Background: Effects of drug abuse on LINE-1 retrotransposition (L1-RTP) in somatic cells are unknown.Results: Methamphetamine and cocaine-induced L1-RTP depends on CREB.Conclusion: Methamphetamine and cocaine activate CREB and L1-RTP in neuronal cell lines.Significance: To our knowledge this is the first study to demonstrate the induction of L1-RTP by drugs of abuse. Long interspersed element 1 (L1) is a retroelement constituting ∼17% of the human genome. A single human cell has 80–100 copies of L1 capable of retrotransposition (L1-RTP), ∼10% of which are “hot L1” copies, meaning they are primed for “jumping” within the genome. Recent studies demonstrated induction of L1 activity by drugs of abuse or low molecular weight compounds, but little is known about the underlying mechanism. The aim of this study was to identify the mechanism and effects of methamphetamine (METH) and cocaine on L1-RTP. Our results revealed that METH and cocaine induced L1-RTP in neuronal cell lines. This effect was found to be reverse transcriptase-dependent. However, METH and cocaine did not induce double-strand breaks. RNA interference experiments combined with add-back of siRNA-resistant cDNAs revealed that the induction of L1-RTP by METH or cocaine depends on the activation of cAMP response element-binding protein (CREB). METH or cocaine recruited the L1-encoded open reading frame 1 (ORF1) to chromatin in a CREB-dependent manner. These data suggest that the cellular cascades underlying METH- and cocaine-induced L1-RTP are different from those behind L1-RTP triggered by DNA damage; CREB is involved in drug-induced L1-RTP. L1-RTP caused by drugs of abuse is a novel type of genomic instability, and analysis of this phenomenon might be a novel approach to studying substance-use disorders. Alu (SINE-VNTR-Alu) NTR-Alu) . A singlefective . Nonetheefective , and ∼10efective , 4, and efective , 7. Lateefective –11, and fective ––15. L1 eective ––, 16, whoective ––, 18, wheective –– and reveective ––. ORF1 anective ––, 16, 21.Recent observations suggest that L1-RTP occurs in somatic cells and that copy numbers of L1 are increased in human brain tissues , 22. In e.g. Ser133 phosphorylation) is mediated by a number of kinases, including phosphorylated extracellular signal-regulated kinase (pERK) (28–The cAMP response element-binding protein (CREB), as the name suggests, binds to cAMP-responsive element (CRE), which is found in most cAMP-regulated promoters –30. CREB–e (pERK) . CREB ispERK) 28–, 33. CREpERK) 28–.Methamphetamine (METH) and cocaine are recreational drugs that cause addiction and are among the most widely used illegal drugs worldwide –38. Thes–42–In this study we found that METH and cocaine induce L1-RTP and that the induction of this activity by METH and cocaine depends on CREB. Biochemical analysis revealed that METH or cocaine recruits ORF1 to chromatin. To the best of our knowledge, this is the first study demonstrating the induction of L1-RTP by drugs of abuse. These data support the existence of CREB-mediated genome shuffling via L1-RTP; in this paper we discuss its possible involvement in psychiatric disorders associated with METH and cocaine use.2 in Eagle's minimum essential medium plus Ham's F-12 medium containing 1% nonessential amino acids and 15% fetal bovine serum . PC12, HeLa, HT1080, and SK-N-SH cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The transfection efficiency was ∼70% for HeLa and HT1080 and ∼30% for PC12, SH-SY5Y, and SK-N-SH cells as determined using fluorescence-activated cell sorting on day 2 after transfection of plasmid DNA encoding enhanced green fluorescent protein . Protease and phosphatase inhibitors were purchased from Roche Diagnostics . METH and cocaine were purchased from Dainippon Sumitomo Pharma Co., Ltd., and Sanseidou (Japan). A license for METH (no. 25-013) and cocaine (no. 1040) was issued by the governor of Hyogo. We prepared a 100 mm stock solution of the RT inhibitor, stavudine (d4T), in DMSO. About 30 min before the addition of METH or cocaine, d4T was added to a final concentration of 50 μm to the cell culture medium.SH-SY5Y (EC94030304), PC12 (EC88022401), HeLa (EC93021013), HT1080 (EC85111505), and SK-N-SH cells (EC86012802) were obtained from DS Pharma Biomedical. SH-SY5Y cells were maintained at 37 °C and 5% CO133, Cell Signaling Technology), and glyceraldehyde-3-phosphate dehydrogenase  were used as primary antibodies. A rabbit polyclonal antibody against human ORF1 was generated using the peptide MGKKQNRKTGNSKTQSAC as an immunogen  , γ-H2AX (Millipore), CREB , phospho-CREB (Seratories) . As secoR) . Cells were transfected with either pEF06R or pL1-NeoR using Lipofectamine 2000 (Invitrogen). The cells were selected for 2 days with puromycin (0.5 μg/ml) for pEF06R or with hygromycin (50 μg/ml) for pL1-NeoR. Next the cells were incubated for an additional 2 or 3 days with the indicated amounts of chemicals. For the PCR-based assay, genomic DNA was prepared from harvested cells using a DNA extraction system . For the semiquantitative PCR, primers that were designed to cover exons would amplify a product of ∼1040 bp (without L1-RTP), whereas they would generate a product of ∼340 bp (with L1-RTP) after L1-RTP (A). Thus, the occurrence of L1-RTP was determined by evaluating the size of the amplified product. After staining the amplified DNA with SYBR Green I , the signal intensity of the 340-bp bands was measured using a molecular imager  and normalized to β-actin (ACTB) bands, used as an internal control. EFGP was PCR-amplified using 5′-CTGGTAGTGGTCGGCGAGCTG-3′ and 5′-GACCACCCTGACCTACGGCGT-3′ as the forward and reverse primers, respectively. Amplification reactions were performed using the Ex TaqTM polymerase (TaKaRa) and the following cycling conditions: initial step for 15 min at 94 °C followed by 28–32 cycles of 30 s at 94 °C and 15 s at 68 °C. To detect ACTB as the internal control, 5′-GAGGGAAATCGTGCGTGA-3′ and 5′-AGAAGGAAGGCTGGAAAA-3′ were used as the forward and reverse primers, respectively. PCR amplification was performed using the following cycling conditions: initial 5 min at 94 °C, 20 cycles of 30 s at 94 °C, 30 s at 62 °C, and 1 min at 72 °C. The PCR product was analyzed in agarose gels and visualized with SYBR Green I Nucleic Acid Gel Stain.L1-RTP was assayed as described previously –49. For –R) , 51, forr L1-RTP A. Thus, 6 cells transfected with pL1-NeoR and selected with hygromycin (50 μg/ml) were transferred to 100-mm plates at ∼105 per plate. Next, the cells were incubated for 3 days with the selection and further cultured in the presence of G418 (270 μg/ml). After 3–4 weeks, cell aggregates were stained with methylene blue, and the number of colonies was counted. To determine the occurrence of L1-RTP, each of the six plates was incubated with chemicals or a buffer control, and the number of resulting colonies in each plate was counted.In the colony formation assay, ∼2.0 × 10R vector has a modified mneoI indicator cassette with a bacterial/mammalian promoter and the ColE1 origin of replication (R-Asp702Ala (D702A) contains a missense mutation in the reverse transcriptase domain of ORF2. The reverse transcriptase activity of ORF2 is attenuated by the D702A mutation  was used for reverse transcription, and amplification of cDNA was monitored and quantified using SYBR Premix Ex Taq (TaKaRa) on an Applied Biosystems 7900HT Fast Real-time PCR system (Invitrogen) according to the manufacturer's instructions. All data were normalized to ACTB. For the quantification of both endogenous and exogenous ORF2 mRNA, a primer set of L1-EGFP+5653F (5′-CCAAATGTCCAACAATGATAGACTG-3′) and L1-EGFP+5762R (5′-CCATGTCCCTACAAAGGATATGAAC-3′) was used. For the quantification of mature or precursor mRNA of EGFP, primer sets of L1-EGFP+6342F (5′- TAGTGGTTGTCGGGCAGCAG-3′) plus L1-EGFP+7351R (5′-TTCAAGATCCGCCACAACATC-3′) or L1-EGFP+7222F (5′-TGGAAGCTGGGTGTGTAGTTATCTG-3′) plus L1-EGFP+7365R (5′-GGCATCAAGGTGAACTTCAAGATC-3′) were used, respectively. For the amplification of ACTB-derived DNA, a primer set of 5′-GAGGGAAATCGTGCGTGA-3′ plus 5′-AGAAGGAAGGCTGGAAAA-3′ was used. To evaluate the levels of mRNA, each sample was analyzed in triplicate, and statistical analysis was performed based on the relative intensity of the mRNA levels normalized to ACTB levels.qRT-PCR was performed as described previously . Total RE). SH-SY5Y cells were transfected with pEF06R▵3′CMV, and qRT-PCR was performed. The results of 24-h incubation with either METH or cocaine were analyzed.To correctly assess the mature form of L1 mRNA in the case of pEF06R, it was necessary to exclude the effects of the antisense mRNA transcribed from the inverted 3′ CMV promoter . For thiFig. 2m Tris-HCl (pH 7.6), 150 mm NaCl, 1 mm EDTA, 0.1% SDS, 0.5% deoxycholic acid, 1% Nonidet P-40 (radioimmune precipitation assay buffer), and protease inhibitors. After ultrasonication using Bioruptor  for 12.5 min  at the middle level of output (250 W) at 4 °C, the soluble cellular extracts were recovered after centrifugation for 10 min at 16,000 × g. The protein concentration of each sample was determined using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific), and cell extracts were subjected to WB analysis. The blots were probed with the primary antibody followed by a horseradish peroxidase-conjugated secondary antibody. The immune complexes were visualized using PierceTM Western blotting Substrate Plus (Thermo Fisher Scientific). WB results were documented and quantified using ImageQuant LAS 4000 mini and Amersham Biosciences Imager 600 .The cells were washed with phosphate-buffered saline (PBS) and resuspended in 50 mm METH or 0.5 mm or 0.25 mm cocaine. The extracts were subjected to WB analysis with α-ORF1 antibody as the primary antibody.SH-SY5Y cells were transfected with pEF06R, and either METH or cocaine was added to the culture medium on day 2. Total cell extracts were prepared 24 h after the addition of 0.13 or 0.06 mm siRNA using Lipofectamine RNAiMAX in SH-SY5Y and SK-N-SH. The nucleotide sequences of each siRNA are shown in R. Two days later, the cells were replated, incubated for 2 days with METH or cocaine, and subjected to analysis. As a control, Silencer Negative Control siRNAs  were used.Two small interfering RNAs (siRNAs) were prepared (Invitrogen), and their functions were evaluated by transfection into cells followed by WB analysis. We transfected 10 nB. To evaluate the roles of the target gene products, cells were transfected with CREB siRNA using Lipofectamine RNAiMAX for each gene on day 3 after the first transfection of pL1-NeoR. On day 4, pSiR-CREB  was isolated using the Subcellular Protein Fractionation kit (Thermo Fisher Scientific) with micrococcal nuclease as described previously (We used the pORF1-TAP (tandem affinity purification) construct , which eeviously , 48, 49.SH-SY5Y cells were washed with PBS and fixed with 4% paraformaldehyde in PBS. The fixed cells were permeabilized with 0.5% Triton X-100 in PBS for 5 min. After incubation with PBS and 5% bovine serum albumin for 30 min, the cells were incubated with α-γ-H2AX antibodies. After 1 h of incubation at 37 °C, secondary antibodies conjugated with Alexa Fluor 555 (Invitrogen) were added for 1 h at 37 °C. Nuclei were stained with Hoechst 33258 (Invitrogen). The slides were mounted in an anti-fade solution and examined under a fluorescence microscope .U test. A p value of <0.05 was assumed to mean statistical significance.Statistical significance was evaluated using the Mann-Whitney R; A and B)  and a rat pheochromocytoma cell line . As shown in Fig. 1C, METH induced L1-RTP in different cell lines, including SH-SY5Y and PC12, but did not induce L1-RTP in a human fibrosarcoma cell line .We initially performed PCR-based and colony formation assays using the filtered drug of abuse and the plasmids pEF06R and pCEP4/L1mneoI/ColE1 (pL1-NeoA and B) , 50, 51.A and B) –49 in whm were detected (data not shown). We then tested if high doses of METH could induce L1-RTP in HT1080 and SH-SY5Y cells and whether low doses of METH could induce L1-RTP in PC12 cells (D). When METH induced L1-RTP, SH-SY5Y cells expressed EGFP (E). Next, we evaluated METH-induced L1-RTP using a colony formation assay in which L1-RTP activated the expression of a functional neomycin resistance gene and supported the growth of cells cultured in the presence of the cytotoxic drug, neomycin. According to the experimental procedures shown in Fig. 1F, HT1080 and SH-SY5Y cells were transfected with pCEP4/L1mneol/ColE1 , selected on hygromycin, and then treated with METH on day 3 after the transfection. Approximately 105 cells were incubated initially with 0.25–0.015 mm METH. After neomycin selection, we observed 50–100 colonies in each plate , indicating that the frequency of L1-RTP induced by METH was ∼1 per 1.0–2.0 × 103 SH-SY5Y cells . METH induced L1-RTP at a low concentration . In contrast, METH did not induce L1-RTP in nonneuronal HT1080 and HeLa cells .No cytotoxic effects of METH at concentrations up to 1 m12 cells D. When Msed EGFP E. Next, m using the colony formation assay in SH-SY5Y cells (A). After neomycin selection, we observed 50–100 colonies on each plate (A and B), indicating that the frequency of L1-RTP induced by cocaine was ∼1 per 1.0–2.0 × 103 in SH-SY5Y cells. On the other hand, ethanol and sodium barbital did not induce L1-RTP in SH-SY5Y cells (B). No cytotoxic effects of cocaine, ethanol, or sodium barbital were observed at the concentrations tested (data not shown). Next, using a mutant vector, we tested whether colonies induced by METH and cocaine were dependent on L1-RTP or if these colonies formed nonspecifically. The mutant pL1-NeoR vector used in this experiment was reverse transcriptase-negative (due to a D702A mutation). The results revealed that the colonies did not form at all (C). We conducted another experiment using an RT inhibitor (d4T). Previous studies reported a suppressive effect of d4T on L1-RTP . Thus, the results suggested that L1-RTP induction by METH and cocaine is a phenomenon dependent on RT.We next tested whether other drugs of abuse induced L1-RTP, such as cocaine, ethanol, and sodium barbital. We evaluated cocaine-induced L1-RTP at 0.13–0.5 m5Y cells A. After ch plate , A and B5Y cells B. No cytm at all C. We conn L1-RTP , 54. TheE, F, and G) or ORF2 mRNA (H) or the expression levels of the ORF1 protein (I).Moreover, we ruled out the possibility that METH and cocaine induce L1-RTP by up-regulating the expression level of L1 mRNA. qRT-PCR analysis revealed that neither METH nor cocaine increased the splicing efficiency of the immature L1 transcript , and we detected no focus formation of γ-H2AX after treatment of cells with METH at 0.13 mm or cocaine at 0.25 mm (C). These observations indicated that induction of L1-RTP by METH and cocaine was attributable to the nongenotoxic effects of these compounds.Farkash et al.  reportedf γ-H2AX , A and B 0.25 mm C. These A). Next we examined the involvement of CREB and found that two CREB siRNAs (CREB-1 and -2) efficiently suppressed the expression of endogenous CREB  and that both siRNAs abolished METH- or cocaine-induced L1-RTP . These data suggested that the siRNA abrogated the activity of endogenous CREB and inhibited L1-RTP. To confirm this hypothesis, we carried out an add-back experiment using siRNA-resistant CREB cDNA. We confirmed that the transfection of a plasmid DNA expressing a siRNA-resistant FLAG-tagged CREB mRNA (pSiR-CREB) could restore protein expression that had been reduced by the siRNA . Subsequently, we found that pSiR-CREB restored the formation of NeoR colonies, which had been suppressed by the siRNA . Moreover, we confirmed these experiments on another cell line. When the experiments with CREB siRNA and added-back siRNA-resistant CREB were performed on the SK-N-SH human neuroblast cell line, the results were the same as with SH-SY5Y cells (data not shown). These data indicated that CREB was involved in METH- or cocaine-induced L1-RTP.To determine the mechanism behind METH- and cocaine-induced L1-RTP, we assessed the involvement of CREB. According to a recent report, METH and cocaine induce the phosphorylation of CREB . To confFig. 4et al. . The METH- or cocaine-induced chromatin recruitment of ORF1 was blocked by both types of CREB siRNA . These data indicated that METH and cocaine separately mobilized the L1 component to chromatin via a mechanism involving the activation of CREB.Goodier et al.  reportedin vitro. In this study the induction of L1-RTP was found to be induced by METH or cocaine in neuronal cell lines such as SH-SY5, PC12, and SK-N-SH but not in nonneuronal cell lines such as HeLa and HT1080. Therefore, it appears that METH or cocaine induce L1-RTP only in neuronal cells. METH induced L1-RTP at low concentrations  and caused fluctuations in the levels of intracellular calcium. When the intracellular calcium concentration increases, calcium interacts with intracellular calcium-binding proteins and produces various physiological effects. Hondebrink et al. (m) stimulate an acetylcholine-evoked calcium increase. Moreover, depolarization-evoked calcium increase is inhibited after exposure to high METH concentrations (1 mm) (C and D).Recently, the incidence of psychiatric disorders associated with abuse of drugs such as alcohol and METH has been increasing worldwide . It is nk et al.  showed ts (1 mm) . The chas (1 mm) . Thus, Ms (1 mm) , C and DWe found that METH- or cocaine-induced L1-RTP are not involved in the mechanisms of DSBs. METH or cocaine did not up-regulate γ-H2AX (H2AX phosphorylated at serine 139) , which iCREB cDNA does not induce L1-RTP (F–I). These findings suggest that CREB functions as the mediator for METH- and cocaine-induced L1-RTP. It is possible that this specific factor induces changes in chromatin structure and relevant transcriptional network, thereby leading to psychiatric disorders.In a variety of species and types of memory, the process of consolidation of a new memory requires gene expression that begins during training. The proteins of the CREB transcription factor family are evolutionarily conserved regulators of this gene expression , 34. CREe L1-RTP . CREB, ae L1-RTP , F–I. Thet al. (Recent evidence demonstrated that neurons actually support high levels of L1-RTP ; for exaet al.  reported


In addition, the extent of PD-1/PD-L1 down-regulation is closely related with AF burden. More importantly, Allogeneic mixed leukocyte reactions (MLR) shows that the mDCs PD-L1 down-regulation is associated with increased T cell (CD4+ and CD8+) proliferation, increased type 1 effector cytokines (IL-2 and IFN-γ) secretion, and decreased type 2 effector cytokine (IL-10) secretion. Then, PD-L1 up-regulation by the stimulation of IFN-α can significantly convert this representation. Collectively, our report suggest that T(CD4+)/mDCs-associated PD-1/PD-L1 pathway plays a key role in AF immune regulation. PD-1/PD-L1 down-regulation in AF patients promotes T cells function and may contribute to AF pathogenesis.Although immuno-inflammatory response contributes to pathogenesis of AF, molecular and cellular mechanism in this process remains poorly understood. Recently, increasing evidence suggests that Programmed death-1 (PD-1)/PD-1 ligand (PD-L) pathway may be a potential pathway participating in AF pathogenesis. In this study, we detected the PD-1 and PD-L1, 2 expression on peripheral blood function cells by flow cytometry in 91 atrial fibrillation (AF) patients and 35 healthy volunteers. The expression of PD-1 on CD Atrial fibrillation (AF) is the most common arrhythmia, but the pathophysiological mechanism leading to this condition remains poorly understood. However, increasing evidence suggests the influence of an immune-mediated inflammatory response in AF pathogenesis 4+ and CD8+ T cells, NK cells, B cells and monocytes upon activation. It is engaged primarily by its ligands PD-L1  and PD-L2  4+ and CD8+ T cells Programmed death-1  is a CD28 homolog and costimulatory molecule expressed on CD4+ T cells or mDCs in AF patients, and that the extent of the down-regulation is closely related with the AF burden. Additionally, PD-1/PD-L1 down-regulation significantly enhanced CD4+ and CD8+ T cell proliferation and influenced cytokines secretion in vitro. Our study indicates that the PD-L1 may be induced by IFN-α. We further up-regulated expression of PD-L1 on mDCs by IFN-α treatment and found that mDC-mediated T cell proliferation was suppressed and the pattern of cytokine secretion was converted. This suggests that PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs may modulate immune regulation and play a role in AF pathogenesis.In this study, we examined the expression profiles of PD-1 on T cells and PD-L1 and 2 on T cells, myeloid dendritic cells (mDCs) and macrophages in patients with paroxysmal and persistent AF, to assess their roles in AF pathogenesis. We found that PD-1/PD-L1 expression is substantially down-regulated on CDn = 42) and the persistent AF group (n = 49). The diagnosis of AF was adopted in the ACC/AHA/ESC 2006 guidelines on AF i) episodes of paroxysmal AF lasting less than 6 hrs each and (ii) a sum of paroxysmal AF lasting largely less than 30% of the total time. The frequent paroxysms group (group F) consisted of 15 patients with at least one of the following characteristics: (i) a total time of recurrent paroxysmal AF lasting largely longer than 30% of the total time and (ii) at least one episode of AF lasting more than 6 hrs. The other 10 patients who did not meet the above criteria were excluded. The time of paroxysms was mainly judged by ECG, holter monitor or symptoms of palpitations. This method of grouping can depict the arrhythmia burden and the risk for adverse events Between May 2010 and January 2012, a total of 91 patients with a diagnosis of AF were enrolled in the Department of Cardiology of Tangdu Hospital, Xi'an, China. The AF patients were divided into two groups: the paroxysmal AF group , lymphocytes and white blood cell counting according to routine protocols. Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized blood by Ficoll–Hypaque  density gradient centrifugation, and were viably frozen in 80% foetal calf serum , 10% dimethylsulphoxide and 10% RPMI 1640 medium (Gibco) in liquid nitrogen for subsequent analysis.5 cells were counted in each analysis. The results were processed with FCS express V3 software and described with the percentage of positive cells.The following monoclonal antibodies (mAbs) were used: CD3-FITC , CD8-PE , CD4-APC , PD-1-PE-cy7 , PD-L1-PE , PD-L1-PE-cy7 , PD-L2-PE , lineage-1-FITC, HLA-DR-PerCP , CD11c-APC , CD14-APC . All Abs were purchased from BD Biosciences . PD-1 expression on T cells, PD-L1 and PD-L2 expression on mDCs, macrophages and T cells were measured, respectively, using fresh heparinized peripheral blood. In brief, blood samples were stained with a mixture of Abs , as well as isotypecontrol antibodies, for 30 min. at 4°C. The cells were then processed with FACS lysing solution  to remove RBC, fixed and analysed by four-colour flow cytometry on a FACSCalibur (BD Biosciences). A total of 1 × 104+ T cells were positively isolated from PBMCs using the CD4 Positive Isolation Kit . MDCs were obtained from PBMCs by CD19-negative selection, followed by CD1c-positive selection using the CD1c (BDCA-1) Dendritic Cell Isolation Kit (Miltenyi Biotech) with a MidiMACS separator unit. All procedures were performed according to the manufacturer's instructions. CD4+ T cells or mDCs purity exceeded 90%. Unless otherwise stated, freshly isolated cells were carefully collected and suspended in RPMI 1640 medium containing 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin. MDCs were treated for 72 hrs in a 96-well plate in RPMI 1640 medium containing 10% FCS only or with saturating doses of IFN-α . Cells were collected at 0, 24, 48 and 72 hrs of in vitro cell culture. The expression of PD-L1 was analysed immediately on a FACSCalibur (BD Biosciences).CD5 cells/200 μl) for 24 hrs. In some cases, IFN-α  was added in the culture medium. On the following day, mDCs were mixed at different concentrations  with purified CD4 or CD8 T cells (1 × 105 cells/200 μl) from a healthy individual in the presence of 2 μg/ml anti-B7-H1  or control antibodies  for 5 days. Cells were pulsed with 1 μCi/well of [3H]thymidine  for 18 hrs before harvest. Before measurement of incorporated [3H]thymidine, part of the culture supernatants was collected for cytokine detection.Purified mDCs cultured with poly(I:C)  were performed in 96-well flat-bottom culture plates (at 1 × 10Cytokine assessment was carried out using ELISA Kit (eBioscience) for three cytokines  of culture supernatants. All procedures were performed according to the manufacturer's instructions. Absorbance was measured on an automatic plate reader. The sensitivity of these ELISA kits were 1 pg/ml for IL-10, 2 pg/ml for IL-2 and 4 pg/ml for IFN-γ.anova was employed for comparing means of multiple groups, followed least significant difference (LSD) for paired comparison. The Mann–Whitney U-non-parametric test was used for differences between non-parametric analyses. To evaluate differences in categorical variables between study groups Pearson's chi-squares test was used. All P-values were two-tailed and values <0.05 were considered statistically significant.All experiments were repeated three times. Quantitative data were expressed as mean ± SD, and categorical variables as absolute and relative frequencies (percentages). The statistic software SPSS 19.0 was employed for statistical processing. Continuous data were tested for normal distribution by the Kolmogorov–Smirnov test and were found to be suitable for parametric analyses. One-way P > 0.05). β-blocker as heart rate-slowing agent was used significantly more in AF patients groups than control group (P < 0.001). AF patients groups had higher CRP levels compared with control group and CRP level in persistent AF group was further higher than paroxysmal AF group .The characteristics of the study participants in this study are presented in Table4+ and CD8+ T cells in paroxysmal AF patients, persistent AF patients and control group. Representative dot plots of PD-1+ distinct subpopulations were shown in Figure4+ T cells in AF patients was significantly decreased (P < 0.001) than healthy controls (12.04 ± 1.95). Compared the two groups, we found persistent AF patients (1.79 ± 0.32) expressed a lower level of PD-1 than paroxysmal AF patients , persistent AF group (3.61 ± 0.54) and control group  than healthy controls (0.95 ± 0.19), and persistent AF group (0.25 ± 0.08) was lower than paroxysmal AF group /mDCs interactions might participate in the pathogenesis of AF.We further detected PD-L1 and PD-L2 expression on the function cells. Representative dot plots of PD-L1+ and PD-L2+ distinct subpopulations were shown in Figuresoups Fig., F and H4+ T cells. To further explore this phenomenon, we examined a paroxysmal AF group with a 3-month follow-up. We divided them into two subgroups (group F and group O) and measured PD-1/PD-L1 expression on CD4+ T cells and mDCs. The results showed that PD-1+ distinct subpopulations of CD4+ T cells and PD-L1+ distinct subpopulations of mDCs in group F  were significantly lower than group O  were more efficient in inducing CD4+ and CD8+ T cell proliferation at all ratios tested compared with mDCs from controls . It suggested that the T cell-stimulating capacity of mDCs from AF patients was enhanced. Then, we examined the effects of mDC-associated PD-L1 up-regulation on allogeneic T cell proliferation in the presence of IFN-α. We found that the upgrade capacity of mDCs from AF patients to stimulate CD4+ and CD8+ T cells proliferations was restored, leading to results similar to that of normal mDCs . Furthermore, we examined the effects on allogeneic MLR in the presence of anti-PD-L1. As expected, we found that the restored capacity of mDCs from AF patients to stimulate CD4+ and CD8+ T cells proliferations was up-regulated again by treated with anti-PD-L1 . These data suggested that the down-regulation of PD-L1 on mDCs appears to transmit stimulating signal to CD4+ and CD8+ T cells in AF patients, resulting in an increase of proliferation and PD-L1 up-regulation on mDCs can reduce the allostimulatory capacity of mDCs from AF patients.In the allogeneic mixed leukocyte reactions (MLR), we cocultured mature mDCs from persistent AF patients and healthy volunteers at different ratios with T cells from a third healthy individual and studied how the decreased PD-L1 expression on mDCs in AF patients influence the T cells proliferation. The results showed that mDCs of AF patients /mDCs-associated PD-1/PD-L1 pathway in immune regulation of AF. Our results suggest that down-regulation of PD-1/PD-L1 on CD4+ T cells or mDCs might, at least in part, take part in AF pathogenesis. Firstly, PD-1 expression on CD4+ T cells and PD-L1 on mDCs is down-regulated in AF patients compared with controls. Secondly, down-regulation of PD-1/PD-L1 was more significant in persistent AF patients, those considered to have a more pronounced inflammatory state compared with paroxysmal AF patients. This suggests that the extent of PD-1/PD-L1 down-regulation may be closely related with AF burden, which is further supported by the subsequent follow-up investigation. Thirdly, allogeneic MLR experiments indicate that low-level mDCs PD-L1 expression promotes T cell proliferation, suggesting that the PD-L1 levels may be linked with the inflammatory state. Cytokine secretion changed with mDC PD-L1 levels, further supporting this assertion. Collectively, PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs may participate in the process of immune regulation and play a role in AF pathogenesis.In this study, we comprehensively investigated the expression of PD-1 and PD-L1 and 2 on key regulatory cells including mDCs, macrophages, and CDRegarding the underlying mechanisms about how PD-1/PD-L1 down-regulation modulates AF pathogenesis, we speculate on the following possibilities. Firstly, PD-1/PD-L1-associated T cell proliferation may contribute to atrial electrical remodelling. T cell proliferation may also increase secretion of cytokines, such as IL-6 and MCP-1, which may affect the contractility and electrical stability of myocytes differently 4+ and CD8+ T cells in AF patients was not statistically different compared with controls, suggesting that PD-1/PD-L1 inhibitory signals between T-T cells may not participate in regulating T cells. Importantly, the expression of PD-L2 on mDCs, macrophages, and CD4+ and CD8+ T cells in AF patients was not statistically different compared with controls. Our study suggests that PD-L2 indeed is expressed on CD4+ T and CD8+ T cells, consistent with earlier studies 4+)/mDCs-associated PD-1/PD-L1-independent mechanism in AF regulation.In this study, we investigated PD-L1 expression on several cell types, and mDCs have been proposed to be responsible for specific T cell regulation in AF. As a subpopulation of DCs, mDCs express elevated levels of MHC and a range of accessory molecules involved in cell migration, adhesion and co-signalling It is important to note that several limitations that need to be addressed regarding this study. First, to supporting the suggestion that the PD-L1 levels may be linked with the inflammatory state, more cytokines except IL-2, IL-10 and IFN-γ need to be examined. In addition, TNF-α as another important inflammatory cytokines may be not relevant to PD-L1 change in MLR (data not shown). The specific mechanisms is unclear and need further research. Second, although the PD-1/PD-L1 pathway appears to be important, additional co-inhibitory pathways such as those involving CTLA-4 and B7 interactions cannot be excluded in AF pathogenesis. Further study will be needed to determine whether the PD-1/PD-L1 pathway is the only mechanism or if it is just one that regulates the immune response in AF. Third, the specific role of the PD-1/PD-L1 pathway in AF is not clear and it remains unclear whether PD-1/PD-L1 down-regulation is a cause of AF, or merely a consequence.4+ T cells and PD-L1 on mDCs in AF patients. The extent of PD-1/PD-L1 down-regulation was closely related with AF burden. Furthermore, PD-1/PD-L1 down-regulation could improve, at least in part, T cell proliferation and cytokine secretion change, which reflect the inflammatory state and contribute to AF pathogenesis. Therefore, PD-1/PD-L1 down-regulation on CD4+ T cells or mDCs participates in the pathogenesis of AF and up-regulating the expression of PD-1/PD-L1 molecules may represent a new therapeutic option for AF.In summary, the present study begins to shed light on the role of immune pathogenesis in AF, showing that PD-1 is down-regulated on CD


Graphene, in single layer or multi-layer forms, holds great promise for future electronics and high-temperature applications. Resistance to oxidation, an important property for high-temperature applications, has not yet been extensively investigated. Controlled thinning of multi-layer graphene (MLG), e.g., by plasma or laser processing is another challenge, since the existing methods produce non-uniform thinning or introduce undesirable defects in the basal plane. We report here that heating to extremely high temperatures (exceeding 2000 K) and controllable layer-by-layer burning (thinning) can be achieved by low-power laser processing of suspended high-quality MLG in air in “cold-wall” reactor configuration. In contrast, localized laser heating of supported samples results in non-uniform graphene burning at much higher rates. Fully atomistic molecular dynamics simulations were also performed to reveal details of oxidation mechanisms leading to uniform layer-by-layer graphene gasification. The extraordinary resistance of MLG to oxidation paves the way to novel high-temperature applications as continuum light source or scaffolding material. Graphene has been considered one of the most promising materials for generating novel technological applications in near future. This exciting material presents exceptional electrical1357Most of these studies have been so far focused on single layer graphene (SLG) structures. However, large-scale production of high quality SLG and its manipulation have remained technologically challenging91112In particular, SLG is a flexible material that can be easily deformed when deposited on rough surfaces and it tends to fold up during processing in liquids, e.g., during transfer to other substrates16171920221920Recently, we have observed26MLG ~ 2000 K) without instantaneous burning out the samples. This strong heating is possible for suspended samples since the main heat losses in this case are due to heat conduction to air (<105 W/m2 K)−5 W. Losses due to continuum radiation emission are much smaller, estimated to be ~10−8 W, so that TMLG up to 2000 K or even higher can be achieved, for laser power of 1 mW and samples with surface area ~10 μm2.Here, we further exploit these ideas comparing exposure of supported (deposited over solid substrates) and suspended MLG samples to localized laser heating in air. It is important that experiments were performed under cold-wall reactor conditions using a low-power (a few mW) laser focused at the sample, where only a small solid sample is heated by the laser, while the surrounding gas is maintained basically at room temperature. Low gas temperature implies extremely low dissociation degreeExperimental configurations for laser heating of suspended and supported nanoplatelets are shown schematically in The observed uniform and gradual thinning of suspended platelets under localized low-power laser heating is in striking contrast to most of reports published so far considering burning of graphitic materials exposed to air or oxygen under high temperatures, as well as to our experiments with supported samples. Extremely high thermal conductivity of high-quality graphitic materials  in graphitic sheets3031193536373839The temperatures estimated from the Raman spectra are surprisingly high, up to 2000 K or even more. On the other hand, we also performed radiation spectra measurements in visible and near-infrared spectral region that confirmed strong increase of the radiation background corresponding to very high MLG surface temperatures see , and theAs can be seen from 19In MD simulations show that the etching process starts when atomic oxygen binds to a graphene layer forming epoxy structures. The presence of the epoxy groups allows carbon atoms to be extracted when more atomic oxygen reacts with the group, forming vacancies. These observations are in excellent agreement with the ones reported by Duin and co-workers3 levels are kept at very small values and tend to zero as temperature reaches even higher values. For the highest temperature values analyzed, CO formation is once again favored causing a drop on the CO2 levels, as the burning process becomes considerably faster as seen in It is important to stress that different gasification regimes are observed at different temperature ranges. Carbon dioxide formation is favored at low temperatures, characterizing a complete burning process. As the temperature reaches higher values, carbon monoxide formation becomes favored and carbon dioxide levels decrease. At this point, the formation of carbon trioxide can also be observed, but COThe reasons for the extraordinary oxidation resistance observed here in the suspended multi-layer graphene and burning under high-temperature laser heating in air are due to differences between our set-up and the ones usually employed in hot-wall reactor experimentsIn summary, we presented a novel approach for processing of suspended high quality multi-layer graphene platelets by irradiation using focused low-power laser in air. For comparison, processing of supported MLG was also studied. The gas was kept at room temperature due to the “cold-wall” reactor set-up. Irradiation by laser was performed using a confocal Raman spectroscopy configuration allowing for simultaneous monitoring of the temperature and quality of samples. We have shown that laser heating results in very high temperatures (up to 2000 K or even more) and the burning process happens in completely different ways for suspended and supported samples. When MLG flakes were supported on a solid (oxidized silicon) substrate, holes through many layers are formed in the heated region. However, for suspended graphene samples, a uniform thinning through selective layer-by-layer etching has been achieved, without significant changes in lateral dimensions of the samples. The analysis of data obtained using Raman spectroscopy during processing of suspended samples indicates also that formation of initial defects within the basal planes exposed to gas is followed immediately by their complete removal through fast in-plane etching. In other words, under the present hot-sample/cold-gas conditions the time required for the formation of initial defects in a basal plane is much larger than that required for burning out of a damaged graphitic layer. Reactive molecular dynamics simulations were used to gain further insights on these processes and are fully consistent with these interpretations.We have shown that with using a simple technique it is possible to controllably burn graphene layer by layer, thus allowing to obtain few layers or even single layer graphene. We would like to remark that using suspended multi-layer graphene samples  as proposed here, two challenging graphene problems can be solved: high quality controlled thinning of multi-layer graphene and achieving high graphene temperatures, even when exposing samples to atmospheric air. The results can provide new ways to achieve even stronger heating of multi-layer graphene in vacuum or in different controlled gas environments. The new methodology presented here can be adapted to the fabrication of few and multi-layer graphene of controlled thickness with remarkable low defect density. At the same time it paves the way for a new series of graphene applications, such as continuous radiation source and/or as scaffolding material stable at extremely high temperatures. We hope the present work will stimulate further investigations along these lines.MLG flakes were produced from high quality natural graphite  using mild ultrasound processing (sonication) in N,N-dimethylformamide (DMF) solutionsThe gradual and uniform thinning of samples was confirmed in a large number (>30) of experiments with different sizes of samples , applied laser power (from 0.1 to 1 mW) and exposure time .−1/K was measured, while for single layer and bi-layer graphene higher values were obtained (−0.016 cm−1/K and −0.015 cm−1/K). No data are available in literature for conversion coefficients at higher temperatures. Maximum G band downshift values observed here were as high as 30 cm−1, corresponding to near 3000 K or 2000 K maximum sample temperatures if −0.011 cm−1/K or −0.016 cm−1/K conversion coefficients  are used. Alternative method of graphene temperature determination is based on comparison of intensities for Stokes and anti-Stokes components of the G bandFrom In heating experiments, we usually kept the laser power below 1 mW. When elevated laser power (>1 mW) was used, samples were observed completely burned out in a few seconds , and we were able to observe the exact moment of the sample disappearance through the backscattered Raman signal. Gradual decrease of the Raman bands was evident for thickness of samples smaller than 10 nm allowing avoiding the complete sample burning.To characterize changes in the shape and thickness of MLG platelets we used high resolution SEM images  taken before and after heating procedure. For MLG flakes deposited onto Si-substrate we also made AFM (atomic force microscopy) measurements (NT MDT) to see profile change at the place where laser beam was focused.We processed a large number of suspended samples, and measurable changes in the thickness and/or lateral size were detected for 24 of them (some of flakes were burnt out completely or fell down as a result of processing). Significant decreases of the surface area and of the thickness during the heating procedure were observed and measured for 18 and 8 flakes, respectively.et al.For the supported flakes, the in-plane etching rates were calculated by estimating the change on the area before and after laser heating from SEM images, normalizing it by the average perimeter of the flake and then dividing this value by the time of laser heating. The cross-plane etching rate was calculated by dividing the thickness change by treatment time. For the supported flakes the cross-plane etching rate was calculated following the approach proposed by Pop 2) exposed to an atomic oxygen atmosphere .


Medicinal products that have been developed and approved for one disease may be the object of additional clinical development in other disease areas or of additional pharmaceutical development for new and different formulations. The newly developed products can be named as repositioned or reformulated products, respectively. Market access of repositioned or reformulated products in Europe and the United States is an interesting object of study as it may provide clarity about which parameters are assessed and considered to bring added value, other than the molecule itself. As such, we aim to evaluate if the added value of repositioned or reformulated medicinal products can be systematically described, quantified, and predicted. As a first step toward investigating the impact of market access on drug research and development trends for repositioned and reformulated products, it is necessary to have consistency in the designations for the case studies evaluated in this project. In an attempt to achieve that consistency, the current study aims to propose harmonized definitions for the repositioning and reformulation strategies and to propose a taxonomy for the medicinal products derived thereof.A systematic literature review was conducted to collect information on existing cases of repositioning or reformulation. A search strategy was developed by defining the search objectives, targeted data sources, search keywords, and inclusion/exclusion criteria for the retrieved documents.A total of 505 publications were retrieved through a search of the main data sources. The screenings and the ad hoc search led to a total of 56 publications to be used for the case study data extraction. In total, 87 repositioning and/or reformulation cases were found described in the literature, 23 of which presented different definitions and/or classifications by different authors.Given the disparity and inconsistency of terminologies and classifications in the literature, a harmonized nomenclature for drug repositioning, reformulation, and combination cases will allow for a robust analysis of the added value and market access conditions attributed for each strategy and case type as assessed by regulators and payors in Europe and the United States. After evaluation of the existing terminologies and given the absence of clear and consistent definitions for drug reformulation and repositioning in the literature, we propose a global terminology and taxonomy in order to cover all of the previously unclear definitions and classifications for repositioned and reformulated products. De novo drug development is costly ($1.3 billion) and time-consuming (10–17 years) (ostly $1. billion 7 years) .As a result of such market forces, pharmaceutical companies are looking for cost-effective and reduced-risk strategies for developing drug products and protecting existing products from competition as well as extending their patent protection time . Develop‘Reformulation’ is the development of different formulations for the same pharmaceutical drug , whereasThe study of the added value attributed by regulators and payors to potential new development directions for existing drugs is of interest to developers who wish to maximize the potential of their products and sustain their pipelines. As such, identifying opportunities and a rationale for market access of ‘repositioned’ and ‘reformulated’ drugs is a subject of high importance. In addition, market access of repositioned and reformulated products can help to illustrate the parameters that are assessed and considered to bring added value, other than the molecule itself. The regulatory strategies and regulatory pathway trends selected for repurposed drugs might vary considerably and also impact the market access conditions attributted for such products. However, the lack of consensus regarding a harmonized taxonomy for repositioned and reformulated products makes it difficult to identify and review existing cases.Part I: Reassessment of nomenclaturePart II: Regulatory pathPart III: Market access implicationsTherefore, we propose a three-part publication with the ultimate goal of understanding the rationale and eventually predicting the impact on market access and conditions attributed to drug reformulation and repositioning in the pharmaceutical industry in Europe and the United States:As a first step toward investigating the market access association with drug research and development trends for repositioned and reformulated products, it is necessary to have consistency in the designations for the case studies that are underlying this project. In an attempt to achieve that consistency, the current study aims to propose clear definitions and classifications for repositioning and reformulation strategies and to propose a harmonized taxonomy for the medicinal products derived thereof.Collect and describe potential cases of drug repositioning and reformulation mentioned in the literature.Describe and evaluate the circumstances that lead to drug repositioning and reformulation.Describe and evaluate the classification and nomenclature used for drug repositioning and reformulation in the literature.Evaluate the possible implications of existing regulations and guidelines for the development and market approval of drug reformulation/repositioning.Describe the success criteria to be achieved for drug repositioning and reformulation, from a payor perspective.Evaluate the impact of pricing and reimbursement requirements, regulations, and evaluations for the market access of repositioned drugs and reformulated drugs, and if these can be forecasted.Evaluate the potential lost benefit for patients and the society as a whole in cases where pricing or reimbursement cannot be achieved.A systematic literature review was conducted to collect information on the identified repositioning and reformulation cases. In line with the project's main goal, which is to research reformulation and repositioning cases in order to evaluate the impact on pricing and reimbursement decisions in Europe and the United States, the following objectives were set for the literature research:The present study focuses on objectives 1, 2, and 3 in order to evaluate the consistency of nomenclature of drug reformulation and repositioning cases.ad hoc search facilitated the retrieval of publications covering some of the topics of interest. In line with the objectives of the project, a list of keywords and a search algorithm were established  were considered for the search. For the initial selection, it was deliberately decided not to have a time limit regarding the time of publication. Any literature that mentioned only potential candidates for drug repositioning or reformulation were excluded given the impossibility to assess the impact on market access for these products.All the sourced references were imported to a Data Manager file. The list of titles and abstracts were screened (1st selection process) and once the full publications were collected, according to the process previously described, they were thoroughly screened (2nd selection process).ad hoc search allowed for the inclusion of eight more pertinent articles and four proprietary reports, which resulted in a total of 56 publications for data extraction.The search in the Embase database sourced 505 publications. After screening for duplicates and analyzing titles and abstracts, 87 publications were selected. The reasons for rejection were recorded . From thThe selected documents were published between 1995 and 2012. As shown in Our results included 125 cases of reformulation and repositioning. By using basic definitions for reformulation, ‘a new formulation of existing product’, and for repositioning, ‘a new indication for a known drug’, the number of cases was reduced to 87 after eliminating duplicates. In particular, some cases of repositioning such as sildenafil, thalidomide, and imatinib were frequently found. Timing of launch of the new productRepositioning or reformulation can be done with drugs that were never commercialized for their originally developed goals. This was the case with sildenafil for its first repositioning from angina to erectile dysfunction. Several cases were found for marketed products, including specific situations of discontinuations. A notorious example of that situation is that of thalidomide, which was repositioned twice after being tragically withdrawn from the market , 5, 7.Availability of generics of the original product when the change occurred® were already available when the new product was launched. It is common that line-extension drugs are introduced prior to the introduction of generics (®) was reformulated 1.5 years prior to patent expiry  and Paxil® (paroxetine), respectively  and minoxidil , 7, 18.In contrast, a rational approach relies on the understanding of the disease physiopathology and/or the drug mechanism. Imatinib is an illustrative example: the understanding of the role of KIT tyrosine kinase was the trigger for initiating the preclinical studies for gastrointestinal stromal tumors , 18, 23.Pharmacological target compared to the original productSeveral drugs, including duloxetine, sunitinib, and everolimus, were found to be effective in another disease via the same pathway or protein interaction. This is what some authors refer to as ‘on-target repositioning’ , 5.‘Off-target repositioning’ is defined as a new therapeutic use based on a newly discovered (or previously unexplored) additional pharmacological mechanism for a known drug . This waTherapeutic area compared to the original productIn many cases, the new and original indications are within the same therapeutic area [trastuzumab, sunitinib , and pacThe same drug product can be found effective in a different therapeutic area. There are many examples of on-target repositioning that fit in this category, including that of finasteride , 17, 18.The events and processes leading to the repositioning of a product are diverse. Repositioned products are classified based on three main criteria.Modified release formulations®)  technology for mesalazine tablets (Mezavant XL®) .Change in pharmaceutical form and/or change in administration routeChanges in the pharmaceutical form can be varied and can include no change in the administration route, such as the microemulsion form of cyclosporine A and the syrup form of valproic acid, both for oral use , 16. A cExcipients change without impact on pharmacokinetic parameters®) when reformulated to Astepro®   can be modified to form a new formulation. This modification ranges from a ‘simple’ chiral switch, such as escitalopram and esomeprazole , 12, 26,®) .The change of specific ingredient and structural change of the active compound may involve the change of chemical, physical, or clinical parameters as illustrated in Cases of reformulation are particularly frequent in the realm of psychiatry because non-adherence to treatment is a key issue in the treatment of mental illnesses , 12, 25.®) (®)   or in th®) (®) .Many definitions were available in the literature for drug repositioning and reformulation. There was an unclear definition and limitation on what is to be considered ‘true’ repositioning or reformulation, if these can include or exclude ‘simple’ line extensions and indicate how to define and denominate the cases when the drug is simultaneously developed through a repositioning and a reformulation process. Additionally, it wasn’t clear in the literature in which classification category can drug combinations be included. This was also the situation for the cases with structural changes of the active ingredient such as chiral switch, prodrugs or use of active metabolites.This lack of consistency was also observed in the classification criteria of the cases. Some authors based their classification on the change of pharmacokinetic properties, while others used a classification based on the timing of the original product's lifecycle. Furthermore, other authors based their classification on the differences in the approach used for discovery of indication or on the commercial success of the repositioned product.Our research findings confirmed the non-availability of a complete and homogeneous definition for both drug repositioning and reformulation. Thus, different authors have been developing and using different, and sometimes, even contradictory definitions , 28, 29.One of the common points in the different definitions used by different authors was that drug repositioning is defined as the process of finding a new use for an already known drug. However, definitions diverged in several parameters, such as the exact meaning of ‘new use’ and ‘known drug’. Some authors consider a new indication within the same/similar therapeutic use as repositioning, while others exclude this category from the repositioning definition. Another case was the so-called ‘geographic repositioning’, which is considered by some authors as a repositioning category while this particular case does not meet the definition criteria of discovery of a new use.Some authors considered reformulation as part of drug repositioning efforts. Also, differences regarding the classification of types of reformulation were found in the literature.Drug repurposingThe term repurposing includes all the re-development strategies based on the same chemical structure of the therapeutically active ingredient as in the original product. More precisely, under this term, we regroup drug repositioning, reformulation, and combination strategies.Drug repositioningRepositioning is the process of finding a new indication for a drug or compound. By this, we assume that the new indication is distinct from the already approved/intended indication of the original product, where ‘distinct’ implies an anatomical and/or therapeutically distinct indication referring to the 10th version of the International Classification of Diseases (ICD-10). The situation where the new indication involves a different pharmacological target (off-target repositioning) is the only exception where a new use in a similar indication will be covered by the actual definition.The original product candidate to drug repositioning should fall into one of the following categories:Drugs that were never commercializedDrugs in clinical developmentDrugs with finalized but failed/negative clinical development due to poor efficacy profile and/or less frequency due to efficacy issuesDrugs that were not completely developed, particularly from academic institutions and public sector laboratoriesDrugs that are/were commercializedDrugs that have been marketed but discontinued for commercial reasonsDrugs that have been marketed but discontinued for safety/public health reasonsMarketed drugs for which IP rights are still in placeMarketed drugs for which patents are already expired or when generic versions are already available in the market.Some authors also consider drugs that have been discovered, developed, and marketed in small or emerging markets but not widely launched nor launched in larger pharmaceutical markets, especially in the United States and Europe, as potential candidates for drug repositioning , 7, 33. ReformulationsReformulation is, by the simple definition of the term, making a particular change in the formulation of the original drug. This can be achieved by exploiting advances in formulation technology to change the release of the active substance, pharmaceutical forms, and/or route of administration but it can also concern some excipients with no impact on the pharmacokinetic parameters. No change should be incurred in the structure of the API except when it is a chiral switch. By this, we assume that approaches modifying the chemical structure of the API, such as prodrugs or active metabolites, are not included in our definition. Cases where the development of a new product does not include a change in the original formulation  should also be excluded.Repositioning aided by reformulationRepositioning aided by reformulation is a particular situation wherein a new formulation  is developed for a new therapeutic use of a candidate for repositioning. To be considered as a repositioned and reformulated product, the change incurred to the original product should adhere to both aforementioned definitions.Drug combinationRegarding the specific case of drug combinations, we concluded that drug combinations can be considered a separate strategy, and where criteria of definitions are met, they can fall under the classification of reformulation and/or repositioning. By this, combinations can be classified as cases of reformulation or repositioning provided there was a change in formulation or indication, respectively.The previously described absence of a common, clear, and concise definition for repositioning and reformulation of drugs prompted us to propose a global definition that attempts to cover all of the previously unclear and sometimes contradictory criteria for the definitions and classification of cases of repositioning and reformulation.Based on the proposed definitions and from our understanding of actual classifications, we propose a complete classification algorithm  for drugThe chosen parameters for classification reflect our evaluation of the attributes having a direct or indirect impact on the development of products, particularly on their market access conditions. The combination of factors such as the commercialization and genericization status of the original product at the time of launching the new product, being developed by the same or a different company, under the same or a different brand name as for the original product, leads to a large set of development scenarios.More specifically, cases of repositioning  and 4 arGroup 0: Chiral switch, excipient change without pharmacokinetic impact and cases where none of the other classification of groups  is applicable;Group 1: Same pharmaceutical form, same administration route, and different pharmacokinetic parameters ;Group 2: Different pharmaceutical form, same or similar administration route, and same pharmacokinetic parameters;Group 3: Different pharmaceutical form and different administration route.Concerning drug reformulation  and 5, iFinally, the classification of drug combinations  and 6 isAlthough our proposal aims to be universal yet precise in its content, some cases may be beyond its purview. Due to the ultimate goal of analyzing the association of pricing and reimbursement regulations on development of repurposed drugs, we have excluded in-development or non-approved cases and cases for which data could not be retrieved. As such, we cannot extrapolate the utilization of our proposed nomenclature to all possible existing cases of repositioning or reformulation. For all new cases not included due to date limitation inherent to the current literature review, we suggest that these can be analyzed and categorized for their nomenclature as soon as they are known and eventually the proposed nomenclature be re-evaluated in light of these future findings, if necessary.Given the disparity and inconsistency of terminologies and classifications found in the literature, a harmonized nomenclature for repositioning, reformulation, and combinations will allow a robust and consistent analysis of the added value and market access conditions attributed for each strategy and case type as assessed by regulators and payors in Europe and the United States.Click here for additional data file.Click here for additional data file.


During conversations participants alternate smoothly between speaker and hearer roles with only brief pauses and overlaps. There are two competing types of accounts about how conversationalists accomplish this: (a) the signaling approach and (b) the anticipatory (‘projection’) approach. We wanted to investigate, first, the relative merits of these two accounts, and second, the relative contribution of semantic and syntactic information to the timing of next turn initiation. We performed three button-press experiments using turn fragments taken from natural conversations to address the following questions: (a) Is turn-taking predominantly based on anticipation or on reaction, and (b) what is the relative contribution of semantic and syntactic information to accurate turn-taking. In our first experiment we gradually manipulated the information available for anticipation of the turn end (providing information about the turn end in advance to completely removing linguistic information). The results of our first experiment show that the distribution of the participants’ estimation of turn-endings for natural turns is very similar to the distribution for pure anticipation. We conclude that listeners are indeed able to anticipate a turn-end and that this strategy is predominantly used in turn-taking. In Experiment 2 we collected purely reacted responses. We used the distributions from Experiments 1 and 2 together to estimate a new dependent variable called Reaction Anticipation Proportion. We used this variable in our third experiment where we manipulated the presence vs. absence of semantic and syntactic information by low-pass filtering open-class and closed class words in the turn. The results suggest that for turn-end anticipation, both semantic and syntactic information are needed, but that the semantic information is a more important anticipation cue than syntactic information. Participants in a conversation have a number of tasks that they have to perform simultaneously. They have to comprehend the speaker’s utterance while at the same time they need to prepare their response to that utterance, preferably before the current speaker ends their turn. Despite the complexity of these processes the alternation between the speaker and the hearer roles is generally timed with only short pauses and overlaps . This coanticipatory approach, in which it is assumed that participants are able to predict the end of a turn in advance, and the signaling approach, which assumes that listeners perceive specific signals to detect the end of a turn.There are two competing main approaches providing an explanation for the turn-taking organization: the The aim of this study was first to determine the relative contribution of these two proposed mechanisms to turn-taking and second, to investigate which linguistic information sources listeners predominantly use for end-of-turn anticipation. To this end, we conducted a series of button-press experiments with turns from natural conversations while manipulating both the respective critical information sources and the task.The anticipatory approach argues that the precise timing in conversations can only be explained by the listeners’ ability to make accurate predictions about the end of the speaker’s utterances. Depending on the assumed anticipatory model listeners use various kinds of information to anticipate. The first to claim that listeners are able to anticipate a turn ending were anticipate these signals but react to them after having perceived them. Influential proponents of the signaling approach who did numerous studies on finding explicit turn taking signals are In contrast, the signaling approach assumes that turn transitions are regulated by an exchange of conventional vocal or gestural signals e.g., . So in tThe standard argument against the signaling approach is that the relevant cues occur too late in the speaker’s turn to enable timely speaker changes. As a counter-argument, We want to suggest that the presence of gaps longer than 200 ms does not necessarily mean that the turn before the gap was reacted to. Speakers often intentionally delay the production of so-called ‘dispreferred’ responses, which leads to longer pauses .The stimulus collection procedure is the same as the one described in For the audio recordings we put the participants in two separate rooms and required them to wear closed headphones. Directional microphones were placed on a table in front of them. We established a telephone-like connection between them, such that both participants could hear both themselves and their interlocutor. The speech of each of the two participants was recorded separately on the two channels of a stereo recording device. This way, we avoided cross talk between the participants in our recordings. The participants rapidly got used to the recording situation and the resulting conversations appeared natural and lively.Figure 1 shows the distribution of the FTO values.After recording the corpus, the conversations were transcribed, registering overlaps, pauses, laughter, turn beginnings and endings, assessments , and conTable 1) 1.Although the general shape of the FTO distribution resulting from the German telephone corpus looks similar to the Dutch FTO distribution from Table 2 presents some descriptive statistics of the target turns.From this corpus we randomly selected 100 target turns and an additional 16 turns for practice purposes. We took care that the turns contained at least five words so that the participants in the planned button-press experiments obtained enough information content to potentially base their reaction on. Furthermore, we made sure that the random selection reflected the distribution of pauses and overlaps of the natural conversations. Furthermore we balanced the sex of the speaker in the target turns . The total number of different speakers in our target stimuli was 16. After selecting the target turns, we extracted them into individual sound files using Praat  and creaNatural-Turn. The target turn was presented as it occurred in the natural conversations. In this condition the participants had access to all potentially relevant information to base their anticipation orreaction on.Advance-Knowledge. The participants could first read the content  of the turn before they heard the target stimulus. Because the participants knew in advance how the turn was going to end, they were, in principle, maximally capable to anticipate the turn end. In this condition the response distribution of anticipated responses was measured.Scrambled-Word-Order. We randomly changed the order of the words within the target turn using Praat. The pauses between the words in the original were assigned to the subsequent word. The resulting stimuli therefore had the same duration as the Natural-Turn stimuli. In this condition there was no sequential word-order information to base the anticipation on, but there were still words present. Thus, the predictability of a word on the basis of its preceding words is switched off, i.e., the cloze probability  each containing 25 target turns. Within each block there were four practice trials followed by the 25 target turns. We created eight different experimental lists. In the first four lists we permutated the block order according to a Latin-square design. The remaining four lists were the same as the first four lists with the block order as well as the presentation order of the stimuli reversed. Each of the target turns appeared in all four conditions across the lists but none appeared twice within the same experimental list.The participants received a written instruction that they had to listen to short audio fragments, taken from real conversations, and to press a button as soon as they thought the speaker in the fragments would finish speaking. They were informed that they would be presented with four different blocks, and that in one of these blocks they had to first read the content of the fragment before they heard the corresponding audio fragment. Furthermore, they were informed that in two blocks the stimuli were manipulated acoustically. The stimuli were presented to them via closed headphones. We randomly assigned the participants to one of the eight experimental lists (10 per list).The participants were presented first with the four practice trials and after that with the corresponding trial block. After each practice block the participants got the chance to ask the experimenter questions. Each experimental block contained a visual countdown from 3 to 1 followed by the auditory presentation of the stimuli. As soon as the participants pressed the button the sound was immediately cut off. In this way we made sure that the participants got no feedback about their performance. The trial block Advance-Knowledge differed from the other trial blocks because after the visual countdown the participants were presented with a written sentence, representing the content of the turn. After pressing the button the sentence disappeared and the acoustic presentation of the stimulus started.For the presentation of the stimuli we used the E-Prime software package ,b, whichFigure 2 shows the BIAS distributions for the four different conditions. Figure 3 shows an overview of the average BIAS per condition. The average BIAS is negative in all conditions, which gives a first hint that participants tried to anticipate the turn ending, rather than wait until the turn fragment was over.We first calculated the BIAS, which is defined as response time minus the duration of the target turn. F1 = 23.259, p < 0.001; by items: F2 = 18.82, p < 0.001). Bonferroni-corrected paired t-tests, pairing over identical turn fragments from the two conditions under comparison, revealed that the Natural turn condition led to significantly more negative BIAS than the Noise and the Scrambled-Word-Order condition. The latter condition led to significantly more negative BIAS than the Noise condition. Whereas the BIAS in the Advance-Knowledge and the Natural turn condition did not differ significantly from each other.An ANOVA for the dependent variable BIAS showed a significant effect for presentation condition . This provides “strong” evidence for the null hypothesis.The Bayesian paired t-test resulted in “substantial” evidence (BF = 0.1) for the null hypothesis. This analysis allows us to conclude that there is no statistically reliable difference between the BIAS in the Advanced-Knowledge and the Natural Turn condition. So the participants’ button press accuracy with the natural turns was just as good as when they had advance information about the content of the turn. This finding suggests that participants are indeed able to anticipate a turn ending, and that they are using this strategy to predict when a turn is going to end.Comparing the subject means of the BIAS variable with the Bayesian paired The significant difference between Scrambled-Word-Order and Noise condition indicates that having access to words (even though they were in the wrong order) still allowed them to anticipate better than chance.response consistency. To investigate the response consistency over conditions we computed the Entropy for every stimulus/condition pair  = 62.5, p < 0.001). Bonferroni-corrected paired t-tests revealed that all differences between individual conditions were significant (p < 0.001), the exception being the difference between Advance-Knowledge and Natural-Turn.As in the BIAS analysis, an ANOVA of the Entropy showed a main effect for condition t-test. The analysis (BF = 0.2) provided “substantial” evidence for the null hypothesis .Again we compared the Entropy values in the Advance-Knowledge and Natural-Turn condition using a Bayesian paired The analysis of the participants’ button press consistency supports the interpretation of the BIAS results. The results showed that the Entropy in the Natural-Turn condition and the Advance-Knowledge condition was comparable. Thus, in the Natural-Turn condition the participants were able to consistently and accurately anticipate the turn-end and consequently used anticipation as a strategy to tell when a turn was over.In contrast, in the Scrambled-Word and the Noise condition the Entropy values were significantly higher than in the other two conditions. This suggests that the participants tried to anticipate the turn-end rather than just waited for the end of the fragment, which lead to significantly broader distributed responses. In addition, the average Entropy in the Scrambled-Word order condition was significantly lower than in the Noise condition. This corresponds to the BIAS analysis above where participants in the Scrambled-Words condition were significantly more accurate in detecting the turn end. Hence, the participants are more consistent and accurate in the end-of-turn projection when they have access to words compared to when they only hear noise. One explanation of this finding could be that even with the scrambled word order listeners are able to recognize the basic meaning of the turn, enabling them to roughly guess when the turn finishes. Additionally, it is possible that once the participants “gambled” that a certain word was the last word, they could anticipate the end of that word, as suggested by research on auditory word recognitions .We showed in Experiment 1 that listeners in dialog are indeed able to anticipate the end of the speaker’s turn and that they consistently use this ability to predict when a turn is going to end. When listening to the natural turns the participants showed the same response accuracy and consistency as when they knew the end of the turn in advance. Our results are in line with earlier findings that listeners anticipate turn endings and that natural language is predictable to a certain degree . Hence, We now have an empirically derived distribution of anticipation times, from a task in which the participants were asked to anticipate turn-ends, and had the information to do so. To find out about the distributional properties of the reaction process, which we assume also plays a role, we need to study the reaction time distribution of participants that had no information to anticipate (as in the Noise condition of Experiment 1) but in addition, were not instructed to anticipate, but rather to respond to the end of the stimulus. To this end, we conducted Experiment 2.An empirically estimated anticipation distribution is provided by the Advanced-Knowledge condition of Experiment 1. In Experiment 2 we want to find the other distribution based on pure reaction time. To this end, we used the Noise stimuli from Experiment 1, but now explicitly instructed the participants to respond only after they perceived the end of the fragment.Twenty native speakers of German participated in the second experiment . None of the participants in Experiment 2 had taken part in Experiment 1.Each participant was presented with all of the 100 noise target stimuli created in Experiment 1. In addition we took four stimuli from the practice block for practice purpose. There were two experimental lists, whereas in the second list the presentation order of the stimuli  was reversed.The participants received a written instruction that they had to listen to noise fragments and press a button as soon as the noise stopped.Within the experiment the participants were presented first with the four practice trials followed by the 100 target stimuli. After the practice trials the participants got the opportunity to ask questions. After the presentation of the first 50 target stimuli there was a break. The participants had to start the presentation of the remaining 50 stimuli by pressing a button, so that they could determine the length of the break by themselves. The participants were randomly assigned to one of the two experimental lists (10 per list).Figure 5. As expected, the reaction time distribution shows a pronounced sharp peak at a positive FTO  whereas the anticipation distribution is broader and extends into the negative FTO range. In addition, the mode of the anticipation distribution is at a negative FTO value. The intersection of the two distributions characterizing the response time at which anticipation and reaction are equally probable is in good agreement with the 200 ms cut-off value proposed by The reaction time distribution obtained in this experiment is presented together with the anticipation distribution from Experiment 1 in relative probability of anticipation and reaction. The so-called Reaction Anticipation Proportion (RAP) value is defined as the natural logarithm of the ratio of anticipation and reaction probability.This is why we define a new measure designed to capture the Ant(t) and reaction probabilities PReac(t) at time t.Equation 1: Definition of the RAP value as logarithmized ratio of the anticipation PAnt(t)P and Reac(t)P denote the probability that a response at time t was an anticipation or reaction, respectively. These probabilities were computed in R  ted in R  using thFigure 6. The RAP is positive for a broad FTO interval ranging from about -750–200 ms and negative for FTO values in the interval from about 200–550 ms. Hence, reaction is more probable only in a relatively brief time interval. In addition, the influence of the pronounced sharp peak of the reaction distribution on the RAP value is weakened by the non-vanishing anticipation probability in the corresponding FTO range.The RAP as a function of FTO is presented in To demonstrate and validate the use of the RAP measure we applied it to the data analysis of the Natural turn and Noise conditions of the first experiment. The mean RAP value of the Natural turn condition was 0.60 and of the Noise condition -0.53. This supports our interpretation of the results of Experiment 1 that in the Natural turn condition the participants anticipated the end of the turn. In contrast, the responses in the Noise condition were predominantly based on reaction. It is noteworthy that the absolute value of the mean RAP of the two conditions are comparable, indicating that anticipation and reaction are about equally probable in the correspondingconditions.We used the RAP measure to study the relative contribution of semantic and syntactic information in end of turn anticipation in Experiment 3.In Experiment 3 we investigated the relative role of syntax and semantics as a cue for end-of-turn anticipation. Experimental as well as corpus-based studies  suggest 2.To tease apart semantic and syntactic information in natural communication we used the widely used distinction between open and closed class words in linguistics and psycholinguistics. Open class words in German contain nouns, verbs, adjectives, adverbs, and proper names. The open class words are “rich in referential meaning” , p. 539 Therefore, in this experiment, we operationalized semantic information as open class words, and syntactic information as closed class words. To address the question which information source listeners use for anticipation, we conducted a similar reaction time experiment as in Experiment 1. We manipulated the presence of semantic and syntactic information in the turn fragments from Experiment 1 by acoustically manipulating the recognizability of open- and closed class words. To evaluate the influence of these manipulations on the anticipation and reaction probability we used the RAP measure introduced before.If only syntax is used for end-of-turn prediction, then the absence of closed class words should result in a decrease in anticipated and an increase of reacted responses. On the other hand, if semantic information is used for the anticipation of a turn ending, we expect a deteriorated anticipation performance in absence of open class words. However, if both semantic and syntactic information are used to the same extent, then the effect should be similar in absence of content as well as closed class words.Eighty native speakers of German who neither participated in Experiment 1 nor in Experiment 2 participated in Experiment 3 .Table 4 for an example of one experimental stimuli in all conditions). Natural Turn: the target turn was presented as it occurred in the natural conversation. Closed-Class-Words-Removed: the closed class words were “removed” by low-pass filtering (at 500 Hz Hanning Window). Open-Class-Words-Removed: by low-pass filtering we “removed” the open class words (at 500 Hz Hanning Window). Intonation-Only: the whole turn was low-pass filtered (at 500 Hz Hanning Window) so that no words could be recognized, but intonation remained intact. This condition served as a comparative baseline since neither syntactic nor semantic information were left in the turn fragment.The same 100 target and 16 practice turns as in Experiment 1 were used. We created four different versions of each turn fragment  Natural-Turn, (2) Intonation-Only, (3) The stimuli from the Closed-Class-Words-Removed and Open-Class-Words-Removed condition. The latter were presented within one block. The blocks Natural-Turn and Intonation-Only contained 25 and the combined block Closed-Class-Words-Removed and Open-Class-Words-Removed 50 target turns (25 stimuli from Closed-Class-Words-Removed and 25 stimuli from Open-Class-Words-Removed). Within each block there were four practice trials followed by 25 and 50 target turns, respectively. We created eight experimental lists. As in Experiment 1, we permutated the block order in four of these lists according to a Latin-square design. The remaining four lists were the same as the first four lists with the presentation order of the target stimuli and the practice trials reversed. As in Experiment 1 the lists were constructed so that all of the 100 target stimuli appeared in all four conditions across the lists but none appeared twice within the same experimental list.We used the same procedure as in Experiment 1.Figure 7 shows the response distributions for the four different conditions. Figure 8 shows the average RAP values for the different conditions. The average positive RAP values in the Natural-Turn and the Closed-Class-Words-Removed condition indicate that the participants anticipated more frequently than reacted to the end of the turn in these conditions. In the Open-Class-Words-Removed and the Intonation-Only condition the participants reacted more often to the end of the turn.F1 = 47.85, p < 0.001, by items: F2 = 74.11, p < 0.001). Bonferroni-corrected paired t-tests revealed that all differences between individual conditions were significant (p < 0.001).An ANOVA on the RAP values showed a significant effect for presentation condition  they reacted significantly more frequently to the turn end than when only open class words were identifiably.The results suggest that semantic information is more important than syntactic information for end-of-turn anticipation. If no semantic information is available, it is less likely for the listeners to anticipate the turn ending. This also means that only knowing the syntactic frame and number of slots is not sufficient to estimate the timing of the turn. Nevertheless, the anticipation performance increased significantly when both open class words as well as closed class words were available. This could be explained by the fact that by removing closed class words the prediction of the content of the turn is also hampered. So for maximal anticipation performance listeners need semantic as well as syntactic information, probably because they need to be able to project the content of the turn. These results support the findings of Another interesting finding is that participants’ anticipation performance was significantly higher when they got only closed class words compared to when only intonational and rhythmical information was available. This indicates that on top of the prosodic properties the syntactic structure provides additional anticipation cues.Taken together these results suggest that semantic information is the most essential cue for anticipation. But to be maximally capable to anticipate a turn ending listeners need both semantic and syntactic information, since only the combination of both information sources allows for a correct projection of the content of the turn.In this study we addressed three different questions. First we investigated whether the observed accuracy in natural turn-taking was primarily due to anticipation or reaction to signals. Second, we wanted to quantitatively estimate the relative contribution of anticipation and reaction processes to the observed distribution of floor transfer timings. Finally, we studied the relative contribution of semantic and syntax in the timing of turn transitions.In Experiment 1 we conducted a button press experiment in which we manipulated the information necessary for anticipation. The results showed that the listeners’ response accuracy and consistency are similar when they (a) heard the natural turn and (b) when they are maximally able to anticipate the turn end by having advance information about the turn content. We conclude that listeners are indeed able to anticipate a turn ending andthat they use this strategy consistently to tell when a turn is going to end. Thus, our findings support the functioning of the turn-taking mechanism proposed by The data collected in Experiment 1 allowed us to estimate an empirical distribution for pure anticipation, sowe proceeded to assess the counterpart distribution for pure reaction in Experiment 2 by explicitly instructingparticipants to react to the end of noise signals. We combined the two distributions to estimate the RAP, whichrepresents the relative probability for a turn transition to have been guided by anticipation or reaction. By instructing the participants to react to the offset of a noise signal we estimated the ‘other extreme’ of anticipation, namely responding to the very end of a stimulus. We assume that a reaction to the offset of noise and a reaction to possible signals occurring at the very end of the turns  are comparable from a reaction time point of view. It should bepointed out that it is also possible that conversationalists react to signals that occur before the very end ofthe turn. Because in our approach we assessed only the extreme opposites of pure  anticipation and reaction, our data do not allow for an estimation of the possible contribution of such responses to the relative proportion of anticipation and reaction.In Experiment 3 we investigated the effect of the presence or absence of semantic and syntactic information on the anticipation and reaction probability using the RAP measure. The results showed that the participants werestill able to anticipate the end of the turn when they got access to semantic information. With only syntactic information available, the participants started to rely more on reaction. However, we found that to be maximallyable to anticipate, listeners need syntactic information as well as semantic information. The absence of syntactic information hampers the projection of the content of the turn. We concluded that for anticipation both semantic and syntactic information are needed. Nevertheless, it appears that semantic information is a more important cue than syntactic information.stochastic view of the turn taking process. By empirically estimating, for the first time, separate probability distributions for anticipation and reaction processes in end-of-turn detection, we were able to estimate the relative probability for a turn transition to be caused by anticipation or reaction at a given FTO value. This differs from the approach by The RAP measure introduced in Experiment 2 is not only an analysis tool for the characterization of turn transitions but implies an inherently Despite the mentioned advantages of the RAP measure over the strict threshold value suggested by We showed in Experiment 3 that semantic information is a more important cue for anticipation than syntax. This finding contradicts former studies  which asAnother explanation for the importance of semantic information in turn-taking could be that in naturalistic contexts, the semantics may provide stricter constraints on the turn construction than syntax does. Syntax theoretically allows for an infinite extension of a turn by the addition of new constituents. Furthermore, non-sense sentences like the famous “Colorless beautiful green ideas sleep furiously” , p. 15 aBy presenting isolated turns from natural conversations and letting the participants respond to the end of the turn by a button press we could both keep the characteristics of natural speech and at the same time systematically manipulate the turn fragments in order to test our specific hypotheses. Nevertheless, by isolating the turns we are not able to consider the impact of dialog context on anticipation. The discourse context could add information about the speaker’s illocutionary intentions in the turn that is being produced, which in turn could help the listener anticipate its content. It is an interesting issue for future research whether, and if so, how, the discourse context can improve the anticipation of a turn ending.The authors contributed to the following activities. Carina Riest and Jan P. de Ruiter designed the studies. Carina Riest and Annett B. Jorschick collected the data. Carina Riest, Annett B. Jorschick, and Jan P. de Ruiter analyzed the data. The paper was drafted by Carina Riest and revised by Annett B. Jorschick and Jan P. de Ruiter. All authors approved the final version of the manuscript and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.