Fear conditioning, in which a cue is conditioned to elicit a fear response, and extinction, in which a previously conditioned cue no longer elicits a fear response, depend on neural plasticity occurring within the amygdala. Projection neurons in the basolateral amygdala (BLA) learn to respond to the cue during fear conditioning, and they mediate fear responding by transferring cue signals to the output stage of the amygdala. Some BLA projection neurons retain their cue responses after extinction. Recent work shows that activation of the endocannabinoid system is necessary for extinction, and it leads to long-term depression (LTD) of the GABAergic synapses that inhibitory interneurons make onto BLA projection neurons. Such GABAergic LTD would enhance the responses of the BLA projection neurons that mediate fear responding, so it would seem to oppose, rather than promote, extinction. To address this paradox, a computational analysis of two well-known conceptual models of amygdaloid plasticity was undertaken. The analysis employed exhaustive state-space search conducted within a declarative programming environment. The analysis reveals that GABAergic LTD actually increases the number of synaptic strength configurations that achieve extinction while preserving the cue responses of some BLA projection neurons in both models. The results suggest that GABAergic LTD helps the amygdala retain cue memory during extinction even as the amygdala learns to suppress the previously conditioned response. The analysis also reveals which features of both models are essential for their ability to achieve extinction with some cue memory preservation, and suggests experimental tests of those features. Neurobiologically, fear conditioning and its extinction are better understood than any other forms of emotional learning Maren, . Fear coAlthough several brain regions are involved in these processes, fear conditioning and extinction depend most heavily on synaptic plasticity occurring within the amygdala  of the GABAergic synapses of inhibitory interneurons onto certain amygdaloid projection neurons , henceforth referred to as Model 1 and Model 2, which are relatively simple but capture the basic features of amygdaloid connectivity and plasticity. Their relative simplicity enables them to be analyzed using exhaustive searches of their state spaces.The neural elements in both Model 1 and Model 2 can be interpreted either as single neurons, or as groups of neurons within the same subregion that all have the same connectivity and behavior (see Discussion). The responses of the neural elements in the models depend entirely on the weights of the connections between them, so the state of either model is completely determined by the configuration (combination) of its connection weights (representing synaptic strengths). As these weights change, according to rules of plasticity, the state changes, and model analysis involves searching the space of all possible states  for configurations that produce fear conditioning followed by extinction but that also conform to certain constraints as determined by experimental findings. The analysis of each model begins with minimal constraints. Subsequent searches with progressively more restrictive constraints reveal the possible role of the endocannabinoid system in extinction of fear conditioning, and also reveal which features of each model are the most critical for the performance of that role.A schematic illustrating the known synaptic connectivity within the amygdala, and between the amygdala and certain other brain regions, is shown in Figure The forgoing scheme is based on the following anatomical and physiological findings from mammals, mainly rodents. Thalamic nuclei including the medial geniculate and the posterior intralaminar project to LA  part of the prefrontal cortex projects to several amygdaloid nuclei including LA and both ITCl and ITCm , which includes LA and BA, undergo both LTP and LTD  spaces. Such an approach is useful for “proof of concept” studies, but would not be appropriate in the present context where the analytical goal is to determine which weight configurations are compatible with both LTD of GABAergic interneuron synapses and extinction, and which of those also conform to known neurobiological constraints. Any combinatorial search must avoid a combinatorial explosion, and the present analysis will proceed from simplified versions of the framework depicted in Figure all possible orders , and the entire tree of state transitions can be searched for states of interest  indicate what operations can occur without dictating their order. Using a declarative specification, applicable declarations can execute in any order. For the purposes of exploring the possible states of a system modeled using a declarative specification, applicable declarations can execute in Declarative programming has been used for decades to model and analyze complex manmade systems, but its use in biology is quite recent , Maude executes every applicable rule. If r rules apply in the initial state, then there are r new states at depth d = 1. If r rules apply from any state at any depth, then there are rd states at any depth d. The tree of state transitions widens rapidly until rule execution ceases. Maude then searches the state-transition tree for states of interest in a breadth-first manner. States of interest have explicit properties  and may be subject to certain conditions . In this analysis almost all states of interest needed to satisfy certain conditions, which represented experimentally determined constraints.In Maude a declaration is either an equation or a rule. An applicable equation must always execute, and in so doing it simplifies but does not change the state of the model system. In contrast, an applicable rule may execute or not, but by executing it changes the state of the model system. In the Maude specifications for both Model 1 and Model 2, rules produce allowed weight modifications while equations determine the effects of each weight change on model element responses. Thus, rule executions produce allowed connection weight changes and cause the model system to transition from one state to another. State-space search in Maude involves searching the state-transition tree, which Maude first constructs through rule executions as follows. From the initial state , can be found by an unconditional search for all terminal states. The configurations that produce extinction following fear conditioning with additional constraints were found by conditional searches. Each of the weight configurations reported resulted from a unique series of rule executions, but all series terminated before a tree depth of The structures of Model 1 and Model 2 were different but the procedures for making individual weight changes were the same in both. Generally, LTP occurred during simulated fear conditioning , after which LTD occurred during simulated extinction . In both models, individual weight changes were of absolute value 1 and all weights were bounded from 0 to 2, so that excitatory weights could only take values of 0, +1, or +2 while inhibitory weights could only take values of 0, −1, or −2. These levels correspond to the observed ranges of LTP and LTD, which can respectively double a synaptic strength or reduce it to 0 . There is some uncertainty as to whether the endocannabinoid system facilitates extinction through associative or non-associative mechanisms  that are uniformly distributed over the constrained space of weight changes.−6. MATLAB was used to make 1000 searches for each of Model 1 and Model 2. For either model, the number of searches achieving zero error with LTD of inhibitory interneuron connections was expressed as a percentage of the number of searches achieving zero error with or without LTD of inhibitory interneuron connections. This percentage provided a gauge, using MATLAB directed search, on a random sample of real-valued weight configurations that was compared with an analogous percentage based on exhaustive search of integer-valued weight configurations using Maude. Comparison of the results of exhaustive, integer-valued search vs. random, real-valued search provides some assurance that the necessary restriction to integers for state-space searches did not limit the main findings (see Results).A directed search in MATLAB proceeds by perturbing, in turn, each weight change by an amount Δ, keeping all other weight changes unperturbed, and finding the error associated with each new set of weight changes. The error for any set is the sum of the absolute differences between the desired and actual model outputs  in the unconditioned, conditioned, and extinguished cases. If a perturbed set has a lower error than the current  set, then the perturbed set with the lowest error becomes the new current set, Δ is doubled, and the perturbation/evaluation process is repeated. If no perturbed set has a lower error than the current set, then the current set is unchanged, Δ is halved, and the process is repeated. The whole procedure continues until Δ falls below 10Calculations were performed on a Pentium PC with dual, 2.8 GHz processors and with 2 GB of RAM. Programs were run under the Windows XP operating system. Maude calculations were performed using Core Maude version 2.4 as part of the Maude for Windows package, downloadable for free from the MOMENT website: moment.dsic.upc.es. The Maude search of integer space for all 19,273 of the terminal states for Model 1 required 4 min 52 s, while that for all 8394 terminal states for Model 2 required 2 min 24 s. For both models all constrained Maude searches required less time. Directed searches of continuous space were performed using MATLAB version R2010a. Each individual MATLAB directed search for Model 1 and Model 2 required 0.7 and 0.9 s, respectively . These methods are directly scalable to larger networks, subject to computational resources.Model 1 is based on a scheme proposed by Lafenêtre, Chaouloff, and Marsicano . For example, BA1 represents the firing rate of the BA neuron in the No Fear pathway. Connection weights, which represent synaptic strengths, are denoted descriptively. For example, wLA1toBA1 is the weight of the connection from LA1 to BA1 in the No Fear pathway. Initially, all connection weights have absolute value 1 except wCStoLA1 and wCStoLA2, which are initially 0. All connection weights in Model 1 are modifiable except wUStoLA2 and wCEmtoPAG. The modifiable weights in Model 1 can undergo LTP and LTD, which are increases and decreases in weight absolute values, respectively (see previous subsection). The most straightforward scheme for weight modification in Model 1 is for all modifiable weights to undergo LTP and LTD during fear conditioning and extinction, respectively, but many departures from this scheme will be required as part of Model 1 analysis.The activity of any element in Model 1 is determined simply as the sum of its weighted inputs plus its bias. For example, the activity of CEm is computed as follows: CEm = bCEm + wBA2toCEm × BA2 + wITCmtoCEm × ITCm. Note that the CEm bias (bCEm) is 0 and wITCmtoCEm is negative. Except for CEm, all amygdaloid elements in Model 1 have initial spontaneous rates of 1. Note that US and CS are 0 under spontaneous conditions. The bias values for each element  and from CEm (wCEmtoPAG) are not modifiable. Unlike Model 1, wITCmtoCEm is not modifiable in Model 2, and neither is wITCltoITCm, because plasticity at those synapses has not been demonstrated experimentally . All other connection weights in Model 2 are modifiable. As in Model 1, weight modification in Model 2 involves LTP and LTD, which are increases and decreases in weight absolute values, respectively. In Model 2, wCStoLAi, wCStoLA1, wCStoLA2, wLAitoLA1, wLA1toITCl, and wLA2toITCl can undergo LTP during fear conditioning and LTD during extinction. In contrast, wILtoLAi, wILtoITCl, and wILtoITCm can undergo LTP during extinction with no weight modification allowed during fear conditioning.Initially, all connection weights in Model 2 have absolute value 1 except those from CS  and those from IL , which are initially 0. All elements in Model 2 except US, CS, CEm, PAG, and IL have non-zero initial spontaneous activity. Biases ensure that all non-zero initial spontaneous activities equal 1  alone can activate PAG. When fear conditioning has ended, different allowed weight modifications continue during extinction training until CS activity can no longer activate PAG. For both phases of learning, connection weights are modified one at a time, and every individual weight change is followed by evaluation of the effects of that weight change on the responses to CS of all neural elements. Weight modification terminates if PAG goes to 1 during fear conditioning or to 0 during extinction.In Model 2, IL takes value 1 during extinction training but is otherwise 0. Note that Model 1 lacks element IL. Because the Maude specification exhaustively explores the model state space, extinction occurs from every possible weight configuration that supports fear conditioning in either model. These conditioned configurations include those in which the weighs of the connections of inhibitory interneurons onto LA projection neurons have undergone LTP. This is consistent with experiment because LTP of inhibitory interneuron synapses onto LA projection neurons can accompany the LTP of thalamic and cortical synapses onto LA projection neurons that is associated with fear conditioning . Because the focus is on extinction, weight configurations that support fear conditioning are not analyzed, but those that support extinction following fear conditioning are analyzed in detail.The data on the effects of endocannabinoids on extinction present a puzzle. They indicate (see Introduction and Methods) that extinction is accompanied by LTD of the synapses onto LA projection neurons from inhibitory interneurons . As in the original LCM scheme, Model 1 is organized into Fear and No Fear pathways. In the Fear pathway, LA2 is excited by US, and can be excited by CS after fear conditioning. Then LA2 excites BA2, BA2 excites CEm, and CEm excites PAG, producing UR or CR. In the No Fear pathway, LA1 can be excited by CS or disinhibited by LAi1 after extinction training. Then LA1 excites BA1, BA1 excites ITCm, and ITCm inhibits CEm. Inhibition by ITCm can cancel excitation by BA2, thereby preventing the issue from CEm of commands to PAG for CR. Thus, Fear or No Fear behavior in Model 1 depends on the responses of its elements, and those depend on the weights of the connections between the elements. Model 1 was implemented by programs written in both Maude and MATLAB (see Methods), and a battery of identical weight adjustments were made in both. For all weight adjustments, model behavior was the same in both programs (not shown). While this does not prove that the Maude specification is free of bugs, it does reduce the odds that any of the results of the analysis have been corrupted by programming error.9 or 19,683 possible weight combinations. Of interest for the analysis are those combinations that produce extinction following fear conditioning, and especially those that produce extinction in conjunction with LTD of the weights of the connections of inhibitory interneurons LAi1 and LAi2 onto LA projection neurons LA1 and LA2, respectively. To determine whether wLAi1toLA1 or wLAi2toLA2 have undergone LTD during extinction training, their values immediately following fear condition must be recorded. This introduces two further weight parameters. Because each of wLAi1toLA1 and wLAi2toLA2 can either get more negative during fear conditioning  or stay at their initial value (−1) each of the two weight-record parameters has two possible values, so the total number of possible weight (and weight-record) combinations increases by a factor of 22 to 78,732.Analysis of Model 1 begins by assuming that the weights of all connections are modifiable except wUStoLA2 and wCEmtoPAG. In that case there are 9 modifiable weights, and each weight has 3 possible values , so there are 3In the original LCM scheme, extinction is thought to be mediated by potentiation of the No Fear pathway. This, in turn, is thought to occur through CB1-mediated LTD of the synapses of inhibitory interneurons onto the BLA projection neurons composing the No Fear pathway. That corresponds to LTD of wLAi1toLA1 in Model 1. Perhaps because LCM was designed to account for extinction in conjunction with LTD of inhibitory interneuron synapses, state-space search in Maude shows that many of the possible weight configurations are compatible with this condition. Of the total of 78,732 weight (including weight-record) configurations, 19,273 produce extinction following fear conditioning. Of those, 11,310 do so in conjunction with LTD of wLAi1toLA1.In the analysis of Model 1 . Disallowing modification of wITCmtoCEm reduces the total number of possible weight (and weight-record) configurations of Model 1 by a factor of 3 to 26,244. Of those, 777 weight configurations achieve extinction in conjunction with LTD of both wLAi1toLA1 and wLAi2toLA2 and with wLAi1toLA1 equal to wLAi2toLA2 both before and after extinction. Although plasticity of ITCm to CEm synapses may occur in the brain, its lack would not preclude extinction in conjunction with CB1-mediated LTD of inhibitory interneuron synapses, according to the LCM framework.A larger issue concerns plasticity of the synapses of CS inputs onto BLA projection neurons in the LCM scheme. According to LCM  the number of weight configurations that achieve extinction in conjunction with LTD of both wLAi1toLA1 and wLAi2toLA2 and with wLAi1toLA1 equal to wLAi2toLA2 before and after extinction. This number of compatible configurations depends not only on weight changes at the head of each pathway but also on weight changes in the middle and tail of each pathway. The original LCM framework does not seem compatible with this level of flexibility.4 × 22 or 324. Of these only 6 configurations achieve extinction in conjunction with LTD of both wLAi1toLA1 and wLAi2toLA2 and with wLAi1toLA1 equal to wLAi2toLA2 both before and after extinction.The LCM scheme plausibly assumes that potentiation (or depotentiation) can occur all along the two pathways, but changes occurring in the middle and tail of the pathways are secondary to changes occurring at the head. It is therefore of interest to explore the consequences of limiting weight changes to connections at the head of each pathway. Model 1 was again altered to disallow all changes to the downstream pathway weights wLA1toBA1, wBA1toITCm, wLA2toBA2, and wBA2toCEm. Only the following weight changes are still allowed: LTP of wCStoLA2 during fear conditioning, LTP of both wCStoLA1 and wCStoLA2 during extinction, LTP of both wLAi1toLA1 and wLAi2toLA2 during conditioning, and LTD of both wLAi1toLA1 and wLAi2toLA2 during extinction. At this stage there are only 4 modifiable weights in Model 1, reducing the total number of weight (and weight-parameter) configurations to 3In all 6 cases, both LA1 and LA2 respond to CS following extinction. This is consistent with findings that some neurons in LA respond to CS after extinction training  the number of configurations that achieve extinction in Model 1, and implies that CB1-mediated LTD of inhibitory interneuron synapses plays an important role in extinction (see Discussion).The analysis of Model 1 reveals that the LCM scheme , LTP of both wLAi1toLA1 and wLAi2toLA2 during conditioning, and LTD of both wLAi1toLA1 and wLAi2toLA2 during extinction. Now there are only 3 modifiable weights in Model 1, reducing the total number of weight (and weight-parameter) configurations to 3This result reveals that the parallel Fear and No Fear architecture is essential to the LCM scheme. Weight adjustments accomplish fear conditioning when CS responding is greater in the Fear than in the No Fear pathway, and accomplish extinction when CS responding in the No Fear pathway increases enough to balance that in the Fear pathway. Given the progressively more restrictive constraints applied in Model 1 analysis, balancing the Fear and No Fear pathways required that wCStoLA1 equaled wCStoLA2 and that wLAi1toLA1 equaled wLAi2toLA2 after extinction. Achievement of this Fear and No Fear balancing requires LTP of the synapses of CS inputs onto some LA neurons during extinction training, which could be verified experimentally. Yet Model 1 analysis reveals that the feature of the LCM framework that is critical for it to accomplish extinction in conjunction with GABAergic LTD is the existence of parallel Fear and No Fear pathways. Model 1 analysis also shows that certain LCM assumptions, including that CB1-mediated LTD affects some GABAergic synapses but not others, are not critical to it. The analysis suggests that experimental verification of Fear and No Fear pathway connectivity would be the most effective way to test the validity of the LCM framework .The LCM architecture hinges on the fact that BA can both excite CEm directly and inhibit CEm indirectly via ITCm (see Methods). However, lesion studies call into question the idea that BA is essential for all forms of fear conditioning and extinction. Although BA lesions disrupt context conditioning  projection neurons , and a battery of identical weight adjustments were made in both. For all weight adjustments, model behavior was the same in both programs (not shown). As for Model 1, this crosscheck strengthens confidence that the results of analysis of Model 2 have not been corrupted by programming error.9 or 19,683 possible weight combinations. Of critical importance for the analysis is to search for weight combinations (configurations) that achieve extinction in conjunction with LTD of the weight of the connection from LAi to LA1. For the purpose of determining whether wLAitoLA1 has undergone LTD during extinction training, an additional weight-record parameter is added to record its value immediately following fear conditioning. Because wLAitoLA1 can either get more negative during fear conditioning  or stay at its initial value (–1), the weight-record parameter has 2 possible values, so the total number of possible weight (and weight-record) combinations increases by a factor of 2 to 39,366.Analysis of Model 2 begins by assuming that the weights of all connections are modifiable except those from US , from the ITCs (wITCltoITCm and wITCmtoCEm), and from CEm (wCEmtoPAG). Therefore, as has Model 1, Model 2 has 9 modifiable weights, and each weight has 3 possible values , so there are 3Of the total of 39,366 weight (including weight-record) configurations in Model 2, 8394 produce extinction following fear conditioning. Of those, 4659 do so in conjunction with LTD of wLAitoLA1. These and other analysis results for Model 2  are constrained to be 0, and with LTD of neither wLA1toITCl nor wLA2toITCl, and with either LA1 or LA2 excited but neither inhibited by CS after extinction, then the number of Model 2 configurations that achieve extinction in conjunction with LTD of wLAitoLA1 is 0. However, if all the IL connection weights  are constrained to be 0, but the LA to ITCl weights (wLA1toITCl and wLA2toITCl) are free to undergo LTD during extinction training, then the number of Model 2 configurations that achieve extinction with either LA1 or LA2 excited but neither inhibited by CS and in conjunction with LTD of wLAitoLA1 rises to 49.These results can be taken as a prediction of Model 2. Given experimental findings that extinction occurs in conjunction with LTD of the synapses of inhibitory interneurons onto LA projection neurons, and that some LA projection neurons are excited by CS after extinction but none are inhibited, then Model 2 predicts that extinction must be associated with LTD of the synapses of LA neurons onto ITCl, or modification of the synapses of IL neurons onto inhibitory neurons in LA and ITC. This prediction comes with the caveat that Model 2 excludes BA. It also comes with the proviso that the weights of the connections from IL can be modified independently of one another (see below in this subsection). Whether or not they can be is a matter of conjecture, and it points up another way in which Model 2 differs from Model 1.Model 2 also differs from Model 1 in that it does not take a whole-pathway approach but, instead, assumes that the responses of elements along the pathway from LA to ITCl, and from ITCl to ITCm, can be modified by input from IL. Therein lays another conundrum. IL activates LAi, ITCl, and ITCm, and it promotes extinction by activating LAi or ITCm but it promotes the opposite (fear responding) by activating ITCl. It appears that the pattern of IL influence should be of critical importance to extinction in Model 2.Model 2 assumes that the synapses of IL neurons onto amygdala neurons are plastic, but it is not known whether they are (see Methods), much less whether or not they can be differentially modified. For that matter, it is not known whether synapses of CS inputs onto LA neurons, or of LA neurons onto ITCl, can be differentially modified. The most parsimonious assumption is that all synapses onto downstream neurons from the same subregion are plastically modified by the same amount, but making this assumption has serious consequences for Model 2. If the weights of the connections from CS are constrained to be equal (wCStoLAi = wCStoLA1 = wCStoLA2), and if the weights from LA projection neurons are constrained to be equal (wLA1toITCl = wLA2toITCl), and if the weights from IL are constrained to be equal (wILtoLAi = wILtoITCl = wILtoITCm), and if either LA1 or LA2 are excited but neither are inhibited by CS, then the number of configurations that achieve extinction in conjunction with LTD of wLAitoLA1 is 0. Interestingly, the requirement for LTD of wLAitoLA1 is not the culprit here because, given the same equality constraints and restrictions on LA1 and LA2 activity, the number of configurations that achieve extinction with or without LTD of wLAitoLA1 is again 0.These results show that if the weights of the connections from CS are constrained to be equal (wCStoLAi = wCStoLA1 = wCStoLA2), and if the weights from LA projection neurons are constrained to be equal (wLA1toITCl = wLA2toITCl), and if the weights from IL are constrained to be equal (wILtoLAi = wILtoITCl = wILtoITCm), and if either LA1 or LA2 are excited but neither are inhibited by CS, then Model 2 cannot achieve extinction in conjunction with LTD of wLAitoLA1. If the connection weights from CS can be differentially modified but not those from LA and IL, then Model 2 again cannot achieve extinction in conjunction with LTD of wLAitoLA1 and either LA1 or LA2 excited but neither inhibited by CS. However, if the connection weights from LA alone, or if those from IL alone, can be differentially modified, with the others  constrained to be equal, then there are 21 and 42 configurations, respectively, that achieve extinction in conjunction with LTD of wLAitoLA1 and either LA1 or LA2 excited but neither inhibited by CS. Thus, Model 2 predicts that, in preparations in which BA has been lesioned, extinction in conjunction with LTD of the synapses of inhibitory interneurons onto LA projection neurons can occur only if the synapses of LA projection neurons onto ITCl, or those of IL neurons onto LA inhibitory interneurons, ITCl, and ITCm, are not only plastic but differentially modifiable.Differential modifiability means that plastic changes in synapses onto ITCl of LA projection neurons that receive input from inhibitory interneurons can be different from those of LA projection neurons that do not receive such input. It also means that changes in synapses of IL neurons onto ITCl can be different from those onto ITCm or onto LA inhibitory interneurons. Model 2 analysis suggests that the answer to the conundrum of IL innervation is that synapses of IL neurons onto LA inhibitory interneurons, ITCl, and ITCm can all change together by equal amounts, but only if synapses of LA projection neurons onto ITCl can change by unequal amounts. The reverse is also true: synapses of LA projection neurons onto ITCl can change together by equal amounts but only if synapses of IL neurons onto LA inhibitory interneurons, ITCl, and ITCm can change by unequal amounts.disallowed in Model 2. This would correspond to elimination of CB1-mediated LTD in amygdala. With connection weights from CS, LA, and IL all differentially modifiable, and either LA1 or LA2 excited but neither inhibited by CS after extinction, the number of configurations that achieve extinction without LTD of wLAitoLA1 is 303. For direct comparison, a previous search reveals that, with connection weights from CS, LA, and IL all differentially modifiable, and either LA1 or LA2 excited but neither inhibited by CS after extinction, the number of configurations that achieve extinction with LTD of wLAitoLA1 is 957. These results show that LTD of inhibitory interneuron synapses is not only compatible with extinction, but it actually increases  the number of weight configurations that achieve extinction while preserving some LA responses to CS in Model 2 (see Discussion).While the results may go some way toward solving the puzzle of IL innervation, the question remains of how CB1-mediated LTD of the synapses of LA inhibitory interneurons onto LA projection neurons actually contributes to extinction, since such LTD would seem to oppose extinction. This issue is approached using one more search. To make it robust, all equality constraints are dropped . Then LTD of wLAitoLA1 during simulated extinction is Experiments to test the essential features of the LCM and PQL frameworks could employ techniques similar to those that have provided the current body of data on amygdaloid connectivity and plasticity (see Methods), but these would have to be augmented with techniques for accurate assessment of connectivity such as those based on spike-triggered averaging between pairs of extra- and intra-cellularly recorded neurons (Hempel et al., Testing the features that are critical for the PQL framework to account for extinction in conjunction with GABAergic LTD would involve demonstrating the differential modifiability of LA or IL neuron synapses onto postsynaptic neurons, in the absence of influence from BA. Showing that the synapses onto ITCl neurons of at least some LA neurons that receive inhibitory interneuron input undergo LTD during extinction, while those of some other LA neurons that do not receive inhibitory interneuron input do not undergo LTD during extinction, would provide evidence of differential modifiability. Similarly, showing that some synapses from IL onto ITCm undergo LTP while others from IL onto ITCl do not undergo LTP during extinction would also provide evidence of differential modifiability. However, the analysis shows that the weight configurations that are consistent with extinction, along with GABAergic LTD and some preserved LA responding to CS, also encompass a wide range of configurations of the strengths of synapses of LA and IL neurons onto postsynaptic neurons, so the specific differences outlined here are examples of the many synaptic strength change differences that would provide support for the differential modifiability that is critical to the PQL framework.The models analyzed in this study are based on a rich dataset, but the main question addressed in their analysis was prompted by the seminal work of Marsicano et al.  who showModel 1 (based on the LCM scheme; Lafenetre et al., The main finding of the analyses is that CB1-mediated LTD of the GABAergic synapses of LA inhibitory interneurons onto LA projection neurons is not only compatible with extinction, but it increases the number of connection weight configurations that achieve extinction and also preserve some fear memory in amygdala, given model structures and other constraints based on experimental findings. By showing that the number of such configurations is increased twofold for Model 1 and threefold for Model 2, the analysis identifies preservation of fear memory during extinction as a highly likely function of CB1-mediated GABAergic LTD.The increases in numbers of model weight configurations are important for two reasons. The first concerns the relative lack of knowledge concerning mechanisms of neural plasticity in amygdala, compared with the relative abundance of knowledge concerning amygdaloid anatomy and physiology. We know that the amygdala brings about fear conditioning and extinction by making synaptic strength changes but, because we are still unsure about how these synaptic strength changes are made, we could justifiably assume that, given well-established models based on known amygdaloid connectivity, the outcome associated with the most numerous synaptic strength configurations is the one most likely to occur in the real amygdala. Because CB1-mediated GABAergic LTD doubles in Model 1, and triples in Model 2, the number of weight configurations that achieve extinction along with preservation of fear memory in those well-established models, we can conclude that preservation of fear memory in amygdala during extinction is a highly probable function of CB1-mediated GABAergic LTD.The second reason that increases in numbers of weight configurations are important is that the actual plastic mechanisms that bring about fear conditioning and extinction may have substantial random components. Two recent models that suggest possible adaptive mechanisms employ reinforcement signals and/or stochasticity (Krasne et al., In both Model 1 and Model 2, elements could be interpreted either as single neurons or as groups of neurons all having the same connectivity and behavior. While the former interpretation suggests how individual neurons might respond, the latter interpretation suggests how extinction of fear conditioning might result from changes in the strengths of the influences that various cortical and amygdaloid subregions have on one another. Obviously, brain subregions are composed of many more than one or two neurons. Increasing the number of model elements in each subregion would geometrically increase the number of weight configurations that produce any desired outcome, but would not change the relative numbers of those that produce different outcomes if element numbers were increased uniformly. Specifically, uniformly increasing the number of each element in Model 1 or Model 2 would increase the number of weight configurations that produce any particular outcome, but the configurations that achieve extinction and some fear memory preservation with LTD of inhibitory interneuron synapses would still outnumber those that do so without it.An important distinction between the two models is that retention of CS responses in LA emerges automatically in Model 1, as a consequence of its balanced architecture and parsimony assumptions, while retention of CS responses in LA pertain only for a subset of compatible configurations in Model 2. Considering the differences in the LCM and PQL schemes, and so in Model 1 and Model 2, it is remarkable that CB1-mediated LTD of BLA inhibitory interneuron synapses actually increases the options that both Model 1 and Model 2 have available to learn to inhibit a previously conditioned fear response and to preserve some LA responding to CS. Both models, and the LCM and PQL frameworks on which they are based, depend sensitively on specific patterns of connectivity and plasticity, but both also show how extinction of fear conditioning requires an interaction between several amygdaloid subregions and cortical and thalamic inputs to the amygdala.The LCM and PQL frameworks (Pare et al., The computational approach taken here is fundamentally different from that taken in typical modeling studies in neurobiology, which employ imperative programming and are limited to simulation. This approach is based on declarative programming, which readily offers options in addition to simulation such as analysis of model behavior using temporal logic and state-space search (Huth and Ryan, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mechanical ventilation can be perceived as a treatment with a very narrow therapeutic window, i.e., highly efficient but with considerable side effects if not used properly and in a timely manner. Protocols and guidelines have been designed to make mechanical ventilation safer and protective for the lung. However, variable effects and low compliance with use of written protocols have been reported repeatedly. Use of explicit computerized protocols for mechanical ventilation might very soon become a "must." Several closed loop systems are already on the market, and preliminary studies are showing promising results in providing patients with good quality ventilation and eventually weaning them faster from the ventilator. The present paper defines explicit computerized protocols for mechanical ventilation, describes how these protocols are designed, and reports the ones that are available on the market for children. Mechanical ventilation is a sophisticated technique that can keep alive the most severely ill patients; however, it can simultaneously damage the lungs and unfortunately generate unwanted complications . By analIn addition, expertise and human resources are not always available to make sure that patients receive the best ventilation everywhere and all the time. The ability to make timely adjustments of the ventilator according to the patient's condition, without much inter-caregiver variability, would certainly improve the safety and efficiency of mechanical ventilation especially when resources and expertise are not at the bedside 24 hours per day. In the present paper, we will define an explicit computerized protocol, describe how these protocols are designed and developed currently, report the ones that are available on the market for children, and propose some considerations for future developments in this field.A protocol is a document that is designed to guide decisions regarding diagnosis, management, and treatment of specific medical situations. The protocol is based on the medical knowledge acquired from physiological studies, expertise, or evidence and can be generated by individuals or by consensus obtained from a group of physicians or experts. Protocols often are not precise enough to generate a decision at the bedside in a specific situation and thus significant inter-clinician variability in their application may exist.An explicit protocol is designed to provide enough details to generate patient-specific therapy instructions that can be performed by different clinicians with no inter-clinician variability [iability . ImportaAn explicit computerized protocol (ECP) is an explicit protocol supported by computer science to apply the instruction for a given patient at a given time. ECP might be in a laptop or integrated into the ventilator or monitoring device. The medical knowledge is usually implemented in the ECP through "if... then" rules. For example: if the SpO2 is < 88%, then increase FiO2 by 10%. The rules can be more complex and based on a validated physiological equation, such as the Otis equation [equation  as used clinical decision support system (CDSS) is defined as an ECP that uses two or more items of patient data to generate case-specific recommendations through rules that are only in open-loop [A rule can be in an open or closed loop. The rule is an open-loop rule if it results in a therapeutic or diagnostic recommendation displayed on the screen of a device for which caregivers can agree whether to accept the recommendation. According to the previous definitions, a pen-loop .As a step further, a rule that provides a recommendation of modification of the ventilator setting and implements this modification without caregiver intervention is a rule in closed-loop. Currently most of the ECPs used commercially for mechanical ventilation involve both open and closed-loop ventilation rules. A closed-loop ECP is arbitrarily designated as an ECP with at least one rule in closed-loop Figure .The human brain has a limited ability to incorporate data and information in decision making and human memory can simultaneously retain and optimally utilize only seven plus or minus two data constructs . The amoA multidisciplinary approach is needed to generate an ECP; the team should include clinical expert(s) in mechanical ventilation to generate the knowledge and validate ECP in a clinical environment, computer scientists to design the ECP platform, biomedical engineers to implement the ECP into medical devices (monitors and/or ventilators) and test ECP robustness and reliability, and industry to finalize a product that will receive a European Community marking (CE mark) and a U.S. marking (Food and Drug Administration (FDA)) approval.2/FiO2 rule, caregivers modify FiO2 according to SpO2 routinely. An ECP will recommend (open-loop) or do (closed-loop) in the same way, as soon as a valid SpO2 is available. The ECP also will define how often the FiO2 can be changed, the amplitude of change, and add additional rules: for example, define what will occur if FiO2 is 100% and SpO2 still below normal range.The basic component of an ECP is a medical knowledge-based rule. In our clinical practice, we continuously apply rules. If we take the previous example of the SpO2/FiO2 have already been developed for neonates and children and need further clinical validation [The knowledge needed to develop an ECP able to manage the course of mechanical ventilation in any ICU patient is vast and we all know that "the devil is in the details." This knowledge is based on published work on respiratory physiology, clinical observational studies to describe current practice ,13, conslidation ,15.To shorten development and validation times, one option would be to follow the same development and validation process used in aviation by using a simulated flight environment . Medical ECP would need virtual patients with realistic physiological and pathological behaviors for developing and validating ECPs before clinical trials. Several teams are already working on such platforms, although none are currently commercialized for this purpose -20.The five technical components Figure  of an EC2/FiO2 rule already described, the input data is patient SpO2, the control unit analyses SpO2 and selects the rule that corresponds to SpO2 value , or no change if SpO2 is in normal range), the output data is the FiO2 suggestion displayed on a screen (open-loop) or a setting modification on the respirator (closed-loop).In the SpO2 (ETPCO2) into mean values during a 2-minute period. With IntelliVent™ , the ETPCO2 used for the minute ventilation closed-loop is the second highest breath-by-breath ETPCO2 with enough quality index during the last 10 breaths [PCO2 is not always a good surrogate for PaCO2 especially during the acute phase of illness with high dead space. Such situations must be detected by the ECP to avoid any misinterpretation of the input data.The input data, whether entered manually by the clinician or captured by a medical device, needs to be processed to discard artefacts and to be clinically relevant before being sent to the control unit . The key breaths . On the PCO2 [2 and PEEP management according to SpO2 [2 reach threshold values for a certain length of time (mimicking an extubation readiness test) [if... then ..." rules, but some ECPs have been developed using fuzzy logic [The control unit receives clinical information from the patient (input data) and "transforms" this information into orders (output data). The basic structure for processing information is rule-based (see above). All potential paths and situations should be addressed to lead to specific instructions. It usually requires several set of rules to manage two to three input data each. For example, a first set of rules could be for pressure support management according to tidal volume, respiratory rate, and ETPCO2 , a secon to SpO2 , and anoss test) . Rules azy logic , i.e., izy logic ,25. DespOutput data can be recommendations to the caregivers suggesting new ventilator settings, specific orders such as "patient ready for separation from the ventilator," or ventilator settings being automatically adjusted. During the development phase of an ECP, output data are usually recommendations (open-loop). After extensive testing, some rules or sets of rules can be switched to close the loop.A simple, attractively presented, and intuitive user interface is crucial to facilitate the understanding of the ECP decision process and for knowledge transfer at the bedside . IdeallyAs mentioned above, use of a virtual patient mimicking patient-ventilator cardiorespiratory interactions also is important for the development of ECPs. As for aircraft autopilot, a virtual patient may help "debug" the very first ECP versions but also aid understanding the complex interactions between rules and achieving preclinical validation. In addition such virtual patients, which should ideally be incorporated in the medical device , might be more efficient in training and teaching the eventual users Figure . CurrentSeveral barriers exist to the development of ECPs: 1) We do not generate enough medical knowledge in mechanical ventilation and most of the time ECPs are targeting a relatively small and regional scientific community. This can be improved by promoting multicenter international collaborations along the lines of the Pediatric Acute Lung Injury and Sepsis Investigators Network (PALISI), the European Society of Pediatric Intensive Care Medicine and Collaborative Critical Care Research Network (CCCRN) ,27; 2) ISeveral barriers also exist to the acceptance and implementation of ECPs. These barriers include the lack of awareness, lack of familiarity with the protocol, lack of agreement, lack of demonstrated safety and efficacy, lack of known improved outcome, lack of ability to overcome the inertia of previous practice, protocol-related barriers , environment-related barriers (new resources or facilities not accessible) . Among a2 by regulating the negative pressure of an iron lung ventilator in poliomyelitis patients [2 tightly in order to avoid hypo/hyperoxemia and their related side effects [2 adjustment to keep the PaO2 obtained from an indwelling umbilical artery electrode at 10 kPa. The time spent by the neonates with PaO2 at ± 1 kPa of the target PaO2 was 75% with closed-loop control FiO2 compared with 45% with manual adjustment of FiO2. But the invasiveness of the PaO2 monitoring precluded the development of this ECP. Claure et al. conducted a research program designed to develop closed-loop FiO2 adjustment using SpO2 [2 adjusted by caregivers and the other by an automated system, in random sequence. Automated FiO2 adjustment improved maintenance of the intended SpO2 range, and led, significantly, to reduced time with high SpO2 and more frequent episodes with SpO2 between 80% and 86% [Saxton and Myers reported the first ECP to adjust the end tidal PCOpatients . Pediatr effects ,30. Dugd effects  reporteding SpO2 ,33. Thes and 86% . This EC2. These thresholds are defined within acceptable limits as established by a large panel of pediatric intensivists [2 are not automatically adjusted by SmartCare/PS™ but are part of the recommended criteria to initiate a spontaneous breathing trial (SBT).SmartCare/PS™  is an ECP for the closed-loop control of pressure support ventilation. This ECP operates without the need of caregivers intervention but under their supervision Figure , the founsivists ; 2) a stnsivists . In a sinsivists . A multinsivists ,36. The 2 in adaptive support ventilation (ASV). IntelliVent™ manages, with (open-loop) or without the need of caregivers intervention (closed-loop), the four following weaning steps: 1) switch from control ventilation to spontaneous breathing (specific to ASV mode); 2) automatic adaptation of the pressure to maintain the patient in a range of respiratory rate, end tidal PCO2 and SpO2; 3) an automated SBT when children reach minimum ventilation support; and 4) a timer that shows SBT duration  is an ECP for the automated control of minute volume, PEEP, and FiOand SpO2 . Intelliand SpO2 . The majand SpO2 ,38. The PCO2, SpO2, blood gases, and an automatic analysis of chest x-ray. The output data will be ventilation settings modifications and specific recommendations. Several preliminary studies are ongoing to refine the input data [A research program designed to develop and validate an ECP for the management of mechanical ventilation in children with acute lung injury is being conducted by a working group of the PALISI network in collaboration with CCCRN. This program is based on the following principles: the ECP will be an adaptation of a protocol already developed in adults (ARDS Network); the applicable type of ventilation will be pressure control mode and high frequency oscillatory (HFOV) mode. The computerized protocol will start at initiation of ventilation. The basic structure of the protocol will be that of a closed-loop system with the initial stage of design being open-loop, even for HFOV. The input data will include ventilator data, ETput data . The ECPput data  and an EConsidering the current context in the PICU , ECP for mechanical ventilation will very soon be a "must." Open-loop and decision support systems allow too much room for inter-physicians variability and may not achieve the goal of providing the best possible care. As in many other fields, safety and quality will be achieved by closing the loop but allowing physicians to retain the role of supervisor, most directly involved with the most severe or atypical patients. Several closed-loop systems are already on the market, and preliminary studies have shown promising results in providing patients with good quality ventilation and eventually weaning them faster from the ventilator.PJ has a research salary from the "Fonds de Recherche du Québec - Santé" in respiratory critical care. PJ's research on virtual patient (SimulResp) is funded by a grant from the "Natural Sciences and Engineering Research Council of Canada." The clinical research conducted by PJ on SmartCare™ was funded by the Research network of the "Fonds de Recherche du Québec - Santé" and a Ventilator S1 Evita 4 was provided for research purposes by Draeger Medical. The clinical research conducted by PJ on IntelliVent™ was funded by a grant from Hamilton Medical, and a respirator S1 was provided for research purposes by Hamilton Medical. PJ was invited twice to present the results of the clinical research on IntelliVent™ at international meetings organized by Hamilton Medical. PJ also had a respirator Servo i, provided for research purposes by Maquet Medical.MW was the director of the Research and Development Department of Hamilton Medical. MW is cosharing a patent on IntelliVent™ (WO/2007/085110 and WO/2007/085108).PH's salary came from grants from the Research network of the "Fonds de Recherche du Québec - Santé" and the "Natural Sciences and Engineering Research Council of Canada."PJ, PH, and MW drafted the manuscript. All authors read and approved the final manuscript.


To investigate the effect of pelvic floor Neuromuscular Electrical Stimulation (NMES) Therapy in improving endometrial thickness in women with thin endometrium.41 patients undergoing assisted reproduction with a thin endometrium  were recruited and advised to go for a pelvic floor NMES in frozen-thawed embryo transfer cycle. PHENIX Neuromuscular Electrical Stimulation Therapy System was used according to the manufacturer's recommended protocol for 20 to 30 minutes of intermittent vaginal electrical stimulation on the treatment days.A total of 20 and 21 were included in the NMES and non-NMES groups respectively. 12 out of 20 (60%) patients developed endometrial thickness equal to or more than 8 mm after the NMES therapy, which was the primary outcome. The mean thickness of endometrium before and after was respectively 5.60 mm (0.82 mm) and 7.93 mm (1.42 mm) in the therapy group versus 5.50 mm (1.00) and 6.78 mm (0.47) in the control group; the difference was statistically significant (P = 0.002). There was higher pregnancy rate in the NMES group (42% versus 35%) but the difference was not statistically significant.Neuromuscular Electrical stimulation therapy may be effective for the patients with a thin endometrium. Further studies are needed to investigate its effectiveness. By the time of ovulation, many assisted reproductive technologies (ART) practitioners like to see the endometrium at least 8 mm thick. An adequate endometrium thickness and appearance play a key role in the implantation of embryo and achievement of pregnancy -3. ThereUp to date, no study has evaluated the use of Pelvic floor neuromuscular electrical stimulation (NMES) in reproductive medicine. NMES has been used in urogynecology and obstetrics during pregnancy and postpartum period. There is a strong evidence for its effectiveness for treatment of stress urinary incontinence (SUI), pelvic pain, sexual dysfunction, low back pain and constipation in clinical practice . NMES isThe study was reviewed and approved by the Ethical Review Board of the Memorial Hospital of Sun Yat-Sen University. Written informed consent was obtained from all patients.This study was an observational and prospective study. It was carried out in the reproductive center of the Memorial Hospital of Sun Yat-Sen University  from December 2009 to April 2011. Women were eligible for the study if they were 20 to 39 years old, if they were recorded at least 2 previous assisted reproductive treatment cycle failures in which the optimum endometrium thickness was ≤ 7 mm and were undergoing frozen-thawed embryo transfer in hormone replacement therapy (HRT) cycle.Women were excluded from participation if they had a medical history that included pelvic cancer, severe endometriosis, Asherman's syndrome, adenomyosis, or congenital uterine anomaly, use of an intrauterine device, vaginitis, neurologic disorders, systemic diseases, hypertension or diabetes mellitus. Exclusion based on physical examination by the nurse practitioner  occurred if women had vaginal wall prolapse, skin breakdown around the peri-anal region, rectal or vaginal bleeding, or absolute contraindications for pelvic neuromuscular electrical stimulation such as complete denervation of the pelvic floor (will not respond), dementia, cardiac pacemaker, unstable or serious cardiac arrhythmia, unstable seizure disorder, pelvic pain and painful hemorrhoids.A total of 41 women with a history of infertility and who had a thin endometrial  in their previous treatment cycles were recruited into this study. This threshold was chosen based on studies in the literature reported reduced implantation and pregnancy rates when endometrial thickness was 7 mm and less,21,22. AAll the women received previously a standard regime of the GnRH agonist with the use of (Triptorelin) and gonadotropins . Human chorionic gonadotropin (10.000 IU) was administered intramuscularly when lead follicles were ≥ 18 mm in diameter, and there were at least three follicles 16 mm in diameter (IVF/ICSI). 30-36 h after injections of HCG, oocyte was retrieved guided by transvaginal ultrasound. They had a fresh embryo transfer cycle failure and at least one frozen-thawed embryo transfer (FET) cycles on natural cycle, ovulation induction cycle or on artificial cycle without success.Estradiol valerate (E2) 2 mg/day  was commenced orally on day 3 of menstruation cycle without prior pituitary suppression and continued for 5 days. After 5 days (day8), the dose of progynova was changed to 2 mg bid for 3 days. On day10 of menstruation cycle, endometrial thickness was assessed by ultrasound. If the endometrial thickness was ≤8 mm, the dose of progynova was increased to 3 mg bid for 3-5 days, after which the endometrial thickness was evaluated for the second time. If the endometrial thickness was still ≤8 mm, the dose was increased to 5 mg bid until the endometrial thickness reached 8 mm. If the endometrium thickness reached 8 mm, the luteal phase support was started (D0) by providing progesterone  to all patients. If the endometrial thickness failed to reach 7 mm after 20 days of progynova use, the cycle was usually cancelled and restarted in a new protocol , but the final decision was given to patient to go forward for the embryo transfer or cancel the cycle. All the embryos transfer were performed on D3 (72 h after beginning progesterone supplementation).The women were told to lie down in the supine position, lithotomy position, on an examination bench with the legs extended. The nurse practitioner  examined all participants prior to therapy. The physical therapist instructed them how to contract and relax the pelvic floor muscles. A digital vaginal exam was used to help the physical therapist make sure whether the patient successfully contracted the right muscles or not and determine the degree of muscles strength by contraction or compression of examiner's fingers. Each contraction should involve a concentrated effort to get maximum tightening. Patients were instructed to contract only the pelvic floor muscles. If they feel their abdomen, thighs or buttocks tightening then they were asked to relax and aim just for the pelvic muscles by using a less intense muscle contraction. The women were encouraged through detailed and explicit instructions and directions by the physical therapist.The patient still in supine position with one cushion under the head and the legs opened up, both of the legs forming 40° approximately. Two other cushions of about 5-10 cm height were inserted under the knees to prevent the thigh muscles from participating in contraction of pelvic muscles. An ointment  was applied to the tip of the probe before its insertion into the vagina. The probe was inserted to about 6-8 cm deep. The vaginal probe was fixed on the probe box, situated between the two cushions, to avoid squeezing it out of the vagina. All the women had their own vaginal probe . Adhesive electrodes were placed on the skin outside the vagina (on the women's thigh). Vaginal probe was kept in vagina and electrical stimulation was initiated.PHENIX Neuromuscular Electrical Stimulation Therapy System USB 4  was used according to the manufacturer's recommended protocol for 20 to 30 minutes of intermittent vaginal electrical stimulation on the treatment days. Selected parameters included biphasic intermittent current, frequency 40 Hz, pulse width 250 us, and current intensity between 0-120 mA with individually adapted on-off (duty) cycles on the basis of each woman's ability to hold a voluntary contraction. On time ranged from 3 seconds to 10 seconds, and off time from 0 second to 30 seconds. For biofeedback therapy, computer graphs were used to help them locate the pelvic muscles. All patients were encouraged to tolerate as high intensity as possible to get a contraction.After the first therapy, the women were instructed to practice exercises at home. The exercise program consisted on to hold each contraction for 1 second and then to relax the muscles for 5 seconds. This sequence was repeated continuously for 15 minutes. The women were told to do the exercise in lying position so that there is little stress on the muscles. Two training repetitions were performed in the presence of the physical therapist.Endometrial thickness was measured at its thickest part in the longitudinal axis of the uterus before (day9 or day 10) and after NMES therapy  in the two groups. The endometrial pattern visualized was designated as a multilayered or a non-multilayered endometrium. The measurements were performed by one investigator (C.Z) using a computerized vaginal ultrasound with an integrated pulsed Doppler vaginal scanner . CZ was blind to the conditions of the patients.Mean and standard deviation were used for description of variables. A student's t test was used to compare the difference between groups. Comparison of before and after treatment endometrial thickness in the NMES group was performed using Non-parametric Wilcoxon test. Statistical comparison for percentage was carried out by chi-square. All the statistical analyses were carried out using SPSS software  version 16.00. A p value < 0.05 (two tailed) was considered to be statistically significant.2 (range 16.60-27.20). The women in this study had mixed diagnosis. 24/41(58.5%) had primary infertility. 19 women out of 41 were diagnosed polycystic ovary syndrome (PCOS). 31 had tubal occlusion, and 29 with male factor. The mean age and endometrial thickness at baseline (day9) of the two groups showed non-significant difference   which was statistically significant. The majority of patients in the NMES group showed an improvement in the endometrial thickness. 12/20(60%) patients developed endometrial thickness equal to or more than 8 mm after NMES therapy, which was the primary outcome. 3 out of 20 did not respond to the therapy(no change was observed in their endometrium thickness during the cycle)."Regarding the clinical pregnancy rate, 42% 8/19) and 35% (7/20) conceived in the NMES group and nonNMES group respectively. There was no significant difference between the NMES group and nonNMES group (Table 9 and 35%2 women (1 in the NMES group and 1 in the nonNMES group) had embryo transfer cancelled because of a poor embryo quality.NMES is the application of electrical stimulation to a group of muscles through electrodes placed on the skin. It is the elicitation of muscle contraction using electric impulses. The impulses are generated by a device and delivered through electrodes on the skin in direct proximity to the muscles to be stimulated. The impulses mimic the action potential coming from the central nervous system, causing the muscles to contract. It was primarily used by physical therapists to help restore function to injured muscles. Now the utilization of NMES is a routine practice in urology (incontinence) and in gynecology & obstetrics  all over the world -25. The Little information is available regarding the exact mechanisms by which NMES exerts its function on the process of angiogenesis and vascularization in the endometrium. However, based on the data in the literature on the importance of angiogenesis and vascularization for a receptive uterine, we believe that by stimulating uterine smooth muscle to repeated contraction and relaxation, there is an increased in blood supply towards the whole endometrial and the sub-endometrial regions that leads to peripheral tissue trophicity. NMES provides a passive contraction that increases awareness of pelvic floor muscles contractions in general. The etiology of thin endometrium is complex, multifactorial and remains unclear. High blood flow impedance of uterine radial arteries and decreased in vascular endothelial growth factor (VEGF) expression are associated with poor endometrial growth . The inaWith this method, the endometrial thickness was significantly increased and we achieved a pregnancy rate as high as 42%(8/19). Our results are with agreement to those that reported a positive correlation between endometrial thickness and pregnancy rate,5,21,22.In this study, only 3 women did not respond at all to NMES. The uniqueness of this study is that it demonstrated a speedy and significant increase in endometrial thickness. NMES was performed only 3 to 4 times for 20-30 min in the subsequent cycle (once per day consecutively). The number or amount of time of therapy needed to show improvement varies from person to person. NMES therapy was stopped as soon as the endometrial thickness reached 8 mm. Four times therapy was the maximum we prescribed to the women in this study. Other benefits include NMES being safe, it is also a noninvasive technique and it appears as a promising alternative for managing patients with thin endometrium. No known serious adverse effects have been reported by the subjects.Pelvic floor NMES showed a positive effect on the endometrium thickness and the pregnancy rate, but there may be too few subjects in the present study to draw a firm conclusion. Further studies are doubtless needed in this area to investigate this issue.The authors declare that they have no competing interests.MMABD carried out the acquisition of data, analysis and interpretation of data and writing of the manuscript. CZ has been involved in the ultrasound examination and in drafting the manuscript and revising it critically for important intellectual content. SC, physical therapist, carried out the pelvic floor neuromuscular electrical stimulation therapy. DY conceived of the study, and participated in its design and coordination, helped to draft the manuscript and have given final approval of the version to be published. All authors have read and approved the final version of the manuscript.


During acute inflammation, functioning lymphatics are believed to reduce edema and to provide a transiting route for immune cells, but the extent at which the dermal lymphatic remodeling impacts lymphatic transport or the factors regulating these changes remains unclear. Herein we quantify the increase in lymphatic endothelial cells (LECs) and examine the expression of pro-angiogenenic and lymphangiogenic factors during acute cutaneous hypersensitivity (CHS). We found that LECs actively proliferate during CHS but that this proliferation does not affect the lymphatic vessel density. Instead, lymphatic remodeling is accompanied by lymphatic vessel leakiness and lower ejection of lymph fluid, which is observed only in the proximal lymphatic vessel draining the inflamed area. LECs and the immune cells release growth factors and cytokines during inflammation, which impact the lymphatic microenvironment and function. We identified that FGF-2, PLGF-2, HGF, EGF, and KC/CXCL17 are differentially expressed within tissues during acute CHS, but both VEGF-C and VEGF-D levels do not significantly change. Our results indicate that VEGF-C and VEGF-D are not the only players and other factors may be responsible for the LECs proliferation and altered lymphatic function in acute CHS. The processes of angiogenesis and lymphangiogenesis are recognized to play critical roles in cancer progression, metastasis, and many chronic diseases, but also serve non-pathological roles in the normal progression and resolution of inflammation . During Because the vascular endothelial growth factor (VEGF) family of ligands and receptors are known to promote both hemovascular and lymphatic proliferation, several investigations have focused on their role in vascular remodeling during acute and chronic skin inflammation –7. VEGF-To investigate the role of lymphatic function in acute inflammation, we induced acute CHS reactions in normal mice and longitudinally imaged the dermal lymphatics afferent, and the contractile, conducting lymphatics efferent to the regional draining lymph node using NIRF lymphatic imaging. We evaluated the population of cells in inflamed skin, using flow cytometry and histology, as a function of time after induction of acute CHS reaction and assessed lymphatic endothelial cell (LEC) proliferation using a method of incorporation to directly assess proliferation quantitatively. We also assessed the tissue protein expression of growth factors associated to angiogenesis, lymphangiogenesis, and inflammation.Our results show that the LECs proliferate in early onset of CHS, but that this proliferation is not sustained from day 4 to 7 post-CHS. We also determined that the efferent lymph transport is impaired in early CHS as shown by a reduction in lymph ejection, but not by a reduction in lymphatic vessel contractility in both the inflamed and contralateral untreated sides of animals. We then identified several growth factors and inflammatory molecules to be differentially expressed in early inflammation as compared to non-treated animals and animals at later stages of the CHS reaction. VEGF-C and VEGF-D expression were not significantly changed during the course of the CHS reaction. These data may indicate that multiple growth factors other than VEGF-C and VEGF-D work in concordance with VEGF family to promote LECs proliferation, regression, and mediate lymph flow for resolution of CHS inflammation.This study was performed in strict accordance with animal use protocols approved by The Animal Welfare Committee at the University of Texas Health Science Center . Mice were euthanized if they met any early removal criteria  to limit suffering.CHS reactions were induced in wild type C57B/6 mice. For all studies, mice were sensitized by topical application on the shaved upper back of 50µl 2% oxazolone (4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one)  in acetone: olive oil (4:1 vol/vol). Five days after sensitization (day 0), mice were challenged with 100µl 1% oxazolone in solution on the shaved lower back. To assess cell proliferation, 200µg of EdU (5-ethynyl-2’-deoxyuridine)  in 200µl PBS was injected IP in challenged mice and in unchallenged mice every 24 hour for 3 days from day 0. All animal studies were approved by the Animal Welfare Committee at the University of Texas Health Science Center.Mice were humanly euthanized at day 0, 3, or 7 days post-CHS challenge. Skin tissue was removed and placed in 0.1% trypsin in HSBS with 1% FBS for 30 minutes at room temperature. The dermis was then separated from the epidermis using forceps and scissors and minced in 1mm sections before being incubated in 1mg/ml collagenase/dispase  in HSBS with 5% FBS at 37°C for 45 minutes on a shaking platform. After incubation, the dermis samples were placed on ice. The dermis was passed through a 70µm tissue sieve using a rubber plunger. The sieve was rinsed multiple times with PBS. The collected cell mixture was then centrifuged to form a cell pellet. Cells were washed once with staining buffer (5% FBS in HSBS with 5mM EDTA) and then resuspended in staining buffer with of FC block  before incubated with fluorescence conjugated primary antibodies against CD45 (clone 30-F11) , CD31 (clone 390) (Ebioscience), podoplanin (clone eBio8.1.1) (Ebioscience), and Calcein AM 10µM (BD Bioscience) for 30 minutes on ice. We selected the markers CD31 and podoplanin to identify LECs . Th. Th12]. At day 3 after onset of CHS, we observed similar rates of contractile lymphatic propulsion of 5.6 ± 2.1 pulse/minutes, but with a consistent “reflux” of ICG-laden lymph . A typicThus, while the rates of lymphatic contractility were not affected during CHS treatment, lymphatic “reflux” results in less efficient transport of lymph in the inflamed skin lymphatic vessels, resulting in lymphatic insufficiency, with subsequent recovery.We sought to identify which pro-angiogenic and pro-lymphangiogenic factors were present in mice dermal tissues undergoing active LEC proliferation and in which lymphatic function was disrupted. To address the local environment of the lymphatic vasculature, we interrogated the concentration of protein in the dermis and excluded the epidermis which lacks lymphatic vessels. Many growth factors and cytokines are produced by cells in the epidermis (such as keratinocytes), but these factors need to infiltrate the dermis to act directly on LECs. Thus, by assessing the dermis, we measured the growth factors within the local environment of the lymphatic vessels. We performed a milliplex assay for 27 analytes on total protein isolated from mice dermis in non-treated animals, animals at day 3 post-CHS, and day 7 post CHS treatment. We compared the ratios to normal levels between the groups and found 11 factors out of 27 were significantly increased at day 3 compared to both non-treated and day 7 animals . The facThe phenomenon of lymphatic remodeling or change in lymphatic function caused by acute inflammation is not well understood. In this study, we determined that LECs proliferation occurs in early stages of inflammation and is accompanied by infiltration of CD45+ cells and the loss of lymphatic function.We developed a method to identify proliferative LECs in vivo to allow us to determine that the LECs proliferate starting at the onset of the CHS, but do not proliferate after the peak of CHS at day 3. The percentage of non-leukocyte cells decreased significantly at day 3 and 7 post-CHS compared to non-treated animals. This decrease in the relative percentage of non-leukocyte cell population is most probably due to the infiltration of CD45+ leukocytes, which is a hallmark of CHS. The relative percentage of BECs within the leukocyte population decreased from non-treated skin to day 3 post-CHS, but may be an artifact in relative numbers due to the increase in infiltrating leukocytes. Others have shown that angiogenesis does not occurs in the ear during CHS since no changes in vessel density were observed, but that the vessels increase in size and become leaky in response to CHS . Here weWe identified pro-lymphangiogenic factors that are expressed differentially in early CHS, in addition to have tissue expression which parallels the increase and decrease of LEC proliferation and lymphatic function. Our data also suggests that the proliferation of LECs does not lead to the formation of de novo vessels, but rather dilated vessels that could be the cause of the inefficiency of lymphatic contractile pumping. In addition, proliferative LECs may integrate into existing vessels to repair damaged vessels in the inflamed skin. Lymphatic vessels repair possibly requires a dynamic balance between proliferation and vessel regression, similar to that well known and studies in blood vessel angiogenesis –27.We identified that the superficial collecting lymphatic vessels in the inflamed skin regions do not show change in architecture during inflammation, but instead found extravasation of ICG-laden lymph that may be indicative of lymphatic vessel leakiness associated incorporation of with proliferative LECs. Mendez et al. also found that, in acute CHS in the rat foreleg, lymphatic drainage is reduced as shown by interstitial spreading of dye .The peripheral lymphatic system drains excess interstitial fluid back to the blood circulation, but also directs the transport of immune cells. Inflammation impacts lymph drainage and the volume of lymph propelled by lymphatic contraction ,24. HereMost studies of lymphangiogenesis have focused on the role of the VEGF family members in this process. Expression of VEGF-A in the skin was found to cause chronic inflammation after induction of CHS which in turned promoted lymphangiogenesis in the inflamed tissue . Both VEOur data suggest that lymphatic vessels are remodeled and LECs proliferate in CHS. In addition, CHS induces a local reduction in lymphatic transport through the expression of growth factors and chemokines, which promote vessel dilation and leakiness and/or through the changes in local shear forces. Our study indicates that there is a need to investigate further the importance of other factors such as KC/CXCL17 on lymphatic function and proliferation.Figure S1Use of viability Dye Calcein Am is required to identify LECs from skin tissue.Others have used flow cytometry and lymphatic markers to identify LECs in the tissue, but did not use viability dye to discriminate non cellular events. We found that less than 50% of the events that were the CD45-/CD31+/podoplanin+ did not incorporate Calcein Am. This finding indicates that regular discrimination on Forward and Side scatter are not sufficient to eliminate debris and that the use of viability dyes such as Calcein AM is required to correctly quantify and identify LECs in the skin tissue. We show a sample plot of the events that are podoplanin+/CD31+ which we gated on SSC and FSC and for lack of CD45 staining (left plot). We then selected the double positive events and looked for the percentage of cells that also stained for Calcein AM. We observed that only 35.6% of the cells in the podoplanin+/CD31+ gate are actually live cells.(PDF)Click here for additional data file.Figure S2Spatiotemporal plots from multiple animals shows lymphatic backflow in CHS.Plots showing fluorescence intensity along a lymphatic vessel from the inguinal LN to the Axillary LN indicated loss of effective flow at day 3 post-CHS. The plots are from two animals pre-treatment and at day 3 post-CHS. The arrows indicate the direction of the intensity peaks. There is a change in the direction of the intensity peak from always having a forward motion  in the pre-treated animals to both forward and backward flow  in the treated animals. These data are indicative of a change in the effectiveness of the lymphatic flow.(PDF)Click here for additional data file.Table S1Identification of pro-angiogenic.- lymphangiogenic factors in CHS. Table shows the mean ratio ±std of the concentration normalized to non-treated values (N>8) of all the analytes assayed and for which the data was reproducible in multiple assay. We also show the ANOVA results, the FDR for the ANOVA, and the corrected p-value for each comparison made.(PDF)Click here for additional data file.Video S1Video-rate fluorescent imaging of lymphatic flow exposure time of 200 ms.We recorded fluorescent signal after IGG injection as described in the method section. The video shows 200 frames at an exposure time of 200 ms. The video shows ICG dye packets moving within the lymphatic vessels. Fluorescent signals were converted to pseudo-colored images (Green) using MatLab. Elapsed time from the start of recording is shown in the movie and we show 15 frames per second.(AVI)Click here for additional data file.Video S2Video-rate fluorescent imaging of lymphatic flow exposure time of 200 ms.We recorded fluorescent signal after IGG injection as described in the method section. The video shows 200 frames at an exposure time of 200 ms. The video shows ICG dye packets moving within the lymphatic vessels. Fluorescent signals were converted to pseudo-colored images (Green) using MatLab. Elapsed time from the start of recording is shown in the movie and we show 15 frames per second.(AVI)Click here for additional data file.Video S3Video-rate fluorescent imaging of lymphatic flow exposure time of 200 ms.We recorded fluorescent signal after IGG injection as described in the method section. The video shows 200 frames at an exposure time of 200 ms. The video shows ICG dye packets moving within the lymphatic vessels. Fluorescent signals were converted to pseudo-colored images (Green) using MatLab. Elapsed time from the start of recording is shown in the movie and we show 15 frames per second.(AVI)Click here for additional data file.Video S4Video-rate fluorescent imaging of lymphatic flow exposure time of 200 ms.We recorded fluorescent signal after IGG injection as described in the method section. The video shows 200 frames at an exposure time of 200 ms. The video shows ICG dye packets moving within the lymphatic vessels. Fluorescent signals were converted to pseudo-colored images (Green) using MatLab. Elapsed time from the start of recording is shown in the movie and we show 15 frames per second.(AVI)Click here for additional data file.


Over the years, a number of Plk1 inhibitors have been discovered. Many of these inhibitors are substances that compete with ATP for the substrate binding site. The ATP-competitive inhibitor BI 6727 is currently being clinically tested in cancer patients. Another drug in development, poloxin, is the first Polo-box domain inhibitor of Plk1. This compound is a derivative of the natural product, thymoquinone, derived from Nigella sativa. A novel and promising strategy is to synthesize bifunctional inhibitors that combine the high binding affinity of ATP inhibitors with the specificity of competitive inhibitors.Polo-like kinase 1 (Plk1) is an interesting molecule both as a biomarker and as a target for highly specific cancer therapy for several reasons. Firstly, it is over-expressed in many cancers and can serve as a biomarker to monitor treatment efficacy of Plk1 inhibitors. Furthermore, the Plk1 enzyme is expressed only in dividing cells and is a major regulator of the cell cycle. It controls entry into mitosis and regulates the spindle checkpoint. The expression of Plk1 in normal cells is not nearly as strong as that in cancer cells, which makes Plk1 a discriminating tartget for the development of cancer-specific small molecule drugs. RNA interference experiments  Polo-like kinase 1 (Plk1) plays a critical role in cell division and represents a promising target for cancer therapy. Several small molecule inhibitors of polo-like kinase 1 have been described. In the present review, we focus on the biology of the Plk1 enzyme and the significance of small molecule inhibitors as novel candidates for cancer therapy. Polo-like kinases are serine-threonine protein kinases. There are four different polo-like kinases Plk 1-4), of which Plk1 is the best studied. All Plks consist of an amino-terminal catalytic kinase domain, which is responsible for ATP-binding and enzyme activation, and a carboxy-terminal polo box domain (PBD). The PBD is involved in phosphopeptide binding and consists of either one (in Plk 4) or two sequence motifs  which each consist of 80 residues [, of whicPlks possess a conserved catalytic kinase domain, which consists of two lobes, each composed of a polypeptide chain. A hinge region connects these two lobes allowing their rotation. ATP molecules can bind at the gap between the two polypeptide chains. The binding pocket structure is highly conserved among all Plks. In general, all protein kinases reveal similar ATP-binding pockets, which may pose problems in developing highly specific kinase inhibitors ,3.Protein kinases are important for a multitude of cellular signal transduction reactions and are, therefore, subject to strong regulation. They can be activated or inactated by interaction with other proteins, peptides, or small molecules or by translocation in the cell. Protein kinases modify the activity of other proteins by phosphorylation. In a reversible process, the kinase catalyzes the transfer of the γ-phosphate from a donor molecule (ATP) to alcoholic  or phenolic (tyrosine) amino acids, which bear a free hydroxyl group. Plk1 phosphorylates serine and threonine ,6.During mitosis, Plk1 binds and phosphorylates multiple proteins and hence plays a crucial role in cell proliferation. It is therefore not surprising that Plk1 is only expressed in dividing cells. During cell cycle progression, fluctuations of Plk1 expression and activity can be detected. The highest Plk1 levels can be observed during the late G2 and M phases of the cell cycle. Furthermore, Plk1’s cellular localization changes in the course of mitosis .Plk1 is involved in the activation of cyclin B1 and Cdc25C, and thereby triggers entry of the cell into mitosis. Furthermore, Plk1 plays a role in the maturation of centromers, the formation and maintenance of the bipolar spindle, the spindle checkpoint, exit from mitosis, and cytokinesis. During interphase and prophase, Plk1 is localized to the centrosome. In prometaphase and metaphase, it can be detected at the kinetochore and at the spindle poles. During anaphase and telophase, Plk1 is displaced to the central spindles .In addition to promoting proliferation of healthy cells, Plk1 also plays an important role in cancer development. If Plk1 harbors mutations and is overexpressed, the mitotic cell cycle checkpoint is affected. Several cell cycle checkpoints maintain genomic stability in normal cells, including the G1, G2/M- and spindle checkpoints. Upon DNA damage or other cellular injury, the cell cycle is normaly arrested to allow for either damage repair or for induction of apoptosis, if the extent of damage exceeds that which the cell can repair. In cancer cells, cell cycle arrest can be overridden due to defective checkpoints. Frequently, checkpoints are bypassed by overexpression of mitotic kinases involved in the control of the checkpoints. As a consequence, proper cell division is no longer guaranteed. Faulty chromosomal alignment and distribution can occur, leading to aneuploidy. This is a typical phenomenon in cancer cells. After DNA damage, Plk1 activity allows cells in G2/M arrest to re-enter mitosis by activating cyclin-dependent kinase 1 (Cdk1).e.g. breast cancer, ovarian carcinoma colorectal carcinoma, uterine cancer, skin cancer, stomach cancer, and others. To estimate whether or not PLK1 expression is of prognostic value, we have summarized the literature on this topic in Table Compared to normal tissues, Plk1 is overexpressed in many cancers, Whereas most studies used immunohistochemistry to analyze Plk1 expression, a few studies also used RT-PCR to measure mRNA of Plk1 in human tumor samples. This difference is of interest becuase it has long been a matter of discussion which methods are most suitable for clinical routine diagnosis of molecular markers . DespiteTable Protein kinases have garnered great interest as therapeutic targets because they are involved in numerous diseases including inflammatory, neurological, and neoplastic diseases . In maliSince Plk1 is overexpressed in many cancers, it represents an interesting target molecule for the development of specific inhibitors to selectively treat cancer while avoiding toxicity towards normal tissues ,29.PLK1 mRNA have been described that degrade PLK1 mRNA and downregulate Plk1 protein levels [There are several possibilities for suppressing Plk1 expression at the translation level. One mechanism with therapeutic implications is the naturally occurring process of RNA interference (RNAi), which is triggered by double stranded RNA. RNAi leads to suppressed expression of genes with complementary sequences. RNA fragments of about 21 nucleotides, called small interfering RNAs (siRNA), can be enzymatically cleaved into single strands of RNA by an enzyme complex known as RNA-induced silencing complex (RISC). The single-stranded RNA fragments bind to complementary mRNA resulting in its degradation. For the specific inhibition of Plk1, siRNAs complementary to n levels .PLK1 gene expression can be downregulated by single-stranded DNA antisense oligonucleotides. These antisense oligos bind to the complementary mRNA and thereby prevent translation of PLK1[Furthermore, n of PLK1.in vitro resulted in mitotic arrest and subsequent apoptosis [in vivo. Nevertheless, Strebhardt et al. [PLK1 expression by RNAi both in vitro and in vivo, and demonstrated a specific killing of cancer cells while normal cells survived. The reasons for this result are still not completely understood. It is thought that PLK1 siRNA might not only partially inhibit Plk, and that the residual enzyme activity is sufficient for normal cells to survive. Another explanation could be that normal cells activate alternative pathways to respond to siRNA-mediated challenge, allowing them to survive. This may be a siRNA-specific phenomenon, since chemical small molecules such as BI 2536 or Poloxin inhibited both proliferating normal and malignant cells in a consistent manner in vitro[Inhibition of Plk1 by RNA interference in cancer cells t et al.  were ablA detailed knowledge of the exact structural configuration and functional operation of Plk1 is a prerequisite for the synthesis of highly specific polo-like kinase 1 inhibitors. In this context, the phosphorylation reactions play a crucial role, because this is the starting point for the design of specific inhibitors. Different inhibitors can be distinguished depending on their mode of action: inhibitors can compete with either ATP or the substrate or with both ATP and the substrate of Plk1 .Because the ATP binding pocket represents a classic target for the synthesis of kinase inhibitors, the first designed Plk1 inhibitors were ATP analogues that block the ATP binding pocket and thereby prevent access to ATP. For the design of novel inhibitor scaffolds, computer-based methods such as pharmacophore modeling and virtual screening are now used. After identification of candidate compounds in large chemical libraries, wet lab experiments must be used to verify the binding between the compounds and Plk1. Chemical scaffolds may serve as lead compounds which can be subsequently optimized by derivatization to acheive more specific binding to Plk1. In this context, the Plk1 amino acids Arg136 and Leu13 are essential for inhibitor binding, as in the case of BI 2536 [To increase the specificity of Plk1 inhibition, substances binding to the PBD and competing with natural Plk1 substrates have been developed. These compounds are able to block protein-protein interactions and consist of modified peptides, which contain consensus sequences for the corresponding substrates. As proof-of-principle, it was shown for protein kinase A (PKA) that peptide analogs with longer consensus sequences have a greater inhibitory effect than peptide analogs with shorter substrate consensus sequences .Plks are the only protein kinases that cotain PBDs. Therefore, it has been attempted to increase the specificity of inhibitors by using sequences complementary to the PBD. The PBD binds with high affinity peptides containing the consensus sequence S-pS/pT-P/X. The first substrate-competitive Plk1 inhibitor, which binds specifically to the PBD, was discovered using a recently developed fluorescence polarization assay. First, a fluorophore-coupled peptide binds with high affinity to the PBD of Plk1. Next, a chemical library of low molecular weight compounds was tested. These compounds interact with the Plk1 PBD and thus compete with the fluoropore-coupled peptide for access to the kinase, which can be measured by fluorescence polarization. The fluorescence polarization is higher when the labeled peptide is bound to the PBD of Plk1. The polarization is reduced upon displacement of the fluorescing peptide by an inhibitor. Applying this technology resulted in the discovery of synthetic poloxin as well as the natural product, thymoquinone . Other iAs a next step to increase specificity, bifunctional inhibitors have been developed, which inhibit binding of both ATP and substrate via two domains connected by a linker. These compounds compete simultaneously with ATP and the peptide or protein substrate for their respective binding sites. The particular advantage of these bifunctional compounds is that they combine the high affinity of the ATP analogue, and the specificity of the substrate analogue. Ideally, such inhibitors should bind with high affinity to the target enzyme and simultaneously be able to discriminate between closely related specific protein kinases. The structure and the length of the linkage between the ATP and substrate analogues can greatly affect the efficiency of bifunctional inhibitors. As yet, bifunctional Plk1 inhibitors have not been developed ,32.Another type of inhibitor interacts with the kinase substrate to prevent phosphorylation. Since the inhibitor does not directly target the protein kinase itself in this approach, it is quite different from the other known kinase inhibitors. Such inhibitors for Plk1 are not yet known ,33.The aim is to find substances that are able to inhibit Plk1-mediated phosphorylation of proteins by specifically and binding with high affinity to Plk1. Protein kinases are one of the largest protein families. The human proteome contains more than 2,000 different protein kinases of which Plk1 is just one . This ili.e. about one-thousandth of the intracellular ATP concentrations. Therefore, there is a saturation of ATP-binding protein kinases under physiological conditions. A novel ATP analogue must meet two criteria. It has to be specific to distinguish one from the other kinase and its binding affinity has to be high enough to displace the surplus ATP and the donor molecule [Furthermore, ATP is used by all protein kinases as a donor molecule for γ-phosphate. The sequence and structure of the binding site is highly conserved, and therefore similar in all protein kinases, to enable ATP binding to the enzyme. This complicates the specific inhibition of Plk1. Another problem is the intracellular ATP concentrations, which are in the millimolar range. The Km values of protein kinases, however, are significantly lower, molecule .i.e. some phosphorylate tyrosine and serine while others are solely threonine kinases. Therefore, the amino acid sequence of the substrate phosphorylation site is highly conserved. Protein kinases that phosphorylate the same amino acid residues show highly similar sequences, which complicates the design of specific binding inhibitors. The more closely related the proteins, the more similar are their binding sites. Since Plk1 through Plk3 (but not Plk4) contain a two-motif-PBD, they share very similar  amino acid sequences for the substrate binding sites. The challenge is to design a non-phosphorylatable peptide analogue that specifically binds to the high affinity and substrate binding site of Plk1 [Furthermore, protein kinases phosphorylate only certain amino acid residues. One system of kinase classification is based on the nature of the residue phosphorylated,  of Plk1 .2-terminal end and the COOH-terminal end of the catalytic kinase domain. The binding takes place via two hydrogen bonds from the interior of the backbone of the NH- and carbonyl groups of Cys133. Thereby, BI 6727 occupies the ATP binding site of Plk1, which leads to a catalytical inactivation. BI 6727 stops cell division and the arrests the cell cycle in the G2/M phase, which is followed by apoptosis [in vitro and in vivo. This inhibitor was tested on 50 patients suffering from advanced or metastasized solid tumors in a clinical Phase I trial. Thrombocytopenia and neutropenia occurred as side effects [BI 6727 represents an ATP-competing kinase inhibitor belonging to a class called dihydropteridinones. It binds to the hinge region between the NHpoptosis in vitro Nigella sativa volatile. Thymoquinone is known to exert anti-inflammatory, anti-oxidative and anti-neoplastic activities [Poloxin is a synthetical derivative of the phytochemical thymoquinone. It was the first small molecule identified to block Plk1 by specifically binding to the PBD. The identification was based on the screening of a chemical library by means of the above described fluorescence polarization assay . Poloxintivities  and strotivities . Poloxintivities .in vivo. A clinical Phase I trial with patients suffering from advanced solid tumors was performed to determine the maximum tolerated dose and to perform pharmacokinetic analysis. The dose-limiting side effects of GW843682X include sepsis, pulmonary embolism, and neutropenia. GW843682X revealed 400-fold greater inhibitory activity towards Plk1 than towards Plk2 and Plk3 [The substance GW843682X was discovered during chemical optimization of a thiophene. Thiophenes were hypothesized to serve as chemical scaffolds for inhibitors because they are known to have a good inhibitory effect on Plk1. GW843682X is a competitive ATP inhibitor, which occupies the ATP binding site and inhibits the kinase activity. It arrested various cancer cell lines with aberrant Plk1 expression in the G2/M cell cycle phase and induced apoptosis. Furthermore, the anti-cancer effect of this compound was also demonstated PLK1 represents an exquisite prognostic marker and target for cancer therapy because of its strong expression in cancer but weak expression in normal tissues. A number of PLK1 inhibitors have been discovered during the past years, some of which are being tested in clinical trials. Once PLK1 inhibitors are clinically approved, they will represent an attractive novel strategy to improving treatment outcome.The authors declare that they have no competing interests.LW has written the raw draft, TE has corrected and finalized the manuscript. Both authors read and approved the final manuscript.