This paradoxical finding might be explained by the ability of IND6 to act as a mixed-type inhibitor, but also to promote the enzyme activation by adopting two distinct binding modes at the ADaM site. Moreover, functional assays reveal that IND6 increased the eNOS-dependent production of NO and elicited a concentration-dependent vasodilation of endothelium-intact rat aorta due to AMPK and eNOS activation, demonstrating a functional activation of the AMPK–eNOS–NO endothelial pathway. This kinase inhibition profile, combined with the paradoxical AMPK activation in cells and arteries, suggests that these new chemical entities may constitute a valuable starting point for the development of new AMPK modulators with therapeutic potential for the treatment of vascular complications associated with obesity.Endothelial adenosine monophosphate-activated protein kinase (AMPK) plays a critical role in the regulation of vascular tone through stimulating nitric oxide (NO) release in endothelial cells. Since obesity leads to endothelial dysfunction and AMPK dysregulation, AMPK activation might be an important strategy to restore vascular function in cardiometabolic alterations. Here, we report the identification of a novel AMPK modulator, the indolic derivative IND6, which shows affinity for AMPKα1β1γ1, the primary AMPK isoform in human EA.Hy926 endothelial cells. IND6 shows inhibitory action of the enzymatic activity in vitro, but increases the levels of p-Thr Obesity involves changes in body composition as a consequence of an energetic imbalance in which caloric intake is higher than energy expenditure1. The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is responsible for maintaining the energy balance after depletion of energy stores, switching off ATP-consuming anabolic pathways3.Obesity prevalence has increased over the past decades and is now a major public health problem worldwide. It is associated with an enhanced risk of developing cardiometabolic diseases such as hypertension, insulin resistance, type 2 diabetes mellitus, coronary artery disease, myocardial infarction, heart failure, and stroke6. Obesity leads to AMPK dysregulation and endothelial dysfunction, which is the first step in the progression of cardiovascular disease7. We have shown that caloric restriction in young Zucker fa/fa rats has cardiovascular benefits by reducing endothelial dysfunction through AMPK–PI3K–Akt–eNOS activation associated to a reduction in blood pressure, plasma triglyceride levels, and cardiac hypertrophy5. AMPK activation might be thus an important strategy to restore vascular function in cardiometabolic alterations.Endothelial AMPK plays a key role in the regulation of vascular function through the activation of the PI3K-Akt-endothelial nitric oxide synthase (eNOS) pathway and stimulation of nitric oxide (NO) release in endothelial cells8. AMPKα is the catalytic subunit and contains a conventional kinase domain (αKD) located at the N-terminus of the protein, and a C-terminal domain required for interaction with the AMPKβ subunit. The C-terminal region of AMPKα subunit forms a globular domain around which the C-terminal region of the AMPKβ subunit is wrapped. The extreme terminus of the AMPKβ subunit then forms an interaction with the AMPKγ subunit, so that the AMPKβ subunit acts as the scaffold that bridges α and γ subunits. A carbohydrate-binding module (CBM), located within the central region of the AMPKβ subunit, forms a binding site for allosteric activators, termed the allosteric drug and metabolite (ADaM) binding pocket. The AMPKγ subunit contains four repeats in tandem of a structural module called cystathionine β-synthase (CBS) motif. Every pair of CBS repeats provides binding sites for the regulatory adenine nucleotides AXP 9.AMPK is a heterotrimeric Ser/Thr kinase of 1188 amino acids (~ 132 kDa), which is ubiquitously distributed. It is formed by three subunits: α (α1 and α2), β (β1 and β2) and γ , which combine to give 12 different isoforms11, covering from AMP mimetics, such as AICAR and C2, to ADP mimetics, such as O304, which is able to protect against pThr172 or pThr174 dephosphorylation in AMPKα2 and AMPKα1 isoforms, respectively, without allosteric activation of AMPK12. On top, AMPK activator drugs such as A769661, 991, PF-739, etc., directly activate AMPK targeting the ADaM site13. Strikingly, other AMPK modulators with unexpected mechanisms of action have been recently described. MT47-10014 is an allosteric AMPKβ2 inhibitor that simultaneously activates AMPKβ1, whereas SU665615 paradoxically activates AMPK signaling by directly binding at the catalytic site. All these results reveal the complex regulation of this kinase, but at the same time offer the opportunity to be exploited in the search for drugs with novel mechanisms of action.The range of AMPK modulators has been gradually expanded over the last yearsIn this work, we describe novel indolic compounds as modulators of endothelial AMPK. For this purpose, the binding mode of these compounds has been assessed by combining molecular dynamics (MD) simulations, enzymatic and Surface Plasmon Resonance (SPR) assays, together with functional activation studies targeting AMPK, eNOS and Acetyl CoA Carboxylase (ACC) phosphorylation, as well as assessing NO production in human endothelial cells (EA. Hy926) and through vascular function of rat thoracic aorta. Our findings offer new possibilities for regulating endothelial AMPK, as well as exploring the therapeutic implications of this novel mechanism of action.16, we combined surface plasmon resonance (SPR) assays with enzymatic activity studies with the aim to evaluate the ability of the indole derivatives  to bind to AMPKα1β1γ1  with N-iodosuccinimide in dry DMF. From the key intermediate 2, aryl substituted indole derivatives 3–5 and 9 were prepared by reaction with the corresponding boronic acid derivative, Pd(dppf)Cl2 and K2CO3. Compound 10 was prepared by the reaction of ethyl 3-(4-hydroxyphenyl-1H-indole-2 carboxylate 9 with 4-(3-chloropropyl)morpholine hydrochloride. Further, KOH was employed to obtain carboxylic acid derivatives IND6, IND7, IND8, and IND11.The synthetic route employed to prepare IND6, IND7, IND8, and IND11 were screened for binding to the AMPKα1β1γ1 at a single concentration (100 µM) using the SPR technique. Abbott's product A-769662 was used as a positive binding control, and β-cyclodextrin, which binds preferentially to the AMPKβ2 isoform, as a negative binding control. Binding of IND6, IND7, or IND8 to AMPKα1β1γ1 is slightly weaker than A-769662. IND11 showed no response (0.9 RUs), suggesting the importance of the terminal aromatic ring present in the substituent at position 3 for AMPK binding, in contrast with the more polar character of the morpholine unit in IND11, which must be protonated at physiological pH (pKa ~ 8.4).Compounds IND6, IND7, IND8, and IND11 on the enzymatic activity of AMPK was examined through a luminescent assay with the recombinant AMPK isoform α1β1γ1. This assay evaluates the enzymatic activity of AMPK to phosphorylate the SAMS peptide substrate, using A-769662 as positive control. All compounds in the series were found to reduce the activity of AMPK α1β1γ1 at 30 µM and therefore may be initially considered inhibitors . Based on these results, IND6 was selected as representative compound to carry out a detailed evaluation of the biological effect on the enzyme activity.In parallel, the effect of compounds IND6, dose–response assays were performed using different ATP concentrations (from 20 to 1000 µM) and two inhibitor concentrations (10 and 20 µM). The Lineweaver–Burk plot of enzyme kinetics is depicted in Fig. IND6 acts as a mixed-type inhibitor, as noted by the increase in the Ks for ATP and the decrease in Vmax with increasing concentration of IND6. Let us note that this modality of enzyme inhibition was also reported previously for SBI-020696517. Representation of the relationship between Km/Vm and the concentration of IND6 led to inhibition constants Ki and Ki and Ki = 0.26 µM; 17). Overall, although both IND6 and SBI-0206965 exhibit a mixed-type inhibition, which conceptually combines both competitive and uncompetitive inhibition, these results suggest that the inhibitory activity may reflect different mechanisms of action.To investigate the inhibitory mechanism of IND6 binding mode, SPR studies were performed at increasing concentrations (from 10 to 100 µM) of either SBI-0206965 or IND6  . Untreated cells were used as control (CT). AICAR (at 5 mM), a known AMPK canonic activator, and 2-deoxyglucose , a caloric restriction mimetic, were used as positive controls. AICAR, once inside the cell, is phosphorylated by adenosine kinases and is converted to ZMP, an AMP mimetic, which binds the CBS sites in γ-AMPK18. On the other hand, 2-DG is a competitive inhibitor of glucose metabolism14 since it is phosphorylated by hexokinase to DG-PO4, which is trapped in the cell unable to undergo further metabolism19.In order to examine the effect of IND6 in AMPK phosphorylation and some of its targets, such as eNOS and ACC, human endothelial cells of the EA Hy.926 line were treated with IND6 significantly increased the level of p-Thr174AMPK at concentrations of 0.01, 0.1, 1 and 10 µM in a concentration-independent manner  simulations. Four independent simulations were run for the AMPKα1β1γ1 complexes with IND6 and with SBI-020695, which was used as reference system. The X-ray structure of the AMPK–SBI-020695 complex (PDB entry 6BX6) revealed that SBI-0206965 occupies a pocket located between the N- and C-lobes and the hinge region of the enzyme, overlapping with the binding site of compound C, which is a competitive inhibitor of AMPK22. The results obtained from the different MD simulations showed a consistent picture, where SBI-0206965 remains stably bound in the ATP-binding pocket in all simulations , and the carboxylate oxygen of IND6 with the NH group of αGlu96 (average distance of 3.7 ± 0.9 Å), which are larger than those formed by SBI-020695. Overall, these traits agree with the 27-fold lower potency of IND6 relative to SBI-020695 (see above).The MD simulations performed for the AMPK–695 Fig. A. This t695 Fig. B. This iIND6 to the ADaM site, which mediates the activation effect played by several small molecules, such as A-76966223. The ligand was oriented taking advantage of the close alignment exhibited by activators such as A-769662, 991 and SC4 . It is worth noting that the top of the P-loop points to the N-terminus of the αC-helix, leaving the ATP-binding site accessible for the binding of ATP. Indeed, a significant fraction of the conformations sampled by the P-loop superpose well with the conformations adopted in the ternary complex formed by AMPK bound to A-769662 and ATP24 . Remarkably, this binding mode imposes a structural distortion of the P-loop, which occludes the ATP-binding site . Cells treated with DMSO 0.01% [maximum concentration of solvent for IND6 (5 µM)] were used to exclude cytotoxic effects. AICAR (at 5 mM) was used as a positive activation control (see “IND6 (at both 1 and 5 µM) induces a similar increase in DAF-2T fluorescence as AICAR, but at 5000 × lower concentrations . L-NAME abolishes IND6-induced increment of DAF-2T fluorescence indicating that NO increase is a consequence of eNOS activation  in artery segments precontracted with a single dose of NA 10−7 M  or L-NAME (10−4 M)  or AICAR (10–5–8 × 10−3 M) to compare their effect with the one promoted by IND6  and potency (EC50) were calculated. Although the three compounds had a similar Emax, IND6 resulted as potent as A-769662 but significantly more potent than AICAR  Fig. B, which CAR 10–5– × 10−3 MThe results presented in this study point out that the novel indolic derivatives appear to act as paradoxical activators of the endothelial AMPK α1β1γ1, although they exhibit a mixed-type inhibition in the enzymatic assays.1, which is considered the first step in the progression of cardiovascular disease2. Reduced endothelial AMPK phosphorylation leads to down-regulation of the PI3K-Akt-eNOS pathway together with low rates of NO synthesis7. Contrarily, activation of endothelial AMPK restores impaired endothelial function and normalizes systolic blood pressure through the stimulation of the PI3K-Akt-eNOS pathway25. In this context, identification of new chemical entities that can activate endothelial AMPK could be of significant interest for the treatment of obesity-related disorders. Starting with the use of SPR techniques, we selected IND6, which exhibits one of the best affinity values against recombinant AMPKα1β1γ1 . In parallel, the AMPK α1β1γ1 enzymatic activity was assessed by means of a luminescent assay, which revealed inhibitory activity values in the micromolar range against all tested compounds , there was a significant reduction in the ability of SBI-020696 to bind to AMPK, while the affinity of IND6 for AMPK remained at large extent unchanged. All these results suggest that IND6 and SBI-020696 may bind the ATP-binding site, thus leading to competitive inhibition of the enzyme, but also suggest that IND6 may regulate the AMPK activity through binding to an additional pocket.Dysregulation in the AMPK signaling pathway in over-nutrition and obesity contributes to the development of metabolic disorders and endothelial dysfunctionIND6 may be explained from the distinct binding modes observed for IND6 in the ADaM site, and the drastic influence exerted on the structural conformation of the P-loop. Thus, the similar arrangements observed for the P-loop when IND6 is deeply inserted into the ADaM site and in the X-ray structure of the AMPK bound to A-769662 suggest that IND6 may mimic the activating role attributed to this latter compound. Nevertheless, the structural distortion of the P-loop caused by the alternative binding mode, where IND6 protrudes from the ADaM site, might explain the non-competitive component of the mixed-type inhibition, in conjunction with the direct competition exerted by IND6 upon binding to the ATP-binding site. At this point, let us remark that the adoption of two partially overlapping binding modes at the ADaM site may be facilitated by the lack of direct interactions between IND6 and α-Asp90, in contrast to A-769662, which was found to form a hydrogen bond interaction with this residue in our previous studies of the AMPKα2β1–A769662 complex24. In fact, previous experimental studies demonstrated that the interaction between βArg83, αAsp90 and A-769662 is crucial for the enzyme activation10.The biological effect of IND6 promotes AMPK phosphorylation (% p-AMPK/tubulin vs. CT) in the human endothelial cell line, EA Hy.926, which expresses the α1β1 isoform. This suggests a paradoxical activation of AMPK similar to the one described for the indolic compound SU6656, which seems to promote AMPK’s LKB1 dependent phosphorylation26. In any case, the activation of AMPK by IND6 is functional since it translates to ACC, an ubiquitous AMPK target20, as well as to eNOS and NO release. The inhibition elicited by SBI-0206965 on the concentration-dependent relaxation induced by IND6 in arterial rings confirms specific activation of vascular AMPK, whereas its inhibition by L-NAME confirms activation of endothelial eNOS. This is in accordance with the AMPK-dependent eNOS activation described in endothelial cells21, as well as in arteries associated to a reduction in blood pressure, plasma triglyceride levels, and cardiac hypertrophy5. Activation of eNOS by AMPK is a well-described pathway, although an attenuated NO production in response to AMPK activation has also been reported27.Despite the mixed-type inhibition, cellular assays showed that IND6 to promote AMPK stimulation is comparable to AICAR (5 mM), 2-DG (1 mM) or A-769662 (0.1 mM). However, the potency of IND6 is in the micromolar range, one and three orders of magnitude higher than A-769662 and AICAR, respectively, as clearly observed in the relaxation curves. To note, the apparent discrepancy between the concentrations of IND6 tested in the in vitro assays with recombinant proteins (SPR or inhibition studies) and the active concentrations observed in cell culture, which are much lower than in the former case. This could be explained by the synergic effect of the intracellular machinery, which leads to signal amplification28.The efficacy of IND6 binding profile provides a basis to rationalize the activating behavior of IND6 in EA Hy.926 cells and arteries by increasing AMPK activity in a functional manner, as demonstrated by the increment in both ACC and eNOS phosphorylation, two well-known targets of p-Thr172/174AMPK21. Moreover, this study shows that IND6 increases NO levels in both endothelial cells and arteries, demonstrating again a functional activation of AMPK. Our findings provide evidence that IND6 holds potential as treatment of vascular complications associated with obesity, where intracellular ATP levels are high due to the energy surplus and AMPK activity is reduced5. AMPK activation by IND6 might be thus an important strategy to restore vascular function in cardiometabolic alterations.In summary, 3CN and H2O were used as mobile phase . HRMS (EI+) was carried out on Agilent 6520 Accurate-Mass Q-TOF LC/MS equipment. NMR spectra were recorded on a Bruker-AVANCE 300, a Varian-INOVA 400 and VARIAN SYSTEM-500 spectrometer. Melting points were determined on a Mettler MP70 apparatus and are uncorrected.All reagents were of commercial quality. Solvents were dried and purified by standard methods. Analytical TLC was performed on aluminum sheets coated with a 0.2 mm layer of silica gel 60 F254. Silica gel 60 (230–400 mesh) was used for flash chromatography. Analytical HPLC–MS was performed on Waters equipment coupled to a single quadrupole ESI–MS (Waters Micromass ZQ 2000) using a reverse-phase SunFire C18 4.6 × 50 mm column (3.5 μm) at a flow rate of 1 mL/min and by using a diode array UV detector. Mixtures of CHFor experimental details, description of all synthetic intermediates, and characterization of final compounds see the 6) δ 12.79 , 11.73 , 7.55–7.22 , 7.12–7.01 , 5.16 . 13C NMR  δ 163.2; 157.7; 137.6; 136.3; 131.9; 128.8; 128.2; 128.1; 127.4; 126.6; 125.0; 123.6; 122.1; 120.9; 120.5; 114.4; 112.9; 69.6. HRMS (EI+) m/z ([M]+) calcd for C22H17NO3 343.12175; found 343.12179.Synthesis of IND6: 3-(4-(Benzyloxy)phenyl)-1H-indole-2-carboxylic acid: To a solution of ethyl 3-(4-(benzyloxy)phenyl)-1H-indole-2-carboxylate  in EtOH (30 mL), KOH  dissolved in H2O (2 mL) was added. After stirring at 100 °C for 18 h, HCl 1 N (2 mL) was added. The resultant solid was filtrated and dried under reduced pressure to give 20 mg (18%) of IND6. HPLC (SunFire): tR = 9.51 min (gradient: 15–95% of acetonitrile in water). MS : 344 (M+H)+. Mp 194–196 °C. 1H NMR . For this purpose, protein precipitation and digestion with trypsin gel was carried out. Then, LC–MS/MS (Q-EXACTIVE) analysis of triptic peptides was performed , obtaining an m/z MS–MS spectrum that was used for the subsequent database search and identification based on the results. After comparing the information obtained with the data dumped in the databases, it was concluded that the most abundant isoform of AMPK in these cells is AMPKα1β1 with 99.99% accuracy. Although the identification of the subunit γ was not conclusive, we chose the recombinant isoform AMPKα1β1γ1 for the affinity and kinase assay studies.29. The carboxymethyl dextran surface of the flow cell 2 was activated with a 7-min injection of a 1:1 ratio of 0.4 M EDC and 0.1 M NHS. The protein was coupled to the surface with a 300 s injection at several dilutions at 40 µg/mL in 10 mM sodium acetate, pH 5.0. The unreacted N-hydroxysuccinimide esters were quenched by a 7-min injection of 0.1 M ethanolamine-HCl (pH 8.0). The levels of immobilization were around 1000 RUs. Flow cell 1 treated as a flow cell 2 (amine coupling procedure) without protein was used as a reference. Prior to use 10 mM stock solutions of compounds were diluted several times until 100 µM final concentration in the running buffer. Typically, a series of different compounds was injected onto the sensor chip a flow rate of 30 µL/min for a period of 100 s followed by a dissociation period of 200 s. After the dissociation process an extrawash treatment was made over the flow cells with a 50% DMSO solution. No regeneration was needed. For competition measurements between AMPK and different compounds the concentrations used in the mixture were 100 µM for each one. Sensorgrams data were double-referenced and solvent corrected using the Biaevaluation X-100 software .SPR experiments were performed at 25 °C with a Biacore X-100 apparatus  in HBS-EP , with 2% de DMSO, 0.05% Tween 20 and 200 µM ATP when was required, at 25 °C. The protein AMPk was inmobilized on a CM5 sensor chip  following standard amine coupling methodThe AMPK (α1/β1/γ1) Kinase Enzyme System from Promega  was used to screen AMPK inhibitors following the ADP-Glo™ Kinase Assay . The assays were performed in 96-well plates , the assay buffer contains 40 mM Tris, 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 μM DTT. 4 µL of inhibitor was added to each well , followed by 8 µL of enzyme (30 ng), after 5 min incubation at R/T, 8 µL of ATP  and SAMStide (0.2 µg/µL) were added and incubate 60 min at room temperature, then ADP-Glo™ reagent (20 μL) was added allowing to incubate for 40 min at room temperature. Behind the incubation, the kinase detection agent (40 μL) was added and incubated for 30 min at room temperature. Finally, the luminescence was recorded using a FLUO star Optima  multimode reader. The inhibition activities were calculated based on the maximum activity, measured in the absence of inhibitor.For competition assays, the experiments were carried out at four different concentrations of ATP (20–1000 µM) in the absence or presence of the inhibitors, at two concentrations. The results were presented as double reciprocal Lineweaver–Burk plots (1/V vs 1/[ATP]).30, which consists of chains α1, β1 and γ1, was utilized to build up the protein, and the position of SBI-020695 in the ATP-binding pocket was defined after superposition with the X-ray structure 6BX631, which contains SBI-020695 bound to AMPK (isoform α2). To generate the IND6-bound complexes, SBI-020695 and A-769662 (PDB entry 4CFF) were replaced by IND6 in order to build up the simulated systems with IND6 in the ATP-binding and ADaM sites, respectively. Following our previous studies23, the γ-subunit was not considered in MD simulations because it does not participate in the inhibition process by SBI and IND6. Moreover, due to the lack of information of C-terminal tails of α and β subunits in the X-ray structure, these parts were not treated in our simulated systems. Finally, Thr174 was simulated in the phosphorylated form (pThr174).Molecular Dynamics (MD) simulations were used to examine the binding mode of SBI-020695 and IND6 to AMPK, considering the binding to both the ATP-binding site and the ADaM site, which is implicated in the enzyme activation by small molecules, such as A-769662. The X-ray structure of AMPK in the PDB entry 6C9J32 and the Amber ff99SB-ildn force field33 for the protein, whereas the ligand  was parameterized using the GAFF force field in conjunction with restrained electrostatic potential-fitted (RESP) partial atomic charges derived from B3LYP/6-31G(d)34 calculations. The two simulated systems were immersed in an octahedral box of TIP3P water molecules35. The final systems contained around 370 residues, the ligand, around 26,000 water molecules, and one/two Na+ atom for the complexes with SBI/IND6, which were added to maintain the neutrality of the simulated systems.Simulations were performed using the AMBER18 packageSimulations were done in the NPT ensemble for equilibration and NVT for MD productions. The simulations for SBI and IND6 were performed for 4 independent replicas. The minimization of the two systems was performed refining the position of hydrogen atoms in the protein , and subsequently of the whole system (4000 cycles for steepest descent and 1000 cycles of conjugate gradient). Then, the temperature of the system was gradually increased from 100 to 300 K in 5 steps (50 ps each) using the NVT ensemble, followed by an additional 5 ns step performed in the NPT ensemble to equilibrate the density of the system. In this process, restraints were imposed to avoid artefactual changes in the hydrogen bonds between the ligand with Val98, as well as between pThr174 and Arg140. Production MD simulations were run for 250 ns per replica, leading to a total simulation time of 1.0 μs per ligand. Restraints were gradually eliminated during the first 100 ns in order to avoid changes in the ligand binding mode due to structural fluctuations in the ATP-binding pocket, and the analysis of the trajectories was performed on the snapshots sampled in the last 150 ns unrestrained MD simulation.2 flasks with Dulbecco's modification of Eagle's High Glucose Medium , 10% fetal bovine serum , penicillin (100 U/mL) and streptomycin (100 μg/mL) (Biowest) and kept at 37 °C in a humid atmosphere and 5% CO2. Once subconfluent (70–80%) subcultures were carried out using trypsin–EDTA  and used between passages 3 and 7. Cells were incubated for 1 h with either the AMPK activator AICAR (5 mM), IND6 ; or 10 min with 2-DG (1 mM). There were also control cells (CT) without treatment. All the treatments were kept at 37 °C in a humid atmosphere and 5% CO2.Cell culture studies were performed on a human endothelium cell line (EA.Hy926). Briefly, cells were seeded in 75 cm174-AMPKα; 62 kDa), ACC in the residue Ser79 , eNOS in the residue Ser 1177  and tubulin (55 kDa) were determined by WB36  and pSer1177-eNOS  were used as primary antibodies and an anti-rabbit antibody (IgG) marked with peroxidase  was used as secondary antibody. Tubulin was detected with a monoclonal mouse antibody  as primary antibody and an anti-mouse antibody (IgG) marked with peroxidase  as secondary. Quantification was carried out by establishing the relationship between the phosphorylated form of the different proteins and tubulin (p-protein/tubulin) based on the concentration of the modulating compounds administered, in addition to the negative control, on which no treatment was performed. It is worth to be mentioned that in order to obtain the maximum efficiency of the technique, WB gels were cut before the transference following the molecular weight markers depending on where the proteins were expected to appear.For the detection of all the proteins, acrylamide gels at 7% . Polyclonal rabbit antibodies against the pSer–5 M) for 30 min in the dark, at 37 °C in a humid atmosphere and 5% CO2, in DMEM. The DAF-2DA is a non-fluorescent permeable probe capable of diffusing through the cell membrane. Inside the cell it is degraded by esterases to 4.5-diaminofluorescein-2 (2-DAF), which when reacting with intracellular NO gives rise to thiazolofluorescein (DAF-2T), capable of emitting green fluorescence (excitation wavelength 488 nm and emission wavelength 530 nm), so that the intensity of fluorescence emitted will be proportional to the production of NO in the cells. To assess eNOS involvement in DAF-2T fluorescence increase, endothelial cells were also incubated 30 min with the eNOS inhibitor L-NAME (100 µM) before stimulation with 1 and 5 µM IND6. The medium was then removed, the wells were washed twice with PBS for 15 min and then incubated with 4′,6-diamino-2-phenylindol  for 15 min at room temperature and in the dark. The DAPI (excitation wavelength 405 nm and emission wavelength 430 nm) stains the cell nuclei that are visualized by fluorescence microscopy. The cells were fixed in paraformaldehyde (PFA) for 1 h and washed 4 times with PBS 5 min. They were kept in PBS in darkness and at 4 °C until they were used for fluorescence microscopy (LeicaDM 2 000 Led). The quantification of the fluorescence intensity was performed with the ImageJ software and the fluorescence intensity at 530 nm is represented with respect to the control, which are the untreated cells in percentage form.EA. Hy926 cells were seeded on 8-well plates (Sarstedt) at a density of 6000 cells per well and allowed to grow in DMEM until a 60–70% confluence was reached. The medium was then aspirated and the cells for NO detection were incubated for 1 h with no treatment , with DMSO 0.01% (maximum solvent concentration achieved with IND6 5 µM), AICAR 5 mM as a control of activation of NO production, IND6 1 µM and IND6 5 µM. Once the incubation time had elapsed, the medium was aspirated and the cells were incubated with 4.5-diaminofluorescein diacetate (Molecular Probes)  and temperature (25 °C) conditions with standard food and water ad libitum. Animals were housed individually for two weeks. Then, they were anesthetized with ketamine  and xylazine  and sacrificed by exsanguination. Thoracic aorta was dissected and used for vascular function assays. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health  and was approved by the Ethics Committee of Universidad Complutense de Madrid (PROEX 206/18). All efforts were made to avoid animal suffering in accordance with the ARRIVE guidelines for reporting experiments involving animals.37. Once isolated, it was placed in a Petri dish containing Krebs Henseleit (KH) solution  at 4 °C. With the aid of a binocular lens (Leica GZ4), the blood was carefully removed from inside the vessel and the connective tissue and perivascular adipose tissue were separated. The artery was then cut into segments of 2 mm in length. The experiments were carried out in intact arteries with endothelium.Vascular function studies were performed on the thoracic aorta, as previously described37. Arterial segments were placed in an organ bath containing a KH solution at 37 °C continuously bubbled with a mixture of 95% O2 and 5% CO2, maintaining a physiological pH between 7.3 and 7.4 constantly. These were subjected to an initial tension of 1.5 g; which was periodically readjusted for 45 min until stabilization. Once the preparations were stabilized, their functional integrity was checked with KCl (75 mM). This value represented 100% of vascular contraction for each of the arterial segments. Next, concentration–response relaxation curves were performed after contraction to a single dose of NA (10−7 M), both with acetylcholine to test the integrity of endothelial function and with the different AMPK activators  to assess vasodilatation. To test the possible involvement of both AMPK and eNOS in the IND6-dependent vasodilation, arteries were preincubated for 30 min with the AMPK and eNOS inhibitors, SBI-0206965 (10−4 M) and L-NAME (10−4 M), respectively. After each curve, the preparations were washed 3 times with KH solution, and a 20-min rest period was left between each curve to ensure that the effects observed in each curve were not due to the agents used previously. The relaxation results were expressed as percentage relaxation with respect to the previous contraction obtained with NA (10−7 M). The analysis of the recordings obtained was performed with the aid of ACQ Knowledge 3.9 software (BioPac Systems INC).Each of these segments was inserted between two horizontal rigid steel wires (300 μm in diameter) according to the method described previously−1 M in distilled water and kept at − 20 °C until use. 2-deoxyglucose (Sigma) was prepared at a concentration of 10−1 M in distilled water and used immediately. SBI-0206965 (Sigma-Aldrich), A-769662 (Tocris Bioscience) and the different modulator candidates were prepared in DMSO (Sigma). NA (Sigma Aldrich) was prepared in a saline-ascorbic solution (0.9% NaCl/0.01% ascorbic acid), Ach and L-NAME (Sigma Aldrich) were prepared in 0.9% NaCl solution. A stock solution (10−2 M) was prepared for each of them and stored at − 20 °C until use (maximum 3 months).AICAR  was prepared at a concentration of 1.3 × 10The results obtained were expressed as the arithmetic mean ± the standard error of the arithmetic mean (S.M.E.). Comparisons of the results obtained between individual groups were made using Shapiro Wilk's analysis of variance followed by a one or two-way ANOVA and a Dunnett test (parametric) or a Kruskal–Wallis (non-parametric) to analyze differences between experimental groups with the untreated control. Significantly different groups were considered when p < 0.05. GraphPad Prism 9 software (San Diego) was used for statistical analysis. The free software ImageJ was used for image analysis.Supplementary Information.


Understanding the mechanisms of modulators’ action on enzymes is crucial for optimizing and designing pharmaceutical substances. The acute inflammatory response, in particular, is regulated mainly by a disintegrin and metalloproteinase (ADAM) 17. ADAM17 processes several disease mediators such as TNFα and APP, releasing their soluble ectodomains (shedding). A malfunction of this process leads to a disturbed inflammatory response. Chemical protease inhibitors such as TAPI-1 were used in the past to inhibit ADAM17 proteolytic activity. However, due to ADAM17′s broad expression and activity profile, the development of active-site-directed ADAM17 inhibitor was discontinued. New ‘exosite’ (secondary substrate binding site) inhibitors with substrate selectivity raised the hope of a substrate-selective modulation as a promising approach for inflammatory disease therapy. This work aimed to develop a high-throughput screen for potential ADAM17 modulators as therapeutic drugs. By combining experimental and in silico methods , we modeled the kinetics of ADAM17 inhibitor. The results explain ADAM17 inhibition mechanisms and give a methodology for studying selective inhibition towards the design of pharmaceutical substances with higher selectivity. A disintegrin and metalloproteinases (ADAMs) are multidomain proteins consisting of an N-terminal metalloproteinase domain, a disintegrin domain, an epidermal growth factor (EGF)-like domain, a cysteine-rich domain, a transmembrane domain, and an intracellular C-terminal domain. As ADAM10 and ADAM17 lack the EGF-like domain, the membrane-proximal domain is referred to as stalk region ,3,4). FuThere exist several approaches to address ADAMs as therapeutic targets, including targeting expression, maturation, and activation, the use of conformation-specific antibodies, inhibition of the active site by small molecule inhibitors, or changes of the conformation by so-called exosite inhibitors ,9,10. HyOne computational method for identification of potential inhibitor or ligand binding sites and a rough estimation of the relative binding strength is the molecular docking, which is commonly used in different fields, especially in the field of drug development ,19,20,21In the current study, we aimed to develop a high-throughput screen for potential ADAM17 modulators and their potential as therapeutic drugs. We built a highly reproducible and easy-to-handle experimental kinetic model for ADAM17 modulators using an artificial substrate and a common cell line. We compared a hydroxamate-based small molecule inhibitor to an exosite inhibitor to evaluate different modes of action. The combination within in silico structural modeling allowed for specific prediction of binding sites of these inhibitors, and the clarification of the mode of action. Thus, we established a screening tool, which can be further translated to a different cell/substrate system. Together with an automated process may be used as a high-throughput assay for drug development.M and kcat, however, would exclude these concerns and ensure the comparability of different experiments. For kinetic measurements with quantification in the linear portion, excess of the substrate is required (referred to as substrate saturation), ensuring that the amount of substrate is not the rate-limiting process. Therefore, we performed kinetic substrate cleavage measurements with varying protein amounts of HEK cell lysates. As the endpoint values after reaching the plateau phase displayed linearity between different protein amounts , whereas KM remained statistically unchanged (M (approx. 28%), which did not reach significance and could point toward an uncompetitive or at least mixed-type mode of action.As mentioned above, determining the mode of action of a potential modulator requires a combination of experimentation and structural modeling. It is well known that TAPI-1 as a hydroxamate-based small molecule inhibitor complexes the Zn ion in the active site of ADAM17. Furthermore, it was described that the exosite inhibitor 2155-17  , uncompetitive (reduction of both KM and Vmax) or mixed-type inhibitor, whereas competitive inhibition can already be excluded at this stage due to the kinetic properties [M (approx. 28%), which did not reach significance and could point toward an uncompetitive or at least mixed-type mode of action.The inhibitory function of TAPI-1 is exerted by the metal-chelating properties of the hydroxamate residues and thus the binding in the active center. Therefore, one could expect a competitive inhibition of ADAM17 activity by TAPI-1 with competitive inhibition being characterized by an increase of Koperties . In caseoperties . Howevercat/KM) of an enzyme. Calculation of the turnover number kcat requires the concentration of the enzyme itself. ADAM17 has two distinct forms: the 130 kDa pro-form carrying the inhibitory pro-domain and the 100 kDa mature form after removing the pro-domain. It was already shown in previous publications that the amount of mature and pro-form ADAM17 equals  and weak uncompetitive action (binding only enzyme-substrate complexes) of inhibitors or to distinguish a non-competitive inhibitor from mixed type inhibitors. Therefore, molecular docking simulations with the extracellular domain and the catalytic domain of ADAM17 were performed. As ligands, TAPI-1, the secretase substrate II and the proposed exosite inhibitor CID17 were used. The exosite inhibitor were used for blind docking, where the exact docking site is not known. In this way, a possible exosite could be determined. For docking of hydroxamate-based inhibitors like TAPI-1, the binding site is already known and docking site could be specified around the zin ion (10 Å around zinc ion) ,37. FurtTAPI-1 is a well-described inhibitor that supposedly binds with its hydroxamate group toward the zinc ion, thereby chelating the metal ion leading to inhibition of the proteolytic activity . The exaM and Vmax) are only bound after initial substrate binding. Consecutive docking revealed that TAPI-1 is not able to enter the active site due to the bulkiness of an already bound substrate. Thus, in line with the lack of statistical significance of the observed slight KM changes, an action as an un-competitive inhibitor can be excluded based on this model. Non-competitive inhibitors bind at sites distinct from the substrate binding’s site. Mixed-type inhibitors differ from non-competitive inhibitors by decreasing the enzyme’s binding affinity for the substrate upon inhibitor binding and vice versa. This is well in line with the fact that a pre-incubation is required in experimental settings, with TAPI-1 potentially binding while the substrate is constitutively associating and dissociating. One could argue that mixed-type inhibitors bind to an allosteric site. As evident from the docking, TAPI-1 binds (a) to one edge of the substrate-binding site, and (b) functions as chelator, thereby disabling for catalysis. Thus, based on the in silico and kinetic modeling, we assume that TAPI-1 is a mixed type non-competitive/competitive inhibitor.Per definition, un-competitive inhibitors  was carried out, the substrate had a more compact form and was not as stretched and ordered F,H as wiThe hypothesis here is that the exosite inhibitor (CID17) binds to the extracellular domain or the outer layer of the metalloproteinase domain of ADAM17, changing its structure or folding to induce an inhibition without chelating the zinc like TAPI-1. Docking the exosite inhibitor with the extracellular domain of ADAM17 model showed a favored docking of inhibitor between the linker arm and the globular form of the catalytic domain  D,G. The max was reduced, whereas KM was not changed  and theAll compounds were >95% pure by HPLC.g and 4 °C. The protein content in the supernatant was determined by Nanodrop  and adjusted to 0.8 mg/mL. It is important to note, that the final protein concentration should be at least 0.5 mg/mL. The samples were two-fold diluted with reaction buffer  and pre-incubated with either inhibitors (10 µM) or vehicle control (0.1% DMSO). The fluorogenic alpha-secretase substrate II  was added to final concentrations as indicated in the figure legends, and subsequently the increase in fluorescence  was measured each 5 min for 36 cycles at 37 °C using a Tecan plate reader . In case of the subtilisin protease 1ST3, protein concentration was determined by bicinchoninic acid (BCA) assay  and used at a final concentration of 8 µg/mL. As a linearity control, a two-fold dilution series of the initial protein extract or 1ST3 was measured in parallel. To exclude effects of varying substrate lot numbers, background activity and bleaching processes on the experimental results, a blank control containing no lysate was measured in parallel and subtracted in each experiment. The maximum velocity (Vmax) and the concentration at half-maximal velocity  were determined by enzyme kinetic modeling. The mode of action was determined in a combination of experimentation and structural modelling with the following assumption: non-competitive inhibition, constant KM with reduced Vmax; competitive inhibition, increased KM with constant Vmax; un-competitive inhibition and mixed-type models, reduced KM with reduced Vmax.Human embryonic kidney cells were cultured in DMEM high glucose supplemented with 10% FBS and sub-cultured at 80% confluence. Cells were lysed in extraction buffer  and centrifuged for 10 min at 16,000× The concentration of ADAM17 in cell lysate was determined by ELISA using a commercial kit . Different dilutions of cell lysates were measured to check for accuracy and linearity.w/v) non-fat dry milk in tris buffered saline with 0.05% Tween for 1 h and probed overnight at 4 °C against ADAM17 , followed by incubation with a POD-coupled secondary antibodies (diluted 1:20.000). After addition of the chemiluminescent substrate , signals were recorded using a luminescent image analyzer LAS3000 and quantified using AIDA image analyzer v.4.27 .HEK cell lysates were probed for ADAM17 pro-form and mature form expression as described before . BrieflyBacillus subtilis (B. subtilis) DB104.1 was done in three 500 mL flasks with each 100 mL buffered LB-Media (pH 8.6) (1% (m/v) tryptone, 0.5% (m/v) yeast extract and 1% (m/v) sodium chloride) supplemented with kanamycin (100 µg/mL). After culturing for 72 h at 250 rpm and 37 °C, bacterial cultures were centrifuged at 14,000× g and the supernatant was transferred for further use. 250 mL of the supernatant were dialyzed against 20 mM HEPES (pH 7) overnight. The dialyzed fraction was then loaded on a 5 mL cationic exchange chromatography column  with a flow rate of 2 mL/min. The protease was eluted via gradient (4 mL/min) with 20 mM HEPES (pH 8.0) supplemented with 1 M NaCl. The purified enzyme (47 mL) was again dialyzed against 75 mM sodium citrate buffer with 12.5 mM Tris-HCl, 0.005% Triton X-100 at pH 8.0. The expression resulted in 47 mL purified enzyme and 50 mL unpurified enzyme in buffered LB-media. Protein concentration was determined by bicinchoninic acid (BCA) assay  and used at a final concentration of 8 µg/mL.The expression of 1ST3 with http://www.ncbi.nlm.nih.gov/protein (accessed on 11 January 2022) , which consists of 824 amino acids (AAs), was retrieved from the National Center for Biotechnology Information (NCBI) at −1 per atom during 200 steps) were reached. MD simulation was performed for 50 ns at 298 K by rescaling the time-averaged atom velocities using a Berendsen thermostat [−1. Snapshots were taken every 25 ps and the recorded trajectories were statistically analyzed using YASARA. RMSD and RMSF can be found in To validate the predicted hybrid extracellular domain model, MD simulation in YASARA was performed as follows. For preparation the model was neutralized and solvated in a periodic box containing TIP3P  water anermostat  and a soMolecular docking of substrate and inhibitor was carried out using VINA implemented in the YASARA software package ,82. Dockp-value < 0.05 was considered significant. Details are included within the figure legends.Quantitative data are shown as mean +/± SD calculated from at least three independent experiments and cell isolates. The data were analyzed using PRISM 8.2 . A In summary, we modeled the complete extracellular domain of ADAM17 and the catalytic domain, which were docked with an artificial substrate and two different types of inhibitors (TAPI-1 and exosite inhibitor). Through the combination of in silico and experimental and kinetic modeling, we differentiated between distinct modes of inhibitor action distinguishing subtypes of mixed type inhibitors. Furthermore, we found a folding mechanism with the exosite inhibitor, which blocks the binding site and leads to inhibition in this way. We uncovered how the catalytic reaction functions, the charge distribution during cleavage, and the Zinc ion participation in this charge distribution. Thus, the proposed model could be used to screen, e.g., sizeable natural compound libraries for selective inhibitors, which would be of interest for several diseases, including viral and bacterial infections. Nevertheless, for the final validation of an inhibitor, the system would have to be tested in a ‘total cell’ setting resembling the membrane-bound form of ADAM17 and the respective substrate.


This retrospective comparative study aimed to compare the efficacy of selective caudal fixed screw constructs with all variable screw constructs in anterior cervical discectomy and fusion (ACDF). Thirty-five patients who underwent surgery using selective caudal fixed screw construct (SF group) were compared with 44 patients who underwent surgery using all variable constructs (AV group). The fusion rate, subsidence, adjacent level ossification development (ALOD), adjacent segmental disease (ASD), and plate-adjacent disc space distance were assessed. The one-year fusion rates assessed by computed tomography bone bridging and interspinous motion as well as the significant subsidence rate did not differ significantly between the AV and SF groups. The ALOD and ASD rates and plate-adjacent disc space distances did not significantly differ between the two groups at both the cranial and caudal adjacent levels. The number of operated levels was significantly associated with pseudarthrosis in the logistic regression analysis. The stability provided by the locking mechanism of the fixed screw did not lead to an increased fusion rate at the caudal level. Therefore, the screw type should be selected based on individual patient’s anatomy and surgeon’s experience without concern for increased complications caused by screw type. However, the anterior cervical plate is not without implant-related complications, including screw migration and fracture8. Furthermore, a plate placed proximal to the adjacent disc space reportedly increases the incidence of adjacent level ossification development (ALOD), which can adversely affect the range of motion and degeneration of unoperated levels11. Therefore, plating techniques that can minimize the chance of complications while taking advantages are needed13.Anterior cervical plating has been widely applied in anterior cervical discectomy and fusion (ACDF) to enhance fusion rate, improve cervical alignment, and prevent graft subsidence14. The fixed and variable screws are two types of screws used for dynamic plating. Because the insertion angle is more freely adjustable with variable types, endplate injury can be avoided with a variable screw even when the screw insertion point is near the operated disc space. Therefore, a variable screw is advantageous when keeping the plate-adjacent disc space distance to > 5 mm, which is needed to avoid ALOD11. Fixed screws provide additional stability due to the stable grabbing at the screw-plate interface. Whereas fixed screws allow rigid fixation, variable screws allow toggling or rotational movement, which demonstrates the advantages of dynamic plating14.Dynamic plating has been commonly used because it can avoid distraction force at the graft–bone interface and stress shielding13. Previous reports have demonstrated that favorable clinical outcomes with a high fusion rate can be achieved using diverse types of screw constructs13. Park et al. reported a fusion rate of 100% by using fixed screws only at the cranial vertebra and variable screws at the middle and caudal vertebrae15. A fusion rate of 83% has also been reported using all variable screw constructs6. However, most studies did not directly compare the results of different screw constructs. Although it has been reported that the fusion rate is not affected by the screw type, there is little evidence regarding the rate of ALOD, ASD, or subsidence according to screw type16. Therefore, whether using different types of screw constructs could lead to different outcomes needs further evaluation.Diverse anterior cervical plating constructs such as fixed construct, hybrid construct, and unconstrained constructs using different types of screws are being utilized10. Furthermore, pseudarthrosis or implant failure most commonly occur at the caudal-most level18. Therefore, we attempted a hybrid construct using fixed screws at the caudal-most instrumented level and variable screws at the cranial and middle levels to prevent ALOD at the proximal adjacent level while minimizing the pseudarthrosis rate at the caudal level. This selective caudal fixed screw construct would maintain a cranial plate-disc space distance of > 5 mm to prevent ALOD and provide further stability at the caudal-most level by using the locking screw mechanism of fixed screw. We hypothesized that a selective caudal fixed screw construct would result in a higher fusion rate and a similar rate of ALOD or adjacent segment degeneration (ASD) as the all variable constructs.Previous reports have demonstrated that ALOD more commonly occurs at the proximal adjacent level compared to the distal adjacent level19.All procedures were performed in compliance with the standards of our department. The study protocol was approved by the Institutional Review Board of Dongguk University Ilsan Hospital (01-019). The requirement for informed consent was waived due to the retrospective nature of the study. The methods were performed in accordance with relevant guidelines and regulations. This study was designed and reported in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement for cohort studies, which provides guidance for strengthening observational studiesWe retrospectively reviewed 101 patients who underwent ACDF with plate augmentation between September 2012 and March 2018 who met the inclusion/exclusion criteria. Inclusion criteria were: (1) patients who had surgery due to degenerative cervical myelopathy/radiculopathy at levels between C2 and C7; (2) patients who underwent surgery using allograft as an interbody spacer; (3) those with a number of operations between one and three; and (4) those who were followed-up for at least 2 years postoperatively. Exclusion criteria were: (1) patients who underwent surgery due to tumor, infection, or trauma; (2) and those who had previous cervical operation. All operations were performed by a single surgeon (JJY).Patients who underwent surgery using all the variable screw non-constrained constructs were categorized as the all variable group (AV group). Patients who underwent surgery using the hybrid construct with fixed screws at the lowermost instrumented vertebra and variable screws at rest of the levels were categorized as the selective fixed group (SF group) Fig. . In our Clinical characteristics and radiological data of patients were collected from medical chart reviews. The neck and arm pain VAS and NDI scores were recorded preoperatively, 1-year postoperatively, and at the final follow-up.20. Fusion was assessed at 12 months postoperatively by two methods using dynamic lateral radiographs and computed tomography (CT) images using the following criteria: (1) interspinous motion (ISM) < 2 mm on a 150% magnified flexion/extension lateral radiograph  bone bridging formation on sagittal and coronal reconstructed CT images  with an appropriate size was inserted into the disc space. Local autogenous bone grafts were inserted into the remaining empty disc spaceFor the AV group, cranial and caudal screws were inserted at the anterior endplate corners and angled away from the endplate to use the shortest cervical plate  and longest screw to keep the cranial and caudal ends of the plate as far away from the adjacent discs as possible. All screws were inserted using the variable angle type. In the SF group, cranial screws were inserted using the same technique as described for the AV group. The fixed screws were inserted at the caudal level and variable screws were used at the rest of the levels. The insertion angle of the fixed-angle screw was 12° caudal. Screw length was determined based on preoperative CT measurements. The patients wore a neck collar for 6–12 weeks.Categorical variables were analyzed using the chi-square test, whereas continuous variables were analyzed using the Student’s t-test. A logistic regression analysis was performed to identify factors associated with pseudarthrosis. Further subgroup analysis comparing single-level operation and multi-level operation by chi-square test was also performed. Intraobserver agreements were assessed using the intraclass correlation coefficient (ICC) and Kappa coefficient. All data management and analyses were performed using SPSS version 21.0 software . P-values < 0.05 were considered significant.Of the 101 patients reviewed, 79 met the inclusion criteria, and were included in the study. Forty-four patients were included in the AV group  and 35 patients were included in the SF group . The AV group involved more radiculopathy patients than the SF group (p = 0.02). In contrast, there were no baseline differences between the two groups. Number of levels operated was 1.6 ± 0.7 levels for the AV group and 1.7 ± 0.7 levels for the SF group (p = 0.75)  and ISM  did not differ significantly between the AV and SF groups  and SF groups . Subsidence also most commonly occurred in the lowermost level in the AV  and SF groups . The distribution of the location of pseudarthrosis (p = 0.93) or subsidence (p = 0.29) did not significantly differ between the two groups  ALODs and 2 (4.5%) ASDs were detected in the AV group, whereas 3 (8.6%) ALODs and 2 (5.7%) ASDs were identified in the SF group. The rates of ALOD (p = 1.00) and ASD (p = 1.00) did not significantly differ between the two groups. Plate-adjacent disc space distance did not significantly differ between the AV and SF groups . Furthermore, the rate of plate-adjacent disc distance < 5 mm did not significantly differ between the two groups   ALODs were noted in the AV group, and 2 (5.7%) ALODs in the SF group (p = 1.00). There were no cases of ASD at the distal adjacent level in both groups. Plate-adjacent disc space distance did not significantly differ between the AV and SF groups . The rate of plate-adjacent disc distance < 5 nm also did not differ significantly between the two groups   and final follow-up  did not significantly differ between the AV and SF groups  Table . A subgr16. One issue regarding the safe plating method is the decreasing incidence of ALOD. Lee et al. reported that the plate-disc space distance should be > 5 mm to decrease ALOD incidence10. This technique involves inserting cranial and caudal screws from the corners immediately adjacent to their respective operative-level end plate and placing the shortest plate that fits this screw placement11. This technique, by limiting anterior longitudinal ligament dissection, is reported to decrease ALOD incidence with no additional complications11.Many previous studies have been performed to determine the optimal plating method for ACDF12. A screw length greater than 75% of the antero-posterior vertebral body diameter is recommended to decrease the pseudarthrosis rate13.Other points to consider in anterior cervical plating are decreasing the amount of subsidence and the rate of pseudarthrosis. Park et al. reported that a short plate with an oblique screw trajectory construct is effective for decreasing the incidence of ALOD and subsidence10. The ALOD limits motion at the adjacent segment and accelerates the degeneration of the level next to the adjacent level11. This justifies the need to increase the plate-disc space distance at the cranial level. In contrast, pseudarthrosis or implant failure most commonly occurs at the caudal level18. This can be explained by increased shear stress at the level near the cervicothoracic junction25. However, the ALOD risk is relatively low at the distal adjacent segment, which emphasizes the need for additional stability at the caudal level, whereas there is less need to increase plate-disc space distance10. Based on these previous findings, we attempted a hybrid construct using fixed screws selectively at the caudal level to increase its stability while inserting the variable screws at the cranial level. Another potential advantage of selective caudal screw construction is that endplate injury caused by screws can be avoided. Since the lower endplate of the vertebral body has concavity in the sagittal plane, inserting a fixed screw with less angulation has the potential to injure the lower endplate of the cranial vertebra. Furthermore, due to the lordotic shape of the cervical spine, screws are often inserted with greater angulation than intended, with a variable screw at the caudal level, which could injure the lower endplate of the caudal vertebra. Surgeons can decrease the risk of these endplate injuries by using variable screws at the cranial level and fixed screws at the caudal level (Fig. The ALOD more commonly occurs at the proximal adjacent segment, and a plate-adjacent distance of < 5 mm is known as a risk factorvel Fig. .Figure 426. Song et al. also demonstrated that bone bridging assessed on CT is a reliable marker of fusion22. Furthermore, subsidence was assessed by measuring the change of distance between the endplate of vertebral body and edge of the allograft in CT performed at 2 days and 1 year, postoperatively. Although subsidence is commonly assessed by the change in total interbody height or disc space height, this method would not be accurate when using allografts as interbody spacers because allografts themselves can change in height. Therefore, the change in the distance between the endplate of the vertebral body and the edge of the allograft was used for a more accurate assessment of subsidence.In the present study, ISM evaluated by dynamic radiography and bone bridging identified on CT was used to assess solid union. Riew et al. reported that the sensitivity and specificity for diagnosing pseudarthrosis were 87.1% and. 91.4%, respectively, with ISM18. However, the fusion rates of selective fixed constructs and all variable constructs did not differ significantly. Although the locking mechanism at the screw-plate interface of the fixed screw was expected to increase the stability of the caudal segment, it did not lead to increased fusion rate. The ALOD and ASD rates were not significantly different between the SF and AV groups. The cranial and caudal plate-adjacent disc space distance and rate of patients with a plate-adjacent disc space distance of < 5 mm, which is a risk factor of ALOD, did not significantly differ between the two groups. Furthermore, clinical results, such as neck and arm pain VAS and NDI scores, did not significantly differ between the two groups.The results of this study demonstrated that pseudarthrosis and subsidence most commonly occur at the caudal level. These results are consistent with the results of previous reports16.Since the rates of pseudarthrosis, subsidence, ALOD, and ASD were similar in both groups, the selective fixed screw construct did not seem to provide additional advantage over the all variable screw constructs. The variable screws are more advantageous than the fixed screws at the point where the insertion angle is freely modifiable and a longer screw can be inserted with increased angle. In contrast, the advantage of the fixed screw is that it can be easily inserted with constant angulation. Based on the results of this study, screw types can be selected based on individual patient’s anatomy and surgeon’s experience, without concern for increased pseudarthrosis or subsidence caused by screw type. Oh et al. also reported that fusion rates of using the fixed and variable screws are similar. This is consistent with the results of this study18.Further studies should be conducted to clarify the method to decrease the rate of pseudarthrosis at the caudal level, especially for multi-level surgery, since the results of the current study did not demonstrate significant results by screw construct difference. Although Lu et al. reported that pseudarthrosis at the caudal level can be decreased by selectively using low-dose bone morphogenic protein at the caudal level, there is still concern regarding complications caused by bone morphogenic protein for anterior cervical surgery30. The result of the logistic regression analysis in this study also demonstrates that multi-level operation is a risk factor of pseudarthrosis, consistent with the results of previous studies. With increased fusion level, micromotion and contract stress also would have increased at the graft–bone interface, which could lead to pseudarthrosis31.Previous studies have demonstrated that the number of fusion levels, bone graft type, plating, sex, age, smoking, greater preoperative segmental motion, and greater T1 sagittal slopes are related factors associated with pseudarthrosis after ACDF16. Finally, this study has a potential bias due to the retrospective nature of this study.Our study has some limitations. First, this study had a limited sample size to assess the rate of ALOD or ASD. Second, there was a temporal difference in the type of operation performed. Although all operations were performed by a single surgeon at a single institute, unidentifiable factors due to time difference could have affected the results. Third, insertion angles and lengths of the screw were not considered as factors. However, a previous study demonstrated that screw insertion angle does not affect subsidence or fusion rateIn conclusion, the fusion rates, subsidence, patient-reported outcome measurements, plate-adjacent disc space distance, ALOD, and ASD were not significantly different between the selective caudal fixed screw and all variable screw constructs. The stability provided by the locking mechanism of the fixed screw did not lead to an increased fusion rate. Therefore, it would be better to select screws based on individual patient’s anatomy and surgeon’s experience without concern for increased complications caused by screw type. This study demonstrates provisional results of comparison between all variable screw constructs and selective caudal fixed screw constructs in ACDF. Further clarification with a larger sample size is warranted.


Runoff from the Greenland Ice Sheet has increased over recent decades affecting global sea level, regional ocean circulation, and coastal marine ecosystems, and it now accounts for most of the contemporary mass imbalance. Estimates of runoff are typically derived from regional climate models because satellite records have been limited to assessments of melting extent. Here, we use CryoSat-2 satellite altimetry to produce direct measurements of Greenland’s runoff variability, based on seasonal changes in the ice sheet’s surface elevation. Between 2011 and 2020, Greenland’s ablation zone thinned on average by 1.4 ± 0.4 m each summer and thickened by 0.9 ± 0.4 m each winter. By adjusting for the steady-state divergence of ice, we estimate that runoff was 357 ± 58 Gt/yr on average – in close agreement with regional climate model simulations (root mean square difference of 47 to 60 Gt/yr). As well as being 21 % higher between 2011 and 2020 than over the preceding three decades, runoff is now also 60 % more variable from year-to-year as a consequence of large-scale fluctuations in atmospheric circulation. Because this variability is not captured in global climate model simulations, our satellite record of runoff should help to refine them and improve confidence in their projections. Accurate assessments of ice-sheet runoff are essential for sea-level projections. A new method using satellite altimeter observations can provide near real-time surface mass balance measurements across an entire ice sheet and reveal runoff variability not captured by global climate models. Surface processes have been the principal driver of this imbalance because net SMB has declined as the regional climate has warmed4. In part, this is due to changes in atmospheric circulation5 leading to increased runoff6, behaviour which is likely to continue as global temperatures rise7. As well as raising global sea level, ice sheet runoff also delivers freshwater into the regional ocean8, weakening deepwater convection9 and reducing the strength of the Atlantic meridional overturning circulation10. Runoff also influences coastal marine ecosystem productivity in adjacent fjords11 and sediment delivery to the North Atlantic and Arctic oceans12. En-route to the ocean, runoff also supplies water to surface13, englacial14 and subglacial15 hydrological networks, which are known to influence rates of ice flow16. Although automatic weather stations provide point measurements of SMB components18 and satellite observations are able to monitor trends in the extent of surface melting19, regional climate models  have been the principal source of ice-sheet wide estimates of runoff.Ice sheets are impacted by the atmosphere through a variety of surface mass balance (SMB) processes—primarily snowfall and runoff—and also lose mass to the oceans via glacier discharge. The Greenland Ice Sheet has lost mass over recent decades because ice discharge has exceeded its SMB23 with net ablation  and net accumulation  confined to summer and winter months, respectively, measurements of ice sheet elevation change offer an opportunity to monitor each process. Long-term signals of ice sheet surface lowering brought about by increases in runoff, firn densification and ice discharge have been resolved by satellite altimeters; in particular CryoSat-226 and ICESat-227 thanks to their fine spatial sampling. At the same time, ice sheet elevation changes arising from SMB and firn processes alone have also been reconstructed using RCMs and firn density models29, and satellite altimetry and firn density models have been combined to partition ice sheet elevation changes into signals due to ice-dynamical and meteorological processes30. While RCMs have been able to capture recent interannual variability in runoff, global climate models (GCMs) often used in sea level projections have not, and have underestimated Greenland’s contribution to sea-level rise as a consequence31. To date, however, the potential of satellite altimetry to directly monitor SMB has yet to be exploited. In this study, we use CryoSat-2 radar altimetry to separate long-term and seasonal elevation changes in the ablation zone of the Greenland Ice Sheet between 2011 and 2020, to directly measure runoff at the scale of the Greenland Ice Sheet and characterise its recent interannual variability.Because Greenland SMB follows a strong seasonal cycle26 applied within 5 × 5 km grid cells to separate elevation fluctuations evolving over time from those resulting from local topography and temporal variations in radar backscatter (see Methods). To evaluate the accuracy of the CryoSat-2 elevation trends, we compare them to 15,380 contemporaneous and independent estimates determined from airborne laser altimetry32 and find a mean difference of 15 cm/yr. Thinning is concentrated at the ice sheet margin, consistent with the findings of previous surveys26, likely as a result of increased surface melting26 and the speedup of marine-terminating glaciers35. There is a notable signal of thickening at Storstrømmen in northeastern Greenland, which is known to have slowed after surging between 1978 and 198436. We suggest a band of no elevation change just inland of the ice sheet margin in the southwest  than firn densification modelling (−30 cm/yr)28. In addition to elevation trends, we also compute the time-varying evolution of surface height . The IMAU-FDM output is restricted to the period January 2011 to December 2016, which provides 5 years of overlap with our CryoSat-2 data set. To allow the firn model to be compared to the satellite observations 20 simulations (which compare favourably to observations of the current climate21) to define the spatial extents of the ice sheet ablation, runoff and dry snow zones  in the majority of the years for which IMAU-FDM and CryoSat-2 data are available (between 2011 and 2017). The ‘runoff zone’ is taken as the 418,325 km2 area for which runoff is predicted in the majority of years, and includes the ablation zone. The dry snow zone is taken as the 802,350 km2 area for which no runoff emerges in the majority of years , and excludes the percolation zone where meltwater percolates and refreezes within the snowpack. On average, between 1980 and 2010, 80 % of annual total runoff emerges from the ablation zone and the rest arises from a 207,100 km2 area just inland20. The ablation, runoff, and dry snow zones receive 12 %, 37 % and 27 % of the annual total snow accumulation, respectively20.To examine elevation changes associated with different meteorological processes, we use RACMO2.3p2R2 = 0.98) and winter (R2 = 0.96), and agree to within (r.m.s. difference of) 23 cm overall  and snowfall in winter (September–April). We take advantage of this distinct characteristic by computing the average seasonal rates of elevation change between 2011 and 2020 by fitting piecewise linear trends to the altimeter time-series in summer Fig.  and wintall Fig. . We alsoson Fig.   and 2019 (1.91 ± 0.50 m), both record-high melt years. In 2012 and 2019 a strongly negative North Atlantic Oscillation (NAO) increased the prevalence of near-stationary high-pressure systems39. We exclude the southeast (Basin 4) from our overall statistics, because the ablation zone is narrow  and because the sector has a rugged terrain that is challenging for both altimetry and regional climate modelling. At the basin scale, seasonal elevation changes from CryoSat-2 altimetry agree with those derived from IMAU-FDM to within 45 cm in winter and 36 cm in summer . Potential sources of error in the IMAU-FDM include, but are not limited to, an inadequate choice of reference period selected for initialisation, or the reduced ability of the RCM forcing data to sufficiently capture changes in ice albedo and sensible heat flux in low-lying regions45. Satellite altimetry also struggles when monitoring rugged terrain, which is particularly apparent in the eastern and southern ice sheet sectors ; the same is true for RCMs when resolving steep slopes46. Seasonal changes in the scattering properties of the firn layer could also lead to a poorer agreement between the satellite altimetry and IMAU-FDM, especially in the southeast sector where spurious signals associated with tracking of submerging ice lenses have been detected47, and there is some evidence of these effects in our elevation trends in the south-east inland from the ablation zone , because their elevation changes are not solely due to SMB. These areas represent just 5 %  of the runoff zone  as well as dynamic thinning of up to 2 m/yr. Across the remaining area, we adjust the altimeter summer height changes within each 5 × 5 km grid cell to remove the elevation signal associated with the steady-state divergence of ice and thereby isolate the contribution due to SMB anomalies alone (see Methods). We then use the resulting elevation change anomalies to estimate summertime changes in mass by aggregating them across two regions and applying fixed densities.Because seasonal changes in ice sheet elevation are primarily driven by SMB, and because the dominant process during summer is ice meltingone Fig.  and coin3), and across the inland runoff zone, and when the elevation changes are negative, we assume they occur at the density of firn. We obtained a firn density of 684 kg/m3 from shallow cores collected recently in the region56 rather than ice-sheet wide models as they are known to be biased high in the inland runoff zone56, though the choice has only a small (< 10 %) impact on our runoff solution because the region makes a small overall contribution (see Methods). Our approach is a simplification because it neglects mass accumulated during the summer period. However, although this simplification could in principle lead to an underestimate of runoff, our choice of ice and firn densities mitigate this because they represent upper and contemporary bounds, respectively, and in any case, the simplification has only a minor impact because snowfall is a small fraction (15 %) of the summertime SMB across the runoff zone (see Methods). To verify this, we compared model estimates of SMB and runoff within the ablation and runoff zones, and find them to be highly correlated 20 — indicating that other processes play a minor role.In the ablation zone, we assume that elevation change anomalies occur at the density of ice  average simulated by RCMs (296 Gt/yr)20. Our record shows interannual variability in Greenland runoff between 2011 and 2020 has been 60 % higher than in the previous three decades . We find the largest relative departure from both models in 2020, when runoff recorded by CryoSat-2 is 97 and 90 Gt/yr lower than RACMO2.3p2 and MARv3.11, respectively, but still agree within their respective uncertainties (which is estimated to be 15–20% for the RCMs57) for this and all years surveyed by CryoSat-2. Over the longer term, RCMs22 show that there has been a progressive increase in summertime melting and thinning across the ice sheet ablation zone  mean20, runoff during the period 2011–2020 has been 21% higher, on average. However, the past decade has also seen a marked increase in interannual runoff variability, with a standard deviation of 99 Gt/yr — 60% higher than the previous three decades as determined from RCMs, and twice as high as the preceding one — and a minimum of 247 ± 56 Gt/yr in 2017 when the melt season was short in comparison and snowfall in June reduced melt-albedo feedbacks. This suggests that the freshwater outflow and ice discharge from the ice sheet may become more variable as the climate warms, as well as more intense, which may have implications for ocean circulation10 and ecosystem productivity58. While not captured in the GCMs used in sea-level projections to date, this variability should be accounted for to accurately predict a sea-level rise in the future31; our observational-based approach can help improve descriptions of the ice sheet–atmosphere interactions in climate models and improve confidence in their projections.Our method provides a record of seasonal elevation changes across the Greenland Ice Sheet, and the first observational and satellite-based estimate of ice sheet runoff at scale. Between 2011 and 2020, the ablation zone thinned by 0.5 ± 0.1 m/yr, on average, because summer thinning was more than twice as large as winter thickening. In total, the ice sheet lost 3571 ± 182 Gt of ice through runoff over the 10-year survey period, with record-breaking losses of 527 ± 56 Gt/yr first in 2012 and then 496 ± 53 Gt/yr in 2019. These highs occurred in summers when atmospheric conditions promoted strong runoff from the ablation zone, in concert with widespread melting across much of the ice sheet surface57, general improvements in SMB models21, and assessments of climate projection skill59. Our method demonstrates the exciting potential of satellite altimetry to monitor ice sheet SMB processes, and the next step is to extend our method to include an assessment of snowfall variability.Because our approach is based on satellite observations, it allows runoff to be measured across the entire ice sheet and in near real-time and, potentially, backwards in time using measurements acquired by older satellite altimeter missions. It also provides an independent method for monitoring SMB processes based upon satellite observations, which can support improvements in RCM and GCM capability60. Using a model fit method  we generate time-series and trends of ice sheet surface elevation change on a 5 × 5 km grid, allowing for elevation fluctuations within each grid cell caused by topography  the threshold offset centre of gravity retracker we use67 is less sensitive to changes in volume scatter than physically based algorithms, and (ii) we only examine elevation trends in the ice sheet interior, which are less affected by the melt event when determined over longer time periods68. At the ice sheet margins where CryoSat-2 operates in SARIn mode, we use the ESA Level 2 SARIn retracker, which fits an analytical model to individual SAR waveforms69.Although previous studies have also adapted backscatter corrections to account for the effects of an episodic change in snowpack characteristics on the shape of altimeter echoes25Across a small proportion (9 %) of the ice sheet area our method fails to retrieve a solution, and we estimate elevation trends in this region instead of using an empirical model based upon latitude  and dynamic elevation trends determined from satellite altimetry and IMAU-FDM (2011–2017). To identify areas of long-term dynamical imbalance from the ice velocity data, we fit linear trends to time series grouped within 240 m pixels which are flowing faster than 50 m/yr and contain measurements on at least 10 occasions. From these trends, we define areas where changes in ice speed are significant in both their magnitude  and in their variability  to be in a state of dynamical imbalance . In this way, we assume that (1) in the runoff zone and away from areas of dynamical imbalance, thickness changes due to ice divergence are approximately equal to the long term SMB mean and (2) because the dynamic response to SMB changes occurs over multi-decadal timescales72, present-day observed departures in thickness in these areas are dominated by changes in SMB. In the absence of direct observations, we assess the validity of this assumption by comparing dynamic and SMB-related ice sheet thickness changes derived from an ensemble of ISMIP6 model experiments, forced by a high-end emission scenario (RCP8.5)72 20, lending confidence to this assumption. Nevertheless, this is a simplification because our solution does not account for the effects of summertime snowfall, sublimation and drifting snow erosion on observed surface height changes. Sublimation and windblown divergence represent limited contributions to SMB 20. During our observational period, summertime snowfall amounts to 22 and 32 Gt within the ablation and inland runoff zones on average; 7 % and 33 % of the modelled runoff, respectively, and 13 % of the total runoff20. The effects of this on our solution will be dependent on the snowfall conditions within a given year: the melting of a thick surface snow layer at the start of the runoff season, present due to heavy snowfall in the preceding winter, for example, could bias our runoff estimates high due to our choice of higher densities in both the ablation and inland runoff zones. We note that in the inland runoff zone, where snowfall is higher, our choice of a lower density acts to reduce this bias in part, and that modelled surface densities typically vary by 14 and 9 kg/m3 at the beginning of May in the ablation and inland runoff zones, respectively28. Conversely, any snowfall in the summer which runs off the ice sheet will not be captured by our observations, and our solution could underestimate the total runoff. Taking both of these effects into account, snowfall equal to the modelled average will bias our runoff by 5 % and 21 % within the ablation and inland runoff zones, respectively, and 8 % overall; however these effects may be larger for years with heavy snowfall.In the runoff zone, and outside areas of dynamical imbalance, we assume the observed elevation changes represent the surface height change due to all SMB processes integrated over the summer, and that during our chosen summer period the SMB is dominated by runoff. Within the ablation and inland runoff zones, we find that modelled summer SMB and annual runoff are highly correlated , before aggregating across the ablation zone and within each calendar year to estimate the annual runoff. As with our estimates of elevation change, we quantify the uncertainty on the altimeter runoff estimates at each epoch by computing the regional average of the standard error of the mass change within all contributing pixels and assume there is no error associated with our choice of density. To estimate the uncertainty in the annual runoff, we sum the uncertainties from each summer epoch within each calendar year in quadrature.Because CryoSat-2 does not fully sample the ablation zone during a given 60-day period, we scale our mass change estimates at each epoch according to the proportion of the observed area within 500 m elevation bands  and temporal (10 days) resolution74. IMAU-FDM uses an expression for firn-densification75 adapted to fit in situ density profiles retrieved from the Greenland Ice Sheet28 and covers the period 1960–2017. The surface layer of the firn column is forced by SMB components , surface temperature and 10 m wind speed at 11 km horizontal and sub-daily (3–6 h) temporal resolution from the RACMO2.3p2 RCM20. Surface elevation changes are computed with respect to a spin-up period (1960–1979), over which zero surface elevation change due to firn and SMB processes is assumed28. In the ablation zone, where there is no persistent firn layer, elevation changes are modelled due to SMB processes alone.We simulate surface elevation changes due to firn compaction and SMB processes during the period of our satellite altimeter record using the Institute for Marine and Atmospheric Research Utrecht Firn Densification Model (IMAU-FDM)To compare firn model outputs to the satellite observations, we resample the gridded firn model time-series to a spatial (5 × 5 km) and temporal (60-day) domain common to that used for the radar altimetry through bilinear interpolation. We then obtain average rates of elevation change due to SMB and firn processes through fitting a linear trend to the cumulative firn height anomaly time series in each grid cell. In a similar manner to the Cryosat-2 data, we compute time-varying surface height evolution in the ice sheet ablation zone at 60-day intervals by spatially averaging gridded monthly elevation anomalies.38 (Fig. 39 where the airborne surveys are extensive enough to allow averages to be representative of the whole area (Supplementary Fig. 76, which may induce corresponding elevation changes not considered by the firn model. Basin 6.2, on the other hand, is a land terminating sector where seasonal elevation changes are likely to be dominated by surface processes.We compared time series of height evolution and seasonal elevation changes from both satellite altimetry and IMAU-FDM to independent estimates derived from airborne laser altimeter measurementsSupplementary informationPeer Review File


Draxin belongs to the family of inhibitory axon-guiding factors that regulate neuronal migration and axonal spreading in the developing brain. This glycoprotein has recently been considered to play an important role both in hippocampal differentiation and adult neurogenesis in the dentate gyrus. Given that it has been reported that antipsychotic drugs may affect neurite growth and neurogenesis, we have therefore investigated whether chronic treatment with olanzapine modulates draxin immunoreactivity in the adult rat hippocampus. After analysis of local fluorescence intensity, we found a significant increase of draxin immunoexpression both in the subgranular zone (SGZ) and granular zone of the rat hippocampus following long-term olanzapine administration. This study reveals, for the first time, the modulatory effect of the atypical antipsychotic medication olanzapine on expression of the novel chemorepulsive protein draxin in the context of adult neurogenesis regulation. Moreover, this is the first report dealing with pharmacological aspects of draxin signaling. An elevated draxin expression may indirectly support a recently formulated hypothesis that olanzapine may drive adult neurogenesis via paracrine draxin-related signaling. This action of draxin is a new element in the neurogenesis mechanism that may be part of the action of second-generation antipsychotics in the treatment of schizophrenia, indicating more detailed molecular studies are urgently required to fully investigate these potential novel mechanisms of neurogenesis. Draxin  is an inhibitory axon guiding factor and local chemorepulsive glycoprotein involved in neuronal migration and neurite growth in the developing brain ,2,3. ThiOlanzapine, an atypical antipsychotic agent, acts as an antagonist of brain dopaminergic receptors (D1-D5), with some affinity to others, including serotoninergic , α1-adrenergic, muscarinic, and histaminergic . Olanzapp = 0.0005; F, DFn, Dfd: 2.322, 6, 6). In the analyzed brain sections, draxin immunoexpression was very dispersed within the subgranular and granular layers of the dentate gyrus, with diverse intensity of the fluorescent signal  male Sprague–Dawley rats from the Medical University of Silesia Experimental Center housed at 22 °C with a regular 12/12 light–darkness cycle with access to standard Murigran chow and water ad libitum. All procedures were approved by the Local Bioethic Committee at the Medical University of Silesia (decision no. 36/2012) and were conducted in a manner consistent with NIH Guidelines for Care and Use of Laboratory Animals.Two groups of rats  received control vehicle or olanzapine  by intraperitoneal injection for 4 weeks. This optimal, nontoxic dose was established on the basis of pharmacological standards developed in preclinical studies on antipsychotics in the context of adult neurogenesis ,29. 24 hp ≤ 0.05.For morphometric assay of draxin immunoreactivity, 5 sections per rat were used. Due to the highly dispersed and diffuse draxin immunofluorescence in densely arranged granular cell clusters, the number of cells was not calculated. All images (2 per section) were captured with Nikon Coolpix optic systems and processed using Image ProPlus software . Histologically analogous serial sections were analyzed densitometrically with use of ImageJ (v1.51j8). Pictures were normalized and background was extracted . Further, the green channel was isolated as a BW (8-bit) picture. At this stage, five ROIs (250 × 250 pix) were selected in the region of staining to calculate mean grey value, then the average staining intensity was calculated for the picture. For control and treated groups, seven pictures were analyzed . The average gray value within the selection was the sum of the gray values of all the pixels in the selection divided by the number of pixels, reported in calibrated units  if Analyze> Calibrate was used to calibrate the image. For RGB images, the mean was calculated by converting each pixel to grayscale using the formula gray = 0.299 red + 0.587 green + 0.114 or: gray = (red + green + blue)/3 if " Unweighted RGB to Grayscale Conversion" is checked in Edit> Options> Conversions. The total number of TUC-4-positive cells in the comparable SGZ areas was additionally estimated. Cells were counted from standardized frames of the specimen . Statistical analyses were performed using Statistica . Mean differences between the groups were analyzed using an unpaired two-tailed t-test and a nonparametric Kruskall–Wallis test. Differences were considered statistically significant at We have shown for the first time an increase of draxin immunoexpression in the adult rat dentate gyrus after long-term treatment with the atypical antipsychotic medication olanzapine. This may indirectly support a recently formulated suggestion that olanzapine may increase adult neurogenesis in both the canonical and hypothalamic sites via paracrine draxin-related signaling; however, further molecular studies are urgently required to confirm this possible regulatory interplay.