We present a new approach to model dose rate effects on cell killing after photon radiation based on the spatio-temporal clustering of DNA double strand breaks (DSBs) within higher order chromatin structures of approximately 1–2 Mbp size, so called giant loops. The main concept of this approach consists of a distinction of two classes of lesions, isolated and clustered DSBs, characterized by the number of double strand breaks induced in a giant loop. We assume a low lethality and fast component of repair for isolated DSBs and a high lethality and slow component of repair for clustered DSBs. With appropriate rates, the temporal transition between the different lesion classes is expressed in terms of five differential equations. These allow formulating the dynamics involved in the competition of damage induction and repair for arbitrary dose rates and fractionation schemes. Final cell survival probabilities are computable with a cell line specific set of three parameters: The lethality for isolated DSBs, the lethality for clustered DSBs and the half-life time of isolated DSBs.By comparison with larger sets of published experimental data it is demonstrated that the model describes the cell line dependent response to treatments using either continuous irradiation at a constant dose rate or to split dose irradiation well. Furthermore, an analytic investigation of the formulation concerning single fraction treatments with constant dose rates in the limiting cases of extremely high or low dose rates is presented. The approach is consistent with the Linear-Quadratic model extended by the Lea-Catcheside factor up to the second moment in dose. Finally, it is shown that the model correctly predicts empirical findings about the dose rate dependence of incidence probabilities for deterministic radiation effects like pneumonitis and the bone marrow syndrome. These findings further support the general concepts on which the approach is based. Understanding the dose and dose rate dependence of the cellular response to radiation is of key interest for risk estimations after occupational or accidental radiation exposure as well as for medical applications in radiation oncology. In general, significantly reduced effects are observed after protracted irradiation as compared to acute irradiation where the same total dose is given in a short time of just a few seconds or minutes. Typical dose rates cover a broad range from mGy/year, that are particularly relevant for everyday radiation risk, up to several Gy/min, which are of interest for effects as a consequence of medical applications or severe radiation accidents.Many studies thus aim to elucidate the impact of specific temporal patterns of dose delivery like e.g. low dose rates, pulsed dose rates or high dose rates Induction of DNA damage, in particular double strand breaks (DSBs), has been identified as the key initial event causing observable radiation effects like e.g. cell killing. However, intriguingly the mere number of DSBs is not sufficient to characterize the extent of the effects, since cells in general are able to process and to repair large fractions of the initially induced DNA damage Compartmentalization resulting from higher order chromatin structure represents a potential framework allowing for the classification of DSBs with respect to the multiplicity of DSBs in individual substructures. Chromatin loops of 1–2 Mbp genomic length have been identified as relevant for the processing of DSBs The aim of this paper is to present a kinetic model for the assessment of dose response curves based on the approach reported in In the following, the basic concepts of the model as presented in The Giant LOop Binary LEsion model (GLOBLE) i) which is much smaller than the probability for lethal events after clustered DSBs (εc). The term “lethal event” comprises a variety of events that lead to cell death, e.g. the inability of a cell to rejoin loose ends or any kind of misrepair of a DSB – potentially involving loop interactions - which has no viable outcome etc.The existence of giant loops suggests two categories of radiation induced DNA lesions (binary lesions) with different severity. In case that a single DSB is produced within a loop the two ends of the DNA strand always remain in proximity due to the attachment to their original site. In contrast, if multiple DSBs coexist in a loop, different sizes of DNA fragments might diffuse away and in some instances larger gaps might be opened in the chromatin. Consequently, although the loss of smaller fragments of the DNA might be tolerable for a cell, it should be expected that in the average cellular repair mechanisms run with a much lower fidelity after the induction of multiple DSBs than after the induction of single DSBs. This assumption that the average lethality significantly increases if a second DSB is produced in a loop but only weakly changes with every further DSB, motivates the common classification of multiple DSBs as a “clustered DSB” in contrast to “isolated DSB” in the GLOBLE. Isolated DSBs go in hand with a cell line dependent probability for lethal events (εi and εc the knowledge about the average number of isolated and clustered DSBs (in and cn) after irradiation is sufficient to calculate the corresponding cell survival probability S. It is the Poissonian probability for no lethal event: iL and cL denominate the average number of lethal events due to isolated and clustered DSBs, respectively.With εLN equally sized domains within a nucleus. Given a genomic length of about 6000 Mbp and an approximate loop size of 2 Mbp one can roughly deduce that LN≈3000.In order to assess the numbers of isolated and clustered DSBs after irradiation in the GLOBLE, the DNA giant loops are identified with small target volumes in the cellular nucleus – so called domains i and εc.As a first working hypothesis with respect to the lethality of DSB classes, domains are supposed to be closed objects. That is, the lethality of damage is fully determined by the number of initial DSBs induced within an individual loop and it does not explicitly depend on the damage induced in the neighboring domains. This does not exclude per se the interaction of DNA ends from different domains and thus e.g. the formation of chromosome aberrations as a consequence of misrepair or misrejoining processes. Lethal events as result of domain interactions are comprised in the mean values εD with a yield of αDSB = 30/Gy/cell. Thus, the average number λ of DSBs per domain is If instantaneous photon radiation is applied, there is a homogeneous dose deposition over the cellular nucleus. In consistency with our previous studies Exploiting Poissonian statistics to get the probabilities for 0,1,2,… DSBs per domain after the irradiation procedure it holds that: For the average number of isolated and clustered DSBs it follows that: i and εc of the GLOBLE in the origin (D = 0) and the coefficients from the Linear-Quadratic model  has been derived: In According to these equations, isolated DSBs are responsible for the initial linear slope of cell survival curves in the GLOBLE and isolated and clustered DSBs affect the quadratic component.In the derivation of all presented formalisms a constant radiosensitivity over all the phases of the cell cycle has been assumed as a first approach.For the kinetic extension of the GLOBLE, a concept of levels comparable e.g. to laser theory is introduced. In the dynamic process during irradiation, domains transit from one level to another one. To quantify the time dependent occupation of a level, the cell population averaged fraction of domains in a nucleus on the respective level is considered. From the presented premises of the GLOBLE one can derive five levels with corresponding occupations:0f: Average fraction of domains with no DSB and no lethal eventif: Average fraction of domains with one DSB which has not been processed yetcf: Average fraction of domains with more than one DSB which have not been processed yetil: Average fraction of domains with lethal event(s) after processing an isolated DSBcl: Average fraction of domains with lethal event(s) after processing a clustered DSBiL, is calculated separately from the number of domains cL which have suffered a lethal event after a clustered DSB. To be consistent with the working hypothesis that domains are closed objects with respect to the lethality attributed to the different DSB classes, the transition of one domain to another level is independent of the other domains on the same initial level. Since the lethalities comprise lethal events after interaction, there are no quadratic terms but only linear terms involved in the temporal change of the occupation of the levels.The distinction of two levels which incorporate domains which have suffered a lethal event is for the sake of comparability with the static formulation of the GLOBLE. Here, the number of domains with a lethal event due to an isolated DSB, 0f a domain migrates to the if level after the induction of a first DSB and afterwards into the cf level if a next DSB is produced in coexistence with the first one  or they migrate to the il and cl level respectively (red arrows) which are “absorbing” (no arrows leave these levels). The former transition means that some repair process has rejoined the DSB(s) within a DNA giant loop with a viable outcome . The latter transition means that misrepair has led to at least one lethal event in the corresponding loop and that a viable outcome is no longer possible.From the two levels where damages have not been processed yet, cf to the if level.In the kinetically extended GLOBLE, different repair processes with varying repair fidelities are involved in the rejoining of isolated and clustered DSBs respectively. A last DSB remaining after all the other DSBs from a clustered one have been rejoined should not evoke a more effective repair mechanism. Therefore, there is no way to transform a clustered DSB to an isolated one and so there is no transition (arrow) from the Setup of differential equations: To quantify the time development of the five GLOBLE levels and to finally assess the interference of DSB induction and repair, adequate transition rates have to be found. Motivated by the supposition that DSBs are produced linearly in dose it holds for an arbitrary dose rate 0f to if and from if to cf  is followed by a much slower one  iHLT and the half-life time of the slow phase of repair with the half-life time of clustered DSBs cHLT. The individual rates ir, for the transition from the if to the 0f level (if → 0f), cr for cf → 0, mif for if → il and cm for cf → cl can be gained from the total repair rates : The processing of DNA DSBs in the nucleus during cellular repair mechanisms should define how the levels xm and the corresponding total repair rate is the probability for a lethal event (εx) it holds that Due to the fact that the ratio between the lethal misrepair rate Putting all the previous statements together yields five coupled differential equations (DEs) in the kinetic extension of the GLOBLE: The survival after an arbitrary irradiation schedule which imposes the adequate initial conditions for the solution of the DEs is defined by D which is delivered with a constant dose rate T: In case that an irradiation schedule consists in the application of a single dose il and cl after an infinite amount of time as One can express the occupation of the two levels T where the irradiation ceases. i and εc define how much more domains will suffer lethal events until infinity. Finally, the occupation of the levels which represent the domains with lethal events after an infinite amount of time is given by the sum of the replenishment during and after the irradiation.That is, in order to calculate the average fractions of domains which have suffered at least one lethal event after an infinite amount of time d separated by a time 1t. If a cellular nucleus contains LN domains, an acute photon irradiation with dose d will produce an average of t = 0: In the split dose experiments considered in this publication cells are treated with two equally sized acute doses r domain . Therefop(0 DSB), p(1 DSB) and p(≥2 DSBs) can be calculated with D by d). Between the two doses there is no induction of lesions but a repair of DSBs takes place. So at time 1t there areHere, the upper script “+” denotes “after irradiation” and an upper script “–” will denote “before irradiation” in the following. The probabilities isolated andclustered DSBs left in the average. The expression for the average number of domains without DSB and lethal events i and εc, one can compute the average number of domains that have suffered a lethal event during the repair procedure  or  are presented graphically in the chosen publications they were read in with “GetData Graph Digitalizer” (further information to be found athttp://getdata-graph-digitizer.com/). For stability reasons, the natural logarithm of the survival data and of the computed survival were taken for the execution of fit procedures with the GLOBLE. To find the optimal set of parameters {εi, εc and iHLT} for each of the examined cell lines the least squares method was used. For this purpose, the sum of squared residuals over all the data points collected for one cell line was expressed in dependence of εi, εc and iHLT. The resulting function was minimized with the “NMinimize” function and the “DifferentialEvolution” method in Wolfram Mathematica 8.0.0 (http://www.wolfram.com/mathematica/).In order to validate the concept of the kinetic extension of the GLOBLE, we first assessed the quality of the model in the description of larger sets of experimental data for the two cases of irradiation with constant dose rate and of split dose experiments. We then analyzed the limiting cases of dose rate Dose rate experiments:i, εc and iHLT} which – according to the concept - allows for the prediction of cell survival probabilities over the whole range of applied dose rates. In other words, for each cell line, there is a characteristic set of parameters {εi, εc and iHLT} which determines the effect of any single dose treatment with constant dose rate on this cell line.The measured data in the two panels in i, εc and iHLT} describes all the survival curves taken under the application of different dose rates reasonably well. There are no systematic under- or overestimations. The derived parameters εi, εc and iHLT reflecting the radiation response of the investigated cell lines are presented in In fact, this statement is generally manifested by the other 15 dose rate experiments. In every case a common set of parameters {εi, εc and iHLT are reasonable if they are judged by their proposed biological meaning. The two probabilities for lethal events are i is always << εc and the half-life time of isolated DSBs is smaller than 3 h. The HX32 and the CHO 10B2 cell lines make exceptions featuring half-life times of isolated DSBs which are higher than one would expect from experiments (5.7 h and 6.1 h respectively). However, since peculiar parameter values occur only in these two cases this should not constitute a serious shortcoming. If the two outliers are not accounted for, the median of iHLT is 0.48 h which is in good agreement with experimental observations.As it can be seen in In the end, there is only one systematic problem in the application of the GLOBLE in dose rate experiments: if cell lines show a dose rate effect despite a linear acute survival curve this can hardly be reproduced or predicted with the kinetically extended GLOBLE. In the model, the linear component of survival curves is dose rate independent and thus linear survival curves with dose rate dependent slopes corresponding to one cell line are not compatible. Optimal fits to the amount of available data sets for such cell lines– in this investigation HX138, HX142 and SB - show too pronounced shoulders at high dose rates to compensate for an initial slope which is too small.Split dose experiments:i, εc and iHLT}. There is no systematic deviation between the modeled graphs and the measured points in terms of an under- or overestimation of the survival in certain sections of the curves.i is always << εc and the half-life times for isolated DSBs are in the range of experimental observations with a median of 0.458 h. There are no exceptions or peculiarities which would argue against the applicability of the GLOBLE in split dose experiments.Fits to split dose experiments in four other cell lines verify the good performance of the GLOBLE in terms of a distinct agreement of measured data points with the describing graphs. Furthermore, Limiting case: Dose rate: If the applied constant dose rate T) this means that the dose D is given in an instant to the target. Thus, in the limit For cf = 1−0f − if, the solution of these differential equations and the expression of cf is the same as equations Exploiting the fact that Limiting case: Dose rate → 0: If a single dose is applied with a very low constant dose rate there is a large amount of time for the processing of a first DSB before the next lesion occurs within the same domain. Since there will be no coexistence of two or more DSBs in one domain, no clustered DSBs will be produced during the irradiation. For the system of differential Given no DSBs at the beginning of irradiation, the survival probability derived from the solution of this system of differential equations is implied by: iαDSB which is consistent with the static case and −6/Gy2), the upwards bending of survival curves predicted with the GLOBLE is of no practical relevance.The first order Taylor expansion coefficient of the solution equals εDose rate effect in cell survival probabilities: In cell survival studies, treatment planning and many other areas it is common to quote parameters of the Linear-Quadratic model (LQ) for the characterization of a treated cell line. Therefore, comparing the kinetically extended GLOBLE with the LQ and relating the newly developed parameters with the established ones is instructive.The introduction of the Linear-Quadratic model (LQ) for the description of cell survival after instantaneous irradiation was mainly motivated by the empirical observation that cell survival curves typically show a linear and a quadratic component if they are plotted on a logarithmic scale. Consequently, in the LQ, the negative natural logarithm of the survival is a polynomial of second order in the dose: D is stretched over a protraction time TIt can be adjusted for a reproduction of dose rate effects in cell survival probabilities occurring when a single dose r represents the linear restitution of chromosome aberrations in time in the original publication. Therefore, in a more general sense, only the kind of lesions which is responsible for the linear slope of the LQ survival curves enters the Lea-Catcheside factor G.In the following, we will always refer to this adjusted version if we consider the LQ. The rate T. This implies an increase in cell survival probabilities until maximum values are reached with straight survival curves. G (dotted lines) from 1 at small to 0 at large protraction times. G = 1 implies a full quadratic bending of survival curves after acute irradiation  and G = 0 implies straight survival curves.Through the introduction of the Lea-Catcheside factor the quadratic contribution to modeled cell survival curves diminishes with an increase in the protraction time T with equation D = 0) up to second order which are equivalent to α (first order coefficient) and 2Gβ (second order coefficient) were derived. Independent of the protraction time, the first order coefficient equals the one that has already been found in the examination of the static GLOBLE. That is, no matter which protraction time is chosen for an irradiation, the initial linear slope of cell survival curves remains constant. This is comparable to the LQ and the identification of the first order coefficient at D = 0 with α yields α  =  εiαDSB in agreement with equation For a comparison with the LQ model, the dose rate −ln(S) at D = 0 varies with T in the kinetically extended GLOBLE. Since its mathematical formulation is longish it is not presented explicitly at this point. It is dominant in the prediction of the dose rate effect – i.e. the disappearance of shoulders in cell survival curves if one increases the protraction time of the irradiation. To find an equivalent for the Lea-Catcheside factor G the calculated second order coefficient of the GLOBLE was multiplied with 0.5 and normalized with the GLOBLE expression for β implied by equations r in the kinetic extension of the Linear-Quadratic model incarnates only the lesions which are responsible for the linear component of cell survival curves this rate r was identified with i + mi)r  =  (r was set to ln(2)/(0.5 h) and c + mc)(r was chosen to be ln(2)/(5 h). The upper panel (A) makes clear that the differences between the Lea-Catcheside factor and its GLOBLE-equivalent are hardly noticeable by eye. Therefore the lower panel (B) additionally gives the relative deviation of the two factors. It starts to increase rapidly to infinity at large protraction times – sooner for high α/β ratios and later for low α/β ratios. This is because the GLOBLE features a finite second moment at low dose rates  whereas the LQ second moment approaches 0. However, it has to be emphasized that the large relative errors are of no practical relevance as they affect only the values which are anyway almost identical to zero.Although the GLOBLE and the Linear-Quadratic model are almost equal up to second order there are distinct differences in the reproduction or prediction of cell survival probabilities as it was already stated in Incidence of deterministic effects of radiation: Cell killing is considered to be a major determinant of deterministic effects that are observed in tissues after radiation insult 50D at which 50% of the exposed persons show defined clinical symptoms of deterministic effects linearly increases with a decrease of the dose rate. This can be expressed in terms of a linear function of the inverse of the dose rate Empirically, it has been observed that the dose 50D after an acute irradiation and 50D for two extreme cases, namely pneumonitis and the bone marrow syndrome (black and red solid line). For pneumonitis values of 2/h are reported, whereas the bone marrow syndrome is represented by 2/h. Accordingly, one sees a large increase of 50D for pneumonitis if the dose rate is lowered but only a small shift of 50D in case of the bone marrow syndrome.The parameters i and εc were derived by means of equations i/εc and with that via equations i = 0.00333 and εc = 0.229 for pneumonitis and εi = 0.00333 and εc = 0.09 for the bone marrow syndrome. To find an endpoint equivalent to 50D with the GLOBLE, the dose leading to the same cell survival probability as the instantaneous application of 10 Gy (for pneumonitis) and 3 Gy (for the bone marrow syndrome) was taken (isoeffective dose).For a comparison with the GLOBLE, ε50D in In In the previous sections it has been shown that the kinetically extended GLOBLE is an appropriate model for the assessment of cell survival after photon irradiation with arbitrary dose rates. In the following we will briefly discuss the results amongst others by pointing out conceptual differences to other models and by comparing the repair rates found in the results section to other published data.The GLOBLE differs from other approaches in the modeling of cell survival due to a strong commitment to empirical findings. Not only the input parameters but as well the introduced classes of lesions are strongly linked to biological mechanisms which were deduced from experimental observations. In particular the introduction of two discrete lesion classes namely isolated and clustered DSBs that are characterized on the basis of the multiplicity of DSBs in higher order chromatin structure represents a distinct feature of the model. In the GLOBLE cell survival thus is not directly expressed in dependence of the dose, as it is common in the setup of many other cell survival models. Instead, it is primarily the spatio-temporal damage distribution pattern that is used to determine the cell killing probability. At first, the distinction between dose deposition and damage distribution pattern might appear to be subtle. However, actual differences in the dose deposition pattern do not necessarily lead to differences in the damage distribution pattern. That is, completely different local and temporal dose depositions might produce the same amount of isolated and clustered DSBs and therefore evoke the same radiation effect.In this regard, the kinetic approach proposed in this publication suggests an extension to other radiation qualities. Whereas we have been focusing here on applications to photon radiation, an extension to high-LET radiation is straightforward and will thus be an essential part of future investigations. Since parts of the presented concepts are also implemented in the Local Effect Model (LEM) As already indicated, there might be some clustered DSBs composed of fragments which are too small to allow for a distinction from isolated DSBs in terms of their individual lethality. However, in the special case of photon radiation which has been considered in this publication there is a homogenous dose deposition over the cellular nucleus which implies an almost homogenous damage distribution. Therefore, the yield of extremely small fragments should be negligible in a first approximation. With regard to modeling cell survival after ion radiation the introduction of a threshold for fragment sizes defining a clustered DSB might be necessary due to the high density of lesions along the particle tracks.iHLT from the GLOBLE are directly at hand  by Oliver et al. In the GLOBLE approach presented in this publication the clustering of DSBs within DNA giant loops is considered to be decisive for cell survival probabilities. That is, the existence of multiple DSBs within chromatin structures of about 2 Mbp or within a few micrometers is supposed to drastically increase the risk for cell death compared to single lesions within such orders of magnitude. This hypothesis is strengthened by findings of Johnston et al. who state that chromatin loops in the order Mbp are the “critical targets for induction of DSBs” On the other hand, there are indications that the clustering of DNA damage including single base damages, strand breaks and double strand breaks within a few nanometers is important for the effectiveness of radiation. Nikjoo et al. Modeling work directly comparing short scale and long scale clustering effects as reported by Friedland and co-workers i << 1, which thus has to be regarded as an effective lethality for all  isolated DSBs. However, for photon radiation the fraction potentially attributable to clustered lesions on the nm scale is unlikely to change within the experimental relevant range of doses and dose rates, since presumably all DSBs are induced by single track effects as indicated by the linear dependence of the yield on dose. When the concept of the GLOBLE will be used to model ion radiation effects, the lesion induction concept of the LEM may be used. There an enhanced DSB yield compared to photons is resulting from an explicit simulation of single strand break clustering on the nm scale. In conclusion, clustering of damage on the nanometer versus micrometer scale is not contradictory in the concept of the GLOBLE but rather complementary.Although damage clustering on the nm scale is not explicitly accounted for in the GLOBLE, this should not reject the possibility for its influence on cell survival. Since in the GLOBLE we refer to measured DSB yields, these implicitly include the whole spectrum of complexities  that can be attributed to individual DSBs, and the fact that only a certain fraction of DSBs has a high impact on cell killing might be reflected in the value of εAs demonstrated in the results section, the GLOBLE allows for a reproduction of low LET cell survival probabilities after application of protracted or split doses. Since there are no systematic deviations, the simplifications made as a first approach seem to be reasonable. For instance, the assumption of a constant, average radiosensitivity over all phases of the cell cycle does not strongly bias the final results although in principle cell cycle dependent radiosensitivities have to be expected. We found that there are no significant differences in the quality of the description of the chosen experiments where cell cycle effects can be excluded with varying confidence or cannot be excluded at all.One reason why distortions due to cell cycle effects should not be expected in in most of the considered experiments anyway is given by the relatively long population doubling time of the investigated cell lines in comparison to the maximum irradiation time. This statement is amongst others supported by Kelland and Steel i and εc; the same holds true for a variation of the DSB yield.A next point that might be discussed is the assumption of a constant DSB yield of 30/Gy per cell. In experiments the range of measured DSB yields goes from about 25/Gy per cell In order to further assess the consistency of the mechanistic ideas behind the GLOBLE approach, we will compare the corresponding half-life times with other observations reported in the literature.iHLT) can be compared to published half-life times of the fast component of repair. For instance, the half-life times for isolated DSBs (iHLT is determined with GLOBLE fits to split dose experiments (column 4), these values are much closer to the values gained by an exponential recovery fit to the same experimental data – as it should be expected. There is no systematic under- or overestimation.Furthermore, Obviously, the half-life times found with the IR model and the LPL model in dose rate experiments (column 5 and 6) underestimate the values found in split dose experiments in the same order of magnitude as the ones determined with the GLOBLE. There seems to be a common disagreement of theory and measurements which amongst others Stephens et al. and Steel et al. already pointed out in their publications of 1987 iHLT from the half-life times gained with an exponential recovery fit might be reasoned with the different approaches in taking into account cellular repair processes. In the GLOBLE there are two classes of lesions which are repaired with different fidelities and kinetics whereas in the exponential recovery there is only one kind of lesion with a purely exponential decrease in the time after irradiation. Although the magnitude of the half-life time of clustered DSBs cHLT is of little importance , the recovery takes less time and the maximum survival probability is reached at smaller times between the fractions, consequently.The impact of the diverging half-life times computed on the basis of either dose rate or split dose experiments is visualized in iHLT found in dose rate and split dose experiments do not falsify the hypothesis that the characteristics of one cell line can be described with one set of parameters in the GLOBLE. The experimental uncertainties have to be taken into account here. Furthermore, the values of εi found with fits to the different experimental approaches are in the same order of magnitude Click here for additional data file.


The aim of this article is to propose several methods that allow to investigate how and whether the shape of the hazard ratio after an intermediate event depends on the waiting time to occurrence of this event and/or the sojourn time in this state.A simple multi-state model, the illness-death model, is used as a framework to investigate the occurrence of this intermediate event. Several approaches are shown and their advantages and disadvantages are discussed. All these approaches are based on Cox regression. As different time-scales are used, these models go beyond Markov models. Different estimation methods for the transition hazards are presented. Additionally, time-varying covariates are included into the model using an approach based on fractional polynomials. The different methods of this article are then applied to a dataset consisting of four studies conducted by the German Breast Cancer Study Group (GBSG). The occurrence of the first isolated locoregional recurrence (ILRR) is studied. The results contribute to the debate on the role of the ILRR with respect to the course of the breast cancer disease and the resulting prognosis.We have investigated different modelling strategies for the transition hazard after ILRR or in general after an intermediate event. Including time-dependent structures altered the resulting hazard functions considerably and it was shown that this time-dependent structure has to be taken into account in the case of our breast cancer dataset. The results indicate that an early recurrence increases the risk of death. A late ILRR increases the hazard function much less and after the successful removal of the second tumour the risk of death is almost the same as before the recurrence. With respect to distant disease, the appearance of the ILRR only slightly increases the risk of death if the recurrence was treated successfully.It is important to realize that there are several modelling strategies for the intermediate event and that each of these strategies has restrictions and may lead to different results. Especially in the medical literature considering breast cancer development, the time-dependency is often neglected in the statistical analyses. We show that the time-varying variables cannot be neglected in the case of ILRR and that fractional polynomials are a useful tool for finding the functional form of these time-varying variables. In this article the disease course of primary, non-metastatic breast cancer patients is analysed taking first isolated locoregional recurrence (ILRR) of the tumour into account as an intermediate event. In this context, usually separate analyses are carried out for each endpoint and also for the intermediate events. As pointed out by  “these se.g.11]. The J and the separate transition hazards approach S.The following two paragraphs discuss the joint transition hazards approach J models transitions 1 →3 and 2 →3 jointly. A time-varying covariate indicates whether the patient is in state 1 or 2 and therefore if the first or the second hazard function is “active”. The model is: Approach 1It≥d}{ is the time-varying indicator variable and β the corresponding parameter. The transition 1 →2 is modelled apart from the other transitions. Therefore the resulting hazard functions are: where z are assumed to have the same effect on both transitions to the absorbing state (common parameter γ). For transition 1 →2 the effects of the covariates are modelled separately (γ12 independent of γ). The relation between the hazards λ13 and λ23 is defined as c:= exp ). If only the time-varying indicator is used, i.e.f≡1, the relation c = exp (β) is constant and the transition hazards 1 →3 and 2 →3 are assumed to be proportional. If c = 1, the hazard function is not changed by the intermediate event. If assumption of proportionality is not realistic, time-dependent structures can be added to the function c as shown in Table If this approach is used, the time-invariant covariates Models J.III (1 and 2) and model J.IV violate the Markov assumption. The estimated parameters of the time-varying covariates allow for testing directly the difference between the hazards before and after the intermediate event. The models are nested and it is therefore possible to use directly a criterion (AIC for example) to assess the goodness of fit. This constitutes an advantage of this approach.S models all transition hazards separately. The transition λ12 is modelled like in approach J (eq.(1)). Therefore we obtain : Approach d and the time since recurrence t - d is modelled via the function f. Approach J is a special case of model S.The time-invariant covariates are allowed to have different effects on all hazards. The time-dependency of the hazard 2 →3 on the time of recurrence λ0,23 what is not self-evident. In equation (2) the time since the first diagnosis t is chosen. This approach is often called “clock forward” approach. Figure t - d can also be used . If the time-scale t is chosen, only individuals who have already changed to state 2 can be used for the estimation of λ0,23. It is therefore necessary to account for left-truncation. The implementation of the time-scale t - d is easier. However, when using time since recurrence, the comparison of the hazard rates before and after recurrence is no longer straightforward.Using this approach, one has to chose the time-scale for the function S is a special case of the stratified model 32]. These.g.[This article demonstrates that the shape of the hazard ratio in the “illness-death” model depends on the inclusion or exclusion of time depending covariates. Comparisons between model J.I up to model J.IV with “death” as outcome variable confirm that the ILRR should be modelled as a time-dependent intermediate event. The results allow the determination of the impact of the ILRR on the course of the disease. Comparing the results of model J.I and J.II it is obvious that the ILRR is a time-dependent phenomenon and affects the hazard rate. Entering the study, the patients are under risk of ILRR or death. After having experienced an ILRR they are only at risk of death. Therefore a model has to be chosen where the assignment of the observations to different risks is not fixed in advance but dynamic in time. This is ignored in many publications when the patients are grouped retrospectively by recurrence as in . Models e.g.. The reacf. Figure J restricts too much the shape of the hazard ratio. The relation between the hazards in approach J does not model the difference between the hazard rates satisfyingly and is therefore not the appropriate model. Approach S shows that an early ILRR leads to a sudden increase of the risk of death which decreases relatively fast after the recurrence, but that a late ILRR (after 2 years) only increases the hazard by a factor of about 2.5 which does not longer depend on time since recurrence t - d. From a medical point of view, it is interesting to know whether the appearance of an ILRR is due to the first disease and expresses the declining health of the patient, or if the ILRR is independent from the first disease. The results show that the ILRR is not completely independent from the first disease. Indeed, the risk of death remains on a higher level after an ILRR (ratio >1). But there is also an independent part: approach S indicates that an early recurrence leads to an additional risk, but this risk diminishes after successful removal of the second tumour.The comparison of model J.II and J.III.1 indicates that the time of recurrence affects overall survival. The later the recurrence appears, the lower is the risk of death. The comparison between model J.III.1 and J.IV implies that the hazard function depends also on time since ILRR. With respect to overall survival (J models the shape of the ratio correctly, but the dependency on time of recurrence d could not be analysed. In fact, in all J models this variable was not significant. The model S however shows this dependency on d via the baseline hazard. This proves that the proportionality assumption between the hazard functions λ13 and λ23 in model J does not match the data. Whenever the ILRR appears, the risk of distant disease has a peak thereafter. This peak is more pronounced if the ILRR occurs early (Figure With respect to distant disease free survival, approach y Figure . After tThe results also show that fractional polynomials are useful to determinate the functional form of time-varying covariates. Even if the results of the J.IV model using a predefined exponential function are almost equivalent in the case of overall survival, the results show that the use of FPs is the best choice for distant disease free survival.Summing up, we have investigated different modelling strategies for the transition hazard after ILRR or in general after an intermediate event. An example is given in which including time-dependent structures alters the resulting hazard function considerably. It is important to realize that there are several modelling strategies and that each of these strategies has certain restrictions and may lead to different results.It was also shown that fractional polynomials are a useful tool for finding the functional form of the time-varying variables.Diagnostic methods have changed over the last 20 years. The aim of the paper was to show that the time-varying effect of the ILRR has not to be neglected. The methods developed for doing this are still valid nowadays. The aim of this article was not to reanalyse the four studies of the German Breast-Cancer Study Group.We declare that we have no competing interests.MS conceived the research topic. CMH explored that idea, performed the statistical analysis and drafted the manuscript. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/13/80/prepub


The results of cognate docking with the prepared Astex dataset provided by the organizers of the “Docking and Scoring: A Review of Docking Programs” session at the 241st ACS national meeting are presented. The MOE software with the newly developed GBVI/WSA dG scoring function is used throughout the study. For 80 % of the Astex targets, the MOE docker produces a top-scoring pose within 2 Å of the X-ray structure. For 91 % of the targets a pose within 2 Å of the X-ray structure is produced in the top 30 poses. Docking failures, defined as cases where the top scoring pose is greater than 2 Å from the experimental structure, are shown to be largely due to the absence of bound waters in the source dataset, highlighting the need to include these and other crucial information in future standardized sets. Docking success is shown to depend heavily on data preparation. A “dataset preparation” error of 0.5 kcal/mol is shown to cause fluctuations of over 20 % in docking success rates. Since then many docking programs have been developed [Docking methods have now been in existence for over 35 years, starting with Levinthal et aln fibers . Since teveloped , primarieveloped . To monieveloped –10. Howeeveloped , the tareveloped , the inpeveloped , 10, andeveloped . As a reOne major stumbling block to the advancement of protein–ligand docking validation has been the lack of a standard test set agreed upon and used by the entire community. The absence of such a set is one reason why it can be difficult to compare or even reproduce published docking results, because access to the primary data used in the computational experiments is often limited . The neeTo overcome the lack of organized competitions and standardized test sets in protein–ligand docking, efforts have been made to publish datasets, such as Astex , and DUDWith no standing organization to produce standardized datasets and to run competitions for protein–ligand docking, it is left up to individuals to organize fair and un-biased events , 31–33. Development of a new scoring function, GBVI/WSA dG.The results of cognate docking with MOE using the ACS-Astex set input ‘as-given’ and after in-house manipulations.Detailed analysis of docking failure cases, which point out errors and inconsistencies in source test datasets.The effect of dataset preparation on docking error, and the effect of error on docking success rates.This paper presents the results of our participation in this session and covers four major points of discussion:not in the ACS-Astex set,  and (b) we wanted a simple force field-based scoring function with fewer terms, which will have a smaller probability of being over-fitted compared to more complex scoring functions [The MOE docking architecture provides a standardized docking workflow that divides docking into a series of protocols, each of which can be modified and adjusted independently of the others. Thus it provides a good starting platform to compare the effect of each aspect of the docking workflow on the final results. To this effect, we developed a new scoring function, which was easily plugged into the existing MOE architecture for this study. We elected to develop a new scoring function instead of using existing functions because (a) we wanted to test a scoring function developed and trained on data unctions .The results of cognate docking to the ACS-Astex set are presented, both using the data ‘as-given’, as suggested by the competition organizers and after in-house manipulations. The performance improvements along with details and justification of the in-house input manipulations will be discussed.Special attention is given to the docking failures, especially in cases where failure should be expected because of problems with the source data, such as bad contacts and incorrect chirality. We show that even the highly-curated ACS-Astex dataset used in this study has several problems, despite being of modest size and having been examined by experts in the field. This highlights the real technical and scientific difficulties in preparing protein–ligand validation test sets. Based on experiences in this study, recommendations for dataset preparation are put forth.Finally, the estimated errors in docking that result from differences in structure preparation are shown to significantly affects docking, giving rise to differences in success rates greater than 20 %.For training and testing of the GBVI/WSA dG  scoring function, the SIE  and CSAREach set was downloaded from their respective sources , 35. TheThe protein–ligand binding free energy is calculated using a formalism similar to that of SIE.ng GB/VI  which esmentclass2pt{minimc are constants that were fit to the affinity values of the 99 complexes of the SIE dataset. Fits were done using partial least squares regression in the MOE QSAR module. Additionally the scaling factor for electrostatic interaction was empirically set to 2/3 which yielded higher accuracy then the ideal theoretical value of 1/2 [α, γ and e of 1/2 .The initial dataset was prepared by the organizers of the ACS session and given to participants to use “as-given” . DespiteUpon closer examination of some of the complexes it was noted that the hydrogen bond network was not optimal and therefore further optimization was warranted. Two examples demonstrating the need for re-optimization are 1MMV and 1V4S  were identified as containing an inverted stereocenter in the original dataset given to the participants compared to the original PDB structure. Thus, docking results that use the corrected version of these ligands (where the stereocenters were set to be the same stereochemistry as in the PDB) will be referred as the “corrected” set.The MOE-Dock architecture consists of four major components: (1) ligand-conformation generation (2) optional pharmacophore filtering (3) ligand placement and scoring in the pocket, and (4) flexible receptor and ligand refinement with re-scoring. In this study, ligand conformation generation was accomplished by supplying the docking engine with an ensemble of prepared ligand conformations generated using the Conformation Import application , with deThe binding site region was defined using the crystallographic ligand for all datasets. Since the purpose of the study was to demonstrate the upper limit of docking accuracy on the simple problem of self-docking, the default “Rigid Receptor” protocol was used , as oppoc, chb and cm are constants which have been trained on over 400 protein ligand complexes. Eflex is a topological estimate of ligand entropy. Both fhb and fm are measures of geometric imperfections of protein–ligand and metal–ligand interactions. The top 1,000 poses produced from placement were then scored using the London dG scoring function .\documenin GB/VI . The topLigand placements were assessed with the root-mean-squared-deviation (RMSD) between the heavy atoms of the predicted pose and those of the crystal structure. The percent success (% success) for placement was defined as the number of systems where the RMSDs to the crystal structure of a docked pose is less than a given threshold.The SIE set was selected for training the scoring function because the SIE scoring function formalism is similar to that of the proposed GBVI/WSA dG scoring function. Additionally, the SIE scoring function has been shown to be predictive in various tests and applications –51, demoTraining of the GBVI/WSA dG scoring function on the SIE dataset resulted in a mean-unsigned error (MUE) of 1.35 kcal/mol which is significantly better than the null model, where the average affinity of the set is used as the predictor  had inverted ligand stereochemistry when compared to the PDB structures. These inversions were present in the input data given to us by the organizers . IncorpoThe improvement in docking results going from the ‘as-given’ to the ‘modified’ and ‘corrected’ datasets is also reflected in the pose RMSD statistics give in Table The decrease in median RMSD for the top 1 pose between the ‘as-given’ and ‘corrected’ data-sets reflects improvements in scoring achieved by using the ‘corrected’ versus the ‘as-given’ data-set. This increase in docking accuracy by using optimized structures as input has been seen with other docking programs  which reFor purposes of this study a docking failure is defined as cases where the top scoring pose has an RMSD to the crystal structure of greater than 2 Å. Based on this criterion, 17 out of 85 ACS-Astex complexes (20 %) would be considered failures. The PDB codes and details of the failure are reported in Table Nearly half of the failure cases (8) were due to placement failure, where the docker was unable to generate any pose under 2 Å RMSD. In six other cases, failure was due to scoring, because a pose was generated under 2 Å but it was not scored as the top pose.The organizers identified many structural errors present in 22 complexes which normally exclude them being selected as part of a docking set. These problems include complexes which contain poor electron density of the ligand and/or binding pocket residues, alternate location of residues close to binding pocket and possible crystal packing interactions with the ligand. Surprisingly only 2 of the 22 problem structures docked unsuccessfully and made it to the failure list. This included 1HVY which was identified as having crystal packing interactions with the ligand and 1Y6B which was identified as having an alternate location of a residue in the binding site. To see if 1HVY failed due to exclusion of the crystal packing interactions, symmetry-related residues with at least one atom within 10 Å of any atom in the asymmetric unit were created. This new structure was then used for re-docking of the 1HVY ligand. In the case of 1Y6B, it was re-docked using the lower occupancy conformation of CYS1043. In both cases re-docking did not yield a successful result. This suggests that the identified structural problems were not causing the failures to produce successful docking results.Further examination of the 17 failures suggested that many could be attributed to a lack of bridging water molecules, metal binding interactions and predictions in solvent-exposed regions of ligands.Of the 17 complexes which failed to dock successfully, 8 fail due to the lack of bridging water molecules  but it scores 0.5 kcal/mol higher in energy than the best scoring pose (Top 1 = 2.40 Å RMSD) which binds to the zinc. The exclusion of the crystallographic water from this complex is the root of this docking failure even though it is not bridging between the ligand and receptor. To accurately model this system one should not only include the water but be able to accurately model the displacement of the water and the associated displacement cost.Predicting metal interactions is a recognized issue in the field of docking and scoring . While tAnother common docking failure is the scoring of solvent exposed regions of the ligand such as with 1N2V. In the crystal structure the butyl group of the ligand is solvent exposed making no strong interactions with the protein. While we can generated a good pose (Top 30 = 1.14 Å RMSD) it is over 0.5 kcal/mol higher in energy than the best scoring pose (Top 1 = 2.23 Å RMSD). The docked pose is favoured because the butyl group is placed into a pocket which is filled with water in the crystal structure.Overall, many of the failures cases can be attributed either missing information in the curated dataset or the accuracy of the scoring function. Although the competition and the dataset produced are steps in the right direction, the results here suggest that future curated docking datasets need to contain all the information from the crystal structure. The results also demonstrate the necessity of including crystallographic waters to predict the correct pose in some cases. While the addition of crystallographic waters have been shown to increase docking accuracy, it must be noted that there is no improvement when including them for binding affinity predictions .. found that correcting multiple structural problems in the CSAR set resulted in decreasing the MUE by 0.5 kcal/mol for SIE [Even though a respectable success rate was achieved on ACS-Astex set, this was only achieved by preforming additional preparation to the structures that were initially given. This additional preparation accounted for an increase in 11 % accuracy when compared to running on the ‘as-given’ set. This suggests that the GBVI/WSA dG scoring function is sensitive to minor modifications in the protein environment which is typical of any force-field based scoring function. This sensitivity can have a dramatic effect on the accuracy of docking and binding affinity predictions. As an example, Sulea et al for SIE .To accurately distinguish between active and decoy ligands in virtual screening, there must be good separation in predicted binding affinities between actives and decoys. In the case of pose prediction one needs significant separation in energy between good (≤2 Å RMSD) and bad (>2 Å RMSD) poses. In other words the further the pose is from the crystal structure the higher in energy it should be. On average, in reference to docking using MOE on the ACS-Astex set, good poses are within 1.07 kcal/mol of best scoring pose, while bad poses are 2.74 kcal/mol. This difference of 1.67 kcal/mol may seem large when compared to the 0.5 kcal/mol error associated with dataset preparation but is close to the limit of accuracy of the GBVI/WSA dG scoring function . In fact by examining the number of docking poses versus their relative energy difference with the best scoring pose, the good and bad poses overlap significantly . The error perturbation allows estimation of the effect of data preparation or force field error on docking success rate. Within one standard deviation of the average RMSD for a complex, the success rate at 2 Å RMSD can vary from 72 to 95 %  it can be to compile, curate and maintain a docking test set. The number of errors and the details surrounding each error case suggest that the probability of a single person flawlessly preparing and maintaining an entire docking dataset is low, especially as the dataset becomes large. Other datasets used as docking standards are also known to contain errors , 66, 67,adding additional data, such as correcting chain termini, capping chain breaks and adding missing side chains. Information such as alternate conformations of residues and crystallographic waters should also be retained. Any decision to remove information from the structure should be decided by the researcher, after retrieving the curated set.Future datasets should consist of only The curated dataset must be updated when an error is identified and not allowed to propagate. If the problem is with the experimental data and cannot be updated, the structure should be removed from the set.Future datasets should move beyond self-docking (which was done in this study) since proteins are dynamic objects and therefore is a best case scenario only and include multiple structures of the same protein with different ligands (cross-docking).Benchmarking sets should be prepared, curated and stored by the community as a whole, such as the multiple revisions to the CSAR set. Until a common community built dataset is created, it will be difficult to draw conclusions from a comparative docking study.Until a universal docking data preparation protocol is developed and accepted by the community, docking studies should always consider the effect of dataset preparation on docking performance, including estimates of the magnitude of dataset preparation error, and its effect on the reported docking performance.The results of this study suggest a series of recommendations for future docking dataset preparation:When a docking dataset created with these recommendations is available we can better assess docking methods and move towards creating blinded competitions such as CSAR, CASP and CAPRI. Through competitions like these the field of docking will hopefully become more robust and reliable.


The article aims to test the hypothesis that audiovisual integration can improve spatial hearing in monaural conditions when interaural difference cues are not available. We trained one group of subjects with an audiovisual task, where a flash was presented in parallel with the sound and another group in an auditory task, where only sound from different spatial locations was presented. To check whether the observed audiovisual effect was similar to feedback, the third group was trained using the visual feedback paradigm. Training sessions were administered once per day, for 5 days. The performance level in each group was compared for auditory only stimulation on the first and the last day of practice. Improvement after audiovisual training was several times higher than after auditory practice. The group trained with visual feedback demonstrated a different effect of training with the improvement smaller than the group with audiovisual training. We conclude that cross-modal facilitation is highly important to improve spatial hearing in monaural conditions and may be applied to the rehabilitation of patients with unilateral deafness and after unilateral cochlear implantation. Binaural localization of sounds includes several strategies which are based on interaural difference cues in the sound pressure levels and in the times of arrival of the sound Adaptive cross-modal brain plasticity is known to be a common case after sensory loss Recent psychophysical studies have shown that audiovisual training can increase the rate of learning and can improve perceptual performance in the auditory or visual modality alone The aim of our study was to find out whether enhanced audiovisual integration induced by training can improve the localization of a sound source in monaural conditions. To test our hypothesis, we trained one group of subjects in an auditory-only protocol, another group of subjects with spatially and temporally congruent audiovisual stimuli . BesidesThree days before the training session, all the subjects were tested in bi- and monaural conditions for auditory localisation of sounds. When combining all the subjects, the binaural performances were relatively precise see  with meaIn all groups, the subjects went through a daily session of monaural sound localization during 5 consecutive days. A direct comparison between pre- and post-training performance when the sound was presented alone, showed that the accuracy of the subjects increased after 5 days of practice. However, in spite of a daily training, the subjects never reach the performance level observed in binaural conditions during the pre-training test and in all groups the unsigned errors (in degree) remained statistically higher than that observed in the normal situation (bootstrap).The improvement in sound localization varies according to the conditions of practice  and according to the side of stimulation with respect to the plugged ear. When comparing the unsigned error before and after training , one canIn the group which received a visual indication of their performance accuracy (group FB), subjects also present an overall improvement in their sound localization performances expressed as a 10.8° (±0.1°) reduction in their mean unsigned error of sound localization.In the audiovisual group (group AV), a spatially congruent visual stimulation was presented simultaneously to the sound. After the 5 training sessions, when tested in auditory alone conditions, this group showed the highest improvement in monaural sound localization with a global decrease in unsigned errors of 13.6° (±0.1°). Such increase in accuracy is statistically greater than that observed in the FB and A-only groups . Of importance is that such an improvement by audiovisual training can be observed for all the spatial fields in the azimuth. The highest reduction in spatial errors was observed when the sound was presented in the 30–70° ispilateral to the plugged ear (20.6°±0.4° reduction). The improvement was the smallest in the −30°/+30° central region (6.3°±0.2° decrease) and intermediate when the sound appeared in the side ipsilateral to the unplugged ear (12.2°±0.4°). In the A-only and the FB groups, the amelioration of the performances of the subjects is also the strongest for sound location in the azimuth ipsilateral to the plugged ear. In the A-only and FB group the subjects tend to be worse in localizing the auditory stimuli when it appears in the central 30 degrees from the central fixation point on each side. On the opposite, in the AV group, the subjects showed a fairly significant improvement in sound localization for sound located in the central region. This can be also seen in Finally, we compared the performance of the audiovisually trained group for the audiovisual trials in the beginning of the training and in the end. This analysis was performed to check if the subjects were guided by the visual cues to localize the sound source. The errors during the audiovisual stimulation were minimal  and were much lower than that observed for the same group in the binaural pre-training session (p>0.5). Further, no significant difference was found for any loudspeaker in the comparison of the training days (p>0.7). Such analysis suggests that the subjects were highly influenced by the visual stimuli to localize the sound but failed to reveal an improvement due to the training in the visual modality.To conclude, we observed the highest significant improvement in monaural sound localization when the sound is simultaneously accompanied by the visual cue; such improvement can be obtained for all azimuth location of the sound source.We used signal detection measures to separate perceptual (d′) and decision-level (ß) effects. On the basis of the similar performances between groups during the pre-training test, we searched for an effect of the training protocols  on the evolution of the d′ values with the hypothesis of a larger increase of the perceptual sensitivity following the bimodal training. As explained in the method section, we considered as “hits” the responses located within 5° from the centre of the sound source. This method for the hits corresponds to the one applied in the feedback group to indicate the correct response during the experiment, thus we can compare directly the d′ values of all our groups. The direct comparison of pre- and post-training values of d′  did not It is worth mentioning that the different training protocols are also expressed by different variations in the decision criteria of the subjects when pre- and post-training data are compared . SubjectIn normal binaural conditions, sound localization in the horizontal plane is performed by computing the differences in intensity level or time of arrival of the sound (ILD and ITD respectively) that are present between the two ears There are numerous evidences that normal hearing subjects can learn to localize a sound source when the spatial cues are experimentally modified Several hypotheses have been proposed to explain how subjects can adapt to the altered interaural cues induced by the earplug including internal representation However, in the AV and FB groups, additional mechanisms have to be considered because these subjects present a much higher level of adaptation to the altered binaural cues when compared to the A-only group. Perceptual learning One conceptual question which emerges from our results concerns the theoretical differences between the “feedback” inputs and multisensory interactions in the present conditions. In the group that received a bimodal visuo-auditory stimulation in half of the trials (AV group), the improvement can be also interpreted as resulting from the feedback mechanisms provided by the azimuth and time congruent LED. The temporal order of feedback inputs is important in the efficiency of perceptual learning There is a large body of evidence for the importance of synergy between sensory modalities in our global perception and the associated behaviour Our results showed that the repetitive presentation of the visual stimulus in the temporal and spatial congruence with the auditory stimulus can improve the performance for the auditory stimulus presented in isolation. Such results imply a convergence of the visual and auditory spatial representations in the brain. Several studies have pointed out the role of the tectum in merging auditory and visual spatial maps  helping them to restore the auditory modality. We have shown that in post-lingual CI recipient, patients maintain the high skill in lip-reading acquired during the prolonged period of deafness, even after several years of auditory recovery Eighteen normally-hearing subjects  participated in the protocol. They were divided into 3 groups with no distinction of age and gender between the groups (3 men and 3 women per group). All subjects reported no auditory or neurological disease and had normal or corrected to normal vision. All participants gave their full-informed consent prior to their participation in this study in accordance with the Declaration of Helsinki (1968). The study was approved by the local research ethics committee . The subjects were financially compensated for their participation.The experiment was conducted in a dark soundproof anechoic room. A subject sat on a chair with his chin stabilized on a special framework (UHCOTech HeadSpot). During the experiment, the subjects were asked to fixate upon a green light-emitting diode in front of them which corresponded to the central loudspeaker. The study was realized in monaural conditions; one ear of the subject was plugged with an ear plug (average noise reduction 30 dB) and covered with an ear muff (average noise reduction 20 dB). The opposite muff was taken off, a sponge was glued to the resulting free end of the muffing device and it was comfortably placed behind the subject's open ear during the experiment.The apparatus consisted of 15 piezoelectric loudspeakers arranged horizontally in semicircle with a radius of 0.5 m in front of the subject, the subject being in the centre of the semicircle . LoudspeHaving perceived the sound from a loudspeaker , the subject had to indicate the source of the stimulation with a laser beam. This beam was projected from a rotating emitter in the centre of the semicircle of loudspeakers which can be manipulated by the subject through a manual knob. A home-made device, using a numerical potentiometer was allowed to record the position of the laser beam on the semicircle with a precision of 0.3°. A knob for rotating the laser emitter was on both the right and the left sides of the support so that the subjects could use either hand. Near each knob, there was a button to confirm the response. The subjects turned the knob with their preferred hand and pressed the button on the other side with the other hand. At time the response button was pressed, the laser position in degrees was registered. Having confirmed the response with the button, the subject repositioned the laser beam at the centre of the semicircle and waited for the next trail to start. The inter-trials time interval was random in the interval of 0.5–1.5 sec.Auditory stimuli were the rectangular white noise  generated by Adobe Audition 3.0 lasting 50 ms and presented at the intensity of 60 dB SPL (measured at the centre of the semicircle of loudspeakers). Visual stimuli (red LEDs) of same duration (50 ms) were delivered simultaneously to the sound in cases of visuo-auditory conditions (see below). The LEDs were located above each loudspeaker (and above the black fabric covering the loudspeakers).pre-training sound localization abilities. The subjects were firstly engaged in 15 trials to familiarize them with the device, then 5 trials per loudspeaker were presented binaurally . Then the presentation of the monaural (to the left ear) auditory stimulation followed with 10 trials per loudspeaker resulting in 150 random trials. Having perceived the sound from a loudspeaker, the subject had to indicate the source of the stimulation with a laser beam as explained above. The pre-training session as well as the training sessions lasted about 1 hour including 2 pauses of 5 minutes each.Three groups of 6 subjects participated to this protocol during which they underwent a testing session once a day during five consecutive days. Three days before (pre-training session), subjects had to perform the task in binaural and in monaural conditions to get familiar with the apparatus and to asses their During the training sessions, the subjects were divided into three comparable groups. In one group, the conditions of stimulation were auditory only (Group A). In the second group, auditory stimuli were accompanied in half of the cases by an azimuth spatially congruent visual LED (Group AV). Audiovisual trials in this second group were presented randomly among auditory stimuli. In a third group of 6 subjects, stimulation was only auditory but in half of the cases a feedback was given to the subjects on the accuracy of their performance (Group FB). After pressing the button a small screen fixed above the central loudspeaker indicated “correct” or “incorrect”. A “Correct” message was given when the response was ±5° from the centre of the correct loudspeaker. If the response was outside this 5° range, the “incorrect” indication was presented. All the experimental conditions were similar to the ones of the auditory group (Group A).Each day during 5 consecutive days, subjects were presented a session of 20 trials per loudspeaker . The performance of the subjects of all groups were analyzed and compared before (pre-training) and after this 5 days practice (post-training) during an auditory-alone presentation. Thus, by comparing the pre- and post-training performance, in Group A we assessed the effect of auditory practice, while in group AV we could observe the effect of audiovisual training on spatial monaural hearing in the horizontal plane.Direct comparisons of the post- and pre-training performance, as well as between groups, were performed using the bootstrap method with bias-corrected and accelerated confidence intervals First, we considered the difference in unsigned error (deviance of the response from the source of the sound in degrees) before and after training for each group. For each loudspeaker, the errors were re-sampled 60 times, then we calculated the mean post- pre-training difference for each sample and re-sampled the difference 10000 times to obtain confidence intervals per loudspeaker.We also calculated the post- pre-training difference after dividing the semicircle into three sectors: the “plugged sector” corresponding to the responses to sound locations at 70°, 60°, 50°, 40°, and 30° on the side ipsilateral to the plugged ear, the central sector corresponding to the positions at 10° and 20° on both sides, the “unplugged” sector that encompass the locations at 70°, 60°, 50°, 40°, and 30° ipsilateral to the unplugged ear. For each sector, we re-sampled the error 300 times, calculated the mean post- pre-training difference for each sample and re-sampled the difference 10000 times to obtain confidence intervals per sector.Finally, we applied SDT to analyse the performance of the subjects


To return to their feet, inverted click-beetles (Elateridae) jump without using their legs. When a beetle is resting on its dorsal side, a hinge mechanism is locked to store elastic energy in the body and releases it abruptly to launch the beetle into the air. While the functional morphology of the jumping mechanism is well known, the level of control that the beetle has over this jumping technique and the mechanical constraints governing the jumps are not entirely clear. Here we show that while body rotations in air are highly variable, the jumps are morphologically constrained to a constant “takeoff” angle  that directs 98% of the jumping force vertically against gravity. A physical-mathematical model of the jumping action, combined with measurements from live beetle, imply that the beetle may control the speed at takeoff but not the jumping angle. In addition, the model shows that very subtle changes in the exact point of contact with the ground can explain the vigorous rotations of the body seen while the beetle is airborne. These findings suggest that the evolution of this unique non-legged jumping mechanism resulted in a jumping technique that is capable of launching the body high into the air but it is too constrained and unstable to allow control of body orientation at landing. Click-beetles (Elateridae) are among the record holding insects that apply powerful catapult leverages to jump to heights (or distances) of many body lengths Athous haemorrhoidalis, with body length 10–12 mm) jumped to a height of up to 30 cm (i.e. >25 body lengths) and performed up to six somersaults in the air before landing Evans Lanelater judaicus from movies showing the trajectory of the beetles in air. The kinematics data and beetle morphology were used to construct a biomechanical model of the jumping mechanism. The model was then used to evaluate the jumping constraints from the mechanical relation between the flexion dynamics of the body and the resulting aerial maneuver.Intrigued by the evolution of a unique righting mechanism that seems to be deprived of means to control the orientation of the body during the jump and landing, we investigated how a fixed elastic action results in different body orientation at landing every time. We analyzed the jumps of Lanelater judaicus  and two mirrors (26×30 cm each) or later at 250 frames s−1 using two Photron SA3 cameras. The mirrors were placed perpendicular to each other and angled at 45° to the line of sight of the camera. When a beetle was positioned between the mirrors, it was visible to the camera from the front directly and from two sides through the mirrors. To position the beetle on its back between the mirrors with minimal handling interference we used a piece of smooth cellulose paper placed in the path of a walking beetle. Once the beetle walked over the paper, the paper was gently raised and made vertical above the desired location. The beetle lost foot-hold on the slippery surface and slid off the paper onto its back. Typically, the beetle first attempted to find a foot hold that could aid in righting by swinging all legs through the air. After several futile trials they tucked their appendages close to the body, assumed the pre-jump posture The aerial maneuver of 9 individuals of The shape and dimensions of the body extracted from orthogonal images of 4 dead beetles were used to construct geometrically simplified models of the body . We treaM) that rotates the subunits towards each other. The moment flexing the body was assumed not to vary over the short time of the flexion (dM/dt = 0). Consequently, the angular acceleration of each subunit was taken as constant and each subunit rotated according to oI is the moment of inertia of the subunit about the hinge.Manipulation of the dead beetles revealed that rotation of the subunits about the hinge was constrained by the morphology of the rigid exoskeleton. The flexion angle of the body (θ), measured between the long axes of the subunits , was appA and P denote the anterior and posterior subunits respectively. After rearranging:B, which is a constant - the ratio of the moment-of-inertia of the two subunits. Writing the constant acceleration equations for α and β for the duration of the flexion (ft):B . The four modeled beetles were used to calculate an estimate for B (Both subunits are rotated by the same moment (but in opposite directions) so that:te for B .When the body, resting against the ground, changes from the pre-jump to the flexed posture the center of mass is accelerated upwards launching the beetle into the air The live beetles were observed somersaulting and/or rolling in the air. B calculated from our modeled beetles  in equation 6 yields α = 48.93° and β = 6.07°. Using these angles to rotate the two subunits about the hinge in the models, we found that the translation of the center of mass (cmd), as the body changed from the pre-jump to the flexed posture, was cmd = 0.7±0.06 mm. The translation was towards the posterior and up  at an angle of 77.6±1.33° (mean ± s.d. of n = 4) to the horizontal. This angle constitutes the jump angle predicted by the models. The predicted angle (77.6°) was only 2.3° smaller than the mean angle observed in live beetles  about the long axis by 1° (from 1° to 2°) increased the speed of roll from 8.6 to 19.91 revolutions s−1. The variability of observed rotation rates in live beetles (The simulations of angular speed at takeoff  showed t beetles  is withiAdjusting the flexion angle of the body after takeoff has limited effect on the moment of inertia of the entire body about the center of mass. For both pitch and roll the changes to the estimated moment of inertia is only 10% when the flexion angle is changed between θ = 0° to θ = 55° .cmd) during the flexion action, we obtain that the size of cmd and its angle relative to the horizontal are set to a constant by the rigid morphology the beetle. The direction of the force that acts on the center of mass and launches the beetle into the air, has the same direction as cmd. Hence, the beetles are constrained to a constant jump angle and this prediction by the model is strongly supported by the observed jump angles in live beetles , resulting in reduced acceleration and takeoff speed. Hence, the beetles may control the speed at takeoff by adjusting muscle contraction in the pre-jump posture. Indeed the takeoff speed observed in live beetles  value. Adjusting takeoff speed to increase jump height always leads to an increase in the horizontal distance as well.Regarding the control of rotations of the body while airborne, not only are the rotations inaccurate and often excessive for righting purposes, it also seems that each jump has its own combination of somersaults and/or rolls . Spring-−1 (or 20 rolls s−1), the 10% adjustment on rotation rate is probably insufficient to slow the rotation of the beetle enough to allow controlled landing in the right orientation.Since the beetles often leave the ground with very high angular velocity, the conservation of angular momentum requires that in the absence of significant air resistance they will continue to rotate while airborne. However after leaving the ground the angular speed of rotation can be somewhat altered without changing the angular momentum by changing the moment of inertia of the body. Human athletes and falling cats adjust body configuration in the air to adjust the moment of inertia thus controlling the rotation of the body to some extent Click-beetles will jump as a defensive response as well as for righting Text S1A) Analysis of high-speed movies. B) Estimating the center of mass and mass moment of inertia from modeled beetles. C) Estimating the force, torques and angular velocities of body rotation developed by the jumping action.(DOC)Click here for additional data file.Figure S1Example of body contours used to model the beetle geometry. A) Planform view, the body is modeled as two halves of ellipses with a common minor axis at the location of the hinge . B) Side view based on images of the body in the pre-jump posture. The position of the Center of mass (cm) is denoted by the encircled×symbol. C) Notations of diameters and axes for each volume element see also .(DOC)Click here for additional data file.


Modern Science has, over four centuries, made strides un-witnessed probably in the history of mankind. Scanning through the physical world with all its complications and complexities, the classical mechanics, relativistic mechanics, and quantum mechanics, we have understood the world around us with mathematical precision. At one time, a few decades back, we thought that matter and energy are two independent building bricks of our physical world.2. As Fritj of Capra has put it, the science is in transition to move to understand the deeper and subtler dimensions of creation to bring into its fold consciousness. Attempts are continuously made to understand the facets of consciousness, spectrum of consciousness, etc. It is in this context, Prof Josephson tells often in his lectures that we need to go beyond quantum physics. He has great expectations in the ancient wisdom of the East in general and Upanishads in particular.However, we progressed to find the matter–energy continuum. Quarks as the packets of energy have shown us that everything in the physical world can be conceived as essential energy and is governed by the equation E= mcThe 10 Upaniúads  forming the wisdom base for which India is known all over the world has the other dimensions of creation presented with such vividity that any scientist would get fascinated about it. The other specialty is that the techniques of Yoga provide the necessary skills to realize the truth or reality or pure consciousness by gaining mastery over the mind. Reality is at the base of all creation and this state of deepest silence of mind is also the supreme bliss, total knowledge, and the power unparalleled. The Taittèreya Upaniúat has promoted this wisdom base through it spaòca koúa viveka—the five layered existence of the whole creation of which the physical world is the grossest. Next to that are the subtle layers unseen by the eyes called práïamaya, Manomaya, and Vijòánamaya Koúás. The causal layer of consciousness is the ánandamaya koúa from where all creation emerges. A schematic of the same is self-explanatory. As a manifest of bliss, as waves in an ocean. The layer of bliss is a state where mind has gone to its deepest levels of silence with all pervasive expanses.


The Centro de Investigação em Saúde de Manhiça (CISM) is a research institution whose mission is to promote and conduct biomedical research in priority health areas. The CISM is located in Manhiça, 80km from Maputo (Mozambique).2 with close to 84,000 inhabitants. In this area, all houses are geo-positioned (GPS) and the population is under demographic surveillance. The morbidity surveillance system collects information on all paediatric outpatient visits and admissions to the Manhiça District Hospital. The CISM research agenda is directed at the priority health problems in Mozambique. The CISM maintains stable research collaborations with the HCB/UB and the Barcelona Centre for International Health Research (CRESIB). We are conducting a project to develop a prevalence survey system for HCRI, based on the Spanish EPINE study with more than 20 years of experience. Questionnaires as well as definitions will be tested in a pilot study at the CISM this summer, and data collected and analyzed following the standard protocol. The CISM contributes to the strengthening of human resources in the country through the training of researchers and other technical personnel. Physicians and Nurses from the CISM will be trained is the use of the surveillance tool, to ensure that the community benefits from research results.The CISM has 3 well developed platforms  crucial. These platforms cover a study area of 500 kmImproving healthcare provision in the Manhiça District is one of the CISM’s priorities. There is no data regarding the rates and types of HCRI in Mozambique, and simple, easy to do prevalence studies could be a way to approach this health problem.None declared.


A hybrid atomic force microscopic (AFM) measurement system combined with white light scanning interferometry for micro/nanometer dimensional measurement is developed. The system is based on a high precision large-range positioning platform with nanometer accuracy on which a white light scanning interferometric module and an AFM head are built. A compact AFM head is developed using a self-sensing tuning fork probe. The head need no external optical sensors to detect the deflection of the cantilever, which saves room on the head, and it can be directly fixed under an optical microscopic interferometric system. To enhance the system’s dynamic response, the frequency modulation (FM) mode is adopted for the AFM head. The measuring data can be traceable through three laser interferometers in the system. The lateral scanning range can reach 25 mm × 25 mm by using a large-range positioning platform. A hybrid method combining AFM and white light scanning interferometry is proposed to improve the AFM measurement efficiency. In this method, the sample is measured firstly by white light scanning interferometry to get an overall coarse morphology, and then, further measured with higher resolution by AFM. Several measuring experiments on standard samples demonstrate the system’s good measurement performance and feasibility of the hybrid measurement method. Atomic force microscopy (AFM) is one of the most important micro/nanometer dimensional measurement methods. Since its invention in 1986  are the calibration parameters. Because the A and cu have linear relationship, kaz [kaz [In the hip, kaz , 3 are skaz [kaz  = −208 n3.2.To test the measuring performance of the AFM system, a step standard sample is measured. The standard step height is produced by VLSI Corporation. The substrate material is quartz with dimensions of 25 mm × 25 mm × 3 mm and the step height material is made of silicon dioxide with a height of 454.8 ± 2.7 nm calibrated by NIST. The measurement results in The evaluation of the step height standard is according to ISO5436-1: 2000. The measurement data needs to be leveled before being evaluated. The evaluation results are shown in 3.3.The lateral measurement range of the AFM system can reach millimeter scale; however, the measuring efficiency is limited as a result of the point-to-point scanning approach. Consequently, the optical microscopic measuring method is used to complement the AFM to realize hybrid measurement.Regarding the benefits of the optical measuring method, on the one hand, due to the use of the field measuring mode, the measuring efficiency of white light scanning interferometry is much higher than that of the AFM; on the other hand, white light scanning interferometry belongs to the non-contact measurement methods, providing pre-inspection of the sample to avoid dangerous areas for the AFM system.Based on the features of the large-range positioning platform, a hybrid measuring method combing the AFM and the white light scanning interferometry is proposed in order to enhance the measuring efficiency of the AFM by means of the localization of white light scanning interferometry. The basic procedure is to use white light scanning interferometry to conduct quick measurements on sample surfaces with a relatively low lateral resolution. Then, according to the measuring results of the white light scanning interferometry the regions of interest (ROI) on the sample are chosen. The dangerous region which is beyond the AFM system’s measuring ability can also be found and excluded from the ROI. Finally, the selected ROI is moved to the position under the AFM head by the NMM and then measured more accurately by the AFM. The method can not only improve the measuring efficiency of the AFM but also protect AFM head.A critical problem to be solved is how to unify the measuring coordinate system of the AFM and the white light scanning interferometer. The white light scanning interferometer can be used as a normal optical microscope which has the same lateral coordinate system determined by the magnification of objective. Hence, the solution is to unify the AFM plane coordinates into the white light scanning interferometer’s imaging coordinate system. As depicted in As shown in The 450 nm step height standard is chosen to perform measurement experiments. The NMM platform is moved precisely to make the sample imaged in the field of view. Then, the ROI is selected, as shown in The sample is measured by the white light scanning interferometer. In the measurement, moving interference fringes can be observed, as shown in The sample is moved in lateral directions by the NMM until the position of the top-left of the ROI coincides with the one of the cantilever’s end, as shown in As depicted in In the experiment, a 5× objective is selected to collect more topography information of the sample surface. The imaging area of CCD is 1,000 pixel × 900 pixel corresponding to 1,250 μm × 1,125 μm in the measuring coordinate system which means that the pixel length is 1.25 μm. The white light interference peak is determined by using the center of gravity method. By adjusting the NMM, the length of vertical scanning step is set as 50 nm, and the number of steps is set as 140 corresponding to a 7 μm scanning range. The measuring process of the white light scanning interferometry takes about 2 minutes. The specific experimental procedures are as follows:4.A hybrid AFM measurement system combined with white light scanning interferometry based on a precise large range positioning platform is developed. The AFM head has a self-sensing behavior and can be conveniently fixed under the objective of the optical interference system. The FM operation mode is adopted to overcome the dynamic response limitation by using a probe’s high quality factor. The AFM head is set up on a large-range positioning platform capable of performing millimeter scale lateral scanning. The measurement of a step standard demonstrates a good measurement accuracy and repeatability of the system. The measurement experiments illustrated that the efficiency of the AFM can be improved through this method.