented throughout the paper. Intention-to-treat (ITT) analysis was performed to validate these results; all participants who were randomized were analyzed despite deviations from the protocol. To perform ITT, missing data were imputed using the baseline observation carried forward approach.η2 coefficients ranging from 0.01 to 0.05 were regarded as small, from 0.06 to 0.12 moderate, and ≥0.13 large.Repeated measures ANOVA was used to test for changes in outcome variables across time and between the groups. Cohen's Regression analysis was employed to test directional relations between depressive symptoms and reduction in inflammation and oxidative stress.p value of <0.05 was considered significant in all analyses.A Statistical analyses were performed by using the SPSS 17 .The study was approved by the Vuk Vrhovac University Clinic for Diabetes Ethics Committee on June 30, 2010. A written informed consent was obtained from all the participants.Among the 1740 patients assessed for eligibility, 365 were considered eligible and 265 agreed to participate in the interventions (72%). Of these 209 completed baseline laboratory and psychological assessments (79%), with 74 randomized to psychoeducational intervention, 66 to physical exercise, and 69 to diabetes reeducation. Attrition rate was 3% during the interventions (6 of 209 patients), 3% from baseline to six months (7 of 203 patients), and 2% from 6-month to 12-month follow-up period (4 of 196 patients). Of the seven dropouts who completed the intervention but missed both follow-up assessments, two missed the follow-up appointments due to health problems, four were unwilling to come, and one patient died. No harmful incidents were recorded throughout the interventions. Four patients were excluded from per-protocol analyses due to the initiation of pharmacological therapy. No differences between the participants and dropouts across the three study groups were observed.1c) and BMI, based on repeated measures ANOVA are given in The effects of interventions on depressive symptoms and biomarkers of oxidative damage and inflammation, as well as glucoregulation , indicating comparable efficacy of psychoeducation, physical exercise, and enhanced treatment as usual on the primary study outcome.No significant between-group differences over time were obtained with respect to depressive symptoms . The size of the time effect indicated a moderate effect of the three treatments on self-assessed depressive symptoms.Significant reductions in depressive symptoms occurred across all three study groups from baseline to 12 months  as did u-8-oxodG  and sialic acid . The latter showed small but statistically significant time x group effect . However, after adjusting the results for multiple comparisons by Bonferroni test, it did not remain significant. Uric acid increased significantly over one year in all three groups . The effects of the interventions on sialic acid can be regarded as large (0.32) and on leukocytes, u-8-oxodG, and uric acid as moderate .Total leukocytes significantly decreased in all three groups (No significant changes were registered with respect to adiponectin and hs-CRP.1c nor BMI were significantly changed in any of the study groups from baseline to 12 months.Neither HbAp > 0.05).No between-group effects were obtained with respect to one-year changes in biomarkers of inflammation and oxidative damage , while the results for sialic and uric acid were close to statistical significance  (results not shown in tables).The results obtained by using regression analyses indicated that improvement in depressive symptoms at six months predicted reductions in u-8-oxodG at twelve-month follow-up  by the interventions performed.Beneficial effect of cognitive-behavioral intervention in reducing oxidative stress and inflammatory response has been reported in hemodialysis patients suffering from insomnia , but rat9/L or hs-CRP < 5.0 g/L.Sialic acids comprise a family of heterogenous monosaccharides abundantly present on the cell membranes, with complex and multiple biological roles in cellular signaling and immunity . TSA is The ability of psychological intervention to reduce both depressive symptoms and inflammation has been evidenced in depressed breast-cancer patients . Further1c, clinically considered to reflect a fair glucoregulation (It is well known that the intensity of oxidative damage and inflammation in diabetes is remarkably and positively associated with the severity of hyperglycemia . Howevergulation . ObesityChronic stress, with deregulation of stress-response adaptive mechanisms, is considered to be a common feature of both diabetes and depression . There iThe strengths of this study are in examining a highly prevalent population of type 2 patients with depressive symptoms but no clinical depression, using a randomized comparative design, employing structured behavioral interventions and analyzing clinically relevant changes of simple-to-measure oxidative/inflammatory biomarkers. Its limitation is the lack of treatment as usual control intervention, precluding the possibility to investigate the course and outcomes of subclinical depression without any treatment. Also, the study was carried out in only one center—a tertiary diabetes clinic—which does not allow concluding about its feasibility in other clinical settings. Per-protocol analyses are presented in the study results which were confirmed by the intention to treat analysis based on baseline-observation-carried-forward approach. A lack of estimation derived from multiple predictors may be considered a limitation of the study.This study has demonstrated that improvement in depressive symptoms is accompanied with reduced oxidative damage and inflammatory response in type 2 diabetic patients with subsyndromal depression. Testing mediating effects has indicated that medium-term improvement in depressive symptoms predicts long-term-reduction in oxidative damage, while mediating factors for observed reduction in inflammatory response should be further investigated. Clinical benefits of treating subsyndromal depression were shown to be comparable in all three treatment arms.Regardless of the mechanisms involved, our study clearly indicates that enhanced treatment as usual, defined as brief patient-centred diabetes reeducation, is not inferior to psychoeducation and exercise in providing beneficial outcomes in subsyndromally depressed type 2 diabetic patients. Simple behavioral interventions are capable not only of alleviating depressive symptoms, but also of reducing the intensity of damaging oxidative/inflammatory processes, thereby mitigating the risk of development of diabetic complications.


Delayed-release dimethyl fumarate (DMF) demonstrated efficacy and safety in the Phase 3 DEFINE and CONFIRM trials.To evaluate delayed-release DMF in newly diagnosed relapsing–remitting multiple sclerosis (RRMS) patients, in a post-hoc analysis of integrated data from DEFINE and CONFIRM.Patients included in the analysis were diagnosed with RRMS within 1 year prior to study entry and naive to MS disease-modifying therapy.n = 223) or delayed-release DMF 240 mg BID (n = 221) or TID (n = 234). At 2 years, delayed-release DMF BID and TID reduced the annualized relapse rate by 56% and 60% (both p < 0.0001), risk of relapse by 54% and 57% (both p < 0.0001), and risk of 12-week confirmed disability progression by 71% (p < 0.0001) and 47% (p = 0.0085) versus placebo. In a subset of patients (MRI cohort), delayed-release DMF BID and TID reduced the mean number of new or enlarging T2-hyperintense lesions by 80% and 81%, gadolinium-enhancing lesion activity by 92% and 92%, and mean number of new non-enhancing T1-hypointense lesions by 68% and 70% . Flushing and gastrointestinal events were associated with delayed-release DMF.The newly diagnosed population comprised 678 patients treated with placebo (Delayed-release DMF improved clinical and neuroradiological outcomes relative to placebo in newly diagnosed RRMS patients. Consistent with this, previous studies with interferon beta and glatiramer acetate (GA) demonstrated an association between early treatment and improved outcomes, such as a prolonged time to conversion from clinically isolated syndrome (CIS) to clinically definite MS (CDMS) and a reduction in the number and volume of lesions on MRI.39The pathological course of multiple sclerosis (MS) is believed to evolve over time. In the early stages of the disease, autoreactive lymphocytes gain access to the central nervous system, initiating a cascade of events leading to demyelination, axonal transection, and neurodegeneration.11 delayed-release DMF 240 mg twice (BID) and three times daily (TID) resulted in significant reductions in clinical and magnetic resonance imaging (MRI) activity and demonstrated an acceptable safety profile in RRMS patients over 2 years.12 These effects were generally consistent across subgroups of patients stratified by baseline demographic and disease characteristics.13Delayed-release dimethyl fumarate (DMF) is a novel, oral MS therapeutic studied in people with relapsing–remitting MS (RRMS). In a pre-specified integrated analysis of the Phase 3 DEFINE and CONFIRM trials,15 and modulation of the expression of pro- and anti-inflammatory cytokines1619 and phase 2 detoxification enzymes.18 Inflammation and neurodegenerative processes are prominent early in the course of MS, so agents with putative dual anti-inflammatory and neuroprotective effects, such as delayed-release DMF, may be particularly useful.The mechanism by which delayed-release DMF exerts its therapeutic effect in MS is unknown. However, clinical benefits are believed to be related, in part, to activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathwayTo examine the efficacy of delayed-release DMF in newly diagnosed patients, a post-hoc analysis of integrated data from DEFINE and CONFIRM was conducted. The newly diagnosed population included patients who had been diagnosed with RRMS within 1 year prior to study entry and were naïve to MS disease-modifying therapy. The analysis included clinical and neuroradiological efficacy endpoints as well as basic safety data (adverse events).11 Briefly, eligible adult patients (18–55 years) had a diagnosis of RRMS per McDonald diagnostic criteria20 and an Expanded Disability Status Scale (EDSS) score of 0–5.0, inclusive.21 Further, patients had experienced at least one clinically documented relapse within one year prior to randomization, with a prior brain MRI demonstrating lesion(s) consistent with MS, or at least one Gd+ lesion on a brain MRI scan obtained within 6 weeks prior to randomization.The designs of the DEFINE and CONFIRM trials have been described in detail elsewhere.The primary endpoint of DEFINE was the proportion of patients relapsed at 2 years. The primary endpoint of CONFIRM was the annualized relapse rate (ARR) at 2 years. Additional endpoints included the time to 12-week sustained disability progression, number of Gd+ lesions, number of new or enlarging T2-hyperintense lesions, and number of new T1-hypointense lesions, all at 2 years.The integrated analysis of DEFINE and CONFIRM was pre-specified prior to the unblinding of CONFIRM and was to be conducted only if the patient populations and treatment effects were similar between the studies. The integrated analysis was considered valid due to the many similarities between DEFINE and CONFIRM, including inclusion/exclusion criteria, regions from which patients were recruited, overall design, measurement criteria, and observed efficacy. Roughly equal proportions of newly diagnosed patients were drawn from each of the pivotal studies.20 and who were naïve to MS disease-modifying therapy. The 1-year criterion was defined prior to the analysis being conducted and was chosen because the median time since diagnosis of RRMS in the overall treatment-naïve population was 1 year.The newly diagnosed population was defined as patients who had been diagnosed with RRMS per McDonald diagnostic criteria within 1 year prior to study entryThis post-hoc analysis was performed on data from the integrated DEFINE and CONFIRM dataset. The pre-planned integrated analysis was finalized prior to unblinding of CONFIRM and required baseline characteristics and treatment effects to be homogeneous across the studies.The analysis included data from patients in the intent-to-treat (ITT) population (defined as patients who underwent randomization and received at least one dose of study drug) who were randomized to receive placebo or delayed-release DMF BID or TID. Patients randomized to receive GA were excluded because there was no GA comparator arm in DEFINE, and because CONFIRM was not designed to test the superiority or non-inferiority of delayed-release DMF to GA. In general, the analyses were based on all observed data before patients switched to alternative therapies. MRI endpoints were analyzed using ITT patients in the MRI cohort for whom at least one MRI scan was available for analysis. MRI lesion count data post-early withdrawal or post-alternative MS treatment usage were imputed using a constant rate assumption.Annualized relapse rate  was analyzed with the use of a negative binomial regression model adjusted for baseline EDSS score (≤2.0 vs. >2.0), baseline age (<40 vs. ≥40), study, region  and number of relapses in the year prior to study entry. Regions were pre-defined based on geography, type of health care system, and access to health care in each country. The proportion of patients relapsed was derived using Kaplan–Meier analysis and analyzed with the use of a Cox proportional hazards model with study as a stratifying factor, and adjusted for baseline age (<40 vs. ≥40), region, baseline EDSS score (≤2.0 vs. >2.0), and number of relapses in the year prior to study entry. Disability as measured by time to 12-week confirmed EDSS progression was analyzed using a Cox proportional hazards model with study as a stratifying factor, and adjusted for the following covariates: baseline EDSS score (as a continuous variable), baseline age (<40 vs. ≥40), and region. The odds of having more Gd+ lesions were analyzed using ordinal logistic regression adjusted for study, region, and baseline number of Gd+ lesions. The mean number of new or enlarging T2-hyperintense lesions was analyzed using negative binomial regression adjusted for study, region, and baseline T2-hyperintense volume. The mean number of new non-enhancing T1-hypointense lesions was analyzed using negative binomial regression adjusted for study, region, and baseline volume of T1-hypointense lesions.n = 223, 221, and 234 in the placebo, delayed-release DMF BID, and delayed-release DMF TID groups, respectively). A subset of these patients  comprised the MRI cohort. Baseline demographic and disease characteristics were similar across treatment groups  and 60%  and 47%  and 81% , delayed-release DMF BID (97%), and delayed-release DMF TID (95%) groups . Adverse13 consistent with findings from previous studies that early intervention with interferons or GA is associated with improved outcomes.39In this post-hoc analysis of integrated data from DEFINE and CONFIRM, delayed-release DMF demonstrated strong efficacy across a broad range of clinical and neuroradiological outcome measures in patients newly diagnosed with RRMS. Over 2 years, both dosing regimens of delayed-release DMF (240 mg BID and TID) significantly reduced the ARR, risk of relapse, proportion of patients with 12-week confirmed disability progression, odds of having more Gd+ lesions, mean number of new or enlarging T2-hyperintense lesions, and mean number of new non-enhancing T1-hypointense lesions, compared with placebo. The effects of delayed-release DMF in the newly diagnosed population were numerically stronger than those seen in the overall ITT population of DEFINE and CONFIRM,2 and greater frequency of relapse and higher lesion load in early MS are associated with poorer long-term outcomes2225, immediate intervention in newly diagnosed patients may slow the accumulation of damage and progression of disability. Indeed, associations between MS disease activity and long-term clinical prognosis seem to become weaker over time, suggesting an early window of maximal therapeutic opportunity.2325From a neuropathological and clinical perspective, the rationale for early intervention is strong. As neurodegenerative effects including axonal transection are observed from the early stages of the disease26 to within 6 months,28 2–5 years,30 or even 8–10 years of diagnosis.32 As we sought to conduct a robust analysis of a newly diagnosed population, we selected 1 year from diagnosis as our criterion to ensure our study was adequately powered. One year was the median time since diagnosis for the treatment-naïve population of DEFINE and CONFIRM, and it fell clearly within the range of criteria used to characterize the newly diagnosed population in the literature. It should be noted, however, that the patient subgroup evaluated in this report represents patients with early RRMS and not patients with CIS at disease onset.There is no accepted universal criterion for newly diagnosed or “early” RRMS. Among the criteria that have been used previously are time from symptom onset, time from diagnosis, EDSS score, clinical presentation consistent with CIS, conversion from CIS to CDMS, or a combination of these. In those studies that have used time from diagnosis, the critical interval has varied widely, from immediately following diagnosisThe recommended dosing regimen of delayed-release DMF is 240 mg BID. The data for 240 mg TID were included here to explore the general consistency of the effects between the doses. In the newly diagnosed population, similar to the ITT population, the effect sizes with delayed-release DMF BID and TID were broadly similar. However, both BID and TID showed a treatment effect on reducing disability progression in the same direction. Although the effect size was different between BID and TID (71% vs. 47% reduction), the confidence intervals of the point estimates overlapped. Therefore, the different effect size was mainly due to data variations of the newly diagnosed population.12 Flushing, nasopharyngitis, and gastrointestinal events including diarrhea, nausea, and abdominal pain were among the most common adverse events reported by patients treated with delayed-release DMF in both populations.12 The incidence of adverse events leading to discontinuation of study treatment in the newly diagnosed subgroup was 5%, 12%, and 11% in the placebo, delayed-release DMF BID, and delayed-release DMF TID groups, respectively, compared with 12%, 14%, and 14% in the overall safety population.12The safety and tolerability profile of delayed-release DMF in newly diagnosed patients presented here, while limited in scope, is acceptable and comparable to that seen in the overall integrated safety population of DEFINE and CONFIRM. For example, the overall incidence of adverse events in the newly diagnosed subgroup was 92%, 97%, and 95% in the placebo, delayed-release DMF BID, and delayed-release DMF TID groups, respectively, compared with 93%, 95%, and 94% in the overall safety population.Finally, it should be borne in mind that this is a post-hoc analysis, and, as such, the results should be interpreted cautiously. The study was not powered a priori to analyze the endpoints presented herein in this subgroup of newly diagnosed patients. Therefore, further prospective confirmation is necessary to support our findings.This integrated post-hoc analysis suggests strong treatment efficacy of delayed-release DMF in patients with newly diagnosed RRMS, and further supports the use of delayed-release DMF as an oral treatment option in a broad spectrum of people with relapsing MS.


We demonstrate super-resolution imaging of proteins and nucleic acids that were densely labelled with fluorophores using the concept of “click chemistry”. E. coli.We demonstrate high-density labelling of cellular DNA and RNA using click chemistry and perform confocal and super-resolution microscopy. We visualize the crescent and ring-like structure of densely packed RNA in nucleoli. We further demonstrate click chemistry with unnatural amino acids for super-resolution imaging of outer-membrane proteins of  All fluorescence microscopy-based methods require suitable and specific strategies for labelling a target structure. An additional request for super-resolution microscopy is a particularly high labelling density, which is reasoned by the sampling theorem.4Various fluorescence microscopy techniques which can bypass the spatial resolution limit have been developed in recent years. Commonly summarized as “super-resolution microscopy” techniques, they constitute a new and valuable toolbox for cell biology -catalysed Huisgen 1,3-dipolar cycloadditions,5 which is one example of a chemical reaction today referred to as “click chemistry”.7 This technique was initially introduced to fluorescence microscopy for labelling nucleic acids9 and phospholipids.10 In the recent years, the field has evolved dramatically, and new developments include alternative chemical strategies for click-like reactions, copper-free click chemistry, live cell labelling as well as dual-colour labelling with two orthogonal click reactions.17 Click chemistry has also entered the field of super-resolution microscopy, and has been applied to visualize chromosomal DNA in eukaryotes19 and Escherichia coli (E. coli)20 as well as proteins in the plasma membrane.13 A recent approach has also reported optical mapping of DNA sequences by methyltransferase-directed click chemistry and sequence readout by super-resolution microscopy.21An elegant strategy to covalently bind small synthetic fluorophores to cellular target structures with high efficiency and specificity makes use of copper of the fluorophore influences the photophysical properties23 or may induce quenching.24 So far, azide-substituted and red-emitting carbocyanines (e.g. Alexa Fluor 647)18 or custom-synthesized rhodamines19 were used for super-resolution imaging of DNA. Biological applications, however, require multiple targets labelled. Here, we present click chemistry labelling of cellular nucleic acids using a variety of spectrally distinct fluorophores and demonstrate confocal and single-molecule localization-based super-resolution fluorescence microscopy,28 facilitating new dye combinations for multi-colour imaging. Furthermore, we present a simple strategy to label the outer membrane proteins of E. coli using click chemistry in combination with an unnatural amino acid, and visualize the boundaries of bacterial cells with super-resolution imaging.Single-molecule localization microscopy demands bright fluorophores that allow robust and well controllable photoswitching.8 After fixation and permeabilisation, the alkyne group of EdU is conjugated to an azide-substituted fluorophore through a copper-catalysed click reaction  to the growth medium, followed by natural uptake by the cells and incorporation into the chromosomal DNA during replication.reaction . Here, wreaction  or purchreaction  and reporeaction .20  to pulse-label RNA of HeLa cells for 10 min and used click chemistry to label nascent RNA with Alexa Fluor 647. We visualized the nuclear RNA using confocal  are required, which limit the application in crowded or densely packed structures such as nuclei and bacteria. Second, high labelling densities as they are required for sub-diffraction microscopy techniques can be achieved. Third, virtually any fluorophore can be conjugated to nucleic acids. This allows users to choose the optimal fluorophore for the particular application, e.g. photoswitchable fluorophores for single-molecule localization microscopy. DNA labelling with intercalator fluorophores compatible with super-resolution imaging has also been demonstrated.38 However, the photon yields and photostabilities of intercalators are lower than those of bright organic fluorophores. In some cases, the low quantum yield of unbound intercalators can also be the source of additional background signal.In comparison to other labelling techniques for nucleic acids, click chemistry with alkyne-modified nucleobases has several advantages. First, incorporation of EdU and EU leads to specific labelling only of newly synthesized DNA or RNA; nucleic acids which were already synthesized prior to EdU/EU addition will not be labelled, allowing studies of nascent replication or transcription sites similar as with BrdU or BrUTP.9 and are widely used for confocal microscopy of eukaryotic cells. Here, we applied EdU/EU treatment in eukaryotic cells for 10 to 15 minutes. Furthermore, we monitored the growth rate of E. coli in the presence of EdU . During cell growth, HPG is incorporated into newly synthesized proteins, replacing methionine, which facilitates the subsequent labelling of all newly synthesized proteins with an azide–fluorophore17 (40 In addition, this approach is in principle live-cell compatible; incorporated azide-containing amino acids could be labelled in copper-free click-reactions.12Click chemistry has also entered the field of protein labelling through the use of unnatural amino acids.ophore17 . In ordeophore17 . The resIn summary, we have demonstrated click chemistry as versatile tool for super-resolution fluorescence microscopy of cellular structures. Click chemistry on the one hand can provide the high labelling densities typically required for super-resolution microscopy. On the other hand, it is a flexible strategy to label nucleic acids with virtually any synthetic fluorophore, which allows choosing the best-suited one for a specific super-resolution technology. Furthermore, this approach can be combined with pulse labelling and thus visualize particular states of the cell cycle. Finally, a whole new area of applications opened with the introduction of click chemistry into protein labelling through unnatural amino acids. In the future, click chemistry will certainly develop into a widespread and generally used toolbox for cell and tissue labelling.


One-in-ten Sri Lankans are employed abroad as International Labor Migrants (ILM), mainly as domestic maids or low-skilled laborers. Little is known about the impact their migration has on the health status of the children they ‘leave behind’. This national study explored associations between the health status of ‘left-behind’ children of ILM’s with those from comparative non-migrant families.A cross-sectional study design with multi-stage random sampling was used to survey a total of 820 children matched for both age and sex. Socio-demographic and health status data were derived using standardized pre-validated instruments. Univariate and multivariate analyses were used to estimate the differences in mental health outcomes between children of migrant vs. non-migrant families.Two in every five left-behind children were shown to have mental disorders , suggesting that socio-emotional maladjustment and behavioural problems may occur in absence of a parent in left-behind children. Male left-behind children were more vulnerable to psychopathology. In the adjusted analyses, significant associations between child psychopathological outcomes, child gender and parent’s mental health status were observed. Over a quarter (30%) of the left-behind children aged 6–59 months were ‘underweight or severely underweight’ compared to 17.7% of non-migrant children.Findings provide evidence on health consequences for children of migrant worker families in a country experiencing heavy out-migration of labour, where remittances from ILM’s remain as the single highest contributor to the economy. These findings may be relevant for other labour ‘sending countries’ in Asia relying on contractual labor migration for economic gain. Further studies are needed to assess longitudinal health impacts on the children left-behind. International labor migration has become a crucial engine for economic development for many countries worldwide ,2. The gILMs from Sri Lanka has grown ten-fold during the past decade, with 90% employed in the Gulf, and 730 registered workers departing Sri Lanka each day . In whatUnited Nations (UN) agencies have articulated the need for migration-related determinants of health to be explicitly included in the post-2015 Millennium Development Agenda through the UN general assembly’s High Level Dialogue on International Migration and Development, and through the Global Migration Group . The IntThe adverse effects on the psychological well-being and development outcomes of children due to separation from a parent for extended periods has been characterized by a number of researchers -17. HoweThree studies from Sri Lanka examined health status of left-behind children utilizing standardized psychometric measures -24. ResuA number of studies have investigated the relationship between international labor migration and child malnutrition -27. RemiWith high rates of malnutrition in developing nations that are also major source countries for ILMs, and with the growing evidence base linking child nutritional deficiencies with mental health problems ,31 exploMigration & Health in 2011 prompted public health attention and called for an evidence-based research agenda on health of migrants  and hyperactivity disorders  is highest in children from migrant families than from children in comparative families ] and non-migrant families .The ethnic profile of the study sample closely matched national population ratios from the 2001 national population census, with 74.5% of migrant families and 78.2% of non-migrant groups being of Sinhalese ethnicity . The meaTypology of employment of the migrant worker was assessed according to the Sri Lanka Bureau of Foreign Employment (SLBFE) classification of occupations . The majGeneral health was perceived to be three fold poorer in parents of migrant children (12.7%) than compared to the non-migrant families (3.9%), p < 0.001. There were also nearly twice as many migrant parents currently diagnosed with illness (34.7%) than comparative parents from non-migrant households (18.2%) p < 0.001.Overall prevalence of common mental disorders in adults  was higher in left-behind parents/caregivers of migrant children [19.2% (95%CI 15.3-23.2)] than the non-migrant parent group [9.1% (95%CI 6.2-11.9)]. Prevalence of depression was doubled in the migrant group , compared with non-migrant parents . Prevalence of somatoform disorder (5.7% 95%CI 3.4-8.0) and anxiety disorder (2.1% 95%CI 0.6-3.5) was marginally higher in spouses of migrant families than that of non-migrant counterparts .Table The adjusted odds ratio showed presentation of any child psychiatric diagnosis is 40% less likely in female children as compared to male children in left-behind families . A similar association was observed in children from non-migrant families . Educational status and employment status of the parents/giver of the child also influenced child psychopathological outcomes. The adjusted odds ratio of having any psychiatric diagnosis was 2.67 times more likely in left-behind children whose parents had not attended school .In children from migrant families whose left-behind parents were unemployed, the likelihood of having any psychiatric diagnosis was 1.13 times higher than those employed [OR 1.13 (95%CI:1.00-1.27)]. However, this result was observed only before adjustment for child age and gender.This study addresses the question of whether left-behind children of migrant workers are at increased risk for mental health problems and adverse nutritional status. Whilst observational studies cannot determine causality, the evidence suggests overall negative associations at the intersection of labor migration and child mental health, but a weak association with nutritional status.A number of researchers have theorized that migration of a parent for extended periods may transform family relationships and functioning ,43. For Studies from other Asian countries show mixed patterns of psychological well-being of left-behind children. A study by Graham and Jordan  revealedThe Philippines has been recognized for its pro-active policy and program efforts in protecting the rights of its labour migrants and enabling a culture of support to families through a network of civil society and non-governmental organizations . These iOver a quarter of children aged 6–59 months of migrant households were found to be underweight or severely underweight. The findings of our study revealed that the comparative non-migrant child groups also have a high risk of being underweight 32.7%). These finding are consistent with the underlying nutritional trend in Sri Lankan children that shows an overall prevalence of underweight children in 2009 to be 22.1% . The few.7%. ThesA major strength of the study is its representativeness. Previous small scale studies focussed on only male-headed migrant households, derived from a single ethnic group , and from an urban setting within a capital city (Colombo). Our sample was derived from a true cross-section of the left-behind families of migrant workers in Sri Lanka. The generalizability of our findings is therefore enhanced. Our sample was also reflective of the true pattern of work categories published by the SLFBE data: seventy-five percent of labour migrants worked in the ‘unskilled’ labour sector, with professional categories comprising of less than ten-percent of departures.Our study primarily focused on child psychopathology, however, resilience and protective factors were not adequately explored. Identifying such enabling factors and resilience trajectories in left-behind children is crucial for formulating policies and programs to effectively manage migration and address health and social impact.Whilst this study provides an insight into how migration effects health status of left-behind children, further research is needed to explore how intra-household power dynamics, transnational parenting, relationship outcomes, and whether male versus female headed households have an effect on child health outcomes. Research is needed to assess how factors such as the duration and frequency of an often cyclical pattern of migration affects health outcomes; how household remittances are actually spent to promote child development outcomes; children’s own experiences/expectations; abuse and violence within migrant families; and, how left-behind families access support services at community level.Cross-sectional studies may only suggest but not determine causality. Prospective cohort and longitudinal studies are needed to assess if children left-behind truly recover from the experience of parental migration. The re-integration of migrant parents after a long-term absence from child may cause problems due to acculturation issues, family conflicts and re-establishment of livelihoods . The impThe findings from this study contribute to an evidence-based approach to developing Sri Lanka’s Migration Health policy and program processors. In our study, two in every five left-behind children were shown to have mental disorders, with a significant burden of socio-emotional maladjustment and behavioural problems. Concerns centre on how separation from parents may effect nutritional, behavioural and psychological development of left-behind children. Community programs to strengthen the capacity of relevant government workers such as public health midwifes , child protection officers, school counsellors and foreign employment agency welfare officers to identify and address social, health and nutrition issues of families left behind are needed. Programs may involve undertaking mapping and vulnerability assessments of migrant families through a coordinated network of such village level workers; development of case management or care plans for left-behind children using community participatory approaches; providing awareness and information to prospective migrant families; and providing guidance for primary caregivers of left-behind children.As contractual labour migration to places like the Middle East remain a pervasive phenomenon, the impact on children left-behind leaves many unanswered questions. The consequences of long-term migration on family relationship structures, parenting and health vulnerabilities have complex associations that require further longitudinal analysis.health of migrants promotes a ‘safe, dignified and healthy migration’ process for the benefit of both migrants and their families [rights, remittances and responsibilities for both State and ILM. The high levels of malnutrition in many labour sending countries within the developing world and the complex interplay between migrant remittances and child nutrition also form an important yet unexplored policy area. Balancing human rights discourses , in the context of social and health impact to both families and remittance dependent economies form formidable policy challenges for governments seeking to ‘manage’ migration and development.The World Health Assembly resolution on families . Though


Individuals with a history of suicide attempt have a high risk for subsequent labour market marginalization. This study aimed at assessing the effect of individual and parental factors on different measures of marginalization.Prospective cohort study based on register linkage of 5 649 individuals who in 1994 were 16–30 years old, lived in Sweden and were treated in inpatient care for suicide attempt during 1992–1994. Hazard ratios (HRs) for labour market marginalization defined as long-term unemployment (>180 days), sickness absence (>90 days), or disability pension in 1995–2010 were calculated with Cox regression.Medical risk factors, particularly any earlier diagnosed specific mental disorders , personality disorders: HR 3.9, 95% CI: 3.1, 4.9), repetitive suicide attempts  were associated with a higher relative risk of disability pension. Individual medical factors were of smaller importance for long-term sickness absence, and of only marginal relevance to long-term unemployment. Country of birth outside Europe had an opposite effect on disability pension  and long-term unemployment . Female sex was positively correlated with long-term sickness absence , and negatively associated with long-term unemployment .As compared to disability pension, long-term sickness absence and unemployment was more strongly related to socio-economic variables. Marginalization pathways seemed to vary with migration status and sex. These findings may contribute to the development of intervention strategies which take the individual risk for marginalization into account. Disability pension and sickness absence have increased in Sweden and other member countries of the Organisation for Economic Co-operation and Development (OECD) over the last decades, particularly in young people ,2. MentaSocial insurance policies such as sickness absence and disability pensions are intended to provide financial support for individuals with impaired work capacity due to disease or injury . Due to Suicide attempt can be viewed as one of the most detrimental consequences of mental disorders , and conDespite the magnitude of this problem and the consensus on the need to address it, studies on determinants of labour market marginalization in young people with suicide attempts are sparse. One study found that young suicide attempters, compared to non-attempters had significantly longer durations of unemployment later on in life, as well as a longer duration of welfare benefits . With reIt is currently unknown if the determinants of labour market marginalization vary across various labour market outcomes such as disability pension, long-term sickness absence and long-term unemployment. An earlier study showed that a diagnosis of schizophrenia and past violent suicide attempts were risk factors for subsequent sickness absence and disability pension in young suicide attempters , but thiThis study aimed to 1) investigate determinants of labour market marginalization  in young suicide attempters, focusing on aspects related to the suicide attempt, medical risk factors, and social characteristics and 2) identify potential sex differences in the association between the specific risk factors and the three outcome measures.st, 1994, who did not have disability pension at baseline, with at least one parent born in Sweden and treated in inpatient care following attempted suicide during the three years preceding study entry, i.e. 1992, 1993 or 1994 (N = 5 649).The baseline study population comprised all individuals alive, resident in Sweden, between 16 and 30 years of age on December 31Suicide attempts were coded in accordance with the International Classification of Diseases 10th Revision (ICD-10). Among the 5 649 individuals with inpatient care due to attempted suicide between 1992 and 1994 when aged 14 to 30 years, 4 434 (78.5%) were determined suicide attempts, and 1 215 (21.5%) were of undetermined intent. In accordance with earlier studies both events of determined intent (X60-84) and of undetermined intent (Y10-34) according to the ICD-10 were combined. This procedure intends to limit temporal and geographic differences in ascertainment ,18,19. Ast December 2010. Statistics Sweden provided data on age, sex, country of birth, education, and year and length of unemployment. Data on dates of sickness absence and disability pension from 1994 and onwards were derived from the Social Insurance Agency. Information on date and diagnosis of inpatient care starting from 1973 and the date and cause of death from 1961 and onwards were obtained from the National Board of Health and Welfare. The multi-generation register was used to identify the parents of the index subjects.Register data was available for each individual retrospectively and prospectively up to 31Characteristics of index subjects include the repetition of suicide attempt hospitalizations, mental (diagnosis-specific) and somatic inpatient care, and socio-demographics .The following classification was used with regard to diagnosis-specific mental disorders: Schizophrenia and non-affective psychoses (F20-F29); affective disorders (F30-39); substance abuse disorders (F10-F19); neurotic, stress-related and somatoform disorders (F40-F48); organic disorders including mental retardation ; behavioural, emotional and developmental disorders ; personality disorders (F60) .Parental characteristics included parental disability pension, mental and somatic inpatient care, suicide attempt, suicide, death other than suicide, as well as parental educational level . PaternaAll individual variables and parental markers were measured from the start of the respective register (see previous paragraph) up until the year before the index event for individuals with suicide attempt.The socio-demographic characteristics  of index persons and parents were measured on December 31st 1994.Three different outcome measures were analysed: 1) disability pension, 2) long-term sickness absence , and 3) long-term unemployment .All residents with a permanently reduced work capacity due to injury or disease can be granted permanent disability pension. Since 2003, temporary disability pension (“aktivitetsersättning”) can be granted for young individuals between 19 and 29 years of age in case of reduced work capacity or in case that compulsory education is not completed at 19 years . Sick paAll individuals who live in Sweden, aged over 16 years and have an income from work or unemployment benefits are eligible for sickness benefits by the Social Insurance Agency if they have a reduced work capacity due to disease or injuries.In order to be eligible for unemployment benefits, the individual needs to be unemployed, able to work, and ready to accept work that is offered. In addition, prior to becoming unemployed the applicant shall have worked at least six months . The datFor all individual and parental determinants, missing values were coded as a separate category and included in the analysis. Cox regression with time dependent covariates was used in order to adjust for changes in individual risk factors during follow-up. Time dependent covariates measured during follow-up included suicide attempt, inpatient care due to mental/somatic diagnoses and inclusion in the labour market . The following additional time-dependent variables during follow-up were considered for specific outcomes as reported in brackets: long-term unemployment/year  and long-term sickness absence (unemployment). All time-dependent covariates were dichotomized and measured annually.Follow up lasted for as long as 16 years (1995–2010). Censoring was due to emigration to a foreign country, death, or end of follow up, whichever occurred first. In the analyses of unemployment and sickness absence as outcome measures, censoring was also due to disability pension because individuals with disability pension are not eligible for unemployment or sickness absence benefits.Interaction of sex with the other risk factors was tested with the partial likelihood ratio test. If there was a significant interaction effect (p<0.05), analyses were stratified by sex.Ethical approval for this study was obtained from the Regional Ethical Review Board, Karolinska Institutet, Stockholm (review number 2007/762-31). The ethical review board approved the study and waived the requirement that informed consent of research subjects should be obtained. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008.Of individuals with a history of suicide attempts (n = 5 649), 4052 (71.0%) had one attempt, 990 (17.5%) had 2–3 registered attempts, and 607 (10.7%) had >3 attempts. A clear majority of attempts were due to poisoning . Cutting  was the second most frequent method used. During the follow-up 194 (3.4%) individuals died by suicide and 150 (2.7%) by other causes of death.In the crude analyses, granting of disability pension was positively associated with older age , negatively associated with country of birth outside the European Union , and the hazard ratio increased with number of suicide attempts reaching a three-fold HR for individuals with three or more attempts. Suicide attempters with previous psychiatric inpatient care had increased risk for disability pension, with HRs being highest for schizophrenia , followed by personality disorders . Earlier somatic inpatient care was associated with an HR of 1.7 . Among the parental characteristics, low educational level , psychiatric inpatient care , disability pension , and parental non-suicidal death  were associated with a higher risk for disability pension in offspring .In the fully adjusted model, female sex , older age  and low educational level  remained positively associated with subsequent disability pension, while country of birth outside the European Union decreased the relative risk of disability pension .Repeated suicide attempts were associated with a higher risk of disability pension . Schizophrenia  and personality disorders  remained the diagnoses with the largest risk estimates related to disability pension. Earlier somatic inpatient care  and parental disability pension  were also significantly associated with disability pension .In the crude analyses, long-term sickness absence was positively associated with female sex  and older age , and negatively associated with country of birth in the European Union but outside of Scandinavia . The hazard ratio increased in a dose-response pattern with number of suicide attempts, reaching a 1.5-fold HR for individuals with three or more attempts . Most psychiatric diagnostic groups of individuals with previous psychiatric inpatient care had increased HR for long-term sickness absence, with HRs being highest for affective disorders , followed by developmental disorders . Earlier somatic inpatient care was associated with a HR of 1.5 . Among the parental characteristics, low educational level , psychiatric inpatient care , disability pension , and parental non-suicidal death  were all associated with a higher risk for sickness absence in offspring .In the fully adjusted model, female sex , older age  and residence in small cities or villages  were positively associated with subsequent sickness absence. Repeated suicide attempts were associated with a higher risk , and there was no additional risk from previous psychiatric diagnoses on the risk of long-term sickness absence. Earlier somatic inpatient care  and parental disability pension  had some effect on long-term sickness absence in offspring, and sickness absence was also associated with parental education, with a 1.2-fold risk  for offspring of parents with low educational level .An interaction with sex and repetitive suicide attempts was found with regard to subsequent sickness absence. Men with two and more than two suicide attempts had higher HRs (1.4 (1.2–1.7); 1. 5 (1.1–1.9) for subsequent sickness absence, respectively. Related estimates for women were 1.1 (1.0–1.3) and 1.1 (1.0–1.3).There was an additional interaction with sex and previous mental diagnosis-specific inpatient care with regard to subsequent sickness absence. Men had generally higher HRs than women. For example, the HRs and CIs for inpatient care due to substance abuse and behavioural disorders for males were 1.2 (1.0–1.5) and 1.4 (1.0–2.1), respectively. The corresponding estimates among females were 0.9 (0.8–1.1) and 1.1 (0.9–1.3), respectively.In the crude analyses, long-term unemployment was negatively associated with female sex , and positively associated with older age , residence in villages  or medium-sized cities , country of birth outside of the European Union , and low educational level . Among previous psychiatric medical diagnostic groups, only a diagnosis of substance abuse  was significantly associated with long-term unemployment. Earlier somatic inpatient care , and parental characteristics including low educational level , psychiatric inpatient care , disability pension  and non-suicidal death  were also associated with long-term unemployment in the crude analyses .In the adjusted model, female sex was negatively associated with subsequent long-term unemployment . Residence in villages  or medium-sized cities , country of birth outside the European Union  and low  or medium educational level  remained significant predictors of subsequent long-term unemployment. Also low  or medium  parental educational level, and parental psychiatric inpatient care  remained associated with later long-term unemployment .We examined the association between various medical, socio-demographic, and parental risk factors among persons who had inpatient care due to suicide attempt with subsequent labour market marginalization, which was defined as long-term unemployment, long-term sickness absence or granting of disability pension. Medical risk factors were most important for the subsequent granting of disability pension and also for long-term sickness absence. With regard to long-term unemployment, medical risk factors were of marginal importance. In contrast, all socio-demographic factors were associated with higher risk for unemployment. This included male sex and non-European migrant status. On the other hand, male sex and birth outside Europe were associated with decreased risk of being granted disability pension. Interaction tests revealed that men with previously diagnosed mental disorders or repeated attempts had higher risk of long-term sickness absence than women.This study revealed clear variation in the relevance of socio-demographic and medical risk factors for various markers of labour market marginalization. We found that repeated suicide attempts and somatic comorbidity in young suicide attempters were predictive for subsequent sickness absence and disability pension but not for unemployment. Previous inpatient care due to mental disorders was only predictive of subsequent disability pension. Consistent with earlier studies, schizophrenia and personality disorders were most strongly associated with disability pension ,22. PoteBeing born in a country outside Europe was associated with decreased risk of disability pension but increased risk for unemployment. This finding may signal that immigrants with suicide attempts who were born outside Europe enter a labour market marginalization track that typically involves long-term unemployment, but not granting of disability pension. In contrast to this finding, previous studies reported an increased risk of disability pension for immigrants from outside the EU who experienced sickness absence due to stress-related mental disorders or depressive episodes ,25. BesiLower socio-economic status and older age was predictive for all outcome measures, though to varying degrees. This is in line with previous findings, that labour market marginalization is socially determined ,29. LiviWomen had higher risk of being granted disability pension or long-term sickness absence, whereas men had a higher relative risk for long-term unemployment which is in line with previous studies in individuals with sickness absence or outpatient care due to mental disorders . Men hadIn contrast, parental disability pension was associated with a higher risk of disability pension and long-term sickness absence in offspring suicide attempters. Both intra-familial morbidity and social patterns that may influence granting of disability pension may play a role in this finding. A recent study identified low labour market participation in the parents as risk factors for sickness absence in the offspring . ParentaStrengths of the present study include its prospective design and the use of national register data which have practically no dropout rate. Evaluations of several registers have shown high data quality. For example, 1.2% and 0.8% missing main diagnoses were reported for the National Patient Register and the Causes of Death Register, respectively . Data onWith regard to study limitations, the present study was based on individuals who were hospitalized for suicide attempts. As it is known that only a quarter of suicide attempters receive inpatient care, the present findings can only be generalized to individuals who have more serious medical conditions that result in hospitalization . Follow-Labour market marginalization is not a homogenous construct, as evidenced by the different associations of medical and socio-demographic factors with disability pension, sickness absence and long-term unemployment. While medical factors played a detrimental role in the receipt of disability pension and sickness absence, their role in long-term unemployment was only marginal. Moreover, marginalization processes seemed to differ by sex and migration status, with birth country having a different effect on disability pension and long-term unemployment, and repeated suicide attempts having a stronger effect for men than women with regard to long-term sickness-absence. Because labour market marginalization could result in a worsening of risk for suicide due to social isolation and social marginalization, it would be ideal to be able to establish programs designed to keep those with labour market marginalization engaged in the labour market. These findings may contribute to the design of clinical and public health intervention strategies which have to take the identified individual variations in risk for marginalization into account.


Lifespan and the proportion of older people in the population are increasing, with far reaching consequences for the social, political and economic landscape. Unless accompanied by an increase in health span, increases in age-related diseases will increase the burden on health care resources. Intervention studies to enhance healthy ageing need appropriate outcome measures, such as blood-borne biomarkers, which are easily obtainable, cost-effective, and widely accepted. To date there have been no systematic reviews of blood-borne biomarkers of mortality.To conduct a systematic review to identify available blood-borne biomarkers of mortality that can be used to predict healthy ageing post-retirement.Four databases  were searched. We included prospective cohort studies with a minimum of two years follow up and data available for participants with a mean age of 50 to 75 years at baseline.From a total of 11,555 studies identified in initial searches, 23 fulfilled the inclusion criteria. Fifty-one blood borne biomarkers potentially predictive of mortality risk were identified. In total, 20 biomarkers were associated with mortality risk. Meta-analyses of mortality risk showed significant associations with C-reactive protein , N Terminal-pro brain natriuretic peptide  and white blood cell count . There was also evidence that brain natriuretic peptide, cholesterol fractions, erythrocyte sedimentation rate, fibrinogen, granulocytes, homocysteine, intercellular adhesion molecule-1, neutrophils, osteoprotegerin, procollagen type III aminoterminal peptide, serum uric acid, soluble urokinase plasminogen activator receptor, tissue inhibitor of metalloproteinases 1 and tumour necrosis factor receptor II may predict mortality risk. There was equivocal evidence for the utility of 14 biomarkers and no association with mortality risk for CD40 ligand, cortisol, dehydroepiandrosterone, ferritin, haemoglobin, interleukin-12, monocyte chemoattractant protein 1, matrix metalloproteinase 9, myelopereoxidase, P-selectin, receptor activator of nuclear factor KappaB ligand, sex hormone binding globulin, testosterone, transferrin, and thyroid stimulating hormone and thyroxine.Twenty biomarkers should be prioritised as potential predictors of mortality in future studies. More studies using standardised protocols and reporting methods, and which focus on mortality rather than risk of disease or health status as an outcome, are needed. Population demographics are changing worldwide, as people are living longer and the birth-rate is falling in many countries. In the UK the number of adults over 60 years of age is expected to increase by 7 million over the next 20 years . The proResearch is beginning to focus on interventions to help health span to keep pace with lifespan. Most interventions focus on lifestyle factors including physical activity and diet  and is Studies with a prospective cohort design and at least 2 years follow up were included to allow the predictive value of biomarkers to be assessed. Where studies investigated disease prevention, only primary prevention studies were included. For Medline and Embase, standardised filters from BMJ Evidence  which reStudies that included human participants, of either sex, with blood-borne biomarker data available for participants with a mean age of 50–75 years at baseline were included. Studies that recruited participants based on risk factors for a disease, rather than presence of the disease itself, were included as these studies were likely to focus on primary prevention.This review was restricted to studies examining biomarkers that are blood-borne and where a relationship with mortality was reported. Studies that examined genetic factors or biomarkers diagnostic of a particular disease rather than mortality risk were not included in this review.A systematic review of the literature was conducted across Medline, Embase, Scopus and Web of Science. The search strategy was created in Medline and translated for the three other databases. The specific search strategy  includedTwo investigators (EB and JL) conducted title and abstract screening to identify articles justifying full text screening according to the selection criteria. Any disagreements were resolved by discussion. The reference lists of studies accepted after full text screening were cross-checked by hand to identify other relevant publications. Studies identified from reference cross-checking were subjected to the same process. Data were extracted from the full text article of all studies accepted after full text screening using a customised data extraction form . The datComprehensive meta-analysis  and Stata  were used to pool and to meta-analyse results from the individual studies. Pooled results were extracted and analysed as Hazard Ratios (HR) with 95% confidence intervals. Hazard ratios for biomarkers were analysed by subgroup, where possible. Overall Hazard ratios are also presented. A random-effects model was used and 2-sided P values from an inverse variance statistical method are reported.2 statistic [2 were calculated using Higgins et al’s method [2 was >50%, the degree of heterogeneity was considered substantial. Potential publication bias was appraised by visual inspection of the funnel plot of effect size against the SE for each study, with asymmetry formally assessed with Egger’s regression test.Statistical heterogeneity was evaluated using the Itatistic , 27, whis method , 29. WheThe characteristics of the participants in the cohorts included in the review varied widely, as did sample size , length of follow up (range: 2.7 to 19 years) and gender balance see . The maj2 = 64.38; Q 28.1; DF 10; p = 0.002) and cardiovascular disease (CVD) mortality , but baseline CRP has no relationship with risk of coronary heart disease (CHD) mortality . Regarding other specific causes of mortality, higher CRP concentrations at baseline were associated with greater risk of cancer mortality . A funnel plot of all meta-analysed studies on CRP did not show asymmetry and Egger’s regression test was not significant (p = 0.44) indicating likely absence of publication bias.C-reactive protein was examined in 12 studies , 49–51. 2 = 66.81; Q 6.02; DF 2; p = 0.049) and studies with follow-ups over 5 years  the association between CRP and all-cause mortality remained significant. Comparison of these subgroups showed no significant differences (p = 0.540).Subgroup analysis by follow-up length showed that among studies with follow up of 5 years or less , CHD mortality  and CVD mortality . One study reported an association with non-CVD mortality.Overall, higher concentrations of NT-proBNP at baseline were associated with greater subsequent mortality . Kistorp2 = 85.28 (95%CI 57 to 95); Q = 13.56, DF = 2; p = 0.0001).Baune et al , Baylis 2 was significant  Q = 14.95, df = 3, p = 0.0001). Although the Egger test was non-significant (p = 0.73), with only 4 studies the possibility of publication bias cannot be excluded.Four studies examine the association between WBC count and all-cause mortality , 44, 52.For the remaining putative biomarkers of mortality risk, there were too few studies to warrant meta-analysis.Overall, there was no association between 25(OH)D and all-cause, CV and non-CV mortality but higher 25(OH)D concentrations were protective in men with intermediate to high coronary risk scores for all-cause  and CV mortality  .There was a significant positive association between baseline BNP concentrations and all-cause mortality risk  .Carriere et al  examinedMenke et al  found thBaylis et al  reportedCarriere et al examined the association between TTR and all-cause mortality at two time points. Those participants in the lowest quartile of TTR had a greater risk of mortality at ≤5 years follow up  but this apparently greater risk was not present at 5 to 9 years follow up .Schnabel et al  examinedIn contrast, Schnabel et al  found noCarriere et al  divided HDL-C was negatively associated with all-cause mortality with a significantly lower risk (p = 0.01) in those with the highest baseline HDL-C concentrations . When stratified by sex, this trend was significant in females  but not males  . This neRisk of all-cause mortality was higher in those males and females with the highest baseline LDL-C concentrations  compared with the lowest concentrations . A similar pattern was seen for cardiovascular mortality  .There was a positive association between non HDL-C and all-cause mortality for both males  and females . Cardiovascular mortality risk was also increased with higher non HDL-C concentrations in both sexes  .Cui et al  comparedWu et al  divided In addition to IL-6, Baune et al  investigThere were no associations between matrix metalloproteinase 9 (MMP-9)   or myeloBaylis et al  reportedCarriere et al (2008) reported that AAG concentrations in the highest quartile were associated with significantly increased mortality risk at ≤5 years follow up . At 5 to 9 years follow up, compared with the middle quartiles, mortality rates for men in the lowest quartile were reduced significantly  but there was no such effect in women .Wu  reportedHigher levels of TIMP-1 were associated with greater risk of mortality  .There was no evidence that SHBG levels were associated with mortality risk  .Menke et al  reportedAmong American participants in the Third National Health and Nutrition Examination Survey (NHANES III), higher plasma homocysteine concentrations were associated with increasing CV mortality risk  .Baylis et al  found noAmong 3250 Framingham Study participants (54% of whom were women), there was a positive association between OPG and all-cause mortality  . HoweverHigher baseline concentrations of suPAR were associated with increased risk of mortality at 13 years of follow up. Males in the lowest suPAR quartile at baseline survived on average 8.4 years longer than those in the highest quartile (p<.0001) while females in the lowest quartile survived an average of 4.7 years longer (p<.0001) .In a community-based study of 3035 participants, Schnabel et al  reportedSerum creatinine was predictive of CV mortality only in intermediate and high coronary risk groups among NHANES III participants .In community-dwelling older people, there was no association between DHEAS and all-cause mortality , although lower levels of DHEAS were associated with increased risk of frailty at ten years follow up .Three studies investigated the relationship between SUA and mortality and found similar effects , 40, 51.For 922 Framingham Study participants, each SD increase in log-PIIINP was associated with almost 50% increased mortality risk  [Two reported associations between WBC components and mortality. Among almost 10,000 Korean elders, all-cause mortality risk increased significantly in those with the highest compared with the lowest quartiles of granulocytes  . Among pThis systematic review identified 51 blood-borne biomarkers for which relationships between baseline values and subsequent mortality risk were assessed in individuals initially aged 50–75years. The biological role of each of these biomarkers is described briefly in This systematic review is the first to examine the potential utility in predicting mortality risk of blood-borne biomarkers. Whilst a relatively large number of putative risk biomarkers were identified, in most cases there were only a few studies reporting on a given biomarker and these studies differed considerably in terms of participant numbers and characteristics. This limited our ability to undertake meta-analyses and sub-group analyses so caution should be used when interpreting findings from those biomarkers considered in the narrative review. Furthermore, as a sensitivity analysis for CRP where we had the most studies, the meta-analysis was re-run after excluding the data from Makita et al , which hAll of the blood-borne biomarkers identified in this review fulfil some of Johnson’s  criteriaThe main finding of this systematic review was the identification of 20 blood-borne biomarkers that predict mortality risk in middle-aged people. Of these 20, there were sufficient, similar studies reporting on well-established biomarkers such as CRP, NT-proBNP and WBC count to permit meta-analysis which confirmed the potential predictive value of these biomarkers. More evidence is necessary to establish the value of novel biomarkers and whether these add further predictive value to more established ones. One possible use of the biomarkers identified here is as outcome measures in future intervention studies aimed at enhancing healthy ageing. However, it cannot be assumed that blood-borne biomarkers which predict (or are associated with) risk of mortality will be suitable as outcome measures for such intervention studies because healthy ageing has yet to be defined adequately and, as a consequence, surrogate endpoints such as mortality may be inappropriate . In addiThe blood-borne biomarkers identified in this analysis need to be fully evaluated for their predictive capability and responsiveness to lifestyle-based interventions before they can be recommended to assess the utility of such interventions. In addition, it will be important to know i) whether there is redundancy within the 20 biomarkers identified here and ii) which of the biomarkers are the most predictive. Given that ageing is a complex process affecting all body systems, it is possible that a panel of biomarkers (a subset of the 20 identified by this review) would be better than any of the biomarkers individually; this hypothesis also needs to be tested. It may be possible to compare the utility of a range of biomarkers using data from different study designs using the horizontal systematic review method (e.g. ). ExpansS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.