Retrospective analysis of zinc spark profiles revealed that parthenotes and zygotes that developed into blastocysts released more zinc than those that failed to develop. Prospective selection of embryos based on their zinc spark profile significantly improved developmental outcomes and more than doubled the percentage of embryos that reached the blastocyst stage. Moreover, the zinc spark profile was also associated with embryo quality as the total cell number in the resulting morulae and blastocysts positively correlated with the zinc spark amplitude (R = 0.9209). Zinc sparks can thus serve as an early biomarker of zygote quality in mouse model.Upon activation, mammalian eggs release billions of zinc ions in an exocytotic event termed the “zinc spark.” The zinc spark is dependent on and occurs coordinately with intracellular calcium transients, which are tightly associated with embryonic development. Thus, we hypothesized that the zinc spark represents an early extracellular physicochemical marker of the developmental potential of the zygote. To test this hypothesis, we monitored zinc exocytosis in individual mouse eggs following parthenogenetic activation or  We found a significant correlation between the amplitude of the fertilization-induced zinc spark and successful embryo development to the blastocyst stage. Prospective selection of zygotes based on their zinc spark amplitude improved IVF outcomes and more than doubled the blastocyst percentage, indicating that the magnitude of zinc released at the time of fertilization can be used to predict embryo quality. These studies provide strong evidence that the zinc spark, a conserved hallmark of egg activation in multiple mammalian species including human, can be used to predict IVF outcomes.Recently, zinc emerged as an essential element required for the completion of meiosis and egg activation1234567891032To determine whether there is variability in zinc spark profiles that could underlie differences in the quality of individual eggs, we imaged zinc sparks in eggs collected from the same animal following activation with 5 μM Ca-ionomycin (Ca-Iono). Ca-Iono directly delivers exogenous calcium into the cell and induces a single large intracellular calcium transientTo further test the relationship between zinc spark profile and mouse embryonic development, we analyzed zinc spark profiles in individual eggs following parthenogenetic activation in calcium-free media using Ca-Iono or the apo  form of ionomycin (Iono)Using this data, we performed a retrospective analysis to characterize the zinc spark profile associated with each of the three developmental outcomes. In both Ca-Iono and Iono treatments, the unactivated eggs displayed significantly lower zinc spark amplitudes and integrated intensity of all the groups . Zinc spth percentile and those in the bottom 50th percentile. These were then scored on their ability to progress to the blastocyst stage. Published rates of blastocyst formation for this protocol are low (16%)th percentile progressed through early development . These results indicate that the zinc spark profile is a statistically significant marker of mammalian embryo quality that can be used to select embryos with greater developmental potential.To further test these findings, we prospectively selected embryos based on their zinc spark profile and monitored their development. Because the correlation between zinc spark profiles and embryo quality is more robust in Ca-Iono method compared to Iono method, we activated eggs with Ca-Iono and separated and cultured the parthenotes in two groups based on their zinc spark amplitude: those in the top 50We next investigated if zinc spark profiles observed during IVF were correlated with embryo development. We imaged a group of zona pellucida (ZP) intact eggs during fertilization and tracked their long-term development N = 152, . Among tUnder the experimental IVF imaging conditions, 61 out of 152 eggs (40%) were successfully fertilized as confirmed by pronucleus (PN) formation and further embryo development. A portion of these eggs (42 out of 61) was fertilized within the two-hour imaging window and zinc sparks were observed . HoweverTo investigate whether the zinc spark profile is associated with embryo quality in addition to developmental progression, we next compared the fertilization-induced zinc spark profile to the total cell number of embryos that progressed to the morula or blastocyst stages because this parameter is tightly correlated with embryo qualityet al. accompanying manuscript) and mouse but that the magnitude of zinc release as detected by these fluorescence microscopy methods is closely correlated with embryo quality. These results indicate that preimplantation embryo developmental outcomes can be predicted by selecting embryos based on the zinc spark amplitude or potentially by other quantitative measurement of zinc release. It is worth noting that the eggs were exposed to laser scanning and zinc dye-containing medium for 2 hours during zinc imaging. The adverse effects of the operation led to the production of embryos that contained fewer cells at the blastocyst stage than the controls that had not been imaged. Despite this current limitation, embryos with high and low zinc amplitude and integrated intensity could be directly compared to each other as case and control groups. Within these two groups, the data show that developmental outcomes are positively correlated to zinc physiology. Future studies will require the invention of a non-invasive zinc imaging method to further test the utility of zinc release to developmental outcomes including live birth. Ideally, a non-invasive imaging method would avoid introducing any hazardous factors that disturb the physiological condition of the fertilization medium. These studies that will minimize dye and laser exposure to eggs or embryos are underway.Today the use of Assisted Reproductive Technologies (ART) plays a prominent role in fertility treatment242526231415Capturing the moment when a sperm enters an egg in real time is technically challenging as the sperm can approach the egg from any direction at any time and the mammalian egg is approximately 100,000 times the volume of a single sperm cellet al. a manuscript submitted to Scientific Reports). Although we cannot exclude the possibility that subsequent zinc sparks occurred beyond the 2-hour imaging window, this is unlikely because the amplitude of the intracellular calcium transients, a well-known zinc spark trigger, gradually decline to zero after their initiation . Thus a number of molecular pathways and attributes of embryo physiology may be subject to modulation by zinc fluxes. A compromised zinc spark could be an indirect readout of poor cytoplasmic maturation in eggs. This notion is supported by our findings in human that there is a meiotic maturation-dependent acquisition of the ability of the egg to mount a zinc spark, with gametes arrested at prophase I having on average a smaller zinc spark compared to those at the metaphase II stage .The findings that eggs that are able to release more zinc during activation are more likely to support a healthy and competent developing embryo can be interpreted from many different perspectives. One explanation is that the zinc spark is the major mechanism by which eggs export large amount of zinc during activation472in vitro at least for 2 days. Embryo culture not only has negative impacts on embryo development but also has long-term developmental and behavioral consequences37A holy grail in the field of Assisted Reproduction Technology is to identify reliable non-invasive and quantitative markers that can be used to predict embryo development; however, the selection of embryos is still mainly based on morphological observations, such as division rate, degree of fragmentation and blastocyst formationin vitro development or for cryopreservationTaken together, these two studies show that zinc is necessary to the transition of human eggs to parthenotes and points the way to further interrogation of the inorganic signals that underlie the meiosis-to-mitosis transition. Further, the studies suggest that a zinc profile could be used to accept or reject zygotes for further in vitro experiment that used gametes collected from 4–6 weeks old CD-1 mice. We performed a post hoc analysis and the sample size for mice (minimum n = 3) were based on those required to achieve the chosen statistical significance criterion. All post hoc quantitative analysis of zinc sparks was performed identically using Image J and Prism. No outliers were excluded.A retrospective study was designed to evaluate the correlation between zinc spark profiles and embryo development in mouse. We performed an Animals used for gamete collections herein were handled following the National Research Council’s Animal Care and Welfare Guidelines. These procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at Northwestern University. All the experiments were carried out in accordance with the approved guidelines.2 before imaging or activation treatment.To collect MII-arrested eggs, CD-1 female mice, 6–8 weeks old, were injected with 5 IU of pregnant mare serum gonadotropin . MII eggs were obtained from the oviducts 12–14 h after injection of 5 IU hCG, which was administered 46–48 h after PMSG injection. Eggs were released into HEPES-buffered tyrode-lactate solution (TL-HEPES) supplemented with 5% heat-treated fetal calf serum  followed by treatment with 0.1% bovine testes hyaluronidase for 3–5 min to remove cumulus cells. MII eggs were thoroughly washed and transferred into 50 ml drops of KSOM  containing 0.1% BSA under paraffin oil at 36.5 °C and in a humidified atmosphere containing 5% CO2 for 4 hr. After washing, each egg was transferred to individual KSOM droplets (20 μl) and evaluated using phase contrast microscopy for signs of activation and embryo development. The eggs that did not show pronuclear formation or underwent apoptosis within 8 hr post activation were defined as “unactivated eggs”. The activated eggs were divided into two groups according to their developmental outcomes at 120 hr: (1) the “non-blastocyst” group, consisted of embryos that were activated but not able to reach blastocyst stage; (2) the “blastocyst” group, consisted of embryos that were able to reach blastocyst stage 120 hr post activation.For Ionomycin (Iono) activation experiment, mouse eggs were placed in a 360 μl drop of 20 μM FZ-3 (for zinc monitoring) in Ca-free hCZB medium under oil in a coverslip-bottom imaging dish . Imaging was performed at 37 °C on a TCS SP5 (Leica) confocal microscope  equipped with a stage top incubator , 20× objective and an Ar (488 nm) laser line. Initial fluorescence images were obtained prior to activation. A 40 μl 10× stock solution of 50 μM Iono was introduced to the imaging drop 1 minute after the start of zinc imaging. Images were collected every 4 seconds for 5 minutes. Eggs were then kept individually in cycloheximide (10 mg/ml) and cytochalasin B (5 mg/ml) in KSOM (0.1% PVA) at 36.5 °C and in a humidified atmosphere containing 5% COIn the Ca-Ionomycin (Ca-Iono) activation experiments, mouse eggs were placed in a 360 μl drop of 50 μM FlZ-3 in Ca-free hCZB medium under oil in a coverslip-bottom imaging dish . Imaging was performed at 37 °C on a EVOS cell imaging system  using a GFP light cube with 488 nm excitation or on a Nikon Eclipse system with 488 nm excitation. A 40 μl 10 x stock solution of 50 μM Ca-Iono was introduced to the imaging drop 1 minute after the start of zinc imaging. Images were collected every 4 seconds for 5 minutes. The eggs were then activated and cultured as described in the Ionomycin activation experiment.5 sperm/ml in the HTF medium containing 3 mg/ml BSA. The standard IVF process continued for 6 hours.For the IVF experiments, sperm from the cauda epididymides of proven breeder CD-1 males were collected and purified using a conventional swim up method5 sperm/ml and image acquisition began immediately on a TCS SP5 confocal microscope using 488 nm excitation and an open pinhole. Images were collected every 4 seconds. We shortened the fertilization and imaging time from 6 hours to 2–3 hours to mitigate the damage caused by laser exposure and presence of zinc dye in the medium to both the egg and sperm. Eggs were then kept individually in droplets of potassium simplex optimized medium  and embryo development was evaluated at 8 hours and 120 hours post fertilization. As a control experiment to confirm the fertilizability of the eggs under the imaging condition, we also employed zona pellucida (ZP) free eggs for the zinc imaging and IVF. ZP was removed by brief treatment in acidic Tyrode’s solution .To image zinc sparks during fertilization, mouse MII eggs were allowed to settle in a 50 μl HTF drop that did not contain any serum on a glass-bottom dish . After 5 minutes, regular HTF medium that contained 3 mg/ml BSA and 23 μM FluoZin-3  was slowly added to a final volume of 400 μl, taking care not to disturb the eggs. Capacitated sperm were added to the eggs to a final concentration of 1 × 10To image intracellular calcium and extracellular zinc simultaneously, ZP free eggs were first incubated with 1 μM Fluo4-AM  and 0.02% Pluronic F-127  in TL-HEPES for 20 minutes at 37 °C. Gametes were then washed through dye-free medium and placed in 20 μM FluoZin-3 in HTF medium as described above. Image analysis was performed by defining regions of interest (ROIs) and measuring fluorescence intensity over time using ImageJEmbryos were fixed in 3.8% paraformaldehyde  in PBS for 1 hour at RT. The cells were then permeabilized in PBS containing 0.1% Triton X-100 and 0.3% bovine serum albumin (BSA) for 15 min at room temperature and rinsed twice through blocking solution of PBS containing 0.3% BSA and 0.01% Tween-20 . The eggs were mounted in Vectashield Mounting Media with DAPI . Slides were examined on an SP5 confocal microscope with x40 oil-immersion objective, and near-UV (405 nm) laser lines. Z-stack images were obtained every 2 to 5 μm to span the entire volume of the embryo. Image analysis was performed using ImageJ to count the total cell number of each embryo.p values < 0.05.Values from three or more experiments, performed on different batches of eggs, were used for evaluation of statistical significance. The Prism5.0 software  was used to draw graphs and perform the statistical comparisons using appropriate Student’s t-test, one-way ANOVA or Chi square analysis. Values are shown as means ± S.E.M, and significant differences were considered at How to cite this article: Zhang, N. et al. The fertilization-induced zinc spark is a novel biomarker of mouse embryo quality and early development. Sci. Rep.6, 22772; doi: 10.1038/srep22772 (2016).


Supplemental O2 is prescribed to maintain normoxia and alleviate dyspnea. We sought to better understand the associations between O2 and dyspnea in fILD during the 6-min walk test (6MWT).Exertional dyspnea is a hallmark symptom of fibrosing interstitial lung disease (fILD), and oxygen (O2 (users).1326 fILD patients compose the sample group. Borg dyspnea and other 6MWT variables were compared between subjects who performed the test without (non-users) versus with O2, users perceived greater dyspnea than non-users . Whether subjects became hypoxemic (6-min SpO2 < 89 %) or not during the walk, the results were the same: users perceived greater dyspnea than non-users . Among subjects who did not desaturate (SpO2 drop < 4 %), users walked a shorter distance  but perceived greater dyspnea than non-users . No combination of potentially influential predictor variables entered in multivariate models explained more than 11 % of the variance in dyspnea ratings.There were 812 users and 514 non-users; users were older, more likely to have smoked, had greater body mass index, and had more severe fILD. Despite a similar 6-min SpO2 may lessen, but does not resolve, it. Further research is needed to clarify why fILD patients who use O2 perceive greater levels of dyspnea with activity than O2 non-users.Dyspnea is a complex perception, and in patients with fILD, O Regardless of cause, fibrotic ILD (fILD) is typically progressive and incurable. Exertional dyspnea, the hallmark symptom of fILD, impairs physical functioning and quality of life (QOL) and is often associated with peripheral oxygen desaturation  is commonly used as a measure of submaximal exercise capacity in patients with fILD. Along with distance walked (6MWD), SpO factors . In fILD factors . Investi ratings .2) is commonly prescribed to patients with fILD to maintain normoxia, in hopes of relieving dyspnea , few studies have aimed to decipher the beneficial effects of O2 in these patients [2 compare with those from patients who do not use O2.Although supplemental oxygen  from January 1, 2008 to December 30, 2014. We formed the cohort by querying the NJH research database for patients with fILD who completed at least one 6MWT. Patients with underlying connective tissue disease (CTD) were excluded; thus, the overwhelming majority of subjects had idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia (iNSIP) or chronic hypersensitivity pneumonia (cHP), with diagnoses made in accordance with accepted criteria –10. The 2 drops below 80 %. To maintain reliability in the 6MWT outcome of most interest (distance), we  tried to hold constant as many other variables as possible. Thus, a patient  performed all 6MWT on the same O2 l flow, unless or until he  was unable to walk for a full six minutes without SpO2 falling below 80 %. We included in our analyses data only from patients who walked for a full six minutes. For patients who completed multiple 6MWT, we selected the first test. Ratings for dyspnea and exertion were assessed immediately after completion of the test by the technician and using the CR10 Borg scale  [The 6MWT was conducted similarly in all patients (whether users or non-users), by trained technicians at NJH, according to published guidelines with slight modification . Per staopriate) . The minopriate) .2 was used (users) or not (non-users) during the 6MWT. Student’s t-tests were used for between-groups comparisons of continuous variables. Cochran-Mantel-Haenszel, Chi square or Fisher’s exact tests were used as appropriate for between-groups comparisons of categorical variables. We used Pearson correlation coefficients to express associations between dyspnea ratings and other variables. We used linear regression to examine associations between dyspnea ratings and other variables while controlling for potentially influential predictors. We considered p < 0.05 to represent statistical significance. Analyses were performed using SAS version 9.3 statistical software .Summary statistics were generated for baseline data with the sample stratified on whether O2 at six minutes (88.1 % vs. 88.7 %), users perceived significantly greater dyspnea than non-users , dyspnea ratings among users were significantly higher than in non-users. The same was true for subjects whose SpO2 remained 89 % or greater for the duration of the 6MWT: despite identical mean SpO2 values at six minutes (91.4 % vs. 91.4 %), dyspnea ratings were significantly higher among users than in non-users , and dyspnea ratings were higher among users than in non-users . For the 473 subjects (303 users and 170 non-users) with IPF, the SpO2 values at six minutes were the same (86.7 % vs. 86.9 %), and dyspnea ratings were higher among users than in non-users . Among the 118 subjects whose SpO2 never dropped by more than three points from baseline (rest), although minute-six SpO2 was higher in users than in non-users, dyspnea ratings among users were significantly higher than in non-users (Table Results were similar for the 883 subjects (572 users and 311 non-users) with a history of smoking: the SpO2 use remained a significant predictor of dyspnea rating. As revealed by the R-squared values, none of the combinations of variables explained more than minimal variance in dyspnea ratings.Results from the linear regression analysis are presented in Table 2 during a 6MWT consistently experienced more severe dyspnea than those who did not use O2. Data on the effects of O2 in patients with fILD are surprisingly limited, and much of the information on the potential benefits of O2 that is used in clinical decision-making with fILD patients, is based solely on scientific rationale or borrowed from the COPD literature. In two Letters to the Editor, investigators described the results of retrospective studies in which they examined the within-subject beneficial effects of O2 on various outcome measures collected around 6MWTs [2 was administered according to a semi-quantitative algorithm aimed at maintaining SpO2 at (closer-to) acceptable levels, distance walked, nadir SpO2 and Borg score improved (by one point) over values obtained during a baseline 6MWT. The results were similar for the subgroup of subjects with either idiopathic pulmonary fibrosis (IPF) or NSIP, in whom O2 administration resulted in improved dyspnea (median Borg scores dropped from 4.25 to 3.25) and in the nadir SpO2 increasing from 75 to 83 %. In the other study of 70 subjects , Frank and her colleagues found that, compared to a baseline 6MWT performed on ambient air or with inadequate O2 flow, administering O2 led to increased distance walked and improved nadir SpO2, but dyspnea did not change (mean Borg score 4.8 ± 2.1 vs. 4.5 ± 2.2) [We examined patients with fILD and found that those who used Ond 6MWTs , 6. In o2 affirms it is far from the main contributor. Other contributors include neural inputs arising from receptors in the airways and lung parenchyma, peripheral locomotor and respiratory muscles, and central and peripheral chemoreceptors, along with corollary neuronal discharge arising from the brainstem and cortical motor centers [Dyspnea is a complex perception that depends on the integration of multiple inputs from several sources. Blood oxygen level is but one of those sources, and the weak correlation we observed between dyspnea rating and minute-six SpO centers .2 consistently started with a higher SpO2 than non-users, and although SpO2 declined to a greater degree during the walk in users than non-users (9 % vs. 6 %), minute-six SpO2 was the same in both groups (88 %). Perhaps has dyspnea more to do with SpO2 decline from baseline than the absolute SpO2 at the time of dyspnea rating? Our results suggest this is not the case: the correlation between dyspnea and SpO2 decline was the same (weak) as the correlation between dyspnea and minute-six SpO2. However, in the subgroup of patients who did not desaturate at all during the walk (SpO2 decline < 4 points), despite O2 users having a higher minute-six SpO2 than non-users (95 % vs. 93 %), O2 users perceived greater dyspnea (mean Borg scores 3.4 vs. 2.4).In our study, patients who used O2 , each of these SpO2 measures was a significant predictor of dyspnea, and in each model, O2 use remained an independent predictor of dyspnea. However, each model explained minimal variance in dyspnea scores—again, confirming that dyspnea relies on more inputs than simply blood oxygen.In our statistical models controlling for either minute-six (data not shown) or decline-from-baseline in SpO2 l flow, unless or until he is unable to walk for a full six minutes (in which case flow is reset for subsequent 6MWTs). Because of this practice, on certain occasions, patients at our center may perform their 6MWT on O2 l flows below what they use with exertion at home. In our study, this “intentional under-dosing” of O2 flow—to maintain reliability—could have driven dyspnea ratings up in O2 users. Unfortunately, with this data set, we are unable to determine when this under-dosing might have occurred. Regardless, this “intentional under-dosing” explanation would seem not to apply to subjects whose O2 was dosed adequately enough to maintain an acceptable SpO2 throughout the test, including the over 600-patient subgroup whose SpO2 remained above 88 %, or the greater than 100-patient subgroup whose saturations did not decline at all. In both these subgroups, O2 users rated their dyspnea as more intense than non-users.We suspect users in our study perceived greater dyspnea intensity than non-users because of a complex interaction of elements, including those related to conduct of the 6MWT and perhaps certain neurophysiological factors. At our center, in an attempt to maintain reliability in the 6MWT outcome of most interest (distance), we try to hold constant as many other variables as possible. Thus, a patient performs all 6MWT on the same O2 at home either carry or pull their O2 delivery device while completing their 6MWT. Having to move this excess weight over distance—or altered chest wall mechanics resulting from carrying or pulling the device—could add to dyspnea. To our knowledge, this has yet to be examined, but we believe it  deserves investigation. If carrying or pulling the delivery device is found to add significantly to dyspnea intensity, this could be a target for therapeutic intervention.Also at our center, patients who use O2 was an especially inaccurate reflection of it in this cohort; we doubt this was the case, but if it were, we would expect the inaccuracies to affect both users and non-users equally. We excluded patients with underlying CTD in the hopes of limiting the influence of vascular abnormalities like Raynaud’s or pulmonary hypertension.Another alternative explanation is that dyspnea truly depends greatly on arterial oxygen but SpO2 or other aspects of oxygen delivery also must be considered as potential explanations for our results. While exerting (and into recovery), subnormal lung compliance in patients with ILD induces rapid-shallow breathing. The physical sensation—and mental/emotional impact—of this breathing pattern, which occurs to some degree when patients with ILD exert to any degree, is expected to influence dyspnea ratings. How subjects internally considered, weighed and integrated each component  as they made their ratings for “breathlessness” is unknown. Compared with non-users, O2 users had lower FVC% and, by deduction, lower lung compliance than non-users—a factor expected to hasten and heighten rapid-shallow breathing. We do not measure respiratory rate during the 6MWT at our center, so we are unable to determine whether O2 users had higher respiratory rates than non-users. Additional studies aimed at discerning whether or how much respiratory rate  contributes to exertional dyspnea ratings are needed. Although we are unable to comment on directly-observed respiratory rate, FVC% as a marker of lung compliance could be considered a reasonable surrogate for respiratory rate. In statistical models that included O2 use, and controlled for FVC%, O2 use remained a significant predictor of dyspnea. In a comprehensive appraisal of dyspnea in patients with chronic interstitial lung disease, Faisal and colleagues observed that dyspnea intensity during exertion climbed as inspiratory neural drive increased and tidal volume became constrained  [Various physical factors unrelated to the lungs, SpOxercise) .2 users might have contributed to physical inactivity and deconditioning. Given the practical challenges of using O2 and the possibility that it prohibits patients from living a more active, carefree lifestyle, we suspect that, on average, fILD patients who require O2 are less physically active (and thus less well-conditioned) than patients who do not require O2. De-conditioned skeletal muscles are less efficient and fatigue-resistant than conditioned muscles, and peripheral locomotor [2 users walked shorter distances during the 6MWT; this was true even for the subgroup that did not desaturate to < 89 %. Whether the shorter distance walked was due to deconditioning or some other factor(s) is unknown; however, deconditioning could well explain the interesting and perhaps somewhat paradoxical finding that dyspnea severity was inversely correlated with 6MWD: subjects who walked further, were better conditioned and thus perceived less dyspnea.Another consideration is whether the greater disease severity in Oocomotor  muscle f2: it will likely decrease dyspnea (compared with not using O2), but because dyspnea is driven by so many inputs (with SpO2 being just one), O2 will not resolve dyspnea. Further research is needed to better understand the mechanisms driving dyspnea in patients with fILD and to devise strategies to lessen it.Dyspnea is a complex perception that impacts the lives of patients with fILD. It is important for a patient with fILD to know what to expect when being prescribed O


Quantitative X-ray induced radiation damage studies employing a model protein–DNA complex revealed a striking partition of damage sites. The DNA component was observed to be far more resistant to specific damage compared with the protein. 1—C and sugar-phosphate C—O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein–DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07–44.63 MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N A collection of careful systematic studies has increased awareness of the issues and has served to provide practical solutions, from optimized data-collection strategies , resulting in loss of diffraction data quality from ‘global radiation damage’, observed in reciprocal space as loss of diffraction intensity, loss of resolution, unit-cell expansion and, usually, increased mosaicity  of incoming X-rays. The resulting high-energy photoelectron, typically with a range of 3 to 4 µm for the incident X-ray energies used in MX  inflict this damage  is employed to probe the effects of X-ray induced radiation damage and its quantitative distribution among the various components at the atomic level.via a concentration-dependent ‘on’ and ‘off’ switch. This type of transcriptional regulation is a cornerstone of all biological systems, and requires the recognition and intimate interaction of proteins and specific DNA target sites to form productive complexes.The C-protein C.Esp1396I is a member of a large class of helix-turn-helix proteins that act as transcriptional regulators of gene expression in both prokaryotic and eukaryotic systems. C.Esp1396I finely regulates a bacterial restriction-modification system, providing a delay in endonuclease expression 3clc, resolution 2.8 Å ethanesulfonic acid (MES), pH 7.5, 25% 2-methyl-2,4-pentanediol (v/v) and 40 mM MgCl2. They were cryoloop mounted and vitrified directly under a 100 K N2 gas stream, with no additional cryoprotectant agent being added to the mother liquor.Expression, purification and crystallization of the native C-protein–DNA operator complex C.Esp1396I was performed as described earlier  and an ADSC Q315R mosaic CCD detector. A pinhole designed by R. Ravelli and F. Felisaz (EMBL Grenoble) was utilized to produce a 25 µm circular low-divergence beam at the sample. The beam size before the pinhole was 0.212 mm  × 0.279 mm  (Gaussian profile FWHM). A crystal of C.Esp1396I (30 µm × 30 µm × 10 µm) was oriented with the beam direction parallel to the smallest crystal dimension (10 µm). The crystal–detector distance was fixed to 390 mm throughout.Eight datasets were collected from this C.Esp1396I crystal, each consisting of 100 frames of 1° rotation, each over the same 100° wedge of the crystal, with exposure times per frame and beam attenuation being varied according to the values shown in Table 12.2.a), RADDOSE-3D  and eighth  datasets. The intensity profile of the 25 µm-diameter beam was modelled as a Gaussian with FWHM values as detailed above. The photon flux prior to attenuation was estimated to be 5 × 1010 photons s−1 throughout the experiment, and the resulting diffraction weighted dose (DWD) values . All the results on radiation progression were plotted against the resulting DWD values. A plot of the mean intensity values per whole dataset showing the radiation damage induced decay can be found in Fig. S1 of the supporting information.et al., 2006The calculation of the crystal absorption coefficients in RADDOSE-3D included the water and the heavy-atom content from the crystallization conditions  restraints were used throughout for the four protein subunits present, except in the flexible loop region corresponding to residues 43–46  individually, which were then all scaled using the CCP4 program SCALEIT. A set of seven Fourier difference maps Fno, − Fo,1, for n = 2,…, 8, were then calculated using the CCP4 program FFT  have calculated electron densities (in electrons Å−3) of s times the standard deviation (σ) from the mean electron density of the Fourier difference map. Thus the higher the ±σ-contouring level in Coot at which a difference map peak remained present, the greater the electron density gain/loss from the mean map density was present at that specific point in space.Fourier difference map peaks coincident with the protein–DNA complex indicated potential specific damage regions which were investigated on a case-by-case basis. For a given dose, points within the unit cell situated on an electron density σ-contouring level , representing potential specific damage sites within the C.Esp1396I structure, the Python. This was designed to filter the difference map peak datasets, to (a) remove positive peaks relating to electron density increases between the first and a given later dataset (to isolate sites of electron density loss resulting from X-ray radiation damage), and (b) remove electron density peaks further than a given threshold distance (Å) from the protein–DNA complex (specified by the user). This threshold was initially chosen to be 2 Å since filtering with threshold distances of 2.5 Å or greater was observed to include peaks which were clearly noise, and a ‘by-eye’ investigation of the remaining difference map peaks for the first two difference map datasets (for which the number of filtered peaks was not too high) led to the observation that all the difference map peaks corresponding to clear specific damage were below the 2 Å threshold in these datasets.The seven large datasets of difference map peaks were then processed using a custom-made script, written in the object-oriented scripting language 2⋯CH2) . In the case of covalently bonded atoms, carboxyl acids have C—O bond lengths of ∼1.36 Å, with other C—O bond lengths being ∼1.43 Å, the C—S bond length has been reported to be ∼1.82 Å, the C—N bond length ∼1.47 Å, and the C—C bond length ∼1.54 Å  to facilitate the quick removal of those which were not suspected to be specific damage to the molecules in the C.Esp1396I crystal. For each accepted difference map peak, the closest protein–DNA complex atom was calculated, and the spatially local maximum electron loss per Å3 (as discussed above) for that peak was assigned to this selected atom.The command line operated script allowed a more efficient systematic sweep through the remaining difference map peaks  was not known since it is equal to the total number of electrons in the unit cell, which could in principle be estimated from the amino acid composition and the mM buffer concentration. However, due to the uncertainty in the true solvent concentration within the crystal solvent channels, such a calculation would carry an unknown systematic error. The FFT-output electron density at real-space crystal points is given on an arbitrary fixed scale . For crystals differing in protein, DNA or unit-cell solvent composition, this scale factor would be different  for structural representation and CCP4mg , molecular replacement was performed with A and B, chains C and D) bound to a 35 bp DNA operator sequence (chains E and F) . Non-symmetrical binding between protein chains B and C at the dimer–dimer interface, and a pseudo-symmetrical DNA sequence, prevent true NCS in the complex. The dsDNA is distorted in the complex, due to binding of protein dimers to each operator site, resulting in minor groove compression (leading to a 50° bend at each binding site) and large major groove expansion at the DNA sequence centre  Fig. 3 (McGeeha3.2.a)–5(f)] and DNA [Figs. 5(g)–5(l)The automated scripts provided a means to successfully filter difference-map noise peaks from the system under investigation Fig. 4 but theya)–5(f) reveal the specific damage dynamics of Glu54, Met57 and Asp64 in chain D (left to right) with respect to absorbed dose. Clear loss of electron density localized around the carboxyl groups, due to decarboxylation, is shown for Asp64 and Glu54, and the rate of carboxyl electron density loss with dose appears marginally greater for Glu54 than for Asp64. Figs. 5(a)–5(f) also show specific damage to Met57, where electron density loss and side chain disorder can be observed localized on the methylthio group over increasing doses. Although generally fewer difference peaks are observed on the DNA, there are several locations of specific damage on it too [Figs. 5(g)–5(l)F would generate a single-strand break with significant biological consequences. Additionally, at higher doses [Figs. 5(k) and 5(l)In terms of protein, Figs. 5 and 6(b)specific damage onset is at significantly higher doses for DNA than for protein within the complex, and that specific damage is more evenly distributed between the four base types than amongst the protein residues.The distribution in DNA specific damage between the four nucleotide types is also shown and it is immediately apparent upon comparison with the protein data [Figs. 6 across the protein–DNA complex with respect to accumulating dose  the damage site frequency increases, and (b) the average electron density loss magnitude increases, as expected. Furthermore, it is clearly apparent that lower-dose , and that even at the highest dose . Furthermore, close correspondence is observed in the average isotropic B-factor dose-dynamics between protein chains A and D , protein chains B and C  and DNA strands E and F (both 0.0041 Å2 MGy−1), indicating the rotational near-NCS around the DNA 35 bp sequence centre.Fig. 83.5.3 with respect to accumulated dose was performed for different residue types. For each subplot shown in Fig. 9Analysis of the electron loss per Åa)B and C than for the corresponding residue in chains A and D. Furthermore the dynamics of electron density loss for Met57 are qualitatively similar for chains B and C, and also for chains A and D reflecting the near non-crystallographic symmetry within the complex.It is evident that clear differential specific damage rates are present not only between different residue types but also within a given residue type. For example, in Fig. 9(d) with Figs. 9(a)–9(c)Comparing Fig. 9(B-factor change against dose plots (not shown), the expected correlation was found between the rate of B-factor increase and the rate of electron density loss with respect to dose B-factor increase and the average rate of electron density loss. However, most DNA damage sites are observed to have both low average B-factor increase and electron density loss rates, providing additional evidence of differential specific site dose-dynamics between the protein monomers and DNA strands.In Fig. 8A comparison of the heterogeneous spatial distribution in both protein residue average 4.et al., 2007et al., 2000et al., 1999By using an innovative highly streamlined and automated pipeline for the identification of X-ray induced structural damage patterns, we have established the existence of differential specific damage rates between the protein and DNA components of a model complex C.Esp1396I with respect to dose at 100 K. Whereas other work has studied specific protein and DNA damage in isolation  in the DNA. The location of this SSB is interesting as it correlates with a region of the DNA that is both AT-rich and under significant strain as a consequence of large-scale deformation due to protein binding. It is possible that such strained geometries enhance the radiation damage effects in DNA. This observation merits further investigation since it would have major biological consequences, as the wrapping of eukaryotic DNA around histones to form nucleosomes in part relies on the distortion of DNA around AT-rich sites such as this. The DNA radiation damage observed here could well result from previously suggested mechanisms such as low-energy secondary electron attachment to bases [a suggested precursor to SSBs (Simons, 2006et al., 2000et al., 2003It is again important to note that the low-energy electron damage to DNA components reported by Sanche and co-workers (Boudaïffa B-factor change of a particular chain gives a measure of the real-space averaged disorder present per protein–DNA chain between copies of the C.Esp1396I complex in different unit cells of the crystal. This metric indicates that, on average, the protein components become relatively more disordered with dose than does the DNA, suggesting that the protein is damaged by X-ray radiation at a faster rate than is DNA. Since any global radiation damage effects present would affect the protein and DNA components to the same extent, this could provide a suitable measure to compare specific damage susceptibility between protein and DNA. Our methodology is further validated by the identification of similar damage at palindromically equivalent sites in both protein and DNA components for this particular complex.We have also demonstrated that the normalized mean Further studies on a range of protein–DNA and protein–RNA complexes would allow these metrics to be tested rigorously and would reveal if our observations provide some general rules for X-ray radiation damage to biological nucleoprotein complexes. To aid this goal the custom-made scripts which allow efficient specific damage searching and enable consistent noise peak filtering could be utilized with Fourier difference maps generated from other systems. These investigations are only tractable through the use of a robust semi-automated pipeline such as the one developed here.We can speculate that the molecule that holds our genetic blueprint has evolved to be more radiation-resistant than other cellular components, and this may not be so surprising since sacrificial proteins can be more easily replaced than lost genes. Studies on a wide range of nucleoprotein complexes utilizing these partially automated methods should provide further insight into these intriguing observations.10.1107/S1600577514026289/xh5044sup1.pdf1: An extension of Figure 7 representing specific damage distribution throughout the C.Esp1396I complex for structures derived from all datasets across the full dose range 6.2 MGy to 44.6 MGy. 2: Mean intensity decay for the 8 C.Esp1396I datasets.. DOI: 4x4bPDB reference: 4x4cPDB reference: 4x4dPDB reference: 4x4ePDB reference: 4x4fPDB reference: 4x4gPDB reference: 4x4hPDB reference: 4x4iPDB reference:


E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular & periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of “YQF” triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi’s role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence.Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion.  Most interestingly, structure based phylogeny shows that it falls into porin superfamily. To further understand the functional role of Wzi, beyond being a lectin, we performed molecular dynamics (MD) simulations of Wzi and its mutants embedded in palmitoyloleoylphosphatidyl ethanol (POPE)/Palmitoyloleoylphosphatidyl glycerol (POPG) lipid bilayer hydrated in explicit solvent. We have shown here for the first time that Wzi can also act as a bidirectional passive water specific transporter with a diffusion permeability (pd) of 2.8×10−15 cm3.s−1, suggesting that it may play a role in osmoregulation. Strikingly, this is the first β-barreled porin found to be specific for water molecules owing to its narrow lumen.Here, we aim to explore the conformational dynamics of Wzi to facilitate the understanding of K30CPS. Thus, the dual role for ‘YQF’ triad, as a water entry point at the extracelluar side and as a K30CPS anchorage point, is revealed. Results also offer stereochemical rationale for previous experimental data on the functionality/non-functionality of three H1 helix mutantsWe have identified that conserved residues at five different diffusion points of the extracellular and periplasmic faces of the barrel along with a conserved luminal hydrophobic plug play a key role in regulating Wzi water diffusion. At each entry point, water selective hydrogen bonding network regulates both water entry and exit. Three points at the extracellular side and two at the periplasmic side are found to be involved in regulating the water entry and exit through the barrel. At the periplasmic region, ‘RED’ motif acts as a major exit and entry point. At the extracellular side, while major contribution to barrel entry is through the notch region involving ‘NQQ’ motif, exit is equally through notch region and ‘YQF’ motif. Intriguingly, such a ‘YQF’ motif is observed in sodium-galactose cotransporters12CPS to ‘YQF’ or with its insertion onto the membrane may aid in reducing the bacterial virulence.Results also pinpoint conserved electrostatic, hydrophobic and hydrogen bonding interactions between extracellular L5 residues and the lipid bilayer which play an important role in the insertion of Wzi onto the membrane. Thus, a drug molecule that can either interfere with the water transport of Wzi or the attachment of K30As the similarity between β-barrel motif of Wzi and porins offers a clue to an additional functional role for Wzi apart from being a lectin, we have constructed a structure based phylogenetic tree (SBPT). To our surprise, results show that Wzi comes under outer membrane transporter superfamily  by shariMD simulations carried out to further establish the functional role of Wzi  as a porLikewise, at the periplasmic side, water enters into the barrel through 2 different points that are located at the interspace of the β-barrel and α-helical bundle (marked as points 4 & 5 in d)−15 cm3.s−1.Intriguingly, complete crossing of water molecules across the barrel, from the extracellular side to the periplasmic side  and viceWater diffuses into the barrel in a highly regulated manner mediated by hydrogen bonding interactions both at the extracellular and periplasmic sides through the 5 different water conduction points as detailed below.Tyr…Gln hydrogen bond mediated water entry: Initial diffusion involving point 1 takes place around 5 ns through the interface between Phe(F427) and Tyr(Y380) & Gln(Q102) residues that are in hydrogen bond “locked” state . During Water diffusion through Asn…Gln hydrogen bond network: Water entry via the notch is regulated by the pore formed by the extracellular residues, Asn(N153), Arg(R190), Pro(P191), Asn(N226), Gln(Q227) & Gln(Q378) that is in closed state point 2, . As a waThreonine flipping: At the entry point 3, water diffusion happens through hydroxyl group flipping (NF = 0.92) of T457 . The mecBesides the water diffusion into the barrel from the extracellular side, water also diffuses from the periplasmic side of barrel during the simulation through 2 different entry points.Regulation of water diffusion through bifurcated salt bridge: At the entry point 4a of the periplasmic side, water regulation is mediated by R93 & E474 of the β-barrel and R84 of H3 that together form bifurcated salt bridge. When a water molecule appears in the vicinity of these residues, it hydrogen bonds alternately with the amino groups of R84 & R93 and carbonyl group of E474 and invades slowly into the barrel without disrupting R93 & E474 and R84 & E474 salt bridges (NF = 1) . A seconWater entry through negatively charged periplasmic residues D42 & E303: Water entry through point 5a at the periplasmic side  is initiWater entry through H47-P215-Q247 triad: Point 5b at the periplasmic side favors only 2 water entries into the barrel  through Extracellular water exit: Not surprisingly, mode of water exit at the extracellular side involves same residues as the water entry . At the Periplasmic water exit: Among the periplasmic entry points 4a & 4b, the latter promotes (32 water molecules) more water exit compared to the former (7 water molecules). Points 5a & 5b contribute more towards water exit (31 & 18 molecules respectively) compared to the entry (15 & 2 molecules respectively).Eventually, the extracellular and periplasmic residues that are involved in water entry are involved in exit also .Development of transient water wires  associatA hydrophobic plug present at the barrel center and protruding towards the lumen, governs the water transport across the channel. The plug encompasses conserved aromatic residues  W174 & WTo elucidate the reason behind experimentally observed difference in the functional/nonfunctional forms of pentaglycine helix H1 mutantsIt is noteworthy that all the above pentaglycine mutants retain their α-helical conformation . This isAnother important observation is the tight association of a positively charged potassium ion onto the extracellular surface of Wzi. The ion starts entering into lumen ~58 ns by coordinating with the side chain carbonyl group of Q102 and through subsequent conjoint coordination with the hydroxyl group of Y380. It further moves inside the channel at the interspace between F427 & Y380…Q102 of ‘YQF’ triad  coincidiWzi-WT simulation indicates that L5 is not involved in the water conduction pathway. However, it plays a major role in membrane insertion. Though the L5 residues are initially aligned close to the barrel and are not involved in any interaction with the membrane B and 8A,E. coli outer membrane lectin, Wzi also functions as bidirectional water selective porin, offering justification for its β-barrel fold. Note that although Wzi is specific for water conduction like aquaporins, both are structurally very different, as the latter possesses an alpha helical structure.We have shown here for the first time through structure based phylogeny and MD simulations that −15 cm3.s−1 that is 2 times lower than that of aquaporin monomers as reported in previous MD simulations14Five water diffusion points are identified (two at the periplasmic and three at the extracellular sides), through which, the water entry/exit from extracellular & periplasmic sides occur in a highly regulated manner in Wzi , S3 & S4CPS binding motif. Interestingly, ‘YQF’ triad also participates in potassium ion conduction into the ion-binding pocket at the extracellular side , water molecules move from extracellular to periplasmic side to maintain the osmotic pressure. Thus, water transport across Wzi depends on the concentration of K30CPS on the bacterial surface. Inferred association between additional water diffusion pattern and deleterious nature of periplasmic helix mutants H1mut2 & H1mut3 is in supportive of the role of Wzi in osmoregulation. In fact, relationship between capsular polysaccharide production and osmotic pressure is an already existing fact2021CPS onto Wzi and pinpoints the importance of extracellular L5 in the anchorage of Wzi onto the outer membrane. Thus, disrupting the interaction between K30CPS & ‘YQF’ triad or altering the Wzi water diffusion or disrupting the interaction between L5 & membrane may be attractive strategies to reduce the bacterial virulence.Based on the bidirectional water permeation property of Wzi, we propose here a mechanism by which it may act as an osmosensor. Under hyperosmotic condition, wherein, the K30Besides establishing the role of Wzi as a porin, this study further establishes the importance of molecular dynamics and structure based phylogeny in identifying the unknown function of a protein. Thus, these techniques together can be used to identify the function of hypothetical proteins as well as understand their functional evolution.As Wzi is highly divergent with respect to other family of porins (midnight zone with less than 12% of homology) at the sequence level, structure based alignment is performed to illustrate the role of Wzi as a porin. Using DALIPSI-BLAST29As the 15 residues of extracellular loop 5 L5,  are miss31E. coli, a heterogenous lipid bilayer is used with POPG and POPE in a 1:3 ratio as discussed elsewhere+ ions and 13 Cl−) is added to the solvent using Monte-Carlo method18Wzi systems for molecular dynamics simulations are generated using CHARMM-GUI18333435MD simulation protocol: During the first step of the equilibration run, each system is subjected to 5000 cycles of steepest descent minimization. This is followed by 50 ps of MD simulation using NVT ensemble using leapfrog algorithm with 1 ps time interval, wherein, Berendsen thermostat4450 ns simulations for all the aforementioned systems are also carried out using constant pressure and temperature (CPT dynamics) with CHARMM molecular dynamics program18Quantification of barrel water: To quantify the total number of barrel water molecules per ns, water molecules that fall within the minimum and maximum limits of X & Y coordinates of the entire β-barrel and between −10 Å & 10 Å from the center of the barrel along the Z-axis are summed over every ns.Quantification of barrel permeation events and diffusion permeability: Barrel permeation is calculated at 5 separate points numbered from 1 to 5 (3 at the extracellular side and 2 at the periplasmic side)  as discucm, Ycm, Zcm) is calculated by considering the atoms corresponding to the X, Y & Z coordinates of three amino acids that are involved in the regulation of water diffusion. Subsequently, a local XY-plane (diffusion plane) is defined with the upper and lower bounds of  and  respectively. At the extracellular side, a water entry into the β-barrel  is considered only when a water molecule that has Z coordinate greater than Zcm at nth ps subsequently falls within the defined XY-plane with Z-coordinates between Zcm and (Zcm − 3.5 Å) (Z1) and soon after it crosses (Zcm − 3.5 Å) (cm and (Zcm + 3.5 Å) (Z2) at nth ps, falls within the defined XY-plane with Z-coordinates between Zcm and (Zcm − 3.5 Å) (Z1) and soon after it crosses (Zcm − 3.5 Å)  . Subsequ− 3.5 Å) .cm at nth ps, subsequently falls within the defined XY-plane with Z-coordinates between Zcm and (Zcm + 3.5 Å) (Z2) and soon after it crosses (Zcm + 3.5 Å) (cm and (Zcm − 3.5 Å) (Z1) at nth ps, falls within the defined XY-plane with Z-coordinates between Zcm and (Zcm + 3.5 Å) and soon after it crosses (Zcm + 3.5 Å)  . Subsequ+ 3.5 Å) . Detailed) as described elsewhereCalculated permeation events are used to estimate the diffusion permeability  corresponding to each entry point for every 1 ns trajectory. Subsequently, it is checked whether a specific water molecule is between Zcm and (Zcm + 5 Å) or between Zcm and (Zcm − 5 Å). Water entry and exit is calculated by a protocol similar to diffusion entry/exit calculations, but, only covering ±5 Å distance in the Z-dimension. For instance, if the water molecule is between Zcm and (Zcm + 5 Å), it is counted as an extracellular entry soon after it crosses (Zcm − 5 Å) and the reverse is considered for exit. Presence of the water molecule within the XY plane of the appropriate entry point (i.e Xcm ± 3.5 & Ycm ± 3.5) during the above calculation is also considered. Similarly, at the periplasmic side, water molecule moving from (Zcm − 5 Å) to (Zcm + 5 Å) is counted as the entry and the reverse is taken into account for exit.Quantification of barrel water entry and exit: Calculation of barrel water entry and exit is performed by taking into account the water molecules that fall within 5 Å range of center of mass  between the water oxygen atoms and the side chain/main chain electronegative atoms of the amino acids.Barrel and helix 1 (H1) interfacial water calculation: To calculate the number of water molecules at the interface of H1 and the barrel, the maximum & minimum values corresponding to X & Z coordinates are calculated by considering the appropriate H1 and barrel residues . On the PyMol  is used for the visualization of all the simulated trajectories and VMDHow to cite this article: Sachdeva, S. et al. Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin. Sci. Rep.6, 28157; doi: 10.1038/srep28157 (2016).


Eukaryotic cells commonly use protein kinases in signaling systems that relay information and control a wide range of processes. These enzymes have a fundamentally similar structure, but achieve functional diversity through variable regions that determine how the catalytic core is activated and recruited to phosphorylation targets. “Hippo” pathways are ancient protein kinase signaling systems that control cell proliferation and morphogenesis; the NDR/LATS family protein kinases, which associate with “Mob” coactivator proteins, are central but incompletely understood components of these pathways. Here we describe the crystal structure of budding yeast Cbk1–Mob2, to our knowledge the first of an NDR/LATS kinase–Mob complex. It shows a novel coactivator-organized activation region that may be unique to NDR/LATS kinases, in which a key regulatory motif apparently shifts from an inactive binding mode to an active one upon phosphorylation. We also provide a structural basis for a substrate docking mechanism previously unknown in AGC family kinases, and show that docking interaction provides robustness to Cbk1’s regulation of its two known in vivo substrates. Co-evolution of docking motifs and phosphorylation consensus sites strongly indicates that a protein is an in vivo regulatory target of this hippo pathway, and predicts a new group of high-confidence Cbk1 substrates that function at sites of cytokinesis and cell growth. Moreover, docking peptides arise in unstructured regions of proteins that are probably already kinase substrates, suggesting a broad sequential model for adaptive acquisition of kinase docking in rapidly evolving intrinsically disordered polypeptides. The structure of an ancient signaling complex in the Hippo pathway (an NDR/LATS family kinase) reveals a distinctive activation switch, and shows how this regulator recognizes short and rapidly evolving protein motifs. The core organization of systems that relay information inside cells is preserved across vast evolutionary distances. Thus, detailed characterization of these systems’ crucial modules can provide insight into the emergence and adaptation of signaling pathways, and illuminate broadly relevant mechanisms that control cells’ diverse processes. In this paper we describe the first three-dimensional structure of a protein kinase–coactivator complex from budding yeast that is a key component of “hippo” signaling pathways, which direct cell proliferation, fate, and architecture in a wide range of eukaryotes. We show that this kinase–coactivator complex is a dynamic switch controlled by binding events distant from its active site, and that the kinase recognizes specific short motifs in disordered regions of target proteins by a previously unknown mechanism. This substrate docking interaction provides in vivo robustness to the kinase’s regulation of its known targets, and identifies likely new substrates that expand our view of this hippo pathway’s role in cell division. Moreover, during the course of evolution, the short motif that interacts with the kinase’s docking surface appears in rapidly changing intrinsically disordered regions of a number of proteins that are probably already in vivo substrates. Thus, our findings support the idea that proteins evolve more robust functional links to the signaling networks that control them by acquiring short peptide motifs that interface with key conserved signaling modules. The core organization of these networks is conserved over a billion years of evolution, with related forms described in animals and fungi –3. In thDrosophila, LATS kinases in complex with Mob1 family proteins phosphorylate YAP/Yki family transcriptional coactivators, which promote cell division and resistance to apoptosis  is centrally important for the transition from mitosis to G1 ). GST fusions were purified on glutathione Sepharose beads followed by dialysis into Cbk1 buffer.Cbk1 substrates containing the docking motifs or substrate sites were bacterially expressed as MBP or GST fusions. For pulldown assays, MBP-fusion lysates were incubated with amylose resin (New England Biolabs) on ice for 15 min, followed by washing with PBS  HM motif .2, and 2 mM TCEP. All samples used for crystallization contained an inactivating mutation (D475A) in Cbk1, while some Cbk1 constructs also contained a phosphorylation-mimicking mutation in the HM (T743E). The rational for the D475A mutation was to eliminate the autophosphorylation activity of Cbk1, while the T743E mutation was introduced to capture the kinase in an activated state. Crystallization conditions were found by using a custom in-house PEG crystallization screen in microbatch under oil (1:1 silicon oil:paraffin oil) set-ups at 23°C. All crystals were treated with 10% glycerol before flash freezing in liquid nitrogen. Data on frozen crystals were collected at 100 K on the PXI and PXIII beam lines of the Swiss Light Source  using a beam of 1.00 Å wavelength  complexed with Mob2 were grown in 25% PEG 20000 buffered with 0.1 M sodium citrate (pH 5.5); however, these crystals diffracted only to 4.5 Å resolution. Hoping to improve the diffraction limit of this low diffracting complex by addition of a docking peptide, crystallization trials were set up in the presence of the Ssd1 docking peptide (TTEQSDFKFP) in approximately 4-fold excess. Plate-shaped crystals could be grown under such conditions with both the Cbk1(D475A/T743E)–Mob2 and the Cbk1(D475A)–Mob2 complexes. These crystals diffracted to 3.6 and 3.3 Å resolution . UnfortuData was processed with XDS , and theFor the Cbk1(D475A)–Mob2 model, which was refined to 3.3 Å resolution, 218 of the 745 residues, corresponding to close to one-third of the used protein constructs, are missing in the final model. For example, a long N-terminal Cbk1 region (251–293) and the N-terminal Mob2 region (45–110) could not be located in the electron density map. Apart from these apparently flexible N-terminal protein construct ends, there was no density for the region corresponding to the αC (392–406), the middle part of the Cbk1 activation loop (508–553), the linker region connecting the Cbk1 kinase core with the hydrophobic C-terminal region (714–739), and an internal Mob2 region (149–160) see Figs  and S1. First, we constructed a hybrid model that incorporated regions from Cbk1–Mob2 and PKB/Akt crystal structures. Cbk1’s disordered region containing αC was completed with the ordered region from the PKB/Akt’s crystal structure (PDB ID: 1O6K) using homology modeling followed by energy minimization to remove minor clashes. Furthermore, Cbk1’s short activation loop that contains the S570 autophosphorylation site was remodeled to match the position of the phosphorylated activation loop of PKB. The long activation loop (499–570) and the kinase core–HM connector region (714–739) were built in manually in an extended conformation. We subjected this complete model to MD simulations. Initial analyses suggested that the artificially built-in flexible loop regions (499–570 and 714–739) showed too much structural fluctuation, which precluded free MD simulations. Next, we excluded the activation loop (499–570) and performed a 100-ns MD run on the model where only the HM connector loop region (714–739) was allowed to move freely to pre-equilibrate this loop region. All MD simulations started out from this pre-equilibrated model.−1 nm−1. The energy-minimized systems were subjected to MD calculations. A time step of 2 fs, constant temperature of 300 K, and LINCS bond constraints xFP) might appear in proteins by chance, we also considered proteins that contain conserved docking motifs. In these proteins the enrichments are much stronger. For example, phosphorylation consensus matches are now >8× more like to appear . Similarly, phosphorylated consensus matches and the 50 most conserved phosphorylation consensus matches are >30× and >100× more likely to appear . These tests show that a conserved docking site makes consensus sites (and conserved consensus sites) more likely to appear, and that the phospho-acceptor residues within these consensus sites are significantly more likely to be among the set of phosphorylated positions annotated in the PhosphoGrid database. (B) In the 11 proteins with conserved [YF]xFP docking motifs, 14/27 (51.8%) of the Cbk1 phosphorylation consensus matches are in the 50 most conserved, which is much more than 50/887 (5.6%) in the proteome  Similarly, consensus matches are more likely to be phosphorylated if they are found in proteins with conserved docking motifs  These tests show that a given Cbk1 phosphorylation consensus match is much more likely to be conserved and phosphorylated in vivo if it is found in a protein that contains a conserved docking motif. Therefore, conserved docking motifs point to the functional Cbk1 consensus sites. ConDens data can be found in (A) In proteins that contain matches to the Cbk1 docking motif [YF]xFP, there is a statistically significant enrichment of NDR/LATS phosphorylation consensus sites Hx[RK]xx[ST] compared to proteins that do not contain docking motifs Click here for additional data file.S8 Fig(A) A comparison of Cbk1 bound to the FKFP core docking motif and ERK2 bound to the MNK1 docking motif (2Y9IQ) . The loo(PDF)Click here for additional data file.S9 FigS. cerevisiae (Sc), Sc. pombe (Sp), D. melanogaster (Dm), and Homo sapiens (Hs) bracketed in red. The loop between the D and E helices in the kinase C-lobe and the PxxP motif in the CLT region are enclosed in orange boxes. Bulky hydrophobic amino acids involved in docking motif binding in Cbk1 are boxed in pink. Tree is not drawn to scale. PKA and MAST are used as outgroups to the NDR family.A gene tree of AGC kinases related to the NDR family, with the closest Cbk1 orthologs in (PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


NOS3) gene polymorphisms with respect to coronary artery disease (CAD) have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far , examining association of three polymorphic forms of the NOS3 gene  with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association , followed by T786-C  and 4b/a,  in our pooled analysis. Subgroup analysis revealed that Glu298Asp  and 4b/a  have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry .Several association studies of endothelial nitric oxide synthase ( NOS3 gene. The NOS3 gene is located on chromosome 7q35-36, consists of 26 exons with a total size of 21 kb and it encodes for intracellular NO production Endothelial-derived nitric oxide (NO) has been known to be a major contributor in vascular regulation and has been amply implicated with coronary artery disease (CAD). NO is an important relaxing factor in the human body and is involved in a variety of different important physiological functions. It is synthesized in the human body from L-arginine by at least three isoforms of NO synthase (NOS), viz. inducible NOS, constitutive neuronal NOS and constitutive endothelial NOS (eNOS). NOS3 gene, resulting in the replacement of glutamic acid by aspartic acid at codon 298 . This variant has been shown to be associated with coronary spasm, Hingorani et al., NOS3 gene. NOS3 promoter activity approximately by 50% and has been associated with an increased risk for coronary artery spasm among subjects of Japanese ethnicity. NOS3 promoter region (T786-C) is associated with reduction in the promoter efficiency and the level of expressed enzyme, leading to increased CAD risk. Another common variant of this gene results by a cytosine replacing a thymine at nucleotide −786  at the 5′-flanking region of the NOS3 gene. Among them polymorphic repeats close to the 5′ end of the gene, the 27-base pair (bp) repeat in intron 4 is one of the most studied. The resulting rare 4-repeat allele has been shown to be associated with CAD among subjects belonging to European ancestry. NOS3 gene was found to be associated with MI, among subjects of Turkish descent. There are also several variable number of tandem repeats (VNTRs) with functional significance within NOS3 SNPs with respect to CAD reported association only in recessive genetic model of Glu298Asp, while dominant and allelic comparisons came out to be non-associated in the pooled analysis . NOS3 polymorphisms, which would aid to improve the existing understanding of these polymorphic forms of this gene in relation to CAD was warranted. Taking this as an objective, we conducted a systematic review and updated meta-analysis to ascertain the role of the three most common NOS3 gene polymorphisms in CAD. We also tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries.The only meta-analysis to assess the role of all three The databases of the US National Institutes of Health (PubMed), EMBASE and a MEDLINE Scopus and Web of Knowledge were systematically searched for relevant articles published online by March 2014. Both medical search headings and open text fields were used to identify articles. We systematically searched various databases and the reference lists of the relevant publications using the combination of terms like ‘NOS3’ OR ‘eNOS’ OR ‘constitutive endothelial NOS’ OR ‘ecNOS’ OR ‘endothelial nitric oxide synthase’ OR ‘endothelial NO synthase’ paired with ‘atherosclerotic heart disease’ OR ‘coronary artery disease’ OR ‘CAD’ OR ‘MI’ OR ‘Myocardial Infarction’ OR ‘AMI’ OR Acute Myocardial Infarction OR ‘ACS’ OR ‘Acute Coronary Syndrome’ AND ‘polymorphism’ OR ‘mutation’ OR ‘gene polymorphism’ Or ‘SNP’ OR ‘Single Nucleotide Polymorphism’. The search in these databases was restricted to articles relating to humans, covering all relevant English language publications published up to March 2014. All references cited in the resulting studies and in previously published reviews on this topic were examined to identify additional work not indexed in internet databases. Papers without sufficient information were identified and their corresponding authors were contacted via at least three emails (spaced one week apart) requesting needed information. Publications whose corresponding authors did not respond were thenceforth not included. Since data from unpublished studies is unlikely to be trustworthy, we chose not to include it into this present meta-analysis. The decision to include studies was hierarchical; initially study titles, then abstracts and finally the full body of the text were assessed. The following information was recorded from the retrieved studies: author's names, publication year, country where the study was conducted, study design, inclusion criteria for CAD patients and normal controls, methods used for genotyping and the distribution of polymorphic genotypes and alleles in each group.To be included in the meta-analysis, articles had to assess the association between CAD or MI patients and CAD free controls. Our selection criteria included studies that met all of the following criteria: (1) published in a peer-reviewed journal and independent studies using original data; (2) unrelated case-control or cohort studies; (3) providing complete data with genotype and allele frequencies to calculate the odds ratio (OR) with confidence interval (CI) and p values; (4) CAD patient diagnosis based on coronary angiography/clinical assessment and controls not being CAD patients; (5) all studies included had to be published in English language; and (6) all included studies had to have their genotype distribution among controls consistent with Hardy Weinberg approximations. We excluded the case reports, case studies and the studies not providing adequate information on selection criteria and the actual distribution of genotypes in each group. We also purposefully excluded studies with a surrogate endpoint such as “coronary artery spasm” or “heart failure”. Strict adherence to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses-PRISMA statement http://www.tufts.edu/~mcourt01/Documents/Court%20lab%20-%20HW%20calculator.xls). Studies with genotype frequency among controls yielding a p value of <0.05, did not conform to HWE approximations and were thus excluded.All required data along with genotypic distribution among cases and controls were recorded from publications (in duplicate) on a paper proforma. Later allele frequency was calculated manually and all the data was then transcribed on to a Microsoft Excel program on a computer. We checked departure from Hardy-Weinberg equilibrium (HWE) among control population for each included study using Michael H. Court's (2005–2008) online calculator (RevMan) [Computer program]. Version 5.3. Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2012. The extracted data from all publications were tested using previously described three genetic models i.e. dominant, recessive, and allelic model. Z test . Summary P value and Z value was also calculated (as listed) for each study group, for each genetic model to express the significance and degree of association. Existence of heterogeneity was tested using a Q test. It was performed using Higgins 2I statistics (2I) and Cochran's Q statistics (PQ) for each group of studies. Those resulting with 2I>50% and PQ<0.01 were identified as a heterogeneous group. Low, moderate and high heterogeneity was defined according to previously published estimates 2I values as 25%, 50% and 75%. Using this calculation, 2I value of <50% and PQ>0.01 was considered the criterion for selecting fixed effects for analysis. On the other hand, random effects were used for analyses for groups having 2I values of >50%. In order to define the sources of heterogeneity, subgroup analysis was performed. The subgroups were classified according to predominant ancestry among which the study was conducted. Publication bias was identified by examining Begg's funnel plots All calculations in this meta-analysis were carried out using Review Manager . A total of 60 case control studies  and 2I range = 18–23% and PQ range = 0.26–0.28). All other ancestral groups (apart from African which had only 1 study) showed moderate to high heterogeneity (2I range = 33–78% and PQ range = 0.16 to <0.00001). Among subgroups for Glu298Asp, 17 studies belonged to the European,  and 2I range = 35–69%, PQ range = 0.03 to <0.00001). Twenty nine case-control studies  and 2I range = 50–72%, PQ range = 0.16 to <0.00001). ( and S4)Among the ancestral subgroups, 15 studies constituted the European group, 2I = 17–61%, PQ = 0.17 to <0.00001). Forty three case-control studies, .00001). Table 2 2 rangeI = 0–33%; PQ range = 0.18–0.83), while European and Asian-Indian groups showed very low to very high heterogeneity . A total of 20 studies from European, –0.005). Table 2 Higher degree of heterogeneity was seen amongst almost all ancestral groups (except Asian-Indians) in all comparisons for Glu298Asp polymorphism. All groups were fairly heterogeneous in all comparisons for T786-C polymorphism. Fairly low heterogeneity was seen amongst Middle Eastern and Asian groups in all comparisons for 4b/a polymorphism. Table 2 Potential publication bias among groups of studies for each SNP was assessed by two different statistical tests, viz. Egger's plots S9-A, B). Each point in the figure represents OR of a study plotted against standard error (SE) of OR of that particular study. Different indicators of the studies belonging to each ancestry are used in these plots where they are seen to be generally contained within the inverted cone, indicating no significant publication bias, at least in the pooled analyses.Begg's funnel plots, Sensitivity analysis was performed in each study group in every genetic model, where we excluded studies one after another and conducted the analysis after each omission. The results in none of the groups and studied genetic models altered substantially to change the results from no association to significant association or the other way around (in groups of ≥5 studies). This indicates that the present meta-analysis is robust in nature.NOS3 gene polymorphisms,  with CAD.The present meta-analysis and structured systematic review is the most comprehensive till date and comprises of published studies which investigated the association of three most common  and  and S2) Consistent to the seen pattern, the T allele also showed a significant association with CAD in almost all ancestral groups, barring the Africans which had only 1 study. All comparisons in the pooled analysis for all three SNPs indicated significant association with CAD. The pooled results of Glu298Asp suggested that the presence of even a single T allele in a genotype effects in an increased risk of CAD. The risk of CAD is even higher if the genotype contains more than 1, T allele. This association is evident by the obtained pooled OR in allelic comparison .  and  and S2 CNOS3 SNPs are also now into public domain. NOS3 gene and CAD, Our pooled analysis showed significant associations of all three selected SNPs with CAD in every employed genetic model. Our results are in contrast to the previously published meta-analysis , NOS3 SNPs. Secondly, our study had strict inclusion/exclusion criteria, and we excluded studies with surrogate endpoints, and omitted studies in which controls did not conform to HWE proportions . This methodology ensured that we eliminated studies with potentially biased/inaccurate results, thus possibly presenting a real picture.There are basically two major merits of the present study which makes it superior to all previously published meta-analyses on this subject. NOS3 gene polymorphisms has been studied in detail by several researchers. Most schools of thought suggest that all these SNPs of NOS3 gene viz. Glu298Asp, T786-C and 4b/a 27bp VNTR via various mechanisms affect the serum nitrite/nitrate (NOx) concentrations in the human body. Over a period of time this imbalance in NOx concentrations results in “endothelial dysfunction” which finally blooms into an overt form of CAD. It is plausible that this degree of association with CAD and the resulting NOx concentrations are affected by gene-gene, gene-environment interactions. Compensatory mechanism of the human body against CAD may also increase NO secretion from the vascular endothelium during sudden reperfusion after acute coronary syndromes. x concentrations.The biochemical basis of the association of CAD with various  and , S2) The reason of this differential risk of CAD seen among different ancestral groups carrying this polymorphism is unknown and should be investigated upon. We presume that this difference could had been brought about by the variance in Asp allele frequency (range = 0.04-0.39), as well as several known and unknown gene-gene and gene-environment interactions. The biochemical basis of the association of CAD with Asp allele carriers has although been extensively studied upon however a clear consensus is still lacking. Some studies have reported an association of this SNP with intracellular NO concentration. x concentrations with any of the genotypes or alleles for this SNP. Also, since these associations are often masked, as it is affected by several physiological and pathological conditions, the real effect of NOS3 gene on plasma NOx levels is hard to determine. NOS3 gene.Positive association of Glu298Asp with CAD/MI has been reported over the years in several published studies. We found that along with a significant association for all three genetic models in our pooled analysis, this SNP seen to be associated with CAD separately among all ancestral groups, with the highest degree of association seen amongst the Middle Easterners. The reason behind this dissimilar degree of association among these ancestral groups is unknown. More in depth studies are warranted to answer this pertinent question. In vitro reporter gene assays performed by Nakayama et al., NOS3 mRNA and serum NOx levels in genotypes with ≥1, 786C alleles, NOS3 Glu298Asp and T786-C polymorphism has also been reported to be the cause of increased CAD risk among carriers of this SNP. The T786-C gene variant of NOS3 4b/a VNTR polymorphism with CAD. In contrast to the previous studies,  and , S6) The precise reason of this heightened risk amongst subjects of Middle Eastern ancestry is unknown and can only be speculated upon. Here again the influence of environmental factors such as diet and exercise may well be associated with the increased risk. Conflicting reports between the intron-4 variant and NO pathway activity are there in the public domain. Genotypes carrying ≥1 mutant alleles have been shown to have lower plasma NO levels and reduced protein expression, NOS3 gene. Clear scientific evidence is lacking which can explain the seen association of We believe that the present study has successfully answered the research question that it aimed at the outset and is statistically powered enough to the stand by the results it obtained. Although, there is a scope for limitations in every meta-analysis of genetic association studies and ours was no exception either. Firstly, we believe that there was under representation of the South-Asian region, and we found no published data on the subject from several South-Asian countries like Bangladesh, Sri Lanka and Pakistan etc., which have a sizable population inflicted with CAD. Secondly possibility of errors in genotyping, presence of selection bias and risk of inadequate sample size among different studies cannot be ruled out. It is also noteworthy that in case of polygenic diseases like CAD, an association study cannot test causality, it can merely measure statistical associations. Also, during meta-analysis of association studies we cannot ensure interference of linkage disequilibrium of the selected SNP with another closely linked site and its extent of effect on the overall association. Even after using two statistical tools, we could not detect any hint of publication bias amongst our study groups, at least for Glu298Asp and 4b/a polymorphisms. However, Egger's plot asymmetry was observed, with significant p values for pooled and European subgroup of T786-C polymorphism. Since the accuracy of Egger's test is debatable, and this asymmetry in the aforementioned plots could have possibly been due to factors like inter-study variations or small study effects among European subgroup for T786-C. Even then, the role of existing publication bias cannot be completely ruled out. This also qualifies to be listed as another small limitation in the present study.NOS3 gene polymorphisms i.e. Glu298Asp, T786-C and 4a/b VNTR with CAD transcending all ancestries worldwide. Results amongst different ancestral subgroups were rather illuminating. Amongst the studied NOS3 SNPs, both Glu298Asp and 4b/a showed strongest association among the Middle Eastern subgroup. On the other hand, T786-C and its minor allele seem to carry the highest risk for CAD among subjects of Asian ancestry. Our study significantly improves the understanding of the relation between this gene and CAD and its association amongst subjects of all major ancestries. These findings should thenceforth be assessed clinically for its implications in prevention and treatment strategies of CAD.In conclusion, since the criteria for inclusion and exclusion of studies are critical parts of a meta-analysis and can substantially affect results, we find that the present study is the most comprehensive till date and is systematically planned to funnel out erroneous studies. We thus feel that our study projects the real picture, and confirms the association of the three common Figure S1Forest plot depicting results of meta-analysis of studies reporting NOS3 Glu298Asp polymorphism assessed under dominant (TT+GT vs. GG) genetic model. Effect size estimates for all ancestral groups in this plot were obtained using random effects for analysis. Effect sizes using fixed effects were recalculated for Asian-Indian group which showed homogenous distribution among its included studies. Recalculated effect size estimate for Asian-Indians was, OR, 95%CI = 1.21, 1.01–1.45; Z = 2.07; P = 0.04.(TIF)Click here for additional data file.Figure S2Forest plot depicting results of meta-analysis of studies reporting NOS3 Glu298Asp polymorphism assessed under recessive (TT vs. GG+GT) genetic model. Effect size estimates for all ancestral groups in this plot were obtained using random effects for analysis. Effect sizes using fixed effects were recalculated for Asian and Asian-Indian groups which showed homogenous distribution among its included studies. Recalculated effect size estimates were, OR, 95%CI = 2.22, 1.21–4.08; Z = 2.57; P = 0.01 and OR, 95%CI = 1.85, 1.12–3.04; Z = 2.42; P = 0.02 for Asians and Asian-Indians respectively.(TIF)Click here for additional data file.Figure S3Forest plot depicting results of meta-analysis of studies reporting NOS3 T786-C polymorphism assessed under dominant (CC+CT vs. TT) genetic model. Effect size estimates for all ancestral groups in this plot were obtained using random effects for analysis.(TIF)Click here for additional data file.Figure S4Forest plot depicting results of meta-analysis of studies reporting NOS3 T786-C polymorphism assessed under recessive (CC vs. TT+CT) genetic model. Effect size estimates for all ancestral groups in this plot were obtained using fixed effects for analysis. Effect sizes using random effects were recalculated for three ancestral subgroups viz. Middle Eastern, Asian-Indian and African, which showed homogenous distribution among its included studies. Recalculated effect size estimates were, OR, 95%CI = 1.35, 0.69-2.65; Z = 0.87; P = 0.38 for Middle Easterners; OR, 95%CI = 1.73, 0.38–7.95; Z = 0.71; P = 0.48 for Asian-Indians and OR, 95%CI = 1.85, 0.77–4.44; Z = 1.37; P = 0.17 for Africans.(TIF)Click here for additional data file.Figure S5Forest plot depicting results of meta-analysis of studies reporting NOS3 4b/a VNTR polymorphism assessed under dominant (4a4a+4a4b vs. 4b4b) genetic model. Effect size estimates for all ancestral groups in this plot were obtained using random effects for analysis. Effect sizes using fixed effects were recalculated for Middle Eastern and Asian groups which showed homogenous distribution among its included studies. Recalculated effect size estimates were, OR, 95%CI = 1.73, 1.46–2.05; Z = 6.28; P = <0.00001 and OR, 95%CI = 1.22, 1.04–1.42; Z = 2.45; P = 0.01 for Middle Easterners and Asians respectively.(TIF)Click here for additional data file.Figure S6Forest plots depicting results of meta-analysis of studies reporting NOS3 4b/a VNTR polymorphism assessed under recessive (4a4a vs. 4a4b+4b4b) genetic model. Since all the ancestral groups showed homogenous distribution among its included studies, effect size estimates for all groups in this plot were obtained using fixed effects for analysis.(TIF)Click here for additional data file.Figure S7Begg's funnel plots for testing publication bias in comparisons under different genetic models for NOS3 Glu298Asp polymorphism. Each point in each figure represents OR of a study plotted against the standard error (SE) its OR. Different indicators of the studies belonging to each ancestral group are used in these plots. Figure S7A: Begg's Plot for comparisons under dominant genetic model (TT+GT vs. GG); Figure S7B: Begg's Plot for comparisons under recessive genetic model (TT vs. GG+GT).(TIF)Click here for additional data file.Figure S8Begg's funnel plots for testing publication bias in comparisons under different genetic models for NOS3 T786-C polymorphism. Each point in each figure represents OR of a study plotted against the standard error (SE) its OR. Different indicators of the studies belonging to each ancestral group are used in these plots. Figure S8A: Begg's Plot for comparisons under dominant genetic model (CC+CT vs. TT); Figure S8B: Begg's Plot for comparisons under recessive genetic model (CC vs. TT+CT).(TIF)Click here for additional data file.Figure S9Begg's funnel plots for testing publication bias in comparisons under different genetic models for NOS3 4b/a VNTR gene polymorphism. Each point in each figure represents OR of a study plotted against the standard error (SE) its OR. Different indicators of the studies belonging to each ancestral group are used in these plots. Figure S9A: Begg's Plot for comparisons under dominant genetic model (4a4a+4a4b vs. 4b4b); Figure S9B: Begg's Plot for comparisons under recessive genetic model (4a4a vs. 4a4b+4b4b).(TIF)Click here for additional data file.Data S1The raw data collected from all the published association studies included in the present meta-analysis.(ZIP)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.