Amyloid light-chain (LC) amyloidosis (AL amyloidosis) is a rare and fatal disease for which there are no approved therapies. In patients with AL amyloidosis, LC aggregates progressively accumulate in organs, resulting in organ failure that is particularly lethal when the heart is involved. A significant obstacle in the development of treatments for patients with AL amyloidosis, as well as for those with any disease that is rare, severe and heterogeneous, has been satisfying traditional clinical trial end points . It is for this reason that many organizations, including the United States Food and Drug Administration through its Safety and Innovation Act Accelerated Approval pathway, have recognized the need for biomarkers as surrogate end points. The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. Underlying this consensus is the demonstration that NT-proBNP is an indicator of cardiac response in all interventional studies in which it has been assessed, despite differences in patient population, treatment type and treatment schedule. Furthermore, NT-proBNP expression is directly modulated by amyloidogenic LC-elicited signal transduction pathways in cardiomyocytes. The use of NT-proBNP will greatly facilitate the development of targeted therapies for AL amyloidosis. Here, we review the data supporting the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC and universally accepted by AL amyloidosis specialists, as a surrogate end point for survival. Progressive amyloid deposition and proteotoxic effects of amyloid proteins lead to organ failure, which is especially catastrophic when the heart is affected, and is the primary cause of death.5 As many as 70% of patients with AL amyloidosis have predominantly cardiac amyloid deposition.5 The prevalence of all types of cardiac amyloidosis is assumed to be underestimated because of missed diagnoses, given that the symptoms of cardiac amyloidosis often mimic those of other far more common conditions.8Amyloid light-chain (LC) amyloidosis (AL amyloidosis), a rare, progressive and fatal disease, is the most common form of systemic amyloidosis,10 Overall, plasma cell-directed therapies, also called source therapies, can induce a reduction in the concentration of the toxic LC by ⩾50%  in ~60% of patients, and this translates into cardiac or renal response, or both, in ~20–35% of patients. Cardiac response translates into substantial survival benefit.11There are no approved therapies for AL amyloidosis. That said, frequently used treatment options, such as high-dose chemotherapy in combination with autologous stem cell transplantation, alkylating agents, steroids, proteasome inhibitors and/or immunomodulatory drugs, reduce production of the amyloid-forming precursor protein, immunoglobulin LC, by targeting plasma cells.12 It is for this reason that the use of biomarkers as surrogate end points was proposed by the United States Food and Drug Administration (FDA) with its Safety and Innovation Act Accelerated Approval pathway13 to hasten the approval of new treatments. Furthermore, this position was recently promoted by the FDA: ‘The path to development of promising therapeutics can be enabled by the availability of biomarkers that are analytically validated and clinically qualified for a specific context of use'.14A significant barrier to therapeutic development is posed by all diseases that are serious, heterogeneous and rare; for such diseases, traditional trials with clinical end points are difficult, and sometimes impossible, to conduct.BNP expression, and it makes the use of NT-proBNP, a robust surrogate for survival unique from other applications  in which inconsistencies have justifiably warranted apprehension. This rare and fatal disease should be considered a priority for expediting new treatments. In line with the Accelerated Approval pathway,13 a surrogate end point must be reasonably likely to predict a treatment's clinical benefit so that it will be considered validated and acceptable by regulatory authorities. In accordance with the FDA's criteria for Accelerated Approval, the following qualifying criteria for using a surrogate end point are met for AL amyloidosis:The international AL amyloidosis expert community is in agreement that the N-terminal fragment of the pro-brain natriuretic peptide (NT-proBNP) is analytically validated and clinically qualified as a biomarker for use as a surrogate end point for survival in patients with AL amyloidosis. This consensus is supported by the consistent demonstration of the predictive value of NT-proBNP in all intervention trials to date, which we review here, and is especially striking in light of differences in study design, treatment regimens or combinations of treatment regimens, treatment class, patient population and geographic location. The reliability of NT-proBNP as a biomarker results from a direct intracellular pathway connecting the amyloid-forming precursor protein, immunoglobulin LC, to the induction of kinases known to promote ‘…treats a serious condition…'—this is relevant for the substantial morbidity and mortality associated with AL amyloidosis.‘…demonstrates an effect on a surrogate endpoint that is reasonably likely to predict clinical benefit or on a clinical endpoint that can be measured earlier than irreversible morbidity or mortality (IMM) that is reasonably likely to predict an effect on IMM…'—hematologic response, a commonly used efficacy measure for AL amyloidosis patients, may not capture the specific impact on heart dysfunction, which causes death. Consistent evidence from several independent clinical trials uniformly supports that NT-proBNP not only reasonably, but highly likely predicts clinical benefit to the heart. The use of a survival end point requires unacceptably extended trial durations for initial approval but could be confirmatory.Without acceptance of surrogate end points for survival, a primary obstacle of AL amyloidosis clinical trials is their long duration and large study size, necessary because of the need for end points to demonstrate efficacy  with appropriate statistical power. Furthermore, the availability of several effective lines of plasma cell-directed therapy makes it difficult to evaluate the impact of a specific treatment on overall survival, and significant morbidity caused by organ dysfunction makes it difficult to define progression-free survival. Acceptance by the regulatory authorities of this important surrogate will accelerate drug development by facilitating more rapid clinical development of molecules and will standardize study designs evaluated in clinical trials . This will lead to more drug-industry investment into the study of AL amyloidosis. At present, ‘source therapies' are being used off-label, and few manufacturers are pursuing regulatory approval because of the long development time, difficult trial design and significant investment required for such a small population.15 For this reason, using NT-proBNP as a surrogate end point for pivotal clinical trials in patients with AL amyloidosis will advance therapeutic treatments that save lives. Here, we present the data and rationale for the use of NT-proBNP, a biomarker that is analytically validated, clinically qualified, directly modulated by LC-elicited signal transduction pathways in cardiomyocytes and accepted by AL amyloidosis specialists as a surrogate end point for survival.Although controlling the source of the amyloid precursor protein and factoring hematologic response into efficacy measures is important, they are of limited long-term value to patients in the absence of organ improvement.16 The extent of cardiomyopathy is the most important determinant of outcome in patients with systemic amyloidosis.17 As mentioned, no therapies have been specifically approved for the treatment of patients with AL amyloidosis, and optimal treatment regimens remain undefined.18In AL amyloidosis, misfolded LCs predominantly affect the heart (~70% of patients), kidneys (70% of patients) or both, but can also affect the liver, gastrointestinal tract, soft tissue, and peripheral and autonomic nervous systems (<20% patients each).19 Amyloid-forming LCs also cause rapid and direct myocardial toxicity,21 eliciting oxidative damage,23 possibly through interactions with mitochondrial proteins.24 Amyloid LCs purified from patients with amyloid cardiomyopathy induce p38 mitogen-activated protein kinase (MAPK) signaling,25 resulting in oxidative stress, impaired excitation-contraction coupling26 and, eventually, cardiomyocyte death.26Extracellular amyloid deposits in the heart create anatomic restrictions that contribute to cardiac dysfunction.15 The development of therapies designed to decrease levels of circulating precursor, clear toxic cardiac aggregates and accelerate the clearance of amyloid deposits addresses a critical unmet need to reverse or attenuate cardiomyopathy in patients with AL amyloidosis. The use of NT-proBNP will facilitate the emergence of such novel therapies.Mayo Clinic investigators recently reported that hematologic response alone is not necessarily adequate to result in clinical benefit, and that early organ response predicts improved overall survival after successful therapy in AL.27 In some patients, NT-proBNP assessment may facilitate a better diagnosis than physician review of patient history and other laboratory findings.29 Moreover, the response of NT-proBNP after intervention is often used in clinical trials to assess outcome and cardiac progression. NT-proBNP has been clinically qualified to a great extent in AL amyloidosis, and AL amyloidosis specialists agree that it predicts cardiac response and improved clinical outcome after intervention. Thus, NT-proBNP, in contrast to other cardiac markers , is the focus of this article.NT-proBNP has emerged as an analytically validated, gold standard biomarker for the determination of cardiovascular risk and disease.31 Cardiomyocytes synthesize a pre-propeptide , which is cleaved to the propeptide . After secretion, proBNP is cleaved to the active hormone BNP (amino acids 77–108) and the biologically inactive peptide NT-proBNP (amino acids 1–76).32 Notably for AL amyloidosis, MAPK signaling mediates BNP transcription,35 supporting a direct connection between LC cardiotoxic effects with induced MAPK signaling and BNP levels. This direct modulation of BNP synthesis by cardiotoxic LC amyloid precursor is distinct from the processes proposed to elicit BNP secretion in other cardiovascular diseases and makes BNP levels directly reflective of the LC-induced cardiac pathology in AL amyloidosis. Ventricular dysfunction is indicated by increased serum BNP and NT-proBNP, which are secreted at a 1:1 equimolar basis.32 In the circulatory system, NT-proBNP has a longer half-life than BNP (2 h vs 22 min) and is subject to renal clearance.32BNP is a myocyte-secreted hormone that maintains fluid homeostasis in the body through natriuretic, diuretic and vasodilatory effects. BNP is secreted in response to ventricle distension and stretching caused by volume expansion and pressure overload.36 European Cardiology Society guidelines for the diagnosis and treatment of acute and chronic heart failure include measurement of natriuretic peptides among the essential initial investigations, reporting that a normal natriuretic peptide level in an untreated patient virtually excludes significant cardiac disease, making echocardiography unnecessary.29 Furthermore, in their recent document defining cardiovascular end points for clinical trials, the American College of Cardiology/American Heart Association included NT-proBNP/BNP as laboratory evidence supporting the diagnosis of worsening heart failure.37Measurement of NT-proBNP is a robust, standardized laboratory test than can be easily and rapidly performed worldwide (1 h using electrochemiluminescence immunoassay).39 clinically meaningful response has been defined as a >30% decrease of NT-proBNP, a reduction of ⩾300 ng/l (35.4 pmol/l) in patients with ⩾650 ng/l (76.7 pmol/l) baseline NT-proBNP.40Circulating NT-proBNP levels increase with patient age and renal insufficiency, are higher in women than men and are generally lower in obese patients because of the presence of NT-proBNP receptors on adipocytes and the production of neprilysin in adipose tissue. On the basis of the biological variability, resulting in a critical difference/reference change value of ~25% of circulating NT-proBNP,No clear superiority of BNP compared with NT-proBNP has been established in the biomarker field; however, studies performed in patients with AL amyloidosis have primarily used NT-proBNP. This might be because NT-proBNP has a longer half-life, is more sensitive and more stable  than BNP, which requires stricter pre-analytical requirements.29 and may allow for the detection of meaningful cardiovascular responses in advance of morphologic changes found on echocardiography.41 NT-proBNP is the most sensitive marker of cardiac involvement in AL amyloidosis; it has 100% sensitivity for detecting cardiac involvement estimated by clinical signs, electrocardiography and echocardiography using the validated cutoff of 332 ng/l (0.33 mg/ml or 39.2 pmol/l).42Measuring circulating NT-proBNP is more rapid, more reproducible, more affordable and more accessible than evaluations that require specialized equipment, procedures  and trained personnel,43 Current staging systems for this disease are based on serum levels of NT-proBNP, cardiac troponin T and the concentration of circulating amyloidogenic-free LCs.44 In addition to the relevance of NT-proBNP in prognostic stratification at baseline, changes in NT-proBNP concentration after therapy predict clinical outcomes for patients. Specifically, NT-proBNP response or progression predicts attenuation or progression of cardiac dysfunction, with significant impact on survival.11Several studies demonstrate that cardiac biomarkers, particularly NT-proBNP, are powerful predictors of prognosis in AL amyloidosis.52 demonstrate that baseline NT-proBNP level predicts clinical outcome in patients with newly diagnosed AL amyloidosis. Palladini et al.42 first reported an association between baseline NT-proBNP levels with cardiac involvement and survival in 152 patients with AL amyloidosis .51 Kristen et al.50 demonstrated that baseline NT-proBNP level was an independent predictor of survival in 163 patients with AL amyloidosis. In an analysis of 1998 patients seen over a 30-year period at the Mayo Clinic compared with 313 contemporary patients, Kumar et al.45 reported that NT-proBNP levels consistently identified patients at risk for early death despite improvements in survival during the same period. Wechalekar et al.52 demonstrated that presenting NT-proBNP correlated with survival in a group of 346 patients with newly diagnosed AL amyloidosis who subsequently were treated with hematologic therapies. Kristen et al.46 showed that baseline NT-proBNP level was a univariate predictor of survival in 185 patients with AL amyloidosis. Banypersad et al.47 demonstrated, in 100 patients with AL amyloidosis, that cardiac disease revealed by nuclear magnetic resonance imaging correlated with NT-proBNP levels. Kristen et al.48 confirmed that risk stratification associated with both NT-proBNP and cardiac troponin T predicted 1-year mortality in 125 patients with AL amyloidosis. These 9 independent studies in 3722 treatment-naive patients show that NT-proBNP responses consistently reflect changes in cardiac function and predict survival in patients with AL amyloidosis.Nine studiesloidosis . In sepa56 have established that the NT-proBNP response, defined as a decrease in NT-proBNP of >30% and >300 ng/l (35.4 pmol/l) in evaluable patients , predicts clinical outcome and survival. NT-proBNP is a survival marker independent of therapy type, treatment class, or regimen; these studies represent patients treated with 9 different combinations of therapies and 3 different individual therapies, which include chemotherapies and ASCT as well as steroids, immunomodulatory drugs, proteasome inhibitors, and alkylating agents , thalidomide plus dexamethasone (TDex), dexamethasone (Dex), melphalan plus prednisone (MP) or thalidomide (T), that achievement of NT-proBNP response predicted both overall survival and progression-free survival  response independently predicted survival in 94 patients with AL amyloidosis treated with bortezomib (Bor) or Bor plus dexamethasone (BDex). Palladini et al.49 demonstrated that post-treatment NT-proBNP response independently predicted patient survival in a study of 113 patients with AL amyloidosis treated primarily with MDex, Dex, cyclophosphamide plus thalidomide and dexamethasone (CyTDex) or ASCT. Kastritis et al.56 reported that NT-proBNP response predicted survival in 85 patients with AL amyloidosis treated with BDex or with L-based or risk-adapted BDex. On the basis of these results, in 2012 the International Society of Amyloidosis established and validated NT-proBNP response as an indicator of organ response and as a surrogate marker of survival in AL amyloidosis. NT-proBNP response predicted a significant survival benefit both in testing (n=816) and in validation (n=374) populations treated primarily with MDex, T-based, lenalidomide (L)-based, Bor-based, Dex, MP or ASCT treatments . Although the study is not yet complete and the number of evaluable patients is limited, the preliminary outcome analysis indicates that NT-proBNP response translates into a significant survival benefit  revealed that though decreasing glomerular filtration rate required a higher threshold for detecting heart involvement and predicting survival, NT-proBNP predicted survival in patients with glomerular filtration rates⩾15 ml/min/1.73 m2.58In patients with both renal and cardiac involvement, reductions in glomerular filtration rate can influence NT-proBNP levels. In a study of 248 such patients, stratification by glomerular filtration rate . Similarly, recent studies61 have proposed cardiac magnetic resonance imaging as an additional prognostic tool in cardiac AL amyloidosis, but no study has yet explored cardiac magnetic resonance imaging in response assessment. Notable additional strengths of NT-proBNP assessment are its robust standardization, reproducibility, ease, speed and low-cost compared with more expensive and complex imaging techniques that are frequently operator dependent.Although several cardiac biomarkers and imaging techniques have been reported to have a prognostic value, none have been validated as surrogate markers of cardiac response and clinical outcome in patients with AL amyloidosis. There is no clear correlation between reductions in NT-proBNP with improvements in cardiac anatomy,62 and novel biomarkers such as ST2,63 GDF-15,64 mid-regional pro-adrenomedullin65 and osteoprotegerin66) measured at diagnosis are prognostic factors in patients with AL amyloidosis, they have not yet been predictive of survival based on response after interventional treatment. More evidence is needed to establish their potential value. Only NT-proBNP response after several different classes of treatment indicated patient outcome and could be used as a marker of response. Increased cardiac troponin indicated disease progression and poor survival in patients with AL amyloidosis, but the role for troponin in the definition of response remains less established.67 Finally, NT-proBNP should continue to be evaluated for use in assessing cardiac response in patients with both mutated and wild-type cardiac transthyretin amyloidosis.Although other biomarkers , making NT-proBNP an unambiguous marker of amyloid cardiac disease. The use of NT-proBNP as a surrogate efficacy end point for AL amyloidosis trials using current, validated definitions of response is not controversial. NT-proBNP level should be widely adopted to evaluate the effectiveness of new treatments targeting cardiac dysfunction in patients with AL amyloidosis as an accepted primary outcome. Experts agree that NT-proBNP is the only surrogate end point for survival after treatment, and lowering NT-proBNP and achieving NT-proBNP response are the ultimate treatment objectives. Overall survival as an end point requires a larger study population and a longer assessment period; a trial using NT-proBNP could be half as long . The use68 of the impact of biomarker-based strategies on oncology drug development from clinical trials that led to FDA approval supported the safety and improved efficacy outcomes in FDA-approved anticancer agents using biomarker-based tactics. This approach is now feasible and necessary in AL amyloidosis. Now is a pivotal moment for many novel drugs in the pipeline that promise to address unmet needs in this rapidly evolving, but curable, disease. We owe it to our patients to rapidly assess the efficacy of new drugs. The data presented here establish that the NT-proBNP response, defined as a decrease in NT-proBNP of >30% and >300 ng/l (35.4 pmol/l), predicts clinical outcome and survival and is a validated and qualified surrogate marker of efficacy for interventions for patients with AL amyloidosis.A recent meta-analysis


Allogeneic hematopoietic stem cell transplantation is a curative-intent treatment for patients with high-risk hematologic diseases. However, interstitial pneumonitis (IP) and other toxicities remain major concerns after total body irradiation (TBI). We have proposed using linear accelerators with rice-bag compensators for intensity modulation (IM-TBI), as an alternative to the traditional cobalt-60 teletherapy with lung-shielding technique (Co-TBI). Patients who received a TBI-based myeloablative conditioning regimen between 1995 and 2014 were recruited consecutively. Before March 2007, TBI was delivered using Co-TBI (n = 181); afterward, TBI was administered using IM-TBI (n = 126). Forty-four patients developed IP; of these cases, 19 were idiopathic. The IP-related mortality rate was 50% in the total IP cohort and 63% in the idiopathic subgroup. The 1-year cumulative incidences of IP and idiopathic IP were 16.5% and 7.4%, respectively; both rates were significantly higher in the Co-TBI group than in the IM-TBI group. Multivariate analysis revealed that Co-TBI was an independent prognostic factor for both total and idiopathic IP. In the acute myeloid leukemia subgroup, patients with different TBI techniques had similar outcomes for both overall and relapse-free survival. In conclusion, IM-TBI is an easy and effective TBI technique that could substantially reduce the complication rate of IP without compromising treatment efficacy. Total body irradiation (TBI) has an established role in the conditioning regimens that are administered before allogeneic hematopoietic stem cell transplantation (HSCT)1235678910111291516161515161718202122During the era of cobalt-60 teletherapy (Co-TBI) at our institute (1995 to March 2007), TBI was delivered anteroposteriorly using parallel opposed fields with lung blocks to lower the lung doseAmong the 307 patients enrolled in this study, 181 patients received Co-TBI and 126 patients received IM-TBI. Data on forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1) were available for all patients. Information on total lung capacity (TLC) and carbon monoxide diffusing capacity of the lungs (DLCO) were available for 228 and 195 patients, respectively. The Co-TBI and IM-TBI groups were comparable in terms of age, sex, and the proportion of cases with complete remission (CR) at the time of HSCT . As compared with the Co-TBI group, more of the patients in the IM-TBI group had acute leukemia , initial CNS involvement , and GvHD of either the acute or chronic type . Regarding the pulmonary function test results, only TLC < 85% of predicted was more frequent in the IM-TBI group than in the Co-TBI group  .During the follow-up period , 44 patients developed IP, 19 cases of which were idiopathic  and 2. TRegarding treatments, 41% (18/44) of patients in the total IP group and 53% (10/19) of those in the idiopathic IP group received corticosteroid. Concomitant infection was the main obstacle for the patients who were unable to receive corticosteroid. The IP-related respiratory failure rates were comparable between patients in the total IP cohort and idiopathic IP subgroup . Among the survivors with follow-up lung function tests, 57% (4/7) had moderate to severe restrictive ventilator defect. The total IP-related mortality rate was 50% (22/44), which was similar to the idiopathic IP-related mortality rate .The risk factors for both total IP and idiopathic IP included non-CR at HSCT  and abnormal FVC before HSCT   and 4. AIn a multivariate Cox proportional hazards regression analysis for total IP, non-CR at HSCT, Co-TBI, and pre-HSCT abnormal FVC were independent predictors of higher incidence ; howeverComparisons of the lung doses for Co-TBI and IM-TBI are provided in In order to elucidate the prognostic impacts of different TBI techniques, we analyzed survival in the relative homogeneous group of patients with AML (n = 111). Univariate analyses of overall survival (OS) showed that age greater than 30 years  and non-CR at HSCT  were significant unfavorable prognostic factors. Other clinical parameters, including initial CNS involvement, unfavorable-risk cytogenetics, different TBI techniques, and GvHD (either acute or chronic), did not show any significant associations with survival in the univariate analyses. In a multivariate analysis of OS, only non-CR at HSCT was an unfavorable prognostic factor , age greater than 30 years  and non-CR at HSCT  were significant unfavorable prognostic factors. In resemblance with our multivariate analysis of OS, only non-CR at HSCT was an unfavorable prognostic factor in the multivariate analysis of RFS . Furthermore, 99% (65/66) of the survivors in the IM-TBI group and 97% of the survivors in the Co-TBI group  were observed for more than 1 year, making it possible to detect most of the IP development after HSCT.The possibility of developing IP is always a major concern for conditioning regimens that include TBI. Several patient-specific risk factors for IP were reported previously, including old age151515161718202122151723et al. were the first to describe TBI in transplantation for patients with AML, but their reports were limited to patients in first complete remission2The present investigation is one of the first studies to show that non-CR at HSCT is an unfavorable prognostic factor for IP, using both univariate and multivariate analyses. This might be explained by physicians’ attempts to induce more GvHD in patients without CR at HSCT, who were believed to have dismal outcomes. Previous studies concerning the association of TBI and IP were mainly based on mixed and various hematologic malignancies112151617181920212224252627−1 · day−1 for 2 days) and busulfan  (BuCy)3031Regarding the validity of our comparison of the two TBI techniques, a major concern is the different periods during which the techniques were performed. To eliminate the potential biases that might arise from these different periods, such as improvements in post-HSCT care, we additionally compared the IP rates of patients who received TBI-free conditioning chemotherapy composed of cyclophosphamide One of the limitations to this study was that we did not have complete performance status data. Weiner In conclusion, the rice-bag compensator-containing TBI technique (IM-TBI) may be capable of providing a substantially lower IP rate than the conventional lung-shielding TBI technique (Co-TBI). Additionally, in the subgroup of patients with AML, the IM-TBI technique may be able to provide OS and RFS rates that are similar to those associated with Co-TBI. Further efforts should be invested in developing new and modified techniques of tailoring dose distributions to sites of interest, with the ultimate goal of reducing the rates of late toxicities.This study recruited a total of 307 consecutive patients who received HSCT with a TBI-based myeloablative conditioning regimen from 1995 to 2014 at the National Taiwan University Hospital, including both children and adults. All patients received a pre-HSCT baseline pulmonary function test, routine chest radiograph, and high resolution computed tomography (CT) of the lungs. The patients who received the two different techniques of TBI (IM-TBI and Co-TBI) were compared in terms of the following characteristics: age, definite diagnosis, pre-HSCT disease status, conditioning regimens, and pre-HSCT pulmonary function test results, including FEV1, FVC, TLC, and DLCO. This study was approved by the Research Ethics Committee of the National Taiwan University Hospital and was performed in accordance with the Declaration of Helsinki, including all relevant details. Every patient provided written informed consent.pneumocystis jirovecii pneumonia prophylaxis. After 2007, regular blood sampling tests for serum cytomegalovirus viral load were initiated after conditioning chemotherapy, and lasted for at least 6 months after HSCT. Pre-emptive anti-cytomegalovirus therapy was delivered if the serum cytomegalovirus viral load became elevated.The majority of the patients  received conditioning radiochemotherapy that consisted of cyclophosphamide and TBI. Other conditioning regimens were composed of TBI and melphalan, fludarabine, etoposide, and/or cytarabine. The medications for GvHD prophylaxis included anti-thymocyte globulin, cyclosporine, methotrexate, and/or mycophenolate mofetilTwo techniques of TBI were used during different time periods in this study. After March 2007, the radiotherapy technique for TBI was switched from cobalt-60 teletherapy machines (Co-TBI) to linear accelerators with rice-bag compensators for intensity modulation (IM-TBI). Both techniques included two parallel opposed diagonal fields (40 × 40 cm), which were accomplished through rotation of the fully opened collimator by 45 degrees, to cover the entire body.In the Co-TBI group, patients were treated with a cobalt-60 1.25 MeV teletherapy machine at a source-surface distance equaling 300 cm. Patients received 1.5 Gy per fraction for 3 to 4 days, to a total dose of 12 Gy, and were irradiated anteroposteriorly with a dose rate of 4.4–5.8 cGy/min. Adults were treated in a standing upright position, while children were treated in a reclining positionIn the IM-TBI group, TBIs were delivered with linear accelerators using 10 megavolt photons at source-axis distance equaling 380–470 cm. The total dose was 12 Gy, and was delivered in 8 fractions within 4 consecutive days. The dose rate was 5–10 cGy/min, depending on the patients’ body thickness. Patients lay in a supine position on a 32 cm-wide headboard with both arms positioned on the chest and the legs bent. We used the lateral diameter of the body at the level of the umbilicus as the reference thickness. If the reference thickness was more than 32 cm, additional bolus was applied on the bilateral outer sides of the headboard; on the other hand, polystyrene foam would be placed on the inner side of the headboard if the reference thickness was less than 32 cm. The rice-bag compensators were placed around the head, neck, shoulders, and chest to fill the interspace of these regions, and to decrease the variation of lateral body thickness along the patient axis . The lunTo compare the homogeneity of IM-TBI and Co-TBI, we simulated a three-dimensional plan for Co-TBI by inputting the Co-60 beam data into the Pinnacle Treatment Planning System, version 9.2.0.60009 . This allowed us to compare the dose volume histogram and iso-dose curve of the lung between the Co-TBI and IM-TBI groups.pneumocystis jiroveci pneumonia, tuberculosis36All patients with respiratory symptoms and abnormal chest radiograph findings received high resolution CT of the lungs. IP was defined as multilobar infiltrates on routine chest radiographs and CT in the absence of congestive heart failure, renal failure, or iatrogenic fluid overload17Bone marrow cells were harvested directly or after 1 to 3 days of unstimulated culture, as described previouslyWe compared the characteristics of the Co-TBI and IM-TBI groups using chi-square tests. OS was measured from the date of donor stem cell infusion (described as the date of HSCT elsewhere in this article) to the date of last follow-up or death from any cause, whereas relapse was defined as the reappearance of at least 5% leukemic blasts in bone marrow aspiration smears or new extramedullary leukemia in patients with a previously documented CRHow to cite this article: Chiang, Y. et al. Reduced incidence of interstitial pneumonitis after allogeneic hematopoietic stem cell transplantation using a modified technique of total body irradiation. Sci. Rep. 6, 36730; doi: 10.1038/srep36730 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Astrocytes play important roles in normal brain function and neurological diseases. In vivo two-photon excitation laser scanning microscopy has the potential to reveal rapid, dynamic structural changes in cells in a variety of physiological and pathological conditions. The type of in vivo imaging method has been shown to affect the plasticity of dendritic spines of neurons, but the optimal in vivo imaging methods of astrocytes have not been established. We compared open-skull and thinned-skull imaging methods for two-photon laser microscopy of live astrocytes in neocortex of GFAP-GFP transgenic mice. The thinned-skull method provided stable image intensity and morphological features of astrocytes in vivo over at least one week, with no evidence of astrogliosis. In contrast, the open-skull method resulted in significant changes in image intensity and induced astrogliosis. The thinned-skull method is the preferred approach for in vivo imaging of astrocytes under most conditions involving gross astrocyte modulation or causing astrogliosis. Astrocytes, a subtype of glial cell in the central nervous system, are responsible for maintenance of homeostasis in the brain by regulating local ion concentrations, pH, energetic state, and metabolism . In addiNovel imaging methods for studying astrocytes provide important insights into both the physiological and pathological roles of astrocytes in normal brain function and neurological diseases. In contrast to the fixed, static view provided by conventional pathological studies, in vivo two-photon excitation laser scanning microscopy (2PLSM) is an indispensable method to investigate dynamic changes in cellular and subcellular structure and function in live tissue ,5. SeverTwo primary methods with different merits and drawbacks have been widely used for in vivo cellular imaging of the brain, the open-skull and thinned-skull techniques ,11,12,13Care and use of animals were conducted according to an animal protocol approved by the Washington University School of Medicine Animal Studies Committee  and followed guidelines from the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize animal discomfort and reduce the number of animals used.2, 2 mM MgSO4, 10 mM Glucose, 10 mM HEPES, pH = 7.4) and then covered with a glass coverslip  over the window (Two-to-three month old (~20 g) male GFAP-GFP transgenic mice with a mixed background, expressing enhanced green fluorescent protein (GFP) under a GFAP promoter were bred, originally obtained from Jackson Laboratory. Animal surgeries were performed using aseptic procedures as previously reported with minor modification ,14. Two e window . In thine window . CyanoacImages of astrocytes in neocortex were obtained through the cranial window using a two-photon microscope  with a water immersion objective , IR-adjusted, Zeiss). A Titanium-Sapphire pulsed infrared laser  was used to stimulate GFP at 900 nm. The excitation laser power was manually calibrated to obtain an optimized image determined by saturation of the range indicator in LSM M510 software and was measured below the objective lens using a Coherent Field Master power/energy meter . Low-magnification images approximate 50 to 100 μm below the neocortical surface were first obtained to identify regions with GFP positive astrocytes. At higher magnification , z-stacks of 6 to 10 images with 1 μm steps were taken. Individual images were acquired at 12 bits with frame averaging (2–4 times). Surface vasculature images captured by digital camera were used to identify the same astrocyte for time-lapse imaging at various times  . BaselinPost hoc image analysis was performed using LSM 5 Image Examiner software (Zeiss) and ImageJ software (NIH) in a blinded fashion to evaluate the changes in image intensity, astrocyte number, and morphological features of astrocytes over time. A standard area of 150 μm×150 μm was chosen as the region of interested (ROI) for each mouse, and the same ROI was analyzed at different time points. Image intensity was measured as mean GFP fluorescence intensity under the same excitation laser power and acquisition settings, with follow-up images over time normalized to the baseline image. Astrocyte number was counted in the same ROI at different time points. Morphological features of astrocytes were assessed with respect to total astrocyte size (including processes) and soma size, based on area calculations from the projected Z-stacks .2 area of each side of cerebral cortex was analyzed for GFAP positive astrocyte counting in a blinded fashion.GFAP-GFP transgenic mice were anesthetized with isoflurane during cardiac perfusion of PBS followed by 4% paraformaldehyde . Brains were then dissected out and post-fixed in 4% PFA overnight at 4°C followed by 30% sucrose dehydration. Coronal brain sections (45 μm in thickness) were prepared by frozen sectioning, and were blocked in PBS solution with 10% goat serum/1% BSA/0.3% Triton X-100 for 1 hour at room temperature. Glial fibrillary acid protein (GFAP) is a specific marker for astrocytes. The brains sections were incubated in the primary antibody  overnight at 4°C, and secondary antibody  at room temperature for 4 hr. For GFAP positive astrocyte counting, confocal images were taken with Zeiss LSM 5 PASCAL system coupled to Zeiss Axiovert 200 microscope. Comparable sections from different mice were chosen for comparison. A 0.3 mmAll data were presented as mean ± SEM. Statistical analysis was performed using GraphPad Prism 5 software. Student’s t test was used for comparison of laser power between groups. One-way analysis of variance (ANOVA) with the Tukey’s multiple comparison post-tests was used for comparison of mean fluorescence intensity, astrocytes number, the size of astrocytes and its soma, and soma-to-astrocyte ratio among different groups. P < 0.05 was set as statistical significance.We utilized and compared the open-skull and thinned-skull techniques with repeated 2PLSM of live astrocytes in neocortex of GFAP-GFP transgenic mice. In both methods, about 50% of mice were excluded from analysis due to dura damage, excessive bleeding, opacity of the initial baseline images, or a greater than 50% reduction in mean fluorescent intensity in follow-up images compared with baseline. The open-skull technique, in which all three layers of bone are removed, provides unimpeded optical access to the brain . By compThe open-skull technique, but not the thinned-skull technique, has been reported to induce astrogliosis as assayed by conventional histological methods ,11, but To control for the possibility that the changes in astrocyte number and size after open-skull surgery was an artifact of the changes in image intensity, in separate experiments we performed conventional immunohistochemical staining of GFAP for astrocytes on fixed brain sections at different time points after surgery. Consistent with the in vivo imaging, GFAP-positive cell number did not change over time after thinned-skull surgery, but increased significantly at 1wk after open-skull surgery . HoweverIn vivo imaging has become an important method for investigating normal brain function and neurological disease. The use of 2PLSM in mice with genetically-encoded fluorescent proteins has revolutionized the study of dynamic cellular and subcellular structures in the living brain. For example, in vivo imaging of neurons has revealed a degree of plasticity to dendrites and dendritic spines that was unappreciated with conventional pathological methods ,16. DendIn the open-skull technique, given the unimpeded optical access to the brain, a relatively low excitation laser power (~7 mW) was required to obtain adequate fluorescence intensities compared with the thinned-skull method. In addition, the spatial resolution of the fine astrocytic processes was slightly better with the open-skull method. However, the open-skull method may cause more mechanical disruption of brain parenchymal and meningeal structures and introduce foreign particles to the brain, which may in turn produce inflammatory changes. This may increase opacity of the window, which is consistent with our finding that the image intensity decreased at 24 hr and 3d after open-skull surgery. Thus, it is often recommended that imaging studies start at least 1–2 weeks after open-skull surgery. However, we actually observed an increase in image intensity at 1 week after the surgery, which is likely due to reactive astrogliosis and upregulation of GFAP expression, leading to a direct increase in fluorescence intensity in the GFAP-GFP mice. In contrast, the image intensity stayed constant over 1 week observation after thinned-skull surgery, although a higher absolute laser power (~25 mW) was required due to the residual inner cortical bone barrier. On the other hand, the remaining inner cortical bone may also provide protection against inflammation which may improve image quality. We utilized the newer reinforced thinned skull method with a fused transparent cement and glass window , and eveUnder normal physiological conditions, astrocytes maintain a relatively stable number and morphology, with a bushy appearance and thin processes. No significant changes were detected in astrocytes morphological features after thinned-skull surgery at all observed time points, including after repeated thinning. In contrast, the open-skull surgery led to significant astrogliosis, as evident by both an increase in astrocyte number and changes in size and morphology with time-lapse in vivo imaging. As reported by others ,11, we cThe thinned-skull method provided stable images of astrocytes in vivo over at least one week, and longer when using repeated thinning, with no evidence of astrogliosis. In contrast, the open-skull method resulted in significant changes in image intensity and induced astrogliosis. The thinned-skull method is the preferred approach for in vivo imaging of astrocytes.S1 FigMorphological features of astrocytes were assessed with respect to total astrocyte size (including processes) and soma size, based on area calculations from projected Z-stacks. Areas were measured using ImageJ software. The brightness of the in vivo image was manually adjusted for optimal contrast. To measure the area of astrocyte soma (excluding branches/fine processes) and total area, lines were drawn as shown.(TIF)Click here for additional data file.S2 Fig(A) Schedule for imaging and repetitive thinning for assessment of the thinned-skull method beyond week. Images were obtained for two weeks after using the initial thinned skull surgery. As image intensity significantly decreased at 2 weeks, a second thinning was then performed. (B-I). Representative in vivo images of astrocytes with the repeated thinned skull-method, involving a second thinning at 2 weeks after the initial thinning. The arrows in figures B-I denote the astrocytes that are enlarged as in figure B1-I1, respectively. The effect of repeated thinning of the skull was assessed on image intensity (J) and astrocyte number and size (K-N). (J) Image intensity decreased at 2 weeks after the initial thinning, but repeated thinning restored image intensity at least for another week. (K-N). With repeated thinning, no significant change was observed in astrocyte number, astrocyte or soma size, and the soma-to-astrocyte ratio at all observed time points after surgery. * p<0.05 by one way ANOVA with Tukey's post-test (n = 6 per group).(TIF)Click here for additional data file.S3 FigA) Extension of time-lapse in vivo imaging beyond the initial one week period demonstrates that the astrogliosis that occurred at 1 week after open-skull surgery persisted for at least 7 weeks. B,C) In other animals, surgery was performed, but the first images were not obtained until 2 (B) or 4 (C) weeks after surgery (no baseline or follow-up images prior to 2 weeks). Despite this 2–4 week waiting period after surgery, astrogliosis appeared present with the first imaging session and persisted.(TIF)Click here for additional data file.S4 FigAstrocytes were labeled by GFAP-GFP transgene expression (green) and GFAP immunohistochemical staining (red) in the same sections following open-skull and thinned skull surgery on the left side . Double labeled cells are shown in yellow, indicating astrocytes labeled by both methods. Minimal labeling occurs by either labeling method on the contralateral right side for both open-skull and thinned-skull surgery . On the surgery side (left), astrocytes were increased at 3 days and 1 week after open-skull, but not thinned-skull, surgery with both labeling methods , but interestingly the two labeling methods primarily labeled two different subsets of astrocytes with only modest overlap/double labeling.(TIF)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


Major depressive disorder is often associated with deficits in social and cognitive functioning. Mice transgenic for acid sphingomyelinase (t-ASM) were previously shown to have a depressive-like phenotype, which could be normalized by antidepressant treatment. Here, we investigated whether t-ASM mice show deficits in social behavior and memory performance, and whether these possible deficits might be normalized by amitriptyline treatment. Our results revealed that ASM overexpression altered the behavior of mice in a sex-dependent manner. As such, t-ASM female, but not male, mice showed an impaired social preference and a depressive- and anxiogenic-like phenotype, which could be normalized by amitriptyline treatment. Both male and female t-ASM mice showed unaltered preference for social novelty, novel object recognition, and social and object discrimination abilities. Amitriptyline treatment impaired novel object recognition and object discrimination abilities in female, but not in male, wild-type mice, while female t-ASM mice showed unaltered novel object recognition and object discrimination abilities. This study suggests that female t-ASM mice represent a model of depression with comorbid anxiety and social deficits, without memory impairments. It further suggests that ASM overexpression has a protective role against the detrimental effects of amitriptyline on female, but not on male, non-social (object) memory. Major depressive disorder (MDD) is a severe and chronic mood disorder, with a lifetime prevalence of more than 10%  and cognTricyclic antidepressant drugs, such as desipramine and imipramine, have been shown to induce the proteolytic degradation of the lysosomal glycoprotein acid sphingomyelinase (ASM) ,5, an enConsidering the comorbidity between MDD, social deficits, and memory impairments, we aimed to investigate whether t-ASM mice show deficits in social behavior and memory performance and whether these possible deficits could be normalized by amitriptyline treatment. Given that depression is more prevalent in women and treatment response is often gender-dependent , we perfSmpd1 cDNA under the control of a cytomegalovirus (CMV) immediate early enhancer/chicken beta-actin fusion promoter (CAG) into the Hprt locus (Hprttm1.1(CAG-Smpd1)Jhkh; www.informatics.jax.org/allele/MGI:5523896) [http://www.informatics.jax.org/allele/MGI:2137691). Experiments were conducted with t-ASM and littermate WT controls from the F1 generation.Mice conditionally overexpressing ASM were generated by a targeted integration of a murine ad libitum). Experiments were performed during the light phase, between 09:00 and 14:00, in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Government of Mittelfranken (Permit Number: 54–2532.1-27/11). All efforts were made to minimize animal suffering and to reduce the number of animals used.Male and female WT and t-ASM mice were individually housed for one week before treatment start and remained single-housed throughout the experiments. Age- and sex-matched WT mice were used as social stimuli for the social approach test. Sex-matched 3-week-old juvenile CD1 mice were used as social stimuli for the social discrimination test. Mice were kept under standard laboratory conditions . The initial position of the same mouse varied between experimental mice to prevent for possible place preference. After 5 min, the empty cage was exchanged by an identical cage containing a novel mouse for additional 5 min. Experiments were recorded and the time spent investigating (sniffing) the empty cage, the same and the novel mouse was analyzed by an observer blind to the treatment condition using JWatcher . The percentage of time investigating the empty cage and the same mouse (time investigating empty cage or same mouse/time investigating empty cage + same mouse x 100%) during the first 5 min, and the percentage of time investigating the same and the novel mouse (time investigating same or novel mouse/time investigating same + novel mouse x 100%) during the second 5 min was calculated. A higher investigation time directed toward the same mouse versus the empty cage during the first 5 min was interpreted as social preference. A higher investigation time directed toward the novel versus the same mouse during the second 5 min was interpreted as social recognition and preference for social novelty.The social preference and the preference for social novelty of mice were tested in the social approach test , with sosame) and a novel conspecific was tested in the social discrimination test as previously described [same juvenile was reintroduced along with a novel juvenile for additional 4 min (discrimination period). Experiments were recorded and the time spent investigating the juvenile mice  was analyzed by an observer blind to the treatment condition using JWatcher. The percentage of time investigating the same and the novel juvenile mouse (time investigating same or novel mouse/time investigating same + novel mouse x 100%) during the discrimination period was calculated. A higher investigation time directed toward the novel versus the same juvenile was interpreted as social discrimination and intact social memory. The 3-week-old juvenile mice did not elicit play or aggressive behaviors towards the experimental mice.The ability of mice to discriminate between a previously encountered  were simultaneously placed at opposite side-walls of the arena for 5 min. After 5 min, one object was exchanged by a novel object for additional 5 min. Several plastic objects that differed in color , shape , and size (3–4.5 cm x 3 cm x 1 cm) were used. Objects were cleaned between trials with water containing a small amount of detergent . Experiments were recorded and the time spent investigating the objects (sniffing/touching) was analyzed by an observer blind to the treatment condition using JWatcher. The percentage of time investigating the same and the novel object (time investigating same or novel object/time investigating same + novel object x 100%) during the second 5 min was calculated. A higher investigation time directed toward the novel versus the same object was interpreted as object recognition and preference for novelty.The ability of mice to recognize novelty over familiarity was tested in the novel object recognition test , which wsame) and a novel object was tested in the object discrimination test [same) was placed in one corner of the home cage of the experimental mouse for 4 min (acquisition period). The initial position of the object varied between mice to prevent for possible place preference. After an interval of 60 min, the same object was reintroduced along with a novel object for additional 4 min (discrimination period). The same objects were used as in the novel object recognition test described above. Experiments were recorded and the time spent investigating the objects (sniffing/touching) was analyzed by an observer blind to the treatment condition using JWatcher. The percentage of time investigating the same and the novel object (time investigating same or novel object/time investigating same + novel object x 100%) during the discrimination period was calculated. A higher investigation time directed toward the novel versus the same object was interpreted as object discrimination and intact object memory.The ability of mice to discriminate between a previously encountered  was administered via drinking water at a dose of 180 mg/L, based on previous studies ,15. TreaFor statistical analysis, PASW/SPSS (Version 21) was used. Data were analyzed by two-way ANOVA for repeated measures (factors stimulus x group) or three-way ANOVA (factors genotype x treatment x gender), followed by a Bonferroni’s post-hoc analysis when appropriate. Statistical significance was set at p < 0.05. Overall statistics are shown in same mouse versus the empty cage during the first 5 min of the social approach test, independent of genotype and treatment, reflecting social preference. Whereas water- and amitriptyline-treated WT females showed social preference, water-treated t-ASM females showed lack of social preference, reflected by similar investigation of the same mouse and the empty cage. This lack of social preference could be prevented by amitriptyline treatment, resulting in reinstatement of social preference  memory.We have previously shown a depressive- and anxiogenic-like phenotype in t-ASM mice in the novelty-suppressed feeding paradigm and in the open field test, which was normalized by amitriptyline treatment . The preAccording to our hypothesis, t-ASM mice showed a lack of social preference, indicating deficits in social functioning. Similar to the depressive- and anxiogenic-like phenotype, these deficits were only seen in female t-ASM mice, and could be normalized by amitriptyline treatment. A possible explanation for these sex-dependent effects might be the modulatory effect of estrogens on ceramide synthesis. Ceramide can be generated either through the hydrolysis of sphingomyelin, reaction catalyzed by the enzymes sphingomyelinases, including ASM and the neutral sphingomyelinases 2–4, through de novo synthesis, reaction catalysed by several enzymes, including ceramide synthases (CerS), and through a salvage pathway. Wegner et al.  have shoContrary to our hypothesis, t-ASM mice did not show any signs of memory impairment, neither in social, nor in non-social (object) memory. In more detail, both male and female t-ASM mice showed unaltered preference for social novelty and social discrimination, indicating intact short-term social memory abilities. Similarly, both male and female t-ASM mice showed unaltered novel object recognition and object discrimination, indicating intact short-term non-social memory abilities. Interestingly, amitriptyline treatment impaired both novel object recognition and object discrimination in female WT, but not t-ASM, mice, indicating that ASM overexpression protected female mice against the detrimental effects of amitriptyline on non-social memory. Amitriptyline has been previously shown to impair learning tasks in rodents after acute –21 and cInterestingly, female t-ASM mice were protected against the detrimental effects of amitriptyline on non-social memory performance. We have previously shown that amitriptyline inhibited the activity of ASM in the hippocampus in both WT and t-ASM mice, thereby decreasing ceramide concentration . TabatadTaken together, we have shown that ASM overexpression results in sex-dependent effects on social, depressive- and anxiety-like behavior. Despite these negative effects on behavior, ASM overexpression protected female mice against the detrimental effects of amitriptyline on non-social memory performance. This study suggests that female t-ASM mice represent a model of depression with comorbid anxiety and social deficits, without impairments in social and non-social memory.


Binge drinking (BD), a harmful pattern of alcohol consumption, is common during adolescence. Young adults with alcohol use disorders exhibit hippocampal alterations and episodic memory deficits. However, it is not known how these difficulties progress in community BD adolescents. Our objective was to analyze the relationship between BD trajectory and verbal episodic memory during the developmental period spanning from adolescence and to early adulthood. An initial sample of 155 male and female first-year university students with no other risk factors were followed over six years. Participants were classified as stable non-BDs, stable BDs and ex-BDs according to the third AUDIT item. At baseline, participants comprised 36 ♂/ 40 ♀ non-BDs (18.58 years), 40 ♂/ 39 ♀ BDs (18.87 years), and at the third follow-up, they comprised 8 ♂/ 8 ♀ stable non-BDs (25.49 years), 2 ♂/ 2 ♀ stable BDs (25.40) and 8 ♂/ 12 ♀ ex-BDs (24.97 years). Episodic memory was assessed four times with the Logical Memory subtest (WMS-III) and the Rey Auditory Verbal Learning Test (RAVLT). Generalized linear mixed models were applied. The results showed that, relative to non-BDs, stable BDs presented difficulties in immediate and delayed recall in the Logical Memory subtest. These difficulties remained stable over time. The short-term ex-BDs continued to display difficulties in immediate and delayed recall in the Logical Memory subtest, but long-term ex-BDs did not. The effects were not influenced by age of alcohol onset, frequency of cannabis use, tobacco use or psychopathological distress. In conclusion, BD during adolescence and young adulthood is associated with episodic memory deficits. Abandoning the BD pattern may lead to partial recovery. These findings are consistent with the vulnerability of the adolescent hippocampus to the neurotoxic effects of alcohol. Alcohol is the most widely used psychoactive drug among young people and its excessive consumption represents a serious health challenge . Binge dThe findings of animal studies indicate that, relative to adult rats, adolescent rats are particularly vulnerable to ethanol neurotoxicity , 11. MosRegarding the hippocampus, recent evidence from animal models has revealed long-term reductions in hippocampal neurogenesis  and persAdolescents in the general population engaging in BD also exhibit episodic memory deficits  and execList-learning tasks such as the RAVLT and story recall tasks such as the Logical memory subtest (WMS-III) are some of the most common verbal memory tasks, but they are not interchangeable as each measures different memory processes. The RAVLT is an unstructured word list that requires implementation of strategies  for good performance, which is associated with the frontal lobe , 41. TheUsing a story-learning paradigm, we observed that BDs performed poorly in immediate and delayed recall of verbal information in the Logical Memory subtest (WMS-III), which is a classic hippocampal-dependent task proposed as the “purest” measure of episodic memory as it showed the lowest degree of overlap with other non-episodic memory measures . The defThe aim of the present study was to elucidate the relationship between BD trajectory and verbal episodic memory during the developmental period spanning from adolescence to early adulthood. For this purpose, we analyzed data from a 6-year longitudinal study, following healthy participants between the ages of 18 and 23 years. In addition, we assessed whether the association between BD trajectory and episodic memory is modulated by sex, age of alcohol use onset, psychopathological symptoms or frequency of cannabis use and tobacco use. We thus tested the following hypotheses: (I) in a list-learning paradigm with non-semantically related words (RAVLT), BDs will show difficulties related to prefrontal dysfunction; (II) in a story-learning paradigm  BDs will show difficulties in verbal episodic memory that are compatible with hippocampal dysfunction; (III) the progression of difficulties related to prefrontal regions will improve over time, whereas difficulties related to medial temporal lobe dysfunctions will deteriorate further or remain stable, suggesting different possible mechanisms underlying neurocognitive damage; and finally, (IV) performance of these tasks will improve in subjects who abandon the BD pattern of consumption, indicating partial recovery.The sample initially comprised 155 healthy university students . To select the sample, an anonymous questionnaire, including the Alcohol Use Disorders Identification Test (AUDIT)  and otheParticipants were followed up during a 6-year period (between ages of 18 and 23 years), and four evaluations were made on average every 22 months. As the objective was to assess the effects of the BD trajectory, participants were classified as stable non-BDs , stable BDs (who continued as BDs throughout the 6 years) and ex-BDs . The ex-BDs were further classified into two trajectories: short-term ex-BDs  and long-term ex-BDs  to examine any changes related to the length of time without BD episodes. The non-BDs were defined as those who never consumed 6 drinks on one occasion (or less than monthly) and who consumed alcohol at a speed of two drinks or less per hour. The BDs consumed 6 drinks on one occasion monthly or weekly and the speed of alcohol consumption was three drinks or more per hour. They were excluded if the AUDIT total score was >20. The ex-BDs consumed 6 drinks on one occasion less than monthly and/or they consumed two drinks or less per hour. Abstainers were not included in the sample.The number of participants decreased throughout the study: 155 (76 non-BDs/ 79 BDs) at baseline; 93 (39 stable non-BDs/ 33 stable BDs/ 21 ex-BDs) at the first follow-up; 74 (33 stable non-BDs/ 17 stable BDs/ 24 ex-BDs) at the second and 40 (16 stable non-BDs/ 4 stable BDs/ 20 ex-BDs) at the final follow-up. The total number of data points in each trajectory was 164 stable non-BDs; 133 stable-BDs and 65 ex-BDs (grouped as 33 short-term ex-BDs and 32 long-term ex-BDs). The classification criteria did not allow transitions in the trajectories . Given that this classification implies sample attrition over time, we performed a statistical analysis allowing transitions in consumption trajectory  to ensure that this method of classification did not have any relevant influence on the results. In other words, the statistical model considered the specific pattern of consumption at each time point. The results obtained were almost identical. We used the stable group classification, for the sake of simplicity.The following exclusion criteria were considered in each of the evaluations to reduce relevant confounding factors: history of neurological disorders; psychiatric disorders (DSM-IV-TR), including attention-deficit hyperactivity disorder, conduct disorder or previous diagnosis of depression or anxiety; current psychopathological symptoms as assessed by the Symptom Checklist-90-R (SCL-90-R)   . This taLogical Memory subtest  . In thislme4 package  10% risk) than in immediate trials ([1/0.94] 6% risk). The delayed recall of stories in stable BDs was 10% lower than in non-BDs, and it could be up to 15% lower . The groups did not differ in their responses in the recognition trials.The relationship between trajectory of consumption and episodic memory performance is summarised in p = .158; SidakSD p = .821). Furthermore, the ex-BDs did not differ from the stable non-BDs in immediate recall in the Logical memory test  , However, ex-BDs continued to show deficits  in delayed recall   see . The ex-The sex x group interaction effect was not significant. We examined a possible dose-response effect by considering the speed of consumption (number of drinks per hour) and the effect of BD frequency (item 3 of the AUDIT). Neither of these variables were associated with poorer neuropsychological performance within the stable BDs. Age of drinking onset, frequency of cannabis use and frequency of tobacco use, psychopathological symptoms (GSI score of SCL-90-R) were controlled for, although none of the variables contributed significantly to explain the performance.p< .001). No other significant changes were observed for the other variables in this task in the BDs. In the Logical Memory subtest, there were no significant changes in immediate and delayed recall in stable BDs over time.Stable BDs did not show any significant changes over time in the risk of committing intrusion errors in the RAVLT . Howeverp = .046). In this group, the performance in the Logical Memory did not present any significant variations throughout the study.The average performance of the stable non-BDs group did not differ significantly over time in the RAVLT. Stable non-BDs performed slightly better in delayed recall of stories at the second follow-up than at baseline  relative to the stable non-BDs, whereas long-term ex-BDs performed similarly to the stable non-BDs . Short-term ex-BDs exhibited difficulties in delayed recall ,  while long-term ex-BDs did not show significant difficulties in the same variable , although the difference was almost statistically significant. There were no statistically significant differences between short-term and long-term ex-BDs or relative to BDs in any of the variables.We grouped the ex-BDs into two categories to determine the effect of abandoning the BD pattern of alcohol consumption: short-term ex-BDs and long-term ex-BDs . NeitherThis study examined the relationship between episodic memory and BD trajectory during adolescence and early adulthood (age 18–23 years) in university students with no other risk factors. We first hypothesized that stable BDs would show executive deficits in a list-learning paradigm with non-semantically related words (RAVLT). Stable BDdid not exhibit difficulties in the RAVLT in learning, immediate or delayed recall of words or recognition. However, stable BDs tended to commit more intrusion errors, which may reflect impairment of self-monitoring functions , 54.In a study using a long-term verbal learning task consisting of recalling words from a list of 36 non-semantically related words after a period of 25 minutes, Hartley et al.,  did not We also expected that stable BDs would show difficulties in verbal episodic memory in a story-learning task, compatible with hippocampal dysfunction. In this regard, we found that a stable BD pattern of alcohol consumption is associated with difficulties in recalling stories in the Logical Memory subtest, which has been shown to depend on the left hippocampus . Stable It is widely recognized that chronic alcohol abuse and that reduced hippocampal volume , 12 are Regarding the progression of difficulties, we hypothesized two different trends suggesting different underlying mechanisms. On the one hand, previous studies with part of this sample , 39 showp = .052) in this group. Short-term and long-term ex-BDs performed similarly in immediate and delayed recall and no differences were observed in recalling of stories when both were compared with stable BDs. The findings indicate that consolidation deficits  are particularly resistant to improvement, as long-term BDs displayed a position in performance between non-BDs and BDs but apparently closer to the last group. Overall, the results suggest that verbal episodic deficits continue to occur in the short-term and partial degree of recovery may be observed in the long term for encoding and to a less extent for consolidation of verbal information. Winward et al., [Finally, improved performance was expected in young adults who abandoned the binge drinking pattern, indicating partial recovery. The results showed that relative to stable non-BDs, ex-BDs—both long and short term—did not display executive difficulties. In episodic memory, short-term ex-BDs still had difficulties in immediate (encoding) and delayed (consolidation) recall of stories, relative to stable non-BDs. However, long-term ex-BDs did not show any difficulties in episodic memory relative to stable BDs, although for consolidation the risk of presenting difficulties was almost significant  are less affected by this limitation, as a greater number of data points are included. In addition, these models also consider the response correlation in repeated measures (such as correlated measurement errors and participant´s heterogeneity), resulting in greater statistical power . One posAnother potential limitation of this study is the fact that we did not carry out any neuropsychological assessment before the initiation of BD, which may limit the interpretation of executive deficits, as explained before. However, the age of drinking onset, which constitutes a retrospective factor, did not play a role in explaining the neuropsychological performance in the present study. Regarding hippocampal-related difficulties, participants who abandon the BD seem to show some degree of recovery in the long term, possibly suggesting that the difficulties are a consequence of BD rather a pre-existing feature. In line with this interpretation, a neuroimaging study found that hippocampal volume before the start of alcohol consumption in adolescents (12–14 years old) did not predict AUD four years later . These fWe report the finding of intrusion errors with caution. We have followed a conservative approach and corrected for multiple comparisons (SidakSD), which resulted in a nonsignificant p-value for this variable. Nonetheless, a greater number of intrusion errors in a healthy population with no other risk factors is of clinical relevance and is also consistent with previous findings. Moreover, correcting variables that may be related to different processes  may lead to an overcorrection (Type II error). As a final consideration, no alternative versions were used in the Logical Memory test. Even though there were large intervals between assessments, some minimal practice effects may have influenced the absence of any changes in performance (no further worsening) over time in BDs.In summary, maintaining a BD pattern of alcohol consumption from adolescence through to early adulthood is associated with episodic deficits—especially in memory consolidation—together with possible executive difficulties that also contribute to poor verbal memory performance in healthy university students. Memory plays a crucial role in life, but is particularly important during college years when difficulties in memorizing verbal information may have a great impact on academic life. Abandoning the BD pattern may lead to partial recovery. Nevertheless, there is a pressing need to integrate the scientific evidence to strengthen prevention programmes that raise awareness of the cognitive impacts of BD, principally among university students.


The primary aim was to investigate the role of underlying heart disease on preoperative NT-proBNP levels in patients admitted for adult cardiac surgery, after adjusting for the known confounders age, gender, obesity and renal function. The second aim was to investigate the predictive value of preoperative NT-proBNP with regard to severe postoperative heart failure (SPHF) and postoperative mortality.A retrospective cohort study based on preoperative NT-proBNP measurements in an unselected cohort including all patients undergoing first time surgery for coronary artery disease , aortic stenosis  or mitral regurgitation  from April 2010 to August 2016 in the southeast region of Sweden (n = 2978). Concomitant procedures were not included, with the exception of Maze or tricuspid valve procedures.Preoperative NT-proBNP was 1.67 times (p<0.0001) and 1.41 times (p<0.0001) higher in patients with AS or MR respectively, than in patients with CAD after adjusting for confounders.NT-proBNP demonstrated significant discrimination with regard to SPHF in CAD , MR  and AS . In CAD patients NT-proBNP demonstrated significant discrimination with regard to postoperative 30-day or in-hospital mortality . The number of deaths was too few in the AS and MR group to permit analysis. Elevated NT-proBNP emerged as an independent risk factor for SPHF, and postoperative mortality in CAD.Patients with AS or MR have higher preoperative NT-proBNP than CAD patients even after adjusting for confounders. The predictive value of NT-proBNP with regard to SPHF was confirmed in CAD and MR patients but was less convincing in AS patients. Postoperative heart failure (PHF) remains the major cause of mortality after cardiac surgery –6. The mA limited number of studies have reported that preoperative natriuretic peptide levels differ between patients admitted for coronary artery bypass surgery (CABG), aortic valve replacement (AVR) and mitral valve surgery –17. HoweThe primary aim of this study was to investigate the impact of CAD, AS, and MR on NT-proBNP levels in patients admitted for surgery after adjusting for known confounders not directly related to the heart disease. The second aim was to investigate the predictive value of preoperative NT-proBNP in these cohorts with regard to severe postoperative heart failure (SPHF) and postoperative mortality.Ethical approval (Dnr 2011/ 497–31) was provided for the study by the Regional Ethical Review Board in Linköping, Sweden. The study was performed according to the Helsinki Declaration of Human Rights. Owing to the nature of the study, the ethics committee waived the need for written informed consent.The University Hospital in Linköping is the only referral center in the southeast region of Sweden, serving a population of approximately 1 million. The decision to perform surgery is based on current guidelines , 24. FroThis was a retrospective cohort study. NT-proBNP was routinely measured the day before surgery in elective patients and on the day of surgery in emergency patients. Blood samples were collected in lithium heparin tubes and analyzed within 1 hour (emergency) to 3 hours (elective patients). NT-proBNP was measured in plasma by electro-chemoiluminescence immunoassay on a Roche Elecsys 2010 automated platform  at Linköping University Hospital. The assay had an effective measuring range of 5–35 000 ng/L. The inter-assay coefficient of variation was at 175 ng/L CV = 2.7%, 355 ng/l CV = 2.4% and 1068 ng/L CV = 1.9%. The following upper reference limits (URLs) were applied: 450 ng/L for <50 years, 900 ng/L for 50–75 years, and 1800 ng/L for >75 years. Values < 300 ng/L were considered normal in all age groups and the intervals between 300 ng/Land the URL for the age group were considered a grey zone , 26.Demographic and perioperative data were registered prospectively in a computerized institutional database . All fields were defined in a data dictionary. The variables included in our analysis are shown in the supplementary data . With reThe primary endpoint was preoperative NT-proBNP level in CAD, AS and MR patients after adjusting for the known confounders age, gender, obesity and renal function. The secondary endpoint was the predictive value of preoperative NT-proBNP in CAD, AS and MR patients with regard to SPHF and postoperative 30-day or in-hospital mortality.Emergency operation was defined as a procedure that could not be postponed until the following day and, therefore, was usually performed immediately but not later than 24 hours from acceptance.Preoperative left ventricular (LV) systolic function was assessed by cardiologists dedicated to echocardiography and categorized as normal, mildly depressed, moderately depressed, or severely depressed systolic LV function. Moderate or severe LV dysfunction corresponds to a LV ejection fraction of ≤ 45%. Severe LV dysfunction corresponds to a LV ejection fraction ≤ 30%.Preoperative congestive heart failure was considered in patients with ongoing pharmacological treatment for heart failure who received the diagnosis from a cardiologist.Pulmonary hypertension was defined by a systolic pulmonary artery pressure > 60 mm Hg.Critical preoperative state was defined as ventricular tachycardia, ventricular fibrillation, or cardiopulmonary resuscitation; the need for preoperative ventilator treatment before admission to the operating room; need for preoperative inotropes or IABP; or preoperative acute renal failure (anuria or oliguria < 10ml/h).-1 min-1; need for two inotropes at any dosage; or use of levosimendan at any dosage).Patients were considered to have SPHF if supported by clinical diagnosis in the medical records and/or echocardiographic signs of heart failure and an ICU stay ≥ 72 hours or hospital mortality with one of the following: intra-aortic balloon pump or ventricular assist device, or the use of inotropes . The Mann-Whitney U test was used to compare continuous variables between two groups. The Kruskal-Wallis test and pairwise comparison test were used to compare continuous variables among patients with CAD, AS and MR. The Pearson Chi-square test was used to compare proportions and the Bonferroni correction was used to account for multiple testing. Preoperative NT-proBNP was log10 NT-proBNP was used, adjusting for the known confounders age, renal function, gender and obesity. A multivariable linear regression model was also used to assess the role of underlying heart disease with regard to log10 NT-proBNP, taking into account both the diagnosis and all available data, including the consequences of the disease. Finally, a multivariable linear regression model was used separately in patients with CAD, AS, and MR to determine variables independently associated with log10 NT-proBNP within these diagnostic groups. Potential variables were assessed by guidance of univariate analysis (p<0.25). Collinearity diagnostics were performed using variance inflation factors to exclude unacceptable collinearity between selected variables.To assess the role of underlying heart disease  on NT-proBNP levels at admission to surgery, a multivariable linear regression model with regard to logA receiver operating characteristic (ROC) analysis was performed to calculate the area under the curve (AUC) in order to evaluate the discrimination of preoperative NT-proBNP with regard to SPHF and postoperative mortality. Youden´s index was used to calculate the best cutoff point with regard to sensitivity and specificity. Furthermore, the cutoff point providing a similar sensitivity and specificity in each diagnostic group at or just above 75% was calculated. Univariate preoperative and intra-operative predictors of SPHF and postoperative mortality were analyzed using the Mann-Whitney U-test for non-parametrically distributed continuous data and the Pearson chi-squared test for nominal data. All variables with p < 0.25 were entered into a backward  stepwise multivariable logistic regression to identify preoperative and intra-operative risk factors for SPHF and postoperative mortality. Hosmer-Lemeshow goodness-of-fit statistics were calculated for the final model. Statistical significance was defined as p < 0.05. Statistical analyses were performed in SPSS statistics version 23 (IBM) for Windows and Statistica 13.2 (Dell Inc.).Among all 2978 patients, the median age was 70 63–76] years and 24% were female. The median Additive EuroSCORE was 4[–76 yearsThe distribution of preoperative NT-proBNP of the whole cohort exhibited a positive skewness with a median value of 330 [130–970] ng/L. NT-proBNP was higher in patients with AS than in patients with CAD  or patients with MR . The proportion of patients with NT-proBNP values greater than the age-adjusted URL was higher in patients with AS (32%) or MR (30%) than in patients with CAD (20%). The proportion of patients with NT-proBNP < 300 ng/L was 50% for CAD, 43% for MR, and 30% for AS .After adjusting for age, eGFR, female gender, and obesity, NT-proBNP was 1.67 times higher in patients with AS than in patients with CAD  and 1.41 times higher in patients with MR than patients with CAD . Patients with SPHF had significantly higher level of preoperative NT-proBNP than patients without SPHF in the whole cohort and in the subgroups with CAD, MR, or AS .Preoperative NT-proBNP demonstrated significant discriminatory power with regard to SPHF in patients with CAD , MR  and AS . Age, preoperative dialysis, aortic cross-clamp time in upper quartile (≥ 62min), moderate to severe LV dysfunction, NYHA IV, insulin-treated diabetes, critical preoperative state, and emergency operation were the other variables in the final model . The numFifty-three (2%) patients died postoperatively within 30 days or in-hospital; 43 due to cardiac causes, 3 due to postoperative stroke, and 6 due to other non-cardiac causes, and cause of death was unknown in 2 patients. Further details are given in the supplementary data . PatientIn CAD patients, preoperative NT-proBNP demonstrated significant discrimination with regard to postoperative mortality . Age, NYHA IV, preoperative albumin, preoperative dialysis, and emergency operation also remained in the final model for postoperative mortality .The main and novel finding of this study was that the level of preoperative NT-proBNP in patients with AS or MR was higher than in CAD patients, even after adjusting for confounders. In addition, the predictive value of preoperative NT-proBNP with regard to SPHF was good in CAD and MR patients but less convincing in AS patients. In CAD patients, NT-proBNP had good predictive value with regard to postoperative mortality, whereas the number of deaths was too few to permit analysis in AS and MR. Furthermore, preoperative NT-proBNP emerged as an independent risk factor for SPHF and postoperative mortality in CAD patients.The primary aim of this study was to determine the impact of underlying heart disease  per se on preoperative NT-proBNP levels in patients admitted for cardiac surgery. Among the studied groups, patients with AS had the highest level of preoperative NT-proBNP, whereas patients with CAD had the lowest values. These findings match those of previous studies , 27–31. The link between natriuretic peptides and valvular heart disease has received more attention as echocardiographic findings are easier to associate with natriuretic peptide levels. In MR patients, BNP reflects the hemodynamic consequences of MR, rather than the severity of MR itself . In asymSeveral studies have shown that high levels of preoperative natriuretic peptides are associated with postoperative heart failure, need for inotropic support, in-hospital cardiac events, and postoperative mortality after cardiac surgery –13. The The predictive value of preoperative NT-proBNP with regard to SPHF was less convincing in AS patients. This finding does not support the previous research by Fellahi et al on the prognostic utility of BNP in predicting major adverse cardiac events (MACEs), that apart from heart failure included malignant ventricular arrhythmias, Q-wave infarction, and repeat revascularization, during the first 12 months following CABG or AVR. Fellahi et al reported that preoperative BNP was accurate in predicting MACEs after AVR but not after CABG. PossiblROC analysis revealed a best cutoff for preoperative NT-proBNP of 905 ng/L with regard to postoperative mortality in patients with CAD. This finding is in agreement with a prospective study from our institution on a smaller sample (n = 366) undergoing isolated CABG for acute coronary syndrome, which found the best cutoff for hospital mortality to be 1028 ng/L . SchachnThe indications and timing of surgery will obviously influence the level of natriuretic peptides. In Sweden health care is available for everyone regardless of the patient’s financial situation and the availability of cardiac surgical resources permits most patients to be treated according to current guidelines , 24. ThiThe major limitations of this study are its retrospective nature and that analyses were limited by the data available in our database. For example, data on confounders such as inflammation and pharmacological treatment were unavailable , 42. On Patients with AS or MR admitted for first time cardiac surgery have higher preoperative NT-proBNP levels than CAD patients, even after adjusting for confounders. The predictive value with regard to severe postoperative heart failure was good in CAD and MR patients, but less convincing in AS patients. The predictive value of NT-proBNP with regard to postoperative mortality was confirmed in CAD patients whereas the number of events was too few in the other subgroups.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Under laboratory-conditions, participants performed 16 different activities  for 5 minutes each. ENMO and MAD were computed from the raw acceleration data, and logistic regression and receiver-operating-characteristic (ROC) analyses were implemented to derive thresholds for activity discrimination. Areas under ROC curves (AUROC) were calculated to summarise performances and thresholds were assessed via executing leave-one-out-cross-validations.Thirty-three adults [mean age (standard deviation (SD)) = 27.4 (5.9) years; mean BMI (SD) = 23.9 (3.7) kg/mFor both hip and wrist monitor placements, in comparison to the ActiGraph GT3X+ monitors, the ENMO and MAD values derived from the GENEActiv devices were observed to be slightly higher, particularly for the lower-intensity activities. Monitor-specific hip and wrist ENMO and MAD thresholds showed excellent ability for separating sedentary behaviours from motion-based light-intensity physical activities , with validation indicating robustness. However, poor classification was experienced when attempting to isolate standing still from sedentary behaviours . The ENMO and MAD metrics tended to perform similarly across activities and accelerometer brands.Researchers can utilise these robust monitor-specific hip and wrist ENMO and MAD thresholds, in order to accurately separate sedentary behaviours from common motion-based light-intensity physical activities. However, caution should be taken if isolating sedentary behaviours from standing is of particular interest. There is cumulative evidence that sedentary behaviour, characterised as any waking behaviour with low energy expenditure  while in a sitting or reclining posture –7. This Accelerometers have historically provided data in the form of ‘counts’—an aggregate measure of the intensity and magnitude of accelerations over a given time epoch , 19. CouThe challenge of analysing raw signals revolves around several factors: the management of vast amount of data which are generated; the requirement to remove the gravitational and noise components incorporated within the signals ; and theTo our knowledge, only three studies have methodically investigated the use of the MAD metric with raw acceleration data relating to physical activity –33. VähäAccelerometers were traditionally worn on the hip, however, in recent years wrist-worn accelerometry has emerged and is now also being used in large national health surveys  extend the premise of the ENMO and MAD metrics via developing internally-validated monitor-specific intensity-based thresholds for discriminating between sedentary behaviours and common light-intensity physical activities; and (2) compare and evaluate the performances between the ENMO and MAD metrics. The generation of ENMO and MAD thresholds developed will be sample and protocol specific. Hence, the classifications of sedentary behaviours and light-intensity physical activities should be broadly comparable between studies—irrespective of the metric, accelerometer brand and wear-site used.Investigations were carried out using data from a laboratory-based study which was conducted by the National Institute for Health Research (NIHR) Leicester-Loughborough Diet, Lifestyle and Physical Activity Biomedical Research Unit (BRU). The study was implemented within a bespoke laboratory, located at Loughborough University , which was furnished to enable the sedentary and non-sedentary tasks to be undertaken efficiently. Participants (aged ≥ 18 years) were recruited via email and word of mouth. All participants provided written informed consent, and the study was approved by the Ethics Committee of Loughborough University.g, sampling frequency range: 30–100 Hz ) and the GENEActiv Original monitor ; where g is equal to the Earth’s gravitational pull. The ActiGraph GT3X+ was initialised with a sampling frequency of 100 Hz using ActiLife software V6.10.2. The GENEActiv monitor was initialised with a sampling frequency of 100 Hz using the GENEActiv PC software V2.2. Both devices were initialised using the same computer.Two distinct and commercially available tri-axial accelerometers were utilised: the ActiGraph GT3X+ monitor  and wrist (non-dominant). Consequently, a total of 4 devices were worn by each participant  whilst they performed 16 different activities  in a sequential order for 5 minutes each under laboratory-conditions. The start and end time of each activity was observed (using a clock on a computer) and recorded onto a log sheet. A 30 second break was allocated between each activity. The raw acceleration data from the two ActiGraph GT3X+  and two GENEActiv  devices were downloaded using ActiLife V6.10.2 and GENEActiv PC software V2.2, respectively. For the computation of the MAD metric, the four sets of raw acceleration files were converted to time-stamped .csv files, which were then exported into Stata/IC V13.1  for processing and analysis. The laboratory log sheets (with the observed start and end times of each activity) were utilised for identifying each activity in the time-stamped .csv files.where;MAD is defined as:n = length of the time periodx, y, z) of the raw tri-axial data were first multiplied by 1000 to transform the signals from gravitational units into milligravitational (mg) units. This was implemented in order to ensure that the developed thresholds would be comparable with the prior findings in the literature . The sample consisted of 33 participants [mean age (standard deviation) = 27.4 (5.9) years; mean BMI (standard deviation) = 23.9 (3.7) kg/mTables This is the first methodological study to develop and validate ENMO and MAD intensity-based thresholds for differentiating between sedentary behaviours and common light-intensity physical activities using raw acceleration data collected from both hip- and wrist-worn ActiGraph GT3X+ and GENEActiv tri-axial accelerometers.The monitor-specific hip and wrist ENMO and MAD thresholds showed excellent ability for differentiating between the sedentary behaviours and motion-based light-intensity physical activities . Poor classification was experienced when attempting to isolate standing still from sedentary behaviours. However, these findings are as expected since the magnitude of the acceleration signals is very similar when lying/sitting or standing still, and in order to accurately discriminate between postures, more features of the acceleration signal  need to be considered , 38, 39.Hildebrand and colleagues  recentlyFor both hip and wrist monitor placements, in comparison to the ActiGraph GT3X+ monitors, the ENMO and MAD values derived from the GENEActiv devices were observed to be slightly higher, particularly for the lower-intensity activities. A greater magnitude of accelerations from the GENEActiv has been previously reported when conducting time domain analyses of raw acceleration data using these two accelerometers , 23. ExtEssentially, both the ENMO and MAD metrics offer simple ways of correcting for the gravitational component. Bassett and colleagues recommend that any newly proposed raw acceleration system should be compared to the ones already in use . AlthougOur study has several strengths and some limitations. Strengths include; utilisation of widely-used accelerometers; comprehensive data analysis of raw acceleration data collected at a high sampling frequency; laboratory-based experimental design with usage of both hip- and wrist-worn devices; robust statistical analysis; and the generation of monitor-specific thresholds that distinguish between sedentary and non-sedentary activities. These thresholds are sample and protocol specific, implying that the classifications of sedentary behaviours and light-intensity physical activities should be broadly comparable between studies—irrespective of the metric, accelerometer brand and wear-site used. However, these strengths also have some inherent limitations. The laboratory-based settings may limit generalizability to free-living environments. In particular, it may be more difficult to classify sedentary behaviours in free-living situations using wrist-worn devices since individuals can carry out sedentary tasks involving arm movement as highlighted in this study (e.g. using a computer and a mobile phone whilst in a seated position). Secondly, with the participants instructed to keep their hands on their thighs during the seated activities that involved watching TV (sitting activities 5–9), the real-life positioning and postures of these sedentary behaviours are less likely to be reflected or fully captured. Nevertheless, for both the accelerometer brands, determining the wrist ENMO and MAD thresholds using computer activity as the only sedentary behaviour did not affect the performance or value of the thresholds ). Furthermore, the limited range of sedentary behaviours and light-intensity physical activities can be considered as a weakness of the study. The inclusion of additional activities, such as eating, reading a book or using a mobile phone whilst standing, would have added to the strengths. Lastly, although the thresholds in this study were validated, they were only done so internally. Therefore, it is desirable to cross-validate all available ENMO and MAD thresholds externally; the performance of algorithms developed in laboratory conditions attenuates when applied in field environments .In conclusion, the ENMO and MAD metrics are accessible and increasingly used approaches for analysing hip- and wrist-worn raw accelerometer data, however, well-developed and validated methods utilising outputs from these methods are sparse. Our study provides comprehensible monitor-specific hip and wrist ENMO and MAD thresholds for analysing raw acceleration data, particularly for researchers interested in sedentary behaviour and light-intensity movement. Users can exploit these robust ENMO and MAD thresholds in order to accurately separate sedentary behaviours from common motion-based light-intensity physical activities. However, caution should be taken if separating sedentary behaviours from standing is of specific interest.In terms of making recommendations for future research, due to its’ proven competency and continuous use in the field, this study further supports the use of sensor calibrated ENMO, a well-performing metric which is emerging as the prototypical tool for the analysis of raw acceleration data, to help promote comparability between studies. Nevertheless, the MAD metric also offers an alternative, robust and straightforward technique for analysing raw acceleration data; however, more studies further exploring the MAD metric are required.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.