Nonalcoholic steatohepatitis (NASH) is a condition which can progress to cirrhosis and hepatocellular carcinoma. Markers for NASH diagnosis are still lacking. We performed a comprehensive lipidomic analysis on human liver biopsies including normal liver, nonalcoholic fatty liver and NASH. Random forests-based machine learning approach allowed characterizing a signature of 32 lipids discriminating NASH with 100% sensitivity and specificity. Furthermore, we validated this signature in an independent group of NASH patients. Then, metabolism dysregulations were investigated in both patients and murine models. Alterations of elongase and desaturase activities were observed along the fatty acid synthesis pathway. The decreased activity of the desaturase FADS1 appeared as a bottleneck, leading upstream to an accumulation of fatty acids and downstream to a deficiency of long-chain fatty acids resulting to impaired phospholipid synthesis. In NASH, mass spectrometry imaging on tissue section revealed the spreading into the hepatic parenchyma of selectively accumulated fatty acids. Such lipids constituted a highly toxic mixture to human hepatocytes. In conclusion, this study characterized a specific and sensitive lipid signature of NASH and positioned FADS1 as a significant player in accumulating toxic lipids during NASH progression. Nonalcoholic fatty liver disease (NAFLD) is a pathological condition involving a broad spectrum of lesions ranging from nonalcoholic fatty liver (NAFL) so-called steatosis to nonalcoholic steatohepatitis (NASH). It has been established that NASH may progress to hepatic fibrosis, cirrhosis and hepatocellular carcinoma15The hallmark of fatty liver disease is an intracellular accumulation of lipids, and particularly triglycerides, which causes the formation of lipid droplets in hepatocytes. This accumulation results from an imbalance between the uptake, synthesis, export and oxidation of fatty acids8712131516In this study, we have performed a comparative lipidomic analysis on human liver biopsies from patients with NAFL or NASH. We employed the unbiased mathematical approach that we recently implemented on animal models. Indeed, we have demonstrated that a random forests-based mathematical approach was enabled to characterize hepatic lipid signature specific to NASH in murine modelsA quantitative lipidomic analysis was performed in order to identify lipids discriminating the pathological statuses of the liver. The study was realized on 61 liver biopsies including normal livers as controls (n = 7), nonalcoholic fatty livers  and nonalcoholic steatohepatitis , from the same hospital  . Lipids First, we sought to distinguish different grades of steatosis among the 39 liver biopsies from NAFL. Thus, in order to avoid any bias related to an imbalance and to assure a homogenous repartition of patients between NAFL groups, we employed the classification and regression trees (CART) analysis. Indeed, we have previously demonstrated by using this approach that the total amount of triglycerides (TG) allowed the unbiased discrimination of the grade of steatosis15212223Investigations were further conducted using a random forest-based machine learning approach. This unbiased statistical analysis allows comparing several groups -each exhibiting hundreds variables- thus leading to characterize the predictor variables of a given status18The study was further focused on the validation of the lipid signature of NASH. Additional liver biopsies from 7 patients with NASH called “validation data set” (NASH_Vds) were obtained from another hospital . The characteristics of the study groups were summarized in Among the 32 discriminant lipids, 9 lipids were significantly decreased in NASH, mainly ceramides and phospholipids. On the other hand, 23 lipids exhibited a significant increase in NAFL and/or NASH, including 6 fatty acids, 2 cholesteryl esters, 1 diglyceride, 5 triglycerides and 9 phospholipids .Altogether, these results demonstrated that the random forests mathematical approach allowed characterizing a lipid signature of the grades of fatty liver diseases discriminating NASH patients with 100% accuracy.The metabolic pathways deregulated in NASH were investigated. Interestingly, the 6 fatty acids increased in NASH  belong to the LCFA synthesis pathway . Thus, iFirst, we focused on the two main elongases named ELOVL (elongation of very long chain fatty acids), ELOVL5 and ELOVL6. ELOVL5 activity was significantly increased , whereasThe decrease in FADS1 desaturase activity in NASH may constitute a bottleneck leading to the accumulation of fatty acids upstream. As a consequence, the synthesis of lipids downstream of this enzyme such as the eicosanoid precursors (arachidonic acid (C20:4n-6), eicosapentaenoic acid (C20:5n-3) and docosahexaenoic acid (C22:6n-3)) exhibited a significant decrease in livers of NASH compared to controls . It shouELOVL5, ELOVL6, FADS1, FADS2 and SCD1 were investigated by RT-Q-PCR on NASH compared to normal livers and NAFL matched NASH patients. To avoid any variations of mRNA gene expression levels due to the age, gender, BMI and steatosis grade (i.e. the amount of lipid content), patients from NAFL2 group were matched to NASH patients , fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1)  . As prevto NAFL2 . Interes in NASH  and h.de novo short-chain fatty acids synthesis and in FADS2 and SCD1 activities associated to a decrease in ELOVL6 and FADS1 activities. These results also positioned FADS1 as a bottleneck leading upstream to the accumulation of LCFA, and downstream to the deficiency in VLCFA and thus in phospholipids synthesis.Altogether, these results highlighted the major dysregulation of fatty acid synthesis pathway in NASH. Changes in lipid composition in NASH resulted of the additional effect of increase in The metabolic features observed in human were investigated using animal models. NAFL and NASH can be induced in mice by using specific high-fat diet (HFD) and methionine-choline deficient diet (MCDD), respectively1434353637These observations demonstrated the common metabolic dysregulation along the fatty acid synthesis pathway in both human patients and animal models, leading to the development of NASH.NAFL3 = 2.25 × 105 vs TCNASH = 2.37 × 105), thus strengthening a real difference in terms of distribution.A major feature of NASH revealed by our lipidomic analysis was the failure in total phospholipids . The lowThese results suggested a disruption of cell membrane integrity in NASH, most likely due to a defect in phospholipids, leading to a leak and spreading hepatocyte content out into the parenchyma.The toxicity of the lipids identified in NASH was addressed. Studies were focused on 5 fatty acids accumulated in NASH and available for cell culture. Toxicity of myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1n-7), vaccenic acid (C18:1n-7) and oleic acid (C18:1n-9) were investigated in cell culture on HepG2 cells and human primary hepatocytes (HPH). Lipotoxicity was first addressed for each individual lipid at various concentrations . HepG2 cell line and HPH showed different sensitivities to lipids most likely due to differences in the lipid metabolism of such cellsThese results demonstrated the potent toxicity of the specific mixture of lipids accumulated in NASH.2), severe (between 35 and 40 kg/m2) or morbid (>40 kg/m2) obesity associated or not with type II diabetes assessed by the homeostasis model assessment of insulino-resistance (“low-HOMA-IR” or “High-HOMA-IR”), and/or a metabolic syndrome. In order to find universal lipid markers for the NASH, we intentionally not focused in patients’ medications or systemic complications such as type II diabetes and insulin-resistance as well as their polymorphisms or even gender. Difference in female gender distribution between groups of patients was not linked to the progression of the steatohepatitis but due to the selection between both hospitals due to their specificities (i.e. liver diseases and liver transplantation vs bariatric surgery). Also, the role between genders and steatohepatitis progression is not elucidated yet841The aim of this study was to characterize lipid markers specific to all patients with NASH independently of patient’s background such as mild .Here, we used an innovative unbiased random forests-machine learning statistical analysisi.e. ballooning and Mallory’s hyaline bodies). Therefore, this specific lipid signature is able to discriminate between both steatosis and steatohepatitis.For the first time, this study established a lipid signature of nonalcoholic steatohepatitis based on the quantification of 32 lipids. Such complex signature highlighted the major interest of combining a global approach such as lipidomic with an unbiased RF analysis. Indeed, none of the lipids identified allowed by itself the discrimination of NAFL or NASH, but in contrast the overall lipid signature allowed discriminating between control patients, NAFL groups and NASH patients. The robustness of the lipid signature of NASH was underlined by 100% specificity and 100% sensitivity on samples from two independent hospital centers. Indeed, using two independent groups of patients with NASH from these hospitals emphasizes the sturdiness and the universality of the lipid signature of NASH. Moreover, the main difficulty for a pathologist is to discriminate between steatosis and steatohepatitis, especially before the inflammation and hepatocyte degenerations appeared (i.e. dysregulation of enzyme activities) observed in both human and animal models for this pathology, strengthening the idea that these alterations are universal in NASH. Indeed, we used animal models that developed fatty liver when fed on high fat diet or NASH when fed on methionine choline deficient diet. It should be noted that MCDD mouse model is the most well-known and used model to study NASH182134353637Dysregulations of the metabolic pathway involved in synthesis of fatty acids were highlighted in NASH. The major impact of alterations in this metabolic pathway was reinforced by the similar biochemical features . Recently, it has been demonstrated that fatty acid metabolism was altered in NASH independent of obesity and diet2645464849The concomitant increase of saturated and unsaturated LCFA with the significant decrease of polyunsaturated VLCFA resulted from the decreased activity of FADS1. Impairment of FADS1 activity has created a bottleneck leading to the accumulation upstream of fatty acids up to 20 carbons as also described beforeThus, due to the impairment in FADS1 activity, the deficiency in phospholipid synthesis can damage the cellular membranes in which phospholipids are major components. Indeed, half of the phospholipids from the signature were significantly down in NASH patients Furthermore, we have shown that a surrogate of membrane integrity the PC to PE ratio was significantly decreased in patients with NASH51By using an original approach combining the five fatty acids from the signature, toxicity of lipids accumulated in NASH was demonstrated on the human hepatoma cell line HepG2 and HPH. Although each of the five fatty acids studied showed toxicity individually1213In conclusion, we clearly characterized a specific and sensitive lipid signature, universal for all patients with NASH. This study highlighted dysregulations of the metabolic pathway involved in the synthesis of fatty acids and eicosanoid precursors. In particular, our study positioned ELOVL6 and FADS1 as major players in the progression to NASH. Finally, the current study suggested also a direct role of lipids accumulated in NASH in the progression of the pathology by their toxicity. This opens new avenues for further development of early diagnosis and therapeutic approaches.A total of 68 patients were enrolled in this study selected from two hospital centers . A pathologist expert (CG) reviewed all liver biopsies. Seven patients had a normal liver forming the control group (hepatic steatosis <5%). Fifty four patients have been recorded as affected with NAFLD. To differentiate NAFL and NASH, a histological discrimination was made based on a separate system of scoring the features of NAFLD called the NAFLD Activity Score (NAS)8ad libitum. Animal protocols were accepted by the Institutional Animal Care and Use Committee in Main (Jackson Laboratories) and by the “Comité d’Ethique pour l’Expérimentation Animale” registered to the “Comité National de Réflexion Ethique sur l’Expérimentation Animale 05” (Protocol # Ce5/2012/075). All experiments were performed in accordance with the relevant guidelines and regulation of each country.Male C57Bl/6J mice were fed on chow diet, HFD and MCDD. Mice fed a HFD and MCDD developed NAFL and NASH, respectively as described in our previous publication®-ND1000 (Thermo Scientific). cDNAs were generated by using the RivertAid® First Strand cDNA Synthesis (Thermo Scientific), and Syber Green from FastStart Essential DNA Green Master mixes  were used to quantify hepatic mRNA levels with specific primers of each gene described in Total RNA was extracted from frozen liver biopsies using RNA-STAT 60 reagent (AMS Biotechnology Europe LTD). Quantity and quality of RNA were assessed using NanoDrop® 96 Instrument . Gene expression levels were normalized to actin RNA levels and data analyzed with LightCycler® 96 SW 1.1 software . For each sample, the gene to actin ratio was calculated based on an arbitrary value of copies determined by the standard curve for each gene, as previously describedQ-RT-PCR was performed using LightCyclerin vitro and in vivo, but for practical and ethical reasons is not possible in human studies. Therefore product-to-precursor ratio as surrogate measure to estimate desaturase and elongase activities is assessed in this study as it has been extensively used in different other studies before17212654As standard method to evaluate fatty acid-synthetizing enzyme activities, assay measurement of the rate of radiolabeled precursor FA to their respective products is used 5 viable cells per well onto 96-well plates that had been pre-coated with a solution type I collagen from calf skin between 1 and 10 hours before plaiting cells. The medium was replaced 16–20 hours later with fresh complete hepatocyte medium supplemented with 1 mol/L hydrocortisone hemi-succinate 56562 atmosphere.Normal liver tissue was obtained from adult patients undergoing partial hepatectomy at Saint Antoine Hospital . The first donor was a 63 years old woman treated for liver metastasis for colorectal adenocarcinoma. The second donor was a 36 years old female treated for hepatocellular carcinoma developed on normal liver. The third patient is a 65 years old man treated for liver metastasis of pancreatic cancer. Experimental procedures were performed in accordance with French laws and regulations. Human primary hepatocytes isolation was made based on previous protocol562 atmosphere.HepG2 cells, derived from differentiated human hepatoblastoma® Luminescent Cell Viability Assay, Promega, France).Myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1n-7), vaccenic acid (C18:1n-7) and oleic acid (C18:1n-9) were all obtained from Sigma . Fatty acids were dissolved in absolute ethanol at a concentration of 40 mM stock solutions, sonicated 15 min and then warmed at 70 °C for 15 min, for complete dissolution. FA solutions were filtered through a 0.22 μm filter before use and stored at −20 °C. Then, FA were dissolved in bovine serum albumin  at the final ratio 1/10 (v/v) in William’s E plus Glutamax™ medium or in OptiMEM™  and warmed again at 55 °C for 10 min before useLiver biopsies (5–10 mg) were homogenized in 2 ml of methanol/EGTA (2:1 v/v) with FAST-PREP  tissue lyser for further lipid analyses. Also, the equivalent of 0.5 mg of tissues was evaporated. The dry pellets were dissolved in 0.25 ml of NaOH (0.1 M) overnight and proteins were measured with the Bio-Rad assay. The quantification of the lipids is expressed in nmol/mg of total proteins.Briefly, lipids were extracted from liver tissues according to Bligh and DyerPhospholipids for relative quantification were extracted as neutral lipids but with 2% acetic acid in the presence of the internals standards (Cer(d18:1/15:0) 16 ng; PE(12:0/12:0) 180 ng; PC(13:0/13:0) 16 ng; SM(d18:1/12:0) 16 ng; PI(16:0/17:0) 30 ng; PS(12:0/12:0) 156.25 ng). After centrifugation the organic phase was collected and dried under azote, then dissolved in 50 μL of methanol. Lipids were separated using a Kinetex HILIC column with a mobile phase A of acetonitrile and B with 10 mM ammonium formate in water at pH 3.2 following a gradient and the injection volume was 5 μL. Sample solutions were analyzed using an Agilent 1290 UPLC system coupled to a G6460 triple quadrupole mass spectrometer (Agilent Technologies) and using MassHunter software (Agilent Technologies) for data acquisition and analysis. Data were treated using QqQ Quantitative (version B.05.00) and Qualitative analysis software (version B.04.00).For Fatty Acid Methyl Ester (FAME) analysis, homogenate were extracted as neutral lipids in the presence of the internal standards glyceryl triheptadecanoate (2 μg) and transmethylated 1 h in boron trifluoride methanol solution 10% at 55 °C. After addition of water (1 ml) to the crude, FAMEs were extracted with hexane (3 ml), evaporated to dryness and dissolved in ethyl acetate (20 μl). FAME (1 μl) was analyzed by gas-liquid chromatography on a Clarus 600 Perkin Elmer system using Famewax RESTEK fused silica capillary columns.A subset group of patients with the four grades of liver steatosis (3 patients per group) and a group of patients with NASH (4 patients) underwent for ToF-SIMS imaging analyses.®, Kevley Technologies, OI, US). Before analysis, tissue sections were placed under vacuum at a pressure of a few hPa during 10 min in order to eliminate water. Before analysis, tissue samples were examined and optical images were recorded with a microscope  equipped with a ColorView I camera monitored by CellB software . No further sample preparation was required before introduction in the mass spectrometer.Briefly, each sample was cut at −20 °C with a CM3050-S cryostat . Tissue sections of 10 μm thickness were deposited on a gold coated glass slide , equipped with a Liquid Metal Ion Gun (LMIG) filled with bismuth and allowing delivery of Bi11 ions/cm2 for all the acquisitions, allowing acquisition time of about 10 minutes. Each area was scanned twice in order to record both positive and negative ion images.Briefly, a set of images was acquired without sample stage movement, just by resting the primary ion beam, with a field of view of 500 μm × 500 μm. For these images, the number of pixels was chosen as 256 × 256 to obtain a ~2 μm pixel size. Under these conditions the flow was fixed to 3 × 10m/z is always linear over the whole mass range. Consequently, the mass calibration was made with H−, C−, CH−, CH2−, CH3−, C2−, C3−, and C4H− ions for the negative ion mode, and H+, H2+, H3+, C+, CH+, CH2+, CH3+ and C2H5+ for the positive ion mode, respectively. To refine the mass calibration, ion peaks of cholesterol and vitamin E were used in positive ion mode, and fatty acid carboxylate ions in negative ion mode based on previous studies reported on biological samples616263Due to the very low initial kinetic energy distribution of the secondary ions, the relationship between the time-of-flight and the square root of Data processing was achieved using Surface Lab 6.2 software . This software allows extraction of ion spectra and images from the raw data. In order to compare the relative intensity of species in the first set of experiments, a normalization of their respective mass spectrum intensities had to be performed: the intensity of the mass spectrum from each stage scan was normalized against the area of the smallest one, given that all the data had been acquired under the same experimental conditions616566We selected 3 patients in each group of control, NAFL1, NAFL2 and NAFL3 as well as 4 patients with NASH. The sum of the diacylglycerols (DGs) served to localize the lipid droplets into the liver tissue. C14:0, C16:0, C16:1, C18:0, C18:1, C18:2 and C20:4 were investigated using ToF-SIMS procedure in different areas of the liver. The size of each patch was 500 μm × 500 μmLipidomic data of patients with NASH from Paul Brousse Hospital were used as learning dataset (NASH_Lds) whereas patients with NASH L’Archet Hospital were used as validation dataset (NASH_Vds).2(v) = 0.25), α = 0.05, power (1-β = 0.95), and the number of groups (n = 6).In order to be sure that the number of patients per group is acceptable for further statistical analyses, we performed a statistical parametric test, MANOVA , using GPower software (version 3.1.9.2) and including the following parameters such as: effect size (frpart”) approach was used to build a regression tree based on the predict values from the lipidomic data leading to obtain a classification analysis and a regression tree (CART). CART was applied on lipid families such as total TG, total DG, Total cholesterol, Total CE, Total SFA, Total USFA, Total MUFA, Total PUFA, Total PC, Total PE, Total PI, Total PS, Total Cer and Total SM  and missing values, but could create a random noise which hided the main effects and the relevant predictorsrandomForest” package in R. To determine the best number of predictors (mtry) was used for each split of the tree and tune RF function was used to determine the lowest mtry to the lowest out-of-bag (OOB) error data that was used to get a running unbiased estimate of the classification error as trees were added to the forest melding to determine the confusion matrix (ntree (number of trees to be built) was set up at 1040 corresponding to the number of the columns (variable) of the matrix multiply by ten. During the analysis, the mean decreased accuracy (MDA) and the mean decreased Giny (MDG) were determined. MDA was determined during the OOB error calculation phase and lipids with a large MDA were more important for classification of the data (In order to identify the specific dependent variables (lipids) that contributed to the significant overall effect (between different NAFL groups and between NAFL and NASH groups), a random forests (RF) analysis was used with the following R packages “n matrix  and b. Athe data  and d. Ithe data .FactoMinR” package. The global p-value was calculated using the critical probability associated with the F- test of the analysis of variance along the axes of the first and the second dimensions (α = 0.05). Receiver operating characteristic (ROC) curve was analysed using “pROC” and “Epi” packages and compared control, NAFL1, NAFL2 and NAFL3 versus NASH patients from Paul Brousse and L’Archet Hospitals based on the 32 lipids identified by random forests. Boxplots were drawn using “ggplot2” and “beeswarm” packages. “gplots” and “RColorBrewer” packages were used for graphics.Principal component analysis, 95% confident ellipse centre to the mean and lipids of interest were computed using “t-test. Kruskal-Wallis rank sum test was used to compare gender repartition between the 6 groups of patients. Type I error-set was 5%.Individual variables among the different groups of patients were shown as boxplot or barplot (mean ± standard error of the mean or SEM) and tested with analysis of variance (ANOVA-test) followed by unpaired The institutional review board of each hospital and ethic committee  approved the study and written informed consent was obtained from all patients. Access to this material was in agreement with French ethical laws.Animal protocols were accepted by the Institutional Animal Care and Use Committee (IACUC) in Main (Jackson Laboratories) and by the “Comité d’Ethique pour l’Expérimentation Animale” registered to the “Comité National de Réflexion Ethique sur l’Expérimentation Animale 05” . In accordance to the animal welfare and in the aim to minimize the number of animals, we used data and samples from our previous publicationHow to cite this article: Chiappini, F. et al. Metabolism dysregulation induces a specific lipid signature of nonalcoholic steatohepatitis in patients. Sci. Rep.7, 46658; doi: 10.1038/srep46658 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


O(10 km), associated with bands of near-inertial wave/diurnal internal tide shear of high vertical wavenumber in the upstream Kuroshio. The turbulent kinetic energy dissipation rates within these turbulent layers are >O(10−7 W kg−1), and estimated vertical eddy diffusivity shows >O(10−4 m2 s−1) on average. These results suggest that the high vertical wavenumber near-inertial waves propagating in the upstream Kuroshio could have large impacts on the watermass modifications, momentum mixing, nutrient supply, and associated biogeochemical responses in its downstream.The upstream Kuroshio flows through Okinawa Trough and the Tokara island chain, the region near the continental shelf of the East China Sea and shallow seamounts, where the Kuroshio can induce strong mixing over the shallow topography. Also, tidal currents over the rough topography may produce internal tides, and associated turbulence. The previous observations show energetic high vertical wavenumber near-inertial wave shear in the Kuroshio thermocline, which implies strong turbulent mixing. However, direct turbulence measurements in this region are very scarce. Using high lateral resolution (1–2 km) direct turbulence measurements, we show here, for the first time, that strong turbulent layers form spatially coherent banded structures with lateral scales of > Because the Kuroshio is inherently an ocean dynamic front, where the associated jet flows along the interface between seawaters of different water properties2, mixing processes in the Kuroshio Front play very important roles in watermass formation, transformation, and subduction5. In addition to this physical or hydrographical important role, the mixing in the Kuroshio has profound implications for biogeochemistry because the Kuroshio carries waters of relatively high nutrient concentrations from the tropics through its subsurface layers, as known as the nutrient stream7 similar to the Gulf Stream11. Earlier studies in the Gulf Stream9 and relatively recent studies in the Kuroshio13 have reported that elevated nutrient concentrations are found along these western boundary currents in their subsurface layers compared to that in the ambient waters of the same density. Because along-isopycnal stirring by mesoscale eddies tends to homogenize any tracer anomaly on the density surface in the ocean interior14, the elevated nutrient concentrations at the subsurface of the Kuroshio are most likely formed by across-density/diapycnal fluxes by irreversible diabatic turbulent diffusion. A previous study9 speculated that the turbulent diffusive nutrient flux by geostrophic vertical shear contributes to the concentration of elevated nutrients below the Gulf Stream. However, a limited number of direct turbulence observations at that time did not provide the supporting evidence for this hypothesis17.The Kuroshio, the western boundary current of the subtropical gyer in the North Pacific, has been known to transport large amounts of heat and salt from the tropical ocean20. While a number of previous studies have reported that the beams of the M2 internal tide emanated from the rough topography induced strong turbulence in close proximity to canyons and seamounts22, recent shipboard and lowered Acoustic Doppler Current Profiler (ADCP) measurements in the upstream Kuroshio showed widespread large amplitude near-inertial internal wave shear of high vertical wavenumber in the Kuroshio and regions between continental margins and the Kuroshio23. A very recent field study in Tokara Strait reported near-inertial waves of even shorter vertical wavelengths ~50 m accompanied by pronounced turbulent dissipation rates of O(10−7–10−6 W kg−1)20. One of the most likely mechanisms to form the high vertical wavenumber shear is the near-inertial internal waves generated by the Kuroshio over shallow topography in Okinawa Trough and Tokara Strait19. Because the Kuroshio is forced to turn its direction from northeast to southeast when it approaches Kyushu island, high vertical wavenumber near-inertial internal waves could also be generated spontaneously by the meandering Kuroshio28. Furthermore, the upstream Kuroshio passes the so-called critical latitude, 28.9°N, where M2 internal tide energy can be efficiently converted to that of high vertical wavenumber near-inertial waves through parametric subharmonic instability (hereinafter PSI)30. Near the critical latitude, K1 or O1 diurnal internal tides generated over the rough topography are also near-inertial internal waves. In a strong baroclinic front, where geostrophic shear allows anomalously low frequency internal waves, a recent theoretical study31 showed that PSI of wind-driven near-inertial waves could generate high wavenumber internal waves with a frequency of half the local Coriolis frequency f/2. Regardless of their generation mechanism, these propagating near-inertial internal waves can be trapped on the anticyclonic side of the front where the lowest internal wave frequency is decreased by geostrophic lateral and vertical shears34. The trapped near-inertial waves can break into three-dimensional turbulence and enhance mixing of momentum and tracers37. However, direct turbulence measurements in the upstream Kuroshio are very scarce. Also, the lateral resolutions of previous turbulence measurements are not sufficient to resolve the coherent spatial patterns associated with high wavenumber near-inertial internal wave shear38. As a result, earlier studies did not find elevated turbulence in the thermocline of the Gulf Stream17, and concluded that turbulence in the ocean interior is caused by patchy random internal wave breaking, even in the Gulf Stream. In contrast, a number of recent studies have reported enhanced turbulent mixing in the thermocline of the Kuroshio42 and the Gulf Stream43 caused by propagating internal waves. However, the lateral resolutions of the turbulence data were still insufficient to provide direct evidence that the turbulence is caused coherently by banded internal-wave shear in the thermocline of the western boundary currents. Although a recent extensive microstructure time-series survey at low latitude45 reported that the strong turbulence coincided with the relatively large strain caused by wind-induced near-inertial waves of anomalously low frequency in the anticyclonic vorticity field33, it is not known if near-inertial internal waves form the spatially coherent strong turbulent layers in ocean fronts. In this study, a new turbulence and microstructure observation scheme is developed and utilized to measure turbulence directly with a high lateral resolution of 1–2 km in the upstream Kuroshio near Tokara Strait, where a large amount of energy from the Kuroshio can be dissipated. For the first time, the observations in this study suggest that propagating high vertical wavenumber near-inertial internal waves form spatially coherent banded layers of strong turbulence in the upstream Kuroshio, with O(100 m) and >O(10 km) vertical and lateral scales, respectively.On the other hand, the Kuroshio flows through shallow topography in its upstream along Okinawa Trough and near Tokara Strait, where the Kuroshio over the topography can induce strong turbulence and diapycnal mixingin-situ survey was conducted using the R/T/V Kagoshima-maru during November 12–20, 2016, near Tokara Strait where the upstream Kuroshio flows over the shallow seamounts and through the narrow straits near the Tokara island chain off Kyushu island  in vertical and O(10 km) in lateral direction, similar to Rainville and Pinkel’s study in this region23. With λh and λv, horizontal and vertical wavelengths (m), respectively, −1) (where b = −gρ/ρo is buoyancy (m s−2) with gravitational acceleration g (m s−2), seawater density ρ (kg m−3) and reference density ρo = 1025 (kg m−3), and vertical coordinate z(m)), and f Coriolis frequency (rad s−1), the internal-wave dispersion relation, then implies that the frequency of these waves N~O(10−2 rad s−1), and local f~O(10−4 rad s−1). The internal-wave ray paths computed, assuming a quiescent condition show that the angles of the internal wave rays with near-inertial frequencies are more consistent with the observed shear bands than those with the M2 tidal frequency  as a function of longitude suggests that large shear variance found at 129.3–129.575°E in Leg A is associated with the downward energy propagating internal waves, and that found at 129.4–129.55°E in Leg B is caused by upward energy propagating internal waves . These estimates are comparable to the rate of wind energy input to inertial motions computed by a slab model50  local PSI with anomalously lower minimum internal-wave frequency than f due to mean velocity shears, (3) K1 internal tides generated over rough topography, (4) near-inertial internal waves generated by the Kuroshio over shallow topography in Okinawa Trough and Tokara Strait, (5) spontaneously generated near inertial internal waves by the Kuroshio, and (6) PSI of wind-induced inertial waves to generate high wavenumber anomalously low frequency internal waves of half the Coriolis frequency, f/2 along isopycnal in a strong baroclinic front31.These results indicate that the high vertical wavenumber shear is caused by near-inertial internal waves propagating in the upward and downward directions in the Kuroshio near Tokara Strait. While the wind energy flux to the inertial currents is found to be sufficient to generate an observed amplitude of near-inertial waves, it is not clear how the observed high vertical wavenumber near-inertial internal waves are formed. We speculate that the possible mechanisms are (1) PSI of the M23. Here, we show, for the first time, that these large amplitude near-inertial internal wave shears generate strong turbulence of spatially coherent banded structures, employing a newly developed high-resolution (1–2 km) tow-yo microstructure profiling technique, using 1.5 mm dyneema rope. The thin rope allows the microstructure profiler (VMP-250) to sink relatively smoothly with small drag down to 300 m depth. The measured turbulent shear spectra by the tow-yo profiling show overall good agreement with the Nasmyth empirical shear spectrum51 , because of turbulence within the surface boundary layer, which is forced by momentum and heat fluxes at the surface , and reach O(10−6 W kg−1) at some locations  within the same orders of magnitude  and O(10 km) in the vertical and lateral directions, respectively, are coherently formed along the near-inertial internal-wave shear bands  within the surface boundary layer, which is not surprising considering continuous forcing at the surface with weak stratification in the surface boundary layer  on average, and >O(10−3 m2 s−1) at many locations below 100 m depth , as well as M2 semi-diurnal tidal frequencies. However, these M2 tidal, near-inertial and subinertial peaks in shear spectra are not clear, especially in upper layers, probably because of the Doppler smearing by the strong Kuroshio flow27  is about the half of that estimated for the semi-diurnal component (D2 = M2 + S2 + N2)  (see Methods section) is continuously dissipated by the observed bands of the turbulent dissipation rates, O(10−8–10−7 W kg−1), with the reference seawater density ρo = 1025 kg m−3, the internal waves could be dissipated completely after 1–10 days. Therefore, it is possible that the PSI-induced near-inertial waves can survive ~4 days after the 94-h advection. Even without the tide or PSI, near-inertial internal waves of high vertical wavenumber could be generated by the Kuroshio over the topography19. The globally estimated energy generation rate from geostrophic flow to lee wave by the study of Nikurashin and Ferrari19 in Okinawa Trough, the region near the observation sites, is O(0.1–1 mW m−2), consistent with the vertical energy flux of observed near-inertial internal waves.The observed strong turbulence associated with high vertical wavenumber internal wave shear implies active occurrence of PSI near the critical latitude 28.9°N. The moored ADCP observations during our shipboard experiment suggest that the velocity field is dominantly modulated at Mpth Fig. . Assumin1 diurnal internal tide is the most plausible and likely candidate for the mechanism generating high vertical wavenumber near-inertial internal waves observed near Tokara Strait. The same harmonic analysis for the diurnal and semi-diurnal tidal elevations during the shipboard observations in November 2016 at Tanegashima tidal station  in the Okinawa Trough to be 6.8–21.5 × 10−4 m2 s−1 using a steady-state salinity balance with historical current data and salinity data obtained by the profiling floats. Although the direct turbulence data obtained in this study cover only the upper 300 m depth, and did not reach the layer of σθ = 26.8 kg m−3, strong turbulence just above salinity minimum layer of NPIW, which is equivalent to >O(10−4 m2 s−1) of average eddy diffusivity, is consistent with a previous estimate57. This implies that the observed large amplitude high vertical wavenumber internal waves play very important roles in watermass mixing and modification in the study region.The induced energy dissipations and vertical mixing could have important implications for watermass modifications. A previous study−1 in the subsurface layer, σθ = 24–27 kg m−3 and transports it downstream7, similar to the nutrient stream of the Gulf Stream11. In addition, nitrate concentrations below the Gulf Stream are found to be higher than in the ambient water of the same density9. This along-isopycnal anomaly of nutrients is most likely formed and maintained by diapycnal diffusion, as opposed to homogenizing mesoscale along-isopycnal stirring. Therefore, large average turbulent diffusivity of O(10−4 m2 s−1) in the subsurface layer at 100–300 m depth, σθ = 24–26 kg m−3, reported by this study, indicates that the observed subsurface turbulence induced by the near-inertial internal waves could effectively diffuse up the subsurface nutrient stream of σθ = 24–27 kg m−3 to the shallower layer, generate the elevated nutrient concentrations along the Kuroshio, and increase primary production in its downstream.Not only is the observed pronounced turbulent diapycnal mixing important for the watermass modifications of NPIW, it is also very important for nutrient supply from the lower layer to the surface euphotic layer. Recent studies of the nutrient transport in the upstream Kuroshio showed that despite its low-nutrient oligotrophic surface water, the Kuroshio transports a large amount of nitrate at an average rate of 170.8 kmol s33 catalyzes the breaking and dissipation of the near-inertial/diurnal internal waves, and promoting watermass modification and nutrient supply. Although the contributions of PSI to form observed high vertical wavenumber near-inertial waves were found to be small compared to the diurnal internal tide in our observations, other mechanisms to generate observed inertial waves are not discussed in detail in this study. Okinawa Trough and Tokara Strait are very peculiar regions because of their strong baroclinic current, the Kuroshio , flowing through the shallow seamounts and islands, and through the critical latitude for PSI and the boundary where the diurnal period is identical to the inertial period. More comprehensive field and numerical studies are, therefore, necessary to fully understand the generation, propagation, and dissipation mechanisms of these near-inertial internal waves/diurnal internal tides propagating in the upstream Kuroshio.In this study, for the first time, the spatially coherent banded layers of strong turbulence associated with high vertical wavenumber near-inertial internal waves were directly observed using a new tow-yo microstructure profiler. Our analyses suggest that the high vertical wavenumber near-inertial waves are most likely generated by the diurnal tide over the rough topography in Tokara Strait. Because the observation site is around 30°N latitude, where the inertial period is 24 h, the generated diurnal internal tides are inherently near-inertial waves with slow group velocities. The ADCP and microstructure data suggest that these near-inertial internal waves are trapped and broken into turbulence, preferentially on the anticyclonic side of the upstream Kuroshio, suggesting that the trapping of the near-inertial waves by the Kuroshio due to anticyclonic vorticityZ(t0) = uz(t0) + ivz(t0)  referenced to the time t0, is estimated from the observations Z(t) = uz(t) + ivz(t) at the arbitrary time t asuz(t0) and meridional shear vz(t0) are obtained by taking the real and imaginary parts of (4), respectively.Near-inertial wave velocity and shear rotate clockwise in time in the Northern Hemisphere. When the observation period exceeds or is equivalent to the inertial time scale, this rotation needs to be removed to avoid temporal aliasing of lateral velocity and shear structures. The back-rotated shear −1. The descending speeds of the UVMP, gradually decreasing with depth, were between 0.3–0.8 m s−1. Taking 7 minutes to reach 300 m depth and 5 minutes for recovery, the lateral resolution of the tow-yo survey was 1–2 km, depending on the absolute current velocity. The data were internally recorded and recovered after each transect observation. However, because the both FP07 thermistors mounted initially on the VMP-250 in Leg A malfunctioned, the expendable bathythermographs (XBT-T7s) were deployed six times to obtain temperature profiles. The XBT temperature data were interpolated by objective mapping with decorrelation lengths of 15 km and 40 m along ship-track and vertical directions, respectively, which were then used to estimate the background potential density ρθ and σθ and stratification for Leg A (see below). For Leg B, two functional FP07 thermistors were installed on the VMP-250, so that the potential density ρθ and σθ could be estimated, based on high-resolution FP07 thermistor data. To cross-calibrate the FP07 thermistors, the calibrated UCTD sensor and VMP-250 were clamped together and lowered to 200 m depth with the BT-winch of the Kagoshima-maru.We used a new tow-yo Underway Vertical Microstructure Profiler (Underway-VMP: UVMP) to measure high-resolution spatial structures of microscale turbulence at the Kuroshio Front. The UVMP consists of a vertical microstructure profiler, VMP-250  and a winch for the Underway-CTD . The VMP-250 carries two shear probes, two FP07 thermistors, a pressure sensor, and a vibration sensor to measure microscale velocity shear, microscale temperature gradient, pressure and accelerations, respectively, at a frequency of 512 Hz. The UVMP was tow-yoed at a ship speed relative to water, at 1.5–2.1 m sε, the turbulent shear spectra were computed using the microscale shear data measured by the airfoil shear probe over 8 seconds, at approximately every 5–6 m depth, with an overlap of 4 s (2.5–3 m). The obtained shear spectra were integrated over the wavenumber ranges, where spectra agree with the Nasmyth empirical shear spectrum51, avoiding the integrations of electronic noise at high wavenumber ranges and low frequency instrument vibration noise at low wavenumber ranges. For the wavenumber ranges where the observed and the empirical spectra did not match, we integrated the fitted Nasmyth empirical shear spectrum51  were estimated using the close relationship between potential temperature and potential density found at the observation sites  for σθ > 25 (deeper than 150 m depth), 0.04–0.1 (kg m−3) for 24 < σθ < 25 (100–150 m depth), and ~0.12 (kg m−3) for σθ < 24 ; the estimated density had better accuracy for the denser and deeper layers.Because the UVMP does not carry a conductivity sensor to measure salinity, seawater potential density Cgz, assuming that the near-inertial waves had low potential energy. Using the dispersion relation of the internal waves, the vertical group velocity for the low frequency internal wave isω is the internal wave frequency (rad s−1), and m the internal-wave vertical wavenumber (rad m−1). The horizontal kinetic energy of the internal waves isuz and vz are ADCP zonal and meridional shear (s−1), respectively, and  represents the spatiotemporal average between 100–250 m depth and over the entire transect for Leg A and Leg B. The internal-wave frequency ω was estimated as ω = 1.01 f (rad s−1), based on the internal ray path and the measured velocity shear band  and two components for diurnal (K1 and O1) tidal elevation can explain 99% of the total variance, with the M2 tidal component accounting for 67%, S2 for 13%, N2 for 3%, K1 for 10%, and O1 for 6%. To obtain the time series of subinertial diurnal and semi-diurnal tidal elevation by the harmonic analysis, the computed diurnal D1 = K1 + O1 and semi-diurnal D2 = M2 + S2 + N2 tidal elevation amplitudes were smoothed with a 30-h lowpass filter and compared with the 30-h lowpass near-inertial shear  were analyzed from August 31, 2000 through August 14, 2001. Harmonic analysis with 59 tidal constituents was conducted for the tidal elevation record to compare with the 30-h lowpass near-inertial shear amplitude (see below). The results of the harmonic analysis showed that the variance of the dominant three components for semi-diurnal  was used. The near-inertial amplitude was computed using near-inertial zonal and meridional shear as To compare the obtained 30-h lowpass semi-diurnal and diurnal tidal amplitudes with the near-inertial shear amplitude, the relatively long-term moored ADCP data were analyzed. The moored ADCP  measured the horizontal current at 14 layers with 32 m vertical bins from 480.5 to 64.5 m depth from September 30, 2000 through July 1, 2001, at Stn. TK1 Fig.  in the vThe datasets generated and/or analyzed during the current study are available from the corresponding author upon request.Supplementary Figures


Circulating tumor cells (CTCs) carry independent prognostic information in patients with metastatic breast cancer (MBC) on different lines of therapy. Moreover, CTC clusters are suggested to add prognostic information to CTC enumeration alone but their significance is unknown in patients with newly diagnosed MBC. We aimed to evaluate whether longitudinal enumeration of circulating tumor cells (CTCs) and CTC clusters could improve prognostication and monitoring of patients with metastatic breast cancer (MBC) starting first-line therapy.This prospective study included 156 women with newly diagnosed MBC. CTCs and CTC clusters were detected using CellSearch technology at baseline (BL) and after 1, 3, and 6 months of systemic therapy. The primary end point was progression-free survival (PFS) and the secondary end point overall survival (OS). Median follow-up time was 25 (7–69) months.There were 79 (52%) and 30 (20%) patients with ≥ 5 CTCs and ≥ 1 CTC cluster at baseline, respectively; both factors were significantly associated with impaired survival. Landmark analyses based on follow-up measurements revealed increasing prognostic hazard ratios for ≥ 5 CTCs and CTC clusters during treatment, predicting worse PFS and OS. Both factors added value to a prognostic model based on clinicopathological variables at all time points and ≥ 5 CTCs and presence of CTC clusters enhanced the model’s C-index to > 0.80 at 1, 3, and 6 months. Importantly, changes in CTCs during treatment were significantly correlated with survival and patients with a decline from ≥ 5 CTCs at BL to < 5 CTCs at 1 month had a similar odds ratio for progression to patients with < 5 CTCs at BL and 1 month. Stratification of patients based on CTC count and CTC clusters into four groups  demonstrated that patients with CTC clusters had significantly worse survival compared to patients without clusters.Longitudinal evaluation of CTC and CTC clusters improves prognostication and monitoring in patients with MBC starting first-line systemic therapy. The prognostic value increases over time, suggesting that changes in CTC count are clinically relevant. The presence of CTC clusters adds significant prognostic value to CTC enumeration alone.NCT01322893. Registered on 25 March 2011.The online version of this article (10.1186/s13058-018-0976-0) contains supplementary material, which is available to authorized users. The prognostic value of circulating tumor cell (CTC) enumeration was first shown in patients with metastatic breast cancer (MBC) assessed by the CellSearch system in 2004 . Since tThe aim of this prospective study was to evaluate longitudinal CTC count ≥ 5 cells/7.5 mL blood and CTC clusters using the CellSearch system as a prognostic instrument in women with newly diagnosed MBC from baseline to 6 months, and examine how these relate to progression-free survival (PFS) and overall survival (OS). A secondary aim was to evaluate if early changes in CTC status can predict response at the first radiological evaluation at 3 months.Patients diagnosed with MBC and scheduled for first-line systemic treatment at Skåne University Hospital and Halmstad County Hospital, Sweden, were enrolled into a prospective monitoring trial  conducted by the Department of Oncology and Pathology at Lund University, Sweden. The study was approved by the Lund University Ethics Committee (LU 2010/135). Inclusion criteria were age ≥ 18 years, Eastern Cooperative Oncology Group (ECOG) performance status score 0–2, and predicted life expectancy of > 2 months. Exclusion criteria were prior systemic therapy for metastatic disease, inability to understand the study information, and other malignant disease in the preceding 5 years. After selection, the participating patients started first-line systemic therapy for MBC according to national guidelines; the treating physicians were blind to the CTC results. Patients underwent structured clinical and radiological evaluation every 3 months or at the discretion of the treating physician. Progression versus non-progression was defined according to clinical practice based on clinical and radiological evaluation using the modified Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 . Using tSamples of whole blood and serum were collected at baseline and after approximately 1, 3, and 6 months of treatment. The serum marker CA15-3 was analyzed at the Department of Clinical Chemistry at Skåne University Hospital with an accredited method used in clinical practice . Twenty-three of the participating patients experienced treatment failure within 6 months of commencement; therefore, they were started on second-line therapy, for which blood sampling was repeated (at baseline after treatment failure (baseline 2) and after 1, 3, and 6 months).Blood samples were collected in 10 mL CellSave Preservation tubes , stored between 15 and 30 °C and processed within 96 h of collection. CTCs were isolated and enumerated using the Food and Drug Administration (FDA)-approved CellSearch system (Menarini Silicon Biosystems) as has been described in detail previously . Two invCTC clusters were defined as groups consisting of ≥ 2 CTCs clustered together and with non-overlapping nuclei. Presence of other cell types in addition to CTCs was not documented. Two independent assessors evaluated CTC clusters in CTC galleries exported from the CellTracks Analyzer II system, as described previously . No addiStatistical power calculations based on estimated PFS, fraction of patients with a CTC count above the predefined threshold (≥ 5 CTCs), the inclusion period, and the estimated follow-up time determined the required study sample size to be 154 patients  criteria . Time frIn addition, Cox models with time-dependent covariates were used to estimate the effects of the longitudinally measured binary variables ≥ 5 CTCs and CTC clusters on OS. Briefly, the follow up of each patient was split into multiple non-overlapping episodes for which each of the two covariates were constant. The number of such episodes per patient varied between 1 and 6: 1 episode was sufficient for patients with the same CTC and CTC-cluster status at BL and all follow-up visits. With three follow-up visits , change in one or both of these variables can be observed up to three times for patients staying on first-line treatment. Patients who switched to second-line treatment within 6 months from BL can have up to three additional episodes with change in one or both variables. Missing values at follow-up visits were imputed using the principle of last observation carried forward. For example, the HR for ≥ 5 CTCs in models of this kind should be interpreted as the ratio of the mortality during episodes with ≥ 5 CTCs to that of episodes with < 5 CTCs. The method of last observation carried forward is reportedly less prone to selection bias than deletion by list , but it P values in the exploratory analyses were not adjusted for multiple testing and should therefore not be compared to the 5% cutoff. Statistical analysis was with IBM SPSS Statistics  and STATA .The association between CTC count and the outcome of the first evaluation was assessed by logistic regression. The value added of CTC count and CTC clusters to a prognostic clinicopathological model was evaluated using LR statistics in Cox regression models, based on a model previously described by Bidard et al. . P valueIn total, 156 patients with newly diagnosed MBC were enrolled in the study between April 2011 and June 2016. There were 31 patients with stage IV disease at initial diagnosis and 125 patients were diagnosed with distant recurrence. Patient and tumor characteristics are summarized in Table Blood samples from 115 patients at all time-points  were analyzed. In total, 591 blood samples were collected and analyzed; two sampling errors and four technical errors were encountered. At baseline, 79 (52%) of 152 evaluable patients had ≥ 5 CTCs (predefined cutoff). The fraction of patients with ≥ 5 CTCs decreased during first-line treatment from baseline to 1, 3, and 6 months, and patients receiving subsequent second-line systemic therapy had on average higher CTC counts at all time points, as depicted in Fig. There were 30 patients with CTC clusters at baseline. During first-line systemic therapy, 39 patients had CTC clusters at any time point  and during second-line therapy, 10 patients had CTC clusters Fig. . HoweverPFS 1.68; 95% confidence interval (CI) 1.17–2.42; P = 0.005) and OS   Fig. . These rHRs increased time-dependently during treatment in longitudinal landmark analysis of CTC count, predicting worse PFS and OS from all follow-up sample time points in patients with CTCs ≥ 5 in the sample Table . A reducPatients with presence of ≥ 1 CTC cluster at baseline had inferior OS and PFS compared to patients without CTC clusters Table . HRs forStratifying patients based on CTC count and presence of CTC clusters revealed four risk groups  where patients with CTC clusters had the worst PFS and OS from all evaluated time points Fig. . When apP = 0.7, Additional file To evaluate CTCs as an early predictor of progression in MBC, changes in CTC count from baseline to 1 month or 3 months were analyzed in relation to the outcome of the first evaluation at 3 months (progression versus non-progression). In logistic regression models, patients with a rapid decrease in CTC count (from baseline to 1 month) had an odds of progression that did not differ significantly compared to patients with a consistently low < 5) CTC count (odds ratio (OR) 1.27;  CTC counTo evaluate the value added by CTC count and CTC clusters compared to the currently used clinical prognostic variables, we built a prognostic model based on the previously published model by Bidard et al. . The basBL, cutoff ≥ 5 CTCs) and baseline CTC clusters  to the baseline clinicopathological model constructed in this cohort, revealed a significant improvement in survival prognostication for both PFS and OS when the variables were added separately  as a separate group to further explore CTC count and CTC cluster detection. This revealed that 6 months after therapy initiation, patients with 1–4 CTCs had PFS and OS closer to patients with ≥ 5 CTCs than to patients with no CTCs, indicating that even a small number of CTCs in addition to the predefined prognostic cutoff ≥ 5 CTCs  might beA strength of this study is the prospective design of serial CTC and CTC cluster evaluation over 6 months in women with newly diagnosed MBC, including sampling before the start of first-line systemic therapy and structured evaluation at pre-specified intervals. The median follow-up time from baseline was 25 (range 7–69) months and the follow-up data were extensive as few patients terminated the study prematurely. Molecular data to determine breast cancer subtype were available from metastasis biopsies from 73% of patients. Furthermore, the treating physicians were blinded to the CTC results, which avoided treatment bias. This study thus enabled investigation of the presence and dynamics of CTCs and CTC clusters during the first 6 months of treatment, and we applied structured monitoring and blood sampling at predefined time points. A potential weakness of this study is the long inclusion period related to the strict inclusion criteria that included only newly diagnosed cases of MBC before the start of first-line therapy and ECOG performance status score between 0 and 2. Moreover, we included patients irrespective of type of systemic therapy and thus we can draw no conclusion on treatment response related to a specific type of therapy.The results of this study support the clinical utility of longitudinal CTC and CTC cluster evaluation for prognostication and treatment monitoring in patients with MBC, who are starting first-line systemic therapy. The prognostic value of CTC count ≥ 5 CTCs and CTC cluster evaluation increased over time, suggesting that the dynamic changes in CTCs and CTC clusters are more relevant to prognosis than a single baseline enumeration. Presence of CTC clusters added significant prognostic value to CTC enumeration alone and standard clinicopathological characteristics at all time points and could identify a subgroup of patients with a notably worse prognosis. These findings are highly relevant for improving prognostication in MBC and in helping clinicians monitor patients with MBC during systemic therapy.Additional file 1:Supplementary information: power calculation performed before initiation of the study. (PDF 483 kb)Additional file 2:Figure S1. CTC count as a continuous variable in relation to presence of CTC clusters. (PDF 182 kb)Additional file 3:Table S1. Multivariable Cox regression analysis of prognostic variables. (PDF 426 kb)Additional file 4:Table S2. Unadjusted Cox regression HRs for CTC count ≥ 5 vs < 5 CTCs, and CTC count ≥ 20 vs < 20 CTCs. (PDF 114 kb)Additional file 5:Table S3. Change in CTC count in relation to progression versus non-progression at first radiological evaluation. (PDF 131 kb)Additional file 6:Table S4. Unadjusted Cox regression analyses of patient and tumor characteristics at baseline. (PDF 146 kb)


Different from the upright pyramid obtained by alkaline solutions, the formation of inverted pyramid results from the coexistence of anisotropic etching and localized etching process. The obtained structure is bounded by Si {111} planes which have the lowest etching rate, no matter what orientation of Si substrate is. The Si etching rate and (100)/(111) etching ratio are quantitatively analyzed. The different behaviors of anisotropic etching of Si by alkaline and Cu based acid etchant have been systematically investigated.The so called inverted pyramid arrays, outperforming conventional upright pyramid textures, have been successfully achieved by one-step Cu assisted chemical etching (CACE) for light reflection minimization in silicon solar cells. Due to the lower reduction potential of Cu Currently, industrialized techniques for texturing Si wafers are generally based on alkaline solutions for single-crystalline Si (c-Si) by the anisotropic etching1 or acid solutions for multi-crystalline Si (mc-Si) by the isotropic etching2. Usually, upright random sized pyramidal structures4 for c-Si and ‘worm like’ structures5 for mc-Si will be obtained, respectively. More specifically, the texturization of c-Si is usually based on alkali/isopropyl alcohol (IPA) mixture required processing at high temperatures (75 °C–85 °C) for about 20 minutes7, which had already been commercialized on (100) oriented Si wafers decades ago. It is well-known that each atom of Si {100} surface has two dangling bonds and two back bonds in contrast to one dangling and three back ones for each atom of {111} surface. Therefore, the activation energy to remove an atom from {100} surface is smaller than that from {111} surface because it only needs breaking two back bonds rather than three ones in the case of {111} surface. Thus, in alkaline etchant, the etching rate is faster along the directions of [100] than that along other directions due to the lower activation energy of the atoms on {100} surface8. Thereby, the etching mainly occurs along the [100] directions and stops at the {111} planes, leading to the formation of upright pyramidal structures.Silicon (Si) surface texturing is an indispensable step for the fabrication of Si solar cells due to the high reflection of planar Si wafers. Thus, surface texture and antireflection coating such as SiN12. Due to the complexity and high cost of the photolithographic based patterning processes, such technique is not compatible with industrial solar cell manufacture. Recently, Lu et al. reported Cu assisted chemical etching (CACE) to prepare nanopore-type inverted pyramidal antireflective layers which took hours etching time13. Very recently, Y. Wang et al. discovered a new technical solution to fabricate micrometer sized inverted pyramidal structure by maskless Cu-nanoparticles assisted chemical etching in Cu(NO3)2/HF/H2O2/H2O solutions16, which had lower reflectivity and higher light absorption, along with a shorter etching time and a lower etching temperature during the texturing process compared with the industrial pyramidal structure texturization. Commonly, both upright pyramids and inverted pyramids are the consequence of anisotropic etching of Si (100) substrate, which are bounded by {111} crystallographic planes due to the slowest etching rate of {111} planes. The aim of this paper is to explain why alkaline solutions form upright pyramid while Cu(NO3)2/HF/H2O2 mixtures form inverted pyramid, though both methods are anisotropic etching.The inverted pyramid texture, outperformed the pyramid texture because of their superior light-trapping and structure characteristics, were typically fabricated by a combination of alkali wet chemical etching and photolithography2O2 process with the assistance of Cu nanoparticles by taking advantage of the anisotropic electrochemical behavior of a Si crystal. The etching mechanism of different oriented Si surfaces is systematically studied. The Si etching rate and (100)/(111) etching ratio are investigated as well. Additionally, surface morphologies and reflectivity after etching are characterized.In this paper, more detailed analysis will be proposed for the anisotropic etching of Si based on a HF/H3)2, 4.6 M HF and 0.55 M H2O2 mixtures for fifteen minutes at 50 °C followed by removing of Cu nanoparticles  comparing to Ag+/Ag (EAg+/Ag = 0.8 V) and different electronic properties of (100) and (111) planes in c-Si. More Cu nanoparticles nucleate on Si (100) plane because of the higher probability of Cu2+ ions to capture electrons. Si underneath the Cu nanoparticles are preferentially oxidized and then etched by HF, leading to a faster etching along Si [100] directions15. Thus, both the lower reduction potential of Cu2+/Cu and different electronic properties of (100) and (111) planes in c-Si contribute to the anisotropic etching of CACE.Figure les Fig. . Microsi25. Therefore, less inverted pyramids were obtained in comparison with those on the surfaces of raw Si wafers used in industry due to the less defects on polished Si surface at the early time. With the prolongation of reaction time, the region without defects could also be etched, and the size of inverted pyramid became larger, as shown in Fig. 27. The opening masks and the laser induced pits were used to localize the alkali etching inside the masks or pits, just like the localized electrochemical anisotropic etching of CACE. However, as the alkali etching time increased, the regular inverted pyramidal structure collapsed and tiny upright pyramids began to appear on the surface. Because the laser induced holes became shallow and the opening masks were destroyed, leading to the etching process was no longer localized inside the holes or masks. Hence, the formation of inverted pyramids not only need the anisotropic etching, but also the localized etching process.Figure In order to further verify the mechanism of CACE, the commonly used (110) and (111) oriented wafers are etched in the Cu based acid solutions. Figure 3)2, 4.6 M HF and 0.55 M H2O2 mixtures at 50 °C for different time and simulated schematic of Si (111) etched structure. The method of simulated schematic is also using (111) plane to incise the {111} crystallographic planes. Due to a lower surface dangling bond density, the Si (111) planes had less “available” electrons than Si (100) planes, Cu nanoparticles just distributed dispersedly on Si (111) surface15. The etching started at the defect sites, and the geometries of the etched structure is also bounded by Si {111} crystallographic planes. The < 111 > axis of a cubic system is an axis of hexagonal symmetry. Thus, hexagonal and triangular structure are obtained. For the hexagonal structure, as depict in Fig. 29. As the etching time extended to 15 minutes, the hexagonal structure turns into equilateral triangular structure due to the continue etching of another three {111} planes. As described above, the structure obtained by the Cu-assisted chemical etching method used in this paper is bounded by Si {111} crystallographic planes whatever orientation of the Si substrate is. Besides, the etched structure can be predicted by using the substrate plane to incise the {111} crystallographic planes.The (111) oriented Si is rarely used since it etches slowly in the alkaline solutions. In contrast, it is much easier to be etched by CACE. Figure 30S is the Si surface area, and t is the etching time. Figure 15/cm231. Higher surface bond density leading to higher probability of Cu2+ ions to capture electrons, which makes faster etching rate. The In order to thoroughly understand the anisotropic etching mechanism of CACE, the etching rate of the three oriented wafers is characterized. The average etching rate , (110) and (111) substrate and the upright pyramids obtained by alkaline etchant are shown in Fig. 2+/Cu and different electronic properties of different Si planes, the deposition of Cu nanoparticles shows orientation-dependent, which leads to the anisotropic etching of Si substrate. Besides, both anisotropic etching and localized etching process contribute to the formation of inverted pyramid. The obtained structure is bounded by the Si {111} planes, no matter what orientation of Si substrate is. Different from alkali etching, the value of anisotropic factor of CACE is almost the same value as that of the ratio between the free bond density of Si (100) and (111) planes. Much faster etching rate (0.54 μm/min) on Si (111) plane has been realized by Cu based acid solutions compared to almost zero etching rate by alkaline solutions. What’s more, the superior structural characteristic fabricated on Si (100) surface has huge application in photovoltaic cell mass production.In summary, the different behaviors of anisotropic etching of Si by alkaline and Cu based acid etchant have been systematically investigated. Thanks to the lower reduction potential of Cu3)2, 4.6 M HF, and 0.55 M H2O2 at 50 °C. The residual Cu nanoparticles were removed away using concentrated nitric acid in a sonication bath. The morphologies and structures of the wafers were characterized by a Hitachi S-4800 scanning electron microscope (SEM). The hemispheric total reflectance for normal incidence in wavelength from 300 nm to 1000 nm was measured using a Varian Cary 5000 spectrophotometer with an integrating sphere.Boron-doped (1–3 Ω·cm), 500 μm thick, (100), (110) and (111) oriented, double polished Si wafers were thoroughly rinsed in acetone to remove any organic contaminants and then rinsed with deionized water before etching. The upright pyramid texture was obtained by etching in alkaline solutions containing 2 wt% potassium hydroxide (KOH) and 10 vol% IPA. Meanwhile, we got the inverted pyramid texture by using Cu based acid solutions containing 5 mM Cu(NO


Finally, there is a paradoxical relationship between serum 25[OH]D concentration, ethnicity, and markers of bone health: Black athletes often present with low serum 25[OH]D without physiological consequences. One explanation for this could be genetic differences in vitamin D binding protein due to ethnicity, resulting in greater concentrations of bioavailable (or free) vitamin D in some ethnic groups. In the absence of any pathology, screening may be unnecessary and could result in incorrect supplementation. Data must now be re-examined, taking into consideration bioavailable or “free” vitamin D in ethnically diverse groups to enable new thresholds and target concentrations to be established; perhaps, for now, it is time to “set vitamin D free”.The last decade has seen a dramatic increase in general interest in and research into vitamin D, with many athletes now taking vitamin D supplements as part of their everyday dietary regimen. The most recognized role of vitamin D is its regulation of calcium homeostasis; there is a strong relationship between vitamin D and bone health in non-athletic individuals. In contrast, data have consistently failed to demonstrate any relationship between serum 25[OH]D and bone health, which may in part be due to the osteogenic stimulus of exercise. Vitamin D may interact with extra-skeletal tissues such as muscle and the immune system to modulate recovery from damaging exercise and infection risk. Given that many athletes now engage in supplementation, often consuming extreme doses of vitamin D, it is important to assess whether excessive vitamin D can be detrimental to health. It has been argued that toxic effects only occur when serum 25[OH]D concentrations are greater than 180 nmol·l Vitamin D was first identified in the early twentieth century by a forerunner of nutritional biochemistry, McCollum . His pioWith advancement of new technologies, our understanding of the vitamin D endocrine system and its biological significance has grown exponentially. Generation of a vitamin D receptor knockout mouse  and highThe aim of this review is to provide a current perspective on the functions of vitamin D that may influence athletic performance, to clarify current guidelines for vitamin D intake, and to highlight crucial aspects of future work that must be tackled. We will address key areas of common confusion that surround vitamin D in the sports world, such as defining and measuring vitamin D and what physiological functions relevant to athletic performers can be optimized by maintenance of adequate vitamin D status.Exactly what constitutes vitamin D deficiency is subject to intense debate. Moreover, a lack of understanding of the key metabolites in the vitamin D pathway can lead to erroneous recommendations . For thi2 and 25[OH]D3, which are biologically inactive metabolites produced by hydroxylation of the vitamin D2 and D3 precursors  for vitamin D centered on its role in bone health . AlthougMore importantly, as we explore in more detail, the correlation between 25[OH]D and bone health is grossly misleading. Although it is unequivocal that vitamin D is required for calcium deposition and that 25[OH]D has been the best marker of identifying vitamin D exposure, consideration for what fraction of total 25[OH]D is measured has been largely overlooked for many years . MoreoveThe misinterpretation of medical guidelines and suggestions of new, non-validated sets of guidelines (such as those suggested by Heaney and Holick  and Zitt−1).Many studies have assessed 25[OH]D concentrations across the world in elite and sub-elite athletes throughout different months of the year –28. Larg3 should be sourced that can deliver the correct dose. Recommendations regarding supplementation dose vary widely and can often be confusing. From the authors’ combined applied experience, dosing strategies for vitamin D in elite sport range from 1000 IU/day to blanket supplementation of up to a 100,000 IU bolus per week. This review should serve to direct practitioners towards a need for a supplementation decision system that should be implemented on an individual basis and provide the most current advice for safe and effective supplementation protocols.When a need to supplement has been identified, an appropriately screened supplemental form of vitamin DThe purpose of athletic training is to provide a stimulus that disrupts homeostasis to bring about an adaptive response that improves competition performance. For athletes, maximizing the training stimulus is therefore a core principle of the training program. Nutrition strategies to complement the adaptive response to a physical/metabolic challenge are intensely researched. Recently, on the basis of animal trials and in vitro basic biology studies, data have emerged suggestive of a beneficial role for vitamin D in skeletal muscle repair and remodeling.−1 with supplemental vitamin D3 at 4000 IU/day has a positive effect on the recovery of force following a bout of damaging eccentric exercise D concentrations to > 75 nmol·lexercise . Similarexercise , 33. Theexercise . The aut2D3 D and induced by activation of the toll-like receptor cascade in the presence of pathogenic microbiota . In the in vitro .−1, such that all athletes with circulating concentrations lower than this breakpoint had one or more episodes of illness. Those with higher concentrations had one or fewer episodes. A similar study in endurance athletes reported that a greater proportion of athletes maintaining circulating 25[OH]D concentration < 30 nmol·l−1 presented with URTI symptoms, with the fewest symptoms reported in those with 25(OH)D concentrations > 120 nmol·l−1 oduction , 67. An oduction .−1 and 1.80 (95% CI 1.05–3.08) for levels < 25 nmol·l−1. However, this relationship was found only among participants who were hypertensive at baseline [−1) and elevated risk of myocardial infarction [Human trials have produced some evidence that vitamin D deficiency may be related to an increased risk of cardiometabolic outcomes. The Framingham Offspring Study found an association between lower serum 25[OH]D concentrations and increased risk of cardiovascular events . There wbaseline . Data frfarction . Scragg farction  also shofarction , 72, serfarction . The rel−1) athletes were significantly smaller than those of insufficient and sufficient athletes D-deficient state remain unclear. However, what is understood, is that the remodeling mechanisms associated with cardiac disease and chronic overloading  differ considerably from the physiological adaptations seen in athletes induced through prolonged and intensive exercise.Studies in rodent models show that the VDR and 1α-hydroxylase mediate arterial hardening and endothelial function. Also, the endothelial dysfunction observed in VDR-knockout mice is caused by a reduction in nitric oxide bioavailability . FurtherThe relationship between vitamin D and cardiac function remains highly controversial. Despite the growing body of evidence demonstrating a link between vitamin D deficiency and cardiovascular risk factors, very few studies have examined the association between vitamin D status and cardiac structure and function in healthy athletes. Future research should look to identify the precise mechanisms causing cardiac hypertrophy with increases in vitamin D status in healthy athletes.Vitamin D status is indicative of calcium absorption and bone mineralization , and a c2D3 and parathyroid hormone (PTH). The active form of vitamin D, 1,25[OH]2D3 affects osteoblast function via different mechanisms. It controls remodeling via induction of receptor activator of nuclear factor (NF)-κB ligand (RANKL) [2D3 from the 25[OH]D3 precursor and that this activity is likely to account for the skeletal effects of circulating 25[OH]D3 D3 .Athletes undertake mechanical loading from training or competition that is associated with an increase in bone mineral density (BMD) , 103. An2D pathway may potentially account for some of these differences [Recent research shows no association between serum 25[OH]D concentration and measures of bone health in an ethnically diverse athletic population, irrespective of exercise type (weight/non-weight bearing) . This drferences , 111. Thferences , 113. Fu2D3 on osteoblasts.Optimum concentrations of serum 25[OH]D for the best possible skeletal health are still debated. Many investigators define the threshold for vitamin D sufficiency as the lowest serum 25[OH]D concentration that maximally suppresses PTH secretion and/or optimizes BMD –116. ObsThere appears to be a paradoxical relationship between ethnicity and vitamin D concentration that has largely been ignored. When examining 25[OH]D deficiency in ethnically diverse populations, studies demonstrate that Black and Hispanic men are at elevated risk of 25[OH]D deficiency but at lower risk of osteoporosis, rapid bone loss, and associated fractures , 113 thaVitamin D-binding protein (VDBP) provides insight into why certain ethnic groups may have distinct 25[OH]D and BMD relationships . VDBP is3 2D3], the biologically active form of vitamin D, and the remaining unbound 25[OH]D is considered bioavailable . About 10–15% of total 25[OH]D is bound to albumin, in contrast to free 25[OH]D, which accounts for < 1% of total circulating vitamin D D D and 1,25-dihydroxyvitamin Ditamin D . Since t 25[OH]D .VDBP gene (rs4588 and rs7041) D concentrations, very few studies have examined whether high concentrations of 25[OH]D that do not result in toxicity are actually beneficial. Our group recently began to address this question by examining the effects of high-dose vitamin D3 supplementation  on all major vitamin D metabolites  in a cohort of elite athletes D. This metabolite is thought to exert a negative effect on 1,25[OH]2D3 signaling and may inhibit the conversion of 25[OH]D to 1,25[OH]2D3 in a negative feedback loop. Interestingly, when athletes were withdrawn from supplementation, the 24,25[OH]D metabolite remained elevated even though 25[OH]D and 1,25[OH]2D3 fell. One could speculate that persistent elevation of 24,25[OH]D in the face of declining active 1,25[OH]2D3 could result in the opposite effect than what was intended. Recent evidence supporting the examination of all vitamin D metabolites has emerged. Pleiotropic effects of the vitamin D metabolome were observed in a study of vitamin D status and muscle function and gene expression in the elderly, suggesting that future supplementation studies should not be restricted to usual analysis of the major circulating form of vitamin D, 25[OH]D [Figure athletes . Our fin 25[OH]D .3 exposure . Despite being highly relevant to total and bioavailable vitamin D concentrations, VDBP is not included in most studies examining vitamin D deficiency and measures of health in athletes. Nevertheless, racial differences in VDBP have been explored in the general population to some degree. A recent study demonstrated that community-dwelling Black subjects had lower levels of VDBP and serum 25[OH]D (38.9 ± 0.5 nmol·l−1) than White subjects (64.4 ± 0.9 nmol·l−1) D but higher BMD compared with White subjects D concentration is measured as a marker of vitamin D status because of its long half-life and close relationship to vitamin Dmol·l−1) . Howeversubjects .This questions the validity of the commonly used laboratory test for serum 25[OH]D concentrations in assessing vitamin D deficiency in ethnically diverse groups. Consistent with the “free hormone” hypothesis , severalAs our understanding of vitamin D biology develops, it is becoming clearer that determining true vitamin D status is multifactorial. Systematic screening to determine 25[OH]D concentrations in isolation is expensive and demonstrates a poor relationship to bone health. If testing is warranted, practitioners should use the appropriate assays to determine bioavailable (free) vitamin D concentration rather than total serum 25[OH]D and VDBP genotype, if possible.The emerging body of evidence surrounding vitamin D and athletic performance is bolstering support for the need to control vitamin D concentration in athletes. Undoubtedly, adverse risks are associated with vitamin D deficiency that will affect athletic performance directly and indirectly. New insights into the responses of the vitamin D metabolome to supplementation, and emerging evidence suggestive that free 25[OH]D may be a more useful marker of vitamin D status, add new complexities to the area. Nevertheless, the field moves closer to a more complete understanding of the vitamin D endocrine system. The purpose of our review was to collate the most recent advances in this field and provide suggestions, based on current understanding, as to how vitamin D can be managed in practice. To this end, we have structured our thoughts into a decision tree  . In addi


Glioblastoma multiforme (GBM) remains one of the most incurable cancers. Although the genomic era has produced massive quantities of data in an attempt to characterize its molecular drivers, 2, thesThe clinical development and testing of the αv integrin antagonist cilengitide provides an interesting case study to show how a personalized medicine approach may be able to resurrect a therapeutic that failed to provide a survival benefit in an “unselected” GBM patient population. Cilengitide EMD 121974), is a cyclic peptide designed to block the function of αvβ3 and αvβ5 integrins that had been implicated as key drivers of tumor angiogenesis and glioblastoma progression. Althoug4, is a cWe therefore reconsidered whether all GBM tumors are equally addicted to integrin αvβ3 during progression. Remarkably, we discovered that sensitivity to αvβ3 blockade could not simply be predicted by high αvβ3 expression, but rather on a state of addiction to the high-affinity glucose transporter, Glut3, that provides tumors with a critical glucose-uptake advantage in the nutrient-poor environment of the brain. MechaniFurthermore, we identified a genetic signature capable of predicting which GBM tumors would show this Glut3-addicted phenotype. In particular, we found that GBM tumors with markers defining the proneural and classical molecular subtypes were addicted to αvβ3/Glut3. In contrast, tumors with markers defining the mesenchymal subtype were not addicted, even when they displayed high expression of both αvβ3 and Glut3. This distinction highlights how functionally relevant markers can be incorporated into molecular classification approaches to predict which subsets of patients may respond to a given drug, providing important inclusion criteria for clinical trials. Such an analysis to account for durable responses in individual patients will provide a framework for setting up new molecularly defined clinical trials.Our study also highlights the value of patient-derived gliomaspheres to reflect GBM heterogeneity and addiction status. Indeed, we found the established GBM cell lines U87MG, LN229, and LN18 to be highly sensitive to agents targeting αvβ3/PAK4-YAP/TAZ signaling axis, whereas only a subpopulation of patient-derived GBM stem cell models showed sensitivity. Although the nearly universal effect of cilengitide on established GBM cell lines in vitro and in vivo generated great enthusiasm for its clinical development, only some patients responded to this therapy. In fact, we observed a paradoxical enhancement of viability for several of the “non-addicted” GBM stem cells treated with αvβ3 antagonists. The highly variable response to cilengitide among our panel of patient-derived GBM stem cell models may account for why this drug failed to prolong overall survival in a non-selected GBM patient population. Although our sample size is relatively limited, we predict that less than a quarter of GBM tumors would show sensitivity to cilengitide and that progression may even be accelerated in certain patients. Going forward, the challenge will be to develop rapid and feasible genetic or molecular screens to identify αvβ3/Glut3 addiction in living patients, and to optimize a CLIA-certified screening process to reliably predict responders.Taken together, our recent study highlights new opportunities to exploit the analysis of “big data” to achieve advances in precision medicine and reinforces the importance of profiling individual tumors. Using cilengitide as an example, we suggest that other “failed” drugs may be reconsidered using patient-derived models, allowing them to advance to a more defined style of GBM clinical trial that relies on molecularly-defined inclusion/exclusion criteria to identify a potentially small population of responders.


Dimethyl fumarate (DMF) is an anti-inflammatory and antioxidant drug used to treat multiple sclerosis, but its mechanism(s) of action are not fully understood. In central nervous system (CNS) cells, DMF activates nuclear factor E2-related factor 2 (Nrf2), perhaps ameliorating oxidative stress-induced damage. However, it is not known whether DMF also activates Nrf2 in peripheral immune cells, which are known to participate in CNS demyelination. We conducted a single observation study to determine whether DMF can activate Nrf2 in peripheral immune cells in vitro.max variability is masking Nrf2 activation in individual donors. Additionally, there may be sub-populations of responders, perhaps related to genetic polymorphisms in Nrf2.We performed enzyme-linked immunosorbent assays to measure Nrf2 activation in nuclear extracts of human peripheral blood mononuclear cells treated with DMF at time points from 0 to 6 h, initially determining that DMF did not activate Nrf2, and that the mechanism(s) of action of DMF may thus differ in the periphery compared to the CNS. However, further analyses of our data suggest that high TThe online version of this article (10.1186/s13104-017-2862-8) contains supplementary material, which is available to authorized users. Dimethyl fumarate (DMF) is an anti-inflammatory and antioxidant drug approved for the treatment of relapsing–remitting multiple sclerosis (MS). Treatment with DMF leads to significant reductions in relapse rates, progression of disability, and active brain lesions . The mecWhile MS is a neuro-inflammatory disease, MS likely begins with the peripheral immune system inappropriately activated in response to an environmental antigen, and pathogenesis is immune cell mediated , 4. Peri6 viable cells were used per treatment. In initial time-course experiments, cells were resuspended in serum-free RPMI, then left untreated, treated with vehicle (ethanol), or treated with 200 µg/ml DMF  for 30 min to 6 h at 37 °C/5% CO2. Nuclear extraction was performed according to TransAM Nrf2 kit protocol . Protein concentration for each sample was determined using a bicinchoninic acid (BCA) assay kit, according to manufacturer’s protocol .Apheresis products were purchased from KeyBiologics . The company followed IRB regulations for consenting of human donors. PBMCs were isolated and cryopreserved as previously described . PBMCs wIn a second set of experiments, we used a commercial nuclear extraction kit according to manufacturer’s protocol , added 2.5% FBS  to the samples 15 min after DMF addition in order to improve cell survival , reduced the number of incubation time points to 2 and 4 h (those most likely to show activation based on literature), and added UNT controls for both time points (formerly untreated samples were representative of the longest time point in each assay).To measure Nrf2 activation, we used the TransAM Nrf2 kit, a plate based ELISA kit on which oligonucleotide containing the ARE (antioxidant response element) consensus binding site has been immobilized. The active form of Nrf2 binds to this site, and the detection antibody recognizes an epitope on Nrf2 upon DNA binding. Five micrograms of nuclear extract per sample was assayed. Positive control was Nrf2-transfected COS-7 nuclear extract. Assays were performed according to manufacturer’s protocols in duplicate or triplicate, and plates were read using a SpectraMax M2 microplate reader .6 viable PBMCs (per treatment) were treated for 5 min with 50, 100 and 200 µg/ml of DMF, or treated with 5 µl of vehicle control . Samples were processed and cAMP assay performed according to manufacturer’s protocol .To measure cyclic AMP (cAMP) production as a test of DMF efficacy, 2 × 10Values for each donor were determined as an average of the replicates, which were generated from internal 4 parameter logistic standard curves using Softmax software (Molecular Devices) for cAMP assays, or absorbance at 450 nm for Nrf2 assays. These values were used to determine the arithmetic mean, plus or minus the standard error of the mean, of each condition. Treatments and controls were tested for significant differences in Microsoft Excel 2010 using one-way ANOVAs and Student’s t-tests.Treatment of PBMCs with DMF did not appear to result in Nrf2 activation Fig. a. To ensIn order to eliminate technical difficulties as the reason for failure to achieve Nrf2 activation, we analyzed cell survival post-treatment and added varying concentrations of FBS to optimize survival. UNT and DMF treated samples did not differ significantly in their survival regardless of FBS presence or concentration, but 2.5% FBS resulted in slightly higher survival for both compared to serum-free RPMI at 2 and 4 h (data not shown). We also varied the amount of protein used in ELISAs , and attempted stimulation with monomethyl fumarate (active metabolite of DMF). None of these conditions resulted in consistent Nrf2 activation (data not shown).max, Table max), regardless of time point  of DMF are still under investigation, but activation of Nrf2 and its downstream effectors in the CNS and periphery has been a primary focus. Recently however, DMF treatment was shown to be equally protective in the mouse model of MS in wild type and Nrf2ne cells . Studiesne cells –12. In ane cells .In addition to its widely reported activation of Nrf2 in CNS cells, in vitro DMF treatment of peripheral immune cells has been shown to affect NF-κB activation and production of a variety of inflammatory mediators; we have previously demonstrated that DMF stimulates production of the immunomodulator cAMP via the prostaglandin E2 receptor in PBMCs, but only at relatively high concentrations of DMF . It is pOur individual donor analyses suggest that DMF activation of Nrf2 in PBMCs is highly variable—with subgroups of responders that when aggregated across a time course mask activation. This kind of variability is to be expected in clinical trials since there are many factors in how an oral drug is taken up by patients, but is less expected in in vitro studies. These differences may be due to single nucleotide polymorphisms (SNPs) in Nrf2. There are at least nine (SNPs) in humans; two are in the promoter region of Nrf2 and reduce transcriptional activity. These latter two plus one additional SNP have been associated with variable Nrf2 response to caffeine . AnotherIn future studies, it would be of interest to determine whether specific SNPs are associated with Nrf2 response in PBMCs. If effects on peripheral immune cells are part of the MOA of DMF, determining Nrf2 genotype might be a tool to help decide who should take the drug, which is extremely expensive and has the rare (but serious) side effect of progressive multifocal leukoencephalopathy .There were a number of limitations to this study. The DMF concentration used is relatively high, and addition of a cell type in which DMF is known to activate Nrf2 as a positive control would have strengthened this study. Also, although DMF activates Nrf2 in other cell types within the time points we examined, it is possible that in the “non-responders,” activation is either quickly transient or delayed beyond 6 h in PBMCs. Also, though cell viability was good, the slight drops seen at 2 h (non-responders) and 4 h (non and moderate responders) may contribute to the small reduction in Nrf2 activation compared to UNT. The small sample size for our proposed subgroups makes these results far more indicative than conclusive, though it provides an interesting avenue for analyzing future experiments involving DMF responses. Finally, only healthy control cells were utilized in these studies—it will be very important to compare how cells from MS patients respond in future studies.