Phase-Locked Signals Elucidate Circuit Architecture of an Oscillatory Pathway
Phase-Locked Signals Elucidate Circuit Architecture of an Oscillatory Pathway
Andreja Jovic1,Bryan Howell1,Michelle Cote1,Susan M.Wade2,Khamir Mehta3,Atsushi Miyawaki4, Richard R.Neubig2,Jennifer J.Linderman3*,Shuichi Takayama1,5*
1Biomedical Engineering Department,University of Michigan,Ann Arbor,Michigan,United States of America,2Pharmacology Department,University of Michigan,Ann Arbor,Michigan,United States of America,3Department of Chemical Engineering,University of Michigan,Ann Arbor,Michigan,United States of America,4Laboratory for Cell Function and Dynamics,Advanced Technology Development Center,Brain Science Institute,Wako City,Saitama,Japan,5Macromolecular Science and Engineering Department,University of Michigan,Ann Arbor,Michigan,United States of America
Introduction
Determining the circuit architecture of cellular signaling path-ways is challenging.Analysis using perturbative tools includ-ing siRNA[1,2],protein over-expression[3],chemical inhibitors [4],or caged compounds[5]usually reveal multiple plausible models that require further refinements and clarification,not just one definitive conclusion.Thus,there is always a need for additional tests and readouts that shed light on signaling circuit architecture in a robustly discriminating manner.
Most perturbations applied to biochemical circuit analysis are genetic or chemical in nature and alters the circuit architecture itself.Furthermore,the analysis usually looks at how such perturbations change signaling in response to a single step change with no further time variation in stimulation parameters.While these types of analyses are very useful,the circuit-destructive and temporally non-varying nature limits information that can be obtained concerning dynamic properties of the intact signaling system[6].We hypothesized that analysis of the frequency-dependent response characteristics of the intact biological oscillator circuit to periodic extracellular chemical stimulation would reveal critical activation and recovery properties of biological oscillators to enable elucidation of molecular mecha-nisms.Here we demonstrate and validate this concept for the oscillatory calcium pathway of the G-protein coupled receptor (GPCR)M3muscarinic system.
The biochemical recovery properties of this system were evaluated by reducing the rest period between pulses of the M3 ligand,carbachol(CCh),and observing the resulting calcium responses.We noted the emergence of beat skipping upon periodic stimulation.The phenomenon whereby an oscillatory system becomes synchronized to a periodic stimulation input is referred to as phase-locking.As the rest period between stimulation pulses was decreased,the number of system responses of the signaling pathway of interest became less than the number of stimulatory inputs thereby revealing biochemical pathway recovery properties not attainable by continuous stimulation.Furthermore,the skipped beats often were not completely absent,but instead appeared as small calcium transients that we here termed‘‘sub-threshold’’spikes;these have been observed previously in electrical responses of cellular systems[7].The sub-threshold spikes provided insight into the activation properties of the signaling
system.The complete absence of a sub-threshold spike would suggest that a switch-like mechanism produced calcium spikes; their presence,however,would suggest that a graded mechanism was more plausible.
These experimental observations of phase-locking properties were compared to the activation and recovery properties of nine models of oscillatory calcium signaling;while these models exclusively deal with the temporal dynamics of calcium signaling and we note that more elaborate models that also include spatial dynamics and IP3receptor noise are available[8,9].In the main text we focus upon two highly different models:the Chay et al. model[10],and the positive feedback Politi et al.model[11].The former model is the first that theoretically analyzed calcium dynamics in chemically-induced phase-locking;the latter model was recently published,features experimental work to support its proposed mechanisms,and carries dynamic features from previous models and experiments[12,13,14].In addition,both models are able to account for a wide range of calcium oscillation periods(10s of seconds to minutes)upon continuous stimulation.The activation properties of the Chay et al.model are characterized by switch-like activation of phospholipase C(PLC)by G-protein, and it also features basal inositol triphosphate(IP3)production, which represents a recovery mechanism that ensures that IP3 quickly returns to its pre-stimulus levels.The Politi et al.model does not have such a recovery mechanism,and features graded PLC activation.To produce oscillations in the Chay et al.model, the products of the switch-like activation of PLC(IP3and diacylglycerol)negatively feedback on upstream pathway compo-nents(G-proteins).In the Politi et al.model,IP3,produced by graded activation of PLC,feeds back on downstream elements (IP3receptor)and calcium feeds back upon upstream elements (PLC)to create oscillations.A large number of oscillatory calcium models feature the aforementioned feedback mechanisms [15,16,17,18,19].
Under continuous stimulation,both models exhibit calcium oscillations with increasing frequencies upon increasing stimula-tion concentration,as seen in a host of experimental data [20,21,22].Both models were thus appropriate but indistinguish-able by conventional stimulation methods.The discriminating features provided by phase-locking analysis,however,revealed that neither of the calcium models correctly predicted all the experimental behaviors based upon their activation and recovery dynamics.Furthermore,by analyzing the sources of discrepancy between the predictions and experiments,we were able to propose a mechanism and parameter modification to account for all the experimental observations of phase-locking.
Although phase-locking can be thought of as a general property of biological oscillators[23],it has not been previously explored experimentally in the context of chemical stimulations.While recent reports have claimed that phase-locking events are largely independent of detailed mechanism[24],we show that the properties of phase-locking can be employed for elucidation of some of the activation and recovery properties of an oscillatory calcium system.We demonstrate that phase-locking,which can only be observed using temporally patterned stimulation,comple-ments conventional chemical and genetic tools for elucidating non-linear oscillatory pathways.
Results/Discussion
We assessed cellular responses to square-wave stimulation through use of a microfluidic platform[modification of25],which enabled exploration of phase-locked rhythms induced by chemical input signals(Fig.1a–c).With fixed stimulant concentration(C) and stimulation duration(D),increases in the rest period(R) resulted in increases in the phase-locking ratio(Fig.1d);phase-locking ratios were calculated by dividing the number of system responses by the number of chemical inputs(See Fig.1,2in Text S1).Analysis of the phase-locking rhythms also uncovered the existence of sub-threshold calcium spikes in individual cellular calcium responses(Fig.1b).In addition,we explored the phase-locking trends induced by varying C and D(See Fig.3a,b in Text S1).These observations collectively provided robust discrimination markers for rigorous evaluation of mathematical models of oscillatory calcium signaling in order to elucidate molecular mechanisms.
Nine oscillatory calcium models were chosen as a test set against our experimental results,based upon the inability to discriminate their behaviors using continuous stimulation despite significant differences in their activation and recovery mechanisms.Here we show phase-locking analysis of two of these models:the Chay et al. model[10]and the Politi et al.model[11](Fig.2).Under continuous stimulation,both the Chay et al.and Politi et al. models exhibited oscillatory calcium responses in physiologically relevant frequency ranges(Fig.3a–c);furthermore,both depicted the same behavior as the strength of stimulation was increased,as depicted in Fig.3a and b.We demonstrate that phase-locking analysis is able to effectively dissect the differences in recovery and activation properties between the models(Fig.3d–i).
We first analyzed the Chay et al.model[10](Fig.2a).As depicted in Fig.3d,we found that as the rest period(R)between stimulation events was increased,the phase-locking ratio in-creased.Despite the agreement of the model with the effects of R on phase-locking ratio observed in our system(compare Fig.1d with Fig.3d),it could not account for the presence of sub-threshold calcium spikes(compare Fig.1b with Fig.3g),suggesting inaccuracies in its activation properties.We attributed the lack of sub-threshold spikes to the model mechanisms,and not model
Author Summary
Key to robust discernment of cell circuit architecture is to have as many distinct response features as possible for comparison and evaluation.One under-appreciated char-acteristic of oscillatory circuits is that under periodic stimulation,these systems will exhibit responses synchro-nized to this stimulatory input,a phenomenon termed phase-locking.We demonstrate that phase-locked re-sponse characteristics vary noticeably depending on circuit activation and recovery properties;these response char-acteristics thereby provide a unique set of criteria for oscillatory circuit architecture analysis.The concept is validated through experiments on an oscillatory calcium pathway in mammalian cells;the experimental setup allowed us to explore,for the first time,the properties of chemically induced phase-locking of intracellular signals. Observations of this phenomenon were then used to test the predictions of several existing mathematical models of calcium signaling.Most of the models we evaluated were unable to match all our experimental observations, suggesting that current models are missing mechanistic elements in the context of calcium signaling for the cell type and receptor/stimulant tested.The observations of phase-locking further led us to identify one simple mechanistic modification that would account for all the experimental observations.The techniques and method-ology presented should be broadly applicable to a variety of biological oscillators.
parameter values,as we used a sampling algorithm (Latin Hypercube Sampling (LHS))to survey a range of parameter values and found no parameter set able to result in sub-threshold calcium spikes (Fig.4).The Chay et al.model assumes that G-protein activation of PLC is a switch-like response with a Hill Coefficient of 4.Therefore if activated G-protein levels are not sufficiently high to surpass the threshold for PLC activation,a calcium spike will not result.However,the presence of sub-threshold calcium spikes in our experiments suggested that such a sharp activation threshold does not exist.While some experiments suggest that Gq-protein activation of PLC is graded [26],to our knowledge,there are no studies that have conclusively determined the nature of this interaction;furthermore,these activation properties may be cell type or signaling pathway dependent.When the Hill coefficient of the G-protein/PLC interaction was
reduced below 3.5in the Chay et al.model,calcium oscillations could not be obtained under continuous stimulation (See Fig.4a in Text S1);furthermore,periodic stimulation of the model with Hill coefficients between 3.5and 4did not yield sub-threshold calcium spikes for a wide range of stimulation conditions (See Fig.4b in Text S1).These results have important implications in terms of how extracellular chemical signals are filtered and interpreted by downstream elements.In particular,intracellular calcium is not only frequency encoded [27],but also amplitude encoded [28],which means that sub-threshold calcium responses might affect cellular responses compared to the non-responses that were noted in the Chay et al.model.Therefore,from a mechanistic standpoint,the ability to capture behaviors such as sub-threshold spikes may prove critical.In addition,these findings show that the reaction mechanisms and model parameters need to
be
Figure 1.Individual HEK293cell exhibiting a calcium phase-locking ratio of 0.5upon square-wave carbachol (CCh)stimulation.a)Temporal pattern of CCh stimulation;the cell was addressed with 25nM CCh for 24s,followed by a rest period of 24s.b)Phase-locked calcium response monitored by normalized FRET ratio (I/I 0);I 0is the minimum FRET ratio obtained during an experimental run to which the remaining ratios (I)were normalized.c)FRET images of the cellular calcium responses.(scale bar =10microns)d)Effect of rest period (R)on average phase-locking ratio;cells were exposed to three different rest period values,while the stimulant concentration (C)was fixed at 10nM CCh and stimulation duration (D)was fixed at 24s.Bars indicate the S.E.M.,representative of three experiments for each experimental condition;for each experiment,the responses of least 20cells were recorded,resulting in totals between 85and 106cells for each experimental condition.All pairs of experimental conditions were statistically significant as determined by the unpaired Student t-test (p ,0.05).doi:10.1371/journal.pcbi.1001040.g001
re-evaluated for the Chay et al.model,which has been used for analysis in many other studies [5,29,30,31].
Our experimental observations were then used to evaluate the Politi et al.model (Fig.2b).Individual calcium graphs portrayed sub-threshold calcium spikes upon exposure to square-wave stimulation pulses (Fig.3h).However,the model incorrectly predicted that larger R resulted in smaller phase-locking ratios (Fig.3e),suggesting that the recovery properties of the model are not accurate.LHS analysis indicated that the choice of model parameter values alone could not explain these inaccuracies,suggesting that reaction mechanisms used to formulate the model needed revision.
Thus,neither of the calcium models tested was able to account for all of our experimental observations.We noted that the Politi et al.model showed continued IP3decay between stimulation pulses,while in the Chay et al.model,IP3levels exhibited recovery between stimulation pulses (See Fig.5in Text S1).In the latter model,IP3recovery between stimulation pulses is due to a mechanism for basal IP3production.Addition of basal IP3production to the Politi et al.model was able to correct its deficiencies in recovery dynamics (Fig.3right column);the IP3production value used in our study was similar to that of reference [32].This model revision may provide crucial insight into physiological systems where cells or tissues require fidelity of its calcium signals to periodic chemical stimulation in order to carry out their function [33].Accurate capture of the recovery properties of oscillatory pathways may also play a pivotal role in the entrainment of such systems [34].We note that other mechanisms may be found that can account for our experimental observations,but basal IP3production provides the simplest explanation and is supported by the literature [35,36,37].Collectively,this would suggest that the activation and recovery mechanisms reflected in our revised Politi et al.model (positive feedback mechanism of calcium upon PLC activity,graded PLC activation by G-proteins,and basal IP3production)are a good fit for the pathway studied here.
We also analyzed seven additional calcium oscillation models.We first explicitly included ligand-receptor-G protein dynamics in both the Chay et al.and Politi et al.models analyzed above,to test whether this would affect our predictions.Those modifications did not change the outcomes of the phase-locking analysis (phase-locking ratio vs.C,D,and R and presence of sub-threshold spikes)(See Fig.6in Text S1),suggesting that the discrepancy between model and experiment did not lie in the simplified way stimulation was represented in the original models.We also tested a precursor to the Chay et al.model,a model by Cuthbertson and Chay [38].Like the Politi et al.model described above,it did not contain a basal level of protein activity,and it too yielded a descending staircase as rest period (R)was increased (See Fig.7in Text S1).We next tested the model developed by Atri et al.[16],and found that it produced the correct recovery behavior as well as sub-threshold spikes (See Fig.8in Text S1);these results can be attributed to a basal flux term and graded activation,respectively.However,the calcium oscillation dynamics of the Atri et al.model are significantly faster than the range of oscillation periods we observed experimentally.As a result,we then analyzed a version of the Li and Rinzel model [18]that features slower dynamics,as presented in the study by Sneyd et al.[5].While the model did exhibit calcium oscillation periods closer to what we saw experimentally,it exhibited a decrease in phase-locking ratio as both C and D were increased (See Fig.9in Text S1).This behavior was perhaps due to an augmented inhibitory effect of calcium upon the activation of the IP3receptor;in addition,the model did exhibit sub-threshold spikes and showed the correct recovery properties,which could be attributed to a basal flux term and graded activation,respectively.Finally,we performed phase-locking analysis on the oscillatory calcium models developed by Dupont et al.[17]and Kummer et al.[39].The former model features feedback of calcium upon PLC activity and IP3metabolism,similar to the Politi et al.model,and the latter model features G-protein and PLC dyanmics.While the Dupont et al.model did exhibit sub-threshold spikes,
phase-locking
Figure 2.Mathematical model schematics.a)Mathematical model developed by Chay et al.[10]b)Mathematical model developed by Politi et al.[11].Dashed arrows indicate positive feedback.DAG =diacylglycerol;DAG-DP =DAG-dependent protein;IP3R =IP3Receptor;IP3R(i)=inacti-vated IP3R;Ca2+(ER)=Endoplasmic Reticulum calcium.doi:10.1371/journal.pcbi.1001040.g002
analysis revealed that it exhibited a decrease in phase-locking ratio for increases in R (See Fig.10in Text S1);the Kummer et al.model exhibited sub-threshold spikes as well,but also did not show a change in phase-locking ratio with changes in C (See Fig.11in Text S1).Thus,although we have not performed an exhaustive search,the modified Politi et al.model developed here best describes the qualitative features of our data on the M3pathway.In sum,we employed a combination of microfluidics,real-time imaging,and mathematical modeling in order to probe the circuit architecture of an oscillatory signaling pathway in mammalian cells.Here chemical-induced phase-locking was explored and analysis of its properties was used to test mathematical models and elucidate molecular mechanisms.Previous reports have claimed that phase-locking events are mostly robust to mechanism details [24,40];this study reports that the properties of phase-locking,however,largely depend upon some of the recovery and activation properties of the molecular mechanisms of an oscillatory signaling system.
As microfluidic setups become more elaborate in their ability to generate temporal stimulation patterns,we can expect even more discriminating markers for signaling studies [41];the diverse waveform stimulation patterns generated by microfluidic setups such as the ‘‘chemical waveform synthesizer’’[42]and the ‘‘chemical signal generator’’[43]should prove useful to this end.While a single optical readout (calcium)was employed for this study,the experimental setup is amenable to the use of multiple
real-time readouts of cellular signaling,thereby further enhancing the number of discriminating markers for elucidation of signaling pathways.Finally,although this paper focused on calcium oscillations,we believe our approach would be well-suited for studies on various biological oscillators such as ERK [44],NF k B [45],and components involved in cell cycle [24],circadian [46],and ultradian [47]rhythms.For example,we have performed phase-locking analysis of two popular circadian oscillator models [48,49]and seen dramatic differences in phase locking behavior between the two,despite similar behaviors under conventional stimulation conditions (See Fig.12in Text S1).Thus,these types of phase-locking analyses provide experimentally testable hypoth-eses for elucidating molecular mechanisms and show that the method is applicable to a broad range of oscillatory pathways.
Materials and Methods Cell Culture
HEK293cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)(Invitrogen)supplemented with 10%Fetal Bovine Serum (FBS)(Gibco)and were maintained at 37u C with 5%CO2in 24-well plates.0.25%Trypsin/EDTA (Gibco)was used to detach cells from plates and transfer them to the microfluidic setup.These cells were stably transfected with the M3muscarinic receptor (selected with 0.4mg/mL
Geneticin
Figure 3.Phase-locking analysis of oscillatory calcium models.Behaviors of the Chay et al.model (left column),the Politi et.al.model (middle column),and revised Politi et al.model (basal IP3production =0.3m M/s)(right column)under continuous are depicted in a–c,and behaviors under periodic stimulation are depicted in d–i.a)–c)Oscillation period vs.continuous stimulant concentration for all three models;the shapes of these graphs are highly similar and thus cannot be used to distinguish between the different mechanisms.In a)stimulant concentration has units of 1/s and represents the rate of receptor-mediated G-protein activation.For b)and c)the stimulant concentrations have units of m M/s and represent the maximal rates of IP3production.d)Phase-locking ratio vs.Rest Period (R)(Concentration (C)=0.031/s,Stimulation Duration (D)=10s);as the rest period between stimulation events is increased for the Chay et al.model,the phase-locking ratio increased,as was observed experimentally in Fig.1d.e)Phase-locking ratio vs.R (C =0.8m M/s D =30s);for the Politi et al.model,the phase-locking ratio decreases as the rest period is increased,opposite of experimental results.f)Phase-locking ratio vs.R (C =0.3m M/s D =10s);the revised Politi et al.model exhibits recovery properties consistent with experimental results.Graphs g–i depict individual intracellular calcium vs.time graphs,where the respective models were periodically stimulated with the given values for stimulation parameters C,D,and R;periodic stimulation of these systems revealed the presence or absence of sub-threshold spikes,as observed in Fig.1b.g)Intracellular calcium vs.time,C =0.031/s,D =10s,R =40s.h)Intracellular calcium vs.time,C =0.8m M/s,D =30s,R =95s.i)Intracellular calcium concentration vs.time,with C =0.3m M/s,D =10s,R =30s.doi:10.1371/journal.pcbi.1001040.g003
(Gibco)).Cells were transiently transfected with the calcium FRET probe YC3.60[50].Transfections were carried out with Lipofectamine2000(Invitrogen)using the manufacturer’s protocol.
Microfluidics
Microfluidic device molds were fabricated based upon the ones described in Futai et al.[25].Front-side photolithography [51]was used to construct the outlet channel where cells were cultured;the remaining channels (inlets and ‘‘Braille’’channels)were construct-ed with backside photolithography [52].With the resulting glass mold,PDMS (1:10ratio of curing agent to base)was cast upon the positive relief features and allowed to cure for at least 2hours in a 60u C oven.The resulting device was then irreversibly sealed against a thin (,100m m)PDMS sheet through 30s plasma oxidation.Once sealed,the device was filled with Phosphate Buffered Saline (PBS)and sterilized for 2hrs in a UV oven.To ensure cell adhesion,the chip was subsequently filled with 100m g/mL laminin (Invitrogen)and allowed to incubate at 37u C for two hours.After this,the chip was flushed and refilled with DMEM supplemented with 10%FBS.Transfected HEK293cells were then seeded from the outlet port and were appropriately positioned in the outlet hydrodynamically.The cells were then allowed to attach overnight.
A custom program written in Visual Basic was used to control the dynamic pumping mediated by Braille-actuation [53],and thereby create the various temporal stimulation patterns used in experiments (Fig.1a);experiments with fluorescein solution confirmed the nearly square-wave shape and reproducibility of these patterns.Carbachol (CCh)dissolved in imaging media [54]was added to one of the inlet reservoirs,and the other reservoir was filled with stimulant-free imaging media.Cells in the devices were maintained at 37u C via a transparent indium tin oxide heater [55],situated between the objective and the thin PDMS-sheet
upon which the cells were cultured.Fluid flow did not elicit detectable intracellular calcium responses.
Imaging
Cells were imaged with a TE2000-U Nikon inverted micro-scope,using a 206objective,a standard 100W mercury lamp,and a 490nm long pass dichroic mirror.A CoolSnap HQ2camera (Photometrics,Tucson,AZ)was used to capture fluorescence images of YC3.60-transfected cells.Cells were excited at 450nm and the emission signals were captured at 490and 535nm (filters from Chroma Technology Corp,Rockingham,VT).An ND4neutral density filter was used to reduce photo-bleaching.The excitation and emission filter wheels were controlled by the Lambda 10-3Shutter Controller (Sutter Instruments,Novato,CA).Images were acquired every 3s,and an exposure time of 100ms was used.The program MetaFluor (Molecular Devices,Downington,PA)was used for image acquisition and processing;for each emission image (at 490nm and 535nm)the background was subtracted,ratiometric images were constructed (intensity at 535nm/intensity at 490nm),and calcium FRET ratios of individual cells were generated with this software.These FRET ratios (I)were normalized by the minimum FRET ratio obtained in the experimental run (I 0),and accordingly I/I 0was plotted in our figures,as has been done previously [56].The normalized ratio values of the calcium peaks fell between 1.2and 7.5,which was in accord with previously obtained values using the same FRET indicator [50].
The resulting images were then analyzed to calculate the phase-locking ratios by dividing the number of calcium spike events by the number of CCh stimulation inputs.Since at least several cells always responded to a particular stimulation pulse,we concluded that when cells did not respond,it was due to phase-locking and not a malfunction with the microfluidic setup (Video
S1).
Figure 4.Parameter sampling cannot account for the discrepancy between mathematical models and experiments.Representative individual ‘intracellular calcium concentration vs.time’graphs with unique parameter sets generated by the Latin Hypercube Sampling (LHS)algorithm for the Chay et al.model.Model parameters were sampled from a uniform distribution,where the minimum distribution value was one tenth of the original parameter value and the maximum distribution value was ten times the original parameter value (details provided in Materials and Methods section).Of the 500total unique parameter sets generated by LHS,none could produce sub-threshold spikes upon periodic stimulation (red square waves),contrary to what was observed experimentally (Fig.1,and See Fig.1,2in Text S1);a red ‘X’signifies that the parameter set did not produce sub-threshold calcium spikes.doi:10.1371/journal.pcbi.1001040.g004
Computation of Phase-Locking Ratios
Cells were exposed to9–18stimulation inputs,and the number of calcium responses for each run was recorded.For instance,for a cell that had been exposed to12CCh stimulation pulses and responded with6calcium spikes,the phase-locking ratio was computed as0.5.Calcium spikes that were above levels of background noise(typically more than10%maximum calcium spike height)but did not reach an amplitude greater than33%of the maximum calcium spike height were not counted as true calcium spikes and were deemed sub-threshold calcium spikes(See Fig.1and Fig.2in Text S1).Phase-locking ratios were computed for individual cells,and averages and standard errors of the mean were computed for each experimental condition.Statistics were based upon three experiments(each of no less than20cells)for each experimental condition.Between85–106cells were exam-ined for each experimental condition.The unpaired Student t-test was used to statistically compare pairs of experimental conditions; p,0.05was used as a threshold of statistical significance. Mathematical Models
Nine mathematical models of oscillatory calcium signaling were evaluated in our study:the Chay et al.model[10](Fig.2a),the positive feedback Politi et al.model[11](Fig.2b),the Cuthbertson and Chay model[38],the Li and Rinzel model[13],the Atri et al. model[16],the Chay et al.and Politi et al.models with ligand/ receptor/G-protein dynamics from Ref.[57],the Dupont et al. model[17],and the Kummer et al.model[39].For all these mathematical models,we used the equations and initial conditions defined in the original publications(except for the Li and Rinzel model,for which we used the adaption developed in Sneyd et al.
[5]);model equations,parameters,initial conditions,and brief model descriptions for all models used in this study are provided in Text S1,starting on page13.For the Chay et al.model,it was assumed that receptor-mediated G-protein activation was propor-tional to stimulant concentration.For the Politi et al.model,it was assumed that the maximal rate of PLC-mediated IP3production was proportional to stimulant concentration.These assumptions are based upon those from the original publications.For the Politi et al.model,we used calcium flux strength e=5to reflect the role of extracellular flux in calcium oscillations[58].The mathematical systems were exposed to12square-wave stimulation pulses and the corresponding number of calcium spike responses was counted in order to compute phase-locking ratios;the criteria for assessing the phase-locking ratio were the same as those for experiments,as described earlier in the Materials and Methods Section.To assess the effect of rest period on the phase-locking ratio,this parameter was varied,while stimulant concentration and stimulation duration were fixed;we then plotted the resulting phase-locking ratio against the rest period(Fig.3-middle row).The same procedure was applied to assess the effects of stimulant concentration and stimulation duration on the phase-locking ratio,respectively(See Fig.3in Text S1).Stimulation parameters for the mathematical models were chosen such that the range in behaviors under periodic stimulation matched those observed in experiments.The stimulation concentration‘C’is represented differently for each model,as noted in Text S1.
Original parameters were used for both circadian models [48,49].
All models were coded in MATLAB version7.8.0(MathWorks Inc,Natick,MA)and the system of ODEs was solved with the stiff solver ode15s.
Latin Hypercube Sampling
We used Latin Hypercube Sampling(LHS)to check if inaccuracies in model parameter values alone could account for differences between experimental results and model predictions. LHS is a highly effective method for exploring parameter spaces for mathematical models[59,60,61,62].Using LHS code from Marino et al.[60](https://www.wendangku.net/doc/eb3802714.html,/lab/ usadata/),we varied model parameter values by sampling from a normal distribution with a25%standard deviation;original parameter values were used as the https://www.wendangku.net/doc/eb3802714.html,rger standard deviations(100%)did not yield results different from those at 25%standard deviation.We also sampled parameters from a uniform distribution;the boundaries of the distribution were set by using one tenth of the original parameter value as the minimum and ten times the original parameter value as the maximum.As was the case for sampling from a normal distribution,sampling from a uniform distribution did not yield any parameter sets that could account for the discrepancies between models and experiments.For the Chay et al.model,we varied all twelve independent parameters;for the Politi et al.model,we varied all 17independent parameters,except for b,which represented the ratio of ER to cytoplasm volume.LHS was run for500iterations on each model,and each model output was analyzed to decipher whether the results matched experimental observations(either by constructing‘phase-locking ratio vs.rest period’graphs for the Politi et al.model or by looking at individual model runs for the Chay et al.model,as depicted in Fig.4).
Supporting Information
Text S1Figures1–12,mathematical model equations,param-eters,initial conditions,and brief descriptions for all models used in the study.
Found at:doi:10.1371/journal.pcbi.1001040.s001(0.93MB PDF) Video S1Movie of HEK293cell with phase-locking ratio of0.5 (cell‘A’),and neighboring cells with1:1calcium phase-locking (‘B’).
Found at:doi:10.1371/journal.pcbi.1001040.s002(1.37MB MPG)
Acknowledgments
The authors would like to thank Nobuyuki Futai,Nathan Lanning, Simeone Marino,Antonio Politi,Tom Bersano,and Gary Luker for assistance with various aspects of the experimental or modeling work,and/ or helpful comments.We would like to also thank James Sneyd for providing model equations and parameters for the version of the Li and Rinzel model used in this study.
Author Contributions
Conceived and designed the experiments:AJ JJL ST.Performed the experiments:AJ MC.Analyzed the data:AJ BH MC KM JJL ST. Contributed reagents/materials/analysis tools:AJ SMW AM RRN ST. Wrote the paper:AJ JJL ST.
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KUKA机器人介绍KR16
1、库卡机器人本体、控制柜、机器人编程控制器性能参数具体说明1.1 KR16机器人本体 KR16的外形尺寸及工作范围
KR16性能参数 负载(指第6轴最前端P点负载）16公斤 手臂/第1轴转盘负载10/20 公斤 总负载46公斤 运动轴数 6 法兰盘（第6轴上）DIN ISO 9409-1-A50 安装位置地面/墙壁/天花板 重复精度+/-0.05mm 控制器KRC2 自重235公斤 作业空间范围14.5立方米 每个轴的运动参数运动范围运动速度 轴1+/-185°156°/s 轴2+35°/-155°156°/s 轴3+154°/ -130°156°/s 轴4+/-350°330°/s 轴5+/-130°330°/s 轴6+/-350°615°/s 1.2机器人控制器KRC2 （1）机器人控制器KRC2外形尺寸 控制柜采用高强材料作为结构框架，内部器件布置简洁明了，全部采用总线形式，维护方便、可靠；控制柜内的冷却按欧洲标准设计制造，元器件与冷却回路隔开，冷却可靠，外部灰层不会进入控制柜内部。
（2）KRC2性能参数
1.4 库卡机器人特点 库卡机器人由肘节式结构的机器人本体，KRC2控制柜、示教控制器KCP组成；铝合金机器人本体、高速运动曲线的动态模型优化，使得库卡机器人的加速性能比其它普通机器人高出25%，有利于提高系统寿命、优化工作节拍； KRC2控制柜采用熟悉的个人电脑WINDOWS操作界面，中英文多种语言菜单；标准的工业计算机，硬盘、光驱、软驱、打印接口、I/O信号、多种总线接口，远程诊断； KCP具有示教、编程、安全保护功能； 控制系统具有绝对位置记忆、软PLC（选项）功能； 事故间隔时间长达7万小时---这是其它机器人所无法比拟的。 库卡工业机器人优点描述： （1）标准六轴工业机器人本体： ?合理的机械结构和紧凑化设计 ?6个自由度AC伺服马达 ?绝对位置编码器 ?所有轴都带有抱闸 ?特定的负载和运动惯量的设计，使得速度和运动特性达到最优化 ?臂部的附加负载对额定负载没有运动限制 ?本体和控制器之间7m长电缆, 并可根据需要进行扩展 ?特点描述： ●模块化的机械结构设计，任何部分都可迅速更换 ●高精度电子零点标定，任何人在任何时间所作的零点标定都 是相同的，标定后，程序无需重新校正即可进入生产状态。 ●可调机械手臂，更大的活动空间和柔韧性 ●高速运动曲线中动态模型的优化，加速性能高于普通机器人25%，更利于 提高系统寿命、优化工作节拍。
ppt超级链接的使用
超级链接的使用 教学内容：插入与修改超级链接 教学目标： 1.会使用“插入”→“超级链接”命令插入超级链接。2.会使用“插入”→“超级链接”命令修改超级链接。 教学重、难点：插入与修改超级链接 教具准备:投影机电脑平台 教学设想:在演示文稿中如何实现从这一页跳到另一页。 教学过程： 一、复习提问 1.插入超级链接的基本步骤是怎样的? 2.如何复制带有超级链接的对象？ 3.怎样观看幻灯片的放映效果？ 二、新授在演示文稿中创建超级链接后，就可以跳转到演示文稿中特定的幻灯片、另一份演示文稿、其他office文稿或internet上的某个地址。今天，我们来具体完成超级链接的链接过程。|使用“插入”→“超级链接”命令插入超级链接。首先选定要插入超级链接的对象，使用“插入”→“超级链接”命令，打开“插入超级链接”对话框（P30图）。 1.超级链接到其他文档或Internet地址。a.利用这种超级链接，可以将“通道”指向当前的演示文稿以外的其他文档。b.单击“浏览”，我们可以选择本地硬盘、局域网或Internet地址，也可以在文本框中直接键入目标的信息。c.这里可选的文档的类型多种多样，可以是Office文稿，可以是图片，也可以是声音文件。当使用该超级链接时，程序会自动打开与选定文档相匹配的应用程序。 2.超级链接到本文档中的指定幻灯片利用这种超级链接，右以在当前的演示文稿内的不同幻灯片之间实现沟通。单击“浏览”，我们可以看到当前的演示文稿内的顺序幻灯片。选择符合要求的幻灯片，单击“确定”就可以了。使用“插入”→“超级链接”命令修改超级链接1、单击选中已经建立了超级链接的对象，使用“插入”→“超级链接”命令就可以进行超级链接设置的修改了。 2、在“修改超级链接”的对话框中，多了一个“取消链接”按钮，可以将原链接清除，再修改。 三、教学反思: 插入与修改超级链接的操作一.开机二.入网三.启动PowerPoint 四. 使用“插入”→“超级链接”命令插入超级链接。五. 使用“插入”→“超级链接”命令修改超级链接六.保存文件七.关机 采用对比教学法：从“浏览（B）”与“浏览（W）”之间的区别突出教学
指令设定一览表
指令设定一览表 惯例 x：立即数m：数据存储器地址A：累加器 i：0~7 号位 addr：程序存储器地址 Rev 1.00 66 2011-04-13
注： 1. 对跳转指令而言，如果比较的结果牵涉到跳转即需2个周期，如果没有跳转发生，则只需一个周期即可。 2. 任何指令若要改变PCL的内容将需要2个周期来执行。 3. 对于“CLR WDT1”和“CLR WDT2”指令而言，TO和PDF标志位也许会受执行结果影响，“CLR WDT1” 和“CLR WDT2”被连续执行后，TO和PDF标志位会被清零，除此外TO和PDF标志位保持不变。 Rev 1.00 67 2011-04-13
指令定义 ADC A, [m] Add Data Memory to ACC with Carry 指令说明将指定数据存储器、累加器和进位标志位的内容相加后，把结果储存回累加器。功能表示ACC ← ACC + [m] + C 影响标志位OV , Z , AC , C ADCM A, [m] Add ACC to Data Memory with Carry 指令说明将指定数据存储器、累加器和进位标志位的内容相加后，把结果储存回指定数据存储器。 功能表示[m] ←ACC + [m] + C 影响标志位OV , Z , AC , C ADD A, [m] Add Data Memory to ACC 指令说明将指定数据存储器和累加器的内容相加后，把结果储存回累加器。功能表示ACC ←ACC + [m] 影响标志位OV , Z , AC , C ADD A, x Add immediate data to ACC 指令说明将累加器和立即数的内容相加后，把结果储存回累加器。功能表示ACC ← ACC + x 影响标志位OV , Z , AC , C ADDM A, [m] Add ACC to Data Memory 指令说明将指定数据存储器和累加器的内容相加后，把结果储存回指定数据存储器。功能表示[m] ←ACC + [m] 影响标志位OV , Z , AC , C AND A, [m] Logical AND Data Memory to ACC 指令说明将存在累加器和指定数据存储器中的数据作AND的运算，然后把结果储存回累加器。功能表示ACC ← ACC“AND”[m] 影响标志位Z AND A, x Logical AND immediate data to ACC 指令说明将存在累加器中的数据和立即数作AND的运算，然后把结果储存回累加器。功能表示ACC ← ACC“AND”x 影响标志位Z ANDM A, [m] Logical AND ACC to Data Memory 指令说明将存在指定数据存储器和累加器中的数据作AND的运算，然后把结果储存回数据 存储器。 功能表示[m] ← ACC“AND”[m] 影响标志位Z CALL addr Subroutine call 指令说明无条件地调用指定地址的子程序，此时程序计数器先加1获得下一个要执行的指令地址并压入堆栈，接着载入指定地址并从新地址继续执行程序，由于此指令需要 额外的运算，所以为一个2周期的指令。 Rev 1.00 68 2011-04-13
RAID检测指令
C:\WINNT\Profiles\Administrator>vxdisk list -v Name MediaName Diskgroup DiskStyle Size(MB) FreeSpace(MB) Status Port Target Channel LUN Harddisk0 RAW 502 7 Uninitialized 0 0 0 0 Harddisk1 Disk1 DataDisk MBR 140270 0 Imported 2 0 0 0 Harddisk2 Disk2 DataDisk MBR 140270 140270 Imported 2 4 0 0 Harddisk3 BasicGroup MBR 70135 7 Uninitialized 2 7 0 0 检测出target id. vxdisk list Name MediaName Diskgroup DiskStyle Size(MB) FreeSpace(MB) Status Harddisk0 BasicGroup MBR 502 7 Uninitialized Harddisk1 Disk1 DataDisk RAW 140270 0 Imported Harddisk2 Disk2 DataDisk RAW 140270 0 Imported Harddisk3 BasicGroup MBR 70135 7 Uninitialized C:\>vxdisk diskinfo Harddisk1 Disk information Device Name : Harddisk1 Media Name : Disk1 Disk Group : DataDisk Disk Style : RAW Length : 147084424704 FreeSpace : 0 BusType : 4 Port : 2 Target : 0 Channel : 0 LUN : 0 Signature : 0 Status : Imported Comment : Subdisks : Disk1-01 Disk1-02 C:\>vxdisk diskinfo Harddisk1 Disk information Device Name : Harddisk1 Media Name : Disk1 Disk Group : DataDisk Disk Style : RAW Length : 147084424704 FreeSpace : 0 BusType : 4 Port : 2 Target : 0 Channel : 0 LUN : 0 Signature : 0 Status : Imported
魔兽世界命令大全
/help 列出常用指令帮助 /assist [名字] 协助你当前所选择的目标，或者指定的目标 /cast spell 施放指定的法术，可以包含法术的等级。比如： "/cast Slow Fall", "/cast Polymorph(Rank 2)" /afk [文字] 开启AFK模式显示你要离开一会儿，再输一次/afk关闭AFK模式。 /combatlog 导出你的战斗信息到(wow目录)LogsPlayerCombatLog.txt 文件里。 /dnd [文字] 开启DND模式表示“请勿打扰”，再输一次/dnd关闭DND模式。 /duel [名字] 要求与你锁定的目标决斗，或者要求与指定的目标决斗。 /yield (/forfeit) 在决斗时投降。 /emote 文字 (/em, /me) 表示接下来的文字是动作。 /exit 退出游戏。 /follow (/f) 自动跟随当前目标。 /ignore 名字忽略目标玩家。 /inspect (/ins) 查看目标玩家的装备。 /logout (/camp) 坐下并且登出。 /macro 打开宏设置界面。 /macrohelp 给出关于设置宏的帮助。 /played 显示你游戏人物的在线时间。 /pvp 在接下来的5分钟内开启PVP模式。 /raid 文字 (/r) 在RAID频道里说话。 /random 数字 [数字2] (/rnd, /rand) 扔出一个从1到某个数字范围内的随机数字，或者是两个数字范围之间的随机数字。 /remfriend 名字 (/removefriend) 把一个好友从你的好友列表里去掉。
三星手机测试指令大全
只要不是CDMA的你就按*#06# 记住手机屏幕上的号码！！ 然后你关机取下电池对照后面的号码！ 如果是行货那肯定是回和他一样的， 如果不是行货就会不相同或者不显示 摩托罗拉 （1）产地及生产日期的查询：摩托罗拉手机背后都有一个MSN机械序号，共10位。它代表着“机型代码，厂家代码，生产年份，生产月份和产品系列号”。察看方法是关机，把后盖和电池拿开，在机身背后的条形码下面有一串号码，其中中间的十位就是MSN码，前三位为型号代码，第四码为生产厂家码，每五位为生产年份码，每六位为生产月份码，后四位为序列号。 （2）软件和功能：水货机无移动QQ，无法在机上查版本。港行只有繁体字输入。欧版机，如果没刷中文包，那就是纯粹外文机 MSN码---机械序号： 1。如何查看：关机，把后盖和电池拿开，在机身背后的条形码下面有：如：MC3-41E11 C836DD3P4R 350904805489928 的一串号码。其中“C836DD3P4R”就是MSN码，共十位。 2.代表的意思如下：前三位为型号代码;第四位为生产厂家码，第五，第六位为生产日期码（前面为年份，后面为月份）;后四位为序列号. （1）生产厂家码(第四码)：6-天津3-杭州2-美国R-德国G-美国5-杭州东信W-是新加坡（2）生产年份(第五码)：X-1997年Y-1998年Z-1999年H-2000年B-2001年C-2002年D-2003年E-2004 F-2005（3）生产月份(第六码)：A-B --1月C-D --2月E-F --3月G-H --4月J-K --5月L-M --6月N-P --7月Q-R --8月S-T --9月U-V --10月W-X --11月Y-Z --12月（注：前一个代表上半月，后一个代表下半月） 三. 水货与行货的区别 1.大陆行货,带有移动QQ 功能.可机上查版本. 2. 港行:有简体及繁体界面选择,但只能输入繁体字.无移动QQ.无法在机上查版本. 3. 新加坡版: 简体界面,简体输入.无移动QQ. 可机上查版本.其它信息里的语言列表是002 4. 4. 欧版,如果没刷中文包,那纯粹外文机. 刷了什么版本就跟什么版本的功能一样.不过键盘无笔划印刷。 电池： 摩托罗拉原装电池选购指南
库卡工业机器人运动指令入门知识学员必备)
库卡工业机器人运动指令的入门知识问?学完了KUKA机器人的运动指令后，可以了解到哪些？ 答（1）通过对机器人几种基本运动指令的学习，能够熟练掌握机器人各种轨迹运动的相关编程操作 （2）通过学习PTP运动指令的添加方法，能够掌握机器人的简单编程 机器人的运动方式： 机器人在程序控制下的运动要求编制一个运动指令，有不同的运动方式供运动指令的编辑使用，通过制定的运动方式和运动指令，机器人才会知道如何进行运动，机器人的运动方式有以下几种： （1）按轴坐标的运动（PTP:Point-toPoint，即点到点） （2）沿轨迹的运动：LIN直线运动和CIRC圆周运动 （3）样条运动：SPLINE运动 点到点运动 PTP运动是机器人沿最快的轨道将TCP从起始点引至目标点，这个移动路线不一定是直线，因为机器人轴进行回转运动，所以曲线轨道比直线轨道运动更快。此轨迹无法精确预知，所以在调试及试运行时，应该在阻挡物体附近降低速度来测试机器人的移动特性。线性运动
线性运动是机器人沿一条直线以定义的速度将TCP引至目标点。在线性移动过程中，机器人转轴之间进行配合，是工具或工件参照点沿着一条通往目标点的直线移动，在这个过程中，工具本身的取向按照程序设定的取向变化。 圆周运动 圆周运动是机器人沿圆形轨道以定义的速度将TCP移动至目标点。圆形轨道是通过起点、辅助点和目标点定义的，起始点是上一条运动指令以精确定位方式抵达的目标点，辅助点是圆周所经历的中间点。在机器人移动过程中，工具尖端取向的变化顺应与持续的移动轨迹。 样条运动 样条运动是一种尤其适用于复杂曲线轨迹的运动方式，这种轨迹原则上也可以通过LIN 运动和CIRC运动生成，但是相比下样条运动更具有优势。 创建以优化节拍时间的运动（轴运动） 1?PTP运动 PTP运动方式是时间最快，也是最优化的移动方式。在KPL程序中，机器人的第一个指令必须是PTP或SPTP，因为机器人控制系统仅在PTP或SPTP运动时才会考虑编程设置的状态和转角方向值，以便定义一个唯一的起始位置。 2?轨迹逼近 为了加速运动过程，控制器可以CONT标示的运动指令进行轨迹逼近，轨迹逼近意味着将不精确到达点坐标，只是逼近点坐标，事先便离开精确保持轮廓的轨迹。
蓝牙指令说明
蓝牙指令说明 通过置高PIO6进入设置方式，置低恢复正常状态，进入设置方式后波特率固定为9600，通信状态的波特率可通过指令设置。 指令格式如下： 1、进入设置方式后返回/r/n+OPEN:0/r/n 2、对于设置指令如果指令正确则返回：/r/nOK/r/n，如果错误则返回：/r/nERROR/r/n 3、对于查询类指令 例如AT+BAUD? 如果正确则返回：/r/nOK/r/n/r/n+BAUD:115200/r/n 如果错误则返回：/r/nERROR/r/n 我们所有用到的基本指令如下（以金瓯指令为例）： 1、AT+BAUD 这个指令只设置波特率(同样，查询的话也只返回波特率值)，例如：AT+BAUD=115200停止位和奇偶校验位通过指令AT+UARTMODE设置，模块默认的通讯波特率为 9600,N,8,1，AT模式波特率固定为9600,N,8,1 2、AT+AUTH 这个指令是设置是否需要鉴权的功能，也就是是否需要配对密码的功能 3、AT+PASSWORD 连接密钥 4、AT+NAME 名称中应该能识别空格。 5、AT+CLASS 例如：AT+CLASS=040680，这个直接跟6位数字，返回值也是这种形式 6、AT+ROLE 这个对于我们来说只要有主和从两种模式即可，也就是你们的服务端和客户端 7、AT+CLEARADDR 这条指令实际是配合AT+BIND使用的 8、AT+BIND 绑定地址时：对于从设备, 如果已经记忆地址，则不准被查询和配对，只能被它记忆的设备连接；对于主设备,如果已经记忆地址，则一直试着连接它记忆的设备；所以当绑定地址时，一旦设备记忆了地址，则连接只能在它与它记忆的设备之间建立，而不会与其它设备建立连接。所以，在绑定地址时，如果希望与其它设备建立连接，则必须清除记忆的地址。不绑定地址时：从设备可以被查询和配对；主设备连接记忆设备一定的次数失败后,主设备自动清除记忆的地址，并开始重新查询和配对新的设备。 连接固定的设备，绑定地址。 9、AT+ RADDR 这条指令与AT+LADDR格式相同即可。 10、AT+LADDR 该指令返回值的格式是：/r/nOK/r/n/r/n+LADDR:00025B00A5A5/r/n (地址不要用冒号隔开，或者其他格式) 11、AT+UARTMODE（这个我们一般不会用，默认N,8,1即可） AT+UARTMODE=
测试需要的命令(FTTB)
登录命令： 例子： LOGIN:::CTAG::UN=admin,PWD=admin; ZTE_113.12.238.52 2011-01-06 10:14:45 M CTAG COMPLD EN=0 ENDESC=No Error 握手命令 例子： SHAKEHAND:::CTAG::; ZTE_113.12.238.52 2011-01-06 10:15:39 M CTAG COMPLD EN=0 ENDESC=No Error LST-DEVINFO::ONUIP=222.171.142.73:1288771409262-38::; LST-DEVINFO::ONUIP=222.171.142.74:1288771409262-38::;
ZTE_113.12.238.52 2011-01-06 10:49:58 M CTAG COMPLD total_blocks=1 block_number=1 block_records=1 List of Device Info ----------------------------------------------------- DEVNAME DEVIP DT DEVER MEM CPU TEMPERATURE 技服F820 113.12.238.28 F820 V1.1.0P1 70 44 -- 查询单板信息 例子： LST-BRDINFO::ONUIP=222.171.142.73:CTAG::; LST-BRDINFO::ONUIP=222.171.142.74:CTAG::; ZTE_113.12.238.52 2011-01-06 10:50:30 M CTAG COMPLD
库卡工业机器人运动指令入门知识 学员必备
库卡工业机器人运动指令的入门知识 问?学完了的运动指令后，可以了解到哪些？ 答（1）通过对机器人几种基本运动指令的学习，能够熟练掌握机器人各种轨迹运动的相关编程操作 （2）通过学习PTP运动指令的添加方法，能够掌握机器人的简单编程 机器人的运动方式： 机器人在程序控制下的运动要求编制一个运动指令，有不同的运动方式供运动指令的编辑使用，通过制定的运动方式和运动指令，机器人才会知道如何进行运动，机器人的运动方式有以下几种： （1）按轴坐标的运动（PTP:Point-toPoint，即点到点） （2）沿轨迹的运动：LIN直线运动和CIRC圆周运动 （3）样条运动：SPLINE运动 点到点运动
PTP运动是机器人沿最快的轨道将TCP从起始点引至目标点，这个移动路线不一定是直线，因为机器人轴进行回转运动，所以曲线轨道比直线轨道运动更快。此轨迹无法精确预知，所以在调试及试运行时，应该在阻挡物体附近降低速度来测试机器人的移动特性。 线性运动
线性运动是机器人沿一条直线以定义的速度将TCP引至目标点。在线性移动过程中，机器人转轴之间进行配合，是工具或工件参照点沿着一条通往目标点的直线移动，在这个过程中，工具本身的取向按照程序设定的取向变化。 圆周运动 圆周运动是机器人沿圆形轨道以定义的速度将TCP移动至目标点。圆形轨道是通过起点、辅助点和目标点定义的，起始点是上一条运动指令以精确定位方式抵达的目标点，辅助点是圆周所经历的中间点。在机器人移动过程中，工具尖端取向的变化顺应与持续的移动轨迹。 样条运动
样条运动是一种尤其适用于复杂曲线轨迹的运动方式，这种轨迹原则上也可以通过LIN运动和CIRC运动生成，但是相比下样条运动更具有优势。 创建以优化节拍时间的运动（轴运动） 1?PTP运动 PTP运动方式是时间最快，也是最优化的移动方式。在KPL程序中，机器人的第一个指令必须是PTP或SPTP，因为机器人控制系统仅在PTP或SPTP运动时才会考虑编程设置的状态和转角方向值，以便定义一个唯一的起始位置。 2?轨迹逼近 为了加速运动过程，控制器可以CONT标示的运动指令进行轨迹逼近，轨迹逼近意味着将不精确到达点坐标，只是逼近点坐标，事先便离开精确保持轮廓的轨迹。 PTP运动的轨迹逼近是不可预见的，相比较点的精确暂停，轨迹逼近具有如下的优势： （1）由于这些点之间不再需要制动和加速，所以运动系统受到的磨损减少。（2）节拍时间得以优化，程序可以更快的运行。 创建PTP运动的操作步骤 （1）创建PTP运动的前提条件是机器人的运动方式已经设置为T1运行方式，并且已经选定机器人程序。
以下命令用作于强制连接远程桌面
xp和win 2003远程桌面强制进入命令_远程登 录t人命令 么使用mstsc 在命令他人。我想用我的电脑控制另一太电脑。我们俩个在不同的地方。详细描述下。 服务器的管理经常会遇到这个问题，我们应该怎么处理呢？别着急，这里教大家一个方法： 如果你是在windows xp下面就用这个命令在运行里输入mstsc /admin 如果是在win 2003里面进行T人的话可以用下面的命令mstsc /console 然后再输入你的IP进行连接。你就发现把其它用户给T啦。。爽吧。 下面呢咱们再深入学习一下mstsc 这个命令
远程桌面如果超出最大连接数, 使用命令行mstsc /console登录即可 可以在运行里使用mstsc /console /v:IP:远程端口即可强制登录; 如果直接在远程桌面连接端使用就直接输入/console /v:IP:远程端口. 如：mstsc /console /v:221.221.221.221:34567 远程桌面mstsc参数的了解，可通过mstsc /?学习一下。 远程桌面连接 MSTSC [＜Connection File＞] [/v:＜sever[:port]＞] [/console] [/f[ullscreen]] [/w:＜width＞/h:＜height＞] | /Edit"ConnectionFile" | /Migrate | /? ＜Connection File＞-- 指定连接的 .rdp 文件的名称。 /v:＜sever[:port]＞-- 指定要连接到的终端服务器。 /console -- 连接到服务器的控制台会话。 /f -- 以全屏模式启动客户端。 /w: ＜width＞-- 指定远程桌面屏幕的宽度。 /h:＜height＞-- 指定远程桌面屏幕的亮度。 /edit -- 打开指定的 .rdp 文件来编辑。 /migrate -- 将客户端连接管理器创建的旧版连接文件迁移到新的 .rdp 连接文件。/? -- 生成这个用法消息
设置gm以及gm命令
GM指令，先找到自己的角色 这行就是角色权限 最大权限5 修改之后无需重启服务端，直接游戏 一般修改商城需要重启服务端，gm指令可以无需 /reload_itemmall_db 重新载入商城 一般修改商城需要重启服务端，gm指令可以无需刷物品 /clone_item 物品id 还有个刷物品 /clone_reward_item 物品id 物品数量 上面是单一，下面是可以多刷 刷物品 /clone_item 物品id 还有个刷物品 /clone_reward_item 物品id 物品数量 上面是单一，下面是可以多刷 add_appellation 称号ID 获取称号 /gain_exp 10 //经验 /gain_gold 10 //金币 命令之前都有个/这个 transport_node 地图id //传送到其他地图listarea //列出地图id get_user_info 角色名//获取角色id
invincible 1 //无敌 vanish 1 //隐藏 transport_to_character 角色名//传送到角色身边look 角色id //查看角色详细信息 set_level 60 //设置等级 /banchar
常用的网络测试命令
常用的网络测试命令 在进行各类网络实验和网络故障排除时，经常需要用到相应的测试工具。网络测试工具基本上分为两类：专用测试工具和系统集成的测试命令，其中，专用测试工具虽然功能强大， 但价格较为昂贵，主要用于对网络的专业测试。对于网络实验和平时的网络维护来说，通过熟练掌握由系统（操作系统和网络设备）集成的一些测试命令，就可以判断网络的工作状态和常见的网络故障。我们以 Windows XP 为例，介绍一些常见命令的使用方法。 1 Ping网络连通测试命令 1.1 Ping 命令的功能 Ping 是网络连通测试命令，是一种常见的网络工具。用这种工具可以测试端到端的连 通性，即检查源端到目的端网络是否通畅。该命令主要是用来检查路由是否能够到达，Ping 的原理很简单，就是通过向计算机发送Internet控制信息协议（ICMP ）从源端向目的端发 出一定数量的网络包，然后从目的端返回这些包的响应，以校验与远程计算机或本地计算机 的连接情况。对于每个发送网络包，Ping 最多等待 1 秒并显示发送和接收网络包的数量， 比较每个接收网络包和发送网络包，以校验其有效性。默认情况下，发送四个回应网络包。 由于该命令的包长非常小，所以在网上传递的速度非常快，可以快速的检测要去的站点是否可达，如果在一定的时间内收到响应，则程序返回从包发出到收到的时间间隔，这样根据时间间隔就可以统计网络的延迟。如果网络包的响应在一定时间间隔内没有收到，则程序认为包丢失，返回请求超时的结果。这样如果让Ping 一次发一定数量的包，然后检查收到相应 的包的数量，则可统计出端到端网络的丢包率，而丢包率是检验网络质量的重要参数。 一般在去某一站点是可以先运行一下该命令看看该站点是否可达。如果执行Ping不成功，则可以预测故障出现在以下几个方面： 网线是否连通 网络适配器配置是否正确 IP 地址是否可用 如果执行Ping 成功而网络仍无法使用，那么问题很可能出在网络系统的软件配置方面，Ping 成功只能保证当前主机与目的主机间存在一条连通的物理路径。它的使用格式是在命 令提示符下键入： ping IP 地址或主机名，执行结果显示响应时间，重复执行这个命令，可以 发现 Ping 报告的响应时间是不同的。 如果网络管理员和用户的Ping 命令都失败了， Ping 命令显示的出错信息是很有帮助的， 可以指导进行下一步的测试计划。这时可注意 Ping 命令显示的出错信息，这种出错信息通常分 为三种情况： (1) unknown host（不知名主机），该远程主机的名字不能被DNS（域名服务器）转换成ip 地址。网络故障可能为 DNS 有故障，或者其名字不正确，或者网络管理员的系统与远程 主机之间的通信线路有故障。 (2)network unreachable （网络不能到达），这是本地系统没有到达远程系统的路由，可 用 netstat-rn 检查路由表来确定路由配置情况。
kuka机器人学习报告
kuka机器人学习报告 篇一：工业机器人报告 工业机器人报告 在工业铝深加工可以大幅度增加产值的大势所趋下，集团成立了工业铝深加工分公司。深加工分公司的一个重要的深加工项目就是车体轨道车辆铝型材焊接。为了保证焊接质量，目前国内各大型工业铝型材生产企业深加工工厂都使用自动焊接机器人生产线进行车体轨道车辆铝型材焊接。 一、工业机器人历史 日本是当今的工业机器人王国，既是工业机器人的最大制造国也是最大消费国。但实际上工业机器人的诞生地是美国。美国人英格伯格和德奥尔制造出了世界上第一台工业机器人，他们发现可以让机器人去代替工人一些简单重复的劳动，而且不需要报酬和休息，任劳任怨。接着他们两人合办了世界上第一家机器人制造工厂，生产unimate工业机器人。美国是工业机器人的诞生地，基础雄厚，技术先进。现今美国有着一批具有国际影响力的工业机器人供应商，像Adept Technologe、American Robot、Emersom Industrial Automation等。德国工业机器人的数量占世界第三，仅次于日本和美国，其智能机器人的研究和应用在世界上处于领先地位。目前在普及第一代工业机器人的基础上，第二代工业机器人经推广应用成为主流安装机型，而第三代智能机器人
已占有一定比重并成为发展的方向。世界上的机器人供应商分为日系和欧系。瑞典的ABB公司是世界上最大机器人制造公司之一。1974年研发了世界上第一台全电控式工业机器人IRB6，主要应用于工 件的取放和物料搬运。1975年生产出第一台焊接机器人。到1980年兼并Trallfa喷漆机器人公司后，其机器人产品趋于完备。ABB公司制造的工业机器人广泛应用在焊接、装配铸造、密封涂胶、材料处理、包装、喷漆、水切割等领域。德国的KUKA Roboter Gmbh公司是世界上几家顶级工业机器人制造商之一。1973年研制开发了KUKA的第一台工业机器人。年产量达到一万台左右。所生产的机器人广泛应用在仪器、汽车、航天、食品、制药、医学、铸造、塑料等工业，主要用于材料处理、机床装备、包装、堆垛、焊接、表面休整等领域。意大利COMAU公司从1978年开始研制和生产工业机器人，至今已有30多年的历史。其机器人产品包括Smart 系列多功能机器人和MASK系列龙门焊接机器人。广泛应用于汽车制造、铸造、家具、食品、化工、航天、印刷等领域。（The Comau Smart NS1）日系是工业机器人制造的主要派系，其代表有FANUC、安川、川崎、OTC、松下、不二越等国际知名公司。 二、焊接机器人介绍 焊接机器人是从事焊接（包括切割与喷涂）的工业机器
XP系统远程桌面连接设置：远程桌面连接命令
对于下班后又要加班而不想回办公室的人来说，用远程桌面连接进行控制是个很好的方法，说起远程控制其实很多朋友都已经使用过QQ啲远程协助，也有很多人尝试过PCAnyWhere 等强大啲远程控制软件了。然而，很多朋友却忽略了Windows 系统本身就附带啲一个功能“远程桌面连接，其实它啲功能、性能等一点都不弱，上远程桌面操作控制办公室的电脑就完全和在家里用电脑一模一样的！没有任何区别，而且比其他的远程控制工具好用得多！但是在使用远程控制之前必须对电脑作好相应的设置！下面本站就当前最流行的XP系统，win7系统，VISTA这三种系统的远程桌面连接设置方法及远程桌面命令进行详细的图文讲解 XP系统远程桌面设置教程！ 远程桌面是Windows XP Professional 啲一个标准组件(在 Windows XP Home Edition 家庭版中没有包括啲哟)，它允许你使用任何基于Windows啲客户端从任何位置，通过任何连接来访问Windows XP Professional啲计算机。远程桌面可让你可靠地使用远程计算机上啲所有啲应用程序、文件和网络资源，就如同你本人就坐在远程计算机啲面前一样，不仅如此，本地(办公室)运行啲任何应用程序在你使用远程桌面远程(家、会议室、途中)连接后仍会运行。 远程桌面其实可以为我们做很多事，例如可以让我们坐在家里就可以连接到远程电脑啲桌面，可以任意操作远程啲电脑做任何事，如让它打开迅雷下载、整理硬盘随便，下载BT等等，就像实际操作那台电脑一样，呵呵。心动了吧，那就让我们开始这段操作说明吧！ 远程桌面主要包括客户端和服务器端，每台Windows XP 都同时包括客户端和服务器端，也就是说他既可以当成客户端来连到别台啲装了Windows XP 啲电脑，并控制他，也可以自己当成服务器端，让别啲电脑来控制自己。 除了XP控制XP外，Win98，Win2000，WinME都可以对XP进行控制啲。而且你可以在家里用你啲简体版啲Win2000，控制远处公司或其他地方啲繁体版啲 Windows XP。（呵呵，我喜欢） 下面就让我们跟着说明一步步安装 Windows XP 啲远程桌面连接。 第一步，设服务器 其实Windows xp 装好之后，就可以了，当然如果你啲XP是不用密码自动登录啲，那就不行，没有密码是不让你进行远程桌面连接啲，不然还不任何人都可以操作你啲电脑，那岂不是完蛋。所以我们要先加密码，详细步骤如下：
百度搜索引擎查询外部链接命令
百度搜索引擎查询外部链接命令 百度搜索引擎是没有查询外部链接的命令，只能使用“domain”命令来查询含有当前网址的结果页面。有的朋友会说查询外链可以使用雅虎搜索引擎就可以了，不过大家应该都知道，相关域的数量和质量对网站权重的提升也有作用，而domain命令返回的结果是否是真实的相关域数量呢? 曾经看到过很多网站收录一百万，查询其网站相关域的时候会发现数量远远超过一百万，这是什么情况呢?DOMAIN查询出来的结果中只是显示了包含有您查询的网址的页面，不管这个页面来自哪里，这样查询出来的结果中肯定会包含很多自己的页面，甚至是所有site查询出来的所有结果都在domain查询中出来了，因为域名也是被搜索的对象。而“-”的命令是减去的意思，意思很容易理解，但如何更好的利用才是关键，就比如刚刚的情况，想要得到网站在百度的相关域的准确数据可以使用这个组合命令，命令解释为查询当前域名在百度的相关域的数量并减去当前域名在百度收录的数量，那么得到的结果自然就是当前域名的百度的相关域数量的准确值了。 还有一个问题给大家解释一下，那就是很多站长在DOMAIN自己网站的时候会发现本来查询数量为几千，但是翻页后却发现只有几百个了，这种情况其实很好解释，那就是你在发外链的时候网址局限性比较大，很多查询结果都源自固定的几个或十几个网站，百度为了节省资源而省略了一个网站下的多个查询结果，所以一般在结果的最后一页都有提示：为了提供最相关的结果，我们省略了一些内容相似的条目，点击这里可以看到所有搜索结果。再细心点的网友还发现即使点了那个链接后再来看，翻到后面也不一定会显示跟原来一样的结果，甚至当你翻到最后一页的时候你算下页数计算出总数后跟百度提供的数据还是对不上，我的理解是这样的，因为百度数据库非常大，可能其中一样数据都被分在很多服务器上，所以查询时不同的服务器给出的结果会对不上。
各种数控指令的用法介绍
更多资料请访问.(.....) 数控程序的指令由一系列的程序字组成，而程序字通常由地址（address）和数值（number）两部分组成，地址通常是某个大写字母。数控程序中的地址代码意义如表1所示。 表1
数控程序中的每一个指令都有一定的固定格式，使用不同的数控机床的指令格式也不同，因此需要按照该数控机床的指令格式来编写数控指令。一般的数控机床可以选择公制单位毫M （mm）或者英制单位英寸（inch）为数值单位。公制可以精确到0.001mm，英制可以精确到0.0001in，这也是一般数控机床的最小移动量。表2列出了一般数控机床所能输入的指令数值范围，而数控机床实际使用范围受到机床本身的限制，因此需要参考数控机床的操作手册而定。例如表中X轴可以移动±99999.999mm，但实际上数控机床的X轴行程可能只有650mm，进给速率F最大可输入100000.0mm/min，但实际上数控机床可能限制在 3000mm/min以下。因此在编制数控程序时，一定要参照数控机床的使用说明书。 表2
下面简要介绍各种数控指令的用法。 1．顺序号字 顺序号字也称程序段号。在程序段之首，以字母N开头，其后为一个2～4位的数字。需要注意的是，数控程序是按程序段的排列次序执行的，与顺序段号的大小次序无关，即程序段号实际上只是程序段的名称，而不是程序段执行的先后次序。 2．准备功能字 以字母G开头，后接一个两位数字，因此又称为G指令。它是控制机床运动的主要功能类别。常用的G指令有以下几种。 （1）G00：快速点定位，即刀具快速移动到指定坐标，用于刀具在非切削状态下的快速移动，其移动速度取决于机床本身的技术参数。如刀具快速移动到点（100，100，100）的指令格式为： G00 X100.0 Y100.0 Z100.0 （2）G01：直线插补，即刀具以指定的速度直线运动到指定的坐标位置，是进行切削运动的两种主要方式之一。如刀具以250mm/min的速度直线插补运动到点（100，100，100）的指令格式为： G01 X100.0 Y100.0 Z100.0 F250 （3）G02、G03：顺时针和逆时针圆弧插补，即刀具以指定的速度以圆弧运动到指定的位置。G02/G03有两种表达格式，一种为半径格式，使用参数值R，如G02 X100 Y100 Z100 R50 F250表示刀具以250mm/min的速度沿半径50的顺时针圆弧运动至终点（100，100，100）。其
库卡工业机器人
库卡工业机器人：传送前后轴及车门 当前状况/任务 宝马公司为其在雷根斯堡的工厂寻找一种自动化解决方案用以传送宝马1 系列及 3 系列车型的整个前后轴以及车门。 实施措施/解决方案 传送前后轴及车门 宝马公司选择了三台库卡机器人，包括一台KR 500 及两台KR 360 来传送前后轴。KR 500 从装配系统中取出已装配好的前轴并将其置于装配总成支架上，在那里前轴将被装配到传动杆上。KR 500 的多用夹持器适用于1、3 系列所有车型专有的轴。此外，整个夹持器还满足了宝马公司的要求，即能够在传送过程中使轴的活动部分保持在规定的位置。由此，机器人可将所有需要装配的部件在转配总成支架上准确定位。 两台重载型机器人KR 360 传送后轴。第一台KR 360 从装配系统中取出轴并将其置于多用工件托架的存储器内。第二台KR 360 从存储器中取出轴并将其置于装配总成支架上。如同前轴的情况，放置后轴时所需达到的精确位置可通过一个感知器测量系统得到。为使KR 360 能够在最佳的位置上完成所需的工作，它被安装在一个一米半高的底座之上。特殊的夹持器使机器人能够传送雷根斯堡工厂内生产的车轴。由于机器人控制系统将夹持器作为第七条轴来移动，因此KR 360 就有能力将客车车轴举到轮毂处而不受轮距的限制。 在传送车门方面，四台装配有400 mm 延长臂的KR 150，每两台作为一组，可以替代数目相同的提升站以及所属用以交接的机械装置。在两个机器人小组内部，一台机器人
负责前门，另一台负责后门。当一辆带着空运输吊架的电动钢索吊车停在工位内时，机器人的工作就可以开始了。有关的KR 150 将其夹持器摆动着伸入货物承装工具内部，将其从电动钢索吊车上取下并置于下一层以做好装料准备。两个在此工作的工作人员为吊架的两侧都装上相应车身的车门。之后，机器人将货物承装工具移回上一层并将其重新放回电动钢索吊车。由于机器人重复精度高，就可以避免对车门及电动钢索吊产生损伤。由于对机器人可进行自由编程，因此整个设备也具有很高的灵活性。除此之外，库卡公司还可以满足宝马公司对夹持器的要求：设计简单且安全可靠。 系统部件/合同范围 ·一台库卡重载型机器人KR 500，其承载能力为500 kg ·两台库卡重载型机器人KR 360，每台负重能力为360 kg ·四台库卡载重机器人KR 150，每台承载能力为150 kg ·以PC 为基础的库卡机器人控制系统，包括带有熟悉的视窗操作界面的控制面板 ·夹持器 ·机器人编程 ·投入运行 结果/成效 灵活性高 在适应产品种类变化方面，三台重载型机器人在传送时也具有非常高的灵活性。同样对于宝马公司来说，由于库卡机器人工作范围极大，因此公司就可以灵活自由地选择安装地点。 可用性提高 使用KR 150 替代了已经不再符合技术发展状况的提升站，提高了可用性此外，生产能力强大的标准机器人也能使工艺流程最优化。 集成的紧急对策 宝马公司也能从这个集成的紧急对策中得益。如KR 150 出现故障，第二组机器人就会接替那组机器人的工作。如有必要，使用者可以短期内将可以工作机器人组的生产能力提高到百分之百，这样就可以跟上电动钢索吊车的工作节拍。 投入使用时间短 机器人另外一个优点就是它的投入使用时间短。这归功于上级控制系统与机器人控制系统间的标准化接口，以及经过实践检验的软件模块。