This study presents, for the first time, a two-phase (CO2 and brine) flow visualization under reservoir conditions  for a highly heterogeneous conglomerate core obtained from a real CO2 storage site. Rock heterogeneity and the porosity variation characteristics were evaluated using X-ray computed tomography (CT). Multiphase flow tests with an in-situ imaging technology revealed three distinct CO2 saturation distributions (from homogeneous to non-uniform) dependent on compositional complexity. Dense discontinuity networks within clasts provided well-connected pathways for CO2 flow, potentially helping to reduce overpressure. Two flow tests, one under capillary-dominated conditions and the other in a transition regime between the capillary and viscous limits, indicated that greater injection rates  could be significantly reduced without substantially altering the total stored CO2 mass. Finally, the capillary storage capacity of the reservoir was calculated. Capacity ranged between 0.5 and 4.5%, depending on the initial CO2 saturation.Geologic storage of carbon dioxide (CO Both industry and research communities are currently evaluating the safety and feasibility of long-term CO2 sequestration, and a number of pilot- and demonstration-scale projects have been conducted as a part of this effort to test, monitor, and verify technologies in various subsurface geological environments5.Atmospherically released carbon dioxide  and demonstration- or commercial-scale projects  has been sandstone5. The average reservoir porosity has been 5–35% and reservoir permeabilities have ranged from as low as 5 mD  to 5,000 mD (Sleipner).Previous research related to geologic CO6. In an effort to assess the geologic storage potentials, basalt formations have also been considered as alternative storage formations8.One exception was the MRSCP Gaylord project , where the targeted storage formation consisted of dolomite. This was characterized by interbedded, laminated algal dolomudstone, represented by a mean porosity and permeability of 13% and 22.6 mD, respectively2 storage projects are being conducted worldwide, the need for additional safe storage verification experiments using various geological formations cannot be overemphasized. The fact that only limited data has been collected during the injection and post-injection phases demonstrates the necessity of additional field assessments of the processes leading to plume stabilization and long term trapping9. Followed by on-going international efforts, the first onshore pilot-scale  CO2 storage project is being conducted in South Korea at the Janggi sedimentary formation, located on a south-eastern portion of the Korean Peninsula  distribution of clasts within the matrix was determined utilizing X-ray computed tomography (CT). Additionally, real-time X-ray scanning techniques (conducted during the CO2 and brine flow tests) captured the dynamic distribution of CO2 saturation in the conglomerate core. Multiphase transport simulations were also conducted to assess the effects of heterogeneity on pressure build-up as well as spatial variation of CO2 saturation. Finally, based on the initial-residual (IR) characteristic curve, the storage potential of the conglomerate formation is discussed.Despite these numerous core-scale studies, there has been little investigation on how COf < 0.3). In the midstream portion (70 mm), both medium size of clasts and matrix existed together, whereas larger clast sizes were present in the downstream region (100 mm). Figure A core-plug obtained from the Janggi conglomerate formation was used to investigate the spatial distribution of clasts within a silty sand matrix. Figure c) curve, appearing as a superposition of two uni-modal pore size distribution characteristic curves. The Pc (Sw = 0.5) of the clasts was more than two orders of magnitude greater than that of the matrix  CO2 injection into a brine-saturated core, simulating the CO2 injection period (drainage) conditions; and (ii) brine injection after the CO2 injection test, reproducing the post-injection period (imbibition) conditions.This experimental study aimed to characterize the spatio-temporal distribution of CO2 saturation distribution snapshots revealed the propagation of CO2 saturation throughout the conglomerate core with increases in pore volume (PV)  delineated in Fig. 2 saturation related to small-scale heterogeneity, it clearly presents the increasing tendency within the core sample. All profiles showed logarithmic increases with the PVs. The CO2 saturation profile at 30 mm increased slowly when approaching the asymptote during the flow tests; however, the other profile (such as at 110 mm) approached their asymptotes more rapidly, implying that CO2 saturation was quickly stabilized throughout the experiment. It is interesting to note that CO2 saturation at the downstream side of the core was greater than that at the upstream side in early stages (<1.0 PV). The spatial inversion of CO2 saturation distribution was attributed to the heterogeneous pore space along the conglomerate core, including the presence of complex discontinuities in clasts  were selected, one each located in the upstream (domain I), midstream (domain II), and downstream (domain III) regions as shown in Fig. 2 saturation distribution over time  were located, as seen in Fig. 2 flow, resulting in a rapid increase in CO2 saturation.To perform a detailed assessment of the spatial distribution of COime Fig.  with a srix Fig. . Domain 2 saturation was also plotted for the brine injection tests  immediate mobile CO2 displacement by injected brine (R1), (ii) immobile CO2 preservation as residual trapping (R2), and (iii) the gradual dissolution of residually trapped CO2 into the fresh brine (R3). The dynamic transition of CO2 trapping at 30 mm displayed initial decrease in CO2 saturation from 0.35 to 0.27 during the early stage (0.01–0.1 PV) primarily resulted from the mobile CO2 displacement (Regime 1). In Regime 2, the CO2 saturation was relatively consistent and stabilized at 0.27 between 0.1 and 1.0 PV, which implies the preservation of immobile CO2 . This is possible because the brine that enters the core will initially dissolve a small amount of CO2 at the inlet of the core and becomes fully-saturated preventing it from dissolving any more CO2. However, as more fresh brine was injected into the core, the residually trapped CO2 dissolved into fresh brine (Regime 3).The vertically averaged COsts Fig. . As disc2 saturation snapshots from the three different domains during the brine injection test. In domain I where the matrix dominated, CO2 was distributed uniformly at the end of drainage test. Brine displacement tests induced similar uniform distributions in residually trapped CO2, which eventually dissolved into the brine. However, in domains II and III, a larger fraction of pores was initially filled with CO2, resulting in more snap-off and trapping as brine invaded the pore space. Under these conditions, CO2 dissolution was highlighted only within the matrix and discontinuities.Figure 2 injection from the opposite boundary would cause difference in the distribution of CO2 saturation due to nature in highly heterogeneous conglomerate core. We used TOUGH239 simulator to model multiphase and multicomponent fluid transport to assess directional effect of fluid flow on pressure build-up as well as CO2 saturation distribution along the core sample. A two-dimensional model was constructed based on the CT image of the core sample to reflect the distribution of clasts. Then, two different cases were simulated .The above experiments were conducted with fluid injection at the left boundary, but CO2 saturation map (b and g), profiles for CO2 pressure (c and h) and saturation (d and i) along the center of the model domain, and pressure build-up (e and j) for the two different cases. Both cases showed a significant pressure drop between points A and B, which lie on a boundary of a large sized clast. The heterogeneous pressure distribution resulted in different CO2 saturation within the model for the two cases. For example, case 1 showed relatively high CO2 saturation (Sg~0.5) at point A and low CO2 saturation (Sg~0.3) at point B, while case 2 showed relatively high CO2 saturation (Sg~0.5) at point B. This implies that the distribution of CO2 saturation is dependent on the flow direction. The pressure difference between the inlet and outlet (ΔP) showed ~150 kPa during the early stage of injection and stabilized at ~30 kPa for both cases  were performed in this study, producing associated −1 and ~10−2, respectively. The high −1) reached 2,300 kPa in early injection stages before stabilizing to ~700 kPa; however, the low injection rate (−2) displayed a small buildup of ΔP build-up (~150 kPa) in early injection stages before ΔP stabilized to ~65 kPa for the remainder of the multiphase flow experiment.A dimensionless capillary number (2 saturation distribution maps for the two injection rates (qco2 = 1.0 and 0.1 ml/min) and the CO2 saturation contrast (ΔSco2 = Sq=1.0−Sq=0.1) maps between the high and low injection rate cases. The measured ΔSco2 displayed a strong dependency on core heterogeneity, showing negative values on the upstream side (implying lower CO2 saturation with a greater injection rate (Sq=1.0 < Sq=0.1)). The high injection rate created excessed pressure on CO2 (nonwetting phase), enabling CO2 to transport across the high Pc region on the midstream side. This had a negative impact on the build-up of CO2 mass on the upstream side. At low injection rates, however, additional CO2 occupied the pore-spaces on the upstream side because CO2 transport across the high Pc region on the midstream side was hindered. The CO2 mass within the core was calculated for both 2 mass, suggesting that injection rates, which may cause reservoir overpressure when high, can be significantly reduced without substantially altering the total CO2 mass.Figure 2 phase into the form of blobs or ganglia, becoming effectively immobile41. The capillary trapping potential was evaluated in these conditions based on the performed multiphase flow experiments. The initial-residual (IR) characteristic curves have commonly been used to describe the residual trapping potentials of rock, with numerous developed models describing this relationship. This study applied the Land model42, one of the earliest and most widely used trapping models.During the post-injection stage, capillary forces at the pore scale are responsible for breaking up the CO2,i) and residual saturations , where Sco2,i refers to the initial CO2 saturation prior to brine injection testing whereas Sco2,r refers to the residually trapped CO2 saturation. For analysis, highly heterogeneous conglomerate core was divided into 50 equal subdomains. Then, based on the brine injection testing, the residual CO2 saturation achieved at 0.1 PV (R2) was determined  is a rescaling of the traditional IR saturation plot44. This quantity is of great importance in geologic CO2 storage applications because it states how much CO2 can be stored securely per unit volume of a rock. Figure 2,i and φSco2,r at different porosities . The IR relationships of other core samples (Berea and Pohang sandstones) are plotted alongside for comparison. Core sample details are described in the Supplementary Table 2,i, whereas the φSco2,r values for heterogeneous conglomerate cores were widely spread and dependent on the size of porosity. Although the overall capacity of the conglomerate core was smaller than those of the Berea or Pohang sandstones, the conglomerate high porosity region displayed a similar capacity to the sandstones .The capillary-trapping capacity, defined as φSco2 sequestration, other types of geologic formations are being investigated for this purpose. For example, the target rock formation in MRSCP Gaylord project was dolomite45, and the Midale project (an extension of the Weyburn project) demonstrated the possibility for CO2 containment in a fractured reservoir5. Basaltic formations have also been considered as alternative storage formations8. In South Korea, a conglomerate formation is being tested for geologic CO2 storage.Although sandstone formations are considered to be the most appropriate for CO2 migration behaviours as well as the storage capacity of the highly heterogeneous conglomerate. Variations in clast spatial configurations gave rise to different storage features. Dense networks of discontinuities within clasts were observed to provide well-connected CO2 flow pathways, causing a rapid increase in CO2 saturation, and potentially helping to reduce overpressure. Two flow tests, one in the capillary-dominated condition and the other in a transition regime between the capillary and viscous limits, revealed that a 10× reduction in injection rate caused only a 2% decrease in the CO2 mass. This implies the heterogeneity positively influenced pressure management because high injection rates, which may cause overpressure in the reservoirs, could be significantly reduced without substantially altering the total stored CO2 mass. We note that although chemical reactions are not considered in this study, the complex process of CO2-water-mineral interactions may play important roles on multiphase flow in subsurface reservoirs and trapping mechanisms.This study reported a thorough core-scale investigation of a highly heterogeneous conglomerate formation. The rock heterogeneity was investigated with the use of X-ray CT and MICP analysis revealing that the targeted storage formation was composed of a silty sand matrix and irregularly spaced clasts of various sizes. Laboratory multiphase flow tests with flow visualization under reservoir conditions provided important information on CO2 saturation, the Janggi conglomerate displayed significant variations due to its highly heterogeneous porosity distribution. The conglomerate capacity ranged between 0.5 and 4.5%, depending on the initial saturation (0–0.4): for an initial saturation of 0.4, the conglomerate capacity ranged between 3–4%. Despite the fact that the trapping analyses showed lower overall capacities relative to homogeneous sandstone, the high porosity region of the conglomerate core displayed a similar capacity to sandstones, implying conglomerate formations could be an alternative for CO2 storage formation. CO2 injection and comprehensive monitoring in conglomerate formations will validate the feasibility of CO2 storage in the highly heterogeneous conglomerate formations.Among the four potential trapping mechanisms, capillary trapping is a rapid and effective mechanism for rendering an injected fluid immobile and reducing the need to ensure caprock integrity. The capillary-trapping capacity of the conglomerate core investigated in this study was calculated using imbibition test results and the IR characteristic curve. While the Berea and Pohang sandstone cores showed approximately linear increases in capacity with initial CO2 storage project was launched in South Korea in 2011. The primary tasks  were the site-selection and initial characterization of potential pilot-scale storage sites. The Janggi conglomerate, composed of both clasts and silty sand matrix, was chosen as the target storage formation for the project. More recently, Phase II (2015–2017) was completed, including the drilling of seven boreholes to investigate the lithology of targeted and capping formations. The representative lithological log for this is illustrated in Fig. 1(a)46. The average porosity and permeability of the storage formation were 16.1% and 8.5 mD, respectively47. The overlying Seongdongri formation, composed of thick mudstone and massive dacitic tuffs, forms the regional sealing formation for CO2 storage, and a monitoring well (~40 m up-gradient from the injection well) was drilled. A comprehensive monitoring system was installed including a down-hole pressure and temperature measurements, vertical seismic profile (VSP), electrical resistivity tomography (ERT), distributed temperature sensing (DTS) and distributed acoustic sensing (DAS) along the well via an optical fiber, and U-tube downhole sampler.An onshore pilot-scale geologic COThe dimensions of the core-plug obtained from the Janggi conglomerate formation were 130 × 48 mm (length × diameter). X-ray computed tomography (CT), a non-destructive and non-invasive method, was used to investigate the spatial distribution of clasts within the silty sand matrix. The CT images used in this study were obtained at 140 kV and 200 μA. A total of 2,000 cross-sectional images were taken at 0.065 mm intervals with a micrometer-order pixel size. Subsequently, two-dimensional (2D) images were stacked to generate 3D core sample images.2 and brine) injection, core-holder, temperature controlling, confining, and back-pressure control systems  compared with the contrast between any one of the fluids and the rock48. In the experiments, NaI was used as a dopant to enhance the X-ray attenuation so that the CO2 saturation could be distinguished from the brine in the scanned image.The apparatus for core-flooding experiments was designed to conduct multiphase flow tests. It was comprised of fluid  conditions; and (ii) brine injection following the CO2 injection test, reproducing post-injection period (imbibition) conditions. Downstream pressure was maintained during these two experiments at 10 MPa, and the temperature was set at 50 °C to replicate the subsurface environment at Janggi pilot site. Two different injection rates (q = 1.0 and 0.1 ml/min) were applied. The capillary numbers, −1 and ~10−2, for q = 1.0 and 0.1 ml/min, respectively.Two multiphase flow tests were conducted: (i) CO3) were taken from the conglomerate core each representing either the silty sand matrix or clasts. These subsamples were dried in a vacuum oven at 70 °C for 24 hours prior to testing. The MICP and corresponding mercury saturation measured in the mercury/air system were transformed into a capillary pressure and corresponding CO2 saturation for a CO2/brine system using the following relationship: −1) represents the interfacial tension (IFT) between two fluids, and −1 and −1 was used in the conversion for the geologic CO2 sequestration conditions. The contact angles for the two systems were assumed to be equal29.The pore size distributions and capillary pressure curves for the silty sand matrix and clasts in the conglomerate sample were determined using a mercury injection porosimeter (MICP). To perform MICP tests, two small subsamples (~1 cmSupplementary Information


Computational analysis of protein-protein interaction provided the crucial information to increase the binding affinity without a change in basic conformation. Several docking programs were used to predict the near-native poses of the protein-protein complex in 10 top-rankings. The universal criteria for discriminating the near-native pose are not available since there are several classes of recognition protein. Currently, the explicit criteria for identifying the near-native pose of ankyrin-protein complexes (APKs) have not been reported yet.In this study, we established an ensemble computational model for discriminating the near-native docking pose of APKs named “AnkPlex”. A dataset of APKs was generated from seven X-ray APKs, which consisted of 3 internal domains, using the reliable docking tool ZDOCK. The dataset was composed of 669 and 44,334 near-native and non-near-native poses, respectively, and it was used to generate eleven informative features. Subsequently, a re-scoring rank was generated by AnkPlex using a combination of a decision tree algorithm and logistic regression. AnkPlex achieved superior efficiency with ≥1 near-native complexes in the 10 top-rankings for nine X-ray complexes compared to ZDOCK, which only obtained six X-ray complexes. In addition, feature analysis demonstrated that the van der Waals feature was the dominant near-native pose out of the potential ankyrin-protein docking poses.http://ankplex.ams.cmu.ac.th.The AnkPlex model achieved a success at predicting near-native docking poses and led to the discovery of informative characteristics that could further improve our understanding of the ankyrin-protein complex. Our computational study could be useful for predicting the near-native poses of binding proteins and desired targets, especially for ankyrin-protein complexes. The AnkPlex web server is freely accessible at The online version of this article (doi:10.1186/s12859-017-1628-6) contains supplementary material, which is available to authorized users. Due in vitro  and in somplexes , 10. TheProtein–protein docking is a well-known method for generating protein–protein complexes (poses) using computational methods. The challenging task of identifying the exact bound state of a pair of proteins must consider the following factors: (i) there are several potential ways that a pair of proteins can interact, (ii) the flexibility of the protein, and (iii) changes in the protein conformation after binding . Currentvia assessing the CAPRI criteria in the 10 top-rankings based on the features of ZDock, ZRank, or E_RDock. However, successful predictions do not occur for all cases [ZDOCK has successfully predicted several near-native complexes (poses) of antibody-antigen, enzyme-inhibitor and other pairings ll cases –20. Morell cases . TherefoThe near-native docking pose of Ankyrin-Her2 was successfully predicted using ZDOCK and an extra scoring function . RecentlIn this study, we made a systematic attempt to develop a computational approach for achieving near-native predictors in 10 top-rankings of ankyrin-protein docking poses, which we named AnkPlex. Moreover, this method was generated for (i) analysing and characterizing ankyrin-protein complexes by using a set of informative features that have potential applications and (ii) establishing a user-friendly web server to obtain the desired results without the need to follow complicated mathematical equations generated by the research scientist. The docking poses of seven X-ray complexes of APKs, which had ankyrins with 3 internal domains, were generated using the reliable docking tool ZDOCK. The construction of the docking poses calculated by PSC alone and summation of PSC + DE + ELEC demonstrated there were different numbers of near-native docking poses. The steps for AnkPlex establishment included (i) balancing the near-native and non-near-native poses; (ii) processing the dataset through machine learning of a decision tree algorithm (DT) and a logistic regression (LG) with a combination of 11 features; (iii) selecting the efficient predictive models of DT and LG; and (iv) processing the dataset by combining models of DT and LG.Protein Data Bank (PDB) database for 41 APKs reported up to May 2014. Analyses of the 41 APKs were performed through data pre-processing using the following steps: (i) APKs containing 3-internal-domain were included; (ii) redundant APKs were excluded; (iii) APKs were filter based on the recognition areas [1), complex 2 (C2), complex 3 (C3), complex 4 (C4), complex 5 (C5), complex 6 (C6), and complex 7 (C7). At the same time, the rest of the APKs, including unknown 1 (U1) and unknown 2 (U2), were designated the test group (Ank-TEST). In order to avoid the distinct results from the different selections of training and test sets, other 35 possible datasets were constructed and were used to generate the predictive models for the identification of near-native poses.X-ray crystal structures of ankyrin-protein complexes (APKs) were collected from the on areas ; and  is the Pairwise Shape Complementarity (PSC) score and it was optionally augmented with the electrostatics (ELEC) and the desolvation energy (DE). In this study, the α and β have the default values of 0.01 and 0.06, respectively.ZRankElec (B) is the long-range electrostatic energy and the only fully charged side-chain, as represented in the following equation:qi and qj are the charges on ankyrin and the protein atoms, respectively. The rij in the equation stands for the distance between the atoms of ankyrin and the protein.ZRankSolv (C) is the desolvation term based on the Atomic Contact Energy (ACE).aij is the ACE score.ZRank (D) is a linear combination of ZRankVdw, ZRankElec, and ZRankElec.ZRankVdw score was calculated as follows:ZRankVdw (E) is the van der Waals and short-range electrostatics energy with a distance between the atom pair being less than 5.0 Å. This calculation was based on the parameters of the CHARMm 19 polar hydrogen potential. The εij and σij are the depth and the width, respectively, of the coefficient for the CHARMm 19 polar H.st and the 2nd minimized structure of the ankyrin-protein docking poses, respectively.E_vdw1 (F) and E_vdw2 (H) are the van der Waals energy, as presented in Equation (5), of the 1st and the 2nd minimization, respectively.E_elec1 (G) and E_elec2 (I) are the electrostatic energy, as presented in Equation (2), of the ankyrin-protein docking poses processed for the 1nd minimization of the ankyrin-protein docking poses.E_sol (J) is the desolvation energy, as shown in Equation (4), of the 2E_RDock (K) is the summation of E_sol and (0.9 × E_elec2).Based on observations of the generation of the features, ankyrin-protein docking poses were generated for the energy features using the ZDOCK protocol , 14 (a s1-C7). As shown in Additional file A1: Table S5, SVM yielded 100% near-native poses in the internal testing sets but could not obtain any near-native pose in the external testing sets. The DT and ANN provided the near-native poses from both internal and external testing sets. According to a dataset of C5, the DT was superior in achieving the near-native poses of internal testing sets than the ANN. The LG provided a weighted summation that could rank the docking poses to achieve the near-native poses in the 10 top-rankings. As a consequence, the DT and the LG were selected to construct an ensemble model.Several learning models were constructed including decision tree (DT), logistic regression (LG), artificial neural network (ANN), and support vector machine (SVM) using Ank-TRN  and a logistic method (LG). The decision trees and the logistic regression methods were selected due to the fact that they provide a high number of predicted positive values (true near-native poses). The logistic regression especially provided a weighted summation that was finally ranked to search for near-native poses in 10 top-rankings. All 11 features and all datasets were used to build the DT and LG models. The Ank-TRN (7 APKs) and the Ank-TEST 2 APKs) were evaluated by AnkPlex using the following steps, as shown Fig.  APKs wer1. The number of near-native poses and non-near-native poses were balanced. ZDOCK using PSC + DE + ELEC and regKp provided 699 near-native poses and 44,334 non-near-native poses of Ank-TRN. The non-near-native poses were randomly clustered into 65 groups . Therefore, each training set was composed of the same near-native poses and different groups of non-near-native poses.2. A predictive model using the DT and the LG models was established. All 11 features were combined and generated as feature subsets . The total number of feature subsets was calculated to be 4,095 by following this equation:The DT model was established from 4,095 feature subsets and 65 training sets using the J48 algorithm , 27. The3. An efficient predictive model of the DT and the LG models was selected. Ank-TRN consisted of 7 APKs and was submitted to the learning method for predicting the near-native poses. True positive rates (TPrate) greater than 50% were used as the cut-off value for an efficient learning method. The learning methods that demonstrated a TPrate greater than 50% were selected to further establish an ensemble learning model.DT) and the LG-based learning models (OLMLG) from Step 3 for reducing the number of non-near-native docking poses. The main process of the proposed method, AnkPlex, for increasing the number of TPs (reducing non-near-native poses) consisted of the following steps: (i) only predicted positive samples (PPVDT) derived from OLMDT were select, (ii) a logistic score (LGS) on PPVDT using the LG model was calculated, and (iii) PPVDT was ranked according to LGS and the 10 top-ranking poses that demonstrated the highest LGS were selected. The near-native pose(s) or the true positive (TP) in the 10 top-ranking poses were our targets. The summation score of AnkPlex was defined in the equation given as Equation (7) on Ci, where i = 1, 2, …, 7, and Yi would be set as 1 in case TP was found in the 10 top-ranking poses. Otherwise, Yi would be set as 0. Finally, the score of AnkPlex was the summation product, as defined in the following equation:PP belongs to Ank-TRN containing seven complexes  and Yi would be set as 1 when TP appears in the 10 top-ranking poses. Otherwise, Yi would be set as 0. The number of #PPTRN indicated the sample of Ank-TRN in which the LGS score was among the 10 top-ranking near-native poses. The number of #PPTEST showed the LGS score of the Ank-TEST was among the 10 top-ranking poses.4. Ensemble methods were established. The ensemble learning method, named AnkPlex, was constructed by randomly integrating the DT-based learning models . The method validation parameters, including accuracy (ACC), sensitivity (SEN), and precision (PRES), were calculated using the following equations:There were a few ankyrin-protein complexes (APKs) reported in the PDB database. Forty-one ankyrin complexes, with the number of internal domains ranging from 2–7, have been reported . The highest number, 19 complexes, of ankyrin-proteins contained 3 internal domains. Furthermore, the APKs that reacted with the target using recognition areas were selected. Focusing on target proteins, only proteins with common folding structures, i.e., alpha-, beta- and alpha-beta structures, were considered. Therefore, nine complexes, which included 1SVX, 4ATZ, 3Q9N, 1AWC, 2BKK, 2Y1L, 4DRX, 2P2C, and 4HNA, were used in this study. These nine complexes were randomly divided into two groups, i.e., 7 complexes as Ank-TRN and 2 complexes as Ank-TEST.To optimize the ZDOCK calculation, X-ray crystal structures of APK-TRN, including seven APKs, were calculated with different feature calculations, including the PSC and the PSC + DE + ELEC. The total number of docking poses, including near-native and non-near-native poses, was 54,000 poses (54Kp). Subsequently, the numbers of near-native poses calculated by PSC and PSC + DE + ELEC were compared. As shown in Table p-value <0.001. As presented in Table p = 0.178) and E_vdw2 (p = 0.010). Subsequently, 11 features calculated with the ZDOCK calculation were applied to establish the learning methods.According to the ZDOCK calculations of Ank-TRN, 11 features of near-native poses and non-near-native poses were generated. Univariate statistical approaches were employed to perform exploratory data analysis using average and standard deviations for summarizing important patterns. As shown in Table DT) and the LG-based learning models (OLMLG) were established using the 4,095 feature sets. The learning methods demonstrated the average of the true positive rate to be greater than 50% (TPrate ≥ 50%), and consisted of 4,762 OLMDT and 2,688 OLMLG. The learning models that represented TPrate ≥ 50% with the 10 top-ranking poses of %ACC are shown in Table DT) and Table LG). As a result, ABDEHIJK_g14 of OLMDT exhibited the highest %ACC with %TPrate ≥ 50%. This learning method consisted of sequential combination feature sets that included ZDock (A), ZRankElec (B), ZRank (D), ZRankVdw (E), E_vdw2 (H), E_elec2 (I), E_sol (J), and E_RDock (K) calculated from non-near-native dataset number 13. In addition, CDFGJ_g10 of OLMLG also demonstrated the highest %ACC with %TPrate ≥ 50%. The percentage of precision (%PRES) for all the 10 top-ranking poses of OLMDT and OLMLG was low, which indicated that there was a high number of false positive results (FP). To diminish the number of FP, only the 10 top-ranking poses based on the ZRank score were selected to represent the true positive poses (TP). If the TP were found in 10 top-ranking poses from each Ank-TRN, #PP was designated 1. Thus, the #PP-values of seven Ank-TRN (#PPTRN) were in the range of 0 to 7. As shown in Tables TRN of OLMDT and OLMLG were only 6. Therefore, the individual learning method of OLMDT or OLMLG was not capable of providing the maximum value for #PPTRN.Eleven features of each of the near-native poses (669) and non-near-native poses 44,334) calculated from Ank-TRN based on the recognition areas (regKp) were used to establish the learning methods. Based on the unbalanced number of docking poses, training sets were generated by clustering the non-near-native poses and the near-native poses into 65 sets . Eleven features were calculated from each training set and were ordered to generate 4,095 feature sets. The DT-based learning models  and 2,688 of the LG-based learning models (OLMLG) were randomly combined to generate an ensemble model. Interestingly, the combination of the ensemble model from ABEHIJ_g56 of OLMDT and CDFGHJ_g30 of OLMLG demonstrated superior prediction efficiency due to the fact that this ensemble model (ABEHIJ_g56- CDFGHJ_g30) achieved maximum values for #PPTRN and #PPTEST of 7 and 2, respectively. Therefore, the ensemble model, ABEHIJ_g56- CDFGHJ_g30, was designated to be an ensemble computational model for predicting the near-native docking pose of APKs or “AnkPlex”  and OLMLG (CDFGHJ_g30) provided a #PPTRN value of 6. The first TP of C5 predicted by ABEHIJ_g56 and the C6 predicted by CDFGHJ_g30 were found at pose numbers 14 and 19. This result indicated that a single learning model could not produce all the true positive poses. In the case of the Ank-TEST, OLMDT could not provide the value for the #PPTEST, whereas the #PPTEST of OLMLG was comparable to AnkPlex. Consequently, it can be concluded that the ensemble model, AnkPlex, was capable of including a #PPTRN value of 7 and a #PPTEST value of 2, which suggested that the prediction efficacy of AnkPlex was superior to the single learning model. In addition, the predictive models generated from other 35 possible datasets demonstrated the average number of #PPTRN and #PPTEST value of 6.78 ± 0.42 and 2 ± 0.00, respectively. This indicated that different selections of training and test sets had no effect in the generation of the learning models for predicting the near-native poses.To enhance the prediction efficacy of the generated learning methods, 4,762 of the DT-based learning models  of HIV-1 [GAG1D4 was already constructed. However, the complex structure of AnkGAG1D4-CANTD was not detected [GAG1D4-CANTD and performed re-scoring with AnkPlex. The results revealed that three near-native structures of AnkGAG1D4-CANTD were found in the 10 top-rankings. The recognition residues of AnkGAG1D4-CANTD interactions were further investigated by observing interacting distances ≤ 5 Å. As a result, one docking pose showed that residue R18 was located on the recognition areas of CANTD and two docking poses demonstrated residues R132 and R143 played key roles in the interaction with AnkGAG1D4 (data not shown). This result correlated with previous ELISA results. A point mutation of R18A on helix 1 and R132A and R143A on helix 7 of CANTD showed negative binding to AnkGAG1D4. Thus, R18, R132 and R143 were the key residues of CANTD binding to AnkGAG1D4 [GAG1D4 using this learning model, AnkPlex could not only discriminate the near native docking poses of AnkGAG1D4-CANTD complex but also demonstrated the correct orientation of the recognition area to CANTD.To apply the AnkPlex for investigating the ankyrin-protein complex, AnkR values) were used to identify the correlation between LGS and the weights of the six features to obtain the near-native poses. As shown in Fig. R values of the six features consisted of ZRank (R = 0.60), ZRankSolv (R = −0.56), and E_sol (R = 0.54), which indicated that these three features played an important role in the AnkPlex model for distinguishing near-native poses.Identification of informative features among the 11 features was critical for designing a powerful learning model and for understanding and obtaining insights into the ankyrin-protein docking poses. Based on the six features (CDFGHJ) used in the calculation of the LGS in AnkPlex, the Pearson correlation coefficients . The characteristics of the ZRank score between the near-native and the non-near-native poses were significantly different, with p < 0.001, as shown in Table DT(ABEHIJ_g56)–OLMLG(DFGHJ_g30) was able to obtain #PPTRN = 1 and #PPTEST = 0. However, AnkPlex (OLMDT(ABEHIJ_g56)–OLMLG(DFGHJ_g30) achieved success with #PPTRN = 7 and #PPTEST = 2. Therefore, ZRank was concluded to be an important feature of AnkPlex due to the fact that it could enhance the predictive performance of near-native poses.The ZRank score was ranked as the 1Since ZRank is a linear combination of van der Waals (ZRankVDW), electrostatics (ZRankElec), and desolvation energy (ZRankSolv), one of them had to be identified as the most important. From Additional file nd and the 3rd informative features with R values of −0.56 and 0.54, respectively. Moreover, these two features showed differences in their characteristics between near-native and non-near-native poses, with p < 0.001, as shown in Table DT(ABEHIJ_g56)–OLMLG(CFGHJ_g30) provided #PPTRN = 6 and #PPTEST = 1. Similar to E_sol, the performance of AnkPlex lacked E_sol, i.e., (OLMDT(ABEHIJ_g56)–OLMLG(CDFGH_g30)) yielded #PPTRN = 6 and #PPTEST = 1. This result indicated that the absence of ZRankSolv and E_sol slightly reduced the predictive performance of AnkPlex. However, these two features were required for predicting near-native poses. ZRankSolv was a component of ZRank. This outcome emphasized that desolvation was important for obtaining near-native poses in the ankyrin-protein interaction. Additionally, it was also required for the accuracy of other protein–protein complexes [ZRankSolv and E_sol were the desolvation energies estimated by the summation of the Atomic Contact Energy (ACE) in which the difference between the two features was in the force field calculation and side chain orientation. ZRankSolv and E_sol were the 2LGCFGHJ_g providedomplexes .The LGS score was a combination of the energy determined in the interaction area between ankyrin and proteins of ≤5 Å. In AnkPlex, the interaction area on ankyrin was located on variable and conserved residues of the L-shaped repeat belonging to the internal repeats, the N-terminal repeat and the C-terminal repeat . The funAccording to the hydrophobicity on the interface in AnkPlex  is freely available online.An ensemble method, named AnkPlex, was constructed for fast prediction of near-native states of ankyrin-protein complexes. The AnkPlex model was constructed based on a combination of features generated from the ZDOCK program without using manual inspections. AnkPlex successfully obtained the near-native poses of nine ankyrin-protein complexes in the 10 top-ranking poses. ZRank, which is a combination of electrostatic, desolvation, and van der Waals energy, was the most important feature in AnkPlex. In addition, van der Waals was the dominant feature for obtaining the near-native docking poses. To develop the method for predicting near-native poses of protein complexes, we have implemented easy access to the best models for the scientific community on a web server. AnkPlex  of the nine ankyrin-protein complexes. Table S5. The percentage of predictable true near-native poses of the internal and external testing sets on leaning methods of decision tree (DT), logistic regression (LG), artificial neural network (ANN), and support vector machine (SVM). (DOC 125 kb)Informative characteristics of nine ankyrin-protein complexes. Additional file 2:Supplementary datasets used in this study. (XLS 13646 kb)


Streptococcus suis, a Gram-positive pathogen, is widely recognized as an important agent of swine infection, and it is also known to cause a variety of zoonoses, such as meningitis, polyarthritis and pneumonia. Suilysin (SLY), an extracellular pore-forming toxin that belongs to the cholesterol-dependent cytolysin family, is an essential virulence factor of S. suis capsular type 2 (SS2). Here, we found that morin hydrate (morin), a natural flavonoid that lacks anti-SS2 activity, inhibits the hemolytic activity of SLY, protects J774 cells from SS2-induced injury and protects mice from SS2 infection. Further, by molecular modeling and mutational analysis, we found that morin binds to the “stem” domain 2 in SLY and hinders its transformation from the monomer form to the oligomer form, which causes the loss of SLY activity. Our study demonstrates that morin hinders the cell lysis activity of SLY through a novel mechanism of interrupting the heptamer formation. These findings may lead to the development of promising therapeutic candidates for the treatment of SS2 infections. Streptococcus suis (S. suis) is a Gram-positive coccus and emerging zoonotic agent whose global spread could cause a variety of life-threatening infections, including, but not limited to, sepsis, arthritis, meningitis, encephalitis, endocarditis and pneumonia in swine and humans , a 54 kDa extracellular pore-forming protein that belongs to the family of cholesterol-dependent cytolysin, is a virulence-associated factor secreted by most virulent S. suis strains and is regarded as a virulence marker of S. suis , a yellow natural bioflavonoid compound found in many members of the Moraceae family and various fruits and vegetables, has been shown to possess multiple pharmacological effects, including anti-inflammatory, anti-proliferative and anti-oxidant activities . AdditioS. suis serotype 2) used in this study was a gift kindly provided by Professor Hongjie Fan . SS2 strain ZY05719 was cultured in Todd-Hewitt broth  at 37°C. Morin was commercially obtained from Chengdu Herbpurify CO., LTD  and was dissolved in dimethyl sulfoxide  to make a stock solution (40.96 mg/ml).The bacterial strain ZY05719 (highly virulent Streptococcus suis sly cDNA was subcloned into pET-28a(+) vector  with BamHI and NdeI restriction enzyme cutting sites to construct the prokaryotic expression plasmid pET-28a(+)-SLY. The recombinant plasmid encoding the histidine-tagged SLY protein (rSLY) was transformed in E. coli BL21. Recombinant protein expression was induced by the addition of 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C for 16 h. The supernatant of bacterial cell lysates was loaded onto a Ni-NTA column to purify the resultant rSLY protein.Table 1.The expression vectors for the SLY-Tyr54Ala, SLY-Gln107Ala and SLY-Asp111Ala mutants were constructed using the QuikChange site-directed mutagenesis kit  based on pET-28a(+)-SLY and were verified by DNA sequencing. The expression and purification of these mutants was performed as described above for wildtype SLY (WT-SLY). The primers used in this study for the construction of pET-28a(+)-SLY and its derivatives are listed in Approximately 3 mg rSLY protein was sent to Tianjin Sungene Biotech Co., Ltd.  for the production of anti-SLY rabbit serum, which was confirmed by western blot to detect recombinant SLY protein and secreted SLY in bacterial culture supernatants.5 CFUs/ml. The minimal inhibitory concentration (MIC) of morin for SS2 was measured by a serial dilution method, according to the procedures of the CLSI guideline M31-A2 (SS2 was cultured in THB and diluted at a density of 5 × 10e M31-A2 . Lag-phaStreptococcus suis strain SS2 from an overnight culture was transferred (1:50) to THB with various concentrations of morin  and further cultured to the stationary phase (OD600 = 2.5). Then, the bacteria were pelleted by centrifugation  to obtain culture supernatants. Defibrinated rabbit blood was suspended in PBS to 2.5% vol at a final volume of 1 mL and was incubated with 100 μL culture supernatant at 37°C for 30 min in parallel with controls containing either no culture supernatants (negative control) or 2.5% vol Triton X-100 (positive control). After incubation, the reaction system was centrifuged , and the optical density at 543 nm (OD543) of the supernatant for each sample was determined by ultraviolet spectrophotometry as previously described (escribed .The effect of morin on hemolysis induced by rSLY or its derivatives was measured by incubating 100 μL purified proteins (100 ng/mL) with the indicated concentrations of morin in 1 mL PBS for 30 min at 37°C prior to the addition of 25 μL defibrinated rabbit blood. Cell lysis was determined as described above.A total of 20 μL culture supernatants (as described above) were mixed with Laemmli SDS sample buffer, boiled for 5 min and separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was transferred to polyvinylidene difluoride membranes  and SLY was visualized by primary anti-SLY rabbit serum diluted 1:2000 and secondary horseradish peroxidase-conjugated anti-rabbit antiserum (Sigma–Aldrich) diluted 1:4000 with Amersham ECL Western blotting detection reagents , as described previously .2 atmosphere in complete medium  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin). The cells were seeded at a density of 2 × 104 cells per well in 96-well plates overnight and were subsequently infected with SS2 at the mid-log phase (OD600 = 0.8–1) of bacterial culture and resuspended in FBS-free DMEM medium at a multiplicity of infection (MOI) of 10 with various concentrations of morin for 5 h. Cells treated with 2.5% vol Triton X-100 or DMEM served as the positive and negative controls, respectively. The supernatants were harvested from each well by centrifugation , and the presence of LDH released into supernatants was measured using the Cytotoxicity Detection Kit  according to the manufacturer’s instructions. Microscopic images of stained cells were further obtained under a confocal laser scanning microscope  using live/dead (green/red) reagent (Invitrogen).Mouse J774 macrophage-like cells were cultured at 37°C in a 5% CO4 cells/mL) in complete medium were plated in the upper chamber of 24-well transwell plates  for 3 days, and 500 μL complete medium without antibiotics was added in the lower chamber. At days 4, 6, and 7, the concentrations of FBS in the upper chamber were changed to 5, 1, and 0%, respectively. Upon reaching confluence after 8 days, the cells in each chamber were infected with 200 μL SS2 (1 × 108 CFU/mL) at the mid-log phase (OD600 = 0.8–1) of bacterial culture and resuspended in FBS-free DMEM medium in the presence of 32 μg/mL morin for 15 min. The number of bacteria in the lower chamber was calculated by titrating on THB plates to determine the penetration ability of SS2 through J774 in the presence or absence of morin. The percentage of penetration = 100 × lower chamber CFUs/ upper chamber added CFUs.For the transwell experiment, 1 mL J774 cells . The animal experiments were reviewed and approved by the Animal Care and Use Committee of Jilin University.600 = 0.8-1). Cells were centrifuged , washed once with PBS and suspended in PBS to a final concentration of 1 × 109 CFU/mL. Mice were inoculated intravenously with 100 μL of the bacterial suspension. Two hours after inoculation, morin was administered subcutaneously at 100 mg/kg and at the same dosage at 8-hour intervals. Infected mice administered with DMSO served as the control group. The infected mice (30 per group) were observed for survival analysis for up to 4 days.Overnight cultures of SS2 were transferred into THB (diluted 1:500) and grown at 37°C to mid-log phase  with PDB codes of 3HVN. The free protein obtained from the PDB (3HVN) was first equilibrated by a 100 ns molecular simulation on the solute, which was used for the molecular docking with morin. Then, the geometry of morin was optimized at the B3LYP/6-31Gus paper .0P = 1 bar, coupling time pτ = 0.5 ps). The protein and non-protein systems were coupled separately to the temperature bath. The antechamber programs and AM1-BCC partial atomic charges from the Amber 10 software were used to estimate morin. In this work, the binding free energies between SLY and morin were calculated by the Molecular Mechanis/Poisson-Boltzman Surface Area (MM-PBSA) method supplied with the Amber 10 package. Then, the interaction between inhibitors and each residue in the binding site of SLY was analyzed using the MM-PBSA decomposition process.In this study, complex systems were investigated by MD simulations using the Gromacs 4.5.2 software package based on the Amber99sb force field and TIP3P water model. The electrostatic term was described by using the particle mesh Ewald algorithm method, and all bond lengths were constrained by the LINCS algorithm. The initial velocity was obtained from a Maxwellian distribution. The desired initial temperature was 300 K. The density of the system was adjusted by NPT condition , fD is the free concentration of morin, K is the binding constant and n is the number of binding sites. In this system, due to the morin concentration being far greater than the concentration of protein; the fD is replaced by the total morin concentration. According to experimental results, the linear fitting plots of f vs rr/D between morin and WT-SLY, SLY-Tyr54Ala, SLY-Gln107Ala, or SLY-Asp111Ala could be made. The values of K and n could be obtained based on the plots.To verify the result of MD simulations, the interactions between morin and WT-SLY, SLY-Tyr54Ala, SLY-Gln107Ala, and SLY-Asp111Ala were investigated by fluorescence quenching. The proteins were used as the fluorophore and morin was used as the quencher. Commonly, fluorescence quenching can be described by the following Scatchard equation: n ≥ 3) are expressed as the mean ± SD. GraphPad Prism 5.0 was used for statistical analysis using Student’s t-test. Significance levels of P < 0.05 and P < 0.01 are indicated in the figures.All experimental data . As shown in Figure 1B, the SLY protein secreted by S. suis at the stationary phase could sufficiently induce hemolysis, as 85.38% of red blood cells were lysed by the control sample in the absence of morin. However, upon co-culture with morin, the hemolytic activities of bacterial supernatants were significantly decreased in a concentration-dependent manner . This finding suggests that such inhibition was due to a suppression of SLY activity by morin or a direct neutralization of SLY-mediated hemolysis by morin.The hemolytic activity of culture supernatants from the stationary phase of SS2 was measured by a hemolysis assay in the presence of various concentrations of morin . Additionally, the MIC of morin against SS2 was greater than 1,024 μg/mL, and morin had no influence on SS2 growth at the concentrations required to inhibit the hemolytic activity of culture supernatants . These data indicate that morin has effectively no antimicrobial activity against SS2. Furthermore, the effect of morin on purified recombinant SLY (rSLY)-mediated hemolysis was evaluated, and, consistent with the results described above, the addition of morin significantly reduced the hemolytic activity of rSLY . Taken together, these findings reveal that the inhibition of the hemolytic activity of bacterial culture supernatants by morin was not due to a decreased production of SLY in the supernatants but may be due to the direct binding of morin to SLY to neutralize SLY activity.Western blot analysis was performed to detect the level of secreted SLY protein in culture supernatants and, interestingly, no visible difference was observed between control and morin-treated samples . The release of LDH by J774 was assessed to quantify such protection. In agreement with the above results, morin treatment significantly alleviated SS2-mediated cell injury in a concentration-dependent manner . Additionally, almost no cell injury was observed in the cells treated with only morin, suggesting that this compound had no cytotoxicity in J774 cells .A live/dead (green/red) assay and LDH assay were further employed to verify the potential protection of morin against SS2 Figure 2C), indicating that morin inhibits SLY function in a similar manner to cholesterol, which is consistent with previous findings ; however, this mortality rate was decreased to 56.67% . In summary, our results suggest that morin treatment leads to a substantial inhibition on SS2-induced death in infected mice.To examine whether the protection of morin against Figure 4A. From the binding mode of SLY-morin, it was shown that there are three strong hydrogen bonds between morin and Thr49, Tyr54, and Gln107. Moreover, Asn50 and Asp111 also have a strong hydrophobic interaction with morin . The number of H-bonds between morin and SLY during the 200-ns simulation are plotted in Figure 4B, which is consistent with the above results.Over the 100-ns of MD simulation, it was found that morin localized to domain 2 of SLY, as shown in Figure 4C. Interestingly, our analysis reveals that the flexibilities in the SLY binding sites are different in the presence and absence of morin, which suggests that the residues in the binding sites of the SLY complex are more rigid as a result of ligand binding. In Figure 4C, the residues (50–60 and 100–120) in the protein-morin binding sites show a small degree of flexibility with RMSF of less than 0.1 nm when compared with free protein, indicating that these residues seem to be more rigid as a result of ligand binding.It is widely accepted that the flexibility of the residue can be revealed by the calculation of the root mean square fluctuation (RMSF) of the protein. As such, the RMSF of these residues around the binding sites of the SLY complex and free SLY are shown in Figure 4D, Thr49, Gln107, and Asp111 provide appreciable electrostatic contributions, with ΔEele values of ∼–1.43, ∼–1.28, and ∼–10.34 kcal/mol, respectively. Moreover, Asn50, Tyr54, and Gln107 have the strongest attraction interaction, with van der Waals values of ∼–3.10, ∼–2.08, and ∼–2.65 kcal/mol, respectively. With the exception of Asp111, the majority of the decomposed energy interactions originated from Van der Waals interactions, while electrostatic contributions appeared to be a minor influence on these key residues. Based on the above results, we conclude that the amino acid residues of Thr49, Asn50, Tyr54, Gln107, and Asp111 may be important for morin binding. Hemolysis assay was further performed to detect the inhibitory activity of morin on hemolysis induced by rSLY and its derivatives. Three SLY mutants  remain active for lying defibrinated rabbit blood , however, the inhibition of morin on these mutations was alleviative than WT-SLY .To obtain detailed information of the residues in the SLY-morin binding region and their contribution to the whole system, the ligand-residue interaction decomposition was calculated for the complex system by an MM-PBSA method. As shown in Table 1. According to the theoretical results, the binding free energy (ΔGbind) of the interaction between morin and protein is greater for WT-SLY than mutant SLY, indicating that WT-SLY has the strongest ability to bind with morin. By fluorescence spectroscopy quenching, we measured the binding constants (KA) and the number of binding sites between morin and the three mutants, and the results of this analysis were highly consistent with those obtained by computational biology methods (Table 1). These findings indicate that the information generated by the molecular modeling of the SLY-morin complex is reliable.To confirm this hypothesis, the total binding free energy for the SLY-morin complex and their detailed energy contributions  are summarized in Figure 5A): as a rigid body, there is an extended motion to the entire conformation of the protein, particularly to the “stem” domain 2 . This motion is sufficiently large to meet (but not exceed) the requirements for the conformational transition of SLY from its monomer form to an oligomer. The second principal component (PC2) primarily corresponds to the slight vibration of the protein backbone, as shown in Figure 5B. The PC1 of the complex is composed of several motions, similar to that of the free protein, with the exception of the motion in domain 2 . The second principal component (PC2) was shown in Figure 5D. As shown in Figure 5C, the motion of domain 2 is clearly weaker than that of free SLY. It should be noted that domain 2 is also the binding site of morin, based on previous data. Therefore, it is confirmed that the motion of domain 2 is restricted by the binding of morin. According to previous reports, the toxin protein of the highly conserved CDC family can form the largest transmembrane β barrels. During pore assembly, many aspects of pore formation have been directly observed, including the buckling of domain 2. In this work, the conformational change of domain 2 was clearly blocked upon morin binding, inhibiting the formation of oligomers  and causing the hemolytic activity of SLY to be lost.To explore the mechanism of SLY inhibition by morin, a principal component analysis (PCA) was performed on the MD trajectory of free SLY and the SLY-morin complex to identify the most significant motions of the protein. The first component (PC1) of free SLY is composed of several motions  was performed on the MD trajectory of the free SLY and SLY-morin complex to identify the most significant motions of the protein. Based on our analysis of dynamic trajectory, we can predict that the motion of domain 2 is restricted by the binding of morin which blocks the conformational change of domain 2 from the monomeric to oligomeric form to inhibit the lytic activity of SLY. In addition, the declined sensibility of morin on hemolysis of SLY mutations futher indicated that Tyr54, Gln107, and Asp111 are three important amino acid residues for the engagement of morin with SLY.S. aureus SrtA . The animal experiments were reviewed and approved by the Animal Care and Use Committee of Jilin University.XD and JW conceived and designed the experiments. GL, GjL, ZQ, HL, LW, YW, BL, and XN performed the experiments. JW and XD contributed reagents/materials/analysis tools. JW and XD wrote the paper.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.


Pakistan has one of the highest prevalences of child malnutrition as compared to other developing countries. This narrative review was accomplished to examine the published empirical literature on children’s nutritional status in Pakistan. The objectives of this review were to know about the methodological approaches used in previous studies, to assess the overall situation of childhood malnutrition, and to identify the areas that have not yet been studied. This study was carried out to collect and synthesize the relevant data from previously published papers through different scholarly database search engines. The most relevant and current published papers between 2000–2016 were included in this study. The research papers that contain the data related to child malnutrition in Pakistan were assessed. A total of 28 articles was reviewed and almost similar methodologies were used in all of them. Most of the researchers conducted the cross sectional quantitative and descriptive studies, through structured interviews for identifying the causes of child malnutrition. Only one study used the mix method technique for acquiring data from the respondents. For the assessment of malnutrition among children, out of 28 papers, 20 used the World Health Organization (WHO) weight for age, age for height, and height for weight Z-score method. Early marriages, large family size, high fertility rates with a lack of birth spacing, low income, the lack of breast feeding, and exclusive breastfeeding were found to be the themes that repeatedly emerged in the reviewed literature. There is a dire need of qualitative and mixed method researches to understand and have an insight into the underlying factors of child malnutrition in Pakistan. There are 165 million malnourished children under five years around the globe ,3. ChildIt has been estimated that 170 million (30%) of children under the age of five in the world are moderately or severely stunted, and 110 million (19%) are moderately or severely underweight . AccordiDespite economic and social development, childhood malnutrition still remains a major public health and social problem in less developed countries ,14,15. T(i)to identify about the range of methodologies and methods to access the children malnutrition;(ii)to highlight the emerging themes to address the children malnutrition; (iii)to identify the geographical areas that are neglected in this research in Pakistan; and(iv)to provide recommendations for future work.Malnutrition is the underlying cause of child morbidity and mortality in Pakistan. It has not been the priority issue of government of Pakistan to overcome the malnutrition in children. The aim of this narrative review is to collate and synthesize the published data to understand the problem as a whole. The dearth of studies on child malnutrition in Pakistan has evoked the researcher to search for and review the existing relevant literature. The specific objectives of this review article were:Malnutrition is widely known as under-nutrition until and unless it is not specified . MalnutrAs the topic is public health and anthropological relevance, various databases were utilized, including PubMed, Medline, NCBI, CINAHL, Scopus, and social science citation index. A number of keywords and combination of keywords were used to locate the relevant literature, such as “child malnutrition in Pakistan”, “child undernutrition in Pakistan”, “nutritional status of children in Pakistan”, “child health and nutritional status in Pakistan”, and “child health situation in Pakistan”. Furthermore, the list of references of relevant papers was also reviewed to access for more relevant literature on child malnutrition. The most relevant and current published papers in between 2000–2016 were included in this methodological review article. The research papers that contain the data related to Pakistan were assessed. The review papers, case studies, and letters to the editors were not entertained in this study. At the first stage, through different keywords, 178 papers were searched through different search engines using medical subject heading. At the second stage, titles and abstracts were screened for inclusion in the current study, and duplicate and irrelevant papers  were discarded. At the third stage, 69 papers were selected to review the title of the papers. At the fourth stage, the whole abstracts of the selected papers were reviewed and screened, and at the fifth stage, the decision was made to either include or exclude to review the whole article on the basis of full text papers. At the final stage, 28 papers were considered relevant and included for accomplishing this study. Narrative review is a method used to describe trends in the research field by calculating how many studies have used certain research methodologies, where they were carried out and what are the associated factors with problem under investigation. In a narrative review, the statistical analysis is not possible because the previously published studies were diverse in their objectives and questions. Narrative reviews do connect information into themes. The meta-analysis is only suitable for statistical analysis. Meta-analysis can only be undertaken when studies address the same question and administer the intervention in a similar manner or measure the same outcomes . There wIt was observed that in four papers , body maIn 12 papers, the target population of the children was school-age (more than five years), and 15 papers focused on less than five years’ children in the assessment of malnutrition. Different national and international reports have warned that first 1000 days are the window of the opportunity to strengthen the nutritional status of the children ,27,28,29Different other cross-sectional research and regional studies presented varied results in term of stunting, wasting, and underweight according to their methodology and geographical area. The prevalence of child malnutrition according to the studies under reviewed is discussed in Different studies have highlighted that the severity of malnutrition occurs in a particular age group of children. Laghari  and Gul There is rural and urban disparity in the prevalence of child malnutrition across the world. Mushtaq pointed out that in rural areas the prevalence of child malnutrition was higher compared to urban areas . The burGender is an important indicator of child malnutrition. Khattak found there was no significant difference in stunting and wasting in male and female children . Many otAccording to the latest national survey, 31.5% children were stunned, 45% were underweight, and 10.5% were wasted . SimilarMost of the researchers highlighted that socioeconomic, demographic, and lifestyle factors are responsible of malnutrition in children. Associated factors with child malnutrition according to the studies under review are briefly explained in Ansari  pointed Different national and international reports have warned that the first 1000 days are the window of the opportunity to strengthen the nutritional status of children ,59,60. DThere is dearth of qualitative and mixed-method studies on the causes of child malnutrition in Pakistan. In terms of geographical area covered, most of the studies were conducted in the provinces of Punjab, Sindh, and Khyber Pakhtunkhwa, whereas Baluchistan, Azad Kashmir, Gilgit Baltistan, and Federally Administrated Tribal Areas (FATA) were largely neglected by researchers. The prevalence of all types of malnutrition in Pakistan was found to be higher than the global threshold value. It was found that malnutrition starts at an early age and remains persistent at later stages. Most of the researchers employed the anthropometry technique i.e., wasting (weight-for-height), stunting (height-forage), and underweight (weight-for-age) by the NCHS/WHO for assessment of child nutritional status. The most vulnerable age group of children (6–23 months) was entirely neglected to exclusively study infant and young child feeding practices. It was clearly evident that higher intensity of child malnutrition prevails in the rural areas of Pakistan. Early marriages, large family size, high fertility rates with a lack of birth spacing, low income, and the lack of breastfeeding and exclusive breastfeeding were found to be major determinants of child malnutrition. This is the first study to review the range of methodologies and identify the neglected geographical areas and widely used methods of previously published papers assessing child malnutrition in Pakistan. This research will be helpful for further researchers to design their methodology and target population before conducting their research. This study highlighted methodological gaps: preferred techniques for assessing the child malnutrition, varied study designs, and tagged the geographical areas with human development index rating. However, there are some limitations in this study because the previously published papers were published in different time spans, varied types of populations were under-studied, the varied age groups of the children, the small sample sizes, and different natures of the studies. One-fourth of the studies reviewed in this research had sample sizes of less than 200, and it is very difficult to generalize the result of these small-scale studies. The studies reviewed in this research had the three types of the population groups i.e., community-based, hospital-based, and school-based studies. There was much variation in results of all three types of population, and it was difficult to draw conclusion about the causes of child malnutrition. Therefore, it is suggested that the future studies must be clear in the methodological stage for the selection of the homogeneous studies in term of target population, type and nature of the study, and sample size for better understanding the problem under investigation. There is a need for more research with a more homogeneous approach to know the current real situation of child malnutrition in a socio-cultural and demographic context.It is suggested that further studies should focus on the causes of malnutrition in the infancy stage to strengthen the nutritional status of children and that the neglected areas should also be studied to highlight this problem. The major causes of child malnutrition are also associated with the mother’s reproductive health care behavior, maternal diet, and autonomy. It is therefore suggested that further research also study the maternal factor while exploring the causes of child malnutrition. There is also a need to know about the diet and type of food that mothers prefer to give their children while exploring the causes of child malnutrition. Therefore, it is also suggested that the dietary diversity (DD) recall method should also be utilized to know about the preferred food that mothers give to their children. The component of micronutrients deficiency was entirely omitted under discussion of the previously published research. It is recommended that the micronutrient assessment should be a component of future research for a better understanding of the type of malnutrition. There is also the dire need of qualitative and mixed-method research to understand and highlight the cultural and lifestyle factors that cause child malnutrition in Pakistan.


Maternal deaths from ‘bleeding during and after caesarean section’ (BDACS) have increased in South Africa, and have now become the largest sub-cause of deaths from obstetric haemorrhage. The aim of this study was to describe risk factors and causes of near-miss related to BDACS and interventions used to arrest haemorrhage and treat its effects.Cross-sectional prospective study in 13 urban public hospitals in South Africa, from July to December 2014.There were 93 cases of near-miss related and 7 maternal deaths related to BDACS. The near-miss rate was 2.1/1000 live births, and the case fatality rate was 3.5/10 000 caesarean sections. Associated near-miss risk factors were previous caesarean section in 60% of multiparas, pre-operative anaemia (55%), abruptio placentae (20%) and placenta praevia and/or accreta (20%). Atonic uterus (43%) was the most frequent anatomical cause of bleeding for near-miss, followed by surgical trauma (29%). The median duration of the operations resulting in near-miss was 90 min, with 81% noted as difficult by the surgeon. Interventions in cases of near-miss included second-look laparotomy (46%), hysterectomy (41%), B-Lynch brace suture (9%), intensive care unit admission (32%) and red cell transfusion ≥3 units (21%).Cases from maternal near-miss from BDACS were frequently associated with pre-operative risk factors. Extensive life-saving interventions were required during and after the operations. An important factor in initiating the sequence of interventions is the realisation by the surgeon that the caesarean section is difficult, so that the progression from uneventful operation to near-miss to death can be arrested. The safety of caesarean sections has recently come into question in South Africa. Maternal death resulting from ‘bleeding during and after caesarean section’ (BDACS) is becoming increasingly frequent. The triennial reports of the government’s confidential enquiries into maternal deaths recorded 78 such deaths in 2002–2004, 140 in 2005–2007, 180 in 2008–2010, and 221 in 2011–2013 , failureGlobally, there are insufficient data on maternal deaths related to BDACS, especially in terms of clinical and surgical detail. Even with the recent increase in deaths, these events are so rare that meaningful prospective studies are difficult to do. A helpful alternative is to study ‘near-miss’ related to BDACS. A maternal near-miss is a woman who survives severe complications during pregnancy, childbirth or postpartum. The study of near-miss is an increasingly useful tool in obstetric clinical audit, because of the larger number of cases available for study compared with maternal deaths, and because valuable information can be obtained on how clinical and surgical and clinical management obstacles are overcome after the onset of an acute complication .To our knowledge, there has been no published scientific work focusing on maternal near-miss related to BDACS. Studies or audits on near-miss could improve understanding of the evolution of severe bleeding at caesarean section, and describe opportunities taken and missed to control the bleeding and treat its consequences. The information gained may inform quality improvement and interventions to reduce near-miss and death from BDACS.The aim of this study was to determine the risk factors and causes of near-miss related to BDACS and to describe the clinical and surgical interventions undertaken.This was a cross-sectional prospective study in 13 government hospitals in southern Gauteng province, South Africa, from July to December 2014. Ethical approval was obtained to conduct this study from the University of the Witwatersrand’s Human Research Ethics Committee. Informed written consent was obtained from the participants.The city of Johannesburg is the geographic and economic centre of southern Gauteng province. There are 18 government hospitals with maternity services in southern Gauteng, with about 100 000 births per annum. Three of these hospitals are university-attached tertiary referral hospitals. The 13 hospitals in the study included the three tertiary hospitals, as well as seven regional referral hospitals, and three district hospitals. The district hospitals are staffed by non-specialist doctors and have relatively small case loads. The regional hospitals all have at least one specialist, as well as medical officers who are responsible for most of the daily clinical activities. The tertiary hospitals are better resourced in terms of equipment and facilities, and include university faculty staff and residents training to become specialists.The World Health Organization (WHO) Intervention criteria were used to identify cases of near-miss, and were modified to suit this population . Only neThe researcher trained obstetric staff in each hospital on how to identify a near-miss from BDACS. Each time such a case was identified, a staff member notified the researcher telephonically. The researcher then travelled to the hospital and screened the file for study eligibility. Data were collected from maternal case records, and supplemented, where necessary, from patients themselves or from staff. Informed consent was obtained from the patients. Data collected from the clinical notes were antenatal history, details about the previous pregnancy and blood test results, reason for admission, clinical management, and events leading to the decision for caesarean section. Details about the caesarean section, the findings and the complications were recorded, as well as postnatal observations and interventions. The researcher made regular visits to all hospitals to ensure ongoing surveillance. Numbers of maternal deaths related to BDACS were also recorded.p-value <0.05 was used to determine statistical significance.A sample of 100 near-misses related to BDACS was envisaged to provide reasonable confidence intervals for observed percentage frequencies. With a sample of 100 subjects, an observed percentage has 95% confidence limits that are not more than 10% above and below it. Data analysis employed quantitative techniques, using Stata version 11 software . Descriptive data were analysed using medians, ranges and interquartile ranges for continuous variables, and proportions with percentages for categorical variables. Comparisons of categorical variable frequencies were made using Fisher’s exact test. A The hospitals reported 93 near-misses related to BDACS, as well as 7 maternal deaths. The total number of deliveries at all the hospitals was 46 775, of which 20527 were caesarean sections (43%). The near-miss related to BDACS rate was therefore 2.1 per 1000 (93/43474) live births, and the BDACS- related case fatality rate was 3.4 per 10 000 caesarean sections.n = 2) and parity ≥4 (n = 5) were infrequent. Twenty women were nulliparous and 73 were multiparous. Forty-four (60.3%) of the multiparous women had previous caesarean sections. Anaemia prior to caesarean section (haemoglobin level <11.0 g/dL) was found in 49 of 89 women tested (55.1%). Thirty-six women (40.4%) were HIV infected. Clinical risk factors included hypertensive disorders in pregnancy in 26 (28.0%), abruptio placentae in 19 (20.4%), and placenta praevia and/or accreta in 19 women (20.4%) Nine of the 19 women with abruptio placentae had a hypertensive disorder. The main primary indications for caesarean section were fetal distress , abruptio placentae  and placenta praevia . Eleven women had caesarean section for prolonged labour, 7 in the first stage of labour (7.5%) and 4 in the second stage (4.3%). Twenty-four infants were stillborn, 17 of them in association with abruptio placentae. The surgeons who started the caesarean sections were non-specialists in 29 cases (31.2%), residents in 54 cases (58.1%) and specialists in 7 cases (7.5%). Seven caesarean sections (7.5%) were done in district hospitals, 22 (23.7%) in regional hospitals and 64 (68.9%) in tertiary hospitals.Obstetric data and risk factors for near-misses are shown in Table n = 40; 43.0%), followed by uterine incisional bleeding , placental site bleeding , bleeding at the bladder reflection , pampiniform venous plexus bleeding  and abdominal wall bleeding . Two or more causes of bleeding occurred in some women. In 14 women (15.1%), the cause of haemorrhage was not specified. The causes of bleeding were compared between women with previous caesarean section, placenta praevia/accreta, abruptio placentae, and the remainder . There were significant associations between placenta praevia/accreta and placental site bleeding (p < 0.01), and between abruptio placentae and atonic uterus (p = 0.04). There was a trend to more frequent pampiniform plexus and abdominal wall bleeding in women with previous caesarean section  of haemorrhage was atonic uterus  and 8 had B-Lynch compression sutures . Fourteen women had only resuturing or uterine vessel ligation done, and 5 required no specific haemostatic procedure. Seventy-four women (79.6%) received three or more units of red cell transfusion during their hospital stay. The median lowest postoperative haemoglobin level was 6.05 g/dL (n = 90), with a range of 2.8–12.5 g/dL. Thirty-six of 78 women in whom the platelet count was measured had a postoperative thrombocytopenia <100 000/μL. Seven women had severe acute kidney injury (serum creatinine ≥250 μmol/L), and 6 required dialysis. Thirty women (32.3%) were admitted to intensive care units postoperatively. Eleven women (11.8%) received inotropic drugs. Ten women (10.8%) were transferred by ambulance to higher levels of care.The surgeons noted difficulty in 76 operations (81.7%). The duration of surgery was estimable in 80 cases, and was <60 min in 14 (17.5%), 60–119 min in 34 (42.5%) and ≥120 min in 32 (40.0%). The median duration was 90 min, with a range of 23–345 min. Forty-nine women (52.7%) received blood transfusions during their caesarean sections. Medications and procedures used to arrest bleeding (in addition to routine intravenous oxytocin) included ergometrine, misoprostol, prostaglandin F2-alpha, tranexamic acid, B-Lynch brace suture, systematic uterine vessel ligation (devascularisation), and intrauterine balloons  [The most important common risk factors for near-miss related to BDACS were preoperative anaemia , and previous caesarean section . A rising caesarean section rate increases not only the number of caesarean sections currently, but adds women with previous caesarean section to those who will become pregnant in future. This is one of the reasons for calls to prevent the first caesarean section in women . The HIV = 0.05) . Anaemia = 0.05) . Accordi = 0.05) . A numbeIn 76% of near-misses related to BDACS, the caesarean sections were noted by the surgeons as difficult, and early interventions were made intra-operatively. A range of life-saving options was used, usually in combination, to manage the source and effects of bleeding. Medical management included utero-tonics, tranexamic acid, inotropic support and blood transfusion, with recourse to emergency transport if needed, and intensive care, suggesting the elements of a well-resourced functional health service. Surgical options included frequent use of uterine vessel ligation, B-Lynch compression sutures and hysterectomy, both at caesarean section and at second-look emergency laparotomy, suggesting the presence of skilled obstetric surgeons. The few attempts at intrauterine balloon tamponade were mostly unsuccessful, but this may reflect the characteristics of this group of women. Balloon tamponade works best when placed early in severe postpartum haemorrhage after vaginal birth .This study has certain strengths and limitations. Strengths were a robust near-miss definition and a sample size that allowed description of prevalence and relationships. Another strength is the clinical detail obtained from inspection of all women’s clinical notes by a specialist obstetrician. The reliance on record review for clinical information is also a limitation, with some data missing. Limitations include the possibility that some near-miss cases were not included, because the researcher and the trained contacts at the hospitals could not be in attendance at all births. However, it is certain that all maternal deaths from BDACS were included, because these are subject to compulsory confidential enquiries.Further research should focus on detail in risk factors, surgical findings and interventions. An ideal design would be a case-control study, in which near-miss cases are compared with non-near-miss caesarean sections for risk factors, surgical findings and interventions. A similar study should also be done in a resource-poor setting, where access to technological and specialist interventions is restricted, and where there are likely to be more maternal deaths.Our findings show that maternal near-miss from BDACS is uncommon, but severe enough to necessitate extensive life-saving interventions. These require skilled or specialist health professionals, and a functional obstetric service with access to medication, blood transfusion, emergency transport and intensive care units. An important factor in initiating the sequence of interventions is the realisation by the surgeon that the caesarean section is difficult, so that the progression from uneventful operation to near-miss to death can be arrested.


This study tested whether adults with autism spectrum disorder (ASD) are at higher risk for pathological game use than typically developing (TD) adults. Participants included 119 adults with and without ASD. Participants completed measures assessing daily hours of video game use, percent of free time spent playing video games, and symptoms of pathological game use. The results indicated that adults with ASD endorsed more symptoms of video game pathology than did TD adults. This relationship was strong, enjoying 300,000-to-1 odds in Bayesian model comparison. Results also showed that adults with ASD spent more daily hours playing video games and spent a higher percent of their free time playing video games than did TD adults. Even after adjustment for these differences in daily video game hours and proportion of free time spent on games, model comparisons found evidence for a difference in game pathology scores associated with ASD status. Additionally, escapism motives for playing video games was associated with game pathology scores in both ASD and TD adults, replicating and extending a previous report. In conclusion, the risk for pathological game use appears larger in adults with ASD compared with TD adults. These findings point to pathological game use as a potentially important focus of clinical attention in adults with ASD. Autism spectrum disorder (ASD) is characterized by core deficits in social interaction/communication and by restricted interests and repetitive behaviors . These cAmong these factors, excessive video game play may be an important consideration. Because social interactions are often hyper-stimulating or anxiety-provoking for individuals with ASD , video gPathological video game use is a significant clinical issue in its own right, included as “Internet Gaming Disorder”, a “Condition for Further Study”, in the DSM-5 . AlthougPathological game use is a clinically relevant problem in the general population. Adults who play video games to excess have less time for other activities, including work, educational pursuits, hobbies, socialization with friends and family members, and sleep . In addiThe well-being of adults with ASD could benefit from attention to potential symptoms of pathological game use. Individuals with ASD are at risk for social isolation, preoccupation with specific interests, and repetitive patterns of behavior . As suchd >1) difference between groups . All participants provided written consent prior to participating in the study. Participants received $20 in exchange for their participation. Data and materials are available at Participants included 119 adults (16 women), half of whom had a previous diagnosis of ASD, participating in a larger study on violent video game effects . One indParticipants in the ASD group were recruited through an academic medical center specializing in ASD diagnosis and treatment. These participants had a previous diagnosis of ASD based on the center’s clinical care model. The diagnostic process generally includes structured interviews, behavioral observation, and evaluations conducted by psychologists and/or physicians using standardized assessment tools such as the Autism Diagnostic Observation Schedule (ADOS)  and/or tParticipants in the TD group were recruited through the community and the university campus using email, campus flyers, and face-to-face recruitment. Participants in the TD group had no history of neurological or developmental disorders. Most participants in the TD group were also attending school (95% full-time and 3% part-time), and most were also currently employed (3% full-time and 65% part-time). The ASD and TD groups did not differ in age, gender, or IQ. Demographics are supplied in Engelhardt et al. (2015)r ≥ .85 in both groups), so they were combined to make average time spent playing video games per day, calculated by multiplying the weekday value by 5 and the weekend value by 2, summing these scores, then dividing by 7, consistent with previous research  = 2.46 (1.77) and 1.20 (1.55) for the ASD and TD groups, respectively, d = 0.84 , t(117) = 4.61. Bayesian analysis indicated that the observed difference of transformed values was 300,000 times more likely to reflect a hypothesized difference than no difference.Our primary prediction was that adults with ASD would exhibit higher pathological game use scores than TD adults. Adults with ASD indicated more symptoms than did TD adults (untransformed b = 0.22) and for the percent of free time spent playing video games (b = 0.01). This model was strongly preferred to the null effects model by a factor of 6.4 × 1011  = 42.1).We next explored which features served to predict pathological game use scores through the comparison of multiple regression models. These models could include as predictors each of group diagnosis, average time spent playing video games per day, and/or the percent of free time spent playing video games. This lead to seven candidate models, representing the inclusion of one, two, or all three additive effects. All seven models were preferred to the null effects model. Bayes factor model comparisons suggested that the best-fitting model included parameters for diagnostic group (t(116) = 2.61) in favor of an effect for diagnostic group, even after adjustment for the effect of percentage of free time spent playing video games. Similar model comparisons yielded a Bayes factor of more than 1,400,000-to-1 (t(116) = 6.18) in favor of an effect for the percent of free time spent playing video games, even after adjustment for the effect of diagnostic group.By dropping individual terms from this model, one can assess the evidence for each effect conditional on adjustment for the other effects. For example, we dropped the effect of autism group from the above model, comparing the change in model fit with and without this effect. This model comparison yielded a Bayes factor of 3.0-to-1  = 13.5, BF = 37, b = 0.01. Average daily game play did not strongly predict symptoms whether adjusting for  = 0.26) or not adjusting for  = 1.97) the proportion of free time spent playing games. Within the TD group, the best-fitting model modeled symptoms as a function of the additive effects of both proportion of free time  = 2.84) and daily hours spent playing video games  = 3.07), BF = 1.3 × 107, F = 30.75. For this group, average daily game play did provide additional prediction of symptoms, even when adjusting for the proportion of free time spent playing games, BF = 4.73.These regression models were then performed separately within each group to explore how their influence might differ between ASD and TD adults. Scatterplots are provided in M = 2.80 h, SD = 2.14) and spent a greater percent of their free time playing video games  than did TD adults (Ms = 1.04 h (1.20) and 14.25 (15.94), respectively). These effects were large in magnitude, d = 1.01 , t(117) = 5.53, and d = 1.20 , t(117) = 6.53. Bayes factors found strong evidence of group differences in both of these variables: in each analysis, the model with group diagnosis was preferred to the null effects model by more than a factor of 59,000-to-1. Zero-order correlations between all study variables can be seen in b = 0.21, BF = 19.9, t(57) = 3.29) and TD  = 4.24) groups. Additionally, escapism scores were significantly higher in the ASD group as compared to the TD group, d = 0.81 , BF = 846, t(117) = 4.42.In a previous study, This is the first study to examine pathological game use in adults with and without ASD. Consistent with our primary prediction, we found very strong evidence that adults with ASD are at higher risk for pathological game use symptoms than TD adults. Evidence for this group difference remained even after modeling the average amount of time spent playing video games per day and the percent of free time spent playing video games. Consistent with our second prediction, we found very strong evidence that adults with ASD both spent more time playing video games per day and spent a greater percentage of their free time playing video games than TD adults. These findings are consistent with previous research among children with ASD , suggestAdditionally, some individual differences were found to correlate with PGU symptom counts. Among the TD group, both greater hours per day and greater proportion of free time spent gaming were associated with greater symptom counts. Among the ASD group, however, only proportion of free time spent gaming had a strong correlation with symptom counts. Relationships between these measures and symptom counts were weaker in the ASD group than in the TD group; whether this is because the ASD group has greater difficulty with self-report or less regimented use of time is unclear. Still, both groups showed a relationship between escapism motives and PGU symptoms, replicating and extending the result described in The results here suggest that pathological game use may be an important clinical consideration for this population. Adults with ASD may be at high risk for problematic game use due to particular aspects of the behavioral phenotype of ASD. For example, highly restricted interests, preoccupations, and perseverative interests are key diagnostic features of ASD . For somThese data are cross-sectional and do not permit a causal interpretation of the current results. Longitudinal and experimental studies are necessary to determine both the predictors, mediators, and outcomes of pathological game use among individuals with ASD. Additionally, we did not measure the absolute amount of free time participants had. Individuals with ASD may have fewer responsibilities  than TD peers, which may account in part for their greater use of video games. Whether pathological game use is a cause or consequence of these reduced responsibilities is beyond the scope of this study, but it may be interesting to research in longitudinal designs.Another potential limitation of the study is its reliance on self-report measurements. This strategy could be problematic because previous studies have suggested that individuals with ASD have difficulty understanding and reporting on their own experiences . This maAnother potential limitation of the current study is that the measure of pathological video game use was relatively brief and covered a very wide time span . Although this measure has demonstrated good construct validity in prior research , future Although the current study focused primarily on pathological aspects of video game use, there is also room for research into the potential positive aspects of game use among adults with ASD. Parents, researchers, and practitioners are interested in the potential of games for improving social and behavioral functioning in children with ASD . ExpandiThese results suggest that adults with ASD may be at risk for pathological game use. Future research is needed to identify the prevalence, course, predictors, and outcomes of video game pathology in individuals with ASD. The current findings suggest that clinicians should consider assessing these potential problems in clients. It may be important to consider the potential impact of video game use on emotional, behavioral, and social outcomes among adults with ASD.