Capsicum species), requires specific investigations to understand the physiological and biochemical responses to the aftermath of drought. The fruits and leaves of four chilli pepper cultivars were investigated to elucidate the fruits’ pungency , ascorbic acid content, DPPH  radical scavenging activity, polyphenol content, membrane lipid peroxidation and key protective antioxidant enzyme activity under drought stress (18–28% volumetric water content) as compared to the control (35–60%). Drought increased the chilli pepper fruits’ pungency expressed in Scoville Heat Units (SHU) as well as ascorbic acid content, but this relationship was also dependent on genotype and stress duration. ‘Jolokia’ was marked as most sensitive to drought by increasing content of capsaicinoids and DPPH˙ scavenging activity under stress conditions. Capsaicinoids and Ascorbic acid (AsA) greatly influenced the antioxidant activity of highly pungent chilli pepper fruits, although total phenols played a significant role in the mildly pungent genotypes. Generally, the activities of antioxidant enzymes increased under drought in chilli pepper leaves and fruits, although the intensity of the reaction varied among the cultivars used in the current research. All the investigated biochemical parameters were involved in the drought response of chilli pepper plants, but their significance and effectiveness were highly cultivar-dependent.In many regions of the world, the production of vegetable crops is limited by a deepening water crisis. Drought stress affects productivity and the chemical composition of crops. The variability of drought tolerance between species and cultivars of economically important crops, such as pepper ( Capsicum species. Peppers were domesticated in the Americas and successively introduced into cultivation in almost all the regions of the world [Sweet and hot peppers are among the most important vegetables and spices in the human diet, also used in the pharmaceutical and pesticide industries due to their high content of capsaicinoids, unique alkaloids restricted to the he world . In the he world ,3.Capsicum are capsaicinoids, compounds providing the pungent taste but also of exhibiting antioxidant activity [Chilli pepper is added to dishes in small amounts, but the intake of chilli even as a spice enriches the diet with polyphenols and other bioactive compounds, especially ascorbic acid and other vitamins, carotenoids (α- and β-carotene), capsaicinoids and mineral salts . The mosactivity . There iactivity ,7.2O2 decomposition. Ascorbic acid, an important compound in pepper fruits, acts as a primary substrate for enzymatic detoxification of hydrogen peroxide [Variability in the presence and concentration of phytochemicals in pepper species can be a factor affecting the selection of genotypes for breeding programmes . Fruit cperoxide . A promiperoxide ,15. Manyperoxide .Capsicum spp. are more susceptible to the aftermath of drought at the vegetative stage than at either the fruiting or the flowering stages [Drought is recognised as a stress factor that increases capsaicinoid accumulation in pepper fruits, although low pungency cultivars seem to be more sensitive in this respect , althougg stages . Proper g stages .Since the pepper is considered one of the most sensitive crops to soil water deficit, we hypothesised that application of moderate drought stress to greenhouse-grown, potted plants of different cultivars would allow the analysis of the mechanisms of biochemical and physiological adaptation. The response of leaves and fruits were included to track the pathways of adaptation linked with the fruit’s biochemical profile, the most important component of its quality. Results from analysis of variance (ANOVA)  clearly The SHU values showed ‘Jolokia’ fruits having the highest pungency and ‘Aji Lemon drop’ fruits having the lowest . SHU valInteractions between all experimental treatments on fruit pungency were also evaluated. The ‘Bird´s Eye’ and ‘Puerto Rican’ fruits showed a tendency to have higher SHU levels under drought stress conditions applied for 21 days, while 34-day-long stress treatment caused the reverse effect. ‘Jolokia’ fruits were characterised by significantly higher SHU levels under drought conditions regardless of duration. Differences between the two stress durations in the ‘Aji Lemon Drop’ were not significant. −1 of fresh weight (FW). The analyses of the main effects  content in drought stress and control conditions . The dif‘Jolokia’, ‘Bird’s Eye’ and ‘Puerto Rican’ fruits sampled from drought-stressed plants were characterised by higher DPPH˙ scavenging activity as compared to those collected from control plants. ‘Bird’s Eye’ and ‘Puerto Rican’ leaves collected from drought-stressed plants showed higher DPPH˙ scavenging activity in comparison to the control ones. Generally, higher DPPH˙ scavenging activity was noted in ‘Jolokia’ fruits and leaves while the lowest was in ‘Aji Lemon Drop’ fruits and leaves.Antioxidant enzymes activity and malondialdehyde content were significantly differentiated between cultivars and stress treatment in chilli pepper fruits and leaves as well . CatalasAscorbate peroxidase (APX) activity determined in ‘Aji Lemon Drop’, ‘Bird’s Eye’ and ‘Puerto Rican’ was higher in drought-treated fruits compared to the controls. A similar relationship was observed in the leaves of all cultivars. Generally, ‘Aji Lemon Drop’ was characterised by a higher APX activity measured in fruits and leaves.‘Jolokia’, ‘Bird’s Eye’ and ‘Puerto Rican’ fruits sampled from drought-treated plants showed higher guaiacol peroxidase (GPX) activity as compared to the controls. Similar results were determined in ‘Aji Lemon Drop’, ‘Bird’s Eye’ and ‘Puerto Rican’ leaves. The main effects showed the highest GPX activity in the fruits of ‘Aji Lemon Drop’ and the leaves of ‘Bird’s Eye’ peppers.Malondialdehyde (MDA) content was significantly higher in the fruits and leaves of all cultivars subjected to drought stress. In general, the highest content of this compound was determined in the fruits and leaves of ‘Aji Lemon Drop’.The relationship between experimental traits was determined by Pearson’s correlations analysis  and illuDPPH-radical scavenging activity of chilli pepper leaves was positively correlated with TP and SC contents as well as CAT activity, but negatively with GPX activity . BiplotsIn the dendrograms in Chilli pepper cultivars varied to a high degree in values of Scoville Heat Units (SHU), describing their pungency in terms related to the concentration of capsaicinoids, among which, capsaicin is the predominant component . There aThe mechanisms of crop plants’ reaction to different abiotic stresses are common. Abiotic stresses cause the accelerated production of reactive oxygen species (ROS). Plants have developed common antioxidant defence systems to control the oxidation cascades and to alleviate the cell damage caused by ROS . Non-enzThe cultivars of chilli pepper examined in our research responded to drought stress in different ways. The fruit pungency of ‘Bird´s Eye’ and ‘Puerto Rican’ slightly increased under 21-day-long drought stress application, and then later decreased. ‘Aji Lemon Drop’ showed the opposite behaviour and ‘Jolokia’ had increased fruit pungency under drought stress regardless of its duration. Similar behaviour was described by González-Zamora et al.  for temp−1 of fruit fresh weight, which is consistent with our results. ‘Jolokia’ was mentioned in the research of Kantar et al. [−1 of FW, and this range is considerably narrower than our findings. AsA content in ‘Bird´s Eye’ fruits varied between 530 and 1590 mg 1000 g−1 FW [Chilli pepper fruits are an excellent source of AsA, one of the most effective antioxidants, although the genetic factor is the most important determinant of the final amount of this compound. In the present study, the lowest amount of ascorbic acid was detected in ‘Aji Lemon Drop’ and the highest in ‘Jolokia’ fruits. Kopta et al.  stated tr et al. , who rep0 g−1 FW  and is tGenerally, drought increased AsA content in chilli pepper fruits, and this effect was more significant after 21-day-long stress application. According to Tuteja et al. , it coul−1 FW, and this range corresponds with the results of our research and confirms that the investigated cultivars are valuable sources of phenolics in the human diet. Drought often causes oxidative stress in plants, leading to an increase in the amounts of polyphenols and other secondary metabolites responsible for the acclimation response [Malondialdehyde (MDA) is the final product of lipid peroxidation in the cellular membranes. The increased MDA level reflects oxidative membrane damage . The actresponse ,39. In tresponse , signifiSoluble carbohydrates (SC) play a crucial role, not only in metabolic pathways, and they are important quality parameters of pepper fruits . It is i2 stimulates ribulose-1,5-bisphosphate oxygenation and thus photorespiratory hydrogen peroxide (H2O2) production in the peroxisomes [2O2 into oxygen and water [Drought stress first causes a morphological plant response, namely the closure of stomata in order to minimise transpiration. This causes a decrease of intercellular carbon dioxide concentration due to ongoing photosynthesis in the light. The decreased availability of COoxisomes . Enzymatnd water . Antioxind water , APX, CAThe experiment took place in 2019 in a polycarbonate greenhouse of the Faculty of Horticulture, Mendel University in Brno, Czech Republic, in the municipality of Lednice . The experimental site is located at 176 m above sea level. Meteorological parameters during the vegetation period (June–October) were as follows: the average temperature was 1.6 °C, average relative humidity was 68.9% and the period of sunlight was 1196 h .Structural parameters of the polycarbonate greenhouse were as follows: length 15, width 6 m, height 2.5 m, ultraviolet- stabilised polycarbonate, thickness 5 mm. The experimental protocol was based on the factorial combination between four cultivars and two irrigation regimes, using a split plot design with six replicates. Capsicum chinense Jacq.´Jolokia´ and ´Puerto Rican´, C. annuum L.→´Bird´s Eye´ and C. baccatum L. ´Aji Lemon Drop´. All cultivars were provided by the company World of Chilli, Czech Republic. Seedlings were planted on 10 June in 10 litre pots with the substrate Agro Profimix 2 . The chilli pepper cultivars used for the experiment were The two levels of the irrigation regime corresponded to: (i) the control, 35–60% of volumetric water content (VWC), and (ii) drought stress, 18–28% VWC. Drought conditions were set on the basis of soil daily VWC measurement with ECH2O-TE . VWC setting corresponds to the appropriate level of growing media humidity to assure optimal plant growth. According to the actual VWC, the plants were separately irrigated. Drought conditions were applied for 34 days (2 September–7 October). Measurements of analytical parameters were carried out at 13 and 21 days after drought stress application, respectively. The total amount of water applied to the control plants was 10 L per plant, on average. Drought-treated plants received 60% less water.−1 of Kristalon , containing N:P:K 20:5:10, and Mg 2%.Pests and diseases were monitored and treated according to the standard recommendation for the pepper crops. Prior to transplanting, the fertilisation was practiced by supplying to each plant 68 kg N, 20 P, 75 K, 58 Ca. During the cultivation, fertilisation was regularly applied in a form of foliar spraying with 1g LFruits were harvested from uniform plants at the stage of physiological maturity. Analytical parameters (ascorbic acid and SHU) were evaluated from the first (24 September) and second (7 October) harvests. Stress metabolites  were analysed separately in leaves and fruits sampled on 7 October. Analyses were performed in the Department of Horticulture, University of Agriculture in Krakow, Poland.−1) was multiplied by 15. Only fruit pulp was analysed. The high-performance liquid chromatography (HPLC) method according to Sawant et al.  was used3 of 80% methanol and centrifuged . The supernatant (0.1 cm3) was mixed with 2 cm3 of sodium carbonate. Then, after 2 min, 0.1 cm3 Folin–Ciocalteu’s reagent mixed with deionised water (1:1 v/v) was added to the test tubes. The absorbance of the resulting blue colour was measured at 750 nm using a UV-VIS (Ultraviolet–visible spectroscopy) Helios Beta spectrophotometer  against a reference solution. The results were expressed as gallic acid equivalents (GAE), mg GAE per 1 g FW.Total phenolics (TP) were estimated with the modified Folin–Ciocalteu colorimetric method . Fresh lTotal soluble carbohydrates were determined with the Yemm and Willis  method. 3 of supernatant and 4.9 cm3 of 0.1 mM DPPH˙ dissolved in 80% methanol. The mixture was incubated for 15 min in the dark and at 20 °C, the absorbance was measured using the UV-VIS Helios Beta spectrophotometer at 517 nm. DPPH˙ radical scavenging activity was calculated as DPPH˙ (%) = ((A0 − A1)/A0) × 100, where A0 and A1 are the absorbances of the reference and test solutions, respectively.DPPH˙ radical scavenging activity was determined according to the Bartosz  using th−1 cm−1.The malondialdehyde (MDA) content was measured by the thiobarbituric acid method . Fresh l3 0.05 M potassium phosphate buffer and centrifuged . Supernatant (0.2 cm3) was added to test tubes containing 1.8 cm3 0.05 M phosphate buffer (pH 7.0) and 1.0 cm3 0.05% H2O2 solution in 0.05 M potassium phosphate buffer (pH 7.0). The measurement of H2O2 disappearance was made spectrophotometrically at 240 nm (UV-VIS Helios Beta spectrophotometer).Catalase  activity was determined with the method of Aebi . Samples2O2, and 0.15 cm3 of supernatant. The H2O2-dependent oxidation of ascorbate was measured as the decrease in absorbance at 290 nm (ε = 2.8 mM−1 cm−1) with a UV-VIS Helios Beta spectrophotometer. APX activity was expressed as µg AsA min−1 g−1 FM. The reaction mixture for GPX determination contained 50 mM phosphorus buffer (pH 7.0), H2O2 (1%), and guaiacol (4%) and 0.2 cm3 of supernatant. The increase in absorbance at 470 nm due to guaiacol oxidation was recorded at 25 °C using a UV-VIS Helios Beta spectrophotometer over a period of 3 min (ε = 26.6 mM−1 cm−1). GPX activity was presented as µmol tetraguaiacol min−1 g−1 FM.Ascorbate peroxidase  and guaiacol peroxidase  were determined with the methods of Nakano and Asada  and Zhanp ≤ 0.05. If significant differences were detected between the treatments, the averages were compared and the mean separation was performed using the Scheffe’s test through the Statistica 12.0 software package . The correlation coefficient r was calculated to determine the relation between biochemical traits. Hierarchical cluster analysis was performed on the standardised data using a measure of Euclidean distance and the Ward minimum variance method. Experimental data were also processed using principal component analysis (PCA), in order to evaluate the existing relationships with original variables.Data were statistically processed by analysis of variance (ANOVA) at a significance level of In the present experiment, the evaluation of chilli pepper cultivars in terms of nutritional features and drought tolerance have given interesting hints for commercial use or breeding. It can be stated that SHU and AsA content were significantly affected by all the three experimental factors . The application of moderate drought stress to pot-grown chilli pepper activated biochemical and physiological adaptation mechanisms in cultivar-dependent ways. The drought-induced changes in metabolic pathways were different in the leaves and fruits of investigated genotypes. In some cultivars, short-term drought stress led to an increase in pungency and ascorbic acid content, whereas longer lasting drought resulted in the reverse effect. In ‘Jolokia’, the differences in pungency and ascorbic acid were statistically significant for both drought durations. Moderate stress levels could be an interesting tool for modifying the chemical profile in selected chilli cultivars. Interestingly, drought stress elicited the activation of the antioxidant system which represents an important goal, along with the identification of appropriate metabolic pathways, i.e., specific enzymes synthesis, connected with plant tolerance to drought. Indeed, the expressing of specific enzymes can lead to a beneficial increase in drought tolerance. The analysis of stress-induced metabolites in chilli pepper cultivars, namely capsaicinoids, ascorbic acid and total phenols, as well as antioxidant enzyme activities, can provide detailed information on common or specific mechanisms of drought tolerance and allow us to develop promising genotypes for broad practical application.


Drug combinations that target critical pathways are a mainstay of cancer care. To improve current approaches to combination treatment of chronic lymphocytic leukemia (CLL) and gain insights into the underlying biology, we studied the effect of 352 drug combination pairs in multiple concentrations by analysing ex vivo drug response of 52 primary CLL samples, which were characterized by “omics” profiling. Known synergistic interactions were confirmed for B-cell receptor (BCR) inhibitors with Bcl-2 inhibitors and with chemotherapeutic drugs, suggesting that this approach can identify clinically useful combinations. Moreover, we uncovered synergistic interactions between BCR inhibitors and afatinib, which we attribute to BCR activation by afatinib through BLK upstream of BTK and PI3K. Combinations of multiple inhibitors of BCR components  had effects similar to the single agents. While PI3K and BTK inhibitors produced overall similar effects in combinations with other drugs, we uncovered a larger response heterogeneity of combinations including PI3K inhibitors, predominantly in CLL with mutated IGHV, which we attribute to the target’s position within the BCR-signaling pathway. Taken together, our study shows that drug combination effects can be effectively queried in primary cancer cells, which could aid discovery, triage and clinical development of drug combinations. Ibru the BCR –10 and h the BCR , 12 and  the BCR . Another the BCR  that med the BCR . Althoug the BCR , and BCR the BCR . A promi the BCR , PI3K in the BCR , and BH3 the BCR ) and theMost systematic combinatorial drug studies to date have been based on cell line models. In diffuse large B-cell lymphoma (DLBCL) cell lines, inhibitors of mTOR , PI3K, BHypothesis-driven studies that investigated particular combinations of drugs on primary CLL cells showed synergistic effects for the combination of the Mdm2 inhibitor nutlin-3  and of tPB samples with high lymphocyte fractions  from 52 patients of the University Hospital of Heidelberg fulfilling standard diagnostic criteria for CLL were obtained. Samples from two additional patients were used in a first experiment but not considered for the analysis due to the lack of a suitable control plate. Patients provided written informed consent in accordance with the Declaration of Helsinki and local ethical approval. Mononuclear cells were isolated by centrifugation over Ficoll-Paque Premium  and cryopreserved.2. The drugs and cells were dispensed using an electronic pipette (Millipore) into 384 flat bottom Greiner plates. The plates were covered with breathe easy sheets and Greiner cell culture lids to prevent evaporation and incubated at 37 °C with 5% CO2 for 48 hours.Cells were thawed in RPMI-1640 supplemented with 5% FBS, 1% penicillin/streptomycin, and 1% glutamine. We used 40,000 primary cells per well, cultured in 50 µl RPMI supplemented with 10% pooled and heat inactivated AB-type human serum  at 37 °C, 5% COn = 34, Supplementary Table n = 52 patient samples), idelalisib (n = 30), afatinib (n = 30), and spebrutinib (n = 16)  and 0.2 µM (three samples) for navitoclax, at 0.1 µM for venetoclax, at 5 µM for afatinib (three samples), and at 20 µM for afatinib (two samples) and ibrutinib.To assess cell viability in the primary screen and in the validation experiments, the cells were incubated with the drugs for 48 h , and thn = 52), targeted 454 sequencing (n = 52), assessment of IGHV status (n = 52), assessment of structural variants (n = 52), genome-wide DNA methylation profiles (450k microarrays) (n = 52), and RNA sequencing (n = 52), as previously reported [TP53 mutant samples in our study was 5% . Briefly, primary CLL cells were lysed in M-PER Mammalian Extraction Buffer (Pierce). Cells were treated with 1.67 µM afatinib before or after cell lysis. 2 μg of cleared cellular lysate was mixed with 4 μl 10× protein tyrosine kinase reaction buffer (PK), 0.4 μl 1 M DTT, 0.4 μl 100× BSA, 1 μl 4 mM ATP, and 0.3 μl 1 mg/ml monoclonal antiphosphotyrosine FITC conjugate (clone PY20) adjusted to 40 μl with distilled H2O. All chemicals were provided by PamGene International BV. Each array was blocked with 0.2% BSA and washed with PK solution. Afterwards, kinase reaction was carried out at 30 °C. The reaction mix was pulsed back and forth through the porous material of the PamChip. Every fifth cycle a picture with a built-in CCD camera and after 60 cycles five additional pictures with increasing exposure time were taken.Kinase activities of primary CLL samples were measured using the PamStation® 12 system in combination with PamChip® Tyrosine Kinase Array Chips according to the manufacturer’s instructions  software. To increase the dynamic range of the measurements, the five pictures taken after 60 cycles were summarized into a single value by determining the slope of these signals as a function of exposure time, scaled by a factor of 100. The resulting values of the three biological replicates were logarithm transformed and averaged. The upstream tyrosine kinase responsible for on-chip peptide phosphorylation was predicted by using the “PamApp for PTK upstream Kinase Analysis” within the BioNavigator software (PamGene). This analysis is based on documented kinase-substrate relationships and in silico predictions from the phosphoNET database.A and B, we compared the measured viability vAB for the combination to the expected viability value given by the product of the corresponding single drug effects c of the library drug for each patient sample. In particular, viability measured lower than predicted resulted in SI > 0, indicating synergy. To summarize drug combinations across patient samples, the median SI was used. This approach to assess synergistic effects is strongly related to the Bliss-independence model [vAB . To test significance of synergistic combinations we used a one-sided paired t-test on the viabilities from the independence model and the measured viability from the drug combination across patient samples, separately at each concentration.The luminescence values per well were divided by the median of the DMSO-controls of each plate, resulting in values that indicate relative viability compared with the control; in the following, we term these viability values. As multiple wells on each plate contained the combination drug as a single agent we used the median of their viability values as a measure of the single agent effect for the plated sample. For the library drugs, every plate contained one well per concentration, which provided a viability value for the effect in combination or as a single agent in the plated sample. For four patient samples, we acquired replicate measurements; these were highly similar  approach , 43. Herp values resulting from the t-tests were adjusted for multiple testing across all drug–drug combinations and concentrations (m = 1920 tests) using the Benjamini–Hochberg procedure [The rocedure , separatBoth approaches above are based solely on drug effects at single concentrations and, whilst providing useful screening criteria for potentially interesting combinations, they might also identify less interesting cases. In particular, strongly nonlinear single agent dose–response relationships in the considered concentration range  can result in high values of SI and/or CI for noninteresting combinations, including combination of the drug with itself. To account for this limitation and to complement the effect-based combination scores at single concentrations we used the 10 × 10 validation experiments to assess the synergy for individual patient samples using the ZIP score  (as implTP53 mutation) with drug responses, Student’s t test with equal variance between groups was performed between the corresponding groups for each single drug or drug combination that had at least three samples in each group . Resulting p values were adjusted for multiple testing using the Benjamini–Hochberg procedure [m = 2226 or m = 320 tests.To assess associations of genetic features  measurements . To explore associations of recurrent genetic features (TP53 mutations and IGHV status) with drug sensitivities, we tested for differential drug responses between the sample groups with and without each of these mutations   for all drug–drug combinations that had at least three samples in each group . As shown in Fig. To study heterogeneity of drug responses in the combinatorial screen we first clustered the samples by their response profiles to the 32 agents across concentrations in combination with ibrutinib (52) Fig. . In line52) Fig. . These wTP53 mutations on the response to fludarabine and nutlin-3 as single agents was found to be preserved in combinations with ibrutinib  and idelalisib (n = 10) with inhibitors of BCR and downstream targets as well as CHK inhibitors as well as for afatinib in combination with CHK inhibitors , nutlin-3 (n = 3 out of 12), navitoclax (n = 7 out of 12), venetoclax (n = 4 out of 12), and afatinib (n = 5 out of 12)) and found striking similarities for combinations involving a BCR inhibitor as one of the partners, which suggests strong functional convergence  across patient samples with the 11 combination drugs, including both concentrations for ibrutinib Fig. . We idennce Fig. . For insnce Fig. . In partnce Fig. . Similarnce Fig. . These fL/Bcl-w inhibitor with clinical activity in CLL [Navitoclax is a Bcl-2/Bcl-Xy in CLL  and recey in CLL , 31 suggL inhibition limited its clinical use [As thrombocytopenia caused by Bcl-Xical use , venetocical use . Venetocical use  [1 µM–25In summary, these results confirm that combinations of BH3 mimetics and inhibitors of BCR signaling are effective in CLL, supporting recent clinical studies , 31 and TP53 and IGHV status influenced synergy. While unmutated IGHV positively influenced synergy, TP53 status did not influence synergy . We observed increased viability after 24 and 48 h, an effect blocked by addition of ibrutinib (data not shown). Afatinib was designed to inhibit EGFR at high specificity. Its effect in our study, however, is unlikely to be mediated via EGFR, as EGFR is not expressed in CLL cells . We alsoThe effectiveness of MEK inhibition in CLL has been demonstrated in ex vivo studies . Our scrTo characterize potential cooperative effects among different inhibitors of the BCR pathway, we included inhibitors of BTK , PI3K , and Syk (R406) in the drug library. Combination of these BCRi with one another did not result in synergistic effects. As shown in Fig. r = 0.94). However, we observed drug-specific differences of the combination effects, suggesting that the effects of inhibition of BTK and PI3K are not identical . Taking a global view across all library drugs, we found that combination with ibrutinib resulted in a stronger correlation between the response profiles of the different library drugs compared with idelalisib . Previous work demonstrated that the activity of EGFR inhibitor gefitinib in U-CLL by inhibition of BCR signaling via reduced phosphorylation of Syk/Zap-70/ERK/AKT and decreasing prosurvival proteins such as Mcl-1 . LikewisAlthough previous studies demonstrated that the efficacy of combinations of BCRi in DLBCL cell lines, CLL and MCL , 61, 64,While this screening platform offers a practical systematic approach for the identification of new drug combinations in primary leukemia cells ex vivo, it also has limitations. Cell death was assessed using an ATP-based viability assay (CellTiter Glo), therefore agents with negligible cytotoxicity cannot be judged well. CLL cells taken from PB do not proliferate, so the effect of drugs impeding proliferation cannot be gauged . PreclinDespite these limitations, this study identified potentially promising combination approaches for CLL that may warrant translation into clinical trials. In particular, this study, supported by the prior results , 34, 35,http://mozi.embl.de/public/combiScreen).To foster further research in this area, we provide a Shiny web resource as a resource for the community to explore the results from our screen (Supplementary figures


This distinguishes IST as a switchable infrared plasmonic PCM and enables a programmable nanophotonics material platform. We show how resonant metallic nanostructures can be directly written, modified and erased on and below the meta-atom level in an IST thin film by a pulsed switching laser, facilitating direct laser writing lithography without need for cumbersome multi-step nanofabrication. With this technology, we demonstrate large resonance shifts of nanoantennas of more than 4 µm, a tuneable mid-infrared absorber with nearly 90% absorptance as well as screening and nanoscale “soldering” of metallic nanoantennas. Our concepts can empower improved designs of programmable nanophotonic devices for telecommunications, (bio)sensing and infrared optics, e.g. programmable infrared detectors, emitters and reconfigurable holograms.The high dielectric optical contrast between the amorphous and crystalline structural phases of non-volatile phase-change materials (PCMs) provides a promising route towards tuneable nanophotonic devices. Here, we employ the next-generation PCM In Here, the authors introduce In3SbTe2 (IST) as a programmable material platform for plasmonics and nanophotonics in the infrared. They demonstrate direct optical writing, modifying and erasing of metallic crystalline IST nanoantennas, tuning their resonances, as well as nanoscale screening and soldering. Plasmonics achieves this goal by employing evanescent electromagnetic modes supported by metallic nanostructures to concentrate light fields to deeply subwavelength regions3. Arranging plasmonic nanoantennas (“meta-atoms”) in subwavelength assemblies in so-called metasurfaces allows for realizing ultrathin optical devices with exciting functionalities like beam steering4, lensing6, and holography8. While these functionalities are originally fixed upon fabrication, they can be rendered active by multiple means including antenna geometry change by mechanical deformation11, charge carrier tuning by electric biasing13, and inducing an insulator-metal transition in VO2 by heating17. All of these tuning mechanisms are volatile, i.e., they only persist as long as an external stimulus is provided. In contrast, phase-change materials (PCMs) offer non-volatile tuning. Because of the huge property contrast between their amorphous and crystalline phases resulting from a unique bonding mechanism21, they enable exciting tuneable functionalities like waveguiding24, chemical sensing25, light detection26, and emission27, as well as lensing28. This makes them prime candidates for non-volatile nanophotonic applications such as integrated optical memories, color displays, or active metasurfaces29. The resonance wavelength of antennas in metasurfaces is approximately proportional to the effective refractive index of the antennas’ dielectric environment and size. So far, PCMs have been used in active metasurfaces30 to change the effective refractive index of the dielectric environment, leading to significant resonance shifts32. While PCMs have been successfully used previously to directly optically write dielectric antennas in thin PCM films35, the plasmonic properties of typical PCMs have only recently been reported37. However, they are limited to the near-infrared spectral range by the materials’ band gap, leaving the full infrared spectral range unexplored, up to now. By employing the next-generation non-volatile plasmonic PCM In3SbTe2 (IST)39, we tap here into this unfulfilled potential for PCMs for infrared plasmonics. IST is a semiconductor in the amorphous phase (aIST) but becomes metallic with negative permittivity in the whole-infrared range in the crystalline phase (cIST)40. Compared with other alternative plasmonic materials42, IST’s optical properties can be optically and locally switched. Thus, we propose a programmable nanophotonics material platform in the infrared based on it.Many recent technological advances in key areas like telecommunication, lighting, health care, and solar energy harvesting have benefited from increasing control over light. Confining light waves to ever-smaller length scales and thus overcoming Abbe’s diffraction limit enables ultracompact optical components43).After comparing IST with common PCMs, we show how to write and erase plasmonic antenna arrays by optical switching on the single meta-atom level. Additionally, we demonstrate flexible antenna reconfiguration on the sub-meta-atom level by elongating, shortening and cutting individual rod antennas in half, causing significant resonance shifts. Next, we demonstrate optically tuneable, frequency-selective perfect absorbers based on cIST. Finally, we demonstrate how IST can be used together with conventional, prepatterned plasmonic metasurfaces to either screen individual antennas from incident light or to “solder” them together. Our concepts could lead towards fast and simple fabrication of plasmonic metasurfaces. Moreover, they can empower programmable functionalities of plasmonic devices for infrared optics, sensing and telecommunications. One could devise, for example, real-time (bio)sensing, in-situ pixel reprogramming of infrared detectors and displays, tuneable localized optical heating, and delicate post-fabrication metasurface adjustments to correct fabrication errors or minutely adapt the functionality  like Phosphor and GaAs. Covalently bonded materials share about two electrons between adjacent atoms and are therefore located in the red shaded region of the bonding map44 in Fig. 2Te3 and Ge2Sb2Te5  on the contrary are characterized by an unconventional bonding mechanism coined metavalent bonding45 where about one electron is shared between adjacent atoms. Hence, ordinary phase-change materials like GeSbTe (GST) compounds are all located in the green shaded region of the map .PCMs are characterized by a significant change of their optical properties upon crystallization, which has been attributed to a change in bonding mechanism. Amorphous phase-change materials show the characteristic features of covalently bonded materials . The conductivity of IST at about 104 S/cm46 is correspondingly also about 10–1000 times larger than that of crystalline GST47. This metal-like conductivity in the crystalline state leads to a very different change of optical properties upon crystallization and the potential to exploit extraordinary functionalities, as will be shown below. While GST compounds can be classified as “incipient metals”, IST belongs to the material class of non-volatile “bad metals”48, which form a bridge between the common PCMs and metals. Here, we reveal the opportunities offered by this interesting material class and IST especially for programmable infrared nanophotonics. To emphasize this potential, we denote IST as a plasmonic PCM.Remarkably now, crystalline IST has even less electrons shared between adjacent atomsε‘ at 2.5 µm is plotted for several PCMs49 .However, for crystalline IST the case is different: Because the electrons are delocalized similar to free electrons in a metal, IST changes from a semiconductor to a (bad) metaln of an antenna’s dielectric surrounding, IST can be used to directly change the antenna size by Δl or even totally change the antenna shape itself. While the large refractive index change has been shown to be a powerful tool, it is limited by the naturally occurring refractive indices of PCMs . In addition, the antenna is often not completely surrounded with PCM but, e.g., lies on a substrate, effectively lowering the influence of the PCM’s refractive index change. In contrast, there is almost no limit to the antenna reconfigurations that can be facilitated by IST: Even subdiffraction switching becomes possible when combining crystallizing and amorphizing pulses. Thus, the accessible degrees of freedom for designing infrared properties of antennas significantly exceeds that available with other PCMs.The marked difference of optical properties in the crystalline phase between IST and other PCMs gives access to different antenna resonance tuning mechanisms. Instead of changing the refractive index Δ37, other PCMs, like GeSbTe compounds, usually also have a small spectral window (mostly in the VIS-NIR) where ε‘ < 0 due to interband transitions at energies larger than the band gap energy. Because the charge carriers are not sufficiently delocalized, however, their permittivity does not follow the typical form of the Drude model and stays close to zero there. As a measure for a material’s plasmonic performance in nanoresonators52, in Fig. ν/FWHM, where ν is the resonance frequency. Common metals like Al and Ag have Q factors of about 6. It is noteworthy that Al has a slightly larger Q factor compared to Ag due to its smaller skin depth in the infrared (7.7 nm for Al and 11 nm for Ag at a wavelength of 2.5 µm). The Q factor for IST is about 4, which is significantly larger than that of VO2 and still slightly larger than that of TiN and AZO . Not only for localized but also for propagating surface plasmon polaritons, IST has a superior figure of merit compared to GST in the interband transition region and VO2 . Optical addressing has recently been used with GeSbTe alloys to fabricate thin-film dielectric metasurfaces34 and to program individual meta-atoms in prepatterned PCM-incorporating metallic55 and dielectric metasurfaces56. Going one step further, it is now possible to directly write and erase metallic elements on and below the meta-atom level in a metasurface containing a thin IST film by nanosecond laser pulses. Compared to conventional lithography, no cumbersome additional steps like lift-off or etching are required and the whole metasurface can be erased and rewritten fast and easily at any time. This vastly expands the possibilities of how PCMs can be employed in active metasurfaces.Active infrared metasurfaces based on PCMs can be switched between multiple operation states via heating2 substrate 20 , whereas the resonance is completely gone in the erased state and almost unity transmittance is obtained (blue). To demonstrate the performance over several switching cycles, the transmittance at λ = 4.66 µm is plotted for the first 20 switching steps in Fig. ε‘ < 0 in the visible spectral range (because of interband transitions), which results in high reflectance in the light microscope. In contrast, aIST has ε‘ > 0 in the visible spectral range and thus reflects much less light. In the AFM topography image, however, all three cases can be discerned. The written and rewritten cIST antennas are visible as depressions in the aIST film , while the erased antenna shows a slight elevation in topography (microbump formation60). With s-SNOM, it is possible to measure the local infrared optical properties of the submicrometer crystalline areas with a resolution that is well below the diffraction limit61. The measured near-field optical amplitude signal s3 at a wavelength of λ = 10.57 µm, referenced to the amplitude signal s3(aIST) of the aIST, is shown in the bottom row of Fig. As a platform for the following applications, we investigated how to write and erase plasmonic nanostructures on the meta-atom level in a thin film of 50-nm thick aIST on a CaFsee Fig. . A lightAfter establishing the viable writing and erasing of metallic nanostructures in an IST thin film on the meta-atom level, we performed first proof-of-principle experiments see Fig.  to demon2 antenna array. After the initial writing, the antenna lengths were subsequently changed by switching the IST at the antenna tips. Figure λ = 3.5 µm.We employed the same layer stack as in Fig. Remarkably, the antennas can be made even shorter via reamorphization than the initial cIST antennas. This results in a significantly shorter resonance wavelength: 4.4 µm instead of 5.7 µm. By overlapping slightly shifted (diffraction-limited) crystallizing and reamorphizing laser pulses, it becomes possible to achieve optically switched structures on a scale below the diffraction limit of the VIS switching laser.λ = 4.3 µm, which is more than 20% larger than the tuning range achieved for the static array periods in Fig. The longest antennas in Fig. λ/FWHM ≈ 1.9. Here, the FWHM = 2.3 µm of the smallest antennas was considered in accordance with literature62. This is an exceptionally large TFOM value when compared with common GeSbTe compounds, which facilitate resonance shifts of Δλ ≈ 1 µm and TFOM ≲ 1.232. The resonance tuning with IST of Δλ = 4.3 µm also surpasses other tuning concepts based on charge carrier density tuning  or on phase-transition materials like VO216, where Δλ ≈ 1–2 µm.The large total shift yields a large tuning figure of merit TFOM = Δλres = neff2l + δ, where neff is the effective refractive index of the antenna’s dielectric surrounding and δ is a constant offset parameter65. This linear relation is confirmed in Fig. λres = 1.7·2l + 2.1 µm. Thus, the cIST antennas can be indeed described as plasmonic nanorod antennas. For substrates with a larger refractive index , the effective refractive index neff of the antennas’ dielectric surrounding, and thus their resonance wavelength, is increased. Accordingly, by adjusting the substrate material, one can change the antenna lengths required for a certain spectral resonance position66 and optimize the resonance quality in that wavelength range  of classic metallic nanorod antennas follows the relation Modifying cIST nanoantennas is not limited to adding or removing cIST at the antenna ends. In Fig. The creation of more complex structures also allows to access different resonance modes and to change between them: As shown, one can switch from a single electrical dipole to either two coupled electrical dipoles (cut nanorod) or to a magnetic dipole (split ring resonator). Higher-order modes like quadrupole resonances could also become attainable by reshaping or coupling of individual resonators.80:(SiO2)20, 115 nm aIST, and 70 nm (ZnS)80:(SiO2)20 are deposited , it almost reaches down to 10%. This corresponds to a maximum absorptance of A ≈ 90 % and the tuning enables an absorptance modulation of up to 65%.In another proof-of-principle experiment for optically written plasmonic IST metasurfaces, we fabricated a frequency-selective IR absorber see Fig. . First, ted Fig. . Accordicf. Fig. . The mettra Fig. , there id is plotted against the bar width w. It is determined by a comparison of minimum reflectance between simulations and measurements. The simulated spectra , crystallization starts there and then proceeds downwards. Experimentally, the different crystallization depths are realized by using increasing laser pulse durations for increasing cIST bar widths w (cf. Methods). The optical heating for longer times then allows for crystallization deeper into the aIST layer, resulting in larger crystallization depths. For the given absorber structure, the crystallization depth is an important parameter because it not only defines the total crystallized volume but also the distance between the plasmonic structures and the metallic back-mirror, which is directly related to their electromagnetic coupling and the resulting magnetic dipole moment. Only by optimizing the crystallization depth, the absorptance can be maximized to close to 100% for a given layer stack  and on (antennas excited) by optically switching the IST above the antennas. In the crystalline state, the cIST area acts like a mirror, which screens the antenna from the incident light and thus prevents antenna excitation. This is demonstrated here for the case of an IST-coated Al slit antenna array on a Si substrate see Fig. . In Fig.2-capping are deposited exclusively at the 100 nm dimer gap of 150-nm wide, 30-nm thick Au antennas of different lengths l and period p = 2.4 l. The aIST was then crystallized by heating on a hotplate (see Methods). In the plotted transmittance spectra, the spectral redshift of the transmittance minimum (resonance) from aIST (solid lines) to cIST (dashed lines) patches is observable for each length l. This resonance shift resulting from the conductive connection between the dimer antennas becomes larger with increasing l. For l = 600 nm, it is about 600 nm large, which corresponds to a tuning figure of merit of about 0.7. Here, the resonance shift would originally be even larger, but the Au antenna lengths decreased after annealing in the oven  of a dimer with 600 nm long antennas and a cIST patch are depicted in Fig. Until now, the switching of IST was performed optically and was therefore limited by diffraction of the red light of the switching laser. In case the IST needs to be switched at even smaller scales, it can be prepatterned with nanofabrication methods like electron beam lithography. In this way, ultrasmall IST switches can be strategically arranged on a metasurface and selectively toggled between dielectric and metallic optical properties at the sub-meta-atom level. As an application, we show how the two rods in a gold dimer antenna can be conductively connected by a small patch of IST positioned in the dimer gap Fig. . Thus, t43. On the other hand, one can also realize major reconfigurations like a total change of functionality, e.g., between beam focusing and beam steering. Please note that the switching of prepatterned IST structures is not limited to optical laser pulses but can also be achieved via electrical voltage pulses58.Multiple potential nanoantenna geometries can now be encoded on a single metasurface with strategically positioned IST patches and then switched between by nanoscale soldering. On the one hand, this allows for minor adjustments, which is, e.g., important for compensating tight fabrication tolerances and for adaptive optics3SbTe2 (IST) as a material platform for programmable plasmonics and nanophotonics in the infrared. On the meta-atom level, we demonstrated direct writing and erasing of metallic crystalline IST (cIST) nanoantennas into a dielectric amorphous IST (aIST) thin film for 20 switching steps and characterized them by diffraction-limited far-field as well as super-resolution near-field measurement techniques. On the sub-meta-atom level, we demonstrate flexible antenna reconfiguration by elongating, shortening and cutting individual rod antennas in half, tuning the infrared plasmonic antenna resonances by more than 4 µm. Subsequently, a tuneable broad-band infrared absorber with absorptance of nearly 90%, a mid-infrared spectral tuning range of about 3 µm and an absorption amplitude tuning range of about 65% was shown. Nanoscale screening of prepatterned antennas with a thin IST film results in an on/off functionality, while strategically positioned IST patches allow for nanoscale “soldering” of individual meta-atoms.We introduced the next-generation, switchable, infrared plasmonic phase-change material InThese concepts are fundamentally impossible to achieve with common dielectric PCMs . In addition to a large resonance tuning, IST enables the creation of complex plasmonic structures with different resonance modes. The optical switching can be easily scaled up and parallelized  towards large-area fabrication with high throughput. Our work thus lays the foundation for a programmable nanophotonics material platform for the infrared. It also holds great potential for application in integrated photonics, where, reconfigurable plasmonic components for, e.g., routing, waveguiding and filtering are now made possible by optically or electrically switched IST elements. The extension of our frequency-selective near-perfect IR absorber could lead towards real-time biosensing or the in-situ reprogramming of infrared detectors and displays working in absorption or emission mode.72. Composing dielectric metasurfaces75 out of amorphous IST could enable the switching from narrow Mie resonances to broad plasmonic resonances. Finally, the non-volatile switching of IST could be combined with the dynamic, volatile tuning methods offered by materials like VO2, leading to interesting functionalities like multilevel, intelligent switches77.A thin IST layer on top of negative permittivity materials that host surface waves such as polar crystals (phonon polariton) or doped semiconductors (plasmon polariton) could enable the writing of nanophotonic elements for polaritons like deeply subwavelength resonators or waveguides3SbTe2 (IST) was deposited on the substrates by direct current magnetron sputtering, while (ZnS)80:(SiO2)20 was deposited with radio frequency magnetron sputtering. A LS 320 von Ardenne systems  was operated in constant power mode (24 W for IST and 59 W for ZnS:SiO2) using stoichiometric targets of 99.99% purity. The Al slit antennas were fabricated by first thermally evaporating Aluminum  onto a Si substrate and then patterning the Al thin film with focused ion beam milling with a Helios NanoLab DualBeam 650 by FEI company . The gold dimer antennas with IST in their gaps were fabricated by two-step electron beam lithography with PMMA as resist and Electra92 as conductive polymer. In the first step, gold was evaporated onto the patterned resist. After a lift-off process, the gold dimer antennas are left on the glass substrate. In the second step, the resist was only patterned in the gaps of the dimers. Onto this patterned resist, 50 nm IST and 10 nm (ZnS)80:(SiO2)20 were sputtered (see above). After another lift-off process, the finished gold dimers with IST in their gaps are left on the glass substrate.In55. A pulsed laser beam (wavelength λ = 660 nm) is focused through a 10-fold objective with a numerical aperture of 0.5 on the sample surface. The sample is placed on a Thorlabs NanoMax-TS (Max311/M) stage, which is movable in x-, y-, and z-direction and connected to a Thorlabs closed-loop piezo controller (BPC303). A custom program allows for the automated positioning of pulsed laser shots on the sample surface within 5 nm accuracy. By using long (~500 ns) and low-powered (~10 mW) laser pulses, an IST thin film can be locally crystallized by heating it above the glass transition temperature. The crystallized area has an elliptical shape, whose size can be tuned by changing the laser parameters 55, see e.g., Supplementary Fig. 9 K/s) yields amorphization. The laser pulse parameters  for Fig. px, py] = . For Fig. l, px, py] in µm are measured by light microscopy and given as , ,  and . For Fig. px, py] = . In Fig. w = 0.47 µm, 310 ns for w = 0.65 µm, 340 ns for w = 0.8 µm, and 370 ns for w = 0.9 µm. The switching parameters for screening the slits in Fig. The localized phase change of the IST thin-films was realized with an in-house-built laser setup−1 spectral resolution for Figs. −1 spectral resolution for Fig. The Fourier transform infrared (FTIR) spectroscopy data were collected using a Bruker Vertex 70 interferometer coupled to a Bruker Hyperion 2000 microscope. The FTIR measurement parameters for the data from the Figures is as follows: 1000 scans with 16 cmΩ ≈ 235–250 kHz. This oscillating tip is illuminated by light from a CO2 gas laser at 10.57 µm and the backscattered light from the tip is collected. To suppress background scattering, the detector signals are demodulated at a higher harmonic nΩ(n ≥ 2). By means of pseudo-heterodyne interferometric detection78, the near-field optical amplitude sn and phase φn are obtained. For measurements in Fig. Ω. For measurements in Fig. Ω.Our s-SNOM system (Neaspec GmbH) is based on a tapping-mode AFM. The metallized AFM tip radius ∼25 nm) vibrates with an amplitude of ~40 nm at a tapping frequency 5 nm vibr2, a constant refractive index of about 2.1 and for CaF2 a constant refractive index of 1.4 were assumed.Full-wave 3D simulations have been performed using a commercial solver, CST Studio Suite. Excitation by Floquet Mode Ports has been used to model the experiment setup and to calculate the spectra. Periodic boundary conditions have been used in the lateral directions. Spectra for angles of incidence between 8° and 24° were calculated and averaged according to the intensity distribution of the ×15 Cassegrain objective. The permittivity of IST is shown in Supplementary Fig. Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1


The objective of this study was to investigate peptide YY3–36 plasma concentrations at different stages of anorexia nervosa in a combined cross-sectional and longitudinal design to differentiate between effects of acute undernutrition and more enduring characteristics.The gut–brain axis could be a possible key factor in the pathophysiology of anorexia nervosa. The neuropeptide peptide YY3–36 concentrations in young patients with acute anorexia nervosa (n = 47) and long-term recovered patients (n = 35) cross-sectionally in comparison to healthy control participants (n = 58), and longitudinally over the course of inpatient treatment. Physical activity was controlled as it may modulate peptide YY secretion.We measured fasting plasma peptide YY3–36 concentration among young acutely underweight anorexia nervosa patients, long-term recovered anorexia nervosa patients, and healthy control participants. Longitudinally, there was no change in peptide YY3–36 concentration after short-term weight rehabilitation. For acute anorexia nervosa patients at admission to treatment, there was a negative correlation between peptide YY3–36 concentration and body mass index.There was no group difference in peptide YY3–36 concentration in AN. Future studies should study multiple appetite-regulating peptides and their complex interplay and also use research designs including a food challenge.The current study provides additional evidence for a normal basal PYYThe online version of this article (10.1007/s00394-020-02210-7) contains supplementary material, which is available to authorized users. Anorexia nervosa (AN) is a life-threatening eating disorder typically beginning in adolescence and characterized by an intense fear of weight gain and a relentless pursuit of weight loss, mostly by self-starvation. Only less than half of AN patients fully recover from the disorder, one-third shows improvement with residual symptoms, and one fifth remains long-term chronically ill . The und1–36 is cleaved enzymatically by dipeptidyl peptidase 4 (DPP-4) to the main circulating form PYY3–36 [1–36 and PYY3–36 regarding energy and glucose homeostasis [3–36 is considered to be the one relevant to energy homeostasis [3–36 are mostly mediated through agonism at Y2 receptors on neurons both in the gastrointestinal tract and in the central nervous system [3–36 [3–36 leads to a significant reduction in appetite and food intake in healthy, non-obese volunteers. Evidence from functional magnetic resonance imaging (fMRI) studies points to an influence of PYY concentration on neural activity in brain regions involved in reward processing , implicating that PYY may modulate the reward value of food [Peptide YY (PYY) belongs to the neuropeptide Y family of biologically active peptides that are considered important mediators of the gut–brain axis . It is a PYY3–36 , 9. Thiseostasis , 10, 11.eostasis . The effem [3–36 . Batterhem [3–36  found th of food , 15.3–36 instead of total PYY [Relatively few studies have investigated PYY concentration in AN yielding heterogeneous results, as highlighted in Table otal PYY , 24, 30.3–36 concentrations of patients with acutely underweight AN, before and after partial weight recovery , as well as in long-term recovered patients in comparison to healthy control participants . The inclusion of long-term recovered patients allows the differentiation of state markers  from factors that might confer vulnerability towards AN. To control for possible exercise-related bias on PYY in AN, blood samples were obtained shortly after patients were admitted to an intensive treatment program which helps patients resist the drive to exercise.Gaining a broader understanding of the role of gut hormones such as PYY in the etiology and maintenance of AN holds the potential of identifying prognostic biomarkers and opening up new avenues for treatment. To date, no study on PYY in AN has taken physical activity into account, even though it is likely to be of great importance as a confounding factor in this patient sample. Furthermore, there is a considerably research gap regarding the persistence of possible changes after short-term and long-term weight recovery. Thus, the objective of the current study was to investigate PYY2 (if older than 15.5 years). RecAN participants were required to have had a diagnosis of AN in the past and, for at least 6 months before the study, (1) to have maintained a BMI above the 10th age percentile (if younger than 18 years) or above 18.5 kg/m2 (if older than 18 years), (2) to menstruate, and (3) to not have binged, purged, or engaged in substantial restrictive eating patterns. HC had to be of normal weight, eumenorrhoeic, and without any history of psychiatric illness. All HC were assessed with the SIAB-EX [A total of 140 female subjects participated in this study: 47 underweight patients with acute AN , 35 patients long-term recovered from AN , and 58 healthy control participants . For the longitudinal arm of our study, 32 acAN patients were reassessed after short-term weight rehabilitation. Participants with acAN were admitted to intensive treatment of an eating disorder program at a child and adolescent psychiatry and psychosomatic medicine department of a tertiary care university hospital. Newly admitted patients spent their first days under close supervision by specialized nursing staff and continuous heart rate monitoring at night. Therefore, we can ensure an exercise-free time period of at least 18 h before blood sample collection. Diagnosis of AN was established using the expert form of the Structured Interview for Anorexia and Bulimia Nervosa (SIAB-EX)  and requ SIAB-EX  and exclInformation regarding exclusion criteria and possible confounding variables, including menstrual cycle and use of contraceptive medication, was obtained from all participants  using the SIAB-EX , supplemAdditional to the evaluation with the SIAB-EX , eating g for 15 min) in a pre-cooled centrifuge (5 °C), aliquoted, and stored at − 80 °C. Plasma PYY3–36 and leptin concentrations were measured according to the manufacturer's instructions. To prevent enzymatic in-vitro degradation of PYY, DPP-4 inhibitor  was added before thawing of samples in a concentration of 10 µl/ml. Plasma PYY3–36 concentration was analyzed in duplicate using the commercially available human PYY3–36-specific radioimmunoassay  with an intra-assay coefficient of variation (CV) of 6–11%, inter-assay CV of 7–15%, and a lower limit of detection of 20 pg/ml. The employed RIA utilizes 125I-labeled PYY and a PYY3–36 antiserum to determine the PYY3–36 concentrations in the research samples and is specific to human PYY3–36, while PYY1–36 is not detectable. Plasma leptin concentration was measured using a commercially available enzyme-linked immunoabsorbent assay with an intra-assay CV of 4.2%, inter-assay CV of 6.7%, and a lower limit of detection of 0.2 ng/ml  and served as a control variable.Venous blood samples were collected into vacutainer tubes between 7 and 9 a.m. after an overnight fast, for the acAN group at the first timepoint within 96 h after initiating intensive treatment. Aprotinin  was added during blood sampling to prevent protein degradation by serine proteases. Samples were immediately centrifuged . The Wilcoxon signed-rank test was employed to analyze PYY3–36 concentrations longitudinally in the acAN group . Correlation analyses were performed using Spearman correlation and p values were corrected for multiple comparisons using the False Discovery Rate correction method of Benjamini and Hochberg [For all samples, PYYHochberg . StatistHochberg  with theDemographic and clinical characteristics are summarized in Table 3–36 concentration among the acAN-T1, recAN, and HC groups, H(2) = 4.04, p = 0.133 , duration of recovery (for the recAN group), and measures of psychopathology  were tested for each group using Spearman correlations. For the acAN-T1 group, there was a negative correlation between PYY3–36 and BMI , but no significant correlation of PYY3–36 with any of the other parameters including age-adjusted BMI-SDS and pre-treatment physical activity. There were no significant associations between PYY3–36 and demographic and clinical variables for the acAN group at T2, the recAN, or the HC groups. An exploratory analysis to test a possible influence of the duration of realimentation and BMI change over the course of treatment on PYY3–36 concentrations in the longitudinal acAN study population revealed no significant influence of these factors .Associations of PYY3–36 concentrations in patients with acute AN and individuals after long-term recovery from AN cross-sectionally in comparison to healthy control participants, and longitudinally over the course of inpatient treatment. To specifically measure the isoform most relevant to energy homeostasis [3–36-specific RIA. There was no group difference in PYY3–36 concentrations. Our longitudinal analysis in acute AN patients also yielded no change in PYY3–36 concentration after short-term weight rehabilitation. At admission to treatment, we found a negative correlation between PYY3–36 concentration and BMI.In the present study, we investigated plasma PYYeostasis , we chos1–36 and PYY3–36) instead of PYY3–36 [1–36 and PYY3–36 is further underlined by a recent study demonstrating different physiological effects of the two isoforms [The results of the present study are in line with a number of studies reporting a normal PYY concentration in acute AN –20. Howe PYY3–36 , 42, 43.isoforms . Last buisoforms –28. The n = 7, recAN n = 4) and no healthy control group was included in the study. Gendall et al. [Two longitudinal studies, which reported no significant change in PYY concentrations after weight gain, are in accordance with our findings , 25. To l et al.  also repOur finding of a negative correlation between PYY concentration and BMI in the acute AN group before treatment is consistent with the results of a number of studies , 43, 45.3–36 concentrations in AN. Second, taking into account the complexity of the function of PYY as a regulator of energy homeostasis, measurement of fasting concentrations is only one approach to investigate its role in the etiology and progression of AN and future studies in acute AN should also target the PYY3–36 response to standard meals. To date, only four studies investigated PYY in response to meal intake in AN with mixed results [3–36 instead of total PYY [1–36 and PYY3–36, we chose an assay that targets only the isoform PYY3–36, which is assumed to be more relevant for energy homeostasis. Thus, our results allow no conclusions regarding the physiological effects of PYY1–36 and measurement of both, total PYY (the sum of PYY1–36 and PYY3–36) and PYY3–36, yielding their ratio , would allow for a more comprehensive picture of the peptide’s metabolism in AN.When considering our findings, some limitations have to be taken into account. First, the sample size may not have been sufficient to draw definite conclusions regarding basal PYY results –20, 24 a3–36 concentration in AN. Future studies should further measure both PYY1–36 and PYY3–36, explore their levels in response to food intake in AN, and take physical activity as a possible confounding factor into consideration. Another promising approach would be to simultaneously study multiple appetite-regulating peptides and their complex interplay.In conclusion, the current study provides additional evidence for a normal basal PYYSupplementary file1 (PDF 384 kb)Below is the link to the electronic supplementary material.


CFC bind to the aromatic peptides FF and YY (K ≈ 229–702 M−1), while AA binds significantly weaker (K ≈ 65–71 M−1).Small peptides are involved in countless biological processes. Hence selective binding motifs for peptides can be powerful tools for labeling or inhibition. Finding those binding motifs, especially in water which competes for intermolecular H-bonds, poses an enormous challenge. A dynamic combinatorial library can be a powerful method to overcome this issue. We previously reported artificial receptors emerging form a dynamic combinatorial library of peptide building blocks. In this study we aimed to broaden this scope towards recognition of small peptides. Employing CXC peptide building blocks, we found that cyclic dimers of oxidized  K2 = 3.1 × 104 M−1) with a roughly 10-fold higher affinity than Ac-GG. However, constructing binders in this fashion requires a design on a case by case basis, as well as a high synthetical effort for each new iteration. A more general approach for the development of artificial receptors can be the use of a dynamic combinatorial library (DCL). In a DCL a large variety of molecules is generated by a dynamic exchange of building blocks. The dynamic nature of those exchange reactions is essential since it allows the library to be under thermodynamic control. Therefore, the equilibrium responds to stimuli such as addition of a template, which would stabilize a suitable receptor and thus amplifies its formation. This strategy has led to the discovery of many artificial receptors [K ≈ 104–105 M−1) first and proved that it can emerge from a DCL afterwards. Despite these encouraging results no further research was done [2+, Ni2+ complexes. The addition of dipeptides caused characteristic spectral changes by ligand-exchange reactions [Peptides are one of the most abundant and structurally versatile motifs in nature. Thus, selective binders for peptides are extremely desirable but also highly challenging to design. Early developments in artificial peptide recognition go back to the 1970’s when it was discovered that crown ethers can bind to ammonium functions, such as protonated amines in peptides –3. Schmueceptors –14. Notaeceptors ,15–16 vewas done . Additioeactions .CHC (p(CHC)2), which binds two molecules of the neurotransmitter N-acetylneuraminic acid (NANA) in a cooperative fashion . Recently we rationalized that since our peptide building blocks consist of the same binding motifs as binding proteins , our DCL should be applicable to a similar scope. In order to show that our system offers a general approach to find binders for biomolecules in water we choose to test our DCL against peptide templates.We previously used a peptide derived DCL for the development of artificial carbohydrate receptors –20. An iWe started from our established CXC peptide building block design (single-letter code) were the terminal amino acids are cysteine (C) and the X can be any amino acid . This alCFC, CEC, CSC and CAC  in an equimolar ratio in a slightly alkaline buffer (pH 7.4). An excess of our target molecules, the simple dipeptides AA, FF and YY (Y for tyrosine), were added to one sample each. An additional sample without dipeptide addition served as reference. After several hours of stirring in an open vial the thiol functions of the cysteines are converted quantitively to disulfides by oxidation with oxygen. Until fully oxidized library members can exchange building blocks in a thiol–disulfide exchange reaction. While a closed monomer can be observed in the beginning 2 and p(CFC)2 were synthesized using a suitable protecting group strategy (CFC(Acm)), which is stable under the cleaving conditions of the SPPS. Thus one cysteine moiety is left deprotected and can be addressed selectively either by dimerization with another CFC(Acm) or with inversely substituted tripeptide (C(Acm)FC). These linear peptide dimers were subsequently cyclized by oxidative cleavage of the Acm groups to give a(CFC)2 and p(CFC)2, respectively. The binding affinities and thermodynamic data were quantified by ITC measurements 2 and p(CFC)2 are very similar and no selectivity is observed.All investigated interactions show a negative enthalpy and are therefore exothermal. Though the magnitude of these values differs based on the dipeptide. The binding constants of FF with a(CFC)2 and p(CFC)2 the enthalpy is very low, which is cause for a poor signal-to-noise ratio. Other values for these particular interactions should therefore be met with some caution. FF seems to bind stronger to a(CFC)2 than to p(CFC)2, though again the values show some uncertainty and real values of the binding constants are likely in the same order of magnitude as for YY. Unlike for the interaction with FF, the entropy change of interaction with YY appears to be highly positive, which might be due to hydrophobic interactions. We do not attribute this behavior to a fundamentally different mode interaction compared to FF though, as both dipeptides behave very similar in the following NMR experiments. The effect may be attributed to the intermolecular interaction of the dipeptides with themselves. For example, FF is a popular self-assembly motif, which also explains its poor solubility (≈5 mM) [a(CFC)2/p(CFC)2 towards FF, but also the dissociation of FF assemblies. The aromatic side groups of FF and YY seem to play an important role in the binding, since AA binds significantly weaker. Each of the aromatic units contributes to the binding to a roughly equal amount, as is shown by a comparison with AF and FA  . Hence tF and FA , which bYY or FF to a(CFC)2 or p(CFC)2 hinted at formation of H-bonds due to shifts of protons at α-carbon atoms between δ = 3.85 to 4.00. However, no binding constants could be determined from those measurements as even in a 10-fold excess the system showed no signs of saturation 2 or p(CFC)2 2 , since tFC)2 (2O .a(CFC)2 and p(CFC)2 are stronger binders for the aromatic dipeptides FF and YY (K ≈ 229–702 M−1), then for the non aromatic AA, which is bound less efficiently with a factor of about 3–10 times weaker (K ≈ 65–71 M−1).In summary, we demonstrated that artificial peptide receptors can emerge from a peptide-based DCL under competitive conditions in water. In agreement with our previous works on carbohydrate recognition, this supports our initial assumption that dynamic peptides, due to their similarity towards enzymes, can bind a broad scope of biomolecules. ITC measurements suggest that the cyclic tripeptide dimers 4CO3 buffer . 50 µL stock solution of each was given to a 1.5 mL vial. Another 100 µL of a template stock solution (90 mM) was added. For a reference sample the template solution was substituted for buffer. Small magnetic stirring bars were added, and the vials were covered by a layer of perforated Parafilm to slow down drying due to fast air exchange. After stirring overnight (100 rpm), the samples were acidified with a 5% solution of formic acid in water. The samples were diluted 1:10 with water and measured with HPLC–MS setup I.Stock solutions of each CXC building blocks (7.5 mM each) were prepared in NHThe HPLC setup was based on a Shimadzu system and consisted of a CBM-20A controller, a SIL-HTA auto sampler, two 10ADVP pumps, a DGU-14A degasser, a CTO-10AVP column oven and an SPD-10AVP variable wavelength detector. The setup was coupled to a micrOTOF time of flight mass analyzer (Bruker) and controlled by Compass HyStar (Bruker) version 3.2 and microTOFControl (Bruker) version 3.0. Measurement data was analyzed using Compass DataAnalysis version 5.0 R1 (Bruker).4FA, pH 3.2) and acetonitrile. The injection volume was 5 µL. The following gradient was used with a flow rate of 150 µL/min  with a SeQunant ZIC-HILIC  precolumn was used as stationary phase. The mobile phase was a mixture of water . Concentration were used as noted. In general, the titrant concentration was 20 times the concentration of the other peptide. ITC was performed on a Nano ITC low volume titration calorimeter (TA Instruments) with a cell volume of 170 µL. The program used 20 injections of 2.5 µL each in an interval of 2.5 min and a stirring rate of 350 rpm. The device was operated using ITCRun version 2.1.7.0 (TA Instruments). For each experiment a correction for the dilution was measured by titrating the titrant to pure solvent. For initial analysis of the heat rates NanoAnalyze version 3.8.0 (TA Instruments) was used. Data fits and thermodynamic data was obtained using a numerical approximation using Microsoft Excel.1H NMR of a(CFC)2 was recorded in pure D2O.For the Job plots 3 mM stock solutions of each component in deuterated buffer were mixed in different ratios with an interval of 10% or 20%. Spectra were recorded on a DD2 500 (Agilent) or a DD2 600 (Agilent) spectrometer. Small amounts of DMSO (83 nM) were used as reference (δ = 2.62 ppm). Variable temperature 4·2H2O  was dissolved in D2O (1 mL) and afterwards dried in vacuo. This step was repeated for a total number of three times. The salt was dissolved again in D2O (10 mL) and the pD was adjusted with NaOD (40 wt % in D2O) using a pH-calibrated glass electrode and the following relation [Deuterated buffer was prepared by the following procedure. Regular non-deuterated NaHPOrelation .File 1Synthesis, additional data, and NMR spectra.