The model is based on 3D spheroids from primary human hepatocytes from five different donors. Across these donors, we observed highly reproducible differences in the extent of steatosis induction, demonstrating that inter-donor variability is reflected in the in vitro model. Importantly, our data indicates that the genetic variant TM6SF2 E167K, previously associated with increased risk for NAFLD, induces increased hepatocyte fat content by reducing APOB particle secretion. Finally, differences in gene expression pathways involved in cholesterol, fatty acid and glucose metabolism between wild type and TM6SF2 E167K mutation carriers (N = 125) were confirmed in the in vitro model. Our data suggest that the 3D in vitro spheroids can be used to investigate the mechanisms underlying the association of human genetic variants associated with NAFLD. This model may also be suitable to discover new treatments against NAFLD.There is a high unmet need for developing treatments for nonalcoholic fatty liver disease (NAFLD), for which there are no approved drugs today. Here, we used a human  It comprises a range of conditions, from relatively benign simple steatosis to more progressed forms, including nonalcoholic steatohepatitis (NASH) and liver fibrosis which may progress to cirrhosis and hepatocellular carcinoma6 as well as cardiovascular morbidities8. NAFLD has a strong genetic component9, with the patatin-like phospholipase domain containing 3 I148M  and transmembrane 6 superfamily member 2 E167K  genetic variants having the largest effect on the susceptibility to the entire spectrum of liver disease9.Nonalcoholic fatty liver disease (NAFLD) is considered a global epidemic10 suggesting a causal role of fat accumulation in causing liver fibrosis, the mechanism behind the toxicity of intracellular neutral lipid is not clear. The increase in hepatocyte intracellular fat can be attributed to derangements of at least four main metabolic pathways: increased triglyceride (TG) synthesis, increased TG uptake, decreased fat secretion or decreased fatty acid (FA) oxidation11. In genetic association studies carriers of the TM6SF2 E167K variant have substantially higher risk of NAFLD and at the same time lower risk for cardiovascular disease14. Human TM6SF2 E167K carriers have lower circulating apolipoprotein B100 (APOB) levels15 suggesting a reduction in the number of secreted lipoproteins. The TM6SF2 E167K genetic variant may result in a misfolded protein that is prone to intracellular degradation resulting in reduced protein levels16. Tm6sf2 inactivation in mice is, like in humans, associated with hypocholesterolemia and increased liver fat accumulation. In addition, hepatic VLDL-TG secretion rate is reduced while the APOB secretion is unaffected in the Tm6sf2 knock-out (KO) mice, indicating no change in the number of secreted lipoprotein particles from the liver17 in discordance with the human data15.Accumulation of intrahepatic fat is the hallmark of NAFLD. Despite a Mendelian Randomization study19. The indisputable advantage of in vivo models is the representation of the (patho)physiological interplay of organs in the body. However, animal models have also clear limitations such as interspecies differences across liver pathways21, disease characteristics22, high costs and ethical concerns. Thus, human in vitro systems are commonly used to complement animal experimentation23. To address the limitations of simple human in vitro models culturing primary hepatocytes in a classical 2D monolayer, 3D spheroids generated from primary human hepatocytes have proven to be excellent surrogates for human physiology24 by providing an in vivo-like cellular microenvironment25. However, spheroids composed of primary human hepatocytes have not been exploited as a model to investigate molecular genetic mechanisms involved in the development of NAFLD.At present, there is no effective and approved pharmacotherapy available for the treatment of NAFLD. For development efforts aiming at novel treatments, disease models reflecting human NAFLD are required. Numerous dietary and genetic animal models are used for investigating the disease and its progression, of which most are rodent modelsTM6SF2 E167K genetic variant using human data from hepatic transcriptome. Next, we used a recent in vitro 3D spheroid NAFLD model from primary human hepatocytes26. This in vitro system reflects the liver physiology of WT and TM6SF2 E167K donors.Aim of this work was to generate a 3D spheroid model to study molecular genetics of NAFLD. To achieve this goal we first examined differences in hepatic lipid metabolism between wild type (WT) and carriers of the TM6SF2 E167K mutation (N = 9) compared to WT (N = 116). Therefore, to understand the metabolic changes in TM6SF2 E167K carriers, we performed enrichment for genes with a nominal p-value < 0.05 which are related to hepatic glucose and lipid metabolism using Gene Ontology (GO) biological process (BP) terms and KEGG biological pathways , the strongest common genetic variant increasing liver fat content27. Furthermore, donors were genotyped for the PNPLA3 rs2294918 (E434K), GCKR rs1260326 (P446L) and MBOAT7 rs641738 (C > T) NAFLD risk alleles  and 320 µM total FA concentration, conjugated to bovine serum albumin (BSA), as described previouslyTo assess the viability of cultured spheroids, we determined intracellular ATP content after incubation with and without FAs using two different concentrations at treatment day two, four, six and ten Fig. . We obseNext, we evaluated differentiation of hepatocytes in spheroids by determining intracellular and extracellular levels of APOB, a protein only expressed in hepatocytes and involved in hepatocyte TG secretion by very low-density lipoprotein (VLDL), a pathway present exclusively in hepatocytes. We found that APOB protein synthesis was preserved Figs  and 7A,BAll these data suggest that spheroids are viable, well differentiated and functional up to 10 days of incubation with FAs.To test whether FA incubation results in an increased fat content in the spheroids, we measured total intracellular TG content in single cell suspension obtained from disintegrated spheroids by fluorescence intensity using AdipoRed™ assay reagent Fig.  and by cTM6SF2 E167K-2, WT-1 and WT-2, using low and high FA concentrations  and WT (pooled from 3 donors) spheroids after 2 days of incubation with high FA concentration , and examined the directional regulation (up or down) of these genes , de novo lipogenesis (FASN and ACSS2) and phospholipid dephosphorylation (PLPP3) were upregulated in the hepatic TM6SF2 E167K spheroids, while gluconeogenesis (FBP1) was upregulated in the spheroids and downregulated in humans carrying the E167K risk variant  than the WT-1 (−1.87 fold) and WT-2 donor (−2.56 fold). APOB expression in WT-3 was unchanged , a protein necessary for the assembly of APOB-containing lipoproteins, in the transcriptome of patients at risk for NAFLD carrying the TM6SF2 E167K protein.To understand the consequences of carriage of the POB Fig. . Since tged Fig. . We alsoTM6SF2 E167K donors have lower secretion of APOB compared to spheroids formed from hepatocytes of three wild type (WT) hepatocyte donors investigated.In this work we show in both human liver tissue samples and in 3D spheroid cultures of primary human hepatocytes that carriage of the TM6SF2 E167K mutant protein is associated with an upregulation of the cholesterol and FA biosynthesis pathways. We also demonstrate that spheroid cultures with human primary hepatocytes obtained from two TM6SF2 E167K sequence variant, as compared to the WT. We found a striking upregulation of most of the enzymes involved in lipid synthesis and specifically of cholesterol and FA metabolism with both GO and KEGG. Interestingly, KEGG analyses found an enrichment of genes which are classified as involved in the pathogenesis of viral hepatitis and NAFLD. This is in line with the human genetic association data showing that TM6SF2 E167K carriers have higher risk not only for NAFLD but also for progression of liver damage in patients infected by viral hepatitis B and C30.We started by examining enrichment for significantly differentially expressed genes  in human liver from individuals at risk for liver disease carrying the 24 from five individual donors, two carrying the TM6SF2 E167K protein and three the WT protein. In both TM6SF2 E167K and WT the ATP levels, as well as APOB synthesis and secretion were preserved up to ten days after formation, indicating that spheroids were viable, well differentiated and functional within this time frame. Next, to induce intracellular lipid accumulation, we exposed the spheroids to increasing doses of a mixture of saturated and monounsaturated FAs for up to 10 days. FA load did not affect spheroid viability and differentiation. Most importantly, in the spheroids derived from the TM6SF2 E167K donor we consistently found a larger increase in the intracellular fat content as compared to the WTs, which was dose- and time-dependent. This suggests that the spheroids may represent a reliable model to study the mechanism underpinning predisposition to lipid accumulation in carriers of the TM6SF2 E167K variant. Furthermore, the phenotypic expression of the mutation in the presence of excess FAs is consistent with the recent gene environment interaction observed for this gene variant31.Next, we used a novel 3D spheroid NAFLD model utilizing primary human hepatocytesTM6SF2 E167K spheroids had upregulated genes involved in the cholesterol, FA and glucose metabolism. Overall, the directional regulation of genes involved in cholesterol metabolism was consistent in humans and the spheroid model. We found a discordance in enzymes involved in lipolysis (PNPLA2), gluconeogenesis (FBP1) and β-oxidation (ACADS). While PNPLA2 is a key enzyme in lipolysis, we did not observe any significant change in the expression of other lipolytic enzymes (LIPE and MGLL) involved in TG hydrolysis in humans.We examined the intracellular biological pathways by differential expression analysis and found that, consistent with human data, TM6SF2 E167K primary hepatocytes have similar metabolic changes present as in livers of human carriers. These changes, namely increased cholesterol and FA biosynthesis, may contribute to the increase in intracellular fat. Interestingly, excess in free cholesterol is known to cause hepatic damage33. Our data are consistent with increased cholesterol esters in the liver of TM6SF2 E167K carriers34. Therefore, although we could not evaluate protein expression and directly measure de novo lipid synthesis, our data suggest that the progression of NAFLD in TM6SF2 E167K carriers could be ascribed not only to reduced lipid secretion13, but also to the excess synthesis of cholesterol or other lipotoxic lipid species33. Interestingly, the mouse knockout (KO) model and in vitro overexpression of the TM6SF2 E167K sequence variant accumulates intrahepatic fat, but this was associated with reduced cholesterol biosynthesis35, possibly suggesting that the mouse model may not closely mirror human physiology. However, in contrast to our results, a previous study reported downregulation of lipid synthesis in the liver of TM6SF2 E167K carriers34.Taken all this together, our data indicate that the spheroids generated from TM6SF2 E167K spheroids show a lower secretion of APOB, coupled to lower intracellular APOB protein levels. The Tm6sf2 KO mouse was found to have unchanged secretion of APOB particle number17, while a human genetic association study showed lower levels of circulating APOB15. Our data are consistent with the human genetic association study, indicating a defect in secretion of APOB particles, which may cause intracellular lipid increase. Regarding the cause of reduction in APOB secretion, we found lower intracellular levels of APOB. This could be due to an increase in APOB degradation, possibly due to unlipidated or underlipidated forms of APOB36. These data are consistent with the notion that decreased transfer of FAs on nascent VLDL in the endoplasmic reticulum (lipidation) contributes to intrahepatic fat accumulation in TM6SF2 E167K carriers17. However, differently than in the mice model, we showed that in human hepatocytes this defect also results in decreased APOB protein levels and secretion. A limitation of our study is that we cannot take into account the effect of intestinal uptake of lipids in chylomicrons which is also reduced in the presence of the TM6SF2 mutant protein in the postprandial state37. However, the data presented in this study and in previous genetic studies were in a fasting state, minimizing the interference of lipoproteins of the fed state15.Next, to investigate on the mechanism of intracellular fat retention we examined the APOB secretion as well as intracellular synthesis in spheroids. We found that TM6SF2 E167K hepatocyte donors because of the relatively low allele frequency of this sequence variant. It should also be taken into consideration that the two mutant donors were of younger age  and LifeTechnologies .PPP1R3B)38. Informed consent was obtained from each patient and the study protocol was approved by the Ethical Committee of the Fondazione IRCCS Ca’ Granda, Milan and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Individuals with increased alcohol intake , viral and autoimmune hepatitis or other causes of liver disease were excluded. Steatosis was graded based on the percentage of affected hepatocytes as 0: 0–4%, 1: 5–32%, 2: 33–65%, and 3: 66–100%. NAFLD Activity Score (NAS) was assessed by systematic evaluation of hepatocellular ballooning and necroinflammation. Fibrosis was also staged according to the recommendations of the NAFLD clinical research network39.RNASeq was performed in 125 severely obese individuals who underwent to percutaneous liver biopsy performed during bariatric surgery, as described in a previous study assessing the role of a genetic variant in the protein phosphatase 1 regulatory subunit 3B gene  were excluded. Per gene reads were counted according to the ENSEMBL human transcript reference assembly version 75 exploiting RSEM package43. Raw counts normalization and differential gene expression analysis were performed exploiting DESeq2 package44 according to the standard workflow. P-values were corrected for testing multiplicity by Benjamini-Hochberg false discovery rate45.RNA extraction was performed using miRNeasy mini-kit , according to the manufacturer’s instructions. Agilent 2100 Bioanalyzer was used for RNA qualitative assessment, high-quality samples (RNA integrity number >7) were used for library preparation. RNA was sequenced in paired-end mode using an Illumina HiSeq 4000 . Reads quality evaluation and trimming was conducted using FastQC  and TrimmomaticTM6SF2 rs58542926 wild type and E167K mutant carriers . The tests were carried out using Gene Ontology (GO) Biological Processes (BP) and KEGG pathways. Hypergeometric distribution was used to calculate p-values, which were subsequently adjusted for multiple hypothesis testing using Benjamini and Hochberg false discovery rate (FDR) correction45.We used GeneTrail2 tool (26787660) to test for the enrichment of functional annotations among differentially expressed genes between TM6SF2 E167K donors. The sourcing details and demographics of the donors are available in Supplementary Table et al.24 and Kozyra et al.26 were mainly employed, using slight variations. In short, 2,000 viable cells per well were seeded into ultra-low adhesion 384-well plates  or ultra-low adhesion 96-well plates . Total viabilities of the cell preparations after thawing ranged between 90–95% for hepatocytes from all donors, except for the TM6SF2 E167K-1 donor (84%). Since lower total cell viability is frequently associated with reduced spheroid formation capability, brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK was used to support spheroid formation, as described recently46. The same treatment was done for the WT-3 donor to allow for direct comparisons of both preparations , supplemented with 5.5 mM glucose, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 100 pM insulin, 5.5 µg/ml transferrin, 6.7 ng/ml sodium selenite, 100 nM dexamethasone and 10% fetal bovine serum. The cells were allowed to aggregate and form spheroids for 7 days, with daily medium change starting on the fourth day of culture. Medium changes were performed with FBS-free medium of the otherwise same formulation as described above (maintenance medium). After the start of fatty acid incubation, medium was changed every 48 hours  and thawed according to the supplier’s instructions. It was not possible to identify additional ons Fig. . After s26. Exposure started 7 days after seeding  and the saturated FA palmitic acid (PA) in a ratio of 2:1, mimicking human plasma concentrations of free fatty acids (FFAs), as described previously2+, Ca2+) per well. Subsequently, 50 µl of assay reagent were added following vigorous mixing for further promoting penetration of the 3D reagent into the spheroid matrix. The plate was incubated in the dark for 25 min at room temperature and luminescence was read using a SpectraMax Gemini XS  reader.Total intracellular adenosine triphosphate (ATP) content was quantified by luminescence using CellTiter-Glo® 3D Cell Viability Assay  with a slightly modified protocol. In brief, for each replicate one individual spheroid was transferred by sedimentation into a white 96-well assay plate  containing 50 µl PBS . Prior to staining, spheroids were fixed in a 4% paraformaldehyde solution at room temperature for 1 hour, followed by storage at 4 °C in PBS . Fixed spheroids were incubated with double-staining solution at room temperature for 90 min, followed by incubation at 37 °C for 30 min and washing with PBS . Confocal imaging was performed in a black 96-well assay plate  using an LSM880 Airyscan  microscope.Intracellular neutral lipid droplets were stained using Nile Red staining, nuclei were stained using Hoechst 33342 . In brief, a double-staining solution containing 2 µM Nile Red and 16 µM Hoechst 33342 was prepared in PBS , of which 200 µl were used per assay in a black 96-well assay plate . For each spheroid replicate , one individual spheroid was taken up in 20 µl of its maintenance or treatment medium, respectively, and the complete volume transferred into the trypsin solution. For determining the culture medium-derived background signal, for each replicate  20 µl of maintenance or treatment medium supernatant, respectively, without spheroid were added to the trypsin solution. The fully prepared assay plate was incubated at 37 °C for 10 min. Following, 7 µl of AdipoRed reagent was added to each replicate of spheroid sample and culture medium background assay, respectively. For full dissociation of the 3D spheroid cell aggregates, repeated and vigorous resuspending of the suspension was performed. Subsequently, the assay plate was incubated in the dark at room temperature for 10 min and fluorescence intensity was measured  on an infinite 200 reader . The culture medium-derived signal for each condition was subtracted from all respective spheroid samples prior to determining the relative fold change of fluorescence signal intensity between FA-free BSA control and FA-incubated conditions.Intracellular TG content was determined using a fluorescent probe that specifically partitions into fat droplets , following the manufacturer’s instructions with slight modifications. For facilitating access of the reagent to all cells, the spheroids were disintegrated using trypsin treatment. Trypsin solution was prepared by a 1:1 dilution 0.5% trypsin-EDTA with PBS . Cell lysis was accomplished by repeated vigorous resuspension of the spheroids at 4 °C in a mixture of M-PER Mammalian Protein Extraction Reagent  and cOmpleteTM Mini Protease Inhibitor  working solution (prepared according to the manufacturer’s instructions), in a ratio of 9:1. The homogenate was sonicated 5 times for 15 s in ice cold water and subsequently centrifuged at 12000 × g and 4 °C for 15 min. The supernatant was extracted and total protein concentration determined on a Nanodrop 1000 spectrophotometer . Samples were stored at −80 °C until further analysis.For protein isolation, spheroids were pooled and washed twice with PBS  for the APOB-100 detection. Cell lysates as well as concentrated and diluted cell media samples were mixed with Laemmli buffer containing 2-mercaptoethanol (ratio sample: Laemmli buffer = 4:1 v/v) and incubated for 5 min at 95 °C. Proteins were size-separated by SDS-page  and transferred onto a nitrocellulose membrane . Nitrocellulose membranes were incubated overnight with primary antibodies, washed 3 times for 10 min with tris-buffered saline containing 0.2% tween (0.2% TBS-T buffer), incubated 1 h with HRP-conjugated secondary antibodies, washed 3 times for 10 min with 0.2% TBS-T buffer, incubated with Immobilon Western chemiluminescent HRP substrate . Bands were visualized using Chemidoc XRS System . The following antibodies were used: mouse anti-APOB , rabbit anti-calnexin , mouse anti-albumin .TM6SF2_rs58542926 (E167K), PNPLA3 rs738409 (I148M), PNPLA3 rs2294918 (E434K), GCKR rs1260326 (P446L) and MBOAT7 rs641738 (C > T) polymorphisms by TaqMan™ assays  using CFX384 Real Time PCR detection system . Data analysis was performed using Bio-Rad CFX manager software.DNA was extracted from hepatocytes using DNeasy Blood & Tissue Kit . DNA from all donors was undergone to genotyping for TM 7 Flex Real-Time PCR System . QuantStudioTM Real-Time PCR Software, version 1.3,  was used for data analysis.Total RNA was isolated using miRNeasy Mini Kit . RNA was reverse-transcribed into cDNA using High Capacity cDNA Reverse Transcription . RT-qPCR analysis was performed on customized microfluidic TaqMan™ array 384-well cards using TaqMan™ Fast Advanced Master Mix  on a QuantStudioSupplementary InformationSupplementary Table S2Supplementary Table S3Supplementary Table S4


The STAT signaling pathway is important in dendritic cell (DC) development and function. Tumor cells can induce STAT signaling, thereby inhibiting DC maturation and immunostimulatory functions, leading to hampered efficacy of DC-based immunotherapies. Platinum-based chemotherapeutics can inhibit STAT signaling, thereby making them an interesting tool to improve DC development and function. In this study, we provide a comprehensive overview of STAT expression and phosphorylation during DC differentiation and maturation and investigate the effects of platinum drugs on STAT signaling during these processes. Monocytes were differentiated into monocyte-derived DCs (moDCs) with IL-4 and GM-CSF and matured with cytokines or TLR ligands. STAT expression and phosphorylation were analyzed by western blotting, and moDC viability and phenotype were analyzed by flow cytometry. Platinum drugs were added at day 3 of differentiation or at the start of maturation to investigate regulation of the STAT signaling pathway. All STAT proteins were expressed during moDC differentiation and STAT1, STAT5, and STAT6 were phosphorylated. No significant changes occurred in the expression and phosphorylation state of the STAT proteins during differentiation. After maturation with TLR ligands, the expression of STAT1 increased, but other STAT proteins were not affected. Phosphorylation of STAT1 and STAT3 increased during maturation, where TLR ligands induced significantly higher levels of phosphorylation than cytokines. Platinum drugs cisplatin and oxaliplatin significantly inhibited phosphorylation of STAT6 during differentiation and maturation. Treatment did not affect the phenotype or viability of the cells. As STAT6 is an important regulator of DC function, these findings suggest a role for platinum-based chemotherapeutics to enhance DC function via inhibition of STAT signaling, thereby potentially enhancing efficacy of DC-based immunotherapies. The signal transducer and activator of transcription (STAT) signaling pathway is regulated by a family of 7 STAT proteins that can be induced by over 50 cytokines, growth factors, and hormones. Binding of these factors to their receptors initiates the phosphorylation of receptor-associated Janus kinases (JAKs). Phosphorylated JAKs subsequently form a docking site for STAT proteins where they are phosphorylated. Phosphorylated STAT proteins can then directly bind to DNA and regulate gene transcription . Additioα  , and R848 , respectively, for 24 hours.Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood  by density gradient centrifugation using lymphoprep (Axis-Shield). Adherent cells were obtained as previously described . Immaturμg/ml or 7 μg/ml, or cisplatin , at a concentration of 2.5 μg/ml or 5 μg/ml, was added to the culture medium, either at day 3 to day 4 or at day 6 to day 7, to investigate the effect on differentiation and maturation, respectively. Cells were harvested by adding 4°C PBS and were washed with PBS. Around 1 million cells were stored at 4°C for further flow cytometry analysis the same day and all other cells were snapfrozen and stored at -80°C until lysis. Purity of the harvested moDCs was on average 88%.MoDCs were treated with clinically relevant concentrations of platinum drugs during differentiation or maturation . OxaliplExpression levels of maturation markers and costimulatory molecules were evaluated using flow cytometry. Between 50,000 and 80,000 cells per well were first stained for cell viability using Fixable Viability Dye eFluor450  in PBS for 20 minutes. Thereafter, single stainings were performed with the antibodies shown in μg of protein for STAT1, 3, 5, and 6, 100 μg for pSTAT1, 3, and 6, or 200 μg for pSTAT5. Samples were fractioned by electrophoresis in 8% SDS-PAGE gels, using 30% 37.5 : 1 Acrylamide/Bis solution (Bio-Rad) and further processed for western blot analysis [STAT protein expression and phosphorylation status was assessed by western blotting. Cell pellets were lysed in lysis buffer (pH 7.8) containing 50 mM Tris base, 1 mM EDTA, 150 mM NaCl , 1% NP40 (Roche Diagnostics), 1 : 100 phosphatase inhibitor cocktail 2 and 3 (Merck), 1X protease inhibitor cocktail, and 1X PhosSTOP (both Roche Diagnostics). Before polyacrylamide gel electrophoresis, Laemmli sample buffer (Bio-Rad) was added 1 : 5 to cell lysates containing 25 analysis . After bAfter washing, membranes were incubated with 1 : 5000 goat-anti-rabbit IRDye800 , 1 : 5000 goat-anti-mouse IRDye680 , and 1 : 5000 goat-anti-rat IRDye680  as secondary antibodies and analyzed with the Odyssey Imaging system (LI-COR Biosciences).Western blot quantification and phenotype data are normalized to the loading control and are depicted as relative expression to day 2 or day 6 + SEM Figures  and 2 orα-isoform, increased during differentiation. Stable levels of phosphorylated STAT1 were observed from day 2 to day 6 and phosphorylation of STAT3 was weak or not detectable. Phosphorylation of STAT5 slightly decreased on days 4, 5, and 6 when compared to day 3 and STAT6 phosphorylation moderately increased at day 6, but these changes were not statistically significant. Furthermore, phosphorylation of STAT2 and STAT4 was not observed during differentiation as well as maturation and is therefore not shown  or cisplatin (2.5 μg/ml or 5 μg/ml) at day 3 of differentiation. μg/ml compared to control, and a similar trend was observed for the lowest concentration of cisplatin. The increase observed in the expression of total STAT6 emphasizes the potency of especially cisplatin to specifically inhibit phosphorylation of STAT6. In addition, to investigate the effect of platinum drug treatment on the phenotype of the immature moDCs, expression of HLA-ABC and HLA-DR/DP/DQ and costimulatory molecules CD80 and CD86 was analyzed  or cisplatin (2.5 μg/ml or 5 μg/ml) was added at day 6, together with the maturation stimuli. μg/ml) or cisplatin significantly inhibited the phosphorylation of STAT6 at day 7 compared to untreated matured moDCs. The lowest concentration of oxaliplatin significantly decreased phosphorylation of STAT6 in cytokine-matured moDCs, but not in TLR-matured moDCs. The expression levels of HLA-ABC and HLA-DR/DP/DQ and costimulatory marker CD86 were not affected by treatment of the mature moDCs . Therefore, future research is needed to assess if platinum drugs stimulate blood-DC subsets similar to moDCs and what adjuvants are needed to create the optimal environment for platinum drugs to potentiate DC function. Ultimately, platinum drugs could be an interesting potential tool to regulate the STAT signaling pathway in order to improve DC function and enhance the efficacy of DC-based immunotherapies.Our findings in moDCs indicate that combination therapy with platinum compounds possibly also enhances the efficacy of naturally occurring blood-DC vaccines, which are a promising alternative to moDC vaccines . Howeverotoxin B , 44. Howotoxin B . Furthertivation . However


N-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide)); formerly STA-4783) is a mitochondria-targeted chemotherapeutic agent that has demonstrated efficacy in selective cancer cell killing in pre-clinical and clinical testing. The biologically active form of elesclomol is a deprotonated copper chelate , which has been shown to enhance reactive oxygen species (ROS) production and induce a transcriptional gene profile characteristic of an oxidative stress response in vitro. Previous studies suggest that E:C interacts with the electron transport chain (ETC) to generate high levels of ROS within the organelle and ultimately induce cell death. The purpose of this study was to further explore the mechanism of cellular and mitochondrial toxicity of E:C by examining its direct effect on mitochondrial bioenergetic function. The results obtained indicate that E:C treatment in whole cells of non-tumorigenic origin at high concentrations (40 μM and higher) induces a rapid and substantial increase in mitochondrial superoxide levels and dissipation of mitochondrial membrane potential. Furthermore, similar higher concentrations of E:C act as a direct uncoupler of oxidative phosphorylation and generalized inhibitor of electron transport activity in isolated, intact mitochondria, and induce a dose-dependent inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity in freeze-thawed mitochondrial preparations. The results of this study are important in that they are the first to demonstrate a direct effect of the E:C chelate on bioenergetic function in isolated mammalian mitochondria, and suggest the possibility that the increase in ROS production and cytotoxicity induced by E:C may in part be due to uncoupling of mitochondrial oxidative phosphorylation and/or inhibition of electron transport activity. These results also provide important information about the mechanisms of mitochondrial and cellular toxicity induced by E:C and will ultimately contribute to a better understanding of the therapeutic potential of elesclomol as an anticancer compound. Elesclomol (( N-malonyl-bis(N′-methyl-N′-thiobenzoylhydrazide)); formerly STA-4783; Elesclomol  and a gene transcription profile characteristic of an oxidative stress response in vitro . Interesthe drug . 2+ in the bloodstream. However, in vitro studies investigating the mechanism of action of elesclomol have demonstrated that the addition of a pre-formed elesclomol:copper (E:C) complex is necessary to induce cytotoxicity [The biologically active form of elesclomol is a deprotonated copper chelate . Upon thtoxicity . In one toxicity . The resThe purpose of this study was to further explore the mechanism of cellular and mitochondrial toxicity of E:C by examining its direct effect on mitochondrial bioenergetic function in whole cells of non-tumorigenic origin and in isolated mammalian mitochondria. The results obtained indicate that E:C treatment in whole cells induces a rapid and substantial increase in mitochondrial superoxide levels and dissipation of mitochondrial membrane potential. Furthermore, E:C acts as an uncoupler of oxidative phosphorylation and generalized inhibitor of electron transport activity in isolated, intact mitochondria, and induces a dose-dependent inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity in freeze-thawed mitochondrial preparations. The results of this study are important in that they are the first to demonstrate a direct effect of the E:C chelate on bioenergetic function in isolated mammalian mitochondria, and suggest the possibility that the increase in ROS production and cytotoxicity induced by E:C may in part be due to uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport activity, or both.https://www.medchemexpress.com/) and made fresh daily at a concentration ranging from 1–10 mM in dimethyl sulfoxide (DMSO). The E:C complex was formed by mixing equimolar and equivolume additions of elesclomol and copper chloride (dissolved in H2O). MitoSOX Red mitochondrial superoxide indicator for live-cell imaging was obtained from Molecular Probes ; the mitochondrial superoxide scavenger MitoTEMPO was obtained from Sigma Aldrich ; the mitochondrial membrane potential probe TMRE  was obtained from Biotium ; and the live cell nuclear stain, NucBlue Live, and the cell viability stain, NucGreen Dead, were obtained from ThermoFisher Scientific . All solutions were used according to the manufacturer’s specifications.Elesclomol was obtained from MedChemExpress (® CCL-70™) were grown in Eagle′s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum. All cells were maintained at 37 °C in a 5% CO2 atmosphere.The CV-1 African green monkey kidney epithelial cells . The CV-1 cells were seeded in glass bottom 35 mm plates (MatTek) and grown to 90% confluency. To initiate an experiment, cells were incubated for 10 min with 5 μM MitoSOX in phenol red free EMEM, washed twice with sterile phosphate buffered saline, and replenished with phenol red free EMEM and 2 drops/mL media of NucBlue or NucGreen. Cells were then treated with either DMSO (control) or 70 μM E:C, and MitoSOX, NucBlue, and/or NucGreen fluorescence was monitored over a period of 1 h. To block mitochondrial superoxide accumulation, some cells underwent a 1-h pre-treatment with 5 μM of the free radical scavenger MitoTEMPO prior to addition of DMSO or E:C. 4-(trifluoromethoxy)phenylhydrazone; 100 μM). Mitochondrial membrane potential was assessed by measuring TMRE fluorescence using a Zeiss 800 confocal microscope. The CV-1 cells were seeded in glass bottom 35 mm plates (MatTek) and grown to 90% confluency. To initiate an experiment, cells were washed twice with sterile phosphate buffered saline and incubated for 10 min with 100 nM TMRE in phenol red free EMEM and two drops/ml media of NucBlue or NucGreen. Cells were then treated with either DMSO (control) or 70 μM E:C, and TMRE and NucBlue and/or NucGreen fluorescence was monitored over a period of 1 h. As a positive control for uncoupling of oxidative phosphorylation and dissipation of mitochondrial membrane potential, cells were incubated for 10 min in the presence of the ionophore FCCP ). The final pellet was re-suspended in ST and the protein concentration of the mitochondrial suspension was determined by the method of Lowry [Mitochondria were isolated from bovine or rat liver by a process of differential centrifugation at 4 °C . Brieflyof Lowry .2HPO4-KH2PO4, 5 mM MgCl2, and 10 mM Tris-HCl (pH 7.4). An initial rate of oxygen consumption (state 2 rate) was recorded after the addition of mitochondria (approximately 1.5 mg) and respiratory substrate . The ADP (120 nmol) was then added to obtain a state 3 rate, which is a measure of oxygen consumption that is coupled to the process of ADP phosphorylation to ATP. Following a measurable state 4 rate , 80 µM of the chemical uncoupler 2,4-dinitrophenol was added to obtain a respiratory rate in the absence of coupled oxidative phosphorylation. Respiratory rates were measured in the absence or presence of varying concentrations of E:C (25–100 μM).Oxygen consumption was measured in freshly isolated rat liver mitochondria using a Clark-type polarographic oxygen electrode  in a solid-state Peltier temperature controlled chamber maintained at 30 °C. The basic respiratory assay medium consisted of 225 mM sucrose, 10 mM KCl, 1 mg/mL 10 mM KSpectrophotometric measurements of mitochondrial electron transport enzyme activities were obtained using a single-wavelength, temperature-controlled spectrophotometer at 37 °C essentially as described previously ,12. All 1 to the cuvette, and the change in the absorbance was recorded over time in the presence and absence of 10 μM rotenone. The rotenone sensitive activity of complex I of the mitochondrial ETC was determined by subtracting the rate measured in the presence of rotenone from the rate obtained in the absence of rotenone. The NADH-ubiquinone oxidoreductase activity (a measure of electron transport through respiratory complex I) was determined by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. Mitochondria (300 µg) that had been subjected to three cycles of freeze-thawing in liquid nitrogen were added to a cuvette containing 50 mM potassium phosphate, 3 mg/mL fatty acid free BSA, 300 μM KCN, 100 μM NADH in the absence or presence of varying concentrations of E:C (25–100 μM). The reaction was initiated by adding 10 μM ubiquinone2O, 50 mM potassium phosphate , 30 μM KCN, in the absence or presence of varying concentrations of E:C (25–100 μM). The sample was pre-incubated with the 20 mM succinate for 10 min at 37 °C in order to fully activate the enzyme. The reaction was initiated by adding 50 μM oxidized cytochrome c to the cuvette, and a change in the absorbance was recorded over time. Succinate-cytochrome c reductase activity (a measure of electron transport through respiratory complexes II and III) was determined by measuring the rate of increase in absorbance at 550 nm due to the reduction of oxidized cytochrome c. Freeze-thawed (−20 °C) preparations of isolated mitochondria (300 μg) were added to a cuvette containing dH2O, 40 mM potassium phosphate , in the absence or presence of varying concentrations of E:C (25–100 μM). The reaction was initiated by adding 50 μM reduced cytochrome c, and a linear rate was recorded. Cytochrome c oxidase activity (a measure of electron transport through complex IV) was determined by measuring the decrease in absorbance at 550 nm due to the oxidation of reduced cytochrome c. Freeze-thawed (−20 °C) preparations of isolated mitochondria (300 μg) were added to a cuvette containing dHThe effect of E:C treatment on mitochondrial superoxide production in whole cells was determined using the live-cell permeant superoxide indicator, MitoSOX, which is selectively targeted to the mitochondria and fluoresces red upon oxidization by superoxide (but not by other ROS or reactive nitrogen species). To assess the effect of E:C treatment on mitochondrial membrane potential in whole cells, CV-1 cells were treated with 70 μM E:C in the presence of TMRE. The data presented in To assess the relationship between E:C-induced superoxide generation and loss of mitochondrial membrane potential in whole cells, CV-1 cells were treated with varying concentrations of E:C (5–70 μM) for a constant exposure time (20 min) in the presence of MitoSOX or TMRE. The data presented in Additionally, 2 consumption. The results show that when either glutamate/malate (a complex I electron donor) or succinate (a complex II electron donor) was used as the respiratory substrate, E:C stimulated the S2 respiratory rates in a dose dependent manner over a concentration range from 0–50 μM. These data suggest that E:C acts as an uncoupler of oxidative phosphorylation. However, when either glutamate/malate or succinate was used as the respiratory substrate, a significant decrease in O2 consumption was observed at the highest concentration of E:C tested (100 μM).Measurements of oxygen consumption in intact, isolated rat liver mitochondria were made in the absence or presence of varying concentrations of E:C and presented in In the presence of ADP, the oxygen consumption rate measures oxidative phosphorylation; that is, the respiratory rate in which substrate oxidation (via electron transfer) is coupled to ATP synthesis. The state 3 (S3) respiratory rate is that which is measured in the presence of substrate and added ADP, while the ADP-stimulated rate is calculated by subtracting the S2 respiratory rate (substrate only) from the S3 rate. The results show that when either glutamate/malate or succinate was used as the respiratory substrate, 25 μM E:C had no significant effect on S3 or ADP-stimulated respiratory rates A,B. HoweThe uncoupler-stimulated respiratory rate is induced by addition of the proton ionophore 2,4-dinitrophenol  and is essentially a measure of overall electron transfer activity that is no longer coupled to ATP synthesis. Results in 2 or elesclomol as single agents. The results in 2 acted as an uncoupler of oxidative phosphorylation, inducing a dose-dependent increase in S2 respiratory activity over the range of concentrations tested (25–100 μM E:C), but having no significant effect on the S3 respiratory rate. The observed decrease in the ADP-stimulated (S3-S2) rate therefore occurred as a consequence of (and in proportion to) the increase in the S2 rate, and not as a consequence of inhibition of ATP synthesis and/or electron transport activity. A dose dependent inhibition of the DNP-stimulated respiratory rate was also observed over the range of concentrations tested (25–100 μM). When succinate was used as the respiratory substrate, the uncoupling effect by CuCl2 was more immediate and pronounced (2 and maintained up to the highest concentration tested (100 μM). The S3 respiratory rate was not affected at a concentration of 25 μM CuCl2, but was inhibited by approximately 60% at both the 50 and 100 μM CuCl2 concentrations. The DNP-stimulated respiratory rate was inhibited approximately 75% compared to the control value at each concentration of E:C tested (25–100 μM E:C). This decrease in both the S3 and DNP-stimulated respiratory rates is likely due to either a general membrane disruption of the ETC activities, or to an excess of uncoupling agents  rather than to a site-specific inhibition of electron transport activity. Polarographic measurement of oxygen consumption in intact, isolated rat liver mitochondria was also made in the absence or presence of varying concentrations of either CuClonounced D. Maxima2) had no significant effect on S2, S3 ADP-stimulated, nor DNP-stimulated rates when either glutamate/malate or succinate was used as the respiratory substrate (Over the range of concentrations tested (25–100 μM) elesclomol as a single agent , through complex II and III, to cytochrome c; and, cytochrome c oxidase activity measures the rate of complex IV directly by monitoring its rate of oxidation of reduced cytochrome c. The effect of varying concentrations of E:C on electron transfer enzyme activities was investigated using freeze-thawed preparations of isolated mitochondria. In order to discern any potential site-specific action of the E:C chelate, assays were chosen such that only limited segments of the respiratory chain were engaged in electron transfer activity at any one time. For example, NADH-ubiquinone oxidoreductase activity is a measure of the rate of electron transfer from the oxidizable substrate NADH, through complex I; succinate-cytochrome c reductase activity is a measure of electron transfer from an oxidizable FADH50) achieved at a concentration of approximately 70 μM. The effect of E:C on succinate cytochrome c reductase and cytochrome oxidase activities are shown in The effect of E:C on NADH-ubiquinone oxidoreductase activity is shown in 2−•. The mitochondrial enzyme superoxide dismutase converts O2−• to H2O2, which can then acquire an additional electron from a reduced transition metal to generate the highly reactive hydroxyl radical, .OH. Under controlled conditions, ROS play an important role as signaling molecules that mediate changes in cell proliferation, differentiation, and gene transcription [The results of this study indicate that E:C treatment in non-tumorigenic cells induces a rapid and substantial increase in mitochondrial superoxide levels . This iscription ,15. Howecription . Interescription ,18. It hcription ). In faccription ,20.Additionally, the data presented here show that treatment of non-tumorigenic cells with the same concentration of E:C that induces a rapid and substantial increase in mitochondrial superoxide levels, also dissipates the mitochondrial membrane potential to an extent sufficient to cause changes in cell morphology suggestive of a loss of membrane integrity or dying/dead cells . Further2 as a single agent also acts as an uncoupler mitochondrial oxidative phosphorylation irrespective of whether a complex I or complex II electron donor is used as a respiratory substrate  in freeze-thawed mitochondrial preparations A. Compleferation . It is sferation . Interesferation ,20,23,24m), and that uncoupling agents, which lower ΔΨm, will decrease mitochondrial ROS production [m, and raised the possibility that FCCP induced ROS production may be from a source other than the mitochondria. In another study using rat liver mitochondria, the results provided evidence that FCCP caused a burst of H2O2 production that preceded membrane permeabilization [It is of interest to consider the relationship between the uncoupling and ROS generating effects shown to be induced by E:C in this study. It is generally accepted that ROS production is dependent upon the mitochondrial transmembrane electric potential difference , and to display additive interactions with known tuberculosis drugs, such as isoniazid and ethambutol, and a synergistic interaction with rifampicin [Mtb in the presence of copper compared to non-copper supplemented growth medium. The authors suggest that the ability of elesclomol to enhance ROS generation and accelerate uptake and intracellular accumulation of copper, which is toxic to Mtb at high concentrations, as possible mechanisms for the compound’s bactericidal efficacy. Additionally, a very recent report elucidated a unique mechanism of action of cytotoxicity by elesclomol in cancer cells that exhibit proteasome inhibitor resistance or a forced shift away from glycolysis and toward a state of enhanced mitochondrial metabolism, or both [Earlier reports have clearly established the ability of elesclomol to generate high levels of mitochondrial ROS and ultimately induce cell death in a variety of cancer cell lines. Several recent studies show that elesclomol can produce a number of other biological effects in different models, suggesting the possibility of multiple mechanisms of action and cellular targets for the compound. For example, it has been shown that low nanomolar concentrations of elesclomol can be used to shuttle sub-lethal concentrations of copper into the mitochondria as a therapeutic strategy to restore mitochondrial respiratory activity in copper deficient yeast mutants . Using mfampicin . Interes or both . In thesThe results of the study presented herein provide important information about the mechanisms of mitochondrial and cellular toxicity induced by E:C. First, they reveal at least two additional E:C targets directly affecting mitochondrial bioenergetic function. The data indicate that E:C treatment in non-tumorigenic cells can induce a rapid and substantial increase in mitochondrial superoxide levels and dissipate the mitochondrial membrane potential. Furthermore, the data demonstrate that over the range of concentrations tested, E:C acts as both an uncoupler of oxidative phosphorylation and a generalized inhibitor of electron transport activity in isolated, intact mammalian mitochondria, and induces a dose-dependent inhibition of mitochondrial NADH-ubiquinone oxidoreductase activity in freeze-thawed mitochondrial preparations. These results suggest the possibility that the increase in ROS production and cytotoxicity induced by E:C may at least in part be a result of the uncoupling of mitochondrial oxidative phosphorylation or inhibition of electron transport activity, or both. The results also suggest the possibility that differences between the mitochondria of normal versus cancer cells  might somehow contribute to the selective cytotoxicity exhibited by elesclomol for certain cancer cell types. Indeed, nearly a century of scientific research has revealed a number of notable differences in the structure and function of mitochondria between normal and cancer cells, including differences in mtDNA sequence, molecular composition, and metabolic activity (for review see ). Additi


The application of chopped-fiber reinforced polymers in screwed connections at high temperatures raises the question of creep under long-term loading. While up to now thermoplastic materials have mainly been the focus of attention when it comes to creep, this paper shows that thermoset carbon-fiber SMCs (sheet mold compounds) can also be affected by this phenomenon. Screwed connections were investigated regarding their loss of preload force at 120 °C ambient temperature. Additionally, strain–time diagrams were recorded at different stress levels at 120 °C in a creep test setup of a universal testing machine by using optical strain tracking of SMC coupons. The transverse modulus under compression in thickness direction was determined in the same test setup. For data application within a FEA  software power law curves according to Norton–Bailey creep law were fitted in the strain–time graphs. The applicability of the obtained creep law was crosschecked with a test carried out on the loss of preload force of a screwed connection. The developed simulative methodology offers the possibility to simulate various mounting situations of the bolted connection and to investigate measures against the loss of preload force easily. A promising possibility to limit the loss of preload force due to creep was simulatively evaluated.  Usually, a screw in mechanical engineering is used to clamp two materials with a force, which is high enough to induce friction and thus avoid sliding between the surfaces of the clamped parts. The factors influencing the sliding of the surfaces against each other are the clamping force of the screw and the coefficient of friction between the screwed surfaces. Attention in this study is put on the clamping force. The clamping force may not be too high, to avoid unnecessary high stress in the screw or yielding of the clamped material. If the clamping force decreases too much, the screwed surfaces can move relative to each other, due to an external force. Both limit cases should be avoided. Therefore, a change of screw preload force, after the tightening of the screw, is not desired or at least has to be within well-known limits ,2.Screw connections suffer from loss of preload, due to different reasons: dynamic forces can cause settlement of surface roughnesses, rotational self-loosening of the bolt/nut, and/or fatigue of the clamped/bolt material . Plenty There are several approaches on prediction of preload loss for screw assemblies ,4,5,6,7.Alternatively, it is also possible to develop a universal creep law for a material in the FEA, which allows a simulative problem analysis in different application scenarios. This procedure, with full implementation in the FEA, has been reported for unreinforced epoxy specimens, i.e. isotropic material . The creIn this paper the work of  was pickSMC sheets typically contained chopped fiber-rovings, with lengths up to 50 mm with random orientations. These chopped fiber-rovings fell onto a continuous resin mat in a production line and were then covered with another resin mat. Both surfaces were then covered with a protective foil. The material was rolled up afterwards .To ease readability in the following, the acronym “C-SMC” is used for the material “Polynt SMCarbon 80 CF60-3K/2”. The material is shown in According to the datasheet 3000 filaments per roving were used and 50% of them were chopped into 25 mm length and 50% into 50 mm length. The C-SMC uses a fast curing epoxy resin. The cure cycle was 5 min at 145 °C and 100 bar internal mold pressure, according to the data sheet. Test plates with a thickness of 3.6 mm were produced with a covered mold area of 80% by the raw material, so material flow effects were kept on a low level. Every test plate was post cured at 160 °C for 2 h in an oven. The tensile modulus and strength were tested according to ISO 527-4, averaging 20 test specimens . Table 1The elastic modulus in the thickness direction is necessary for modelling creep in the thickness direction. For this, C-SMC specimens with the dimensions 31.7 mm × 31.7 mm were prepared from a 3.6 mm thick coupon plate . A tableTwenty-four specimens were stacked, resulting in a 86.4 mm high stack. The stack was placed between pressure discs of a Zwick Roell 50 kN universal testing machine, equipped with an oven chamber . Before For determination of the transverse modulus in thickness direction (z direction) the universal testing machine applied alternating forces between 5 kN and 15 kN  at 2 MPa stress level and around 100,000 s (27.8 h) at 20 MPa stress level .Development of a Norton–Bailey creep law approximation was the next step to fit the obtained data for the simulation. Three temperature dependent regression constants are needed for application of the power law. They can be determined by curve fitting of measured data via the method of least error squares. This method was used here, within a spreadsheet program. ,25The Norton–Bailey creep law is shown in Equation (2). For the measured creep strain data see , the regIt should be noted that the obtained Norton–Bailey creep constants for the material are only valid, if the creep takes place in the first two creep stages. If the pressure is much higher than the 20 MPa mentioned above, a tertiary creep stage should be questioned, which would significantly accelerate the creep process. To rank at which pressure damage is to be expected in the laminate: A previous publication stated, that CFRP has a permissible surface pressure of 140 MPa or higher in static testing [A screw assembly with a force measuring washer was set up to measure the loss of preload force over time at a real setup. The force measuring washer contains strain gauges, which are used to measure the applied compression force. Large-series steel washers had direct contact with the composite specimens with an inner and outer diameter of 9 and 24 mm (according to ISO 7093 ). A tablA 9-mm wide borehole was milled into the C-SMC specimens to have a loose fit with the M8 screw. The screw assembly consisted of 15 stacked specimens (screw assembly A). Every C-SMC plate was constrained with a washer at the top and on the bottom. The washers have one levelled side, which is placed in contact with the C-SMC coupons. All steel parts of the screw assembly are made from the stainless steel A2-70 with an expected le level ,28.a = 1 µm and the C-SMC coupons to Ra ≤ 1 µm. In previous publications a correlation between the surface roughness of clamped parts and the loss of preload was stated . A high The screw assemblies were stored without any preload force in an oven at 120 °C for 12 h to ensure a constant temperature distribution. Afterwards, the screws were tightened to 10 kN preload relatively fast (around 1 min) manually from outside of the oven. A wrench with extension was placed in a feedthrough, which is usually used to get wiring out of the oven. Inside the oven, a frame was placed, which is made from aluminum profiles, with a vice attached to it. The vice was holding the screw assembly. This test setup, where tightening of the screw can be done inside the closed oven, ensures a constant temperature through the assembly at all times of the experiment.2 to the CFRP. With the preload of 10 kN a mean stress of 25 MPa was applied in z direction of the CFRP, when measurement started. Therefore, plastic deformation or damage in the C-SMC, due to too high preload on the screw, should not be an issue, following this rough estimation. As mentioned, a previous publication stated that CFRP has a permissible surface pressure of 140 MPa or higher . The M8 6 s (1000 h). The change of the measured screw force over time is displayed in The time resolved force changes of both screw assemblies A and B were tracked over 3.6 × 10It can be seen, that screw force loss at screw assembly A is dominant in the primary creep stage within the first 360,000 s (100 h) and is then significantly reduced in the secondary creep stage. The graphs are staggered due to the discrete resolution of the measured screw preload force .For testing of plausibility of the obtained data  a simulaThe obtained regression constants from The simulation was built up as a transient solution with solver 601. Fixed timesteps, which increase logarithmic over time, were defined. The logarithmic increase of the time step size seemed reasonable, since the strain changes at the beginning of the simulation are still greatest and will become smaller and smaller later. The first timestep sizes were only fractions of a second. The block was modeled solid and no contact formulations were used. Since there is a uniform stress state in all test coupons, contact modelling should not lead to a different result at all. The model contained a total of about 11,000 finite elements. Linear hexahedral elements were used.The applied force was chosen as described in The effective stress, which is calculated with the deviatoric stresses, is used for the calculation of g guide” .The simulation revealed a good approximation to the fitted Norton–Bailey curve at both stress levels 2 MPa and 20 MPa . This caAnother publication dealt also with the simulation of creep behavior of composites in the thickness direction . An offsA CAD (computer-aided design)  and FEA All contact areas of the screw assembly were provided with nonlinear contacts with a typical coefficient of friction of 0.15 between CFRP and steel. The coefficient of friction between metal and CFRP is discussed in various publications, e.g., ,33, and 5 1/K was used for all steel parts. The C-SMC had the same material card, as described in The simulation was built up as a nonlinear solution 601 in SIEMENS NX Simcenter 12. Fixed time steps, which increase logarithmic over time, were defined. The pretension of the screw was modelled by cooling of the screw neck and thus use of the material’s thermal contraction. In the first 0.01 s of the simulation, the screw neck was cooled down. A simple isotropic steel material card with 200 GPa Young’s modulus, Poisson’s ratio of 0.25, and a thermal expansion of 11 × 10At 0.01 s a pretension of 2.5 kN, or respective 10 kN for a full model, was applied on the bolt. There was no larger time delay implemented, which could represent the actual tightening process, because measurement of creep strain also started just after the desired stress level was reached . The bolThere is a high agreement between simulation and measurement data , even whThere are different options for prevention of screw preload losses. When designing with polymers, the use of expansion bolts or conical spring washers is conceivable to compensate creep-induced preload losses. Both methods reduce the amount of preload loss, when the screw strain is lowering due to creep of the clamped material. Another possibility is to transfer the preload force of the screw not into the creeping material, but into an adapter sleeve made out of steel .However, the last-named method is often entailed with higher implementation effort at part design and higher manufacturing costs. In the following, the first approach of compensating the bolt preload loss by conical spring washers is examined in more detail.The obtained creep data from 7 s (7777 h or 324 days).The screws were tightened to 2.5 kN in the first 0.01 s of the simulation . The scrIt can be seen, that the screw assembly with the conical spring washer was able to maintain 87% of its preload force (8.7 kN) at the last time step, whereas the screw assembly without conical spring washer maintained only 46% (4.6 kN) of its original preload force. These values are not universal applicable for different screws, but have to be crosschecked at every use case. The percentage loss in preload force can be further reduced by using multiple spring washers in a bolted joint. The developed modelling approach proves its high effectiveness and enables long-term prediction of the reliability of the chosen screw design .This paper provides a simulative modelling approach of predicting the creep behavior of composite materials, that are clamped in screwed connections. 6 s (1000 h), which is a great advantage in terms of experimental effort. The test coupons for determining the Norton–Bailey regression constants as well as the screwed samples had a laminate thickness of 3.6 mm. It is expected, that the agreement between simulation and experiment is the highest, when both tests are done with the same laminate thickness. One carbon-fiber SMC with one material thickness was investigated in this paper. Testing more materials and different material thicknesses would be interesting aspects for future investigations. A material card was created for the SMC material “Polynt SMCarbon 80 CF-60 3K/2” to simulate creep in thickness direction at 120 °C. A very good agreement was found between a bolt preload loss test and its simulation. The measured time of 300,000 s (83.3 h) as a basis for the Norton–Bailey creep law regression constants was not much, compared to a typical product lifetime. An extrapolation of the obtained data revealed a very good agreement even with a much longer run of the experiment of 3.6 × 10The lack of operability of the Norton–Bailey creep law in orthotropic material cards in current FEA software is a major drawback for composite material creep modeling. Nevertheless, as the main load at the screw assembly is applied in thickness direction, the FE simulation with an isotropic material card revealed still an accurate correlation with measured results .The high creep rate at the temperature of 120 °C was not expected for the investigated C-SMC. The phenomenon creep in thickness direction seems to be challenging for SMCs, even with a gap of 30 °C to the glass transition temperature. From design point of view, it was shown, that conical spring washers can be an effective way of limiting creep-induced loss of screw preload force.


Cecropia have been used by Latin-American traditional medicine to treat metabolic disorders and diabetes. Previous results have shown that roots of Cecropia telenitida contain pentacyclic triterpenes and these molecules display a hypoglycemic effect in an insulin-resistant murine model. The pharmacological target of these molecules, however, remains unknown. Several lines of evidence indicate that pentacyclic triterpenes inhibit the 11β-hydroxysteroid dehydrogenase type 1 enzyme, which highlights the potential use of this type of natural product as phytotherapeutic or botanical dietary supplements. The main goal of the study was the evaluation of the inhibitory effect of Cecropia telenitida molecules on 11β-hydroxysteroid dehydrogenase type 1 enzyme activity. A pre-fractionated chemical library was obtained from the roots of Cecropia telenitida using several automated chromatography separation steps and a homogeneous time resolved fluorescence assay was used for the bio-guided isolation of inhibiting molecules. The screening of a chemical library consisting of 125 chemical purified fractions obtained from Cecropia telenitida roots identified one fraction displaying 82% inhibition of the formation of cortisol by the 11β-hydroxysteroid dehydrogenase type 1 enzyme. Furthermore, a molecule displaying IC50 of 0.95 ± 0.09 µM was isolated from this purified fraction and structurally characterized, which confirms that a pentacyclic triterpene scaffold was responsible for the observed inhibition. Our results support the hypothesis that pentacyclic triterpene molecules from Cecropia telenitida can inhibit 11β-hydroxysteroid dehydrogenase type 1 enzyme activity. These findings highlight the potential ethnopharmacological use of plants from the genus Cecropia for the treatment of metabolic disorders and diabetes.Plant extracts from the genus  Obesity is increasing worldwide with recent estimated figures of 1.4 billion individuals . HyperteRecent evidence suggests that an imbalance in glucocorticoid homeostasis plays a major role in obesity and MetS . PartialSeveral lines of evidence support a connection between 11β-HSD1 and MetS. Studies utilizing 11β-HSD1 knockout mice revealed improved glucose tolerance, lipid profiles, insulin sensitivity, and a reduction in body weight ,8. In coSeveral pharmaceutical companies have identified numerous 11β-HSD1 inhibitors and many of these have entered Phase I trials . High thhttps://www.ebi.ac.uk/chembl/) using 11β-HSD1 as a target, revealed that 410 molecules have been reported as 11β-HSD1 inhibitors. Among these molecules, close to 15% are PTs or steroids. For example, PTs in the leaves of the medicinal plant loquat (Eriobotrya japonica) displayed an additive low micro-molar inhibitory effect on 11β-HSD1 in cellular lysates . The signal is inversely proportional to the concentration of cortisol in the sample or calibrator. In each assay plate, cortisol concentration was determined from a calibration curve determined for delta F versus cortisol standards. Inhibitor IC50 values were determined for concentration-dependent inhibition curves using GraphPad Prism version 7.00 for Windows .Enzymatically produced cortisol was quantitated by an HTRF-based competition assay ,25,26. IC. telenitida chemical fractions and the isolated molecule. Cortisol production was quantitated by HTRF in 100 µL reactions. Each 96-well assay plate included a calibration curve, positive and negative reaction controls, and 11β-HSD1 reactions with library molecules. The well-documented 11β-HSD1 inhibitor known as carbenoxolone was added at 0.9 µM as a positive inhibition control [11 β-HSD1 inhibition was determined by comparing the amount of cortisol produced in the presence and absence of  control . The negi and C0 are the cortisol concentrations in the 11β-HSD1 reactions with and without inhibitors, respectively. The term Sb corrects for non-enzymatically produced cortisol as well as artifacts in the cortisol detection method. The cortisol concentration in the negative reaction control is a reaction with all components minus NADP+/NADPH.Percentage inhibition was calculated using Equation (1) where CFractions displaying percentage inhibition greater than 73% were considered as “hits”. This inhibition threshold was determined as the average inhibition percentage displayed by the entire library plus three times the standard deviation for all samples tested .+/NADPH), The Z-factor (or Z′) was determined as a measure of the quality control of an assay . This teeviation .(2)Z′=1Nuclear magnetic resonance experiments were performed on a Bruker Ascend III HD 600 MHz spectrometer equipped with a 5 mm cryoprobe -TCI with an effective resolution of 900 MHz . The mono and bidimensional experiments were stored using the standard pulse sequences of the equipment. Chemical shifts (δ) are expressed in ppm and the data analysis was carried out using MestReNova version 11.0 . Mass spectra were obtained in a SQD2 detector coupled to a UPLC H-class chromatographic system in positive and negative modes and data analysis was performed using Waters MassLynx™ software Version 4.1 . The elemental composition and high-resolution measurement were carried out on a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer .C. telenitida contained the greatest concentration of PT molecules [Previous studies indicated that the roots of olecules . TherefoAutomated flash chromatography with pre-packed silica-based columns was utilized to reduce the complexity of an extract. Grouping molecules from different extracts according to the sequential polarity of the fractions, from 21 to 27, was utilized to save costs in further downstream analysis see . The fra+ cofactor regeneration system was used based on glucose 6-phosphate dehydrogenase. This was done to favor the forward reaction during the end-point assay. The reaction utilizing this coupled system was linear for up to 6 h.HTRF based inhibition assays for 11β-HSD have been developed using whole cells, cell lysates, and microsomes . MicrosoColorimetric derivatization of the thin layer chromatography analysis from all fractions presumably revealed a high content of terpene features (data not shown). In accordance with previous results, the majority of PTs were grouped in the DE and E base extracts. Nevertheless, the entire library was assayed for 11β-HSD1 inhibitors in order to completely explore the generated chemical space. Library fractions were diluted to 100 µg/mL in DMSO and tested for precipitation by measuring turbidity. None of the fractions precipitated under the conditions tested. Working dilutions at 1 µg/mL in DMSO were used to test for 11 β-HSD1 inhibition see .The suitability of our methodology for high throughput screening of 11β-HSD1 inhibitors was confirmed by calculating the Z′ value  using alC. telenitida pre-fractionated chemical library, the mean value of inhibition percentage for all of the fractions was 24 ± 16.5. Therefore, we set the threshold for 11 β-HSD1 inhibition at the mean plus three standard deviations. Therefore, fractions eliciting a reduction in cortisol production greater than 73% are inhibitors with a 99% confidence limit.In a well-behaved assay, a true inhibitor should be well above the mean for all other compounds tested. In the case of the C. telenitida roots (125 fractions), only one fraction (DE16) met the established criteria for inhibition. A second round of testing by quadruple independent assays confirmed a positive hit for inhibition. Therefore, only 0.8% of the library showed inhibition, as would be expected for an unbiased diverse library [After screening of the entire chemical library of  library . After sCecropia plants [The capability of triterpene molecules from natural sources to inhibit the enzyme, 11β-HSD1, has been previously reported ,32,33. Ta plants ,34. Due C. telenitida triterpene molecules are responsible for the inhibitory effect observed in fraction DE16, extensive chromatographic separation, and purification steps were performed. Ten sub-fractions were obtained from the DE16 eluent and each one was tested in the 11β-HSD1 inhibition assay  was determined by the Q-Exactive hybrid quadrupole-Orbitrap mass spectrometry and the experimental data were m/z 489.35580 (1.65 ppm error). The chemical structure derived from these data is illustrated in The structure of the molecule isolated in the DE16 sub-fraction 8 was unambiguously determined by The proposed structure of isoyarumic acid is supported by spectral analysis, assignment from HMBC and HSQC correlations, and characteristic chemical shifts see . The douCecropia telenitida bears structural features displayed by other triterpene molecules capable of inhibiting 11β-HSD1. In general, an ursan scaffold, which is also observed in the triterpene corosolic acid, is prominent . The potential effect of structural features unique to isoyarumic acid on the interaction of the inhibitor molecule with 11 β-HSD1 requires further scrutiny. Nevertheless, our results provide additional evidence of the capacity of natural triterpenes to act as 11 β-HSD1 inhibitors and, more importantly, highlight specific structural features that support a positive interaction with the enzyme.Rollinger et al. utilized both experimental and computational data and analyzed several naturally-occurring triterpene molecules . The 11 The structural components that determine the specific inhibition of 11β-HSD type 1 over type 2 are not completely understood, but are likely to be very subtle. Glycyrrhetinic acid see C, which Purified fractions of extracts obtained from some terrestrial plants may potentially be used in a chemically controlled way to favor the additive effect of a natural pharmacophore, which is a premise supported by the ability of plants to biosynthesize structurally-related molecules. The ability to use a natural semi-purified group of substances, with each having a moderate potency, is one of the possible reasons that explains the satisfactory results often obtained with herbal therapies and is supported by the synergistic effect observed in some phytotherapeutic products or botanical dietary supplements ,41.Due to their inherent complexity, obtaining natural products with a defined activity can be problematic. This issue can be somewhat avoided, however, by using pre-fractionated libraries, which offer less chemical complexity but still preserve the chemical diversity inherent in natural products. The degree of fractionation clearly has a direct impact on the obtained results, but greater fractionation also creates a more expensive process ,43. In tCecropia roots for treating Type 2 diabetes may be due to its ability to moderately inhibit 11β-HSD1 enzyme activity. Less potency was observed with increasing sub-fractionation of the primary fraction (DE-16). This observation provides evidence for the syntergistic activity exhibited by combining sub-potent molecules and suggests that the combination of diverse but related molecules may have a stronger pharmacological effect. In conclusion, our findings provide additional evidence that specific fractions obtained from extracts of Cecropia telenitida roots possess moderate inhibitory activity of 11 β-HSD1 and, therefore, can be potentially used to treat metabolic disorders. The study also supports the ethnopharmacological and medicinal application of natural products.In our opinion, one posible mechanism, among others, for the value of the PT expressed in


In the past, a microtubule (MT)-based cortical network had been shown to be indispensable for bcd mRNA transport to the posterior.The formation of the trans-Golgi proteins, we examined the effect of the Drosophila trans-Golgins on bcd localization and gradient formation. Using a genetic approach, we demonstrate that the Drosophila trans-Golgins dGCC88, dGolgin97 and dGCC185 indeed affect bcd mRNA localization during oocyte development. Consequently, the bcd mRNA is already mislocalized before the egg is fertilized. The expression domains of genes downstream of the hierarchy of bcd, e.g. of the gap gene empty spiracles or of the pair-rule gene even-skipped are changed, indicating an altered segmental anlagen, due to a faulty bcd gradient. Thus, at the end of embryogenesis, trans-Golgin mutants show bcd-like cuticle phenotypes.We report the identification of a MT-binding protein CLASP/Chb as the first component associated with this cortical MT network. Since CLASPs in vertebrates were shown to serve as an acentriolar microtubule organization center (aMTOC) in concert with bcd localization which indicates that bcd gradient formation is probably more intricate than previously presumed.Our data provides evidence that the Golgi as a cellular member of the secretory pathway exerts control on The online version of this article (10.1186/s41065-019-0106-8) contains supplementary material, which is available to authorized users. Drosophila, the Bicoid (Bcd) concentration gradient along the cortex provides essential positional information on the development of the anterior-posterior axis where it functions as a morphogen. Bcd as a maternal factor is at the top of the hierarchy of segmentation genes that controls via its gradient, the expression of the gap genes which are expressed in broader domains. The gap genes in turn control genes of the next level of the hierarchy, the pair-rule genes. Members of this class are usually expressed in 7 stripes and provide a double-segment identity. The pair-rule genes control the segmentation genes which then provide cues in every single segment. Once the identity of the segments is determined, cells in every segment are specified by the homeotic genes which ensures that these cells do not lose their identity during embryogenesis.In 2/s was calculated to be required for this process [2/s [In the past, to explain the occurrence of the morphogen gradient, the hypothesis of a diffusion-driven model  resultiess [2/s  to explaess [2/s –6. Theseess [2/s , 8, rathbcd mRNA and Bcd-GFP protein in real-time indicated that the graded mRNA movement made an essential contribution to generating the protein gradient [bcd mRNA could be much higher than that of the Bcd protein.In 2009, an alternative model was reported –10. Heregradient . This fibcd gradient could be established were described, an example involving nucleocytoplasmic shuttling of the Bcd protein [Other models of how the  protein . In this protein , the locbcd mRNA gradient is established within the same short period. In oocytes, substantial evidence exists that MTs are involved in both transportation and localization of the bcd mRNA [bcd, but also the movement of the particles of the posterior determinant oskar (osk), a MT-dependent process that is crucial for its localization at the posterior end [bcd ribonuclear protein (RNP) during the first 2 h of development [bcd mRNA transport was detected at the cortex of early staged embryos [bcd mRNA transport as the primary step for Bcd protein gradient formation is now widely accepted, and consistent with the observation of subtle Bcd protein movement along the cortex [This calls into question of how the bcd mRNA , 14. Notrior end . Besideselopment , 16. The embryos  fulfilli embryos . To conce cortex , 8.bcd mRNA transport. We found that trans-Golgi components affect the formation of the bcd mRNA gradient. Our data demonstrates that the process of bcd gradient formation is probably far more complex than previously anticipated.It should be noted that all MT-arrays that direct axial patterning are disassembled into short and non-oriented MT filaments throughout the last two stages of oogenesis –19, whicbcd mRNA gradient [Drosophila development, a search for a MT-based transportation system was initiated, leading to the discovery of a specific anterior MT network shown to be indispensable for bcd mRNA gradient formation [trans-Golgi network, with the help of the trans-Golgin marker GCC185 [trans-Golgi network and hence could contribute to build up an acentriolar microtubule organizing center (aMTOC). We therefore sought to investigate the role of trans-Golgins in bcd localization and gradient formation using genetic approaches that compromise the function of trans-Golgins.To explain the observation of the gradient  during eormation . Attemptormation , formerlormation , 22, a pr GCC185 , 24. We Additional file 3: Video S1 Chb and Tubulin in the anterior MT network. Video of the 3D-reconstruction of the full confocal stack shown as a representative single section in Fig. Drosophila genome contains four prominent trans-Golgin genes identified as dGCC88, dGolgin97 ), dGCC185 and dGolgin245, which are structurally well conserved compared to their vertebrate counterparts [trans-Golgins were involved in bcd signaling and analyzed the cuticles of trans-Golgin mutants in order to identify bcd-like phenotypes. Since dGolgin245 mutants do not show an overt phenotype and are viable [trans-Golgi proteins were investigated using RNAi fly lines [The terparts . We conse viable , the rolly lines , 27.GAL4-UAS system was used [V32 driver in combination with dGCC88, dGolgin97 and dGCC185 RNAi lines to analyze the cuticle in the knocked-down embryos. The cuticle of two weak alleles of bcd, bcd103–18-5  and head defects were observed. bcd245–35-7 proved to be the stronger allele than bcd103–18-5 and in addition showed fusion of A2 and A3 and more pronounced head defects. In addition, a deletion of thoracic segments 1 and 2  was also observed.The was used , 29, as trans-Golgins varied slightly, but were similar to bcd103–18-5 and bcd245–35-7. In dGCC88i mutants, a fusion of A3-A5 was observed 08155 spanning the entire dGCC185 locus that allowed the establishment of a viable stock, dGCC185c03845/Df(3R)08155, with weak embryonic lethality. dGCC185c03845 is a P-element insertion strain causing a deletion the last 67 aa of dGCC185, thereby removing two thirds of the GRIP domain [trans-Golgi network. Lethal dGCC185c03845/Df(3R)08155 embryos displayed a fairly normal cuticle, however, T2 was lacking, the mouth hooks were placed dorsally and the germband was not fully retracted, forcing the embryo into a slightly curved shape  to analyze the bcd mRNA patterns in knock-down oocytes and embryos. In dGCC88i oocytes, bcd mRNA did not fully localize to the anterior pole 08155 nc embryos, the localization of the bcd mRNA was indistinguishable from that of wild-type embryos (data not shown), as evident from nc 11 embryos 08155 mutant embryo, exemplified by an embryo where no mRNA degradation has yet occurred  and the pair-rule gene even-skipped (eve) were chosen to monitor the activity of bcd, based on their altered expression patterns , 3 (P < 0.01), 4 (P < 0.01), 5 (P < 0.01), 6 (0.01 < P < 0.05), and 7 (P < 0.01) shifted towards the posterior significantly  and Ems (P > 0.05) was an exception  , was used in combination with dGCC185 III. Surprisingly, the results did not show a significant difference compared to the V32 driver (data not shown). However, when embryos from the dGCC185c03845/Df(3R)08155 mutant combination were analyzed, Ems (0.05 < P < 0.01), Eve stripes 1 (P < 0.01), 2 (P < 0.01) and 3 (0.01 < P < 0.05) showed a posterior shift , 4 and 5 (0.01 < P < 0.05) . [trans-Golgi proteins is an important one, the interaction may not be exclusive, suggesting that a single Golgin may not carry a specific function, but rather work together with other Golgins at the trans-Golgi surface. In a situation where a partial-loss of either rab6 or arl1 is achieved, the four trans-Golgins would still not lose their association with the Golgi structure completely. This was demonstrated by [arl1 function leads to mislocalization of dGCC88, dGolgin97 and dGolgin245, but not of dGCC185.ussed by , even unpathways , 41. In pathways , making fficking . Arl1 isfficking . Again,  shown).  pointed rated by , showingdGolgin97i mutants at 0–60% egg length 08155 was utilized to generate embryos with a COOH terminal deletion (the last 67 aa) in dGCC185.Canton-S stock from Bloomington (No. 64349) was used as a control. The maternal °C or 29°C, depending on the efficacy of the maternal driver system.Flies were fed with standard fly food (Bloomington recipe) and were maintained at either 25Embryos in Fig. °C for 3–5 days, as described [Embryos were collected in 24 h. intervals, incubated > 36 h., dechorionated in 50% bleaching solution, fixed in 25% formaldehyde for > 5 h., devitellinized, mounted in Hoyer’s and incubated at 65escribed .1, 2 against tyrosinated tubulin (Thermo Fisher Scientific) was at 1:2000. The rabbit polyclonal antibody against Chb was obtained from Claudio Sunkel and was used at 1:500. The monoclonal antibody 2B8 against Eve (DSHB) was used at 1:250. Rabbit-anti-Ems antibodies were obtained from Uwe Walldorf and were used at 1:1000. DAPI for nuclear staining was used at 1:1000 from a 1 mg/ml stock.The working concentration for mab YLThe protocol for fluorescent in situ hybridization was adopted from , with theve stripe and the posterior border of ems. All data was analyzed with Analysis of Variance (ANOVA) and two-tailed Student’s t-tests. Data are reported as means ± SE.All images were recorded using a Zeiss LSM 710 confocal microscope. Images were post-processed with Adobe Photoshop and Adobe Illustrator. Image J was used to measure the length of the embryos, the distance between the anterior tip, the anterior border of each Additional file 1:Figure S1.bcd mRNA expression in wild-type embryos. bcd expression pattern in wild-type oocytes, as assayed by fluorescent in situ hybridization. (A) bcd mRNA pattern as a single signal in red, (B) merge of the mRNA signal (red) with that of the DAPI channel (blue) denoting the nuclei. (JPG 220 kb)Additional file 2:Figure S2. Statistic analysis of the shifts of the stripes of Even-skipped (Eve) and Empty spiracles (Ems) in trans-Golgin mutants. i: RNAi. ※ 0.05 < P < 0.01; ※※ P < 0.01. The error bars indicate standard deviation. (JPG 1101 kb)


Saururus chinensis (Saururacease), possesses anti-oxidant, anti-inflammatory, and anti-viral activities. In pharmacokinetics of sauchinone, sauchinone is highly distributed to the liver, forming extensive metabolites of sauchinone via UGTs in the liver. Thus, we investigated whether sauchinone inhibited UGTs to explore potential of sauchinone–drug interactions. In human liver microsomes (HLMs), sauchinone inhibited activities of UGT1A1, 1A3, 1A6, and 2B7 with IC50 values of 8.83, 43.9, 0.758, and 0.279 μM, respectively. Sauchinone also noncompetitively inhibited UGT1A6 and 2B7 with Ki values of 1.08 and 0.524 μM, respectively. In in vivo interaction study using mice, sauchinone inhibited UGT2B7-mediated zidovudine metabolism, resulting in increased systemic exposure of zidovudine when sauchinone and zidovudine were co-administered together. Our results indicated that there is potential HDI between sauchinone and drugs undergoing UGT2B7-mediated metabolism, possibly contributing to the safe use of sauchinone and drug combinations.Herb–drug interaction (HDI) limits clinical application of herbs and drugs, and inhibition of herbs towards uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) has gained attention as one of the important reasons to cause HDIs. Sauchinone, an active lignan isolated from aerial parts of  Herb–drug combinations have been steadily increased and encouraged as main medical treatments by the World Health Organization ,2. SinceUGTs are phase II metabolic enzymes that predominantly catalyze glucuronidation of xenobiotics, including approximately 35% of drugs and consequently, facilitating elimination of glucuronidated metabolites through bile and urine ,7. GlucuSaururus chinensis Hort. ex Loudon (Saururaceae), commonly known as Chinese lizard’s tail or Sam-baek-cho in Korea, is a plant with a long history of medical use. It has been used to treat hepatitis in Korea  is the concentration of an herb in the liver (as an enzyme active site), and Ki is an inhibition constant of a herb. This calculation also assumes that metabolic enzyme contributes 100% of a drug metabolism. To determine whether metabolic inhibition in liver can occurs or not, the ratio of [I] to Ki is calculated because sufficient concentration of an inhibitor in the liver is a critical factor that causes metabolic inhibition. Occasionally, Cmax has been used instead of [I], assuming that systemic exposure and tissue exposure of an inhibitor are similar /Ki < 1’ is considered to have median possibility of drug interaction based on [I]/Ki standard [i/AUC of zidovudine and 0.458 for [I]/Ki indicated inhibitory effect of sauchinone on UGT2B7 involved zidovudine metabolism. Also the slower CL and CLNR of zidovudine with sauchinone indicted that sauchinone inhibited metabolic clearance of zidovudine compared to those without sauchinone. Interestingly, except for the metabolism of zidovudine, other pharmacokinetic profiles of zidovudine were not changed by co-administration of sauchinone. Distribution and excretion of zidovudine were not affected by sauchinone, supported by comparable Vss, t1/2, MRT, Ae0–24 h, and GI24 h of zidovudine between with and without sauchinone.With such situation, pharmacokinetic changes of a drug metabolized via Ugt2b7 when co-administered with sauchinone were evaluated in mice. As shown in  1.53 μM  instead There are some limitations to our calculations. First, inhibition of sauchinone or zidovudine metabolites towards UGTs was not investigated. Glucuronidation, oxidation, methylation, and dehydrogenation are involved in sauchionone metabolism . Glucuro®BEH C18 column  and detailed information for structure elucidation and purity experiment were provided in O-glucuronide, serotonin-O-glucuronide, trifluoperazine-N-glucuronide, and zidovudine-5′-glucuronide were purchased from Tokyo Chemical Industry Co. . Chenodeoxy cholic acid-24-acyl-β-d-glucuronide was purchased from Carbosynth . Carbamazepine [an internal standard (IS) of ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)] was purchased from Wako Co. . All other chemicals and reagents used were of analytical grade.Sauchinone was extracted and purified according to a method reported previously . The purTo conduct in vitro  and in vivo (pharmacokinetic study) investigations using mice, animal studies were approved by the Institute of Laboratory Animal Resources of Dongguk University, Seoul, Republic of Korea (IACUC-2016-046). Five-week-old (20–25 g) male Institute of Cancer Research mice were purchased from the Charles River Company of Korea . Upon arrival, mice were randomized and housed at five mice per cage under strictly controlled environmental conditions (22–25 °C and 48–52% relative humidity) with a 12 h light/dark cycle at 150–300 lx luminous intensity. All mice were provided food and water and were maintained during this study.d-glucuronide, trifluoperazine-N-glucuronide, serotonin-O-glucuronide, propofol-O-glucuronide, and zidovudine-5′-glucuronide represented glucuronidation efficacy of UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7, respectively, in HLMs. As UGT selective substrates, β-estradiol, chenodeoxy cholic acid, propofol, and zidovudine were dissolved in methanol, whereas trifluoperazine and serotonin were dissolved in 50% ethanol. These dissolved substrate solutions were serially diluted to required concentrations as a UGT cocktail set containing β-estradiol, chenodeoxy cholic acid, trifluoperazine, serotonin, propofol, and zidovudine. Spiked volume of UGT cocktail set was 1% (v/v) of total volume of mixture. Concentrations of UGT selective substrates were used close to their reported Km values −). Separation was performed on a reversed-phase C18 column  maintained at 30 °C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B) at a ratio of 85:15 (v/v) for 3 min and gradually changed to 10:90 (v/v) for 5.5 min. This composition was maintained until 6.5 min and then back to initially composition at a flow rate of 0.3 mL/min. The total run time was 9 min. Metabolites of UGT-selective substrates were detected using an Waters UPLC-XEVO TQ system  in multiple reaction monitoring (MRM) mode with an ESI interface for positive ions  for each metabolite of UGT-selective substrate was as follows: β-estradiol-3-glucuronide , chenodeoxy cholic acid-24-acyl-β-d-glucuronide , trifluoperazine-N-glucuronide , serotonin-O-glucuronide , propofol-O-glucuronide , and zidovudine-5′-glucuronide .Turbo ion-spray interface was operated in positive ion mode at an ion capillary voltage of 2500 V and a temperature of 350 °C. Operating conditions  were optimized by flow injection of a mixture of all analytes. The 50 as an enzyme inhibition parameter was calculated by fitting the Hill equation to the data using GraphPad Prism 5 .UGT-mediated activities in the presence of sauchinone as an inhibitor were expressed as percentages of corresponding control values (in absence of sauchinone). From percentages of control activity versus inhibitor concentrations, a sigmoid shaped curve was fitted to the data and IC0–24 h). The gastrointestinal tract was cut and extracted with 20 mL of methanol, of which supernatant was collected (GI24 h). Urine and GI samples were then analyzed using UPLC-MS/MS.To investigate the inhibitory effects of sauchinone on UGT2B7-mediated metabolism, zidovudine was chosen as an example drug mainly metabolized via UGT2B7 . Surgica480 min) was calculated using the trapezoidal rule method. Standard methods [max) and time to reach Cmax (Tmax) were read reversibly from the extrapolated data. After the analysis of zidovudine concentrations in plasma by UPLC-MS/MS, pharmacokinetic parameters were calculated as follows. The area under the plasma concentration–time curve from time zero to the last measured time to infinity or last sampling time (480 min) (AUC or AUC methods  were useThe present study investigated the inhibitory potential of sauchinone towards UGTs to cause drug interactions for the first time. In detail, sauchinone inhibited UGT activities in the following order: UGT2B7 > 1A6 > 1A1. It noncompetitively inhibited UGT1A6 and 2B7 activities in vitro. Sauchinone also increased systemic exposure of zidovudine, a substrate of UGT2B7, through UGT2B7 inhibition in vivo mice. All these results provide an early warning for the combination between sauchinone and drugs mainly metabolized by UGT2B7. Due to various contributions of UGT isoforms toward drug metabolism as well as species and individual differences, further clinical investigations should be considered.