This work explores the relationships between a user's choice of a given contraceptive option and the load of steroidal estrogens that can be associated with that choice. Family planning data for the USA served as a basis for the analysis. The results showed that collectively the use of contraception in the USA conservatively averts the release of approximately 4.8 tonnes of estradiol equivalents to the environment. 35% of the estrogenic load released over the course of all experienced pregnancies events and 34% the estrogenic load represented by all resultant legacies are a result of contraception failure and the non-use of contraception. A scenario analysis conducted to explore the impacts of discontinuing the use of ethinylestradiol-based oral contraceptives revealed that this would not only result in a 1.7-fold increase in the estrogenic loading of the users, but the users would also be expected to experience undesired family planning outcomes at a rate that is 3.3 times higher. Additional scenario analyses in which ethinylestradiol-based oral contraceptive users were modeled as having switched entirely to the use of male condoms, diaphragms or copper IUDs suggested that whether a higher or lower estrogenic load can be associated with the switching population depends on the typical failure rates of the options adopted following discontinuation. And, finally, it was estimated that, in the USA, at most 13% of the annual estrogenic load can be averted by fully meeting the contraceptive needs of the population. Therefore, while the issue of estrogen impacts on the environment cannot be addressed solely by meeting the population's contraceptive needs, a significant fraction of the estrogenic mass released to environment can be averted by improving the level with which their contraceptive needs are met. This misinterpretation largely arises from the belief that the use of a particular contraceptive option only results in an estrogenic load if the option itself is estrogen-based To-date, estrogen loads arising from all pregnancy-related events and those arising from the release of EE2 load on the environment would be for some EE2-OC users to switch to non-hormonal methods of contraception such as copper intrauterine devices (copper IUD), diaphragms, or male condoms. As much as this suggestion holds true when considering the direct release of EE2 alone, the recognition that each contraceptive option has an indirect estrogenic load associated with it use  requires that suggestions such as those of Wise et al.'s be revaluated with a renewed focus. That is, it should be asked how the total environmental load of steroidal estrogens would likely change if a given group of users switched from using EE2-OC to such alternative methods. Moreover, the change in the overall load of steroidal estrogens should be estimated for those EE2-OC users who discontinue the use of their current contraceptive option by switching to other methods or by abandoning the use of contraception altogether , the most potent natural estrogen. Specifically, for this evaluation, EE2, E1 and E3 were assumed to be 10 2, respectively. For the rationale behind the selected potencies, see Since estrogens act in an additive manner n, is composed of three distinct contributions represented by the variables Jd,n, Jf,n, and JL,n  and the recently authorized E2-based oral contraceptives (E2-OC). See The most obvious of these and the only one directly acknowledged in the literature to-date is Jf,n, arises from the recognition that all contraceptive options will occasionally fail  of choice, the estrogenic load released over the course of the resultant unintended pregnancies should be attributed to the parents' contraceptive choices. This consideration does not apply to all other outcomes of unintended pregnancies  since these outcomes do not end up satisfying a future need of the parents for a child. See An additional contribution, Jfail see . FailureL,n, due to unintended pregnancies that ultimately lead to the outcome of unwanted births Eq. (1) forms the basis for the evaluation of all contraceptive methods and scenarios presented below.As will be presented below, extensive analyses were conducted to understand how the choice to use a given contraceptive option or contraception collectively relates to the anthropogenic load of steroidal estrogens released to the environment.The Supporting Information sections include details on the development of models that were applied in this study to estimate the:d,n . An analysis is then conducted to evaluate and compare the estrogen loads that are attributable to the choice of individual contraceptive options (Section 1.2). Finally, given that some unintended pregnancies and, hence, births occur even with the use of currently available contraceptive choices, an evaluation is conducted to estimate the estrogenic load as it relates to pregnancy intent and to the effectiveness with which the population's current mix of contraceptive options is used (Section 1.3). All analyses presented are based on data reflecting trends in family planning in the USA.aE, is detailed in The collective use of contraception by a population averts a number of unintended pregnancies aE) can be estimated by adding the estrogenic load that would have been released over the course of 8.8 million unintended pregnancies (pE) to the estrogenic load arising from the legacies of 2.1 million unwanted births (LE) and subtracting from this sum the load that is directly released via excretions of users who currently use estrogen-based methods of contraception (eE) (see It is conservatively estimated that the collective use of contraception over the course of a given year in the USA averts 8.8 million unintended pregnancies (Eq. (S5)); i.e., 2.1, 5.0, 1.6 and 0.07 million unwanted births, abortions, fetal losses and ectopic pregnancies are averted, respectively, as estimated using Eq. (S5a) to (S5d) found in (Ee) see .a, is estimated to be 4.80 tonnes of E2-eq. This averted net load results from:Specifically, using Eq. (S4), the net quantity averted each year by the collective use of contraception in the USA, E2-eq that would have been released over the course of 8.8 million averted pregnancies (Ep)][0.66 tonnes of E2-eq estrogenic legacy of 2.1 million unwanted children whose births are averted (EL)]+[4.44 tonnes of E2-eq estrogenic load directly released due to the current contraceptive mix (Ee)][0.31 tonnes of EaE and pE suggest that, on a per-pregnancy basis, the estrogenic load represented by an unwanted birth's legacy is substantially higher than that released over the course of an unintended pregnancy. The absolute magnitude with which the estrogenic load of an unwanted birth's legacy exceeds that released over the course of an unintended pregnancy is likely higher than what the above estimates would suggest since the manner with which we estimate the estrogenic legacy of an unwanted birth's legacy is highly conservative  since it involves the direct consumption and subsequent excretion of estrogenic content by the user. Since all contraceptive options fail, indirect loads Jf,n (loads arising over the course of the unintended pregnancies) and JL,n (the estrogen legacy of the “extra” children that are the outcome of resultant unintended pregnancies) can be associated with the choice to use each option. As is evident from the results in 2-OC is the only one considered to impose estrogenic loading on the environment 2-OC.Of the commonly used options evaluated, the use of EEn is compared to that of not using any method at all  is the lowest of all contraceptive options. Also, of all reversible contraceptive options, the implant, probably due to its inherent nature, also has one of the highest continuation rates . The results summarized in 2-eq is released; however, only 35% of this load  arises from unintended pregnancies and, hence, from the failure and non-use of contraception. There are two reasons why an average unintended pregnancy event, when compared to an intended one, leads to the release of less estrogenic mass. First, only 45% of unintended pregnancies end up in the outcome of birth, in contrast to 81% of intended pregnancies 2-eq load that is on average 20, 25 and 32 times higher than the levels released had the pregnancy instead concluded in the outcome of spontaneous abortion, induced abortion and ectopic pregnancy, respectively . Due to this and also the growing evidence of the impact of ethinylestradiol, EE2, on aquatic species 2-based contraceptives 2-based contraceptives should be a subject of further discussion 2-OC. To this end, we will explore how the estrogenic flows would be expected to change should a group of EE2-OC users discontinue the use of their method by switching to a range of other contraceptive methods that are currently available to them or by abandoning the use of contraception altogether. This assessment is performed by conducting a scenario analysis. Specifically, in Section 2.1, the estrogenic load under the Status quo scenario for a unit population of a 1,000 first-year EE2-OC users is compared to a 2Discontinue EE scenario, the aim of which is to model the estrogenic load associated with the most likely contraceptive choices made by the user group upon discontinuing the use of the EE2-OC; i.e., upon discontinuing the use of EE2-OC the switching population is expected to either adopt other available contraceptive options or discontinue the use of contraception altogether. The contraceptive choices made by the switching population are modeled using the data of Rosenberg and Waugh 2-OC but still wanted to prevent a pregnancy.To-date there has been a tendency in the literature to only account for the direct estrogen load associated with a given contraceptive method . The results of this analysis are detailed in Section 2.3.Recently, an oral contraceptive preparation that uses estradiol, E2-OC averts an estrogenic load from being released to the environment when compared to using no contraceptive method at all. However, a particularly valuable and, perhaps, more realistic evaluation is one that compares the changes in loads of associated estrogens when EE2-OC users discontinue the use of their method by switching to other contraceptive methods or by abandoning the use of contraception altogether. Such an assessment is performed here by conducting a scenario analysis as described in detail in Changes in Estrogenic Load upon Discontinuation of EE2-OC. It was established above that the use of EE2-OC discontinue the use of their option, they will experience unintended pregnancies at a rate of 297 per 1000 users per year, a level that is nearly 3.3 times higher than the levels they would have experienced had they continued using EE2-OC  of the anticipated 297 unintended pregnancies resulting from the original 1000 EE2-OC users who switched to other options.Overall, it is estimated that, when first-year users of EE2-OC see . Note thStatus quo scenario shown in 2-eq/yr (EE2-OCN) can be associated with the representative user population of 1,000 EE2-OC users. Of this total load, 48% results from the direct excretions of the user population, another 7% is released over the course of unintended pregnancies experienced by the user population, and the remaining 45% is the estrogenic load represented by the legacies of unwanted children born to the representative user population. Upon discontinuing the use of EE2-OC, it is estimated that the user population's total estrogenic load (sN) will increase 1.7-fold to 107 grams of E2-eq/yr, of which 14% would be released over the course of 297 unintended pregnancies experienced by the discontinuing population and the remaining 86% would be the estrogenic load represented by the legacies of unwanted children expected to be born to that population. If the various contributions in Eq. (1) to the loads for the Status quo and 2Discontinue EE scenarios are compared, it can be seen that the increase in total estrogenic load upon discontinuing the use of EE2-OC is driven by a substantial increase in the legacy contribution . Further, over and above the 1.7-fold increase in estrogenic load experienced upon discontinuing EE2-OC use, the user population also experiences undesired family outcomes such as unintended pregnancies, abortions, miscarriages and unplanned births at levels that are approximately 3.3 times higher. Similarly, because the contraceptive needs of the parents could not be met, the number of extra births also increases by 3.3 times and the number of ectopic pregnancies experienced by the user population also increases by an estimated 3.4 times. Further, recognizing that each additional unintended pregnancy presents an added risk of maternal and neo-natal mortality 2-OC, the user population would be expected to experience far higher rates of maternal and neo-natal mortality. Hence, whether the assessment of contraceptive options is performed by considering estrogenic loading, the number of undesired family planning events experienced, or maternal and neo-natal mortality rates, the continued use of EE2-OC appears to be a much-preferred option over discontinuing its use and switching to a range of other options. Notably, nearly one-third of the EE2-OC users discontinue the use of their option within the first year The scenario analysis suggests that under the 2-based oral contraceptive will result in an increase in total estrogen load into the environment hinges on the assumption that users will switch to a particular set of alternative options, as described by the data of Rosenberg and Waugh 2-based oral contraceptives. For this reason, and to concurrently evaluate the suggestion of Wise et al. 2-OC users are modeled as having discontinued the use of EE2-OC by entirely switching to the use of either the male condom, the diaphragm, or the copper IUD. The results for each of these three scenarios are summarized in figures in EE2-OC Users Switch to Specific Non-estrogen-based Contraceptive Options. The above assertion that discontinuing the use of an EEStatus quo scenario, the user population would experience significantly fewer adverse personal, social and public health outcomes ; moreover the level of unintended pregnancies, abortions, miscarriages and unplanned births experienced by the user population would approximately double see . While oomes see . Conside2-OC see . In addi2-OC user population to alternative modes of contraception leads to an increase or decrease in estrogenic load on the environment is inherently dependent on the alternate forms of contraception chosen by the user population. Note that, even though the estrogenic load of the user population is expected to increase significantly for those who currently discontinue the use of EE2-OC as reported above (see Section 2.1), this outcome is inherently dependent on the mix of contraceptive choices chosen by users upon discontinuation. Hence, this would have to be reevaluated should these patterns change in the future. In addition, the results arising from the scenario analyses for users who switch to the male condom and diaphragms clearly suggest that the choice to use a given contraceptive option, or any recommendations that may influence that choice  tonnes +(0.40−0.23) tonnes) can potentially be averted. Viewed another way, the failure and the non-use of contraception by the population of the USA currently represents a potentially preventable annual estrogenic load of 1.3 tonnes of E2-eq. Further, if it is assumed that the contraceptive needs of contraceptive users in the USA can be fully met without the use of EE2-OC, the release of an additional 0.31 tonnes of E2-eq  to switch to those methods that have significantly lower typical use failure rates .Since the potentially preventable load in the USA of 1.3 tonnes of EThe results of this study are intended to inform discussions concerning the relationships between contraception options and their estrogenic impacts on the environment. However, in the interest of clarity, it is also important to point out some limitation to this study, as follows.2-OC use, the higher load of estrogens in the environment for the 2Discontinue EE scenario is expected to be considerably less persistent than that released under the Status Quo scenario. This assertion results from the recognition that EE2 has been reported to be substantially more persistent than natural estrogens Status quo scenario is likely to be more persistent, this assertion would need to be revaluated once sufficient data on the environmental fate, occurrence and relevance of lumiestrone becomes available.With respect to the conclusion drawn in Section 2.1, it is important to note that even though the associated estrogenic load of users is expected to be higher upon discontinuing EE2-OC user increases 2.2-fold when she switches to the use of E2-OC, one must also consider that, although lower in quantity, the total associated estrogenic load of an EE2-OC user may be more persistent than the one released by the user of E2-OC. That is, the excretion of EE2 by the users of EE2-OC are replaced by releases of E1, E2 and E3 by the users of E2-OC, all of which are less persistent than EE2Similarly, with respect to the conclusion drawn in Section 2.3 that the associated loading of a previous EE2-OC, E2-OC, progestin-only pill, Depo-Provera, levonorgestrel-IUS, and the implant) results in the direct release of a number of gestagens to the environment that is analogous to Jd,n for estrogens. Moreover, similar to the situation when evaluating the total associated estrogenic load, the relationships are more complex than the simple consideration that the use of only gestagen-based options only leads to their direct release to the environment. In fact, analogous to the contributions Jf,n and JL,n discussed above for estrogens, the choice to use every single contraceptive option has at least two indirect loads of gestagens associated with their use. First, the gestagen load analogous to Jf,n results from the recognition that every contraceptive option has a failure rate associated with its use and the resultant unintended pregnancy leads to an increased release of progesterone L,n results from the recognition that, had a chosen contraceptive option not failed and further the resultant unintended pregnancy not resulted in the outcome of unwanted pregnancy, the unwanted child's legacy load of gestagens would not have resulted. Therefore, overall, every contraceptive option comes with an associated gestagenic load. Unfortunately, since the eco-toxicological potential for only a handful of gestagens has been explored in sufficient detail in literature Although the focus of this research is to develop a better understanding of the relationship between contraceptive choices and the associated estrogenic loads, it is important to note that the choice to use a given a contraceptive option also has implication for the release of gestagens to the environment. Clearly, a user's choice to use a progestin-based contraceptive option Click here for additional data file.File S2Eco-toxicological Potency of Steroidal Estrogens.(DOC)Click here for additional data file.File S3d,n).Modeling the Steroidal Estrogen Load Released Directly Via the Use of a Particular Contraceptive Option (J(DOC)Click here for additional data file.File S4f,n).Modeling the Load of Natural Estrogens Released over the Course of Unintended Pregnancies that can be Associated with a User's Choice to Use a Particular Contraceptive Option (J(DOC)Click here for additional data file.File S5L,n).Modeling the Legacy Estrogenic Load Arising from an Unwanted Birth and can be Associated with a User's Choice to Use a Particular Contraceptive Option (J(DOC)Click here for additional data file.File S6Pregnancy Events and Estrogenic Load Averted by a Population's Collective Use of Contraception.(DOC)Click here for additional data file.File S7Estimating Relative Contributions as Shown in  (Main Body).(DOC)Click here for additional data file.File S8Modeling Estrogen Loads Arising from Changes in Contraceptive Use.(DOC)Click here for additional data file.File S9Nomenclature and Specific Estimates for All Variables Used.(DOC)Click here for additional data file.File S102-OC Switch to Male Condoms.Changes in Flows of Estrogens When Users of EE(DOC)Click here for additional data file.File S112-OC Switch to Diaphragms.Changes in Flows of Estrogens When Users of EE(DOC)Click here for additional data file.File S122-OC Switch to Copper IUDs.Changes in Flows of Estrogens When Users of EE(DOC)Click here for additional data file.File S132-OC.Flow of Estrogens Associated with the Use of E(DOC)Click here for additional data file.File S14Excretion of Equine Estrogens and their Relevance.(DOC)Click here for additional data file.File S15References for All Supporting Information Sections.(DOC)Click here for additional data file.


Estimating an individual's risk for ventricular tachyarrhythmia (VTA) and sudden cardiac death (SCD) and identifying patients at high risk who would benefit the most from an implantable device remain key challenges for the cardiac electrophysiologist. A sudden onset and broad spectrum of precipitating mechanisms and triggers are the hallmarks of these life-threatening events, making them particularly difficult to foresee. Additional challenges arise because of variations in anatomy and tissue composition when cardiac electrical activity is measured at the body surface, relatively far away from its electrical sources in the heart.2 The proarrhythmic effects of spatial repolarization heterogeneity can be amplified or diminished by the dynamic relationships between cardiac cycle length  and action potential duration (APD).3 The analysis of these relationships, referred to as electrical restitution, proved useful for understanding the mechanisms linking the dynamics of heart-rate dependent APD adaptation to arrhythmogenesis and for determining critical parameter regimes that can be proarrhythmic. In particular, the steepness of the restitution curve has been identified as one of the key factors precipitating T-wave alternans, a proarrhythmic form of repolarization instability.3 The analysis of theoretical properties and experimental relationships between APD restitution and arrhythmias has been the subject of intensive research in the past decade.4 Dynamically induced elevation of spatial repolarization heterogeneity has been shown to promote inducibility of ventricular fibrillation in experimental studies.3 Nevertheless, because of the complexity of restitution relationships in a real-life setting, with freely varying cycle lengths and other confounding factors, analysis of the APD restitution has not translated to the clinical practice of cardiac electrophysiology.Despite these formidable challenges, significant progress has been made in arrhythmia risk stratification. A large body of evidence collected from theoretical, experimental, and clinical studies has shown that spatial repolarization heterogeneity can precipitate regional conduction slowing and unidirectional block and can ultimately lead to the initiation of reentrant arrhythmias.Journal of the American Heart Association (JAHA), Nicolson et al5 present a pilot study of a single-valued composite index, referred to as the Regional Restitution Instability Index (R2I2), which incorporates both (1) an estimated spatial heterogeneity across the 12 surface ECG leads and (2) restitution-related characteristics of repolarization over a range of pacing-induced cardiac cycle lengths. The proposed lumped-parameter metric uses the location of the T wave's peak as a key fiducial point, and thus it is also influenced by the symmetry and waveform characteristics of the T wave, which can undergo changes in the presence of ischemia and repolarization heterogeneity.8In this issue of the peak – Tend interval, and the latter 2 indices had no statistically significant association with the occurrence of arrhythmias in this patient population.To test the accuracy of R2I2, the authors conducted a small but well-designed retrospective study that included 3 groups: (1) a Control group of 29 patients with supraventricular tachyarrhythmias, (2) a Primary Study group in which the method was refined, and (3) a Replication group in which the method was tested. The study showed that R2I2 identified the subjects who experienced VTA/SCD independently of the demographic and clinical factors included in the analysis. Moreover, using cardiac magnetic resonance imaging, the authors showed that R2I2 correlates with the extent of the peri-infarct zone. In contrast, there was no correlation between R2I2 and either QT dispersion or T5 show potential utility of R2I2 for predicting arrhythmic events and warrant further investigation in a larger patient population. In addition, this pilot study raises, as does virtually every novel approach, several nontrivial research questions that deserve discussion.The pilot results reported by Nicolson et al9 introduced it as a measure of activation-independent repolarization heterogeneity. Composite, spatio-temporal descriptors of T-wave morphology in the form of T-wave vector loop and its dispersion have proved useful for VTA/SCD risk stratification after myocardial infarction.10First, we note that R2I2 is a composite index that incorporates both spatial heterogeneity across the 12 leads and restitution-related temporal features. Combining such disparate spatial and temporal features in a single index can be useful for summarizing the most important properties of a complex, multicomponent process in a simple form. This approach is often applied in computational biology and biophysics, despite a tradeoff associated with the difficulties of post hoc analysis, to discern the effect of each participating factor included in a composite metric. In electrocardiography, combining depolarization and repolarization properties in a single index  also has been used for several decades, since Wilson et alpeak (the period from S2 QRS onset to the S2 Tpeak) and TpeakQ (period from Tpeak on the last beat of the drive train to the S2 QRS onset) intervals as a surrogate marker of restitution have practical benefits. In most cases, the location of the T wave's peak on the surface ECG is easier to determine than the end of the T wave, which gradually merges with the isoelectric line, often obscured by noise and baseline wander.11 Selecting the peak of the T wave as a key fiducial point and analyzing the relationship between the QT12 However, the 2-dimensional space of QTpeak and TpeakQ intervals represents a significant departure from the traditional restitution relationship, expressing adaptation of the cardiac APD to changes in the diastolic interval. Indeed, the QTpeak interval entails both depolarization and the early part of repolarization, whereas the TpeakQ interval includes late repolarization and the entire ventricular diastolic interval. Thus, the slope of the relationship between the 2 intervals, utilized in the R2I2 index, is equivalent to a ratio (R):In each individual body-surface ECG lead, the location of the T-wave peak represents a combination of local  gradients and global (apex–base) effects. These locations are clustered around the mean repolarization time, which can be accurately determined by using the time of the T-wave peak on the root-mean-square curve.DRI indicates depolarization interval; ERI, early repolarization interval; LRI, late repolarization interval; and VDI, ventricular diastolic interval. Given that the repolarization interval is usually longer than depolarization and that changes in depolarization time are usually smaller than those in repolarization time, the R ratio could be influenced significantly by changes in T-wave symmetry and waveform pattern. As mentioned earlier, changes in T-wave morphology have been linked to the risk of VTA/SCD in patients with ischemic cardiomyopathy and prior myocardial infarction.10 Further complicating this conundrum, an upsurge in depolarization heterogeneity reported shortly before VTA initiation in patients with heart failure may affect the R ratio as well.2 It remains to be seen how the R2I2 index is affected by depolarization and conduction aberrations and how those changes can be differentiated from enhanced repolarization heterogeneity.where 5 appropriately point out that there is room for improvement and refinement of the R2I2 approach. Perhaps most important for making this method practically useful is the replacement of cardiac pacing by noninvasive maneuvers that can modulate heart rate . Another area for potential improvement that warrants further investigation is the incorporation of experimentally established indices of mean activation and recovery times .12 Hypothetically, this could pave the way for measuring mean repolarization time and quantifying the differences between the global mean and local peaks of the T wave in individual leads. This approach also could provide a validated measure of the global activation-recovery interval.12 Another area for future research is the potential use of the width of the T wave, which correlates with both ventricular dispersion and ischemia.8Nicolson et al13 QT variability,14 T-wave alternans,15 and the width of the T wave, which can be modulated by ischemia.7 It would be particularly interesting to compare the performance of R2I2 with second central moments, which recently have been described as quantifiers of spatial heterogeneity of cardiac depolarization and repolarization in the surface ECG, and which could predict the occurrence of arrhythmic events.2In addition, it will be important to investigate (1) stability of the R2I2 index over time in the same individual; (2) the impact of β-blockers and other medications; (3) the importance of individual differences in heart rate; and (4) the impact of changes in T-wave morphology, particularly its biphasic and notched forms, on the R2I2 behavior. It is also important to examine the effect of anatomic differences in the orientation of the electrical vector of the heart and the associated interlead differences in depolarization and repolarization vectors on the performance of the R2I2 index. Further research also is needed to identify the relationship between R2I2 and other ECG markers of arrhythmia risk, including rhythm instabilities,5 also show that inclusion of restitution properties in composite risk assessment leads to a qualitative improvement in the predictive accuracy as compared with QT dispersion, which by itself has not proved to be a consistent measure of repolarization heterogeneity or its dispersion.12 One can hypothesize that the lumped-parameter approach could be further extended by incorporating a more complete set of nonredundant, proarrhythmic instabilities of rhythm, depolarization, and repolarization, as described above. Adaptive approaches that use an individual's own data as a reference for personalized calibration might also contribute to the improvement of lumped-parameter risk stratification.13To summarize, the novel R2I2 index shows promise in identifying patients who are at risk for VTA/SCD. The authors are to be commended for introducing this innovative approach and conducting a small but well-designed feasibility study that included an independent test group. If the pilot results are replicated in larger studies, this approach may find a place among clinical tools for arrhythmia risk stratification. The data presented by Nicolson et alFinding an optimal combination of nonredundant features within a simple metric to create an accurate, practically useful test for arrhythmia risk stratification remains an open challenge. Because of the complexity and broad spectrum of underlying mechanisms of arrhythmogenesis, the number of possible parameters and their combinations may become prohibitively large for manual analysis. This is a good place and a good time to start thinking about computational methods of combinatorial optimization, which could lead us to further improvements in arrhythmia risk stratification.


Molecular dynamics (MD) simulations are powerful tools to investigate the conformational dynamics of proteins that is often a critical element of their function. Identification of functionally relevant conformations is generally done clustering the large ensemble of structures that are generated. Recently, Self-Organising Maps (SOMs) were reported performing more accurately and providing more consistent results than traditional clustering algorithms in various data mining problems. We present a novel strategy to analyse and compare conformational ensembles of protein domains using a two-level approach that combines SOMs and hierarchical clustering.The conformational dynamics of the α-spectrin SH3 protein domain and six single mutants were analysed by MD simulations. The Cα's Cartesian coordinates of conformations sampled in the essential space were used as input data vectors for SOM training, then complete linkage clustering was performed on the SOM prototype vectors. A specific protocol to optimize a SOM for structural ensembles was proposed: the optimal SOM was selected by means of a Taguchi experimental design plan applied to different data sets, and the optimal sampling rate of the MD trajectory was selected. The proposed two-level approach was applied to single trajectories of the SH3 domain independently as well as to groups of them at the same time. The results demonstrated the potential of this approach in the analysis of large ensembles of molecular structures: the possibility of producing a topological mapping of the conformational space in a simple 2D visualisation, as well as of effectively highlighting differences in the conformational dynamics directly related to biological functions.The use of a two-level approach combining SOMs and hierarchical clustering for conformational analysis of structural ensembles of proteins was proposed. It can easily be extended to other study cases and to conformational ensembles from other sources. Protein dynamics plays a central role in cell life. In many cases biological function involves molecular motion  and it wSignificant contributions in this direction have come from computational methods, in particular from Molecular dynamics (MD) simulations ,4, by whkinetic clustering [geometrical clustering, because only a representative sampling of the accessible conformations is required. The underlying assumption is that structurally similar conformations lie in the same basin of the free energy surface. While often this is an acceptable approximation, a recent study has suggested caution in interpreting the clustering results [As recurrence of and transition between conformations is difficult to extract from the raw ensemble data , groupinustering -11 whereGeometrical clustering for conformational analysis was introduced when simulation time increased up to nanoseconds generating tens of thousands of structures -14 and hSelf-Organising Maps (SOMs) [Among the data-mining algorithms, s (SOMs)  were recs (SOMs) ,24 and as (SOMs) . In the s (SOMs)  comparedIn this contribution, we present the use of a two-level approach  combininThe atomistic dynamics of the set of representative proteins was simulated using the GROMACS package (version 3.3.3) -33 with To evaluate the sampling efficiency the overlap between the conformational spaces spanned by different parts of the simulation ,40 was ctr is the trace of the matrix. When s is equal to 1, the two spanned subspaces are identical, whereas a value of 0 indicates complete orthogonality. In this work, the overlap between the covariance matrix of the atomic coordinates in each half of a simulation and in the overall trajectory [where ajectory  was evali measures its deviation from the time-averaged position during the simulation:Protein flexibility was calculated on a residue basis using the root mean square fluctuation (RMSF) of the positions of the Cα atoms. The RMSF of the Cα atom ri is the coordinate vector for atom i and the < > indicates a time-averaging. To increase the signal/noise ratio, the RMSF calculation was performed after Essential Dynamics (ED) analysis. ED is a widely applied technique based on principal component analysis (PCA) of conformational ensembles [where nsembles , aimed tnsembles .i.e. the RMSD between these atomic distances in a selected conformation, a, and the same distances in a reference structure, b:The geometry of the protein binding site was described by a selected set of pair-wise atomic distances and its conformational changes were measured by distance Root Mean Square Deviation (dRMSD), d is the distance value, i and j are the indices of the selected atoms and N is the total number of computed distances.where In the proposed approach the SOM input data are large ensembles of conformations extracted from the MD trajectories of one or more domains. Each conformation is described by the Cartesian coordinates of the Cα atoms of the protein, after projection of the original data on the eigenvectors that define the Essential Space. At the end of the SOM training process (see "Brief introduction to SOMs" for details) the original conformational ensemble ends up projected on a bi-dimensional feature map where similar elements are associated to neighbour neurons. Therefore the whole input space is described by a limited number of vectors (the prototype vectors of the neurons). These are then submitted to linkage clustering and the representative conformations are extracted for conformational and functional analysis.A optimization of the SOM parameters for the specific type of data is required. The optimization we designed and performed consists of two steps: first, the SOM parameters were characterized and optimized by experimental design to obtain the optimal SOM and then these were used to select the optimal sampling rate of the MD trajectory.A diagram of the overall approach, including the optimization steps performed in this work, is shown in Figure d-dimensional model vector (the prototype vector), where d is the dimension of the input data vectors (in this application d is the number of Cα coordinates). The neurons are connected to adjacent neurons by a neighborhood relation, which dictates the topology of the SOM map.A SOM is a powerful tool for the grouping and visualization of high-dimensional data. It is a specific architecture of artificial neural networks consisting of a low-dimensional  grid of so-called neurons, called map . The numThe SOM learning algorithm could be divided into five steps: a) initial randomization of each prototype vector; b) random selection of an input vector; c) neurons "competition" to "win" the input vector; d) update of the prototype vector of the "winning" neuron and of its topological neighbors by pulling them closer to the "won" input vector; e) repeat from b) for each input vector for a given number of epochs sufficient to ensure a convergent training of the map. As a result, the prototype vector of each output neuron represents a particular feature (conformation) drawn from the input space .hits), black hexagons with size proportional to the neuron population were superimposed to the map. Visual inspection of the map allows the analysis of the final distribution of the original data: neighbor neurons (hexagons) are associated to similar groups of input data (conformations); map regions with highly-populated neurons represent conformations highly sampled during the trajectory.A useful tool to analyse the results is a pictorial representation of the trained map. In this work we choose a topographic map where each neuron is represented by an hexagon. To visualize the number of input data won by each neuron  is a modification of the clustering efficiency index described in [tot Rto account for different SOM sizes. The performance measure (E4) has a unique minimum and was successful against expert-based assessment of clustering [where ribed in  where  mustering .Design of Experiments or Experimental Design [The problem of finding the optimal value of the performance measure is computationally expensive and Artificial Neural Networks are generally affected by the curse of dimensionality. This problem can be solved following different strategies , among wl Design ,51 was sl Design . In the map size, learning algorithm, neighbourhood function, lattice type, shape, alpha type, radius, training length and initial value of the parameter alpha (parameter names as defined in MATLAB). The ranges of tested values are reported in Table map size is based on the dimension of the input data vectors and an estimate of the training data variance  [map sizes was considered for the optimization (100 - 400).Consistent with previous reports  the foll matrix) . InsteadD-Optimality or G-Optimality design criteria. Instead, a Taguchi robust design plan [The particular nature of the parameters does not allow the use of either the widely used ign plan  was usedProb to Enter = 0.05, Prob to Leave = 0.05 and Direction = Mixed and Rules = No Rules. The linear regression model is validated by comparing actual and predicted performance. JMP software was used for data analysis and linear regression fitting [The results of the numerical experiments associated with the design plan are used to find a linear regression model of the performance measure (E4) as function of the SOM's statistically significant parameters. The linear regression is performed in two stages: a stepwise regression  to selec fitting . On the When dealing with a nanoseconds MD trajectory, it is desirable to train the SOM with a minimum number of selected structures while maintaining a reliable picture of the protein dynamics. Therefore, the effect of the sampling rate on the SOM learning process was assessed and the minimum number of frames to extract from a trajectory, without a statistically significant loss of information, was identified.(n) Qis representative of the original data set Q, then the SOM model (n)SOM, learnt by using the training data set (n)Q, when queried with data sets (n) Qand Q must provide SOM hit distributions which are not statistically different.The rationale of the statistical procedure, applied to discover the "minimum number of frames" Figure , is as fn conformations of the trajectory, each belonging to one of n equal width trajectory intervals. To better clarify the statistical procedure, let i(n) Qbe the th irandom sample extracted from Q and i(n) SOMbe the corresponding SOM trained on it by using the optimal parameters determined by experimental design. Furthermore, let i(n) hand i(n) Hbe the SOM hit vectors in the case where i(n) Qand Q are presented to i(n)SOM. Then, the i(n) χChi-square statistic is computed on non-null cells of the SOM hit vectors i(n) Hand i(n) hwhile the null hypothesis is tested by the n Cressie-Read Chi-square test where the significance level has been set to α = 0.05 [In this study such a test was performed on three replicas by randomly selecting α = 0.05 . A diagrcomplete linkage and the average linkage, were tested, following what described in [The optimal SOM resulting from the previous optimization steps summarizes the conformational variability in the input data  through a set of prototype vectors. The next step of our approach consists on combining the prototype vectors (that can be interpreted as "protoclusters" ) in a reribed in .i dbetween the progressively merged clusters are recorded in order; from this set of distances, mean and standard deviation are calculated; the optimal number of clusters is equal to the largest k satisfying the following inequality:Several clustering quality measures and stopping rules have been proposed to select the "optimal" number of clusters. Some of them were empirically shown to be more reliable  but therd sdis the standard deviation, z is a specified constant.where z were tested: 1.25, 1.50, 1.75, 2.00, 2.25, 2.50, and 2.75 for both complete and average linkage. Different values of the optimal number of clusters were obtained: from 5 to 8 for the complete linkage and from 4 to 10 for the average linkage. The most informative cluster separation was obtained with complete linkage and z = 2.75, and with average linkage and z = 2.50, which agrees with that suggested in [A range of values for ested in .vs. average linkage algorithms were verified by the analysis reported in the Methods and Results sections on "Comparison of cluster analysis methods".The stability and better performance of the proposed two-level approach, i.e. SOM and linkage, with respect to other clustering methods, as well as the relative performances of the complete http://ima.udg.edu/~jamf/MARTIN_MF.htm.Hierarchical clustering was performed using the MATLAB Statistics Toolbox  and the c; the point with largest neighbourhood defines the first cluster medoid; this point and its neighbours are removed and the algorithm is iterated until all data have been assigned to a cluster.The proposed two-level approach for clustering structural ensembles (combining SOM with the average or the complete linkage methods) was compared with other clustering methods applied to the original input data vectors: GROMOS nearest neighbour , averageThe quality of the clustering was assessed by the Silhouette (S) index  and the c ranging from 0.01 to 0.40 with increments of 0.005. Then the best solution for each number of clusters was selected according to the relevant quality index.The performance of the methods was compared for the number of clusters ranging from 2 to 15. In the case of GROMOS algorithm, solutions were generated for 1SHG) [10 helix, and three connecting loops: the long RT loop that includes three isolated β-bridges, the n-src and the distal loop  domain and a group of its single-site mutants. The crystal structure of the wild-type domain (PDB code: 1SHG)  is charap Figure . The Spcp Figure . Several2F2W, 2F2X, 2F2V and 2CDT) [1QKX, 1QKW) [The six mutants of the Spc-SH3 domain analysed here (and their PDB codes) are: R21A, R21G, A56G and A56S (nd 2CDT) , N47A anX, 1QKW) . These mX, 1QKW) .10 helix belonging to one of the hydrophobic binding grooves . Analysis of the root mean square distance (RMSD) to the starting structure confirmed a general stability of all trajectories, with an equilibration time around 1 ns . A set of comparative RMSF plots is shown in Additional File Following the protocol described in the Methods section, a SOM was optimized for the analysis of the MD trajectories of the WT SH3 and its six mutants. Each sampled conformation was described by the Cartesian coordinates of the Cα atoms projected on the Essential Space. The 55 Cα atoms corresponding to structurally equivalent positions in all the domains were included in the analysis; therefore, the input data presented to the SOM were vectors of 165 elements.The optimal values for the SOM parameters were found by experimental design using a Taguchi design plan (see Table ○ WT○ R21G○ WT and R21G○ ALL (WT and the six mutants),thus resulting in a total of 432 experiments. The choice of multiple and combined data sets aimed at optimizing the map both for the analysis of a single trajectory and for multiple comparisons of different trajectories.2 and R2adj (R2 = 0.937 and R2adj = 0.936). The following SOM parameters have been judged to be statistically significant at the p-value cut-off of 0.01: Map size, Radius, Training length, Neighbour function and TYPEMOL . The summary of the linear regression model and the significance analysis of the single SOM parameters are reported in Table . The suma posteriori in a test on the impact of different sampling rates (see Methods section for details). The analysis was performed with three replicas for each sampling rate: 1/2 ps, 1/4 ps, 1/8 ps, 1/16 ps, 1/25 ps, 1/50 ps, 1/100 ps, 1/500 ps and 1/1000 ps (corresponding to sampling size from 20000 to 40 conformations): 20000, 10000, 5000, 2500, 1600, 800, 400, 80, and 40 conformations. The results for a test set composed by WT, R21G and N47A, are reported in Table The SOM optimization was performed on conformations sampled with a rate of 1/100 ps, randomly selecting a structure in each interval of 100 ps to avoid a correlation bias. This initial choice was validated and confirmed The two-level approach was compared with three cluster analysis methods: GROMOS , averageThe two-level approach proposed in this work (SOM and linkage) is the best performing: the S (DB) scores are generally higher (lower) for all the number of clusters and the profiles are more stable. This suggests that the SOM preclustering is reducing the noise and generating more compact and well separated clusters. This is also confirmed by the per-cluster values of S  with the SOM parameters and the sampling rate values set in the optimization stage was used to perform a conformational analysis of the MD trajectories of single SH3 domains independently as well as groups of simulations to detect similarities and differences in flexibilities among the systems. We will present the results for the following ensembles: R21G, WT and R21G, WT and six mutants.neuron level, i.e. the ensemble of hit conformations in a neuron; cluster level, i.e. the ensemble of hit conformations closest to the prototype vectors in a cluster; centroid level, i.e. the hit conformation closest to the cluster centroid. Four clusters  between the distances of four selected points in the average conformation of each cluster and in the X-ray structure of the WT SH3 was calculated. The selected points  allows the SOM model to optimally capture the relevant features of a large input space in a smaller set of prototype vectors. These can be interpreted as an intermediate topological reference , which lusters" . This allusters"  where onThe results for the SH3 domains highlighted the specific advantages of a SOM in conformational and functional analysis. The major benefit is the possibility of providing a topological mapping of the conformational space embedded in a simple 2D visualisation that simplifies the identification of differences in the conformational dynamics  are expected to generalize quite well. The Map size parameter is more critical; the optimal value we found (100) allows the summarization of a great number of unique features, but it could be necessary to re-set it specifically for cases with a greater variance in the training data.Our approach can be reliably used on other study-cases. Indeed, the number of statistically significant SOM parameters is small while their optimal values are expected to be suitable for conformational spaces with similar complexity, The approach presented here is independent from the method used to generate the structural ensemble and is reliable to describe both small and large differences. It is therefore suitable to also analyse combinations of ensembles from computational methods with a more extended sampling of the conformational space and from experiments (NMR ensembles or multiple X-ray depositions of the same structure).The test-case presented here includes mutants of a single domain that can easily be aligned. A future development of this study is the identification of alternative representations of protein conformations that do not require the preliminary definition of structurally equivalent positions by structural alignment. This will allow an extension to the comparison of different domains, including distant homologous proteins.The novel approach presented here to analyse conformational ensembles of protein domains resulted in a very efficient way of comparing multiple trajectories of the test case and analysing the role of conformational flexibility in modulating the domain function. The approach can easily be extended to other study cases. Further applications may include the treatment of ensemble of conformations derived from other sources of dynamical data, and the use of a trained map to classify conformational ensembles of similar systems. Future directions involve the extension of the approach to the comparison of protein flexibilities also for distant homologous proteins.DF, AP and LB jointly conceived the study. DF performed all calculations and data analysis. FS participated in the design and set up of the SOM protocol. AP and LB participated in the Molecular Dynamics studies and in the data analysis. DF, AP, FS and LB wrote the manuscript.Plot of RMSD to the starting structure during the MD simulations. The plots report, for all the systems, the RMSD values in the whole MD trajectory and a snapshot of the first 2 ns, to highlight the equilibration time.Click here for fileOverlap of sampling in the MD simulations of the SH3 domains. The values represent the overlap (E1) between the conformational spaces spanned by each half of the simulation and that of the overall trajectory.Click here for fileDistribution of motion in different subspaces for each MD simulation. Values refer to the percentage of total space described by the eigenvectors.Click here for filePlot of RMSF versus residue position in the essential space. MD simulations of: a) WT SH3; b) R21A and R21G mutants, compared to the WT; c) N47G and N47A mutants, compared to the WT; d) A56G and A56S mutants, compared to the WT. Only equivalent residues in the preliminary structure-based alignment are included and their numbering is modified according to the alignment. Secondary structures are reported in the bottom part of each graph for reference; they are attributed according to the DSSP program  and labelled following the nomenclature generally adopted for SH3 domains.Click here for fileComparative Silhouette plots. The panels report the cluster quality for average linkage, complete linkage and the corresponding two-level approach (SOM and average linkage or SOM and complete linkage). In each panel Silhouette profiles are plotted for the number of clusters ranging from 2 to 10. A Silhouette profile is composed by a bar for each identified cluster.Click here for fileDistance matrix and GROMOS clustering. The distance (RMSD) matrix for the combined trajectories of the ALL data set is reported in the upper triangular part of the image. The cluster attribution generated by GROMOS for a number of clusters equal to five is reported in the lower triangular part.Click here for filePlot of RMSD versus time during the MD simulations. From top to bottom, MD trajectories for the WT SH3, A56S and R21G mutants. Conformations attributed to the five clusters obtained from the SOM trained on the entire group of trajectories are coloured according to Figure Click here for file


Since sinomenine is a principle component of the Boiogito, there is a possibility of it being effective on osteoarthritis (OA) of the knee with joint effusion. However, there is no report concerning the effectiveness of Boiogito on knee OA. The objective of the present study is to investigate the therapeutic effect of Boiogito on OA of the knee associated with joint effusion in a comparative study among randomly assigned groups.Boiogito  contains Study was performed using 50 patients who were diagnosed with primary osteoarthritis of the knee with joint effusion. The patients were randomly assigned to two groups: one group (25 patients) using both loxoprofen (2-{4-[(2-oxocyclopentyl) methyl]} propanoic acid) and Boiogito and the other group (25 patients) using loxoprofen, and were evaluated during a 12 week observation period. The assessment parameters including knee scores in the Knee Society Rating System including Knee score and Functional scores, amount of joint effusion by joint puncture in clinically detected cases, the 36-items short form of the Medical Outcome Study Questionnaire (SF-36) as a measurement of health related quality of life were used.The knee scores based on the Knee Society Rating System were improved in both groups. The staircase climbing up and down ability in the Knee society rating system functional score was significantly improved in the group using Boiogito and loxoprofen compared to the loxoprofen group. In the evaluation using SF-36, significant improvements were found in the scores in both groups in physical functioning after 12 weeks. The amount of joint fluid was significantly decreased at 4, 8 and 12 weeks compared to pre-administration baseline in the group using Boiogito and loxoprofen. A side effect of Boiogito, dry mouth, was found in one case. The symptom was mild and improved immediately after discontinuation of administration.The results indicated that Boiogito have a possibility for a treatment modality for joint effusion with osteoarthritis of the knee. Osteoarthritis (OA) of the knee is a degenerative disease of the knee joints which significantly damages the functions of knee joints. The objective of its treatment is to decrease pain while attempting to maintain or increase the range of knee motion and to minimize disabilities in daily living. The majority of osteoarthritic patients visit the clinic with the complaint of pain. As clinical symptoms, joint effusion is frequently associated with pain and limitation in the range of knee motion.Sinomeniuim acutum, which has been utilized by Chinese doctors for over 2000 years to treat various rheumatic diseases [sinomenine inhibits inflammatory reactions [As conservative therapies for OA of the knee, education and physical therapy, oral administration of non-steroidal anti-inflammatory drugs (NSAIDs), intra-articular injection of hyaluronic acid, and use of lateral wedge insole or unloading braces are applied based on the degrees of symptoms . Furtherdiseases . Previoueactions ,6.Sinomenium Stem 5.0 g, Astragalus Root 5.0 g, Atractylodes Lancea Rhizome 3.0 g, Jujube 3.0 g, Glycyrrhiza 1.5 g, and Ginger 1.0 g in a daily dose of 7.5 g. That is constituted of numerous components including sinomenine as a principal component  contains a dry extract 3.75 g of the mixed drug substance consisting of The subjects used were patients with primary OA of the knee associated with clinically detected joint effusion. Those were outpatients having pain in the knee joints while walking and showing a Kellgren-Lawrence  grade IIth week were excluded from the study. As a result, the remaining 47 subjects (24 cases in the concomitant use group and 23 cases in the loxoprofen group) were used in the analysis of the treatment results.The subjects were randomly divided into two groups: one group treated by concomitant use of Boiogito and loxoprofen (thereinafter referred to as the concomitant use group) and loxoprofen administration group (hereinafter referred to as the loxoprofen group). Prior to the study, the subjects signed a written consent of their own free will. The treatment drug, Boiogito, was administered at a daily dose of 7.5 g divided into three portions which were given before meals or between meals. Further, loxoprofen at a single dose of 60 mg was orally administered after meals three times daily. The number of subjects enrolled in the present study was 50 subjects divided into 25 subjects in the concomitant use group and 25 subjects in the loxoprofen group with respect to the treatment drugs. Among them, three cases including two cases (loxoprofen group) who did not come back after the initial examination and one case (concomitant use group) who did not come back after 4The period of administration of the treatment drug was 12 weeks. The assessment parameters including knee scores in the Knee Society Rating System including Knee score and Functional scores , amount 2 tests for ratio of male and female, Wilcoxon's T- test for patients background, change in SF-36 and Knee Society Rating System knee score and functional scores, one-way analysis of variance for change in volume of joint fluid, and then, a Bonferroni analysis was applied to determine the significance of difference for multiple comparison. Significant level was set at p < 0.05.For statistical analyses, according to the types of data, the following methods were used: χRegarding the patients' background, with respect to gender in both groups, the number of females was greater than the number of males. The mean age was 68.3 years in the concomitant use group and 71.5 years in the loxoprofen group. The detailed preoperative comparison between the 2 groups is shown in Table The knee score in the Knee Society Rating System was 75.0 +/- 15.1 before administration in the concomitant use group which improved to 85.8 +/- 11.1 at the time of final observation and 72.8 +/- 16.0 before administration in the loxoprofen group which improved to 81.7 +/- 15.1, demonstrating significant improvements in both groups (p < 0.05). The staircase climbing up and down ability in the Knee Society Rating System of functional score significantly improved from 32.2 +/- 6.3 before administration to 40.2 +/- 4.4 at the time of final observation in the concomitant use group (p < 0.05). In the loxoprofen group, the functional score of stairs in Knee Society Rating System changed from 28.4 +/- 6.4 before administration to 36.6 +/- 6.2 at the final follow up. This change has no significant difference.In the evaluation of SF-36 which is an assessment of the quality of life of the patients, significant improvements were detected in both groups in the physical functions after 12 weeks (p < 0.05) . On the other hand, there were no significant differences in the amount of joint effusion at the time of 4, 8, and 12 weeks after treatment compared to those before treatment in the loxoprofen group  alpha and Interferon gamma in a rat autoimmune myocarditis model, mice dermatitis model and diabetic mice [astragalus which is contained in Boiogito have also possibility for decreasing symptoms of osteoarthritis with joint effusion.Since herbal medicines consist of numerous components instead of a single component, the mechanisms of decreasing the amount of joint effusion are still unknown. However, with respect to the pharmacological action of Boiogito, effectiveness of is model . It was tic mice -18. ThesConcerning the limitation of the present study, we did not investigate the single administration of Boiogito and also there is a limitation that the number of cases is small. The studies using a larger number of subjects and the studies including analysis of pharmacological actions based on the analysis of the characteristic changes in the joint effusion should be needed. Further, it is necessary to have a long-term assessment including the degree of satisfaction by patients in the general evaluation not only from the local results in the knees.In conclusion, this is the first report that the herbal remedy "Boiogito" have a possibility of conservative treatment for joint effusion associated with OA of the knee.Each author certifies that he has no commercial associations  that might pose a conflict of interest in connection with the submitted article.TM has made substantial contributions to conception and design, acquisition of data, analysis and interpretation of data. TM, MI, YK, TO and DT carried out the treatment and followed up the patient and contributed to acquisition of data. AM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Behavioral findings in several strategic games indicate that people punish others if they think they are being treated unequally, even at the cost of minimizing their own material payoff. We investigated the primary driving force behind such non-self-regarding behavior, so-called, altruistic cooperation. In all of our studies, a mini ultimatum game was played either one-shot (in Experiment 1a and 1b) or repeatedly (Experiment 2), and rejections of inequitable distribution were taken as a measure of altruistic cooperation. In Experiment 1a, we replicated previous findings indicating that the key mechanism contributing to the emergence of altruistic cooperation is fairness considerations. In Experiment 1b, we delved into the relative importance of two aspects of fairness considerations  and showed that both aspects were effective in determining the level of altruistic cooperation, with the contribution of intentions being more important. In Experiment 2, we investigated the effect of the opportunity for reputation building and future interaction on altruistic cooperation. We found that these factors became influential only when fairness considerations were weakened, particularly, as a result of the removal of the possible intentions behind an offer. Human altruistic cooperation presents a puzzle from the perspectives of both the standard economic models of the “self-interested actor” and the evolutionary models of the “self-regarding individual” because it involves some characteristics that are difficult to reconcile with the predictions of standard game theoretical and evolutionary analyses. One form of altruistic cooperation is to reward cooperators  and to punish norm violators  at a personal cost, even though the probability that this cost will be repaid (either by third parties or by that specific agent in the future) is very low  /future interaction to the emergence of costly punishment as a form of altruistic cooperation in experimental contexts.outcome proposed by the ProposerSome researchers argue that the underlying mechanism of such non self-regarding behaviors in the UG  is not only to get as much money as possible, but also to maintain fairness norms among players fair However, the available alternative distribution varies in terms of the outcome fairness, sometimes yielding a more equitable outcome  have been a source of much debate  and they will never meet again in another round . Anonymous and one-shot encounters eliminate the possibility of reputation building  and future interaction  respectively, as potential sources of this seemingly fairness-driven behavior  and the possibility of RB and FI (in Experiment 2), are in the emergence of altruistic cooperation in general and costly punishment in particular. Experiments 1a and 1b were designed to understand the role of fairness considerations in costly punishment. Note that, as pointed out previously, fairness considerations have two major aspects, one being related to s Rabin, . Althougs Rabin, , empirics Rabin,  for the Previous studies have already established the importance of intentions behind an action  in the Mini UG: the (8/2) distribution is rejected at different levels depending on whether the alternative distributions are perceived as fair or not . However, findings diverge in terms of rejection rates of the (8/2) distribution when the alternative distribution was more inequitable. More specifically, 9% of the Responders rejected the (8/2) distribution in the 10/0) game in Falk et al.'s  study wh0/0 game Experiment 1b was designed to clarify the relative impact of these two aspects of fairness considerations in the Mini UG. Two special features of this specific version of UG enable us to separate the effect of intentions from that of outcomes , the features of “one-shot-interaction” and “anonymity” are inseparable. Therefore, it is difficult to identify whether the obtained responses could actually be the product of the (mis)perception of one-shot encounters as repeated  or that of the (mis)perception of anonymous encounters as non-anonymous . Thus, incorporation of both possibilities of RB and FI through repeated and non-anonymous game play would make the two previously mentioned explanations (fairness-driven responses via one-shot/anonymous encounter vs. self-regarding responses via misperceived one-shot/anonymous encounter) commensurable. A second and even a more explicit reason was that the possibility of RB and FI are highly interrelated .In Experiment 2, we aimed to understand the combined effect of the misperceived possibility of RB or FI, then an increase in the level of altruistic cooperation should be expected when the actual possibility of RB and FI is added to the context. Although such an additional effect of the possibility of RB and FI has not been investigated in the Mini UG, there are two main reasons for expecting such an increase. First, the importance given to equality is expected to be elevated . In addition, in the Mini UG, there is one special game . Second, the actual offer was indeed determined randomly . UNSW Human Research Ethics Advisory Panel approved the study.Forty-five first year psychology students . The only significant difference in terms of the rate of rejections for the (8/2) distribution within the games was between the (5/5) and the (10/0) games 2  = 4.57, p = 0.033.The rejection rates of the 8/2) distribution across games are shown in Table  distribuThe results demonstrated that when the intentions of the Proposer were not assessable, (1) rejection rates of the (8/2) distribution significantly decreased when its alternative was a more equitable one , and (2) the overall difference in the rejection rates of the (8/2) distribution across four games was not strongly significant. Even then, however, the rejection rates were still not identical across the four games. This is an indication that these rejections were shaped by how unfair the outcome was perceived in comparison to the outcomes yielded by the alternative distributions. Overall, these results support the idea that two aspects of fairness considerations are involved in the emergence of costly punishment, but the contribution of the intention aspect of fairness considerations seems greater, especially when the alternative distribution yields a more equitable outcome .In order to test the effect of the real possibility of RB and FI we changed the structure of the Mini UG from being one-shot and anonymously played to being iterated and non-anonymously played. We predicted that the rejection rates of the (8/2) distribution in the Mini UG should be (1) even higher  when its alternative was the (5/5) distribution because it is adaptive to build the reputation that one is a tough bargainer who rejects unfair offers, and (2) even lower when its alternative was the (10/0) distribution because it is adaptive to give the message for future interactions that one is capable to discern and will reward fair intentions.M age = 19.76, 120 female) at UNSW participated in the experiment as a part of their course requirement and were informed that they would be paid depending on the outcome of their choices. Four participants were tested in each experimental session and there were 48 experimental sessionsOne hundred and ninety-two first year psychology students  and warned against revealing their allocated roles to the others. Individual players were then given detailed verbal instructions  regarding the general structure of the game play, what their roles required them to do, and what the consequences of their accept/reject decisions would be. They were informed that they would play the game for more than one round with the same partner, and that their decision would be announced to other players right before they switched their partners. However, the players were not given any information about (1) the possible distributions available to the Proposer in Mini UGs , (2) how many rounds they would play in total , and (3) when exactly they would switch partners . In order to eliminate a potential wealth effect, the participants were told that the overall amount that they would receive would be determined by a coin flip at the end of the experiment. If the coin toss came up heads, then they would get paid the amount that they earned in the first half of the experiment, and if tails, the amount earned in the second half (please see the Appendix section for the complete instructions). Afterwards, the instructions documents were collected, and the players were taken to the separate rooms to complete a short quiz (included in Appendix) measuring whether all the instructions were understood clearly.Each experimental session consisted of four consecutive rounds and in each round the participants played a different Mini UG game z-Tree p < 0.0001, and p = 0.035, respectively, but not than those in the (8/2) game, p = 0.75All participants passed the quiz distributed before the game play, thus all responses were included in the analysis. We first examined the extent to which the possibility of RB and FI influenced the Responders' overall rejection rates of the 8/2) distribution in each game in order to see how the opportunity of RB and FI could change this overall rejection pattern in each game. Table  distribu2 = 0.83, p = 0.36], the (2/8) games , and the (10/0) games . Contrary to our expectations, the rejection rates of the (8/2) distribution did not increase when the alternative distribution was (5/5), and did not decrease when the alternative distribution was (10/0). However, the (8/2) distribution was rejected in the (8/2) game much more frequently in Experiment 2 than Experiment 1a, χ2 = 15.14, p = 0.0001. Similar patterns of differences were obtained in the comparison of Experiment 1b and Experiment 2. No significant differences were found between Experiment 1b and Experiment 2 in terms of the rejection rates of the (8/2) distribution in the (5/5) 2 = 0.36, p = 0.55], and the (10/0) games . However, rejection rates for the (8/2) distribution were much higher in Experiment 2 than Experiment 1b in the (8/2) game, χ2 = 8.61, p = 0.003. We will return to the interpretation of these results in the General DiscussionA cross-experimental comparison demonstrated that the rejection rates of the (8/2) distribution between Experiment 1a and Experiment 2 did not significantly differ in the (5/5) games . A similar pattern obtained in the (10/0) game as well: except for the difference between Round 1 and Round 3 . The rate of rejections in the (2/8) game in Round 1 was significantly higher than in Round 2 , but not in Round 3 than Round 4 . However, the rejection pattern for the (8/2) game was different: the rejection rates of the (8/2) distribution were especially high in Round 1 and Round 3  = 5.18, p = 0.02], and in Round 3 than Round 4 . This pattern indicates that the effect of RB and FI was especially prevalent in the (8/2) game.For the analysis of round by round rejection patterns, we first conducted a logistic regression by including dummy variables for different rounds, and correcting the standard errors for the clustering on participants . Afterwards, we tested the pairwise round differences for each game. Table d in the 0/0 game p = 0.05). However, for the (8/2) game, rejection rates of the (8/2) distribution, especially in Round 1 and Round 3 were significantly higher in Experiment 2 than those in both Experiment 1a and 1b  distribution in each game in Experiment 2 with the rejection rates in the corresponding games in Experiment 1a and Experiment 1b  respond to intentional unfairness in a Mini UG at a cost to their own material payoff. The difference in the pattern of results between Experiment 1a and 1b showed the relative impact of the aspects of the fairness consideration in shaping altruistically cooperative behaviors. We found that when a distribution yielding an unequal outcome between players was intentionally offered in the presence of a more equitable distribution , that distribution was rejected much more frequently  than when it was unintentionally offered . This pattern of results indicates that from the perspective of the Responder, the intentions of the Proposer matter significantly. However, even when the intentions of the Proposers are not involved, another aspect of the fairness considerations is still present: it is the (un)fairness of the outcome distribution that governs the Responders' rejection behavior. The rejection rates of the unequal distribution were changed depending on the relative equitability of the alternative distribution.In the literature, there are two distinct approaches to the fairness preferences over material benefits  distribution did not change when the Responders were expected to punish unfair offers  or to appreciate fair offers . Cross-experimental comparisons of the rejection rates obtained in Experiment 1a  and Experiment 1b  with the overall rejections rates in Experiment 2  confirmed that there were no changes in the rejections of an inequitable distribution in the (5/5) and the (10/0) games when the possibility of RB and FI was incorporated in to the context.Two potential but competing explanations of this pattern of results can be offered. One is that the possibility of RB and FI is indeed (mis)perceived in one-shot and anonymously played games, and thus did not lead to any differences in the pattern of responses when it was explicitly incorporated into the context  game], but not in the other games in which the intentions were assessable   distribution, especially in the (5/5) and 10/0) games0/0 gamesThe results indicate that the absence of fairness intentions was the primary reason for the possibility of RB and FI becoming effective. However, the comparison between Experiment 1b and Experiment 2 revealed the importance of “outcome fairness” aspect of fairness considerations as well. This is because, except for the (8/2) game, the rejection rates of the (8/2) distribution obtained in none of the games were significantly different between Experiment 2 and Experiment 1b. This finding implies that even the presence of outcome fairness  itself is strong enough to make the rejection rates reach a certain level—a level that could not get increased  or decreased  by the explicit incorporation of RB and FI. The possibility of RB and FI matters only when there is no intention behind the distribution offered, and there is no (more equitable or inequitable) alternative distribution to be offered .These results shed light on when and how the possibility of RB and FI influence the responses in bargaining games. The possibility of RB and FI is normally expected to increase the rejections in the Mini UG as the Responders want to build the reputation of being a “tough bargainer.” This is why such opportunity has been sometimes thought to be the source of conflict  in strategic environments fair intentions, in interactive economic decisions, particularly in the ultimatum bargaining games. The main conclusion drawn from the experiments reported here is that when both outcome and intention fairness considerations are involved in decisions they have a strong combined effect on the emergence of costly punishment as a form of altruistic cooperation, and thus override the potential influence of RB and FI. When an important aspect of fairness concerns, namely intentions, are absent, RB and FI may play an important role.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The sub-micrometer PbS with anisotropic microstructures including fishbone-like dendrites, multipods, cubes, corallines, and hopper cubes were successfully prepared by the solvothermal process. Different morphologies can be obtained by adjusting the reaction temperatures or using the nontoxic controlled reagents which can tune the relative growth rate in the <100> direction and the <111> direction of PbS nuclei. Based on the viewpoint of crystallography about face-centered cubic crystal structure, the possible formation mechanism for sub-micrometer anisotropic structures has been discussed. The difference between the enhanced growth rates on the {100} and {111} planes induced the change of ratio between the growth rates in the <100> and <111> directions, which resulted in the formation of the different PbS anisotropic microstructures. The PbS anisotropic structures exhibited the different visible emission with a peak in the red regions mainly attributed to the variation of shape, size, and the trap state of as-obtained PbS. When the lead acetate (Pb(AC)2) solution was added into 1-pyrrolidine dithiocarboxylic acid ammonium salt (C5H12N2S2) solution, the thick suspension of white precipitate came into being. We separately prepared the precursor with identical solutions and manipulation. The TG-DTA and element analyses of precursor were described in details in the Additional fileIndeed, the route in this work was a thermal decomposition of uni-precursor {[Pb, exposing six {100} facets and eight {111} facets. According to the literature, the final shape of a PbS nanocrystal was mainly determined by the ratio of the growth rate in the <100> direction to that in the <111> direction. From the viewpoint of crystallography, the {111} facets of a rock salt structure intrinsically have a higher surface energy than that of the {100} facets, and the growth kinetic energy barrier of the crystal facet is inversely proportional to the surface energy. This suggests that the growth rate along the <111> direction is higher than that of the <100> direction. However, the growth environment can influence the formation of anisotropic structures, dendrites, multipods, and cube with hoppers. Lifshitz mentioned that the main environmental parameters are as follows: (a) the stabilizing surfactants which can enhance or block the growth of specific facets, (b) the reaction temperature and reaction duration which determined the choice of thermodynamic or kinetic growth, and (c) a large concentration of the precursors to enable a fast kinetic growth and Ostwald ripening effect. In a woIn this work, when the reaction temperature was 165°C and 180°C, the growth rates were fast. This led to a preferential kinetic growth of branches. The difference between the enhanced growth rates on the {100} and {111} planes induced the ratio R to have a value of more than 1.73, which resulted in the formation of the dendrites as shown in FigureIn some corresponding reports, the synthesis of special PbS nanostructures was usually achieved with the stabilizing surfactants. These surfactants are mostly amine compounds, such as ethylene diamine (en). Ma et al. proposed that the amine molecules stabilize the {100} surfaces and, thus, enhance the natural habit of the rock salt structure to react faster along the <111> direction. In our a = 0.5936 nm).The crystallinity of the as-prepared dendrites, multipods, and hopper cubes are clearly illustrated by their distinct XRD patterns shown in Figure2+/S2− source (1:1) were replaced by 2:1 and 3:1. The results showed that the molar ratio of Pb2+/S2− weakly affects the final morphologies of PbS crystals in the present case.In addition, the present works indicated that the compounds of CTAB, EDTA, SDS, CTA, and PEG-400 can also modulate the relative growth rate on the different surfaces of NaCl-type PbS crystals. Some novel anisotropic microstructures have been created through the solvothermal processes with their assistant. When CTAB was added into the system, dendritic PbS microcrystals were obtained Figure. The croSDBS are around 692, 716, and 721 nm with a near equal narrow FWFM of approximately 20 nm, while the intensities of T180 and T180SDBS are stronger than T165. A great deal of investments suggested that the PL performances of nanoparticles are generally impacted by the shape, dimensions, size, size distribution, or density of defects. In the present work, the sharp optical spectra in three PL imply the narrow size distribution and blueshift compared to the bulk counterpart (NIR region) suggesting the strong degree of confinement. The samples T180 and T180SDBS should hold better crystallinity than T165 and possess lower density of defects. The opportunities of nonradiative recombination of photoinduced electron–hole pairs will be depressed, so the intensities of their PL spectra are higher than T165. It is considered that the differences of emission peaks are mainly attributed to the variation of shape, size, and the trap state of as-obtained PbS. The lower wavelengths of T165 and T180 implied the stronger confinement than T180SDBS, which can be understood from the fact that T165 and T180 are composed of one-dimensional units with smaller sizes simultaneously, while the T180SDBS has no one-dimensional structures.FigureIn conclusion, the solvothermal route has been successfully developed to prepare sub-micrometer PbS anisotropic structures with different morphology. The solvothermal synthesis of PbS anisotropic structures including fishbone-like dendrites, multipods, cubes, corallines, and hoppers has been facilely applied by adjusting the reaction temperature or using the different nontoxic reagents. The results indicated that the modulation of reaction condition with different surfactants, which can adjust the ratio between the growth rates in the <100> and <111> directions, is the key for the fabrication of the NaCl-type PbS microstructures.The authors declare that they have no competing interests.YC and PH carried out the preparation of the samples, analysis of the data, the proposition of the mechanism and wrote the manuscript. DJ participated in the design of the experiments and performed the photoluminescence analysis. All authors read and approved the final manuscript.The structure synthesized with different SDBS concentrations was shown in Figure S1. The TG-DTA and element analyses of precursor were described in Figure S2 and Table S1.Click here for file


The present sero-genetic study is the first of its kind to present the baseline data of Bharia tribe of Madhya Pradesh. The main aim of this study is to provide phenotype and allele-frequency data to characterize the population genetically and to fill the void on the genetic map of Madhya Pradesh.For this, blood samples from 92 unrelated healthy individuals of Bharia tribe from Chhindwara district (Tamia block) were collected. Hemolysates prepared were analyzed for two serological (A1A2BO and Rh) and six biochemical  parameters, following the standard electrophoretic techniques.The Chi-square test for goodness of fit revealed no significant deviation between the observed and expected numbers in any of the seven genetic markers, suggesting that the tribe is in genetic equilibrium. A high incidence of B allele in A1A2BO blood group and low incidence of the A1 allele, with presence of A2 in only one individual, and a low frequency of Rh(D)  was observed in serological markers. Also, no rare variant was observed for biochemical markers.Principal Component Analysis done in order to detect the genetic affinity of Bharia tribe with other populations from the adjoining states of Madhya Pradesh based on the allele frequencies, showed a close association of Bharia with Gujarat and Rajasthan. Hence, this study has been helpful in revealing the genetic structure and affinity of Bharia tribe. In the past three decades, a great deal of human genetic data has been generated, but not much work has been carried out on the serological and biochemical markers of the tribal population from the central Indian state of Madhya Pradesh. A large amount of data on ethnic, regional and social distribution of genetic markers has already been reported on Indian populations, but our knowledge about genetic variation of different genetic markers is still limited.–6 Althoudhaya (a system of shifting cultivation) cutters, but nowadays they rear cattle and sell milk and milk products. They are also farmers and mostly produce small millet called Kodo-Kutki. Other areas of activities include forest wealth collection and hunting.Tribes are the assets of a country and the central Indian state of Madhya Pradesh is one of the largest states of India inhabiting the bulk of tribal population of the country , constituting 20.3% of the total population  of the state. Amongst a total of 46 scheduled tribes of the state, the main focus of this study was on Bharia tribe, mainly the inhabitants of the district Chhindwara . Bharia i1A2BO and Rh) and biochemical  data of Bharia tribe from Chhindwara district of Madhya Pradesh.The present study is therefore planned to present the serological (AThe present study comprises blood collection (about 1 ml each) from a total of 92 healthy unrelated individuals of either sex from Tamia block of Chhindwara district, after obtaining informed consent, in accordance with the ethical standards of Institutional Ethical Committee, following ICMR guidelines. Hemolysates, prepared by freezing and thawing technique, were electrophoresed for studying variabilities of different red cell enzymes in Bharia tribe. The electrophoretic typing of the six erythrocyte enzymes was performed following the original techniques for ACP locus 1 (ACP1), PGM locuFrom phenotype data obtained after electrophoresis, allele frequencies were calculated using the gene counting method. Chi-squa12The phenotypic distribution of two serological markers (A1A2BO and Rh) and six erythrocyte enzymes  are presented in In the A1A2BO system, blood group B was found to be the most preponderant and A2 incidence was much lower, whereas the occurrence of A1 was observed to be more than that of O. In terms of allele frequency, highest frequency was that of B (0.2491) and lowest was of A2 (0.0108). In case of Rh system, Rh(D) was observed in all the samples of Bharia tribe. In each of the five enzyme systems considered, all the possible common phenotypes were found, but no example of any rare variant was encountered. For ADA, high allele frequency was recorded for ADA*1 (0.8939), which is found frequently in Indian population. Similar result was obtained for AK1 and PGM1 with high allele frequency of AK1*1 (0.9278) and PGM1*1 (0.6667), while for ACP1, allele ACP1*B showed high occurrence. Interestingly, ESD showed equal value for both the alleles, whereas ESD*1 is the most frequently occurring among the Indian population. Goodness of fit Chi-square test revealed that the distribution of phenotypes in each of the blood groups and enzyme systems typed was in genetic equilibrium in the studied Bharia tribe.By and large, in the case of A1A2BO blood group system, it was observed that the average value of allele B was more as compared to allele A. Among the tribes, the value of B was high as compared to A; however, the difference between A and B frequencies was less among the tribes. Similar 125As far as the erythrocyte enzyme markers are concerned, variations at different enzyme loci were observed. The present ADA*1 frequency in Bharia tribe was found similar to that observed in Keer, while Bh10ESD showed remarkable equal frequency for both the alleles, ESD*1 and ESD*2, despite the fact that the frequency of common allele ESD*1 is reported to be 75% in the Indian population. Low sample size can be a possible reason for this, as earlier publications have shown an average allele frequency of 0.7315 for the other tribal populations of Madhya Pradesh.12 ACP frBoth the PGM1*1 and PGM1*2 alleles are present in all populations in appreciable frequencies  in Indians. No rare variant was observed in Bharia, while so far only Korku tribe from Hoshangabad have shown the occurrence of PGM1*7. For GPI,26Locus-by-locus comparison in Bharia suggests considerable intra-tribal variation in phenotype and allele frequencies. Chi-square value showed a good agreement between the observed and expected values in Bharia tribe. Both serological and biochemical markers were in genetic equilibrium as the differences between observed and expected frequencies were statistically not significant. Also, a considerable range of heterozygosity values across the loci was examined. The highest value of heterozygosity was found for the A1A2BO locus (0.6059) and the lowest for GPI (0.0109), with an overall mean of 0.291. On the other hand, PGM1 and ACP1 showed similar heterozygosity values (0.4444). The variation in average heterozygosity may simply be the genetic consequence of the population structure or it may indicate the effect of polymorphic or directional natural selection operating in the present Bharia tribal population of Madhya Pradesh. This wide variation in heterozygosity values from locus to locus also stresses the need for their estimation at a large number of loci to provide an adequate average heterozygosity estimate. Estimates of average heterozygosity ranging from 0.127 to 0.513 have been reported for Indian populations.29 Some oThe dendrogram  construcThe present study has witnessed variation at different loci in the Bharia tribe, suggesting intra-population genetic differentiation. Every population is different from the other population in terms of its genetic constitution, but this diversity is more at the intra-population level than at the inter-population level. The present study has thus presented a possible illustration of the genetic structure, and the affinity of Bharia tribe of Madhya Pradesh has been revealed. Moreover, genetic studies on Bharia tribe featuring the variations at molecular level, which would help to further support the above presented data, are still under consideration.