Panax notoginseng, has been shown to be efficacious in the prevention and treatment of ischemic stroke in China. The aim of this study is to determine the anti-platelet activity and explore the underlying mechanisms.Increased platelet aggregation is implicated in the pathogenesis of ischemic stroke and anti-platelet strategy may contribute to its therapy. Panaxatriol saponin (PTS), the main components extracted from 2+ concentration ([Ca2+]i) mobilization was detected with fura-2/AM probe. MAPKs phosphorylation was determined by Western blotting.Inhibitory effect of PTS and its main ginsenosides on agonists-induced platelet aggregation was determined using rabbit or human platelets. Intracellular CaOur results showed PTS inhibited the rabbit platelet aggregation induced by various agonists . The three main ginsenosides  existing in PTS also showed anti-platelet activity, while their combination exhibited no synergistic effect on rabbit platelet aggregation. Further study demonstrated that PTS and its main ginsenosides also exhibited inhibitory effect on human platelet aggregation. Mechanism study demonstrated that pre-treatment with PTS inhibited the agonists-induced intracellular calcium mobilization. Moreover, PTS significantly suppressed the activation of both ERK2 and p38 by the agonists via reducing the phosphorylation of ERK2 and p38.We proved that PTS is effective in anti-platelet aggregation, which may, at least in part, be related to the suppression of intracellular calcium mobilization and ERK2/p38 activation. This study may provide one reasonable explanation for the efficacy of PTS on the prevention and treatment of ischemic stroke. Platelets which only exist in mammals mainly function as stopping bleeding at the site of interrupted endothelium. Under normal physiological condition, platelets are quiescent and don’t bind to fibrinogen. Once there is an injury in the endothelium, platelets will adhere to the interrupted endothelium and become activated via changing shape, turning on receptors and secreting chemical messengers. Then, they interconnect through receptor bridges to form aggregation and finally promote the blood coagulation . HoweverPanaxatriol saponin (PTS) is one of the major components of Panax notoginseng and composed of ginsenoside Rg1 (Rg1), notoginsenoside R1 (R1) and ginsenoside Re (Re). It has been clinically used in China for the treatment of cerebral infarction. Previous pharmacological study demonstrated that PTS alleviated the focal cerebral ischemia-induced injury via reducing the cerebral edema, up-regulating HSP70 expression and down-regulating transferring . Our recPTS extracted from Panax notoginseng was obtained from Huasun Group Co., Ltd. . As determined in our previous study , PTS is ad libitum. All experimental protocols involving the use of animals were conducted in accordance with National Institutes of Health (NIH) guidelines and approved by the Committee on Animal Care at the Southwest University.New Zealand albino rabbits (2.0 ± 0.2 kg) used in this study were obtained from Experimental Animal Center of Chengdu University of Traditional Chinese Medicine. The animals were acclimated for 1 week prior to the experiments, and housed in an air-conditioned animal room with a 12/12 h light/dark cycle at a temperature of 25 ± 1 °C and humidity of 50 ± 10 %. The animals were provided with a laboratory diet and water 3C6H5O7 · 2H2O, 16 mM C6H8O7 · H2O, 16 mM NaH2PO4 · H2O, 142 mM dextrose). Platelet-rich plasma (PRP) was obtained by centrifuging rabbit blood samples at 230 × g for 5 min. Platelets were precipitated by centrifugation of the PRP at 800 × g for 15 min and washed twice with the washing buffer  dextrose and pH 7.4). The pellet was re-suspended in HEPES buffer  and the cell dilution was adjusted to 4 × 108 cells · ml-1 for subsequent experiment.The preparation of the platelets has been described previously with minor modifications . The humPlatelet aggregation was performed as previously described . Briefly0 and OD1 indicate the optical density before and after the inducer is added, respectively.Whereas ODThe following equation is used to calculate the inhibition rate (IR) of platelet aggregation.test and PARblank represent the PAR of the tested samples and the blank solvent, respectively.Whereas PAR2+]i was determined with fura-2/AM as described previously i was calculated by the method of Schaeffer [2+]i in cytosol = 224 nM×(F − Fmin)/(Fmax − F), where 224 nM is the dissociation constant of the fura-2-Ca2+ complex, and Fmin and Fmax represent the fluorescence intensity levels at very low and very high Ca2+ concentrations, respectively. In our experiment, Fmax is the fluorescence intensity of the fura-2-Ca2+ complex at 510 nm after the platelet suspension containing 1 mM of CaCl2 had been solubilized by Triton X-100 (0.1 %). Fmin is the fluorescence intensity of the fura-2-Ca2+ complex at 510 nm, after the platelet suspension containing 20 mM Tris/3 mM of EGTA had been solubilized by Triton X-100 (0.1 %). F represents the fluorescence intensity of the fura-2-complex at 510 nm after the platelet suspension was stimulated by inducer, with and without the tested samples, in the presence of 1 mM CaCl2.[Caeviously . Brieflychaeffer : [Ca2+]iβ-actin. The bound primary antibodies were detected by HRP conjugated anti-mouse IgG accordingly. The activity of peroxidase on the blot was visualized by enhanced chemiluminescence (ECL) detection reagents .The cellular proteins were extracted and analyzed for protein expression as previously described . Brieflyt-test. A P-value of less than 0.05 was considered to be statistically significant.All data were presented as mean ± SD from three independent experiments. Statistical analysis was performed by two-tail Student’s -1, with the inhibition rate of 63.9, 69.8 and 71.3 %, respectively. Figure -1 PTS markedly inhibited the aggregation induced by thrombin, with the inhibition rate of 55.5 and 56.4 %, respectively. Similarly, 3 and 10 mg · ml-1 PTS also significantly inhibited ADP-induced rabbit platelet aggregation with the inhibition rate of 55.3 and 55.9 %, respectively.To determine the effect of PTS on rabbit platelet aggregation, we examined its anti-platelet activity against stimulation of different agonists. washed platelets were pre-incubated with different concentrations of PTS and then exposed to collagen, thrombin and ADP, respectively. As shown in Fig. μM. However, both Re and R1 only significantly inhibited the thrombin induced rabbit platelet aggregation at 100 μM within the same tested concentration range , we are then interested in the effect of these single compounds on rabbit platelet inhibition and asked whether there is a synergistic effect when they are used as a combination. As shown in Fig. nge Fig. . When thTo further confirm the anti-platelet aggregation effect of PTS and its main ginsenosides, we then determined their inhibitory effect on human platelet aggregation. As shown in Fig.2+]i is critical for the activation and aggregation of platelet aggregation [2+]i directly inhibits the platelet aggregation. Therefore, we determined the influence of PTS on the calcium mobilization. Figure 2+]i. When pre-incubation of platelets with various concentration of PTS, the [Ca2+]i induced by thrombin dose-dependently decreased by 58.2, 67.3 and 79.4 % at concentrations of 0.5, 1 and 3 mg · ml-1 (p < 0.05), respectively. Similarly, Fig. -1 of PTS significantly reduced collagen-activated [Ca2+]i and 3 mg · ml-1 of PTS significantly reduced ADP-activated [Ca2+]i.It’s well established that i plays an important role in the activation and aggregation of platelets and the thrombus formation. The exogenous or endogenous activation of a membrane receptor with thrombin, collagen or ADP leads to the influx of Ca2+ into the platelets i. While pre-treatment with PTS, the [Ca2+]i in all these agonists-activated platelets remarkably reduced, suggesting that inhibition of calcium mobilization may be one of the common signaling pathways responsible for the inhibitory effect of PTS against platelet aggregation stimulated by these three different agonists.[Calatelets , 21. Thelatelets , 22, 23.MAPKs including ERK2, JNK1 and p38 are present in platelets and activated by various agonists , 24. OurIn summary, we have investigated the potential effect of PTS on platelet aggregation and explored the underlying mechanisms in this study. PTS exhibited anti-platelet activity against stimulation of different agonists, including collagen, thrombin and ADP. The three major ginsenosides used alone also showed anti-platelet activity, whereas their combination didn’t improve the effect synergistically. Further mechanism study revealed that PTS significantly inhibited intracellular calcium mobilization induced by the three different agonists. Moreover, PTS also inhibited the phosphorylation of ERK2 and p38 induced by all the three agonists. Collectively, our study showed PTS has anti-platelet activity and inhibition of intracellular calcium mobilization and p-ERK2/p-p38 may, in part, be as the common signaling pathways responsible for this effect. The anti-platelet activity of PTS may be implicated in its beneficial effect on the prevention and treatment of ischemic stroke.Fmax, the fluorescence intensity levels at very high Ca2+ concentrations; Fmin, the fluorescence intensity levels at very low Ca2+ concentrations; GPCR, G-protein coupled seven transmembrane receptor; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP, horseradish peroxidase; HSP70, heat shock protein 70; IP3, inositol trisphosphate; Nrf2, nuclear factor (erythroid-derived 2)-like 2; OD, optical density; PAR, platelet aggregation rate; PIP2, phosphatidylinositol bisphosphate; PRP, platelet-rich plasma; PTS, panaxatriol saponin; PVDF, polyvinylidene difluoride; R1, notoginsenoside R1; Re, ginsenoside Re; Rg1, ginsenoside Rg1; SDS, sodium dodecyl sulfateADP, adenosine diphosphate; BSA, bovine serum albumin; ECL, enhanced chemiluminescence; EGTA, ethylene glycol tetraacetic acid;


Ebstein's anomaly is a congenital disorder characterized by apical displacement of the septal leaflet of the tricuspid valve. Ebstein's anomaly may be seen in association with other cardiac conditions, including patent foramen ovale, atrial septal defect, and left ventricular noncompaction (LVNC). LVNC is characterized by increased trabeculation within the left ventricular apex. Echocardiography is often used to diagnose LVNC; however, magnetic resonance (MR) imaging offers superior characterization of the myocardium. We report a case of sudden cardiac death in a patient with Ebstein's anomaly with unrecognized LVNC noted on post mortem examination with screening documenting the presence of LVNC in one of the patient's twin sons. Ebstein's anomaly is an uncommon congenital cardiac disorder characterized by apical displacement of the septal leaflet of the tricuspid valve. While Ebstein's anomaly may occur in isolation, it may also be associated with other cardiac conditions. While the presence of an interatrial communication is seen in more than 70% of patients, other associations include left ventricular noncompaction (LVNC) –3. LVNC We report a case of sudden cardiac death in a patient with Ebstein's anomaly with unrecognized LVNC noted on post mortem examination; subsequent family screening documented the presence of LVNC in one of the patient's twin sons. This case demonstrates the important role of MR imaging in patients with Ebstein's anomaly to document the presence of LVNC, highlights difficulties in stratifying risk for sudden cardiac death in patients with complex congenital heart disease, and demonstrates the potential utility of genetic testing.A 45-year-old male with a background of known Ebstein's anomaly died suddenly while at work. Ebstein's anomaly had been diagnosed during early childhood, with significant apical displacement of the tricuspid valve leaflet noted with associated tricuspid valve incompetence. The right atrium was markedly dilated with atrialisation of the right ventricle. The patient maintained a satisfactory exercise capacity until the age of 27 years. At that stage, the patient noted a degree of effort intolerance in the context of severe tricuspid valve regurgitation and intermittent complete AV block. There was no history of syncope or features of tachyarrhythmia. The patient then underwent tricuspid valve repair and permanent pacemaker implantation. The patient had an excellent surgical result with improved effort tolerance and mild to moderate residual tricuspid regurgitation. Over the following 14 years, the patient progressed appropriately with preserved exercise capacity, absence of symptoms of arrhythmia, and no clinical features of right heart failure. Echocardiography consistently demonstrated normal left ventricular size and systolic function. There was persisting right atrial and ventricular enlargement with mild to moderate tricuspid valve incompetence. At the time of review prior to death, the patient remained clinically well with no history of syncope or presyncope. Similarly, there was no history of palpitations. The previously inserted pacemaker had been explanted due to long term maintenance of sinus rhythm.Post mortem examination demonstrated evidence of previous tricuspid valve surgery with marked right atrial dilatation. The right ventricle was thin walled and dilated. The coronary arteries were normal. The left ventricle was mildly dilated with prominent trabeculation at the apex and posterolateral left ventricular wall segments suggestive of left ventricular noncompaction (LVNC) with accompanying fibrosis noted . No otheIn view of the previously undocumented presence of LVNC, the patient's twin sons underwent echocardiographic screening with one child demonstrating features of LVNC .This case outlines the importance of recognizing known disease associations in patients with complex congenital heart disease (CHD) and highlights current limitations in risk stratification for sudden cardiac death in CHD, given the patients preserved left ventricular function. Furthermore, given the limitations of echocardiography in documenting LVNC, there may be a role for routine screening of patients with Ebstein's anomaly with MR imaging to exclude LVNC. Finally, given the variable expression of genetic abnormalities associated with Ebstein's anomaly and LVNC, genetic screening may similarly be important in identifying subtle cardiac disease in relatives of patients with both Ebstein's anomaly and LVNC.LVNC is an uncommon disorder characterized by failure of usual myocardial development with the process of trabecular compaction incomplete. Improvements in imaging techniques have permitted increased recognition of LVNC and allowed for refined diagnostic criteria, utilizing both echocardiography and cardiac MR imaging. The spectrum of disease complicating LVNC is broad, ranging from asymptomatic to severe left ventricular dysfunction with concomitant risks of arrhythmia, systemic embolization, and sudden cardiac death. Predictors of an adverse outcome include earlier age at presentation, impaired functional class, history of ventricular arrhythmia, left ventricular dilatation, and abnormal tissue Doppler parameters . LVNC maThe diagnostic criteria for LVNC have undergone evolution with the advent of MR imaging and improved 2D echocardiography imaging quality and are based on the extent and depth of trabeculation within the left ventricle. MR imaging provides superior myocardial characterization and endocardial definition compared to echocardiography and is a more sensitive imaging modality in detecting LVNC. Current guidelines do not advocate routine MR imaging in patients with Ebstein's anomaly; however, in the setting of a family history of sudden death or in cases where there are concerns regarding left ventricular size, appearance, or function, MR imaging may provide valuable additional information. Given the risk of arrhythmia seen in this case, despite preserved ventricular function, there may be a role for routine screening to exclude LVNC. In this case, the patient has undergone serial echocardiography, with no significant left ventricular abnormalities noted. This reflects the limitations of echocardiography in identifying subtle changes in left ventricular myocardial morphology and highlights the important role of MR imaging in diagnosis.The association between Ebstein's anomaly and LVNC may be related to mutations in the sarcomere gene MYH7. The combination of Ebstein's anomaly and LVNC should prompt formal genetic assessment as well as screening in relatives. As documented in this case, and in previous series, LVNC can manifest without abnormalities of the tricuspid valve.This case highlights the difficulties seen in complex congenital heart disease with regard to issues such as risk stratification for sudden cardiac death, the role of advanced imaging modalities such as MR imaging, genetic testing, and screening of family members. The association between Ebstein's anomaly and LVNC is well established with specific genes implicated, noting incomplete penetrance and variable phenotype. Routine MR imaging of patients with Ebstein's anomaly, and potentially their relatives, may be of value in detecting subtle features of LVNC, which in turn has implications in terms of prognosis.


Background: Surgical treatments for adult spinal deformities often include pelvic fixation, and the feasibility of sacral-2 alar iliac (S2AI) screw fixation has been shown previously. However, sometimes S2AI screw fixation cannot be applied due to the presence of an osteolytic lesion or trauma or because the biomechanical properties of only an S2AI screw is insufficient. Therefore, we questioned the feasibility of using sacral AI screws in other segments and determined whether S3AI and S4AI screws have the potential to be used for sacral fractures. The aim of this study was to investigate the feasibility and radiological features of sacral AI fixation in S1–S4 in an adult population using 3D imaging techniques. Methods: Computed tomography (CT) scans were taken of 45 patients and were imported into Mimics  software to reconstruct the 3D digital images. Next, a cylinder (radius of 3.5 mm) was drawn to imitate the screw trajectory of a S1–4 AI screw, and every imitated screw in each segment was adjusted to a maximum upward and downward angle to acquire the feasible region. The parameters of the S1–4AI screw trajectories were measured. Results: Sacral AI screws could be successfully imitated using 3D digital imaging. The S4AI screw trajectory could be obtained in 19 of 45 patient images (42.2%), while the feasibility rates of S1AI, S2AI, and S3AI screw fixation were 100%, 100%, and 91.1% (41/45), respectively. The feasible regions of S1AI, S2AI, and S3AI screw trajectories were wide enough, while the adjustable angle of S4AI screws was very small. Conclusion: It is feasible to place S1–2AI screws in the entire adult population and S3–4AI screws in some of the adult population. Furthermore, our study suggested that 3D digital images are suitable to study the feasibility of new screw fixation. Surgical treatments for adult spinal deformity often include pelvic fixation, and the high rate of pseudarthrosis in distal fixation in spinal deformity is a challenge for spinal surgeons . A novelHowever, sometimes the S2AI screw cannot be applied due to an osteolytic lesion or trauma  or becauIn this study, we used Mimics 3D imaging software  to investigate the feasibility of S1–4AI screw fixation. This software has previously been shown to provide an accurate visual screw trajectory .This research was performed following the principles described in the Declaration of Helsinki and was approved by the Institutional Ethics Review Board of the Second Affiliated Hospital of Wenzhou Medical University (No. 2015–30). Written informed consent was obtained from all participants.Computed tomography (CT) scans (Dicom format) were taken of the pelvic regions of 45 patients and were imported into Mimics software  for reconstructing the 3D digital images , which oα, the angle between the screw trajectory and the erect line from the anteroposterior view  β, ral view ; and (3)the bone . MoreoveSacral AI screws could be successfully imitated by 3D digital imaging, the ICC between the data from two surgeons was 0.893, which is >0.800, means the data measured by two surgeons was strongly consistent to each other.By observing the 3D images, we found that the ideal screw entry point was at the inner side of the transverse process of the sacrum from S1–S4  and thatWe found that from S1AI to S4AI, the α angle of maximum upward screw trajectory  was gradIn this study, we determined the feasibility of S1AI screw fixation as well as S2AI, S3AI, and S4AI screw fixation and found that they were all inserted successfully in the adult population. By using the “Cut,” “Split,” and “Reposition” functions in the Mimics software, we could observe the screw trajectory clearly . We founEvery sacral AI screw trajectory in each segment had a maximum upward angle and a maximum downward angle. If the screw trajectory overstepped the feasible region between the maximum upward angle and the maximum downward angle, the screw will pierce the cortex of the iliac thin region  or the cAs shown in In this study, more than in previous S2AI screw fixation , we provFirstly, the spine fixation may be extended to pelvic fixation, in S1, surgeons prefer to use an S1 pedicle screw. However, sometimes, the normal osseous structure is destroyed by a lytic lesion or trauma  or the aSecondly, patients with a sacral fracture as shown in Therefore, studying the feasibility of sacral AI screw fixation (including S3–4AI screws) has value, and the technique of sacral AI screw fixation has great potential value in clinical applications as it is theoretically better than traditional pedicle screw fixation and the sacropelvic fixation technique .This is a 3D image study without clinical cases. In a 3D image study, we need some reference line to measure the angles of simulated trajectory. To determine the reference line, we adjusted the 3D image to simulate the patient is left-right symmetry stand position. Then, the erect line was made as the reference line; therefore, this line was subjective, and this is one of the limitation of our this study. In clinical cases, the position was also determined by surgeons subjectively; the angle will be a little different among different surgeons, but this will not influence the feasibility of S1–4 AI Screw fixation on patients. Meanwhile, because of individual variation, we strongly recommend that a preoperative CT scan is obtained and that a detailed preoperative plan is made before sacral AI fixation is applied. Moreover, 3D digital images were suitable to study the feasibility of new screw fixation and could help surgeons to explore the trajectory of screw fixation readily.10.7717/peerj.1587/supp-1Supplemental Information 1The raw data from S1-AI to S40AI.Click here for additional data file.


Real-time three-dimensional echocardiography (RT3DE) provides a more naive realism, which states that the way we observe the world is the way the world actually is. The Necker cube contradicts this claim, because we see one or the other of two cubes  are considered more accurate and better reproducible. However, because of the geometric assumptions of this 2D method, measurements may be inaccurate if the shape of the left ventricle is abnormal or when the acquisition of the 2D images is suboptimal. In other words, like the yellow flower, 2D echocardiographic images are not necessarily a realistic representation of the true left ventricle, but only a perception that is influenced by subjective interpretation and variability in measurements of only a very small 2D portion of the true 3D volume.Let us go back to echocardiography. Visual estimation of wall motion  is highly subjective, imprecise and poorly reproducible. Nevertheless, some sense of The advent of matrix transducers, together with impressive improvements in semi-automated volumetric analysis, have allowed 3D echocardiography to evolve from a complicated and time-consuming research tool into a simple and fast imaging modality ready for everyday clinical use. RT3DE has been extensively demonstrated to be more time-saving, reproducible and accurate than conventional 2D echocardiography [Although contemporary RT3DE analysis software allows assessment of LV volumes and EF in a rather fast and simple way, a certain learning curve still needs to be taken into account. Driessen et al. found that RT3DE software was most accurate in the hands of more experienced observers. Observers were regarded more experienced when they had been working on RT3DE analyses for more than 3 months. Noteworthy, even after 3 months of working on RT3DE, increasing experience may still lead to better results, as shown in one of the landmark papers with respect to quantification of LV volumes by RT3DE . The levRT3DE provides LV volumes smaller than those derived from CMR. CMR is considered the reference technique for assessment of ventricular volumes. However, there are intrinsic limitations of CMR as a reference technique because of its multislice rather than true 3D nature.Usually, as in the study by Driessen et al., CMR-derived volumes are calculated by a disc summation technique . Yet, with in vitro measurements, it was shown that this disc summation may result in an overestimation of 20 % of the true volume. A volumetric 3D CMR analysis resulted in more accurate measurements and RT3DE only slightly underestimated true volumes .The differences between LV volume analysis by RT3DE and CMR have also been attributed to the sometimes insufficient image quality of RT3DE. Blurring of the endocardium may cause difficulty in clearly identifying the endocardial-trabecular border. As a result, with manual tracing of RT3DE images, the human eye tends to trace the inner border of the blurred rim of the left ventricle, which may be the blood-trabecular interface rather than the true endocardial surface. On the other hand, partial volume effects caused by the relatively thick slices in CMR result in the appearance of a fairly smooth surface of the endocardium, which may also be a false representation of the truth.Nevertheless, both the direct 3D volumetric echocardiographic method by Philips and the 3D speckle tracking echocardiographic method by Toshiba, used in the study by Driessen et al., use automated systems to detect the endocardium. This line is supposed to be closer to the centre of the blurred area and these mathematical methods may happen to include more cavity than a manual tracking method, resulting in less underestimation.naive realism of believing everything we measure using 2D echocardiographic images. A sense of representative realism seems to be a welcome alternative to the naive realism of confidence in the accuracy of our eyes and 2D measurements. In my opinion, especially in case of good image quality, RT3DE should be the preferred method for measurement of LV volumes and EF.There is room for improvement. Accurate LV quantification by RT3DE can only be performed using good image quality datasets, usually obtained in 80–85 % of routine patients, a number also found in the ‘daily clinical practice study’ by Driessen et al. Also, regular heart rhythm  and patient cooperation for breath holding are still indispensable. And finally, the relatively low temporal resolution of RT3DE limits the assessment of regional wall motion during exercise and dobutamine stress echocardiography . Neverth


Allosteric partial inhibition of soluble, monomeric proteases can offer major regulatory advantages, but remains a concept on paper to date; although it has been routinely documented for receptors and oligomeric proteins. Thrombin, a key protease of the coagulation cascade, displays significant conformational plasticity, which presents an attractive opportunity to discover small molecule probes that induce sub-maximal allosteric inhibition. We synthesized a focused library of some 36 sulfated coumarins to discover two agents that display sub-maximal efficacy (~50%), high potency (<500 nM) and high selectivity for thrombin (>150-fold). Michaelis-Menten, competitive inhibition, and site-directed mutagenesis studies identified exosite 2 as the site of binding for the most potent sulfated coumarin. Stern-Volmer quenching of active site-labeled fluorophore suggested that the allosteric regulators induce intermediate structural changes in the active site as compared to those that display ~80–100% efficacy. Antithrombin inactivation of thrombin was impaired in the presence of the sulfated coumarins suggesting that allosteric partial inhibition arises from catalytic dysfunction of the active site. Overall, sulfated coumarins represent first-in-class, sub-maximal inhibitors of thrombin. The probes establish the concept of allosteric partial inhibition of soluble, monomeric proteins. This concept may lead to a new class of anticoagulants that are completely devoid of bleeding. Allosterism induced by small or large molecule effectors is increasingly being perceived as a new paradigm to understand molecular pathways and discover new therapeutics1123457191011IC50) and efficacy (% ΔY) – for regulation of activity, whereas orthostery presents only one, i.e., potency, for regulation  and afford the possibility of better regulation of the protease. For example, current orthosteric inhibitors of thrombin  inhibit thrombin with maximal efficacy (ΔY = 100%), which contributes to enhanced risk of bleeding23We have earlier studied a number of sulfated non-saccharide glycosaminoglycan allosteric modulators (NSGMs) of thrombin including sulfated benzofurans26272830In our quest to discover allosteric partial regulators, especially of thrombin, we decided to study the sulfated coumarin scaffold. Our earlier work on designing/discovering allosteric modulators of thrombin led to the sulfated quinazolinone and benzofuran scaffolds, which exhibited allosterism, but induced full inhibition (ΔY = 80–100%) at saturation2627281a–1d and 2a–2v  led to synthesis of head-to-head dimers 3a–3f in yields of 72–90%  was employed, in which selective Sf 72–90% . The strf 72–90% . Likewist yields . The heah 2D NMR . Overallh 2D NMR .This is the first synthesis of a NSGM library based on the coumarin scaffold. The 36 member library contained different locations of the 1 or 2 sulfate group(s), different types of substitutions , different size of molecules (monomer or dimer), different linker length (n = 3 or 4), and different geometry of constituent units (“head-to-head” or “head-to-tail”) see . Thus, aIC50), efficacy of inhibition (∆Y) and Hill slope (HS) for each molecule calculated through non-linear regression analysis , in contrast to nearly all dimeric sulfated coumarins that displayed good inhibition potencies (IC50 < 20 μM). Only those monomeric NSGMs that contained an aromatic group at the 4-position were active, e.g., 2e, 2k and 2p, but not all 4-aryl derivatives were active, e.g., 2d, 2l, 2o, 2w, 2x and 2y  using appropriate chromogenic substrates, as described earlier2933analysis . In termd 2y see . One or 3a–3f, which contained sulfation at the 4′-hydroxyphenyl position (see 3a and 3b were most potent (IC50 ≤ 1 μM). Interestingly, 3a and 3b, and not any other dimer, contain 4-phenyl substitution, which mimics the structure–activity relationship observed for monomers. To assess whether an alternative analog would yield similar activity, we synthesized 3g  human thrombin and followed its fluorescence as a function of 3g levels, as also performed for other sulfated ligands of coagulation factors37KD of 143 ± 14 nM and ΔFMAX of ~28%. Thus, the thermodynamic affinity of 3g is in the same range as its inhibition potency. Finally, inhibitor 3g also displays excellent selectivity for thrombin in comparison to that for factor XIa (~150-fold) and factor Xa (~800-fold) (To characterize 00-fold)  highligh3g was not the most interesting discovery from this study. It was the variable efficacy (ΔY) of thrombin inhibition. All of sulfated coumarins assayed displayed ΔYs ranging from ~20–70% (3g displayed an efficacy of 47% and a Hill slope of 1.2. Other reasonably potent agents displayed ∆Y of 73% (3a), 58% (3b) and 60% (3c). In contrast, sulfated benzofurans and sulfated quinazolinones studied earlier display >80% inhibition2728303340The high potency or selectivity of inhibition noted for  ~20–70% , indicat3g, we performed Michaelis-Menten kinetic studies using Spectrozyme TH as the substrate. 3g. As the concentration of 3g increased, the VMAX decreased from 57.5 to 31.0 mAU/min, whereas the KM remained relatively constant -(SO3−)) for exosite 1 and UFH for exosite 2, the catalytic activity of thrombin was measured at varying concentrations of 3g  increased by a small factor (<1.4-fold) , of which R93A displayed a dramatic 2.3-fold effect. This is an unusual observation and opposes the expectation of a loss in potency. Yet, an analysis of thrombin crystal structure shows that a majority of these residues are located adjacent to hydrophobic sub-sites within/near exosite 2503g’s aliphatic chain and/or aromatic rings resulting in enhancement of potency. This appears to be especially true for the R93A and R101A mutants, and possibly also for others. Earlier work on sulfated low molecular weight lignins has also shown the importance of hydrophobicity in recognition of exosite 2 and support the conclusion that 3g binds in exosite 2 through primarily non-ionic forcesThe inhibition potency of 3g recognition. It is possible that other Arg/Lys, such as R240, K107, K109, and K110 located within or adjacent to exosite 2, may be involved in interacting with the sulfate group of 3g. It is also possible that the 3g interacts with multiple Arg/Lys groups that are near neighbors, e.g., R240, K236, and K235, such that replacement of one does not adversely impact its affinity significantly. This possibility has previously been documented for the thrombin–low molecular weight lignin system and factor XIa–sulfated quinazolinone system, where single mutants showed much weaker changes in activity as compared to triple mutants333g utilizes hydrophobic forces in binding and that one or more Arg/Lys residue within or adjacent to exosite 2 may be the target of its sulfate group.It is not clear why a specific electropositive amino acid residue was not identified as the primary site of 3g induces maximal inhibition efficacies in the range of 28 to 64%, which are comparable to that measured for sulfated coumarins inhibiting plasma thrombin. The retention of the partial inhibition phenomenon by recombinant thrombins shows that changes in the electrostatics of exosite 2 does not impact the intrinsic property of sulfated coumarins, especially 3g.The study of recombinant thrombins also enables further confirmation of the partial inhibition phenomenon. 3g introduces conformational or dynamic allostery.In principle, allosterism may arise from conformational changes or dynamic changes introduced by the allosteric ligand. Conformational changes imply structural changes in the target macromolecule52545511213g and a previously studied sulfated benzofuran trimerBT), which displays inhibition efficacy >80%. Iodide anion is a well-established agent that is known to quench fluorescence through collisional interactions3g induces less conformational changes in the protein in comparison to BT, then access of an active site fluorophore to the iodide anion should be different. Thus, quenching of fluoresceinylated thrombin, i.e., f FPRCK-thrombin, by iodide was studied in the presence and absence of 3g and BT, respectively  and to 0.69 ± 0.02 M−1 in the presence of BT (90% saturation). Thus, 3g reduces iodide quenching of f FPRCK-thrombin by ~50%, while BT reduces by ~71%.To elucidate the nature of allostery, two orthogonal experiments were performed. First, fluorescence-quenching studies were performed on thrombin complexes with ectively  and analectively . The res3g, but not BT, induces partial conformational change in thrombin, a remote possibility of differential quenching by iodide anion is physical shielding by 3g, but not BT. Both 3g and BT bind in exosite 23g binding affinity correlates well with its IC50. If any part of 3g structure was located at the S4 site, the affinity should have been lower. Thus, the results suggest that the observed efficacy (ΔY) of inhibition correlates with the ability of iodide anion to access to the active site fluorescein label, which is modulated by the allosteric inhibitor. Alternatively, 3g induces a much smaller change in the orthosteric site in comparison to that of BT, which suggests a structural basis for its allosterism.Although the above results indicate that 3g indeed induces changes in the orthosteric site, we studied the kinetics of the thrombin–antithrombin reaction in the presence of 3g using a discontinuous spectrophotometric assay, as described earlier593g levels under pseudo-first order conditions, which were analyzed to derive the observed rate constant (kOBS). The intrinsic rate constant of antithrombin inhibition of thrombin, i.e., kINT, which is proportional to the concentration of antithrombin, was calculated from kOBS using kINTversus percent saturation of thrombin with 3g . Therefore, 3g inhibits thrombin by inducing a partial structural change in its active site.To further probe whether  with 3g  shows anhibition . This deThis work presents the first group of small molecules that induce partial allosteric inhibition in a monomeric protease. Mechanistically, this is very interesting and shows that it is possible to regulate monomeric proteases in a manner similar to multimeric proteins and receptors. Our work also shows that partial inhibition arises from a structural change in the catalytic site, just as partial agonism or antagonism is also associated with structural change at the distal site23g, are able to bring about a limited conformational change in the active site of the thrombin, so as to still allow room for binding and cleavage of small substrates. Whether this phenomenon holds true for macromolecular substrates of thrombin19202324Sulfated coumarins, especially 3g selects out one of the conformations from the ensemble present in its native state or induces a new form remains to be determined. Advanced stopped-flow kinetic studies, such as performed for thrombin – hirudin systemOur work adds further support to conclusions derived in the literature that thrombin is a highly plastic enzyme11213g as the most potent small molecule exosite 2 inhibitor of thrombin discovered to date. The sulfated benzofurans developed earlier display potencies in the range of 0.7–200 μM27285050)30363g could be classified as a good scaffold for the discovery of better partial allosteric regulators of hemostasis.In addition to the partial allosteric features, our work presents Overall, this initial discovery is extremely promising from the perspective of pursuing partial allosterism as a novel mechanism for regulators of monomeric proteases. It is possible that partial allosterism of monomeric proteases is a more general phenomenon and further effort will help identify ‘regulators’, rather than ‘inhibitors’, that lead to homeostasis.d6 and DMSO-d6 for NMR were from Sigma Aldrich  or Acros Organics . Chemical reagents including BBr3, SO3•N(CH3)3, Cs2CO3, CuSO4•5H2O, sodium ascorbate, triethylamine (TEA), propargyl bromide, 1-bromo-3-chloropropane, 1-bromo-4-chlorobutane, sodium azide, and TBTA were from Sigma Aldrich , Fisher Scientific , or Acros Organics  and used as purchased. Coumarins were from Indofine Chemical Company Inc. . All other chemicals were analytical reagent grade or higher and obtained from Sigma Aldrich  or Fisher Scientific .Anhydrous and chromatography solvents were from Sigma Aldrich  or Fisher Scientific . Acetone-f FPRCK-)thrombin, antithrombin, factor Xa and factor XIa were from Haematologic Technologies . Recombinant thrombins were gifts from the Rezaie lab (St. Louis University). Spectrozyme TH  and Spectrozyme FXa (methoxycarbonyl-D-cyclohexylglycyl-Gly-Arg-p-nitroanilide) were from Sekisui Diagnostics . S-2366 (L-pyroGlu-Pro-Arg-p-nitroaniline•HCl) was from Diapharma . Bovine unfractionated heparin was from Sigma Aldrich and hirudin peptide -(SO3−)) was from Anaspec .Human plasma proteases including thrombin, fluorescein-labeled  PEG 8000, pH 7.4) was added to each well followed by 5 μL of 240 nM thrombin and 10 μL of sulfated coumarin in DMSO to give 0.002 to 250 μM final concentration . After incubation for 10 min at 25 °C, 5 μL of 2 mM Spectrozyme TH was added to each well simultaneously and allowed to react for 60 seconds at 25 °C while simultaneously monitoring the reaction to obtain the rate of increase in A405. Identical experimental conditions were used for reactions in the presence of antithrombin, except for 100 nM of antithrombin added prior to the addition of the substrate.Chromogenic substrate hydrolysis assay was used to measure potency of inhibition, as previously reported282, 0.1% PEG 8000, 0.02% Tween 80, pH 7.4) was added to each well followed by 5 μL of 43.5 nM enzyme, 10 μL of sulfated coumarin . After a 10 min incubation, 5 μL of 5 mM Spectrozyme FXa was added to each well simultaneously and allowed to react for 300 seconds at 37 °C. The slopes generated from each experiment were used to calculate the fractional residual activity (Y) at each concentration of the inhibitor, and analyzed using logistic YM is the maximum efficacy, Y0 is minimum efficacy and HS is the Hill slope.For factor XIa inhibition studies, 85 μL of buffer  was added to each well, followed by 7 μL of 15.3 nM enzyme and 3 μL of sulfated coumarin  and incubated for 10 min at 37 °C. After the incubation period, 5 μL of 6.9 mM S-2366 was added to each well simultaneously and allowed to react for 300 sec at 37 °C. For factor Xa assay, 180 μL of buffer  at 25 °C. Spectrozyme TH at various concentrations (0–250 μM) was added at each of the four 3g concentrations and the initial rate of hydrolysis was recorded from the increase in A405 as a function of time. This slope was fitted to the standard Michaelis-Menten equation to derive VMAX and KM of the reaction at each of the 3g concentrations.Four concentrations  of 3g was examined in the presence of either HirP (exosite 1 ligand) or UFH (exosite 2 ligand). 3g was prepared in DMSO (0.010–50 μM) and thrombin (4–8 nM) were incubated at 25 °C with HirP (0–187 nM) or UFH (0–22.8 μM) in buffer . After 10 min, thrombin activity was measured using Spectrozyme TH hydrolysis assay, as described above. The slopes obtained (in triplicate) were fitted using IC50,app, ∆Y, and HS. The IC50,app at each concentration of the competitor was also calculated using the Dixon-Webb relationship (Kcomp) of the competitor.Thrombin inhibition by tionship  from the2, 0.1% PEG 8000, pH 7.4) and 20 μL of 2 μM fluorescein-labeled (f FPRCK-) thrombin were added to the cuvette followed by titration with 1 μL additions of 3g. Fluorescence intensities were monitored by excitation at 490 nm and emission at 525 nm using slit widths of 0.5 mm. The KD of 3g–thrombin complex was calculated using the quadratic binding equation . The binding was assumed to possess a stoichiometry of 1:1.Affinity studies were performed in triplicate and conducted using quartz cuvettes. 180 μL of buffer  and 20 μL of 2 μM fluorescein-labeled (f FPRCK-) human thrombin were added to the cuvette; fluorescent intensity readings were taken at the λEX of 490 nm and λEM of 525 nM at 25 °C. A solution of 10 M NaI was made using buffer as the solvent. For the readings where inhibitor was absent, 1 μL increments of 10 M NaI was added directly to the cuvette after initial readings of the buffer-thrombin solution. NaI was added six times, while recording changes in fluorescent intensity each time, giving a concentration range of 0–3.0 M in the cuvette. For the readings where inhibitor was present, after the initial buffer-thrombin read, 1 μL of 80 μM inhibitor solution was added to the cuvette and mixed. An intensity reading was taken and then the addition of 1 μL increments of 10 M NaI was again performed six times, taking reads between each addition. The experiments were performed in triplicate and averaged. Initial fluorescence (F0) was the intensity reading from either the buffer-thrombin solution in the absence of 3g or following addition of 1 μL of 3g. Linear regression was performed using Stern-Volmer relationship  with antithrombin was studied under pseudo-first order conditions such that [AT]0 ≫ [T]0. A fixed concentration of 6 nM plasma α-thrombin in 20 mM Tris-HCl buffer, pH 7.2, containing 100 mM NaCl, 2.5 mM CaCl2 and 0.1% PEG 8000 at 25 °C was incubated for 128 min with final concentrations of 0, 75, 225, or 675 nM of 3g following which a fixed concentration of 100 nM of antithrombin was added and the reaction allowed to proceed. Similarly, 5 nM of plasma factor Xa in 20 mM Tris-HCl buffer, pH 7.2, containing 100 mM NaCl, 2.5 mM CaCl2 and 0.1% PEG 8000 at 25 °C was incubated for 210 min with final concentrations of 0 or 220 nM of 3g following addition of a fixed concentration of 100 nM of antithrombin was added and the reaction allowed to proceed. At a defined time point, a small aliquot of Spectrozyme TH was added to a final concentration of 50 μM for thrombin and Spectrozyme Xa a final concentration of 150 μM for factor Xa. The initial rate of hydrolysis of the Spectrozymes were monitored from the linear increase in A405. The fractional residual enzyme activity at each time point was calculated from the slope, i.e., thrombin activity, measured at the start of the experiment and fitted by the standard exponential decay kOBS, at each concentration of 3g. The intrinsic second-order rate constant of antithrombin inhibition of thrombin (kINT) was calculated using 3g complex, obtained from quadratic kINT of antithrombin inhibition of thrombin–3g complex.The effect of How to cite this article: Verespy III, S. et al. Allosteric Partial Inhibition of Monomeric Proteases. Sulfated Coumarins Induce Regulation, not just Inhibition, of Thrombin. Sci. Rep.6, 24043; doi: 10.1038/srep24043 (2016).


Thermotoga maritima, enables the robust enzymatic production of large quantities of c-di-GMP. To understand the structural correlates of tDGC thermostability, its catalytic mechanism and feedback inhibition, we determined structures of an active-like dimeric conformation with both active (A) sites facing each other and of an inactive dimeric conformation, locked by c-di-GMP bound at the inhibitory (I) site. We also report the structure of a single mutant of tDGC, with the R158A mutation at the I-site, abolishing product inhibition and unproductive dimerization. A comparison with structurally characterized DGC homologues from mesophiles reveals the presence of a higher number of salt bridges in the hyperthermophile enzyme tDGC. Denaturation experiments of mutants disrupting in turn each of the salt bridges unique to tDGC identified three salt-bridges critical to confer thermostability.Large-scale production of bis-3′-5′-cyclic-di-GMP (c-di-GMP) would facilitate biological studies of numerous bacterial signaling pathways and phenotypes controlled by this second messenger molecule, such as virulence and biofilm formation. C-di-GMP constitutes also a potentially interesting molecule as a vaccine adjuvant. Even though chemical synthesis of c-di-GMP can be done, the yields are incompatible with mass-production. tDGC, a stand-alone diguanylate cyclase (DGC or GGDEF domain) from  Gluconobacter xylinusBis-(3′-5′) cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that was initially discovered as an allosteric activator of cellulose synthase in Caulobacter crescentus, DgcZ Escherichia coli K-12 and WspR Pseudomonas aeruginosa and Pseudomonas syringae. Isolated GGDEF domains structurally characterized include YfiN from Pseudomonas aeruginosaXanthomonas campestrisE. coli where binding of Zinc to the N-terminal CZB domain induces an inactive dimeric arrangement of the GGDEF domains 4. Thus, two mechanisms can chiefly account for the feedback inhibition of DGCs mediated by the c-di-GMP product: allostery Marinobacter aquaeoliCatalytically active DGCs bear the GG(D/E)EF consensus amino-acid sequence at their active site (A-site) and a conserved RxxD motif at an inhibitory site (I-site) et al.Thermotoga maritima, hereafter named tDGC, that enables robust enzymatic production of large quantities of c-di-GMP. Enzyme assays showed that tDGC is active even after days of incubation at 30°C but with very low product production, irrespective of substrate concentration, due to feedback inhibition. However, a mutation (R158A) at the I-site of tDGC significantly alleviates production inhibition, enabling synthesis of several hundred milligrams of c-di-GMP with just 10 mg of the mutant enzyme Large-scale production of c-di-GMP would facilitate biological studies of several bacterial signaling pathways and phenotypes controlled by this molecule. Moreover, the immunomodulatory properties of c-di-GMP open up the possibility of using it as a vaccine adjuvant In order to understand the structural determinants of tDGC thermostability and to investigate the catalytic mechanism and feedback inhibition of this biotechnologically important enzyme, we crystallized tDGC (i) in an active-like conformation with two A-sites in proximity, and (ii) in an inactive conformation, with c-di-GMP bound to the I-site. A comparison between these two structures enables us to examine potential conformational changes propagated between the I- and A- site upon c-diGMP binding to the I-site. We also report the structure of the R158A mutant of tDGC. Structural comparison with DGC homologues from mesophilic bacteria reveals the presence of a higher number of salt bridges in tDGC. Using circular dichroism (CD), three key salt bridges responsible for the enhanced thermostability of tDGC were identified.Thermotoga maritima, with codons optimized for bacterial expression, was purchased from Genscript. The gene corresponding to residues 82–248  was cloned into the pET28b vector between Nde1 and Xho1 restriction sites). The R158A feedback inhibition null mutant was purchased from Genscript. All proteins containing six Histidine residues at their N-terminus were overexpressed in E. coli BL21 (DE3) cells by the addition of 0.8 mM IPTG at 37°C for 4 hours. The tDGC protein was initially purified by immobilized metal affinity chromatography (IMAC) using Ni-NTA agarose (Qiagen) and further purified by ion exchange using a HiTrap column (GE Lifesciences) and size exclusion on a Superdex 75 column (GE Lifesciences). The RocR PDE was expressed and purified as described 2. The mixture of RocR, tDGC and pGpG was separated by gel filtration. The identity of the eluted pGpG was confirmed by mass spectrometry.The gene encoding the complete (248 amino-acids) wild type protein TM1788 (Uniprot: Q9X2A8) from Purified tDGC was concentrated to 10 mg/ml and its homogeneity assessed by dynamic light scattering on a DynaPro instrument. Initial crystallization screening was performed by sitting-drop vapor diffusion at 18°C, using commercial crystallization screens from Hampton Research . Intelli 96–3 wells sitting drop plates were used to test three precipitant:protein ratios  using a Phoenix crystallization robot (Art Robbins Instruments). Crystals of the inactive dimer were obtained with 0.1 M monobasic sodium phosphate monohydrate, 0.1 M monobasic potassium phosphate, 0.1 M MES monohydrate (pH 6.5), 2 M NaCl. The R158A mutant was crystallized with 2 M NaCl, 10% (w/v) PEG 6000. The active-like dimer was crystallized with 0.15 M lithium sulfate, 0.1 M citric acid (pH 4.0), 10% (w/v) PEG 6000. The protein used to obtain the latter crystal form was also the c-di-GMP-locked inactive dimer. However, upon mixing with precipitant, the crystallization drops turn milky white, indicating protein precipitation. Upon overnight incubation at 18°C, the crystallization drops become clear again and crystal formation is observed after 4–5 days. Thus, the active-like dimer is likely to originate from a rearrangement following initial protein precipitation.The diffraction properties of these crystals were improved by overnight soaking in a precipitating solution containing 14% (w/v) PEG 6000 prior to flash-freezing in liquid nitrogen, extending the diffraction limits from 3.5 Å to 1.9 Å.Methylococcus capcsulatus (PDB code: 3ICL), which has 40% amino-acid sequence identity with tDGC, as a search probe. The structure was built by iterative cycles of model building at the computer graphics using COOT www.rcsb.org). Buried solvent accessible surface areas upon dimer formation were calculated using the PDBePISA server  and X-ray diffraction data were collected at SLS  and processed using iMosflm GCC ATG AAA GAA GAA TATACC ATT CTG CTG TAT  3′ , 5′ GCC CCG GTG CGT GTG GAACTG AGC ACC TTT CGT  3′ (for E196A) and 5′ GAAGC TAT GTA TAA AGC CAA AG AGT GGC GGA TAT G 3′ (for R233A) along with the respective complementary primers were used. The mutations were confirmed by sequencing and the constructs were overexpressed and purified following the same protocol that was used for the wild type tDGC protein.For thermostability analysis, point mutations were introduced into the wild type tDGC GGDEF domain in the pET22b vector. The forward primers: 5′ 2 were used. The temperature was gradually increased from 25 to 95°C at the rate of 1°C/min and ellipticity was recorded at a wavelength of 220 nm for every 0.2°C increment in temperature. The melting temperature was determined by plotting the CD data at 220 nm against temperature and fitting to a Boltzmann sigmoidal curve (between 60 and 95°C) using the curve fitting software XL Fit4.Circular dichroism (CD) measurements were performed using a Jasco spectropolarimeter (J-810) with a 1 mm path length quartz cuvette. To determine the melting temperature, 200 µl protein samples at a concentration of 0.8 mg/ml, in 20 mM Tris-HCl (pH 8.5), 250 mM NaCl, 5% (v/v) glycerol, 10 mM MgClThermotoga maritima  spanning residues 82–248, hereafter named tDGC. The structures of tDGC and of its R158A mutant were determined in three crystal forms (i) tDGC in an active-like dimeric conformation (ii) tDGC in an inactive dimeric conformation (iii) R158A mutant in a monomeric conformation ( and ). The location of the conserved catalytic 167-GGDEF-171 motif (A-site) and the inhibitory I-site (158-RxxD-161) on loops connecting β2 and β3 and between α3 and β2 respectively, are depicted in ). The four residues responsible for coordinating the Mg2+ ion (Asp126 and Asp169) and for making contact with the guanine moiety (Asn134 and Asp143) are strictly conserved in tDGC, as shown in We expressed and crystallized a domain of the TM1788 protein from ion  and 2. The and2 and their interface is stabilized by one salt bridge and thirteen hydrogen bonds. I-site residues 158-RxxD-161 from the two monomers interact with two molecules of c-di-GMP that are mutually intercalated . However, disruption of the other two salt bridges, K118-D177 and D219-R233, lead to large reductions in the melting temperature of tDGC by 12.3°C and 8.6°C, respectively Caulobacter crescentus (2V0N)7, WspR from Pseudomonas syringae (3I5A) Pseudomonas aeruginosa (3BRE) We report the structure of tDGC from Figure S1 in Based on simulations of bound and free forms of the GGDEF domains, it was previously proposed that product binding at the I-site could induce rearrangements of A-site residues, through a allosteric signal transmitted between these two distant sites Using the tDGC active-like dimer as a guide, we were also able to propose a 2-fold symmetrical dimer conducive to GTP cyclization with a nucleophilic attack by the 3′ oxygen groups of the ribose. Further structural and enzymatic studies are now needed to confirm this model.Thermotoga maritima with protein homologues from mesophilic organisms showed an increased number of salt bridges and compactness of the protein Thermotoga maritima have a higher “contact order”  compared to their mesophilic counterparts m ca 85.0°C), indicating that the nucleotide-mediated dimerization has minimal influence on protein thermostability. The disruption of salt bridges K118-D177 and D219-R233 decreases Tm by 12.3 and 8.6°C, respectively. Interestingly, residues D177 and D219 belong to loop regions of tDGC, suggesting a greater contribution to thermostability of salt bridges located in flexible regions of the protein. In contrast, salt bridge R152-E196, which connects alpha helices α3 and α4 has a more modest influence on thermostability (Tm decrease of 4.2°C). In addition to salt-bridges, a contribution to the thermostability of tDGC could derive from a larger number of hydrophobic interactions Although various structural features could account for thermostability, a study that compared 94 proteins structures from the hyperthermophile In conclusion, our study identified a dimeric active-like conformation for a thermostable GGDEF domain, where the relative orientation of both active sites is compatible with c-di-GMP formation. The R158A mutation in the I-site abolishes feedback inhibition by preventing formation of the inactive c-di-GMP-locked dimer. Our work also points to a few structural features accounting for enhanced thermostability of tDGC including the formation of three surface exposed unique salt bridges. Point mutations introducing salt bridges in solvent-exposed loops could constitute one possible approach to engineer more thermostable proteins.File S1Table 1, Table 2, Figure S1 and Figure S2.(DOCX)Click here for additional data file.


The aim of this study was to investigate the nature of inhibitory processes in early childhood. A confirmatory factor analysis was used to examine the latent structure of inhibitory processes in day-care center children aged 24–32 months and in preschool children aged 36–48 months. The best fit to the data for the younger sample was a single undifferentiated inhibition factor model; in older children, a two-factor model was differently identified in which response inhibition and interference suppression were distinguished. Inhibitory processes are considered important components of cognition that affect an individual's ability to function in everyday life. Using a broad definition, inhibitory processes refer to the ability to control one's mental processes and responses, to ignore an internal or an external prompt, and to perform an alternative action Diamond, .During childhood, inhibitory processes have been found to affect different aspects of child functioning, such as self-regulatory behaviors , in adults  and inhibition of attention (resistance to distractor interference), both of which involve the ability to actively maintain critical goal-related information. Surprisingly, resistance to proactive interference was unrelated to both the prepotent response inhibition and resistance to distractor interference, which suggests that this type of cognitive inhibition acts as an independent dimension.More recently, in a review of the literature, Diamond  suggesteIn contrast, the response inhibition component involves the ability to regulate one's behavior and control one's emotions to support the regulation of a behavior. This ability involves preventing impulsive behaviors when completing a task despite being faced with distractions or other competing stimuli. In children, this behavioral self-control is facilitated if there is sufficient time between the triggering stimulus and the response the child should produce  and the ability to filter out irrelevant information in the environment (interference monitoring and suppression) was also suggested by Bunge et al. , who fouThe presence of inhibitory processes in toddlerhood and preschool-aged children has been established in many studies  was used to investigate the latent structure of inhibitory processes in children aged 24–32 and 36–48 months. Research investigating the underlying construct of inhibition at these age levels is currently absent from the literature.The aim of the present study was to investigate the latent organization of inhibitory processes in early childhood. Following the hypothesis of Bunge et al. , we consresponse inhibition was distinguished from interference suppression. To test these two models, we chose measures that assessed the ability to suppress prepotent but inappropriate responses (response inhibition) and the ability to manage the interference of potentially conflicting features of the task (interference suppression), as suggested by Bunge et al.  in favor of a subdominant response (say “night” when a card with a sun is presented). These tasks are examples of univalent displays in which only a single feature is presented and the conflict is between two response options to the same stimulus feature  in their last year of day-care and 70 children between the ages of 36 and 48 months  in their first year of preschool. The participants were recruited by contacting six day-care centers and four kindergartens in the largest town in a northern region of Italy. Written parental informed consent was obtained before the participating children were admitted to the assessment sessions. Parents also completed a socioeconomic and educational background questionnaire: the mother's education level ranged from 8 to 18 years (mean = 13.7 years), and the father's education level ranged from 5 to 18 years (mean = 11.57 years); the mother's annual income ranged from 0 to 42,000 € , and the father's annual income ranged from 14,000 € to 42,000 € . Children with documented health problems, such as neurological, psychiatric or developmental disorders, or whose primary language spoken at home was not Italian were excluded from the study.The present study involved two samples of 130 typically developing children: 60 children between the ages of 24 and 32 months , followed by an inhibition instruction . The score is calculated as the proportion of the slowdown to the total time using the following formula: T1−T2/T1+T2, where T1 and T2 are the times recoded for the first and the second trials, respectively.The Tower Building , but not to do what the dragon asks. After practicing, there are 10 test trials with the bear and dragon commands in alternating order. The children are seated at a table throughout the task, and all actions involve hand movements. The performances on the bear and dragon trials are considered to be an index of self-control. The tasks are scored as follows: “0 indicates a movement or response when the dragon asks and no movement when the bear asks; 3 indicates no movement when the dragon asks and a movement or response when the bear asks” Also partial credits were scored: 2 indicates a partial movement or response when the bear asks, and a wrong movement when the dragon asks; 1 indicates a wrong movement when the bear asks and a partial movement or response when the dragon asks. The score ranges from 0 to 30.The Day/Night Stroop , for which raw scores were transformed using an arcsine transformation, and the DCCS task, for which raw scores were transformed using a logarithmic transformation [Log10 (max range + 1 − x)]. The transformed descriptive statistics for the Animal House task and the DCCS task were as follows: Animal House: mean = 1.27, SD = 0.38, skewness = −1.06, kurtosis = 0.06; DCCS: mean = 0.85, SD = 0.21, skewness = 0.60, kurtosis = 0.99.All dependent variables displayed adequate distributional characteristics, without substantial skewness or kurtosis. For both age range measures, skewness and kurtosis coefficients were relatively low, except for the t-test. The results showed significantly better task performance for the older children in all tasks, including the Circle Drawing Task , the Fish Task  and the Animal House task .The mean scores obtained by both samples in the common inhibition measures were compared using an independent samples Zero-order (Pearson) and partial correlations controlled for age , the standardized root mean squared residual (SRMR), the Bentler's Comparative Fit Index (CFI), the Non-Normed Fit Index (NNFI) and the Akaike Information Criterion (AIC).To identify which model would be more useful to explain the observed data, a series of CFAs, based on covariance matrices, were performed using EQS 6.1 softwareX2 test was used to evaluate the appropriateness of the CFA model: non-significant X2 values indicated a minor difference between the covariance matrix generated by the model and the observed matrix and, thus, an acceptable fit. The RMSEA and the SRMR are the absolute fit indices, which assess how well an a priori model reproduces the sample data , which is a descriptive measure used to discriminate between competing models, was employed to compare the models. The models with the lowest AICs were considered to be the best.The response inhibition and interference suppression were distinguished. Figure Considering data separately from both age groups, two different theoretical models were performed: an inhibition unitary model and a two-factor model, in which For the 24–32 month sample, the unitary model was the only acceptable solution. The two-factor model results showed that the value of the correlation between the two dimensions was 1; thus, it was not possible to run a model in which the two dimensions are distinguished.X2  and acceptable to good fit indices. Specifically, the NNFI, the CFI and the RMSEA values indicated good fits, and the SRMR showed an acceptable fit.The unitary model showed no significant r = 0.71; the 95% confidence interval for the correlation was ), the two-factor model allows a better explanation of the observed data. As presented in Table X2 was not significant in either solution . Nevertheless, the indices showed the best fit for the two-factor solution: the SRMR was acceptable in both models, however, in the two-factor model, the CFI, the NNFI and the RMSEA indicated good fits, whereas the same indices did not report acceptable values for the unitary model. Finally, the lowest AIC occurred for the two-factor model; thus, it showed the best fit.In contrast, in the oldest sample, the two-factor model fits the data better than the more parsimonious single-factor model. Although the estimate correlation between factors is high  with the exception of the Reverse Categorization task in the 24–32 month sample and the Circle Drawing Task in the 36–48 month sample. The proportion of variability explained by the tasks varied from 0.12 to 0.50 in the youngest children and from 0.21 to 0.42 in the oldest children.As reported in Figure The aim of the current study was to examine the nature of inhibitory processes in early childhood. Although several authors have suggested a multifaceted nature of inhibition Nigg, , an empiThough the growth of inhibitory control during childhood has been largely documented, especially in toddlerhood and preschool years  from various socio-demographic backgrounds were assessed using age-appropriate inhibitory tasks involving different response demands. According to the literature, we considered two different models of inhibition development. First, we examined a unitary factor model, based on earlier studies that indicated a single undifferentiated executive control factor as the most appropriate for describing the executive latent structure in preschoolers ; at a later stage, children develop a more cognitive inhibition that involves the suppression of interfering information or prepotent mental representations (interference suppression).The results suggest that inhibitory processes are not yet differentiated before 36 months of age, after which a distinction between different inhibitory dimensions emerges. We hypothesize a sequential development of inhibitory processes  based on a rule; however, the child must respond to a single stimulus which is clearly indicated by the experimenter. In the Day/Night Stroop task, the child must also suppress the tendency to produce a dominant response in relation to a target. Both tasks are thought to require inhibitory control and WM in remembering the rules. However, memory demands do not significantly influence the performance. With regards to the Day/Night Stroop task, Gerstadt et al.  (response inhibition); however, they also need to manage the visual and attentional interferences to solve the task. In particular, as suggested by Martin-Rhee and Bialystok  in the case of the Reverse Categorization and the color (red or blue) or the shape (rabbit or boat) in the case of the DCCS. Toddlers often fail to classify the blocks in the Reverse Categorization task in the post-switch phase  because they cannot inhibit the rule previously learned (“I've to put the big block in the big box”). Similarly, preschool children have difficulty switching from sorting by color to sorting by shape on the DCCS task because they have difficulty in inhibiting the old way of thinking about the objects. Children in the first year of preschool may remain stuck in thinking about objects according to the objects' initially relevant attribute  converged in the interference suppression factor, which suggests that at this age level, a child's performance is influenced by the ability to filter out irrelevant information, and not only by the ability to suppress a habitual response. Indeed, a specific task may not measure the same ability across different ages , which is traditionally employed to assess planning in adults, proved to measure inhibitory control in young children (Bull et al., The changes that occur in the nature of inhibitory processes from toddlerhood to early childhood may be due to both quantitative and qualitative changes in cognitive processing. For example, the As regards the reasons of the change in the organization of inhibitory processes across the two age-levels, this could be a result of maturational processes, as well as a consequence of the educational experiences of the children. While the 24–32 month sample was recruited from day-care centers, the children in the 36–48 month sample were attending the first year of preschool. In Italy, attendance at preschool is commonly accepted as the first essential stage of the educational system, and over 95% of children between 3 and 5 years of age attend a pre-primary school. Supporting school readiness, the preschool curriculum emphasizes activities that enhance creativity skills, social attitudes, autonomy and the learning process. The transition to preschool provides children with an opportunity to develop cognitive abilities and to improve self-regulation and executive function skills by increasing the children's participation in more structured activities that require more attentional control (Diamond and Lee, However, the current results should be considered in the context of the study limitations.response inhibition at both age levels comprised univalent stimuli associated with a prepotent response that must be overruled. While the tasks used to assess interference suppression comprised stimuli with different features, each was associated with a different response; thus, attention must be selectively focused on the relevant cue. Second, though the models tested were simple, the sample size at each age level was limited, suggesting that further evidence is needed to confirm our findings.First, because of the more limited behavioral repertoire of toddlers compared to preschool children, it was impossible to use exactly the same tasks in both age ranges. The tasks used in the 24–32 month sample are necessarily simpler, though they have similar inhibitory demands as the tasks used to assess the 36- to 48-month-old children. As indicated previously, the tasks used to assess In conclusion, to the best of our knowledge, this is the first study to investigate the latent structure of inhibition at an early age. Because inhibition development is central in several theories of cognitive development (Dempster, 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.


WT) or constitutively active PKD1 (PKD1S744E/S748E) attenuated 6-OHDA-induced apoptosis, suggesting an anti-apoptotic role for PKD1 during oxidative neuronal injury. Collectively, our results demonstrate that PKD1 signaling plays a cell survival role during early stages of oxidative stress in dopaminergic neurons and therefore, positive modulation of the PKD1-mediated signal transduction pathway can provide a novel neuroprotective strategy against PD.Oxidative stress is a major pathophysiological mediator of degenerative processes in many neurodegenerative diseases including Parkinson’s disease (PD). Aberrant cell signaling governed by protein phosphorylation has been linked to oxidative damage of dopaminergic neurons in PD. Although several studies have associated activation of certain protein kinases with apoptotic cell death in PD, very little is known about protein kinase regulation of cell survival and protection against oxidative damage and degeneration in dopaminergic neurons. Here, we characterized the PKD1-mediated protective pathway against oxidative damage in cell culture models of PD. Dopaminergic neurotoxicant 6-hydroxy dopamine (6-OHDA) was used to induce oxidative stress in the N27 dopaminergic cell model and in primary mesencephalic neurons. Our results indicated that 6-OHDA induced the PKD1 activation loop (PKD1S744/S748) phosphorylation during early stages of oxidative stress and that PKD1 activation preceded cell death. We also found that 6-OHDA rapidly increased phosphorylation of the C-terminal S916 in PKD1, which is required for PKD1 activation loop (PKD1S744/748) phosphorylation. Interestingly, negative modulation of PKD1 activation by RNAi knockdown or by the pharmacological inhibition of PKD1 by kbNB-14270 augmented 6-OHDA-induced apoptosis, while positive modulation of PKD1 by the overexpression of full length PKD1 (PKD1 Parkinson’s disease (PD) is a major neurodegenerative disorder affecting over a million Americans at an annual cost of several billion dollars. The incidence of PD is projected to increase dramatically with the advancing median age of the U.S. population. This creates a scientific imperative to improve our understanding of the causes and medical management of PD. Several studies have shown that oxidative stress is one of the major factors underlying the etiology of age-related neurodegeneration. Experimental findings from cell cultures, animal models, and humans indicate that oxidative stress and apoptosis may contribute to the pathophysiological processes in PD Protein kinase D (PKD) belongs to the calcium/calmodulin-dependent protein kinase (CAMK) family and consists of three isoforms that are homologous in structure and function, namely, PKD1, PKD2 and PKD3. Among PKDs, PKD1 is the most studied member of the family, and is widely expressed in many tissues and organs, including thyroid, brain, heart and lungs 3AN27; referred to as N27 cells), which was described previously 2 at 37°C, as described previously The immortalized dopaminergic neuronal cell line obtained from rat mesencephalon, (1RB6 cells) on 0.1 mg/ml poly-D-lysine precoated 12 mm coverslips. Neurobasal medium fortified with B-27 supplements, 500 µM l-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) was used to grow the cells. The cells were maintained in a humidified CO2 incubator (5% CO2 and 37°C). Approximately 5- to 6-day-old cultures were used for experiments. Primary mesencephalic neuronal cells were exposed to 10 µM 6-OHDA for 1–3 h.All of the procedures involving animal handling were approved by the Institutional Animal Care Use Committee (IACUC) at the Iowa State University. The ventral mesencephalon of gestational 16- to 18-day-old mouse embryos was used to prepare primary mesencephalic neuronal cultures N27 cells were exposed to 6-OHDA (100 µM) for 0–9 h at 37°C. Primary neurons were exposed to 6-OHDA (10 µM) for 1–3 h. We determined that 100 µM 6-OHDA is an optimal dose in N27 cells based on our preliminary dose-response studies. Also, we and many other laboratories Cytotoxicity of N27 cells against 6-OHDA (100 µM) was determined using the Sytox green cytotoxicity assay, as described previously After 6-OHDA treatment, primary mesencephalic neurons were fixed using 4% paraformaldehyde. Non-specific sites were blocked with 2% bovine serum albumin, 0.5% Triton and 0.05% Tween-20 in phosphate-buffered saline (PBS). The cells then were incubated with anti-TH, anti-native PKD1 and anti-PKD1-pS744/S748 primary antibodies in PBS containing 1% BSA at 4°C overnight. This was followed by incubation with Alexa 488 and Alexa 568 conjugated secondary antibodies in PBS containing 1% BSA. Further incubation was done with nuclear Hoechst 33342 dye to stain the nucleus. After further washing, the coverslips were mounted on slides, and viewed under an inverted fluorescence microscope . The images were captured with a SPOT digital camera .Cells were lysed in either modified RIPA buffer or M-PER buffer (Thermo Scientific) for Western blot, immunoprecipitation and kinase assays. Nuclear and cytoplasmic extracts were isolated using the NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific), as described previously HA-tagged full-length human PKD1 WT and mutant plasmids were obtained from Addgene, Inc. Predesigned PKD1-siRNA was purchased from IDT, Inc. PKD1-siRNA effectively suppressed >80% of PKD1 protein expression levels after a 36 h post-transfection. N27 cells (50–70% confluence) were transfected with siRNA duplexes using an Amaxa Nucleofector kit (Amaxa), as described in our previous study Data analysis was done using Prism 3.0 software . Bonferroni’s multiple comparison procedure was applied to test for significant differences between treatment and control groups. Differences with p<0.05, p<0.01, and p<0.001 were considered significant from two or more independent experiments (n≥6), and are indicated in the figures.We first determined the time course of N27 dopaminergic neuronal cells to 6-OHDA-induced neurotoxicity using the Sytox cell death assay. The N27 cells were treated with 100 µM 6-OHDA and the cell death was monitored up to 8 h. As shown in Next, we measured PKD1 phosphorylation of Ser744/Ser748 and compared it with cytotoxic cell death. Phosphorylation of Ser744/Ser748 located in the activation loop of PKD1 results in its activation. Therefore, we determined if 6-OHDA induces activation loop phosphorylation of PKD1 in N27 dopaminergic cells. We used antibody directed against phosphorylated Ser744/Ser748 in PKD1 to determine PKD1 activation. As shown in We also performed Western blot analysis following nuclear/cytoplasmic fractionation to determine the subcellular distribution of PKD1 phosphorylation. As depicted in S916A and the native wild type PKD1WT in N27 dopaminergic cells by transient transfection and then exposed the cells to 100 µM 6-OHDA for 1 h. As shown in S916A but not PKD1WT blocked this effect, suggesting that Ser 916 phosphorylation is required for PKD1 activation loop phosphorylation. Together, these results suggest that C-terminal PKD1 Ser916 phosphorylation occurs at very early stages of oxidative stress to positively regulate PKD1 (pS744/pS748) activation loop phosphorylation during 6-OHDA-induced oxidative stress in dopaminergic cells.Following the characterization of activation loop PKD1 (pS744/pS748) phosphorylation, we further examined whether additional phosphorylation occurs during 6-OHDA-induced oxidative stress. Since PKD1 Ser 916 phosphorylation site in the C-terminal region serves as an important regulator of PKD1 activation, we investigated the role of PKD1 Ser 916 phosphorylation during 6-OHDA treatment. As shown in In order to determine the functional role of PKD1 activation during oxidative stress-induced dopaminergic cell death, we first used a pharmacological approach that involved the allosteric PKD inhibitor kbNB 142–70. Briefly, N27 dopaminergic cells were exposed to 100 µM 6-OHDA in the presence or absence of 50 µM kbNB 142–70. We then determined cytotoxic cell death using the Sytox green assay. As shown in WT) and the constitutively active PKD1S744E/S748E in N27 dopaminergic cells, which were then exposed to 6-OHDA. Mutation of serine residues at positions 744 and 748 results in constitutively active PKD1 WT significantly attenuated 6-OHDA-induced cytotoxic cell death in a time-dependent manner compared to vector-transfected dopaminergic cells. Similarly, overexpression of constitutively active PKD1S744E/S748E also significantly attenuated 6-OHDA-induced cytotoxic cell death in a time-dependent manner compared to vector transfected dopaminergic cells  primary dopaminergic neurons obtained from mouse mesencephalon  primary mesencephalic neurons during 10 µM 6-OHDA treatment. Together, these results indicate that activated PKD1 translocates to the nucleus of primary dopaminergic neurons when treated with a Parkinsonian specific toxicant like 6-OHDA, and that 6-OHDA also induces PKD1 S916 phosphorylation in primary dopaminergic neurons.Finally, we examined if PKD1 activation occurs in primary mesencephalic dopaminergic neurons exposed to 6-OHDA. Immunocytochemical staining was performed to examine the subcellular localization of PKD1 in primary mesencephalic neurons. We found a high-level expression of PKD1 red,  in the cn green, . We alson green, . Next, tThis study reveals the existence of a novel defensive signaling mechanism in cell culture models of PD during neurotoxic stress. Our study reports, for the first time, three key findings in an oxidative stress-induced dopaminergic degeneration model using 6-OHDA: (i) PKD1 activation occurs at an early stage of oxidative damage in cell culture models of PD, (ii) phosphorylation of Ser 916 residue regulates activation loop phosphorylation (PKD1- pS744/S748), and (iii) modulation of PKD1 signaling has protective effects against oxidative damage of dopaminergic cell death.Oxidative stress triggers apoptosis and causes neurodegeneration through activation of multiple signaling molecules including kinases and proteases S744E/S748E) or wild-type PKD1 plasmid (PKD1WT) is overexpressed, dopaminergic cells are resistant to 6-OHDA-induced cytotoxicity, even during the late stages of oxidative stress, which is consistent with our hypothesis that PKD1 activation protects against oxidative damage in dopaminergic cells. Furthermore, we also observed 6-OHDA treatment induced nuclear translocation of phosphorylated PKD1 to possibly regulate the transcription of cell survival transcription factors and genes in dopaminergic cell death. Our results are consistent with recent studies demonstrating nuclear translocation of PKD1 in B cells, cardiomyocytes and oestoblasts In the current study, we explored the relationship between PKD1 signaling and dopaminergic degeneration in cell culture models of PD. Several studies have demonstrated DNA fragmentation during late stages of 6-OHDA-induced apoptotic cell death. DNA fragmentation is preceded by ROS generation, mitochondrial dysfunction, and caspase-3 activation In conclusion, our results demonstrate the existence of a novel protective signaling mechanism mediated by PKD1 in dopaminergic neurons. During the early stage of 6-OHDA-induced oxidative stress, PKD1 is activated and protects neurons against oxidative damage. However, prolonged oxidative insult inactivates PKD1 signaling, which might lead to neuronal death. Thus, positive modulation promotes neuronal survival while negative modulation of PKD1 augments cell death. Overall, our findings suggest that PKD1 may function as a cell survival switch in dopaminergic neurons, and its modulation could be a novel neuroprotective strategy against oxidative damage in PD.


SLC12A5) Cl− transporter activity drives the developmental GABA excitatory-inhibitory sequence, but the role of KCC2 in human NDs is essentially unknown. Here, we report two rare, non-synonymous (NS), functionally-impairing variants in the KCC2 C-terminal regulatory domain (CTRD) in human ASD (R952H and R1049C) and SCZ (R952H) previously linked with IGE and familial febrile seizures, and another novel NS KCC2 variant in ASD (R1048W) with highly-predicted pathogenicity. Exome data from 2517 simplex families in the ASD Simon Simplex Collection (SSC) revealed significantly more KCC2 CTRD variants in ASD cases than controls, and interestingly, these were more often synonymous and predicted to disrupt or introduce a CpG site. Furthermore, full gene analysis showed ASD cases are more likely to contain rare KCC2 variants affecting CpG sites than controls. These data suggest genetically-encoded dysregulation of KCC2-dependent GABA signaling may contribute to multiple human NDs.Many encoded gene products responsible for neurodevelopmental disorders (NDs) like autism spectrum disorders (ASD), schizophrenia (SCZ), intellectual disability (ID), and idiopathic generalized epilepsy (IGE) converge on networks controlling synaptic function. An increase in KCC2 ( Neurodevelopmental disorders (NDs) encompass a wide range of diseases, all of which feature some element of impaired brain development, and are associated with cognitive, neurological, and/or psychiatric dysfunction . In immature neurons, [Cl−]i is sufficiently high that GABAAR activation triggers giant depolarizing potentials, which characterize early network activity and stimulate Ca2+-dependent synaptogenesis , a cation-Cl− cotransporter (CCC) mediating Cl- efflux, lowers [Cl−]i in post-synaptic neurons such that GABAAR activation elicits membrane hyperpolarization and fast synaptic inhibition  is a critical region of transporter function, and contains multiple phosphorylated residues  were present in large FC cohorts of ASD, SCZ, or ID by utilizing a similar targeted genetic sequencing approach we successfully utilized for IGE  project initiated by Dr. Guy Rouleau at Montreal Neurological Institute and McGill University in order to identify genes that cause or predispose to numerous disorders of brain development. The sample characteristics of these FC cohorts have previously been described  or NM_001134771 (NP_001128243 amino acids 917–1110)] by following the same protocol outlined in Kahle et al. , Macaca mulatta (XM_001104494.2_prot), Bos Taurus (NP_001193309), Rattus norvegicus (NP_599190), Mus musculus (NP_065066), Tetraodon nigroviridis (ENSTNIT00000021299), and Danio rerio (ENSDART00000009569). Regarding the WebLogo output, the y-axis serves as a means of determining relative conservation and is not an actual measurement; the height of each stack at each amino acid position is relative to the overall conservation at that position, the height of the letters within each stack indicate the relative frequencies for each amino acid possibility, and the width of each stack corresponds to the proportion of valid readings at that position (indicating if sequence gaps exist between the shown amino acids). The effects of amino acid substitutions on protein function were predicted using MutationTaster  to include probands with either two Caucasian parents (EA ASD cases) or two African American parents (AA ASD cases), respectively. According to Supplementary Table 1 in Iossifov et al. , accession number (NM_001134771) to include only v et al. , there wp-values and odds ratios where appropriate.All statistical genetic analysis was carried out using the program R (version 2.15.1). Fisher tests or Mantel-Haenzel Chi Squared tests were carried out to generate the SLC12A5 that encodes the KCC2 CTRD  in three large FC disease cohorts of ASD, SCZ, or ID that were collected as part of the S2D project . In contrast to our previous analysis in IGE which identified an enrichment of KCC2 NS CTRD alleles in IGE cases compared to controls . This limitation is common when identifying rare genetic risk factors, and also relevant for the R1049C variant  or lower the intrinsic activity of transporters at the cell surface (R1049C) , as well as variants that either disrupted or introduced a CpG site  in ASD cases compared to controls.The identification of KCC2 NS genetic variants in ASD and SCZ cases that involve evolutionary conserved residues, are predicted to be pathogenic, and have either previously been shown i in developing CNS neurons that delays the normal ontogenic switch to GABA inhibition (He et al., GABA, but this effect is abolished in the these models (Tyzio et al., − extrusion during the developmental GABA excitatory-inhibitory sequence might be a genetically programmed factor contributing to ASD. Further investigation into the roles of KCC2 in ASD and SCZ, and potentially other NDs, may offer new therapeutic strategies.Lastly, our results are interesting given the recent data from the Fragile X and valproate mouse models of autism, which demonstrate an abnormally prolonged elevation of [ClThe 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.