Py-1-labeled ligand 8 (UR-KAT478) was found to be most suitable in BRET saturation binding experiments with respect to receptor affinity (pKd = 7.35) and signal intensity. Real-time kinetic experiments showed a full association of 8 within approximately 30 min and a slow dissociation of the ligand from the receptor. Investigation of reference compounds in BRET-based competition binding with 8 yielded pKi values in agreement with radioligand binding data. This study shows that the BRET binding assay is a versatile test system for the characterization of putative new ligands at the histamine H2 receptor and represents a valuable fluorescence-based alternative to canonical binding assays.Fluorescence/luminescence-based techniques play an increasingly important role in the development of test systems for the characterization of future drug candidates, especially in terms of receptor binding in the field of G protein-coupled receptors (GPCRs). In this article, we present the establishment of a homogeneous live cell-based BRET binding assay for the histamine H Until now, the characterization of potential ligands in terms of receptor binding is mostly done performing radioligand binding experiments. Despite its high sensitivity, the use of radiolabeled substances is usually connected with some drawbacks. In addition to the constantly increasing costs, the availability as well as the quality of commercial radioligands often declines. Furthermore, the management of radioactive waste is becoming increasingly regulated and expensive. To circumvent these issues, techniques using fluorescently labeled ligands like flow cytometry and the recently described BRET-based binding assay25, is fused to the N-terminus of the GPCR of interest10. After addition of the substrate the enzyme catalyzes an oxidation reaction, which is accompanied by the emission of blue light. Once a suitable fluorescent ligand binds to the tagged receptor, the ligand fluoresces due to bioluminescence resonance energy transfer (BRET). This transfer can only occur when the ligand is in close proximity to the bioluminescent donor, resulting in the observation of a lower non-specific binding as mainly the receptor-bound fraction of the fluorescent ligand is detected. Additionally, the ligand binding process can be followed in real time and not only after equilibrium is reached, which gives deeper insight into the kinetic behavior of the ligand.For the NanoBRET binding assay the NanoLuc, a genetically engineered luciferase2 receptor. Therefore we synthesized three differently labeled fluorescent ligands  of the fluorescent ligands 8–10 is shown in the Supplementary Figures The synthesis of precursor UR-DE36 6, Fig.  was carr2R ligands 8 (UR-KAT478), 9 (UR-KAT515) and 10 (UR-KAT514) was investigated under assay conditions (pH = 7.4) in binding buffer  at room temperature (rt) . Compounds 8 and 9 showed no decomposition during 96 h  and 24 h  incubation, respectively, and exhibited excellent chemical stability. The stability test with compound 10 also showed no chemical degradation. However, after only one hour almost no signal corresponding to compound 10 was detectable  for their use in the BRET-based binding assay, saturation binding experiments were performed at live HEK293T cells, stably expressing the NLuc-H2R fusion protein. As depicted in Fig. Kd/pKd values, respectively) in the nanomolar range could be determined  and 9 (TAMRA) with a pKd of 6.84  after preincubation (60 min) of the cells with fluorescent ligand . A similar behavior was also reported for the structurally related radioligand 7, leading to the assumption that the pharmacological scaffold is responsible for this type of binding28. All kinetic parameters describing the binding of 8 are summarized in Table For the further characterization of ted Fig. . TherefoH2R Fig. , left. Tine c = 1 µM after2 receptor agonists and antagonists, using one fixed concentration of 8 (c = 50 nM) and various concentrations of the respective ligands  in comparison to the other tested competitive ligands, which could suggest the existence of a second receptor affinity state, as previously described32. However, this could not be clearly confirmed in a competition binding experiment using an extended set of concentrations  show comparatively higher affinities at Sf9 membranes, whereas antagonists/inverse agonists (2–4) show lower affinities , ranitidine (3) and famotidine (4) are also often described as inverse agonists at the hH2R, supporting our finding35. Another possibility for the evident discrepancy in the affinity for histamine (1) is the known allosteric effect of sodium on the binding of agonists to several GPCRs36. As the buffer used in radioligand binding assays at Sf9 membranes expressing the H2R is devoid of sodium ions, in contrast to the BRET assay and also to the binding experiments performed at CHO membranes32, we changed the assay procedure by adding sodium in a physiological concentration (c = 145 mM) to the binding buffer. This change resulted in a decrease in affinity for histamine (1) with a pKi of 4.37 ± 0.02  for histamine (1), which is now in good agreement to the binding constant from the BRET binding assay. Taken together the presented BRET-based approach yields comparable binding data for reported histamine H2 receptor ligands, which confirms the suitability of the test system in combination with the fluorescent ligand 8.To show the versatility of the presented assay principle, we additionally performed BRET-based competition binding experiments with different reported Hnds Fig. . Despite2 receptor including the synthesis and characterization of suitable fluorescent H2R ligands. As a homogeneous live cell-based assay, this assay allows for a convenient determination of affinity constants of putative H2 receptor ligands, independent of their mode of action without any washing or separation steps. The results from our BRET binding assay were well comparable to currently used radioactivity- or fluorescence-based (e.g. flow cytometry) binding assays. Furthermore, real-time kinetic measurements can be performed enabling a better resolved monitoring of ligand-receptor interactions. Prerequisite for the establishment of such assays is the availability of suitable fluorescent ligands. Therefore, we synthesized three differently labeled compounds, all of which have proven to be generally usable in BRET saturation binding experiments. Out of those, substance 8 turned out to be the best compromise with regard to receptor affinity and signal strength and was successfully used for further investigations. Until now, BRET binding assays have only been described for the histamine H1,3,4 receptors23, making this study close the gap of NanoBRET assays within the histamine receptor family. Thus, selectivity studies, which are essential for the development of new drug candidates, can be carried out using the same assay principle increasing the comparability of results. All in all, this study shows that the BRET binding assay is a valuable test system for the histamine H2 receptor and provides a novel fluorescence-based alternative to other conventional binding assays.In this study we report the development of a NanoBRET binding assay for the histamine H30. All solvents were of analytical grade.Dulbecco’s modified Eagle’s medium (DMEM) and HEPES were purchased from Sigma-Aldrich . Leibovitz’ L-15 medium (L-15) was from Fisher Scientific . Fetal calf serum (FCS), geneticin and trypsin/EDTA (0.05%/0.02%) were from Biochrom . Bovine serum albumin (BSA) was sourced from SERVA Electrophoresis . Furimazine was from Promega, . Histamine dihydrochloride was from TCI Chemicals . Cimetidine was from Sigma-Aldrich . Famotidine and ranitidine hydrochloride were from Tocris Bioscience . All other chemicals and solvents were purchased from standard commercial suppliers  and were used as received. The fluorescent dyes BODIPY 630/650 X NHS ester and 5-TAMRA NHS ester were purchased from Lumiprobe  or abcr  respectively, the pyrylium dye Py-1 was synthesized as previously published1H: 600 MHz)  with deuterated solvents from Deutero . HRMS was performed on an Agilent 6540 UHD Accurate-Mass Q-TOF LC/MS system  using an ESI source. Preparative HPLC was performed with a system from Waters  consisting of a 2524 binary gradient module, a 2489 detector, a prep inject injector, fraction collector III and the column was a Phenomenex Kinetex  . As mobile phase, mixtures of MeCN and 0.1% aqueous TFA were used. UV detection was carried out at 220 nm. Freeze-drying was carried out using a ScanVac CoolSafe 4-15L freeze dryer from Labogene , equipped with a RZ 6 rotary vane vacuum pump . Analytical HPLC experiments were performed on a 1,100 HPLC system from Agilent Technologies, equipped with Instant Pilot controller, a G1312A Bin Pump, a G1329A ALS autosampler, a G1379A vacuum degasser, a G1316A column compartment and a G1315B DAD detector. The column was a Phenomenex Kinetex XB-C18 column  , tempered at 30 °C. As the mobile phase, mixtures of MeCN and 0.05% aqueous TFA were used. Gradient mode: MeCN/TFA (0.05%) (v/v) 0 min: 10:90, 30 min: 90:10, 33 min: 95:5, 40 min: 95:5, 43 min: 10:90, 50 min: 10:90; flow rate: 0.8 mL/min, t0 = 3.21 min; capacity factor k = (tR − t0)/t0. Absorbance was detected at 220 nm. Purity of the compounds was calculated as the percentage peak area of the analyzed compound by UV detection at 220 nm. The purities of the fluorescent ligands used for pharmacological investigation were ≥ 95%.NMR spectra were recorded on a Bruker Avance 600 amino)-4-((3-(3-(piperidin-1-ylmethyl)phenoxy)propyl)amino)cyclobut-3-ene-1,2-dione × 2 TFA), was synthesized as previously reported29.The amine precursor UR-DE36  was dissolved in 30 µL of DMF, before NEt3 (7.5 or 11 equiv.) was added. The labeling reagents (1 equiv.) were dissolved in 20 µL of DMF, added to the mixture and the vessel was rinsed twice with DMF (20 µL and 10 µL). The mixture was stirred at room temperature for 2 h. Subsequently, the reaction was stopped by addtion of 10% aqueous TFA (20 µL). The crude products were purified by preparative HPLC. The solvent was removed by lyophilization.Following labeling reactions were carried out in 1.5-mL Eppendorf reaction vessels. The amine UR-DE36 , Py-1  and NEt3  according to the general procedure yielding the product as a red solid . RP-HPLC: 96.0% . 1H NMR  δ 9.22 , 7.77 , 7.68 , 7.37 , 7.10 , 7.07 , 7.05–7.00 , 6.88 , 6.51 , 4.34–4.28 , 4.22 , 4.05 , 3.71–3.63 , 3.60–3.51 , 3.25 , 2.85 , 2.72 , 2.69 , 2.03–1.96 , 1.86 , 1.83–1.76 , 1.72–1.55 , 1.40–1.29 . HRMS (ESI–MS): m/z M+ calcd. for C44H56N5O3+: 702.4378; found: 702.4382; C44H56N5O3+ × C4HF6O4- (930.00).The title compound was prepared from amine 6 , 5-TAMRA NHS ester  and NEt3  according to the general procedure yielding the product as a pink solid . RP-HPLC: 96.5% . 1H NMR  δ 8.35 , 8.91 , 8.28 , 8.66 , 7.79–7.44 , 7.38 , 7.22–6.66 , 4.23 , 4.06 , 3.69 , 3.60–3.53 , 3.38–3.34 , 3.33–3.12 , 2.92–2.80 , 2.01 , 1.86–1.76 , 1.72–1.54 , 1.41–1.30 . HRMS (ESI): m/z [M + H]+ calcd. for C48H55N6O7+: 827.4127; found: 827.4126; C48H54N6O7 × C2HF3O2 (941.02).The title compound was prepared from amine 6 , BODIPY 630/650 X NHS ester  and NEt3  according to the general procedure yielding the product as a dark blue solid . RP-HPLC: 98.3% . 1H NMR  δ 9.19 , 8.12 , 8.03 , 7.82 , 7.78–7.70 , 7.62–7.57 , 7.41–7.34 , 7.30–7.25 , 7.08–7.04 , 7.04–6.99 , 6.94 , 4.52 , 4.21 , 4.04 , 3.66 , 3.31–3.26 , 3.10 , 3.02 , 2.88–2.79 , 2.52–2.50 , 2.05–1.95 , 1.84–1.76 , 1.70–1.53 , 1.51–1.28 , 1.27–1.14 . HRMS (ESI): m/z [M + H]+ calcd. for C52H61BF2N7O6S+: 960.4460; found: 960.4471; C52H60BF2N7O6S × C2HF3O2 .The title compound was prepared from amine 2R was purchased from the Missouri cDNA resource centre . The plasmid encoding NanoLuc was kindly provided by Promega . The sequences of the receptor and the luciferase were amplified using standard PCR techniques, introducing restriction sites at their respective 5′ and 3′ ends as well as the membrane signal peptide of the murine 5HT3A receptor upstream of the luciferase gene. These were then cloned in-frame into the pcDNA3.1/myc-HIS (B) vector backbone separated by a flexible linker (-SGGGS-) to generate the plasmid encoding the NLuc-hH2R. All sequences were verified by sequencing .The cDNA coding for the human H2 at 37 °C and regularly monitored for mycoplasm infection using the Venor GeM Mycoplasma Detection Kit . In order to generate stable transfectants, HEK293T wild-type cells were seeded at a density of 3 × 105 cells/mL in a 6-well plate  one day prior to transfection with 2 µg of cDNA using XtremeGene HP transfection reagent  according to the manufacturer’s protocol. After two days of incubation , transfected cells were trypsinized, transferred to a 15 cm cell culture dish  in DMEM and geneticin was added at a final concentration of 1 mg/mL to select for stable transfectants. After stable growth was observed, concentration of geneticin was reduced to 600 µg/mL.All cells were routinely cultivated in DMEM + 10% FCS in a water-saturated atmosphere containing 5% CO2R construct was detached from the cell culture dishes after reaching ~ 80% confluency by treatment with trypsin/EDTA  and was centrifuged . The cell pellet was then resuspended in Leibovitz’ L-15 medium (L-15), supplemented with 5% FCS + 10 mM HEPES, and 1.0 × 105 cells/well were seeded in 70 µL (saturation and competition binding) or 80 µL (kinetic experiments) of assay medium into white 96-well cell-Grade™ plates . The cells were then incubated at 37 °C in a humidified atmosphere  overnight. For saturation binding experiments, serial dilutions (tenfold concentrated) of the fluorescent ligands (8–10) and famotidine  were prepared in dilution buffer (L-15 + 2% BSA + 10 mM HEPES). 10 µL of the fluorescent ligand dilution and 10 µL of L-15  or the dilution of 4 (non-specific binding) were added to the cells. After 60 min incubation time at 27 °C, 10 µL of the substrate furimazine, which was diluted according to manufacturer’s protocol beforehand, were added. After 5 min of equilibration time at 27 °C, the measurement was started. Competition binding experiments were performed as described above using one fixed concentration of fluorescent ligand 8 (c = 50 nM) and varying concentrations of the competitors 1–4, 11, 12, that were added at the same time. Kinetic measurements were performed as follows: 10 µL of L-15  or 4  were added to the cells. After addition of the diluted substrate, the plate was placed inside the reader for 5 min to equilibrate. To start association 50 µL of a threefold concentrated solution of the fluorescent ligand 8  were added to the adherent cells and the plate was measured for 60 min. Dissociation experiments were conducted in wells, which have been preincubated with 8 as described above for association experiments. To initiate dissociation, 50 µL of a fourfold concentrated solution of 4  were added to the cells and the measurement was performed for 4 h. All measurements were performed on a TECAN InfiniteLumi plate reader  at 27 °C using the Blue2 NB  and the Red NB  filter combination with an integration time of 100 ms. For the kinetic experiments, integration time was increased to 500 ms for both channels to reduce noise. BRET ratios were calculated by dividing the acceptor emission (red NB) by the donor luminescence (Blue2 NB).The cell line stably expressing the NLuc-hH50-values. These were transformed into pKi-values using the Cheng–Prusoff equation38.For all BRET binding experiments, specific binding was calculated by subtracting non-specific binding from total binding yielding the “corrected BRET ratio”. For saturation binding experiments, total and non-specific binding were fitted simultaneously using the “one-site total and nonspecific binding” fit. Specific binding was fitted accordingly applying the “one-site specific binding” fit. For competition binding experiments, data were normalized to buffer control (0%) and a 100%-control containing solely fluorescent ligand. The normalized competition binding curves were then fitted with a four-parameter logistic fit yielding pICkobs, whereas the dissociation experiments were fitted assuming a “one-phase decay” model resulting in koff. The association rate constant kon was calculated using the following equation: kon = (kobs − koff)/c(ligand); c(ligand) = 50 nM. Dissociation half-life t1/2 was calculated applying the following formula: t1/2 = ln(2)/koff.The kinetic equilibrium dissociation constant Kd(kin) was calculated as follows: Kd(kin) = koff/kon. The errors for kon,t1/2 and Kd(kin) were calculated according to the Gaussian law of error propagation. All experimental data were analyzed using Prism 8 software .For kinetic experiments, association experiments were fitted with the “one-phase association” fit yielding Supplementary Information


Screening for small-molecule fragments that can lead to potent inhibitors of protein–protein interactions (PPIs) is often a laborious step as the fragments cannot dissociate the targeted PPI due to their low μM–mM affinities. Here, we describe an NMR competition assay called w-AIDA-NMR (weak-antagonist induced dissociation assay-NMR), which is sensitive to weak μM–mM ligand–protein interactions and which can be used in initial fragment screening campaigns. By introducing point mutations in the complex’s protein that is not targeted by the inhibitor, we lower the effective affinity of the complex, allowing for short fragments to dissociate the complex. We illustrate the method with the compounds that block the Mdm2/X-p53 and PD-1/PD-L1 oncogenic interactions. Targeting the PD-/PD-L1 PPI has profoundly advanced the treatment of different types of cancers. Ds between low nM (tight binding) and low µM (weak binding), the PPI complex should be broken up. The “weak” small-molecule binders to the proteins of the PPI, for example, those ligands with the IC50 in high µM and mM range, would not affect the AIDA-NMR . Thus, the strength of the PPI complex determines the weakest binding compounds that may still be measured with the AIDA-NMR.NMR spectroscopy is a highly versatile screening method for drug discovery ,2,3. In Herein, we describe an AIDA-NMR technology that should overcome these disadvantages, allowing for NMR to be used in the primary fragment screening. We use the Mdm2-p53 and MdmX-p53 interactions ,16 as prThe tumor suppressor p53 protein is a key player in protecting the organism from cancer and was therefore given the term "the guardian of the genome". To escape the control system mediated by p53, the majority of human cancers has either mutation within p53 , whereas the rest compromises the effectiveness of the p53 pathway ,20. In tAnother important protein target in cancer is the PD-1/PD-L1 system. PD-1 (programmed cell death protein 1) is expressed on activated T cells and plays a critical role in modulation of the host’s immune response ,24. The D of this interaction is 8.2 and 0.2–0.6 µM, respectively [The interaction of PD-L1/PD-1 and p53-Mdm2/X presents challenging cases in detecting weak binders, because the Kectively ,35. We sectively ,37, suchD values 0.60 µM and 0.24 µM, respectively [D’s determine the weakest inhibitor, which can be tested with the AIDA-NMR methods. For the compounds that weakly bind to Mdm2/X, which still could be good initial scaffolds for further optimization in drug development processes, the method would not be sensitive enough for detecting inhibition of the p53-Mdm2 and p53-MdmX PPI.The complexes of wt-p53 with Mdm2 and MdmX have Kectively . These KThree key residues, namely Phe19, Trp23, and Leu26, of p53 make the highest contribution to the binding energy of p53 with Mdm2/X ,39,40. AD values for the complexes of Mdm2/X with the p53 mutants were established with isothermal titration calorimetry (ITC)  or changing of the amino acids isoform (L→I). Kry (ITC) .D value increased six fold compared to that of the p53-wt/Mdm2 complex. Surprisingly, modifications of Leu26, which is one of the key amino acids of the p53 binding to Mdm2/X, were less efficient than mutations of Leu22. Moreover, the L26I mutant generated a lower KD value (0.31 µM) than in the Mdm2/p53-wt interaction (0.60 µM). Although the interaction with the L26V mutant was weakened, the affinities of the proteins were still too strong to use the preformed complexes for the investigation of the weak binding compounds. Therefore, we designed the double mutants (L22IL26V and L22VL26V), which combined the above-mentioned mutations. The ITC measurements showed a synergistic effect of the double mutations on the binding of the mutant p53s to Mdm2/X  and 3 , showed some weak interactions , partial overlapping , or even the complete overlapping of the Trp23 with Trp53 signals (L22VL26V).To choose proper mutants for the 1D NMR w-AIDA assay, we recorded  mutants . Since t mutants  [13,14,51H NMR spectra of the mutants PD-1/PD-L1 complex, we were not able to reach the top plateau as we could not concentrate PD-L1 solution above 183 µM  . AlthougE. coli  and checE. coli . The affe 183 µM . Therefo1H-15N 2D HMQCs, is so extensive that it is impossible to estimate dissociation constants also for “weaker” binding precursors of the BMS compounds (KD’s double digit μM). One of the NMR techniques that would allow validating the binding and estimating the value of KD is the w-AIDA-NMR assay.A sizeable number of small-molecule binders to PD-L1 that inhibit this PPI has now been described (ca. 1000) ,54. We h5 and 6 in For the start in the PD-1/PD-L1 system, we used the two smallest fragments of the BMS-1166 inhibitor   . After aappeared . This re spectra . This sh spectra . We alsompound 5 .15N-labeled mutated proteins (N66A)PD-1 (13.2 kDa) and wt-PD-L1 . These results correlated with the homogeneous time-resolved fluorescence (HTRF) assay, where the IC50′s for 5 and 6 were determined at 34 µM and above 100 µM, respectively , was 51 µM  RIL . Cells were grown at 37 °C, induced at OD600 of 0.6 with 1 mM IPTG and left for expression for additional 4 h at 37 °C. Cells were harvested by centrifugation, and the protein was purified from inclusion bodies according to previously described methods [g for 30 min at 4 °C. Inclusion bodies were washed twice with PBS supplied with 0.05% TritonX-100 and centrifuged at 12,000× g for 25 min. They were later solubilized overnight in 6 M guanidinium hydrochloride, 100 mM Tris-HCl, pH 8.0, with the addition of 1 mM EDTA, and 10 mM β-mercaptoethanol at 37 °C. Solubilized inclusion bodies were next centrifuged at 60,000× g for 45 min at 4 °C, and the resulting supernatant was dialyzed against 4 M guanidinium hydrochloride, pH 3.5, 10 mM β-mercaptoethanol for 6 h. Refolding was performed in 10 mM Tris-HCl, pH 7.0, including 1 mM EDTA and 10 mM β-mercaptoethanol at 4 °C. Then the mixture was supplemented with 1.5 M (NH4)2SO4. The mixture was centrifuged (3000× g for 35 min at 4 °C) before loading on a butylsepharose 6 Fast Flow column. The protein solution was eluted with 100 mM Tris-HCl, pH 7.2, 10mM β-mercaptoethanol. The fractions containing protein were further purified using a SEC column HiLoad 16/60 Superdex 75 prep grade  previously equilibrated with 50 mM KH2PO4, 50 mM Na2HPO4, pH 7.4, 150 mM NaCl, 5mM β-mercaptoethanol.The recombinant human Mdm2 (1–125 aa) were cloned into pET11a vector ) and expressed in  methods . The pelE. coli BL21 (DE3) . The wt-p53 (1–321 aa) as well as its mutants were cloned into the pET23 vector  and expressed in E. coli BL21 (DE3) RIL . Cells were grown in LB medium (100 µg/mL ampicillin) at 37 °C and induced with 1 mM IPTG (OD600 around 0.6–0.8). The protein expression was performed for an additional 12 h at 20 °C. Cells were harvested by centrifugation. The cell pellet was then resuspended in a lysis buffer  and sonicated as described before. After centrifugation at 60,000× g for 30 min at 4 °C, the supernatant was loaded on the Ni-NTA column and incubated for 2 h at 4 °C. Loaded protein was washed with buffer: 50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole pH 8.0 to elute any unspecifically bound proteins. The target protein was eluted with buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0; relevant fractions were pooled, concentrated, and purified by gel filtration in PBS.The recombinant MdmX (18–111 aa) was cloned into pET46Ek/LIC vector  and expressed in E. coli BL21 (DE3) (New England Biolabs). Bacterial cultures were grown overnight at 37 °C in LB or M9 minimal medium and induced at OD600 of ca. 0.8 with 1 mM IPTG. Following inclusion, purification of bodies was performed using a previously described protocol [The human wt-PD-1  and its mutants  were cloned into pET24d , while recombinant PD-L1 (18–134 aa) and PD-L1-Long (19–238 aa) were cloned into pET-21b and pET-28a , respectively. Each protein was expressed in protocol . SolubilAll ITC experiments were performed on a Microcal iTC200 calorimeter according to the manufacturer’s protocols. To compensate for the heat generated due to protein dilution, a separate experiment was performed in which a protein solution was injected into a sample chamber with a corresponding buffer. The resulting heat was then subtracted from the final signal as a background.2 minimization for a model assuming a single set of sites to calculate the binding affinity KD using ORIGIN 7.0 . p53-wt and mutants with Mdm2 and MdmX titrations settings were as follow: 20 injections, 25 °C cell temperature, 10 µcal/s reference power, 600 s initial delay, 0.012–0.030 mM p53 cell concentration, 0.1–0.3 mM of titrant (Mdm2 or MdmX) syringe concentration, and a stirring speed of 800 rpm. The first injection volume was 0.4 µL with an injection time of 0.2 s, while the rest were 2 µL (4 s injection times). Gaps between injections were set at 150 s, and data points were recorded every 5 s. All experiments were performed in duplicate.Data were fitted using Chi1, 2, 3, and 4 were purchased. Compounds 5 and 6 were synthesized according to the methods described by Guzik et al. [Nutlin-3a and compounds k et al. .Site directed mutagenesis of p53 and PD-1 were performed using PCR. The mutagenic primers for p53  were des15N labeled via expression in minimal medium with 15NH4Cl as the sole nitrogen source. Then 10% (v/v) of D2O was added to the samples to provide lock signal. Spectra were recorded using a Bruker Avance III 600 MHz spectrometer at 300 K equipped with the nitrogen cryo-probe head. By analyzing the line width (relaxation time) of well separated NMR signals, we approximated molecular weights of protein populations present in the sample [1H-15N signals were monitored by the SOFAST HMQC (selective optimized flip-angle short-transient heteronuclear multiple quantum coherence) [Proteins were uniformly e sample . During 2, and 0.05% Tween-20) from 416 nM to 183 µM. Samples were premixed and incubated for 2 h at RT in the dark before loading into capillaries. Data processing is described in the legend of The binding affinities between PD-1 (or mutant) with PD-L1 were analyzed using the MST technique. PD-1 and its mutant were fluorescently labelled according the standard labeling protocol of the NanoTemper protein labeling kit RED-NHS . In MST experiments, the concentration of the labeled PD-1 and mutant was kept constant (20 nM), while the unlabeled PD-L1 were serially diluted in MST buffer  of tested compounds. After mixing all components according to the Cis-Bio protocol, the plate was incubated for 2 h at RT. TR-FRET measurement was performed on the Tecan Spark 20M. Collected data was background subtracted on the negative control, normalized on the positive control, averaged, and fitted with the normalized Hill’s equation to determine the IC50 value using Mathematica 12 .The HTRF assay was performed using the certified Cis-Bio assay kit at 20 µL final volume using their standard protocol as described by Musielak et al. . MeasureThe FP competition assay is based on the displacement of p53 mutant peptide called P2 from the complex with Mdm2/X, as previously described . All mea5 and 6 in a similar manner as the (N66A)PD-1/PD-L1, therefore indicating that the point mutation at the interface of the small molecule non-binding partner indeed does not affect a subsequent target protein/ligand complex. Our NMR results presented here confirmed unambiguously that small-molecule compounds that are able to dissociate the complex between mutant-p53/Mdm2, mutant-p53/MdmX, and the mutated PD-1 and PD-L1 could be fished out, and we could estimate the dissociation constant of the targeted protein/fragment interaction. For the PD-1/PD-L1 small-molecule inhibitor BMS-1166, we could determine that the minimal fragment of BMS-1166 responsible for the PD-L1 binding is fragment 5.Fragment screening is frequently the first step for the identification and development of molecules that modulate the activity of therapeutic targets. Numerous biophysical methods exist for the identification of fragment hits ,63,64,65


NPC800 material shows an excellent surface area 1192 m2/g, total pore volume 0.92 cm3/g and displays a higher CO2 uptake 4.71 mmol/g at 273 k and 1 bar. Furthermore, NPC600 material displays good electrochemical sensing towards H2O2. Under optimized conditions, our sensor exhibited a wide linearity range between 100 µM and 10 mM with a detection limit of 27.5 µM.Nitrogen-doped porous carbon (NPC) materials were successfully synthesized via a Zn-containing metal-organic framework (Zn-MOF). The resulting NPC materials are characterized using various physicochemical techniques which indicated that the NPC materials obtained at different carbonization temperatures exhibited different properties. Pristine MOF morphology and pore size are retained after carbonization at particular temperatures (600 °C-NPC For H2O2 detection, 100 mM of H2O2 prepared from 9.8 M of H2O2 (30 wt%).Zn(NO3)2·6H2O , H2BDC , DABCO  in DMF (60 mL) was taken out into a Teflon-lined autoclave and heated to 120 °C, 2 days later cooling down to room temperature. The Zn-MOF was washed with DMF and dried at room temperature overnight. Further this material was used for the carbonization process.The Zn-MOF was prepared  by, a mi2 atmosphere for a 5 h duration. The resulting materials obtained at 500, 550, 600, 700, 800, and 900 °C are assigned as NPC500, NPC550, NPC600, NPC700, NPC800 and NPC900 respectively.The NPC materials were prepared through a single-step carbonization method . The 0.4T modified SPCE (SPCE/NPCT) was prepared by the following procedure: 10 µL of 2000 ppm (2 mg mL−1) respective NPCT dispersed in acetonitrile suspension was drop coated on precleaned SPCE, and allowed for dry on the hot plate at 40 °C for 15 min. The NPCT prepared by carbonization under the increasing temperature of 500, 550, 600, 700, 800 and 900 °C. The corresponding modified electrode designated as SPCE/NPC500, SPCE/NPC550, SPCE/NPC600, SPCE/NPC700, SPCE/NPC800, SPCE/NPC900, respectively.Prior to electrode modification, the bare SPCE was precleaned electrochemically by potential cycling between −1 and +1 V vs. Ag/AgCl for 6 cycles in 0.1 M pH 7 PBS. NPCλ = 1.54178 Å). The morphology of MOF and NPC materials were observed by high-resolution scanning electron microscopy . The NPC materials morphology was characterized by transmission electron microscopy  at a voltage of 200 KV. The synthesized NPC materials were also recorded with a Raman spectra on a CCD detector  using a He-Ne laser with an excitation wavelength of 632.8 nm. The Zn element presence was investigated by inductively coupled plasma-mass spectrometry . The elemental analysis  was executed by an elementar vario EL III CHN-OS elemental analyzer (Germany). N2 gas adsorption, CO2 gas adsorption of all materials were measured using micrometrics  and the gas sorption analysis purpose, the materials were dried at 120 °C for 12 h under vacuum.The purity of MOF and NPC materials were investigated by powder x-ray diffraction (PXRD) using a Bruker D8 advance instrument  equipped with CuKα radiation  and 44° (101) (The PXRD patterns of the NPCparticle a. The 2°perature . While tperature . The PXR4° (101) b 7]. Th. Th500–7Furthermore, the SEM revealed the morphology partially retained the pristine MOF with distorted graphitic carbon structures , TEM ima−1 to 1700 cm−1 bands were fitted with the spectra, 1180 cm−1 (A1 band), 1340 cm−1 (D band), and 1600 cm−1 (G band) [1, D, G band and fitted after baseline subtraction. The ID/IG ratio increased while increasing the temperature, indicating the formation of disorder with a low degree of graphitization of NPC materials was obtained. The ID/IG ratio between D and G bands revealed the degree of graphitization in carbon-related materials. Temperature 500–700 °C carbonized materials obtained a higher degree of graphitization, due to the ZnO present in the carbon material. Because there is still a definite chemical interaction between ZnO and N atoms, the redshifts of the D bands by approximately 15 cm−1 to ≅1326 were observed [−1 due to the nitrogen present in the NPC materials [Further, synthesized NPC materials were characterized by Raman spectroscopy for the degree of graphitization. The spectrum analyzed range between 1200 cm(G band) . Figure observed . Furtheraterials . Chemica2 sorption analyzer. The 2 uptake isotherm and corresponding pore sizes of the NPC materials. 2 sorption curves of the NPC materials possess type-I isotherms that steeply climb in the low-pressure range (P/P0 = 0−0.10), suggesting that micropores were dominant [0 = 0.40−1.00), there were decent increases in the adsorption in all samples and a slight hysteresis loop between the sorption curves, which revealed that mesopores were also present in the materials. The surface area and total pore volume of the NPC500–700 materials is nearly equal to 273, 287, 289, 296 m2/g and 0.18, 0.20, 0.22, 0.22 cm3/g correspondingly , pore volume (0.45 cm3/g) indicates framework shrinkage and fragmentation throughout the high-temperature carbonization process. The total pore volume of NPC800 material has a better percentage than the NPC500–700 and NPC900 materials. It is noted that the NPC800 material reached 42% of micropores, whereas, the percentage of Vmicro/Vtotal decreased significantly to 13% in the NPC900 sample. CO2 sorption is investigated for the NPC materials at 273 K at 1 bar (800 demonstrated a higher CO2 capture of 4.71 mmol/g than NPC500 (2.85 mmol/g), NPC550 (1.20 mmol/g), NPC600 (1.24 mmol/g), NPC700 (1.71 mmol/g) and NPC900 (2.51 mmol/g) at 273 K and 1 bar. Such a micro-mesoporous structure of NPC800 material provides a fast diffusion of CO2 into the inner pores material. NPC800 material showed CO2 capacity value closely matches/ greater than those of the carbon-related materials  was recorded for SPCE/NPC500, SPCE/NPC550, SPCE/NPC600, SPCE/NPC700, SPCE/NPC800, SPCE/NPC900 of about −99, −88, −29, −12, +83 and +113 µA, respectively. The observation is due to the semiconductor Zn moieties existing up to a carbonization temperature of 700 °C that results in a decrease in FeCN signal. In contrary, carbonization at 800 and 900 °C produced relatively smaller Zn moieties with NPC and hence an increase in signal. This result suggests that the semiconductor Zn content decreased with increasing carbonization temperature. In other words, the FeCN current response is inversely proportional to the Zn content. The obtained results coincide with the elemental analysis, ICP-MS and PXRD results.CV analysis was executed to study the electrochemical behavior of SPCE and SPCE/NPCnversion ,47. Afte2O2 sensing applicability was tested for SPCE/NPC500, SPCE/NPC550, SPCE/NPC600, SPCE/NPC700, SPCE/NPC800, SPCE/NPC900, respectively. 2O2 at various SPCE/NPCT electrodes. CV measurements were done in 0.1 M, pH 7.4 PB solution under the potential sweeping from 0 to −1.2 V at a scan rate of 100 mV s−1. During the cathodic segment, H2O2 reduction peak [T the same reduction peak was noticed with lower over potential (~−0.4 V). Among the various electrodes, detection response are clearer and more explicit at SPCE/NPC600. These results evidently exposed that the SPCE/NPC600 electrode exhibited better electrocatalytic H2O2 reduction than other electrodes. Therefore, SPCE/NPC600 was chosen for further studies.The surface area and porous defective sites have an important role in the electrochemical sensors. Therefore, Hion peak  was noti2O2. The H2O2 reduction current increased linearly with increasing concentration, in which H2O2 was electrochemically reduced at −0.4 V by applying a potential, and thus yielded quantitative current responses corresponding to the content of H2O2  27.5 μM were obtained. In order to access the repeatability of a SPCE/NPC600 modified electrode, 12 repeated injections of 0.5 mM H2O2 were performed and a RSD value of 4.13% was obtained. Compared to a few other Zn based H2O2 sensors Herein, we reported the synthesized NPC materials at various carbonization temperatures at a constant time under a N


Lycoris chejuensis, decreases Aβ production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased β-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver–Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.Accumulation of β-amyloid (Aβ) in the brain has been implicated in the pathology of Alzheimer’s disease (AD). Aβ is produced from the Aβ precursor protein (APP) through the amyloidogenic pathway by β-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aβ. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aβ production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of  Alzheimer’s disease (AD) is characterized by the accumulation of extracellular senile amyloid plaques and intracellular neurofibrillary tangles composed of hyper-phosphorylated tau ,2. HyperThree of A Disintegrin and Metalloproteinases (ADAM) family have been suggested as potential α-secretase: ADAM9, ADAM10, and ADAM17/tumor necrosis factor-α (TNFα) converting enzyme (TACE) . α-SecreSeveral reports have shown that enhancing α-secretase activity might be a potential strategy in AD treatment. The level of sAPPα in the cerebrospinal fluid of the AD patients has been found to be significantly decreased, indicating that α-secretase activity is reduced in AD ,25. OverLycoris chejuensis K. Tae et S. Ko (CJ) originated from Jeju Island in Korea on Aβ generation and spatial memory ability both in vivo and in vitro [We have shown the effect of in vitro . Recentlin vitro . In thisn = 6). The level of sAPPβ was decreased by E144, although the effect was not significant . The minimal effect of E144 on sAPPβ might be explained by the preferential APPsw cleave by β-secretase over α-secretase [n = 5). However, the level of sAPPα was significantly decreased at 8 h after incubation with E144. Apparently, the effect of E144 on sAPPα seemed much larger when we used the Western blot than when we used ELISA. This could be because the conditioned media were concentrated and immunoprecipitated using APP antibody for Western blot. We also tested the effects of E144 on human neuroblastoma SH-SY5Y cells, stably transfected with wild type APP. Even though Aβ42 levels were too low to detect, levels of sAPPα in conditioned media were significantly increased by E144 after 1 h incubation (We tested the effect of E144 on sAPPα production from HeLa cells stably transfected with APP carrying Swedish mutation (APPsw). Cells were incubated with 1 μM E144 for 1, 2, 5, or 8 h. Levels of sAPPα in conditioned media were then measured using a specific ELISA kit a. When cecretase . These recretase . We repocubation . The levn = 4). E144 increased the level of CTFα but decreased the level of CTFβ. These results indicated that E144 increased non-amyloidogenic processing of APP through α-secretase but decreased amyloidogenic processing through β-secretase. Since E144 decreased the amyloidogenic processing of APP, we examined the effect of E144 on Aβ levels. Cells were incubated with 0.5, 1, or 5 μM E144 for 1 h, and levels of Aβ42 and Aβ40 in conditioned media were measured using specific ELISA kits. The level of Aβ42 was significantly decreased by 13.6% ± 3.5%, 18.0% ± 2.1% and 24.6% ± 4% after incubation with 0.5, 1, and 5 μM E144, respectively (n = 4). The level of Aβ40 was also significantly reduced by 9.8 ± 1.1%, 15.2% ± 3.6% and 25.9% ± 5.3% after incubation with 0.5, 1, and 5 μM E144, respectively (n = 4).We then measured levels of CTFα and CTFβ, representing the products of α- and β-secretase, respectively. Cells were incubated with 0.5, 1, or 5 μM E144 for 1 h, and levels of CTF in cell lysates were analyzed using Western blot. A typical Western blot result is shown in ectively e; n = 4.ectively f; n = 4.n = 6), confirming that ADAM17 activity could be measured from cell lysates (n = 6). ADAM17 activity was increased by more than 4-fold after incubation with 5 μM E144. Thus, E144 activated ADAM17 in a concentration-dependent manner. We also performed a cell-free assay for ADAM17 activity using human recombinant ADAM17. E144 increased the activity of human recombinant ADAM17, as shown in n = 4). ADAM17 activity was markedly increased by 2-fold after incubation with 5 μM E144. This result using recombinant ADAM17 in a cell-free environment suggested that E144 directly activated ADAM17.Since E144 increased non-amyloidogenic processing of APP by α-secretase, we investigated whether E144 affected α-secretase activity. First, we performed a cell-based assay for ADAM17 activity using APPsw-transfected HeLa cells. The cell lysate was added into an anti-human ADAM17-coated plate and incubated for 1 h to capture ADAM17. Then, Mca-KPLGL-Dpa-AR, a fluorogenic ADAM17 substrate, was added and incubated for an additional 5 h with 0.1, 0.5, 1, or 5 μM E144. Fluorescence of the quenched substrate was directly related to the enzyme activity. TAPI-1, an ADAM17 inhibitor, inhibited the activity of ADAM17 by 83.1% ( lysates a. E144 aE144 at 1 or 5 μM E144 increased the activity of human recombinant ADAM17 in a time-dependent manner when we used a fluorogenic ADAM17 substrate . We thenn = 4). When the fluorescence intensities were plotted on the X-axis, and the substrate concentrations were plotted on the Y-axis, they showed different slopes that intersected together at an identical point. The Vmax of the enzyme was 26.2 mM/min and Km was 0.02 mM in the presence of 5 μM E144, compared with the Vmax of 11.4 mM/min and Km of 0.58 mM for the control. Thus, E144 decreased Michaelis–Menten constant Km while maximal velocity, Vmax, was apparently not or moderately affected. We examined the substrate-dependent effects of 1 or 5 μM E144, as shown in n = 5). These results indicated that while E144 activated ADAM17 towards APP and a fluorescent substrate, Mca-KPLGL-Dpa-AR, it also inhibited ADAM17 towards another substrate, thereby reducing the secreted TNFα. To clarify this substrate-specific activating effect of E144 on ADAM17, we measured ADAM17 activity using another fluorogenic ADAM17 substrate, QXL520/5-FAM. TAPI-1, an ADAM17 inhibitor, inhibited the activity of human recombinant ADAM17 (n = 5). When recombinant ADAM17 was incubated with 1 or 5 μM E144 for 40 min, ADAM17 activity did not change. Considering these results, the activating effect of E144 was specific for certain substrates. It is known that ADAM17 mediates the cleavage of the substrates involved in brain pathology, inflammation, and cancer . TNFα, ot ADAM17 b; n = 5.n = 4). E144 did not affect human recombinant ADAM10 in a cell-free activity assay either (n = 4). The inhibitor of ADAM10, GM-6001, inhibited ADAM10 activity by 80%. We next tested the effect of E144 on human recombinant ADAM10 activity using another substrate, Mca-KPLGL-Dpa-AR, the same substrate used in n = 4). These results indicated that E144 could activate ADAM10 and ADAM17 in a substrate-specific manner. However, the activating effect of E144 on ADAM10 was less potent than that on ADAM17 although we used the same substrate, Mca-KPLGL-Dpa-AR.We next tested the effect of E144 on ADAM10 activity in both a cell-based and cell-free assay. For the cell-based assay, ADAM10 from the lysate of APPsw-transfected HeLa cells was incubated with 1 or 5 μM E144 for 40 min in the presence of the ADAM10 substrate, 5-FAM/QXL520. Fluorescence of the quenched substrate was directly related to the enzyme activity. ADAM10 activity did not change by 1 and 5 μM of E144 a, n = 4.y either b, n = 4.n = 6). In contrast, the effect of E144 was completely prevented when ADAM17 was down-regulated. These results suggest that the effect of E144 on Aβ might be mostly via ADAM17 in intact cells. ADAM10 is the major physiological α-secretase mediating the cleavage of APP in the neurons. In contrast, ADAM9 and ADAM17 participate in a regulated cleavage of APP . Since wupper panel of n = 4). E144 did not affect the interaction between APP and ADAM9. In contrast, E144 increased both APP-ADAM10 and APP-ADAM17 interactions. We next measured the expression level of ADAMs. Cells were treated with 1 or 5 μM E144 for 1 h. Levels of ADAMs in cell lysates were then analyzed using Western blot. A typical Western blot result is shown in the lower panel of n = 4). ADAMs exist as a pro-enzyme in the trans-Golgi network and undergo maturation through removal of pro-domain, thereby converting to a mature, active form of the enzymes [We showed that E144 directly activated ADAM10 and ADAM17 using recombinant enzymes. We also showed that E144 enhanced the affinity ADAM17 towards the substrate. In addition, APP can bind to the α-/β-secretase binary complex . Thus, w enzymes . Levels Previously, we have confirmed that three active components of CJ, E144, Narciclasine (E2), and 7-Deoxynarciclasine (E3), can decrease the production of Aβ by attenuating APP levels . Since Em of ADAM17. This may indicate that E144 enhances the affinity of ADAM17 towards its substrates. It is possible that E144 can modulate interactions between α-secretase and APP, affecting APP cleavage and Aβ generation. Consistent with this result, E144 increased the interaction of APP with ADAM10 and ADAM17 when we performed co-immunoprecipitation analysis. E2, a structural analog of E144, also increased ADAM10 and ADAL17 activities. Interestingly, however, another analog, E3, did not change the activities of these enzymes. We have recently reported that E144 can attenuate the secretion of Aβ through decreasing the APP level and delaying the maturation of APP . NotablyOne strategy for AD therapeutics is stimulating α-secretase to inhibit the amyloidogenic processing of APP. sAPPα, a product from APP by α-secretase, has been shown to be beneficial for memory function, to have neuroprotective properties, and to be able to stabilize neuronal calcium homeostasis ,40,41,42ADAM proteins can cleave various substrates, including TNF-α, an epidermal growth factor involved in inflammation and cancer ,44. ReceCurcuma longa has antioxidant and anti-inflammatory properties [Some natural compounds, also extracted from plants, have been studied to activate α-secretase for treating AD. Resveratrol, a natural polyphenolic flavonoid extracted from several plants such as grapes, has neuroprotective and antioxidant properties . It can operties . Curcumioperties . In lineoperties . Retinoioperties . Furtheroperties . Importaoperties .Our results indicated that E144 and E2, natural compounds isolated from the plant, could be used as a potential agent for the treatment of AD by activating ADAM10 and ADAM17 in a substrate-specific manner. However, further experiments have to include various substrates of ADAM10 and ADAM17 to avoid many deleterious consequences by activating these enzymes. Moreover, additional experiments are needed to clearly show that the major target of E144 is ADAM17 over ADAM10 since it has been reported that ADAM17-mediated alpha cleavage of APP, referred to as the “regulated” pathway, is mostly occurring when PKC is stimulated ,15,16. S2. Dulbecco’s Modified Eagle Medium (DMEM) was used with 10% heat-inactivated fetal bovine serum (FBS) containing 100 units/mL penicillin, 100 μg/mL streptomycin, 260 μg/mL Zeocin, and 400 μg/mL G418. BV-2 microglia cells were cultured at 37 °C with 5% CO2 in F12/DMEM (1:1) with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. LPS  was dissolved in water.HeLa cells stably transfected with an APP carrying Swedish mutation (APPsw) were cultured at 37 °C with 5% CO6 cells in 100 mm dish. Two 100 mm dishes were prepared for each condition. Conditioned media of 12 mL volume for each condition were collected and concentrated to 100 μL using a kit . Concentrated media were immunoprecipitated with Protein G Agarose (Millipore). APP antibody against N-terminus  was used. For the Western blot, immunoprecipitated samples were washed 3 times with PBS and probed for sAPPα .APPsw-transfected HeLa cells were incubated with 6 mL culture media containing DMSO or E144 at a density of 5 × 10g for 10 min. The Bradford assay  was used to measure protein concentration in the supernatant. SDS-PAGE (8% or 15%) was used to resolve proteins. The resolved proteins were transferred to a polyvinylidene fluoride membrane. Then, it was blocked using nonfat milk powder (5%) in Tris-buffered saline/Tween 20 (TBST) for 1 h at room temperature. The membrane was then incubated overnight at 4 °C with anti-sAPPα (Covance), anti-CTF (Sigma), anti-ADAM9 , anti-ADAM10 (Abcam), anti-ADAM17 (Abcam), anti-β-tubulin (Sigma), or β-actin . After washing membranes using TBST, horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG  were incubated at room temperature for 1 h. Enhanced chemiluminescence was performed to visualize the peroxidase activity. Multi Gauge Software  using a LAS-4000 system was used to quantify the detected signals.APPsw-transfected HeLa cells were washed with PBS and homogenized using lysis buffer . Centrifugation of cell lysates was carried out at 10,000× APPsw-transfected HeLa cells were treated with chemicals dissolved in DMSO. Specific ELISAs were used to measure levels of sAPPα (IBL), Aβ42 , and Aβ40 (Invitrogen) from conditioned medium according to the supplier’s instructions.BV-2 cells were treated with 1 μg/mL LPS (Sigma) for 3 h and then cultured with E144 for 1 h at 37 °C. The cultured medium was collected, and the level of TNFα was measured using specific ELISA (R&D Systems) according to the supplier’s instruction.g for 5 min. After washing with PBS, cells were suspended in extraction buffer  at a final density of 2 × 107 cells/mL. Lysates were incubated on ice for 30 min and centrifuged at 10,000× g for 5 min. The supernatant was diluted 1:1 with a sample buffer. TAPI-1  was dissolved in DMSO. TACE activity was then measured according to the supplier’s instructions using a fluorogenic ADAM17 substrate, Mca-KPLGL-Dpa-AR-NH2. After incubation at 37 °C for 5 h, fluorescence was measured at an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Human recombinant ADAM17 was used to measure ADAM17 activity for a cell-free assay. It was diluted with sample buffer to yield a final concentration of 200 ng/mL. The Lineweaver–Burk plot was obtained using a fluorogenic ADAM17 substrate and recombinant ADAM17. Recombinant ADAM17 was incubated with a ADAM17 substrate at 0.02, 0.4, 1, 2, 4, or 10 mM in the presence or absence of E144. The change in fluorescence intensity was plotted on the Y-axis, and substrate concentration was plotted on the X-axis. ADAM17 activity was also measured using another TACE activity kit . Recombinant ADAM17 (200 ng/mL) was incubated with a different fluorogenic ADAM17 substrate, QXL520/5-FAM, in the presence or absence of E144. Fluorescence intensity was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm.To measure ADAM17 activity for a cell-based assay using the TACE activity kit , APPsw-transfected HeLa cells were collected and centrifuged at 700× 6 cells/mL. Lysates were incubated on ice for 30 min and centrifuged at 10,000× g for 5 min. ADAM10 activity from the supernatant was analyzed using a kit  and the substrate, FAM/QXL520, according to the supplier’s instructions. Human recombinant ADAM10 was used for a cell-free assay. It was diluted with an assay buffer to yield a final concentration of 250 ng/mL. The substrate was incubated in the presence of E144 for 40 min at 37 °C. Fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The relative fluorescence intensity units were calculated compared to the control.To measure ADAM10 activity for a cell-based assay, cells were collected and washed with PBS. Next, cells were suspended in cold assay buffer at a final density of 8 × 10Specific siRNA oligonucleotides against ADAM9, ADAM10, and ADAM17 were designed as follows: ADAM9, 5′-GCTGGGAGGCTCTTCCTCTTCATCCTT-3′, ADAM10, 5′-CGTACACACAATAAACCTT-3, and ADAM17, 5′-AACAAATCTCCAAAGTGGCTCTATGTT-3′. Negative control oligonucleotides (SN-1001) were purchased from Bioneer .v/v). At 24 h post-siRNA treatment, antibiotic-free media with 10 nM siRNA duplexes using Lipofectamine 2000 were added and incubated for an additional 48 h.To knockdown ADAMs, the APPsw-transfected HeLa cells were pre-incubated in a serum-free medium without antibiotics for 1 h, followed by incubation in serum- and antibiotic-free medium with 20 nM siRNA duplexes using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. After 4 h, FBS was added to make 10% FBS in medium . The supernatant was stored at −20 °C until measuring levels of secreted Aβ42 using Human Aβ42 ELISA High sensitivity Kit . The same amount of proteins from total lysates was subjected to Western blot analysis to determine the efficiency of siRNA mediated knockdown of ADAMs.g for 10 min at 4 °C. Then, the supernatant was collected. Total proteins (300 μg) were incubated with 2 μL antibody directed against APP  for 2 h. Immunoprecipitated proteins were washed 3 times with PBS. Western blot was then performed for analysis.APPsw-transfected HeLa cells were incubated with E144 for 1 h at 37 °C. Cells were washed with PBS and homogenized using a lysis buffer. Cell lysates were centrifuged at 10,000× t-test. p < 0.05 was considered statistically significant.Mean ± SEM was used to express all data. Statistical comparisons between controls and treated experimental groups were analyzed using Student’s


Pronounced correlation was described between chlorophyll fluorescence parameters and yield mainly under irrigated conditions. Our result suggested that lower N nutrition may be sufficient mainly under irrigated conditions, and in vivo chlorophyll fluorescence parameters are appropriate for detecting the effect of environmental factors in different growth stages.Efficient nitrogen (N) nutrition has been reported to have the potential to alleviate the drought stress damages by maintaining metabolic activities even at low tissue water potential. The goal of our research was to find a correlation on the genotype level between the effect of different amounts of nitrogen nutrition and water supply at different growth stages. A small-plot experiment was established with three maize hybrids and three levels of nitrogen, and two different amounts of water supply were applied during the vegetation period of 2018 and 2019. Chlorophyll fluorescence parameters were detected, as well as potential and actual photochemical efficiency of PSII, at three growth stages: eight-leaf stage, tasseling, silking. At physiological maturity, the yield of hybrids was also measured. While only genotype differences were described among the investigated parameters in the V8 stage, treatment effects were also realized based on the measured chlorophyll fluorescence parameters during the tasseling and silking stages. Beyond the significant effect of irrigation, a similar impact was declared in the case of 80 kg ha Water is an essential element for living organisms. Water deficiency negatively affects plant growth mainly through the nutrient availability in the soil. In recent years, the amount of annual rainfall has varied considerably all over the world and in Hungary as well. Increasing temperature trends affecting crop production throughout Europe furthermore increase the frequency of drought crop years and negatively affect agriculture in Southern and Central Europe . General−1 of N with the corresponding dose of phosphorus and potassium is optimal, whereas under irrigated conditions, a dose of 120 kg ha−1 is considered appropriate 2 assimiltabolism . Liu [27During the experiment, the most important weather data were recorded at the meteorological station near the experimental site. According to the results, in 2018, more precipitation fell than in 2019 during the examined period .Nevertheless, the weather conditions were favorable in 2019 for maize production. This can be explained by the distribution of precipitation. In May, June, and July of 2018, 157.9 mm rain fell. In contrast, during the same period in 2019, 205.4 mm was the amount of rainfall. The mentioned period is critical from the viewpoint of maize production. To characterize the severity of drought the Gaussen–Banouls xerothermal index was used .Based on the index, only July can be classified as a drought month—when the value is lower than 1—in 2018. However, in 2019, March, June, and August were drought months from the ecological point of view.At the V8 phenological stage, a significant difference could only be detected in Fm values in 2018 . At thisAt the VT phenological stage, significant differences were observed in every examined parameter, but the effect of the treatments and genotypes were also different in 2018 and 2019. In 2018, a difference was observed between Armagnac (0.27 ± 0.004) and Loupiac (0.26 ± 0.003) in the parameter of minimal fluorescence yield (Fo). In maximum fluorescence yield (Fm), a significant difference was noticed between Armagnac (1.32 ± 0.03) and Loupiac (1.23 ± 0.03), as well as between Fornad (1.32 ± 0.03) and Loupiac. Furthermore, there is a statistically significant interaction between fertilizer, irrigation, and genotype. By examining the variable fluorescence (Fv) parameter, similar results were found. A significant difference was noticed between Armagnac (1.06 ± 0.02) and Loupiac (1.06 ± 0.03), as well as between Fornad (0.98 ± 0.03) and Loupiac. Furthermore, there is a statistically significant interaction between fertilizer, irrigation, and genotype. Irrigation has a significant effect on actual photochemical efficiency (yield). Under irrigated condition, the value of the parameter was 0.44 ± 0.03, while the mean of non-irrigated plots was 0.36 ± 0.04. Remarkable differences were found in case of maximum quantum yield of photosystem II (Fv/Fm) in 2018 .−1 level of N has an effect on this parameter. The mentioned level of N was observed as 0.85 ± 0.004, and it was significantly higher than the value at 0 kg ha−1 level (0.79 ± 0.004). There is no significant interaction between any factors. The observation was similar in the case of the Fv/Fo ratio (The 80 kg haFo ratio .−1 (4.19 ± 0.10) than 0 kg ha−1 (3.85 ± 0.10) in 2018. In the case of the Fm/Fo ratio, a similar trend was remarked (−1 fertilizer (5.19 ± 0.10), essentially higher values were noticed than at a level of 0 kg ha−1 (4.85 ± 0.10) N. In 2019, in minimum fluorescence yield (Fo), a significant difference was noticed between Armagnac (0.23 ± 0.003) and Loupiac (0.25 ± 0.004), as well as between Fornad (0.23 ± 0.003) and Loupiac. By examining the maximum fluorescence yield (Fm) values, similar results were found between Armagnac (1.30 ± 0.03) and Loupiac (1.35 ± 0.03). In variable fluorescence (Fv), a significant difference was noticed between Armagnac (1.07 ± 0.02) and Loupiac (1.10 ± 0.02). Additional irrigation also had an effect on this parameter. In the irrigated plots (1.09 ± 0.02) remarkably higher values were noticed than in non-irrigated plots (1.04 ± 0.02).A significantly higher Fv/Fo value was experienced in 80 kg haremarked . At the −1 (0.82 ± 0.002) and 0 kg ha−1 (0.81 ± 0.002) levels of N, a significant difference was found in Fv/Fm values. Irrigation also had remarkable effect on this parameter. The value was 0.82 ± 0.001 in irrigated and 0.81 ± 0.002 in non-irrigated plots. The observation was similar in the case of the Fv/Fo ratio (−1 (4.43 ± 0.09) than 0 kg ha−1 (4.46 ± 0.05) in 2019. In the case of the Fm/Fo ratio, a similar trend was remarked (−1 fertilizer (5.61 ± 0.06), essentially higher values were noticed than at the level of 0 kg ha−1 (5.46 ± 0.05) N. A strongly significant difference was found among irrigated and non-irrigated plots in both the Fv/Fo and the Fm/Fo ratio.Remarkable differences were found in the case of maximum quantum yield of photosystem II (Fv/Fm) in 2019 . Among 1Fo ratio . A signiremarked . At the At the third measuring time, significant differences were evinced in just a few cases. In 2018, a statistically proven difference was found in the case of Fm and Fv values .−1. Furthermore, the effect of fertilizers could be observed only in non-irrigated plots. In 2019, statistically significant interaction was found between fertilizer and irrigation in Fv/Fm, Fv/Fo, and Fm/Fo parameters at the third measurement  and 160 kg ha−1 (206.92 g ± 5.07) of N, significantly higher values were observed than at 0 kg ha−1 (176.09 g ± 9.17).Results of physiological maturity are presented in −1 (185.19 g ± 6.46) and 160 kg ha−1 (204.57 g ± 5.40) levels of N, significantly higher values were observed than at 0 kg ha−1 (159.82 g ± 8.37). A difference was not evidenced at higher levels of N. Irrigation positively affected this parameter in 2019. The difference of means of irrigated and non-irrigated plots was 16.35 g. Kernel weight per ear was also affected by fertilizer level in 2018 and 2019. In 2018, remarkably higher values were noticed at 160 (184.48 g ± 4.57) and 80 kg ha−1 (191.22 g ± 5.13) than at 0 kg ha−1 (156.46 g ± 8.19). In 2019, a significantly higher kernel weight was observed at 80 kg ha−1 (165.21 g ± 5.91) than at 0 kg ha−1 (141.52 g ± 7.38); furthermore, 160 kg ha−1 (182.78 g ± 4.78) caused a powerful deviation compared to the other levels of N. The average kernel weight per ear of irrigated plots (170.37 g ± 5.58) was remarkably higher than that of the non-irrigated plots (155.96 g ± 5.60). The kernel/cob ratio was not affected by irrigation in the examined years. In 2018, only 80 kg ha−1 (8.90 ± 0.24) had an effect on the kernel/cob ratio, but in 2019, just a 160 kg ha−1 (8.51 ± 0.21) dose of N had an effect on the parameter. Remarkable differences were observed between genotypes in 2018 and 2019 as well . In the case of Armagnac, the Fv/Fm (r = −0.95), Fv/Fo (r = −0.97), and Fm/Fo (r = −0.97) parameters correlated with kernel weight at 0 kg ha−1 of N with irrigation. Results were similar in 2019 but then at a 160 kg ha−1 dose of N. In 2018, another correlation was found among fluorescence parameters and kernel weight at 80 and 160 kg ha−1 N levels in the Loupiac genotype  hybrids were sown: Armagnac, Fornad, Loupiac. Treatments at different  levels of N and two irrigations, irrigated (25 mm + 25 mm) and non-irrigated, were used. The repetition number of small blocks was four.Small block field experiments were set up at the trial site (Látókép) of the University of Debrecen  in 2018 and 2019. Three different maize (2O) was 6.58. The soluble element content of the 0–0.3 depth layer of soil was the following: NO3 + NO2 8.04 mg kg−1; P2O5 199 mg kg−1 K2O 451 mg kg−1; Na 332 mg kg−1; Mg 176 mg kg−1; SO42- 6.04 mg kg; Cu 5.79 mg kg−1; Zn 7.9 mg kg−1; and Mn 262 mg kg−1. The organic matter concentration was 3.54%, and the CaCO3 concentration was 0.2%. The precipitation and temperature data were recorded from the meteorological station of the trial field. To describe the severity, the Gaussen–Banouls xerothermal index was used [The soil type of the experimental site is a lowland calcareous chernozem. The main parameters of the soil were the same as described by Nagy . The soiwas used . It was 0) was excited by a weak light (0.1 μmolm−2s−1). The maximal fluorescence (Fm) was induced by a white saturating flash (8000 μmolm−2s−1) (fast phase of chlorophyll fluorescence). The parameters of the fast fluorescence induction phase were investigated: Fo: initial fluorescence, Fm: maximal fluorescence, Fv = Fm−Fo: variable fluorescence, Fv/Fm: potential photochemical efficiency of PSII, Fv/Fo: ratio of the variable and initial fluorescence parameters, Fm/Fo: ratio of the maximal and initial fluorescence parameters. The actual photochemical efficiency of PSII (ΔF/Fm’ = (Fm’−Ft)/Fm’) was measured in a light-acclimated condition under natural light. In vivo chlorophyll fluorescence measurements were carried out in three phenological phases (8 leaf (V8), tasseling (VT), silking (R1)) [The parameters of in vivo chlorophyll fluorescence were detected with a PAM 2100  modulated light fluorometer as described by Schreiber . Samplesng (R1)) . MeasureWe used a completely randomized design with three N treatments, two irrigations, and three genotypes with four replications. For data management and statistical analysis, Microsoft Office Excel 2016 and SigmaPlot for Windows Version 12.0 were used. For the analysis, the methods of three way-ANOVA and Pearson Correlation were used. For all pairwise multiple comparisons, Duncan’s tests were used.−1 is optimal for maize, while in optimal weather conditions, 160 kg ha−1 is optimal. Interaction between irrigation x N x genotype was found only at the VT stage. The parameters were less influenced by genotypes. The chlorophyll fluorescence induction method can provide reliable information during drought stress research. Lower N nutrition may be sufficient mainly under irrigated conditions, and in vivo chlorophyll fluorescence parameters are appropriate for detecting the effect of environmental factors in different growth stages.The optimal fertilizer dose depends on the water supply and temperature. Under dry conditions, 80 kg haKernel weight and ear weight were affected by N dose and irrigation, but the kernel/cob ratio mostly depended on genotypes. According to the results of the correlation analysis, the chlorophyll fluorescence parameters correlated with ear parameters in just a few cases. Although the chlorophyll fluorescence parameters are widely applicable in stress research, the usability in yield estimation is limited.


Objective: The prognostic significance of serum CA19-9 levels in gastric cancer patients remains a matter debate. The aim of this study was to determine the prognostic value of changes in preoperative and postoperative serum CA19-9 levels in patients with gastric cancer.Methods: A total of 1,046 gastric cancer patients who underwent curative gastrectomy in West China Hospital of Sichuan University from January 2011 to December 2016 were analyzed retrospectively. Patients were categorized by minimum P-value using X-tile, while the baseline confounders for CA19-9 changes were balanced through propensity score matching (PSM). The relationships between CA19-9 changes and other clinicopathologic features were measured. Univariate and multivariate analysis were performed to explore the risk factors associated with survival outcomes.Results: We included 653 patients. Changes in CA19-9 levels significantly correlated with age, tumor size, macroscopic type, histological grade, T stage and TNM stage. Kaplan–Meier curves revealed that patients with CA19-9 changes <20% had significant better overall survival than those with changes more than 20% (p < 0.001); Cox regression analysis revealed the CA19-9 change (p = 0.010), gender (p = 0.031), histological grade (p = 0.036) and TNM stage (p < 0.001) were independent risk factors for survival after PSM. Stratification analysis indicated that patients with CA19-9 change more than 20% had worse prognosis that those with CA19-9 change no more than 20% in male (p = 0.002), poorly differentiated or undifferentiated type (p = 0.031) and TNM stage III (p = 0.006).Conclusion: Changes in preoperative and postoperative serum CA19-9 levels were closely associated with clinicopathological traits and was an independent prognostic factor in gastric cancer patients. This parameter may be a reliable marker for prediction of survival. Gastric cancer is one of the most common form of malignancy in China, and it is the second leading cause of cancer deaths, following lung cancer , 2. In CAmong the available tumor markers, carbohydrate antigen (CA) 19-9 is one of the commonly used tumor markers for patients with gastrointestinal malignancies . CarbohyMost published studies focus only on preoperative or postoperative values of serum CA19-9 , 16. ThePatient records were de-identified and anonymized prior to analysis. The Research Ethics Committee of West China Hospital approved this retrospective study and Surgical Gastric Cancer Patient Registry number was (No. WCH-SGCPR-2019-11).We retrospectively enrolled patients who were diagnosed with primary gastric cancer and underwent curative gastrectomy from January 2011 to December 2016 in the Department of Gastrointestinal Surgery, West China Hospital, Sichuan University. The following inclusion criteria were applied: (1) histologically confirmed adenocarcinoma of the stomach; (2) curative R0 according to a pathological diagnosis after surgery; (3) no preoperative treatment; (4) measurement of serum CA19-9 before and after surgery. The following exclusion criteria were applied: (1) metastatic disease; (2) preoperative treatment such as neoadjuvant chemoradiotherapy; (3) incomplete medical records; and (4) no measurement of serum CA19-9 before and after surgery. Finally, 653 patients were included.Clinicopathological traits including extent of resection, tumor location, tumor size, macroscopic type, histological grade, pathological T stage (pT stage), pathological N stage (pN stage), M stage and pathological TNM stage (pTNM stage) were recorded according to the AJCC 8th edition .After undergoing gastrectomy, all patients were periodically followed up in outpatient visits and telephone interviews. The follow-up evaluation was every 3 months during the first 2 postoperative years, every 6 months in the third year, then annually thereafter. Overall survival (OS) was the primary end-point, and the survival time was calculated from the date of operation to the date of death or the last follow-up visit, in January 2019. Of the total patients, 93.3% were followed up.Preoperative serum levels of CA19-9 were measured within 7 days before gastrectomy, while postoperative measurement of CA19-9 levels were usually measured at the outpatient 1–4 weeks after surgery and before administration of adjuvant chemotherapy. The changes of CA19-9 level before and after surgery were labeled as change rate (α):According to the X-tile plots, the optimal cutoff point for CA19-9 change rate (α) was ±.2. As a result, gastric cancer patients were divided into four groups: (1) CA19-9 decreasing more than 20% (α < -0.2); (2) CA19-9 decreasing no more than 20% (−0.2 ≤ α ≤ 0); (3) CA19-9 increasing no more than 20% (0 < α ≤ 0.2); (4) CA19-9 increasing more than 20% (α > 0.2). CA19-9 decreasing or increasing more than 20% means CA19-9 changing more than 20% (α < -0.2 or α >0.2), while CA19-9 decreasing or increasing <20% means CA19-9 changing <20% (−0.2 ≤ α ≤ 0.2).U-test), respectively. The student's t-test was used to analyze continuous data if homogeneity of variance and normal distribution. If not, rank sum test was used. The Kaplan–Meier method and life-table method were used to calculate the cumulative survival rate. Log-rank test and Cox's proportional hazard regression model were applied for univariate and multivariate survival analyses, respectively. To reduce the impact of potential confounding factors and effects of selection bias such as baseline clinicopathologic factors or uneven patient distribution between the CA19-9 changing more than 20% and no more than 20%, 1:1 propensity score matching (PSM) was applied to adjust for age, tumor size, macroscopic type, histological grade, and T stage and TNM stage. A 0.2-width caliper of the standard deviation of the logit was applied to match across the two groups  was used to calculate the optimal cutoff points for the change rate (α) of each group. All statistical analyses were performed using SPSS 19.0 . All continuous variables were expressed as mean ± standard deviation (SD). Unordered categorical variable and ranked data was analyzed using the chi-square test and rank sum test , 125 patients with CA19-9 decreasing no more than 20% (−0.2 ≤ α ≤ 0), 187 patients with CA19-9 increasing no more than 20% (0 < α ≤ 0.2) and 176 patients with CA19-9 increasing more than 20% (α > 0.2); There were 341 patients with CA19-9 changing more than 20% (α < −0.2 and α > 0.2) and 312 patients with CA19-9 changing <20% (−0.2 ≤ α ≤ 0.2).p = 0.405)  and those increasing no more than 20% (0 < α ≤ 0.2) (= 0.405) . We comp= 0.405) .p = 0.106) (A similar result was found between the patients with CA19-9 decreasing more than 20% (α < −0.2) and those with CA19-9 increasing more than 20% (α > 0.2); there was no significant difference of the survival curves (= 0.106)  and clin= 0.106) .These results suggested that patients with CA19-9 decreasing no more than 20% (−0.2 ≤ α ≤ 0) and those increasing no more than 20% (0 < α ≤ 0.2), patients with CA19-9 decreasing more than 20% (α < −0.2) and those with CA19-9 increasing more than 20% (α > 0.2) might have similar biological behaviors. Therefore, we combined patients with CA19-9 decreasing no more than 20% (−0.2 ≤ α ≤ 0) and those increasing no more than 20% (0 < α ≤ 0.2) into one group with CA19-9 change no more than 20% (−0.2 ≤ α ≤ 0.2) and combined patients with CA19-9 decreasing more than 20% (α < −0.2) and those with CA19-9 increasing more than 20% (α > 0.2) into another group with CA19-9 change more than 20% (α < −0.2 or α > 0.2); Then we compared the clinicopathological characteristics and survival of patients with CA19-9 change no more than 20% (−0.2 ≤ α ≤ 0.2) and those with CA19-9 change more than 20% (α < −0.2 or α > 0.2).p = 0.004), tumor size (p = 0.004), macroscopic type (p = 0.012), histological grade (p = 0.038), T stage (p < 0.001), and TNM stage (p < 0.001). Specifically, patients with CA19-9 change more than 20% were more likely to be with large tumor size, macroscopic type III–IV, deep tumor invasion, and more advanced TNM stage than patients with CA19-9 change no more than 20%. However, CA19-9 change was not significantly associated with gender (p = 0.353), extent of resection (p = 0.785), tumor location (p = 0.992), or N stage (p = 0.235) (Clinicopathologic features were compared between patients with the CA19-9 change no more than 20% and those with the CA19-9 change more than 20%. CA19-9 change was found to be significantly associated with age (= 0.235) .p = 0.012), age (p = 0.004), histological grade (p = 0.039), and T stage (p < 0.001). Multivariate analyses found that age (p = 0.004), histological grade (p = 0.039), and T stage (p < 0.001) were independent risk factors for CA19-9 change.As demonstrated in p < 0.001)  .p < 0.001), tumor location (p = 0.002), histological grade (p = 0.002), macroscopic type (p < 0.001), tumor size (p < 0.001), extent of resection (p = 0.021), T stage (p < 0.001), N stage (p < 0.001), and TNM stage (p < 0.001) were closely associated with overall survival. Multivariate analysis with Cox regression indicated that CA19-9 change (p = 0.001) and other clinicopathological factors such as histological grade (p = 0.048) and TNM stage (p < 0.001) were independent prognostic factors for gastric cancer  between the two groups    after map = 0.049), gender (p = 0.021), tumor location (p = 0.010), histological grade (p = 0.001), macroscopic type (p = 0.002), tumor size (p < 0.001), extent of resection (p = 0.042), T stage (p < 0.001), N stage (p < 0.001), TNM stage (p < 0.001) were closely associated with overall survival of gastric cancer patients. Multivariate analysis with Cox regression indicated that CA19-9 change (p = 0.010), gender (p = 0.031), histological grade (p = 0.036) and TNM stage (p < 0.001) were independent prognostic factors for gastric cancer , poorly differentiated or undifferentiated adenocarcinoma (p = 0.031) and TNM stage III (p = 0.006)  .The serum tumor marker CA19-9 have been widely applied in gastrointestinal malignancies. To date, a series of studies have explored the diagnostic and prognostic value of serum tumor markers including CA19-9 in gastric cancer , 13, 14.We found that CA19-9 change significantly correlated with age, tumor size, macroscopic type, histological grade, T stage and TNM stage. Gastric cancer patients with CA19-9 changes <20% had significant better overall survival than those with change more than 20%; Cox regression analysis revealed the CA19-9 change was an independent risk factor for survival before and after PSM.A strong correlation between elevated tumor marker CA19-9 and clinicopathological features has already been reported in gastric cancer. Those studies have demonstrated that preoperative serum CA19-9 was significantly associated with tumor size, differentiated tumor types, the tumor depth, nodal involvement, peritoneal metastases and TNM stage, and elevated preoperative serum CA19-9 was associated with aggressive tumor behavior –25. In tWhether serum CA19-9 levels should be used as prognostic indicators in patients with gastric cancer has long been discussed; however, the results remain controversial. Some studies reported that CA19-9 level was an independent prognostic factor by multivariate analysis in gastric cancer; gastric cancer patients with elevated CA19-9 levels had poorer prognosis than those with normal CA19-9 , 16, 26.Shimada et al. reported that the positive rates for preoperative CA19-9 of gastric cancer increased with TNM stage . Some auThis study had some limitations: First, there was possible selection bias and performance of analysis bias because it was a retrospective study. Second, we only studied the prognostic value of serum CA19-9 change; other serum tumor markers such as CEA, CA72-4 and CA125 need to be further investigated. Finally, we reported the prognostic value of serum CA19-9 changes in our institution and a large-scale, well-designed prospective study is needed in the future.In conclusion, changes in preoperative and postoperative serum CA19-9 levels were closely associated with clinicopathological traits and was an independent prognostic factor in gastric cancer patients, which may be regarded as reliable markers for survival prediction.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Research Ethics Committee of West China Hospital.J-KH contributed to the conception and quality control of the study. X-HS, KL, S-JY, and W-HZ contributed significantly to analysis and manuscript preparation. X-HS, X-LC, and L-YZ performed the data analyses and wrote the manuscript. Z-GZ, X-ZC, and KY helped to perform the analysis with constructive discussions. All authors contributed to the article and approved the submitted version.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.


Study Setting. Single-center retrospective chart review in a tertiary referral hospital. Outcome Measures. The sagittal vertical axis (SVA) (C2-7), T1 slope, and cervical lordosis.  To determine the effect of age on sagittal cervical alignment after cervical laminoplasty procedure so as to identify the group with the greatest degree of variation. t-test and Mann-Whitney U test were used to compare changes between preoperative radiographic cervical sagittal parameters and those 1 year after surgery.  We included patients who underwent cervical laminoplasty between 2014 and 2018 and divided 60 consecutive patients into two groups using the cut-off age of 65 years. The Paired  Mean patient ages in the older and younger groups were 71 years and 52 years, respectively. The difference of C2-7 SVA was greater in the older group.  Postoperative cervical balance can be worse when laminoplasty is performed in elderly patients. As the elderly population grows, there have appeared more patients who require surgery for cervical compressive myelopathy –3. LaminAlthough there are various benefits of laminoplasty, some studies have demonstrated that the procedure often gives rise to cervical sagittal imbalance attributable to injury of posterior structures which include laminae, articular facets, muscles, and ligaments. Postoperative complications of laminoplasty cause persistent neuropathic arm pain, axial neck pain, and postoperative deterioration of cervical alignment due to the progression of kyphosis –12. TherTo our best knowledge, no previous study has evaluated the difference of postlaminoplasty cervical sagittal alignment changes between older and younger patients. Therefore, we are aimed at determining the meaningful difference between these groups by measuring the cervical balance parameters.We predicted that the postoperative changes in cervical sagittal balance will be more severe in elderly patients than in younger patients, and that this difference could be evaluated using sagittal balance parameters. We conducted a retrospective single-center study, including patients who presented to our department for cervical laminoplasty using surgical method both “french door” laminoplasty and “open door” laminoplasty, from 2014 to 2018. We enrolled 98 patients, but excluded patients with tumor, trauma, history of previous cervical spine surgery, and who could not maintain an upright position without assistance. 60 people included and divided them into two groups using age 65 years as the cut-off for two groups even 30 people. We recorded characteristics including age, gender, and diagnosis. Cervical lateral X-ray was obtained using an Infinitt PACS  to measure sagittal balance parameters, including C2-7 sagittal vertical axis (SVA), cervical lordosis (CL), T1 slope, and T1 slope minus cervical lordosis (T1 slope-CL) with accurate position. The follow-up period was set at 1 year to evaluated postoperative changes. We excluded patients who had had previous cervical surgery or cervical vertebral fracture because they were difficult to assess using radiographical parameters.To evaluate cervical sagittal balance, we measured the following parameters on cervical lateral X-ray: C2-7 SVA, T1 slope, CL, and T1 slope-CL. The C2-7 SVA was defined as the total distance from the plumb line of the pedicle center of the C2 vertebra to the posterior superior corner of the C7 vertebra . The CL T-test and Mann-Whitney U test. P values < 0.05 were considered statistically significant.We use SPSS software  to evaluate postoperative changes based on Paired n = 18). The mean age was 70.80 ± 5.03 years. The preoperative data were as follows: C2-7 SVA range -0.60 to 5.10 mm, mean 2.09; CL range 2.70 to 31.60°, mean 13.98; T1 slope range 4.90 to 48.60°, mean 26.16; and T1 slope-CL range 2.10 to 27.10°, mean 13.22 . The mean age was 52.53 ± 7.68 years. The preoperative data were as follows: C2-7 SVA range 0.70 to 4.80 mm, mean 1.97; CL range 5.00 to 33.00°, mean 12.59; T1 slope range 15.00 to 46.00°, mean 25.43; and T1 slope-CL range -1.00 to 29.50°, mean 12.84 . CL changed from 13.98° to 13.04° (P = 0.402), T1 slope changed from 26.16° to 28.84° (P = 0.075), and T1 slope-CL changed from 13.22° to 17.19° (P = 0.017). In the younger group, C2-7 SVA changed from 2.31 to 2.94 (P < 0.01). CL changed from 12.59° to 14. 03° (P = 0.234), T1 slope changed from 25.43° to 28.10° (P < 0.01), and T1 slope-CL changed from 12.91° to 14.73° (P = 0.112)  , Table 2P < 0.01). CL changed from 13.28° to 13.54° (P = 0.758), T1 slope changed from 25.79° to 28.47° (P < 0.01), and T1 slope-CL changed from 13.06° to 15.96° (P < 0.01)  .U test. There was a statistically meaningful result only in postoperative C2-7 SVA minus preoperative C2-7 SVA between older and younger groups (P < 0.01). The other parameters such as T1 slope, CL, and T1 slope-CL showed any statistically significant results (To compare postoperative changes between the two groups, we used Mann-Whitney  results .Laminoplasty is a better procedure than cervical laminectomy because it maintains cervical alignment and postoperative kyphosis , 18. NevThere have been many studies to explain the mechanism of postlaminoplasty sagittal imbalance; nevertheless, those mechanisms remain unclear. Lee et al. stated that the T1 slope was a key factor determining cervical spine sagittal balance . In our We also measured postoperative alignment changes after laminoplasty. We hypothesized that age would be a poor prognostic factor in the postoperative outcome in terms of alignment. We found that changes in C2-7 SVA in the elderly group are more prominent than in the younger group. Koshimizu et al. evaluated the effects of sarcopenia on the sagittal alignment of the cervical spine after cervical laminoplasty . As in oPrior to laminoplasty, surgeons need to advise patients regarding postoperative kyphosis that cause several surgical complications. Regarding decompression of the cervical canal with pathologies such as cervical myelopathy, patients should be carefully selected. Sakai et al. reported that postoperative cervical sagittal alignment tended to deteriorate following laminoplasty, especially in patients with preoperative center-of-gravity-of head-C7 sagittal vertical axis ≥ 40 mm . EspeciaThere are some limitations in our study. First, this was a single-center study with a small sample size. Therefore, despite the fact that we consecutively selected our patients, the results may not be generalizable. Second, our study was a retrospective study, and unintended biases including selection bias and information bias could be present. Furthermore, we did not consider surgical procedures such as single-door laminoplasty or double-door laminoplasty, either or both could be related to postlaminoplasty deformity. Third, our follow-up period was 1 year, which was chosen for reasons such as loss to follow-up; more important changes may emerge if longer follow-up is considered. To our knowledge, there are only few studies comparing cervical sagittal alignment after cervical laminoplasty of older and younger patients. Further studies may validate our findings.There are more significant postoperative changes of C2-7 SVA after cervical laminoplasty in elderly patients than in younger patients. When planning cervical laminoplasty in elderly patients, careful attention should be paid.