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The genome is continuously attacked by a variety of agents that cause DNA damage. Recognition of DNA lesions activates the cellular DNA damage response (DDR), which comprises a network of signal transduction pathways to maintain genome integrity. In response to severe DNA damage, cells undergo apoptosis to avoid transformation into tumour cells, or alternatively, the cells enter permanent cell cycle arrest, called senescence. Most tumour cells have defects in pathways leading to DNA repair or apoptosis. In addition, apoptosis could be counteracted by nuclear factor kappa B (NF-κB), the main anti-apoptotic transcription factor in the DDR. Despite the high clinical relevance, the interplay of the DDR pathways is poorly understood. For therapeutic purposes DNA damage signalling processes are induced to induce apoptosis in tumour cells. However, the efficiency of radio- and chemotherapy is strongly hampered by cell survival pathways in tumour cells. In this study logical modelling was performed to facilitate understanding of the complexity of the signal transduction networks in the DDR and to provide cancer treatment options.
Our comprehensive discrete logical model provided new insights into the dynamics of the DDR in human epithelial tumours. We identified new mechanisms by which the cell regulates the dynamics of the activation of the tumour suppressor p53 and NF-κB. Simulating therapeutic intervention by agents causing DNA single-strand breaks (SSBs) or DNA double-strand breaks (DSBs) we identified candidate target proteins for sensitization of carcinomas to therapeutic intervention. Further, we enlightened the DDR in different genetic diseases, and by failure mode analysis we defined molecular defects putatively contributing to carcinogenesis.
By logic modelling we identified candidate target proteins that could be suitable for radio- and chemotherapy, and contributes to the design of more effective therapies.
DNA damage is of profound biomedical interest, as this type of lesions largely contributes to cancerogenesis [1]. DNA damage is induced by environmental factors, like ionizing radiation [2], but also by intrinsic agents, like metabolically generated reactive oxygen species [3]. Damaged DNA becomes bound by so-called sensor proteins, like replication protein A (RPA) or a complex composed of meiotic recombination 11 (Mre11) - radiation 50 (Rad50) - nijmegen breakage syndrome 1 (Nbs1) (MRN). They trigger a complex network of signal transduction pathways designated as DDR [1]. The DDR causes temporal cell cycle arrest, if the level of DNA damage is low, so the cell can repair it [4]. In response to severe DNA damage, cells undergo apoptosis to avoid transformation into tumour cells [5]. Alternatively, the cells enter permanent cell cycle arrest, called senescence [6]. In presence of DNA damage, the tumour suppressor p53 plays a key role in the decision between survival and death of the cell. Activated p53 either induces cell cycle arrest or apoptosis, mainly by activation of distinct target genes [7]. As shown in biochemical and network modelling studies, p53 levels oscillate in response to DNA damage caused by ionizing radiation. The p53-dependent expression of wild-type p53-induced phosphatase 1 (Wip1) and murine double minute 2 (MDM2) mediates the oscillations [8]. Whereas Wip1 is essential for the generation of oscillations, MDM2 mediates their fine tuning [9]. The duration of the oscillations was proposed to determine, whether p53 acts pro-apoptotic or not [9]. However, apoptosis can be counteracted by activation of NF-κB, the main anti-apoptotic transcription factor in the DDR. Posttranslational modifications of NF-κB essential modulator (NEMO) exert an important role in the signal transduction that links DNA damage in the nucleus with activation of NF-κB in the cytoplasm [10]. Whether DSBs trigger stable oscillations of NF-κB on the level of single cells has not been shown.
SSBs and DSBs are the most lethal types of DNA damage [2]. They can be triggered by ionizing radiation or topoisomerase inhibitors, as therapeutically applied to eliminate tumour cells. Usually, higher proliferation rates of tumour cells render them more susceptible to DNA damage induced apoptosis than normal cells [11]. The efficiency of DNA damaging therapies can be potentiated by blocking cell survival pathways in tumour cells (sensitization) [12]. A strategy to sensitize tumours to DNA damaging agents is adjuvant abolishment of cycle arrest, resulting in necrosis or apoptosis-like cell death by mitotic catastrophy [13]. Other important sensitization targets are components that contribute to NF-κB activation, which otherwise often impedes efficient elimination of cancer cells [14].
However, most tumour cells have a defective DDR [11]. Such molecular defects due to mutations within tumours can be exploited to selectively sensitize tumours to therapy. Inhibitions that result in cell death only in combination with a molecular defect in targeted tumour cells would predominantly eliminate the tumour. Corresponding proteins are thus ideal drug targets [15]. Based on a network modelling approach following this strategy, inhibition targets that sensitize p53-deficient tumours to DNA damaging treatment were found [16].
Despite the high clinical relevance of the DDR, the interplay of the signal transduction involved herein is poorly understood, particularly due to high complexity. Therefore, systems biology approaches are of high value to gain deeper insights. Quantitative modelling requires both, detailed knowledge of kinetic parameters and high computational power. Therefore, such approaches are suitable to model rather small signal transduction modules. Qualitative models provide a better basis for the representation and analysis of large-scale signal transduction networks. Particularly discrete logical modelling is a powerful tool to address important issues, such as detection of network-wide functional interdependencies, identification of intervention targets and predictions on the network dynamics [17,18]. For instance, a literature-based Boolean model has been used to identify cellular myelocytomatosis oncogene (c-Myc) as a putative therapeutic target to treat breast cancer [19]. We previously identified putative therapeutic targets in signal transduction pathways induced by the pathogen Helicobacter pylori[20]. With a Boolean model of the DDR, we predicted candidate targets to abolish NF-κB activation, while leaving apoptotic pathways unaffected. The latter targets might be suitable to sensitize carcinomas to DSBs-inducing therapeutics by promoting apoptosis [21]. A literature-based Boolean model of T cell large granular lymphocyte (T-LGL) leukemia has been used to identify potential therapeutic targets and to investigate the dynamics of the signal transduction underlying this disease [22]. Signal transduction emanating from the death receptor has been studied with a discrete logical model. After inactivating certain proteins, the fraction of pathways that lead to a particular cell fate (apoptosis, necrosis or survival) has been determined in dynamical analyses [23].
Here, we present a comprehensive discrete logical model of the response to SSBs and DSBs based on published experimental data. Our dynamical analysis provided new insights into the regulation of p53 and NF-κB in the DDR. We identified candidate target molecules to sensitize tumour cells to DNA damaging therapeutics. By failure mode analysis, we predicted mutations that might contribute to the formation of carcinomas and validated our model with data from published studies.
Based on quality-controlled literature data, we built a discrete logical model of the response to SSBs and DSBs in human epithelial cells. The model encompasses 96 regulatory components, connected by 98 interactions. It is represented by a logical interaction hypergraph (Figure (Figure1),1), and a list of logical functions describing the interactions (Additional file 1: Table S1). The numbers assigned to interactions in Figure Figure11 correspond to the numbers of the logical functions. The network shows the typical structure of signal transduction networks: the input layer is given by stimuli, which damage the DNA, from where signals are being transmitted to and processed in the intermediate layer, finally reaching the output layer (cell cycle arrest and 'onset of apoptosis'). We chose 'onset of apoptosis' instead of 'apoptosis' as an output, as this output corresponds to the beginning of apoptotic processes, but not to completion of apoptosis, ie. cell death. The activity levels of most regulatory components (nodes) are represented by Boolean state variables, i.e. they can only attain the values '0' (inactive or absent) or '1' (active or present). Ternary (three-valued) variables were only assigned to phosphorylated ataxia telangiectasia mutated (ATM-P), phosphorylated inhibitor of kappa B kinase (IKK-complex-P) and inhibitor of kappa B α (IκBα). In that way, we took account for the fact that each of those components differs in its functions, depending on whether its activity is low ('1') or high ('2'). Specifically, a low activity of ATM (ATM-P = 1) is required for inactivation of the ATM phosphatase protein phosphatase 2 A (PP2A) [24] (interaction 22). Once PP2A is inactivated, DSBs can induce high activity of ATM (ATM-P = 2), which is now able to phosphorylate further substrates [25] (interaction 23). Similarly, the IKK complex has a low basal activity (IKK-complex-P = 1), which is sufficient for partial degradation of IκBα (decreasing IκBα's activity level from '2' to '1'), leading to activation of proto-oncogene c-Rel (c-Rel) in absence of induced DNA damage [26,27] (interaction 67). Upon induction of DNA damage, the IKK complex attains high activity (IKK-complex-P = 2), which enables more degradation of IκBα (IκBα = 0), enabling the activation of the NF-κB dimers p50-p65-P and p50-p50 [10] (interactions 68, 69). For some structural analyses, we took account for the limited knowledge of time-dependent signal transmission by assigning each interaction to one of three time scale values. Interactions composing the signal transduction pathways leading to activation/inactivation of components that are directly linked to the components "CELL CYCLE ARREST" or "ONSET OF APOPTOSIS" were assigned to time scale value 1, as long as literature data did not indicate a distinct delay. Examples of components that are directly linked to "CELL CYCLE ARREST" or "ONSET OF APOPTOSIS" are the transcription factors. Time scale value 2 was assigned to interactions that also lead to cell cycle arrest, apoptosis, or anti-apoptosis, but were shown to occur distinctively later than interactions of time scale value 1. For example, p53-induced protein with a death domain (PIDD) binds to NEMO (interaction 44, time scale 1), and later, PIDD binds to RIP1-associated ICH-1/CED-3 homologous protein with a death domain (RAIDD) (interaction 43, time scale 2) [28]. Time scale value 2 was also assigned to interactions linked directly to the regulatory components "CELL CYCLE ARREST" or "ONSET OF APOPTOSIS". Activation of proteins that initiate switching off parts of the DDR was assigned time scale value 3. This was based on the assumption that these events occur during the latest phase of the DDR. For instance, Wip1 interrupts signal transduction pathways by dephosphorylating ATM and other proteins [29] (e.g. interaction 21). Accordingly, induction of Wip1 expression [30] (interaction 82) has been assigned to time scale value 3. Detailed information on assignments of time scale values are given in Additional file 1: Table S1. For most analyses, we simulated the DDR at time scale value 2, i.e., at a time before feedback inhibition comes into play. Our study focused on inhibitions and molecular defects interfering with cell cycle arrest, apoptosis, or anti-apoptosis. As follows from the considerations above, only time scale value 2 pertains to maximum activity of all components promoting cell cycle arrest, apoptosis, or anti-apoptosis. Hence, for this time scale value, the sensitivity of the simulation results to changes in time scales of interactions should be minimal.
For dynamical analyses, we took account for the knowledge of time-dependent signal transmission by defining three priority classes (see below).
In order to validate the predictive quality of our model, we evaluated simulations on the basis of published studies on epithelial cells (Table (Table1).1). We inactivated certain proteins in the model and then calculated the logical steady state of the model at time scale value '2', i.e. prior to onset of negative Feedback inhibition. Cells can be sensitized to DNA damaging therapy by events that promote cell death [31]. Blockage of cell cycle arrest can cause mitotic catastrophy, a form of cell death [13], whereas blocking of the anti-apoptotic transcription factor NF-κB promotes apoptosis [14].
Inactivation of ATM blocked all pro-survival pathways (cell cycle arrest and NF-κB activation) in the response to DSBs. This is confirmed by studies in which ATM inhibition sensitizes cells to agents causing DSBs [32-34].
Ataxia telangiectasia and rad3-related protein (ATR) inactivation blocked two pathways leading to cell cycle arrest (downregulation of c-Myc, expression of p21) in response to SSBs in our model. This is in agreement with the reported potentiation of SSBs-induced cell death by ATR inactivation in carcinoma cells [35,36].
In our simulation of the response to SSBs, loss of checkpoint kinase 1 (Chk1) blocked one of two pathways promoting cell division cycle 25 A (Cdc25A) degradation. Degradation of Cdc25A leads to cell cycle arrest. Additionally blocked was one pathway leading to activation of p53, a pro-apoptotic and cell cycle arresting protein. Thus, loss of Chk1 suppressed pathways leading to cell cycle arrest and apoptosis. Hence, our results do not indicate, whether Chk1 inhibition sensitizes cells to SSBs inducers. Chk1 inhibition was demonstrated to increase the cytotoxicity to topoisomerase I (TOPI) inhibitors by diminishing cell cycle arrest in carcinoma cells with functional p53 [37-39]. As previously proposed, a partial suppression of p53 activation diminishes predominantly its apoptotic function and to a lesser extent its cell cycle arresting function [15]. This effect might contribute to the sensitization by Chk1 inhibition, but is not captured by the model.
In response to ionizing radiation, absence of Chk2 in our model blocked cell cycle arresting phosphorylation of Cdc25C, and one of two pathways leading to degradation of Cdc25A. On the other hand, activation of the pro-apoptotic effectors promyelocytic leukemia (phosphorylated at serine 117) (PML-PS117) and phosphorylated adenovirus E2 gene promoter region binding factor 1 (E2F-1-P), and one p53 activating pathway are blocked. Hence, the numbers of both, cell cycle arresting and apoptotic pathways were reduced. The simulation did not indicate, whether Chk2 inhibition confers sensitization or protection from cell death caused by ionizing radiation. In most studies, Chk2 inhibition diminished cell death caused by ionizing radiation [37,39]. Correspondingly, Chk2 knockdown protects MIA PaCa-2 carcinoma cells against ionizing radiation [40].
When simulating the response to camptothecin (inhibitor of TOPI) in the model, inhibition of TGF-β activated kinase 1 (TAK1) abolished two cell cycle arresting pathways (c-Myc downregulation and p21-mediated cell cycle arrest). Hence, the model indicates a sensitizing effect of TAK1 knockdown, which was demonstrated in carcinoma cell lines treated with camptothecin [41].
Moreover, putative therapeutic targets for the sensitization of tumours with dysfunctional p53 have been proposed (Table (Table2).2). We compared the response to the topoisomerase II (TOPII) inhibitor doxorubicin in absence of p53 only with the response in absence of p53 and ATM. In the absence of only p53, four cell survival pathways were still active, i.e. activation of anti-apoptotic NF-κB, cell cycle arrest induced by c-Myc downregulation, Cyclin-dependent kinase 2 (Cdk2) inhibition, and phosphorylation of Cdc25C. When p53 and ATM were absent, no cell survival pathway was activated by doxorubicin in the model. Accordingly, the ATM inhibitor KU-55933 sensitizes p53-deficient human carcinoma cells to doxorubicin. Moreover, p53-deficient breast and lung tumours showed higher sensitivity to genotoxic chemotherapy when ATM is inactive as well [16].
In the p53-deficient model, TOPI inhibitors still induced cell cycle arrest. Additional loss of Chk1 abolished one of the pathways leading to degradation of Cdc25A, a phosphatase necessary for cell cycle progression. Apoptotic pathways in p53-deficient cells were not suppressed by inactivation of Chk1. Hence, our model indicated that p53-deficient cells might be sensitized to SSBs inducers by inhibition of Chk1. Indeed, the aforementioned sensitization to TOPI inhibitors by Chk1 inhibition was reported to be more pronounced when p53 is dysfunctional [37-39]. Accordingly, preclinical studies support the combination of Chk1 inhibitors with SSBs inducers particularly for treatment of p53-deficient tumours [37,39].
In the model, inactivation of Chk2 in absence of p53 reduced the number of cell cycle arresting and pro-apoptotic pathways. The sensitivity of tumours with dysfunctional p53 to DSB-causing agents was reported to be potentiated by inactivation of Chk2 [16]. In contrast, another study showed no pronounced potentiation of cell death by Chk2 inhibition in carcinoma cells with a loss of function mutation in p53 [42]. As suggested by our simulations, whether Chk2 inhibition potentiates cell death caused by DSBs might depend on the genetic background, providing a possible explanation for the conflicting experimental data.
In summary, our simulations recapitulated most published studies on the sensitivity of carcinoma cells to DNA damaging agents after inactivation of a certain protein. These results support the suitability of the model for the generation of predictions.
Network-wide causal relationships between all pairs of regulatory components are displayed in the dependency matrix (Figure (Figure2).2). Two components have a causal relationship, if a sequence of adjacent components, a pathway, links them. As the large fraction of yellow matrix elements in Figure Figure22 illustrates, in most causal relationships between two components i and ji is an ambivalent factor for j. In other words, i has an activating as well as an inhibiting influence on another component j. Usually, the activating influence becomes operational at another time scale than the inhibiting influence. ATM for instance phosphorylates, i.e. has an activating influence on Chk2 [43] (interaction 25). However, ATM phosphorylates p53 as well [44,45] (interaction 31), leading to expression of Wip1 later (at time scale value 3) [30] (interaction 82). Wip1 in turn deactivates Chk2 by means of dephosphorylation [46] (interaction 25). Therefore, the activation of Chk2 by ATM is counteracted by the ATM-dependent deactivation of Chk2 by Wip1. Thus, ATM is an ambivalent factor for Chk2, as the yellow matrix element in Figure Figure22 indicates. As the high frequency of coincidences of activating and inhibiting relationships indicates, most pathways become inactivated in a later phase of the DDR. Moreover, these coincidences suggest an important role of crosstalk in the DDR.
Feed-forward loops (FFLs) and Feedback loops (FLs) can play decisive roles in the processing of the signals, which are being transmitted in signal transduction networks. Moreover, they may profoundly influence the dynamics (temporal behaviour) of a signal transduction network [47]. For these reasons, we identified FFLs (Figure (Figure3).3). They appear in two groups, those with 'AND gates' and those with 'OR gates'. For example, 'AND gated' is the activation of sumoylated and phosphorylated IKKϵ (IKKϵ-S-P) by IKKϵ-P and PML-P (Figure (Figure3A),3A), as IKKϵ-S-P activation requires both proteins, i.e. IKKϵ-P AND PML-P. 'OR gated' is for instance the activation of p53-P by either ATM-P or Chk2-P (Figure (Figure3F),3F), as either ATM-P OR Chk2-P phosphorylates p53.
Coherent FFLs of type 1 with 'AND gates' may delay the transmission of activating signals [48]. Such FFLs in the model are shown in Figure Figure3A-E.3A-E. Coherent FFLs of type 4 can have the same function [48]; they are shown in Figure Figure33S-Ä.
As also reported by Mangan and Alon [48], transmission of the fade away of signals ('OFF' signals) in a pathway could be delayed by coherent type 1 FFLs with 'OR gate' (Figure (Figure3F-K),3F-K), by coherent type 2 FFLs with 'AND' gate (Figure (Figure3L3L and M), as well as by the coherent type 3 FFLs (Figure (Figure33N-R).
Incoherent type 2 FFLs with 'AND gate' may accelerate the transmission of 'OFF' signals [48]. We found only one example (Figure (Figure33Ö).
In summary, all but one (Figure (Figure3Ö)3Ö) FFLs identified may delay either 'ON' or 'OFF' signals, thereby transmitting only long-term signals. Moreover, we found that most of these FFLs include either p53, or its regulators. Taken together, short-term signals arising from noise rather than from DNA damage might be filtered out. The same regards signals arising from minor damage of DNA, which becomes rapidly repaired. Only long-term signals from more severe DNA damage would be transmitted to and activate p53. Such a careful regulation seems reasonable in light of the well-known key role of p53 in determining cell fate (survival or apoptosis) after DNA damage. Indeed, such a regulation of the actelyation of p53 involving so far unknown FFLs has been proposed [49]; our results provide evidence for a regulation of p53 phosphorylation by only long term signals and provide candidate FFLs for that mechanism. As we found moreover, the FFL in Figure Figure3A3A might delay 'ON' signals transmitted to IKKϵ-S-P. Similarly, the FFLs in Figure Figure3Z3Z and Ä could delay 'ON' signal transmission to the IKK complex. In both cases, short-term signals could be filtered out. IKKϵ-S-P and the IKK complex mediate activation of NF-κB. Similarly to the mentioned control of p53, such a careful regulation of NF-κB seems reasonable in light of its mayor role in counteracting apoptosis.
Next, we identified FLs that are functional in the logical model (Figure (Figure3a-g).3a-g). All of them are negative. The presence of a negative FL is necessary for stable oscillations [50]. Again, most FLs (Figure (Figure3a-f)3a-f) contain p53, whereas the FL in Figure Figure3g3g contains the NF-κB dimer p50-p65. In the latter FL, NF-κB drives the expression of its own inhibitor IκBα. This FL was shown to cause oscillatory behaviour of NF-κB in a multitude of cells and treatment conditions [51]. Also the FLs in Figure Figure3a-c3a-c have been studied previously with ordinary differential equation or stochastic models as well as experimentally in cells exposed to ionizing radiation [8]. In a logical approach, effects of varied (i) degradation rates of MDM2, (ii) transcriptional activities of p53, and (iii) DNA damage levels on the dynamic behaviour of the MDM2-p53 circuit has been studied. It has been shown that variations in parameter values (e.g. corresponding to mutations) can lead to only four different scenarios of dynamical behaviour of the network [52]. Recently, the feedback-controlled oscillations of p53 were proposed to impact the ultimate cell fate decision [9]. As our results suggest, the negative FLs in Figure Figure3d-f3d-f might cause oscillations of p53 levels in vivo as well.
In order to study the terminal fate (attractors) of the network, we reduced it to a model with conserved attractors. Previously, a method has been proposed to reduce Boolean models to their functional interactions. However, this method is only applicable to models of intermediate dimension (i.e. maximal 20 variables) [53]. Therefore, we used a different network reduction technique, which is applicable to large-scale models (see Methods). The reduced model contains only the regulatory components DSBs early, DSBs late, RPA-ATR-ATRIP-P, ATM-P, p53-P and NF-κB (nuclear p50-p65-P) (Figure (Figure4).4). We calculated the state transition graph of the reduced model by using an asynchronous updating schedule with three priority classes. The state transitions that were assigned to priority classes 1, 2, and 3 coincide with the interactions of time scale values 1, 2, and 3, respectively. Hence, state transitions involving activations of RPA-ATR-ATRIP-P, ATM-P, p53-P or nuclear NF-κB were assigned to priority class 1; priority class 2 embraces the subsequent state transitions leading to activation of 'DSBs late' by 'DSBs early'. State transitions coinciding with the initiation of the inactivation of signal transduction pathways, i.e., the downregulation of RPA-ATR-ATRIP-P, ATM-P, p53-P and NF-κB, constitute priority class 3.
We emphasize that the attractors of the model variants correspond to the fate of the DDR before the cell either completes DNA repair or dies. In response to DSBs, the model (Figure (Figure4A)4A) finally enters a complex cyclic attractor (Figure (Figure4a).4a). This suggests the cellular network might transit through an intertwined cycle of states before completion of either DNA repair or apoptosis. Negative feedbacks are necessary for cyclic attractors [50]. We thus aimed to elucidate in more detail the roles of the identified feedbacks (Figure (Figure3)3) in generating the cyclic attractor. For this purpose, we calculated state transition graphs for model variants with interrupted feedbacks. Models with constitutively active NF-κB (Figure (Figure4B)4B) or deficiency of p53-P (Figure (Figure4C)4C) still enter cyclic attractors (Figure (Figure4b4b and c). Similarly, the model variant with deficiency of NF-κB enters a cyclic attractor as well (not shown). In contrast, the model variant with both p53-deficiency and constitutively active NF-κB (Figure (Figure4D)4D) enters a logical steady state (marked with an ellipse in Figure Figure4d).4d). Even constitutive activation of only p53-P is sufficient to direct the network into a logical steady state (Figure (Figure4e).4e). The network reduction we applied can lead to loss of trajectories in the STG. Therefore, not every trajectory in the STG of the full model might have a counterpart in the STG of the reduced model [54]. Consequently, the reduced model variant's attractors we identified might be different from those of the full model variants (i.e. of the model variants before the reduction was done). Therefore, we checked for each of the five reduced model variant's attractors (Figure (Figure4),4), whether it is equivalent to the attractor of the corresponding full model variant. In general, any attractor is either a logical steady state or a cyclic (simple loop or complex loop) attractor [50,55]. Whereas we were able to identify the logical steady states of the full model variants, their state spaces are too big to identify cyclic attractors. Therefore, if a full model variant has no logical steady state, we inferred the presence of a cyclic attractor. The identified logical steady states are independent of the updating scheme applied [18], and therefore, insensitive to changes in the priority classes. As our aim now was only to check for the type of attractor (steady state or cyclic attractor), there was no need to specify priority classes. As we found, neither the wildtype full model, nor its variant with constitutively active NF-κB or deficiency of p53-P enter a logical steady state (Additional file 1: Table S2, columns A-C). Hence, these full model variants should enter cyclic attractors; the results are in agreement with the identified cyclic attractors of the corresponding reduced model variants (Figure (Figure4a-c).4a-c). The full model variants with both p53-deficiency and constitutively active NF-κB (Additional file 1: Table S2, column D) as well as the full model variant with constitutively activative p53-P (Additional file 1: Table S2, column E) enter logical steady states. Again, the results agree with the results from the analyses of the reduced model counterparts (Figure (Figure4d4d and e). In addition, none of the full model variants contains a functional positive FL (data not shown); thus, this necessary condition for bi- or multistability [50] is not fulfilled. Therefore, each full model variant possesses only a single attractor. Again, our results coincide with the finding that each reduced model variant possesses only a single attractor.
We conclude that all attractors of the reduced model variants correspond to those of the full model variants. Both, the results gained from the analyses of the attractors and the identified functional FLs independently suggest an essential role of p53 and NF-κB in the generation of cyclic attractors of the DDR. This and the prevalence of p53, and NF-κB in the FFLs support the importance of these proteins in governing the dynamics of the DDR.
To identify putative targets for sensitization of carcinomas to therapy, we simulated treatments with agents causing SSBs (inhibitors of TOPI) or only DSBs (e.g. inhibitors of TOPII). p53, homeo-domain interacting protein kinase 2 (HIPK2), ATM or Chk2 are frequently mutated and inactive in carcinoma cells [56-59], therefore, we simulated treatment with inhibitors of TOPI or TOPII in the absence of these proteins. In order to simulate the behaviour of the network before the onset of feedback inhibition, we chose the time scale value '2' of the model. We calculated minimal intervention sets of targets, whose inhibition might sensitize tumours by fulfilling three intervention goals: (i) blocking cell cycle arrest, (ii) blocking activation of anti-apoptotic NF-κB, and (iii) keeping at least one pathway activating 'onset of apoptosis' intact. In presence of (therapeutically induced) severe DNA damage inhibitors that fill out goal (i) would eliminate tumour cells by mitotic catastrophy, and inhibitors fulfilling goals (ii) and (iii) would potentiate apoptosis. We identified 85 sets of molecular targets that could sensitize tumour cells to therapies inducing SSBs or DSBs (Table (Table3),3), and protein sets containing putatively less suitable targets (Additional file 1: Table S3). ATM deficiency in the model already fulfils the intervention goals in presence of DSBs. Hence, we found no sensitization target for such conditions. This result agrees with most studies, showing that ATM inhibition sensitizes cells to therapeutics causing DSBs [32]. Accordingly, cells isolated from Ataxia telangiectasia (dysfunctional ATM) patients show enhanced radiosensitivity [33]. For certain sets, inhibitions of the target proteins might specifically sensitize tumour cells with the indicated mutation, but allow normal cells to survive by entering cell cycle arrest (asterisks in Table Table3).3).
Some predicted target sets include ATR or Chk1, which beside their contributions to the DDR are essential for proliferation. Nevertheless, partial and transient inhibition of ATR or Chk1 during DNA damage diminishes cell cycle arrest rather than proliferation [35,37]. Additionally, some protein target sets that sensitize Chk2-deficient tumours include p53. Although p53 can promote apoptosis, it mediates predominantly cell cycle arrest in Chk2-deficient tumours, resulting in tumour cell survival [15]. Correspondingly, p53 inhibition might sensitize certain tumours to therapeutic treatment [60]. Hence, inhibition of p53 in Chk2-deficient cells seems reasonable. Taken together, we predict putative protein target sets that might sensitize tumours carrying certain mutations to therapeutic interventions. Our candidate target sets in Table Table33 include all published sensitization targets in Tables1 and and2.2. However, with the exception of ATM, inhibiting the published sensitization targets in Tables1 and and2,2, blocks only part of the cell survival pathways of the model in tumours containing certain mutations. In contrast, our proposed target sets (Table (Table3)3) might block all cell survival pathways of the model in tumours containing certain mutations. Hence, our candidate targets might sensitize tumours to DNA damaging therapeutics with higher efficiency.
Next, we aimed to enlighten the DDR in genetic diseases. For this purpose we inactivated in our model the protein whose defect causes a given disease. Then, we simulated the response to SSBs and DSBs simultaneously at time scale value '2', and evaluated our in silico results based on published data. For investigations of the feedback control of the DDR, we simulated at time scale value '3' (Table (Table4).4). The disease Ataxia telangiectasia (ATM-deficiency) has been associated with defects in the activation of p53, G1/S, intra-S, and G2/S cell cycle checkpoints, genomic instability, enhanced radiosensitivity and increased incidence of lymphoid tumours [33,61]. In our simulation, loss of ATM blocked p53 activation and p21 expression, resulting in abolished cell cycle arrest by these proteins. Additionally, the cell cycle promoting protein c-Myc became expressed, and abolished another cell cycle arrest pathway. Cell cycle checkpoint defects are known to contribute to genomic instability, which promotes tumorigenesis [11], and increased cell death by mitotic catastrophy [13]. The abolished activation of NF-κB in the model might further promote apoptosis, although p53-dependent apoptosis was blocked as well. Moreover, in absence of ATM we identified in our model the loss of many signalling pathways involved in the regulation of p53 and NF-κB target genes.
Ataxia telagiectasia-like-disorder (Mre11-deficiency) is also associated with defective induction of cell cycle arrest, genomic instability, and enhanced radiosensitivity [33]. As Mre11 in the model is a subunit of the MRN complex, which solely activates ATM, the blocked pathways are the same as in the Ataxia telangiectasia simulation. The same is true for Nijmegen breakage syndrome (Nbs1-deficiency), as in the model also Nbs1 is only a MRN complex subunit. Nijmegen breakage syndrome has moreover been reported to diminish DNA repair [33,62]. However, DNA damage induced cell cycle arrest promotes DNA repair [11]. Hence, the abolishment of cell cycle arrest by p53 phosphorylation, p21 expression, and c-Myc downregulation in the simulation might contribute to lost repair capabilities. Moreover, breast cancer 1, early onset (BRCA1) is essential for homologous recombination repair of DSBs [1]. ATM-dependent phosphorylation of BRCA1 is averted by loss of Nbs1 in the model, possibly further contributing to DNA repair deficiencies.
Also Rad50 in the model is a subunit of the MRN complex, which solely activates ATM. Hence, the pathways blocked in Nijmegen breakage syndrome-like disorder (Rad50-deficiency) [63,64] are identical to those in the Ataxia telangiectasia simulation.
A reduction of the ATR level causes ATR-Seckel syndrome. Hence, ATR-dependent phosphorylations of Chk1 and p53 are diminished, whereas ATM-dependent responses to ionizing radiation remain intact. There is no pronounced radiosensitivity, and no enhanced incidence of cancer [65]. In line with this report, ATR inactivation in the model did not affect ATM-dependent signalling induced by ionizing radiation. Instead, ATR-deficiency abolished cell cycle arrest mediated by p21 expression and c-Myc downregulation in the model. However, these cell cycle arresting pathways could still be active in presence of an ATR level as low as in ATR-Seckel syndrome cells.
DDR defects that diminish apoptosis and cell cycle arrest are well known to contribute to carcinogenesis by promoting uncontrolled proliferation [11]. We aimed to identify putatively relevant malfunctions in the DDR in epithelial cells. For this purpose, we simulated the response to both SSBs and DSBs simultaneously. Again, we chose the time scale value '2' of the model. By calculating minimal intervention sets, we identified protein defects (i.e. constitutive activations and inactivations that in combination block both, 'onset of apoptosis' and cell cycle arrest in the model. In vivo, constitutive activation of a protein can result from constitutive expression or gain of function mutations of the gene encoding that protein, whereas constitutive inactivation can result from loss of function mutations. Alternatively, such defects of a protein might result from mutations of a gene encoding another protein that regulates the relevant protein. We found 117 combinations of defects that putatively contribute to carcinogenesis by allowing cells with damaged DNA to proliferate (Table (Table5).5). From our search, we excluded activations and inactivations that according to literature data might counteract uncontrolled proliferation (Additional file 1: Table S4). By doing so, we accounted for protein functions being relevant to carcinogenesis, but are not captured by the model. Each row gives an alternative combination of relevant defects (Table (Table5).5). Here, [0] means inactive; [1]: constitutive active at level '1'; [2]: constitutively active at level '2'. Among the results are inactivities of the known or suspected tumour suppressors ATM, the MRN complex subunit Nbs1, fragile histidin triad gene (FHIT), p53, BRCA1, as well as Chk2, and activities of the known oncogene MDM2 [66,67]. In 21 combinations, the NF-κB dimer p50-p65 was found to be constitutively active, as frequently observed in tumours [68]. Carcinogenesis might also be promoted by constitutive active IκBα or the IKK complex (e.g. sets 2, 13). Alternatively, constitutive active IκBα or the IKK complex may promote carcinogenesis in combination with other molecules (e.g. sets 110, 6). To our knowledge, most of the putative carcinogenic mutations we found have not been implicated in tumorigenesis previously. This gap of knowledge could be explained by 'low penetrance genes' assumed to confer cancer susceptibility or resistance [66,69,70]. These were proposed to act in combination in a dosage-dependent manner to incrementally determine cancer predisposition. A major obstacle to their identification is the vast number of possible combinations of mutations to be tackled experimentally [66,71]. However, our results may provide a valuable preselection for follow-up experiments.
It is important to note that the identified mutations might cause cell death by mitotic catastrophy only in response to severe DNA damage, as it is induced in cancer therapies. In contrast, in response to low levels of DNA damage, which is compatible with cell survival, the defects might promote tumorigenesis by enabling uncontrolled proliferation. Such limited DNA damage is caused permanently by cellular insults, like reactive oxygen species generated in metabolic processes [3].
We presented a comprehensive logical model of the DDR including the dynamics of p53 and NF-κB regulation in human epithelial tumours. The large scale of the model and the implementation of posttranslational protein modifications allowed us to account for extensive crosstalk among signal transduction pathways. Our analyses enlightened the dynamics of the DDR and functional consequences of defects underlying cancerogenesis, but also hereditary genetic diseases. We identified candidate target proteins suitable for sensitization of epithelial tumours with different mutations to chemo- and radiotherapy, thus, our predicted target proteins provide a basis for follow up studies to demonstrate their therapeutic usefulness. Overall, the results reflect an approach to facilitate a holistic view on the DDR in health and disease. An important aim of further work is the inclusion of more quantitative data into the model. This would allow to recapitulate observations that lower levels of DNA damage predominantly induce temporary cell cycle arrest and DNA repair, whereas higher levels of DNA damage mainly cause apoptosis.
For network assembly we screened the relevant literature through NCBI/PubMed. Large amounts of published experimental data were evaluated and only high quality data on causal relationships in human epithelial cells were used for modelling. By epithelial cells we refer to either epithelial cell lines in the sense of the American Type Culture Collection[72] or ex vivo epithelial cells. Information on intracellular localization of proteins was retrieved from [73] unless provided in the analyzed publications. Information on oncogenes and tumour suppressors were retrieved from [67,74].
Some structural analyses were based on the representation of the structure underlying the studied model as a directed graph (interaction graph). Such a graph [17,75] consists of a set of nodes representing regulatory components (e.g. proteins, DNA damaging agents, ions), which are connected by arcs representing causal relationships. Signals are propagated from the start node to the end node of an arc. Activations are represented by arrows, whereas inhibitions are symbolized by T-shaped arcs. Each node is associated with a discrete logical state variable, which denotes the activity level of the corresponding regulatory component. The logical model is represented by a list of logical functions defining the target values of a component depending on the activity values of its regulators [18,76]. For combining logical variables in the logical functions we use a special notation of Boolean operators known as 'sum of products'. Thereby we require the operators AND, OR, and NOT for describing any logical relationship [77]. Interactions are described by AND connections of (potentially negated) nodes. Each AND connection describes a sufficient condition for the activity of the target component. Additionally, a component may be activated by several distinct signalling events independently. This is expressed by a logical OR-connection.
The implementation of the 'sum of products' notation allows the representation of the logical model as a logical interaction hypergraph [17,78]. In the logical interaction hypergraph, interactions are represented by hyperarcs. In principle, hyperarcs can connect an arbitrary number of start nodes (regulating components) with an arbitrary number of end notes (regulated components). This distinguishes hyerarcs from arcs, which connect only one start node with one end node. Hyperarcs therefore allow the representation of logical AND-connections between nodes. In our network, each hyperarc points into only one end node. Additionally, a species may be activated by several distinct signalling events independently. Distinct hyperarcs pointing into the same end node represent logical OR connections. For simplicity, we also refer to hyperarcs if an interaction has only one start node. The logical interaction hypergraph was constructed using the software CellDesigner 4.2[79] and subsequently exported to the MATLAB (The MathWorks, Natick, MA, USA) package CellNetAnalyzer 7.0[80] for analyses (Additional file 2). In CellNetAnalyzer, the interaction graph underlying a given logical interaction hypergraph can be generated by splitting each hyperarc into its constituent arcs [80].
Based on the interaction graph the dependency matrix was calculated in CellNetAnalyzer. This matrix reveals functional interdependencies between each pair of species, e.g., it reveals whether a species i is a strong activator (i.e., there are only positive paths, and no species in these paths is regulated by a negative FL), a weak activator (i.e., there are only positive paths, and at least one species in these paths is regulated by a negative FL), a strong inhibitor (i.e., there are only negative paths, and no species in these paths is regulated by a negative FL), a weak inhibitor (i.e., there are only negative paths, and at least one species in these paths is regulated by a negative FL), or an ambivalent factor (i.e, positive and negative paths to the selected species exist) for another species j. This feature facilitates qualitative predictions of the effects of perturbations or knockout experiments [17,80].
FFLs and FLs were identified with the JAVA application MAVisto V 2.7.0[81] on the basis of the interaction graph underlying the logical model (Additional file 2). Negative FLs are a necessary condition for stable oscillations or homeostasis, whereas positive FLs are necessary for multistability [50,82]. The appearance of such dynamical behaviours further requires the loop to be functional. The functionality context of a feedback loop is defined as a set of constraints on the values of the external regulators of that loop [83]. The functionality context of each feedback loop in the logical model was identified on the basis of the logical model with the JAVA tool GINsim 2.4 alpha[84].
By computing logical steady states (LSS) of the logical network upon definition of a time scale value with CellNetAnalyzer[17] we studied the qualitative effects of input stimuli on downstream signalling events and on the outputs. The qualitative effects of loss of function mutations and inhibitions were studied by computing LSS after setting the activity levels of the relevant protein to '0'. Correspondingly, for studying the qualitative effects of constitutive activities, the activity level of the relevant protein was set to its highest possible value ('1' or '2', respectively).
The calculation of LSS also provides the basis for calculations of minimal intervention sets with CellNetAnalyzer on the basis of the logical model [80,85]. These are minimal sets of regulatory components that are to be removed (by knockout, knockdown or inhibition) or to be added (by activation) to achieve a certain intervention goal. The maximum cardinality (maximum number of interventions allowed) of minimal intervention sets was set to 3.
Given a logical model and starting from an initial state of the network, consecutive states of the network can be computed. This is done by updating the activities of all components according to the logical functions. The computed dynamical behaviour of the network can be depicted in a state transition graph [84,86]. Each node in this graph represents a state of the network, i.e. a vector with its vector components representing the activity levels of all network components. The nodes are connected by arcs denoting possible state transitions. Usually, the reaction rates of the model interactions are unknown. Then, there are two basic strategies for dynamical analyses: synchronous and asynchronous updating [55]. In the first case, all activity levels are updated simultaneously. As each state can have at most one successor, the calculation of the state transition graph is quite simple, making it feasible even for large networks. Synchronous updating is based on the assumption that all components make a transition at the same time. This is unrealistic and can result in spurious dynamic behavior [87]. The second, more common strategy is to update only the activity level of one component at a time. The resulting state transition graph captures all possible state transitions, but is bigger than in the synchronous case. Accordingly, the state transition graph is more complex to model and analyse. We therefore restricted the computation of the state transition graph by applying an updating scheme with priority classes [88]. State transitions increasing a components activity are distinguished from state transitions decreasing its activity and were associated to priority classes with different ranks. The ranks were assigned to the priority classes according to the temporal order of interactions in vivo. At any state of the network, among all concurrent state transitions, only those of the class with the highest rank are triggered. As the temporal order of transitions belonging to the same priority class is unknown, we chose an asynchronous updating scheme for transitions belonging to the same class. Since the state space of a discrete logical network is finite, the system finally enters a LSS or a cycle of recurring states, called cyclic attractor [18]. Cyclic attractors are classified into simple loops and complex loops [55]. The former are cycles of network states such that each state can have exactly one successor state, whereas the latter are composed of overlapping simple loops. Dynamical analyses of the logical model were performed with GINsim.
Dynamical analyses of large networks can be very challenging since the size of the state transition graph increases exponentially with network size. We therefore reduced the full model prior to dynamical analyses by removing components in iterative steps. In each of these steps, a component is removed by linking its regulators directly to its target components. Accordingly, the logical functions are properly rewritten. For instance, the cascade, MEK-P→ERK-P→p90-P' can be reduced by removing the component ERK-P. This results in a reduced cascade, in which MEK-P activates p90-P directly. In the course of the model reduction, a FL can be reduced at most to its minimal form, an autoregulation. Autoregulated is a component that can either activate or inhibit itself. In the interaction graph autoregulation is indicated by a self loop, i.e., an arc with the start node and the end node representing the same component. By exclusion of autoregulated components from the reduction process, loss of feedback loops and attractors was avoided [54]. Model reduction was performed with GINsim.
The model was validated on the basis of published experimental studies.
The authors declare that they have no competing interests.
RP constructed the network model and designed the analysis. RP and MN drafted and wrote the manuscript. MN conceived the study and supervised the analysis. All authors have read and approved the final version of the manuscript.
Table S1: Logical functions of the model, Table S2: Logical steady states of the full model variants, Table S3: Excluded targets from search for putative therapeutic targets (see Table Table33 of the main text), Table S4: Excluded targets from search for molecular defects putatively contributing to carcinogenesis (see Table Table55 of the main text).
The CellDesigner file corresponding to the logical interaction hypergraph shown in Figure1, the logical network in the CellNetAnalyzer and GINsim formats and the interaction graph in MAVisto format.
The work was supported in part by the Federal Ministry of Education and Research (FORSYS, BMBF 0313922), (SysTec, BMBF 0101-31P5900), the Ministry of Education of Saxony-Anhalt (Research Centre Dynamic Systems: Biosystems Engineering, XD3639HP/0306) and the German Research Foundation (NA 292/9-1 and SFB854) by grants to M.N. We thank S Klamt, R Franke, J Schaber and A Naldi for helpful suggestions.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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In our July – August 2017 issue
It could be said that Holland is one big art museum and that its illustrious catalogue of past and modern artists is worthy of filling those museums and galleries. Join our author for a stimulating 12-day art appreciation barge cruise through amazing canals and locks to visit the best museums and galleries in the region. Then we're off to Crete for a volunteer vacation teaching students conversational English and exploring this ancient Greek Island. Next our readers may sleep with history in a northeast region of Florida less visited before we cross the vast Pacific Ocean by container ship with an insider look at modern-day freighter travel. We conclude with a challenging hike along New Zealand's famous Milford Track tackled by a group of American seniors, and we learn about the mighty walrus that lives in the planet's northern circumpolar region.
Be sure to visit the magazine's entire Travel Article Library and the handy Theme Pages to browse our growing collection of richly-illustrated features. In addition, we invite you to check our latest TravelWatch Newsletter for fast-breaking travel news … always food for intriguing thought. Finally, our innovative Travel Advertiser Directory is a treat not to miss. See why for yourself!
While showcasing both well-established and freshly-minted alternative travel opportunities throughout the world, this website and its Web magazine promise its readers practical advice and plenty of nuts-and-bolts information to encourage active, educational, value-added vacationing suitable for men and women over 50. The one thing you won't find is an excuse to stay home!
Feature ArticlesClick on title or image button to read each article in full.
Holland is famous for its canals and locks that link landscapes and cities together. Join a 12-day art appreciation cruise aboard a traditional canal boat that takes in historic and modern art museums and teaches painting techniques to participants that were used by well-known Dutch masters.
Combine a trip to one of Greece's most magical, diverse and deeply historic islands with a satisfying opportunity to teach conversational English to young people who will benefit from this gift you can provide not only to get into Greek universities but also to consider careers in tourism.
Wrapped in 400 years of colonial history, Northeast Florida's Amelia Island and the picturesque city of St. Augustine a little further south each offer a host of uniquely-furnished historic sleeps. A welcome bonus is the wide variety of signature breakfast cuisine that leave visitors begging for recipes.
Travel with our author and photographer, Julie and Patrick Lawson, as they board a giant container ship in Seattle to discover the appeal of crossing the Pacific Ocean to Shanghai in a manner quite unlike the experience of a traditional cruise vacation. Could this be a new travel option for you?
Short (four days) but challenging with significant elevation changes, the Milford Track on New Zealand's South Island is a trek that is popular with active mature travelers. MaryAnn Gerst, age 72, shares her ups and downs with seven hiking friends, ages 55 to 65, all from Albuquerque, New Mexico.
The Walrus is eminently recognizable in print photos and on nature shows because of its huge bulk and long protruding tusks. However, few travelers have ever glimpsed it in the wild nor appreciate why this circumpolar marine mammal should be on their viewing bucket list. We offer insights and images that could well change your mind. Photo by Nathalie Lasselin.
Coming in September/October 2017: A senior goes camel trekking in the "Land of Oz".
Check out our "Feature Link of the Month", and the five stories in our latest TravelWatch senior travel newsletter. We invite you to sign up to receive launch notification of each bi-monthly issue of this invaluable newsletter.

subset: commoncrawl


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(The Warriors are going through a giant metallic corridor fighting there way through giant machines. Omi is taking on two with his Elemental Shen Gong Wu, but gets clobbered by a third. Kimiko is fighting a bigger robot with her fire powers, but is no match for it. And soon Clay and Raimundo are brought down too.)
Raimundo: Ok, we lost!
(Then the corridor diminished and turned into the Xiaolin Temple grounds.)
Kimiko: Maybe we should just stick to our level of training instead of going to extremes.
Omi: Besides, whose idea was it to raise it that high?
(Kimiko and Clay pointed at Raimundo.)
Raimundo: Yeah, but so what? Were never gonna face robots that powerful, except for Spicer but he only has puny Jack-Bots.
Clay: Yeah, Rai's got a point. I don't remember the snake ever building robots that can match up to those ones.
Omi: Correct, Clay. But we still need to be in top tip shape.
Kimiko: Tip top shape.
Omi: Most surprising.
(But little do they know they were closely monitored by Jack Spicer with a tracking device while he's in his lair.)
Jack: What? Very well then Xiaolin losers! I'll give you strong robots!
(Then Jack's basement door opened to reveal Wuya.)
Wuya: I got your message saying you wanted me for pure evil purposes.
Jack: Yes my former disembodied head. For I Jack Spicer have created the ultimate, the destructive, the tormenting robot ever to exist.
Wuya: Jack, this is a song I've heard from you countless occasions and they all end up being ultimately, destructively, torment fully destroyed.
Jack: Not this robot, he's got evil written all over him.
(He led Wuya into one of his storerooms.)
Wuya: Wait, I seen you in this room before, you kept on sneaking away into this room doing who knows what.
Jack: You got it. I was sneaking away to perfect this baby.
Wuya: Okay, can I see what your "robot" looks like then?
(Jack pulled away a curtain to reveal a silver coated, human shaped robot.)
Jack: This Wuya is my Destruco-bot! Why I call him that? He is resistant to all Shen Gong Wu, elemental powers from the warriors AND because I've inserted data on the warrior's moves he now fights like a Xiaolin Warrior.
Wuya: Hm, interesting, but this hunk-a-junk won't be able to survive the Kuzusu Atom. It'd be vapourised before you can say, that was a dumb robot.
Jack: Ehhhhh, wrong. This baby is also resistant to the Kuzusu Atom. If you want i can show you a demonstration.
Wuya: Very well. But this is sheer foly.
(Jack pushed some buttons on his remote control watch, and normal Jack-Bots came from no-where.)
Jack: Destructo-Bot, Blaster-Nova!
(Then the robot pulled out a small cannon from it's arm and blasted it at the Jack-Bots and it blasted them into tiny atoms.)
Jack: Ahahahahaha! Impressive eh?
Wuya: I already seen this from your other bots, to totally get me on your side, get me Shen Gong Wu from the Xiaolin Temple.
Jack: As you wish. Destructo-Bot, Let's pay a visit to the Xiaolin Temple!
(The warriors arive from another training-simulation.)
Omi: Well that one was more better than last.
Clay: Yeah, more better than Rai's.
Raimundo: Sheesh it was only a try-out.
Kimiko: Uh-huh.
(Then the bell rang three times that meant danger. The warriors quickly ran to the grounds.)
Kimiko: What's the danger Master Fung?
Master Fung: Jack Spicer approaches. With Wuya.
Clay: Those two palling up together again? I always thought they're more hated than a bad joke at Christmas Eve.
(Jack, Wuya and the Destructo-Bot appeared from the hills, and arrived at the Xiaolin Temple.)
Raimundo: Spicer, what do you, Wuya and your tin can want now?
Jack: Apart from showing off in front of Wuya, i came for some Wu.
Omi: You and which gang?
Raimundo: You and what army!
Omi: Exactly!
Jack: My army's standing before you.
Kimiko: Wuya?
(Jack and Wuya slap their heads.)
Jack: The robot! The robot!
Clay: The feller doesn't seem that powerful.
(Then the Bot tripped him up and kicked him in mid-air, which forced him backwards onto one of the walls.)
Omi: You'll regret that!
(He jumped to face the robot then the bot punched but Omi dodged it and kicked him in the chest and flipped back. Little to Raimundo and Kimiko's astonishment, Omi was on the ground massaging his foot.)
Kimiko: Time to play tough! Star Hanabi!
(The fire shot hit him directly sending huge shockwaves across the grounds. But when the smoke cleared, he was still standing. Untouched and unscratched.)
Jack: This little baby is resistant to all Shen Gong Wu!
Warriors: Even the Kuzusu Atom?
Jack: Even the Kuzusu Atom.
(Then the Bot jumped right in front of Kimiko grabbed her by the face and threw her right against the wall. Causing her to collapse.)
Omi: This fiend is too powerful! We'll have to pull out our secret weapon!
All: Wudai Orion Formation.
(Raimundo and Kimiko were fighting the Bot at the same time which it was handling easily. Then the Bot grabbed both Raimundo and Kimiko's hands with one hand and double kicked them both right against the wall. Then Clay ran forwards and kicked the bot which knocked it back slightly, but he was in pain due to its hard solid armor.)
Omi: There must be a weak spot!
(Then Omi kicked him at the back and noticed that his armor slid off due to the kick.)
Kimiko: That's why he was resistant to all our attacks he had a big armor on!
Clay: That bot is trickier a magician at a magic show!
Raimundo: Well now that we know its weakness, Kimiko can you do the honors?
Kimiko: Will do Rai! Wudai Mars Fire Storm!
(She span round and round causing a huge fire tornado. Which the destruco-bot got sucked into and exploded into tiny pieces. Jack and Wuya were thunder-struck.)
Jack: How can this be possible? Well you may have defeated my super-ultra robot but I still have your wu. (Showing them a sack full of wu. And activating his Heli-bot and flying away.)
Raimundo: Jack bagged six of our wu, the Monkey Staff, Fist of Tebigong, Jet Bootsu, Crouching Cougar, Black Beetle and Monsoon Sandals.
Kimiko: Could have been worse.
(Dojo started writhing and shaking.)
Dojo: We got a new wu, quick to the scroll!
(They reached the grounds with Dojo with the scroll.)
Dojo: The Mechanic Kaiser, whoever wields it can make any machine the user wants. My personal favorite is a duplicating machine. Couldn't get enough of the duplicating machine!
(The warriors and Dojo arrived in what looked like an abandoned factory.)
Raimundo: Yeah great place for a machine Shen Gong Wu, lets make this quick.
(They went inside everything was in ruin.)
Kimiko: Looks like someone was having a really wild party.
Omi: Or worse.
(They reached a giant room which looked like the main room, and on top of a very large machine was the Mechanic Kaiser. But immediately Jack burst through the roof with the Fist of Tebigong.)
Jack: Ahahaha, try and take this wu.
Clay: And why would you want more wu when you snitched ours?
Jack: Hello? Cowboy? I need all wu to rule the Earth remember?
Raimundo: Well can't you give the stealing a break already?
Jack: Sorry. It's in my nature.
Kimiko: Well looks like we'll have to take the wu the old fashioned way. Star Hanabi!
(The blasts hit Jack's Heli-Bot, causing him to crash on the floor.)
Jack: You know what Kimiko, I agree we should fight the old fashioned way. Jack-Bots Attack!
Raimundo: Maybe a bit too old fashioned.
Jack: But this isn't Jet Bootsu.
(He started climbing the machine.)
Kimiko: Not so fast Spicer, Star Hanabi Fire!
Jack: Black Beetle!
(The blasts hit him doing absolutely nothing.)
Jack: Ahahahaha! What's wrong Kimiko? Your old flame die? Ahahaha!
(He continued walking up the statue until Kimiko jumped up and grabbed it along with Jack.)
Kimiko: Jack, I challenge you to a Xiaolin Showdown!
Jack: Right on, I was beginning to think when I was getting in a showdown with you.
Kimiko: Grrrr. My Serpent's Tail for your Monsoon Sandals.
Jack: The game is Race in the Mechanical Maze, first to the Mechanic Kaiser wins!
Both: Let's go! Xiaolin Showdown!
(The place turned into a giant metal maze with a platform on top for the spectators. And Jack and Kimiko were in two separate parts of the maze.
Both: Gong Ye Tenpi!
(Kimiko rushed into the maze and cutting between corners found that she had to cross a guillotine.)
Kimiko: Judolette Flip!
(She hopped right past the guillotine, but it narrowly missed and cut her ribbon off.)
Kimiko: (Pulling off her mask, to let her hair down.) Oh man, that was my favourite one as well, oh well.
(She ran on ahead, meanwhile Jack was making mincemeat out of the maze, since he had the Monsoon Sandals, he used them to make spring-loaded feet to cross the obstacles easily.)
Jack: yawn Too easy.
(Meanwhile Kimiko was hopping in between swords swinging to and fro, and she ran between a corner and saw something glow. It was the Mechanic Kaiser! But further along the other side was Jack.)
Kimiko: You'll never get the Shen Gong Wu!
Jack: Try me!
(Then he jumped up against the wall and pressed his spring-loaded feet against it.)
Kimiko: Serpent's Tail!
(As she darted off towards the Shen Gong Wu, Jack let go and fired faster than she was going. It almost seemed like a tie, but then Kimiko still had the Serpent's Tail and it slid through her hand and span out of control and landed on her butt. While Jack grabbed the Mechanic Kaiser, turning the field back into it's original form.)
Jack: Woohoohooo! I made EIGHT Shen Gong Wu in one day! Ahahahaha. Adios Xiaolin Losers.
(He flew in through the hole he punched at the start.)
Kimiko: (While her hair's still out due to the guillotine.) I'm sorry guys; to be honest I'm still kinda hurt from Jack's invasion.
Raimundo: We all are, don't worry bout it, what can Jack possibly use it for?
Dojo: Don't worry? DON'T WORRY? You just lost me my duplicating machine you...
(The others look at him disgustingly.)
Dojo: Which is a good thing. He.. he.
Clay: Besides, Rai has a point, What CAN Jack use it for?
(Back at Jack's lair.)
Wuya: Most impressive, you got eight Shen Gong Wu, you won a Xiaolin Showdown, I expected worse from a chimpanzee.
Jack: Make fun all you want, but I have a killer idea what to do with this Shen Gong Wu.
Wuya: Create a machine that stops you snoring?
Jack: HA Ha Ha, No I plan on building a machine to build a whole army of my robots, and not just any robots my former-disembodied head, My Destructo-Bots!
Wuya: No! That is the most brilliant idea since... ever!
Jack: Just think Wuya, Two hundred bots a second, twelve-thousand bots a minute, seven hundred and twenty thousand bots an hour, and my favorite part, seventeen million, two hundred and eighty thousand bots a day!
Wuya: Then we leave tomorrow at nightfall!
Jack: Now let's get started! Mechanic Kaiser!
Master Fung: It is Ok Kimiko; a mighty warrior did not win every fight when he was a novice.
Kimiko: I get that part. But losing to Jack is like losing to a clown in a daydream.
(As Raimundo, Omi and Clay laugh at Kimiko's joke, the doors burst open to reveal Chase Young, beaten and weak.)
Omi: Chase Young, what happened to you?
Chase: Jack... thousands of robots...called...destructo-bots.
(He then collapsed in front of Omi and Kimiko.)
Clay: The destructo-bots? But Jack just built the one, which took longer than a 5-stone steak to cook, to build.
Raimundo: The Mechanic Kaiser! Jack must have built a machine that could make hundreds of them.
(They took Chase to the centre table to help him rest, soon he regained consciousness.)
Chase: Ugh... Warriors, I came here to warn you, Jack has mustered the most powerful destructive army he has ever built. He attacked me while I was gathering ingredients for the Lou Mang Long Soup. Just before I fled he said: "First Chase, now the Monks."
Kimiko: So he's now preparing an invasion?
Chase: Looks like it.
Omi: How could we combat them?
Master Fung: Jack must have prepared this army to steal our Shen Gong Wu, we must find a safe place to hide and stop him getting the Shen Gong Wu.
(At that moment the place started to rumble, they went outside and seen Jack on top the mountains.)
Raimundo: You heard Master Fung, quickly gather the Wu. And get to the Heylin Seed's vault! Hurry! I gotta see this army for myself. Falcon's Eye!
(He took a look through the Shen Gong Wu, and seen an army of destructo-bots across the mountains across. He cowered in fear as he saw how much.)
Raimundo: This can't be!
(Over across the mountains.)
Wuya: Our army of destructo-bots is finally complete. No one can defeat us now!
Jack: And now for the final phase to our plan of world domination! Destructo-Bots march to the temple, show no mercy, leave none alive! Once we defeat the monks, nothing can stand in our way of World Domination!
Jack: And now for the final phase to our plan of world domination! Destructo-Bots march to the temple, show no mercy, leave none alive! Once we defeat the monks, nothing can stand in our way of World Domination!
(Back at the temple, Raimundo is still petrified with fear.)
Kimiko: (With a bag of wu to protect.) Rai, come on.
(She then throws him a bag of wu.)
Raimundo: What? Oh right. Sorry.
(He picked up the sack and him and Kimiko ran for the Heylin Seed's vault. At the entrance while Kimiko was unlocking the vault, Raimundo's sack became loose and the Tunnel Armadillo fell out with no one noticing. Soon they were all inside (With Chase Young, since he's still injured.)
Raimundo: I just looked at that army, it is way too big.
Clay: Um how big we talking here? Like Texas swamp big? Or 5 foot steak big?
Kimiko: We can all look before we left; I put up hidden cameras to see what we're up against.
(She typed in a couple of stuff in her laptop and out came a screen with Jack and Wuya being followed by hundreds of Destructo-Bots. And just like Raimundo, Omi Clay and Kimiko cowered in fear.)
Chase: Exactly, even my powers can't stop him. If that were the case we wouldn't be here right now.
Omi: But we can't exactly sit here and just let Jack come...
Kimiko: Shhh, according to my calculations, Jack will discover what's going on in...Three...two...one... (She waved her finger.)
Jack: (From a distance.) HEY, WHERE'S ALL THE WU?
(They all gave a small laugh.)
Omi: As I was saying, we can't sit around and watch Jack invade our temple.
Raimundo: We can't exactly go up there and get our butts kicked and get our wu stolen either.
Master Fung: We must figure out a strategy without the means of fighting the army. Or sitting around and doing nothing.
Clay: We did beat the first one.
Raimundo: But that was hard.
Kimiko: And by dumb luck.
(Omi glared at her.)
Kimiko: Sorry.
Raimundo: So, we know their weakness but doing it's gonna be hard.
Omi: Wait, I grasped it!
Kimiko: Would you be saying you got it?
Omi: Oops, anyway. What if we can get the Mechanic Kaiser?
Raimundo: Then we'd get our butts kicked off Spicer, because he has it.
Kimiko: How do you know that?
Raimundo: ... because er... ok so I don't know.
Omi: But I do know something that might. (He took the Crystal Glasses from the sacks of wu.) Crystal Glasses. Hm... The location of the Shen Gong Wu is hidden in Jack's Lair protected by four Destructo-Bots.
Kimiko: Yes! We're in the clear.
Clay: Er... two things. One how do we rustle them bots?
Chase: How about going in a Xiaolin Showdown that is less physical.
Clay: Ok, and two how do we get outta here without having Jack on our behinds?
Omi: Well we can't exactly use the Serpent's Tail.
Dojo: The Tunnel Armadillo, we can use it here so Jack doesn't see us use it.
Omi: Dojo, you're a genius.
(He jumped into the sacks and buried and buried to find it. He emerged puzzled.)
Omi: Where's the Tunnel Armadillo.
Kimiko: Maybe your looking wrongly, here you gotta tip the sacks over and pile them in one-by-one.
(They did just that putting all the wu in individually, but the Tunnel Armadillo was nowhere to be seen.)
Clay: This is stranger than my pet pig vanishing one day.
Kimiko: D'you think one of us dropped it on the way here?
Raimundo: Probably but even if it was it would be lynched off Spicer.
Omi: (While using the Crystal Glasses.) It is outside this very vault.
(Raimundo opened it and there it was. He swiped it and brought it back in.)
Raimundo: I wonder who dropped it anyway?
Omi: Does it matter? We got it now and that's what matters.
Kimiko: So Omi, What do we do when we have the Mechanic Kaiser.
Omi: We will create a machine that can destroy all machines' that have electricity in it.
Kimiko: An electro-magnetic pulsar?
(Omi stared at her in confusion.)
Kimiko: An electrical component that can scramble all the bots' circuits.
Omi: Exactly.
Raimundo: So what are we waiting for? Let's bring Omi's plan to reality.
(Then the vault door started banging.)
Kimiko: We'd better hurry up because Jack's found us!
Raimundo: Tunnel Armadillo!
(The Shen Gong Wu re-sized to its big form.)
Omi: Wait, Jack will still follow us.
Clay: Yeah well it's better to try partner.
(They all climbed into the Shen Gong Wu except Master Fung and Chase Young.)
Omi: Master Fung, Chase Young, you must come with us.
Chase: We will buy you some time against the robots at hand.
Kimiko: We got no time Omi, come on.
(Then the Armadillo drilled its way through the ground towards Jack's Lair. Then the vault door burst open with Wuya and Jack with hundreds of Destructo-bots.)
Jack: No wu here either, just two losers who've gone brain-dead to think they've got a chance of beating us.
Chase: Come over here and say that.
Jack: Nah, my boys will. Destructo-Bots attack!
(Back at the armadillo.)
Omi: I cannot believe we let Master Fung and Chase sacrifice themselves to save us.
Raimundo: They had to otherwise we'd have Destructo-Bots on our tail right this second. All I'm worried about is that your plan works or not.
Omi: I hope it does. Otherwise Jack has already won.
Clay: Well, we're directly above the snake's place.
Kimiko: Remember what Chase said about going in a Showdown? Who should go?
Raimundo: Maybe we all should.
Kimiko: An eight-way Xiaolin Showdown?
Raimundo: Yeah.
Dojo: Hadn't had one of those in a while.
Omi: Then let's go!
(And they all gave each other a hi-5)
(The armadillo drilled upwards to be in the gardens, luckily there were no destructo-bots.)
Kimiko: They might be guarding the Mechanic Kaiser.
Raimundo: All right let's get it!
(Clay grabbed him before he could jump to the roof of Jack's Lair.)
Clay: Hold on there partner, you can't go waltzing into Jack's lair wuless.
Omi: Correct, and we have nearly all the wu to choose from.
Raimundo: Ok, so who gets what?
Kimiko: Why not choose at random?
Clay: Yeah we could always try that, guess a'll go first.
(He put his hand into the sack and took out the Cannon Blaster.)
Clay: Woowee. Haven't tried this wu yet.
Raimundo: Might as well be me next.
(He stuck his hand into the sack and pulled out the Eye Of Dashi.)
Raimundo: Sweet.
Omi: Now me.
(He took out the Silk Spinner.)
Omi: Amazing!
Kimiko: Now let's see what I get.
(She put her hand in and got the Gills of Hamachi. The others were trying not to laugh.)
Kimiko: Fine. Let's just get this wu and create the EMP bomb.
(The jumped up onto Jack's roof and snuck through the ventilation shafts. Until they found Jack's basement, and the Crystal Glasses was right, there were exactly four Destructo-Bot guards.)
Kimiko: How am I supposed to fight them with only the Gills of Hamachi?
Raimundo: Dunno, maybe you can wag them to death. Just kidding.
(They burst through and the bots noticed them.)
Omi: Hand over the Mechanic Kaiser, or suffer a humiliating defeat!
Bot: Surrender, we will not! Destroy you we will!
(They ran towards each other then collided into each other.)
Clay: Cannon Blaster!
(He turned into a cannonball to crush his bot, but the bot just raised a hand that stops him immediately.)
Kimiko: We can't beat them just get the Mechanic Kaiser!
(All eight of them ran for the Mechanic Kaiser on Jack's table and all touched it at the same time.)
Omi: We challenge you to an eight-way Xiaolin Showdown!
Robot: Our Fist of Tebigong, Jet Bootsu, Serpent's Tail and Changing Chopsticks.
Raimundo: For our Cannon Blaster, Silk Spinner, Eye of Dashi and... Gills of Hamachi!
Omi: The game will be Mechanical Maze.
Kimiko: Omi, I already did that contest.
Omi: Maybe, except this one will be bigger and more challenging.
Robot: We accept!
All: Let's go! Xiaolin Showdown!
(Jack's Lair split up and shifted into a bigger maze than the one Kimiko had to face. Then the eight of them were split up in eight different parts of the maze.)
All: Gong Ye Tenpi!
(Omi ran around three corners before facing a dead end.)
Omi: Darn it.
(He raced back to an alternative road to find a huge pond with poles sticking out of them.)
Omi: Looks like I need to jump across using those poles.
(He jumped three poles along until he noticed it was infested with crocodiles.)
Omi: Gulp
(He jumped a high jump and made it to the other side, but fell into a trap door. Then he got transported to another place in the maze.)
Omi: This was most unpredicted.
(He ran past another corner to find one of the Destructo-Bots racing past. Then further along where the Destructo-Bot was standing was Kimiko. She made a hand gesture meaning that they should attack at the same time. Omi gave a thumbs-up meaning he understood.)
Kimiko: Now!
(They both ran to the Destructo-Bot, but it back-fired as he shrunk itself using the Changing Chopsticks causing Kimiko and Omi to run into each other.)
Robot: Must not waste time fighting, must retrieve Shen Gong Wu!
(Then he raced off where Omi had been.)
Kimiko: Oh... you Ok Omi?
Omi: Yes, I think. Oh...
Kimiko: So you find anything useful up there?
Omi: Only dead ends and a pond with crocodiles in them that a trapdoor at the end.
Kimiko: You said trapdoor?
Omi: Yes, why?
Kimiko: Shhh. 3...2...1...
(They both heard a door opening with a robot screaming. They both laughed.)
Kimiko: Ok we should split up and see what to find next.
Omi: Wait, why not we exchange Shen Gong Wu for a while, I don't mind having the Gills of Hamachi, and you can take the Silk Spinner.
Kimiko: You sure? Cus what would happen if you did fall down another trap door and didn't have the Silk Spinner?
Omi: I am sure one trap door was enough in this maze.
Kimiko: Well, Ok if you're sure.
(Kimiko took the Silk Spinner and Omi took the Gills of Hamachi. And they both took separate directions. Kimiko took the direction the Destructo-Bot was before, and Omi took the one Kimiko went through before.)
(Meanwhile Clay was running along a long narrow part in the maze, only to find himself facing a huge double-guillotine.)
Clay: Whistle that things choppier than a lamb chop on chop Sunday.
(He rolled across it no problem, and then got confronted by one of the Destructo-Bots, the one with the Serpent's Tail he figured since it was in his hand, and luckily the bot didn't notice him. So it zoomed past the walls, giving Clay time to catch his breath.)
Clay: Maybe we shooda made a showdown that was more physical, rather than hide-and-seek.
(He ran past a corner and collided in with Raimundo.)
Raimundo: Clay?
Clay: Raimundo?
Raimundo: gasp thought you were one of the Destructo-Bots, it made mince-meat outta me with the Serpent's Tail.
Clay: Yeah you got lucky, the ol' critter slid past the walls. Probably still looking for the Mechanic Kaiser.
Raimundo: Well we betta find it first, any sign of Omi and Kimiko?
Clay: Nope, they maybe getting more outta this than me 'n you combined.
Raimundo: Alright let's split up.
(They both took their separate directions. Raimundo noticed the double-guillotine.)
Raimundo: Think that's where Clay's been before. Maybe I should go to the right.
(He ran along corners to find a huge-crusher in front of him.)
Raimundo: This is really not my day.
(He timed his run accurately, but when he was about to run he tripped and was about to be crushed when Kimiko jumped in and saved him.)
Kimiko: Jeez Rai, be careful next time okay?
Raimundo: (Blushing) Wow. You saved me.
Kimiko: (Blushing too) Yeah well couldn't let an old friend get crushed with a five-ton crusher could I?
(Then Raimundo noticed Kimiko had the Silk Spinner.)
Raimundo: Hey where'd you get the Silk Spinner? I thought Omi had it.
Kimiko: He did, but me and Omi met up and he wanted to swap wu, said he didn't mind having the Gills of Hamachi.
Raimundo: Okay. So what's the scoop over there? (He pointed past the crusher.)
Kimiko: Absolutely nothing but traps galore. You woulda killed yourself for nothing if I didn't save you.
Raimundo: Well come on, there must be a way to the Shen Gong Wu, or my name isn't Raimundo Pedrosa.
(They both ran in a direction they both never took and cut a corner to find a white light with the pedestal with the Mechanic Kaiser.)
Both: Yes!
(But they didn't know that two other Destructo-Bots had also found the Shen Gong Wu too.)
Raimundo: Come on!
(The four of them ran to the Shen Gong Wu, and then one of them knocked out Raimundo with the Fist of Tebigong.)
Kimiko: (Still running) Raimundo!
Raimundo: (Weakened) Get the Shen Gong Wu!
(She still ran to the Shen Gong Wu until the Bots stopped, meaning they wanted to fight Kimiko.)
Kimiko: You want to fight? Fine by me, it's your scrap-heap.
Bot: You shall not succeed! We will destroy all those that stand in our path!
(One of them tried to punch her with the Fist of Tebigong but she narrowly missed. Then she noticed the other bot was going for the wu.)
Kimiko: Oh no you don't! Silk Spinner!
(The web grabbed onto the bot and she yanked it, making both bots collide. But then the two remaining bots jumped in and knocked Kimiko out.)
Bot: You shall be destroyed!
(It raised the Fist of Tebigong up to crush her. But then she heard a sound, which sounded like water, then a flood of water burst through one of the corners, and Kimiko knew it was Omi who conjured it, since he was in the middle of the water with the Gills of Hamachi in effect. The water washed away the bots but they were still hanging on. Then Raimundo used his wind powers to lift himself and Omi and Kimiko.)
Raimundo: Eye of Dashi!
(The blast hit the water electrocuting the robots. And in the distance was Clay, protected from the electric water with a rock armor round his entire body. He ran up and grabbed the Mechanic Kaiser ending the showdown.)
(They gave a big hug to each other for their victory.)
Dojo: We might have beaten four Destructo-Bots, but we still got an army left.
Omi: Dojo's right we must get back to the Xiaolin Temple and rescue Chase Young and Master Fung.
(Back at the temple, Jack was being entertained with robots fighting each other. And over the hills were the warriors.)
Raimundo: So exactly how are we gonna do this?
Clay: There's no way we can set up the EMP bomb with so many bots there.
Omi: Then I will distract the bots, Jack and Wuya while you set the EMP bomb around the temple.
Kimiko: You had difficulty facing one, what chance you got with over a million bots?
Omi: That Kimiko is a real mystery. Just plant the bombs over the Temple. And activate them to destroy the Bots. Mechanic Kaiser!
(He pointed to the ground and made hundreds of tiny bombs and a small remote control detonator.)
Omi: And if my electronics are correct the bombs won't affect you or the temple. It'll only affect the Robots.
Clay: Well, this is it partners. Time to re-claim our home.
(Raimundo, Kimiko and Clay took some bombs and all of them ran towards the temple.)
(Omi appeared in front of Jack and the bots.)
Jack: Ah, so there's the cue ball. Finally decided to show up have you?
Omi: Jack Spicer! You have invaded the Xiaolin Temple, but you will not invade the world!
Jack: Who says so baldy? You? Then I'll just have to crush you! Destructo-Bots, Attack!
(Soon Omi was surrounded by Destructo-Bots. And in the back round, the others were running across the temple planting the bombs as they went along. Avoiding other Destructo-Bots. Back at Omi, the Bots jumped at him but rolled back, narrowly missing them.)
Jack: Hey, stand and fight coward!
Omi: Well, I do not exactly see you do the same thing.
Jack: My boys are doing my fighting.
(One bot rushed at him, but Omi tripped him up and kicked him away, and used his Shimo Staff to knock off the head of one.)
Jack: Hey, no fair, no weapons.
Omi: This is no weapon Spicer, it's a pole. Haha.
Jack: Doesn't matter. Cus you're still going down.
Omi: Bring it on!
(Meanwhile, Raimundo planted the last bomb.)
Raimundo: That's the last of 'em, let's get back to Omi and get Spicer outta here.
(They rushed back to where Omi was fighting, only to realize that the bots have him captured. Then Jack noticed them.)
Jack: Ah the last exiles. As you can see my boys have made cue ball delight from your pal.
Kimiko: Let him go! Or one button will destroy them.
Jack: Ooooooh, I'm so scared. See the thing is, how long will it take to destroy them all? A minute? Two? Or three? But by that time, cue ball over here will be vaporized by the bots rays. Ahahahaha.
Omi: Never mind about me. Activate the bombs!
(Kimiko looked from Omi to the detonator. Back and forth, them she dropped the detonator, and all three of them raised their hands to surrender.)
Jack: Ahahahahaha, your weakness is this world's doom.
(Then he snapped his fingers for the other bots to seize them. They took their new captive's hands. But then one of the bots had seen that Kimiko had the Mechanic Kaiser.)
Robot: The girl has the Mechanic Kaiser.
Kimiko: Yeah. So?
(All the bots froze not even doing anything.)
Jack: Hey, what's going on? Seize them!
(They did nothing.)
Jack: I said SEIZE THEM!
Robot: We yield, only to the one with the Mechanic Kaiser.
Clay: So all this time we coulda controlled the bots?
(Dojo took out the scroll and examined the Mechanic Kaiser more carefully.)
Dojo: Its right, whoever has control of the Mechanic Kaiser, has control of all it's creations.
Raimundo: So the bots are on our side?
Kimiko: Looks like it.
(Omi broke free from the bots grips and joined the others.)
Jack: Uh-oh. (Bending down.) I SURRENDER! What did I ever do to you?
Kimiko: You created an army ready to destroy us did who knows what to Master Fung and Chase Young, made us go through a hard Xiaolin Showdown AND nearly killed one of my best friends.
Jack: Besides that.
Kimiko: Well then, Destructo-Bots! Get Wuya and Jack outta here!
(They grabbed both of them and threw them all the way over the mountains.)
Raimundo: So what do we do with the bots?
Clay: We can destroy a few and keep some for practice.
Omi: But if Jack were to ever get the Mechanic Kaiser he'd have control of the bots again. I have an idea. Mechanic Kaiser please Kimiko. (She handed him the Shen Gong Wu.) Bots I command you to destroy yourselves.
Bot: As you wish!
(The bots took out their cannons, aimed it towards themselves and vaporized all of them.)
Omi: The storm has passed, the bots are destroyed, and Jack remains unsuccessful in ruling the world.
Kimiko: Master Fung and Chase Young! There still missing.
(They ran back to the Heylin Seed's vault to find the two of them, beaten and unconscious.)
Raimundo: Dojo! Get the Treasure Of The Blind Swordsman.
(He ran to the Temple and brought the treasure with him. And opened it to reveal the Blind Swordsman.)
Swordsman: What is your wish?
Raimundo: Blind Swordsman. Can you heal Master Fung and Chase Young to full health?
Swordsman: Your wish is my command.
(He raised a hand, and two white lights hit the two of them bringing them back to full health.)
Raimundo: Thanks, dude.
(The Blind Swordsman went back into his Treasure.)
Master Fung: Young monks? Was your plan successful?
Omi: Yes master.
Chase: Then that makes us on opposite sides again.
Clay: Too bad.
(Chase Turned into purple smoke and blew out of the vault.)
Omi: We may be on opposite sides Chase, but we'll be ready! For You, for Jack and for Hannibal Bean.


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EDN Members of the Month
In our monthly interview series "EDN Member of the Month", we focus on one of our many members to show both members in the spotlight and the diversity of the EDN membership group. Below you will find an overview of our previous EDN members of the month. Use the navigation bar below to display all Members of the Month for a specific year.
Christian Popp, born in Romania, is a German-French independent producer who started his career as a commissioning editor at ARTE (1997), first in Strasbourg and then in Berlin. He became a producer 12 years ago. Since then he produced for Interscience film, Artline Films and Docdays Productions. In 2012 he founded YUZU Productions, based in Montreuil, France, together with Fabrice Estève ... [read more].
Adam Papliński has been involved in various fields within the film industry for over 25 years. He has worked as assistant director, production manager and producer for feature films, TV spots and documentaries. In 2010-2015, in the framework of Academy of Documentary Arts (ADA) he coordinated the international documentary training and pitching event in Poland ... [read more].
In his 30+ years in documentary television Steven Seidenberg has been a producer, director, head of development & scriptwriter. Steven has wide experience in all documentary genres. As a producer he has worked in history programming (e.g. The Trial of George Washington, Hitler's Death, and Napoleon's Obsession) ... [read more].
Pauline Mazenod is the founder and CEO of Windrose. She holds degrees in political science, music, international cultural management and business administration, and has more than 15 years international experience in the cultural and film sector. Windrose distributes high quality ... [read more].
Victor Correal is the founder and CEO of GuideDoc, an online streaming service for curated documentaries, with new films made available each week. Victor is also himself active in filmmaking and has among other films produced Little World (Marcel Barrena, 2013) and a number of TV shows primarily for Spanish TV ... [read more].


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208 LANDING FERRY Way
Greer, SC 29650
Beds: 4
Baths: 2 | 1
Sq. Ft. Range: 2200-2399
Listing #1337012
Riverside schools and an awesome location convenient to I-85 add to the desirability of this wonderful Eastside home! Situated on a quiet cul-de-sac lot, 208 Landing Ferry Way has an attractive brick & vinyl exterior, new roof installed in 2016, mature landscaping, and a two-car front entry Garage. The interior of the home is warm and welcoming with two story Foyer enhanced by a second story arched window, balcony overlook, half bath and hardwood floors. Beyond the Foyer a Formal Living Room awaits you along with a Formal Dining Room that includes neutral carpet, traditional wainscoting, oversized windows fitted with 2' blinds, and a designer chandelier. The Kitchen offers a bright white color scheme with an abundance of cabinetry, center island workspace, new stainless steel appliances, and a shelved pantry that gives even more storage space for dry goods or small appliances. The adjoining Breakfast Area included pendant style lighting and a glass panel door that allows you easy access to the rear deck. A gas log fireplace with a traditional mantle and marble surround draws you into in the gracious Great Room. It also features neutral carpet and triple windows that overlook the backyard. All of the bedrooms are privately located upstairs. A deep tray ceiling make the luxury Owner's Suite a welcomed retreat at the end of a long day along with a big walk-in closet, tiled shower with glass enclosure, garden tub with tile surround, and an extended cultured marble vanity with dual sinks. All of the secondary bedrooms are spacious in size, have excellent closet space and share a centrally located hall bath that's equipped with a tub/shower combination and extended vanity with dual sinks. Also of note, the fourth bedroom is large enough to be used as a Bonus Room. In addition to its closet, it also offers lighted ceiling fan and easy eave access. A sizeable deck can be found at the rear of the home along with a big backyard that offers plenty of privacy and shade.
Property Features
Location Information
County: Greenville
MLS Area: 022
Latitude: 34.8750190734863
Longitude: -82.2536773681641
Subdivision: Camden Court
Interior Features
Interior: 2 Story Foyer, Attic Stairs Disappearing, Ceiling 9ft+, Ceiling Fan, Ceiling Smooth, Ceiling Trey, Smoke Detector, Some Window Trtmnts, Tub Garden, Walk In Closet, Countertops-Other
Master Bedroom Description: Double Sink, Full Bath, Master on 2nd Lvl, Shower-Separate, Tub-Garden, Tub-Separate, Walk-in Closet
1/2 Baths: 1
Amenities: Common Areas, Lights
Fireplace Description: Gas Logs
Heating: Forced Air, Natural Gas
Cooling: Central Forced, Electric
Floors: Ceramic Tile, Hardwood, Vinyl
Water Heater: Gas
Appliances: Dishwasher, Disposal, Microwave-Stand Alone, Oven-Self Cleaning, Refrigerator, Washer, Oven-Electric, Stand Alone Rng-Smooth Tp
Basement Description: None
Exterior Features
Style: Traditional
Stories: 2
Construction: Brick Veneer-Full
Exterior: Deck, Porch-Front, Windows-Thermal
Foundation: Crawl Space
Roof: Architectural
Sewer: Public
Water: Public
Parking Description: Door Opener
Has Garage: No
Garage Spaces: 0
Driveway: Paved
Lot Description: Cul-de-Sac, Sloped Gently, Underground Utilities
Lot Size in Acres: 0.26
Dimensions: 81X112X104X132
Elementary School: Buena Vista
Jr. High School: Riverside
High School: Riverside
Additional Information
Property Type: SFR
Property SubType: Single Family-Detached
Is a Foreclosure: No
Fees Include: None
$ per month
Year Fixed. % Interest Rate.
|Principal + Interest:
|Monthly Tax:
|Monthly Insurance:
Based on a fully amortized fixed rate loan. Ask your agent for the tax rates in your area. Insurance estimate is based on an average cost, your final premium cost will be determined by the type of coverage you select. This program only provides an estimate.
Listing Agent:
The data relating to real estate on this web site comes in part from the Internet Data Exchange (IDX) program of The MLS of Greenville, S.C., Inc. IDX information is provided exclusively for consumers' personal, noncommercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing. All Data Is Deemed Reliable But Is Not Guaranteed Accurate.
© 2017 The MLS of Greenville, S.C., Inc. All rights reserved.
© 2017 The MLS of Greenville, S.C., Inc. All rights reserved.
GGARMLS data last updated at August 18, 2017 3:06 PM ET
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cut edge wedding veil › two tier cut edge wedding veil › two tier wedding veil › wedding veil review › wedding veils ›
We're delighted with this lovely review of our two tier cut edge wedding veil- Saria
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Why subscribe? 1. We'll keep you up to date on our new designs. 2. We'll let you know when we add more 'How to' guides. 3. Once in awhile we might even send you a special offer ;)
© 2017 Britten Wedding Veils & Accessories, 32 Market Street, Bradford-on-Avon, Wiltshire, BA15 1LL. t 01225 864106


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Red Circle Tea
Mark's question re: Qi
Mark wanted to know about Chi or "Qi" in tea. All tea has qi, but some has more qi than others. Qi is described as elevated blood circulation, whether from the caffine or from an internal body force (think chinese medicine) the increase in circulation causes heat in the body. Chinese medicinal traditions tell us that the lungs act as a cooling system for the body. Since our back is right next to our lungs, people usually feel qi in their upper back first. Qi affects individuals differently so occasionally a person might feel warmth or sweatiness in different parts of their body. All tea has qi to different extents, and paying attention to breathing, general blood circulation and the back are great ways to begin to experience qi. Gradually, you will notice nuances in the effects on your body, realizing, with other teas, you experience a strong qi in extremeties like the head or legs or arms. One of the most interesting qi experiences I've had was sweating between the toes.


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I was in The Boy Child's room earlier and saw something on the bed.
The Brainy One has a dinner/meet up/thing this evening and would be leaving the house round about the time The Boy Child came home from school.
So he took the time to leave a note.
He's a good man.


text:
This recipe is super simple, and widely adaptable – whether you are looking for a way to prepare celeriac, radishes, parsnips, root parsley, kohlrabi, beets, carrots, salsify, potatoes, turnips, rutabaga, winter squash; this recipe can help you.
I usually start by cleaning all of my veggies to be roasted, and cutting them into chunks, roughly the size of your thumb – size doesn't really matter, so long as you remember that larger pieces will take longer to cook, and smaller ones will tender up in the oven more quickly.
Preheat the oven to 350 or so, and lightly oil a baking sheet – for this recipe I usually use grape seed oil. Put your veggie chunks on the baking sheet. You can top them with black pepper, sea salt, or some herbs like savory, thyme, oregano, rosemary, etc.
Bake until you can easily stick a fork through the roots. You could add an extra step of melting cheese over this, but its not necessary. Other nice additions are slices or wedges of tomato, chunks of onion, leeks, shallots or even cabbage, and whole cloves of garlic.
I hope you enjoy 😉


text:
We believe that our patients are the best judge of our quality and service in dental care. Read what our patients are saying about us below. These comments are taken directly from patient satisfaction surveys with their permission.
Thank you for being nice and courteous, and excellent staff.
- S. Carpenter
Great job!! So happy with the two who worked in me!
- Anonymous Patient
I'm so grateful for all of the doctors and staff.
- Anonymous Patient


text:
Spare time in the half-bath's and was breasts "behind dirt" I'm saving up take-Chan found! Mesmerized by the beautiful breasts, and.
Spare time in the half-bath's and gone breasts POV I'm saving up "behind the scenes dirt".
Sense if it says in Japan brought the camera in the bath, half enjoying my friends have taken? www
[Bock note *] "love bath!" or woman saying nature wwwwwwwwwwwwwwwwwwwwwww (image is)
The curvy girl underwear on half of the body after a bath and boobs tits masturbation self portrait photograph