User’s Manual Model CSC 5300 Nano-Isothermal Titration Calorimeter III
Isothermal Titration Calorimeter, User’s Manual Revision 40605 Copyright ©2005 Calorimetry Sciences Corporation All Rights Reserved Printed in the United States of America. No part of the manual may be photocopied or reproduced in any form without the express written permission of Calorimetry Sciences Corporation. This manual is supplied “as is” without any warranty of any kind, and is subject to change without notice.
CSC 5300 N-ITC III i Table of Contents CHAPTER ONE: SPECIFICATIONS AND WARRANTY . 1 Calorimeter Specifications ......................................................1 Warranty...................................................................................1 BindWorks Warranty ..............................................................2 License.......................................................................................3 Overview...............................................................
ii Table of Contents Reinstalling Software...........................................................11 CHAPTER THREE: THEORY OF OPERATION ........... 13 Introduction to Isothermal Titration Calorimetry .............13 Applications ............................................................................14 Batch/Incremental Titration .................................................14 Titration/Data Analysis ........................................................
CSC 5300 N-ITC III iii CHAPTER FIVE: SOFTWARE DESCRIPTION ............ 33 Overview .................................................................................33 ITCRun Software Installation ..............................................33 General....................................................................................33 Main Menu .............................................................................34 File Menu .......................................................................
iv Table of Contents Integration of the peak .........................................................49 Modeling and Fit of the Data ...............................................50 Modeling .................................................................................51 Modeling Overview .............................................................51 Designing an Experiment ......................................................55 CHAPTER SIX: SAMPLE EXPERIMENTS ...................
CSC 5300 N-ITC III 1 Chapter One: Specifications and Warranty Calorimeter Specifications CSC 5300 Nano-Isothermal Titration Calorimeter III Specifications Baseline stability ±0.02 μcal/sec/hr Response Time 15 seconds Injection interval 150 seconds minimum Cell volume 1.0 mL (24K Gold) Precision burette 100 or 250 μL Volume increment 1–15 μL Delivery precision ±0.01 μL Stirring rate 0 to 300 rpm Temperature Range 2 to 80°C Power requirements 95-250 Volts, 50-60 Hz.
2 Chapter 1: Specifications and Warranty • Unauthorized modification or misuse. • Operation outside the electrical specifications for the product. • Improper site or buyer-induced contamination or leaks. • Failure to use proper surge protection. • Improper return packaging. To exercise this warranty, please call or write Calorimetry Sciences Corporation. BindWorks Warranty LIMITED WARRANTY.
CSC 5300 N-ITC III 3 This limited warranty gives you specific legal rights. You may have others, which vary from state/jurisdiction to state/jurisdiction. NO LIABILITY FOR CONSEQUENTIAL DAMAGES.
4 Chapter 1: Specifications and Warranty server) and one person uses that computer more than 80% of the time it is in use, then that person may also use the SOFTWARE on a portable or home computer. 2. UPGRADES. If this SOFTWARE is an upgrade from another BindWorks version, you may use or transfer the SOFTWARE only in conjunction with the upgraded product unless you destroy it.
CSC 5300 N-ITC III 5 or other use expressly permitted by this license. 6. GOVERNING LAWS. This License Agreement is governed by the laws of the State of Utah.
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CSC 5300 N-ITC III 7 Chapter Two: Installation N-ITC III Operating Environment The best results are achieved with the CSC Model 5300 Isothermal Titration Calorimeter when certain environmental requirements are met.
8 Chapter 2: Installation order to obtain optimum performance from the N-ITC III. N-ITC III Setup The following sequence of steps is followed to prepare the N-ITC III for use: 1. Unpack and inspect the instrument and all components. 2. Place the N-ITC III on a suitable bench. 3. Plug the power cord on the back of the N-ITC III. 4. Setup the computer (and printer if applicable) next to the N-ITC III. 5. Connect the USB cable between the N-ITC III and the computer system. 6. Turn on the computer system. 7.
CSC 5300 N-ITC III 9 Product Items • Model 5300 Nano-Isothermal Titration Calorimeter III • User’s guide (this manual) • CSC Proprietary Data Collection and Analysis Software • 1 each 2.
10 Chapter 2: Installation Figure 2-1: Attaching the N-ITC III power cord and USB cable. 3. Plug the loose USB cable into the back of the N-ITC III (Figure 2-1). 4. Plug the power cord of the N-ITC III into a surge suppressor power strip. Do not turn equipment power on at this time. Computer Setup Detailed computer setup instructions are not included here. System configuration may change due to advancements in technology.
CSC 5300 N-ITC III 11 5300 icon located on the desktop or by going to Start⇒Programs⇒CSC 5300. After the software starts set the temperature to 25.0°C and let the system equilibrate for approximately 1 hour to allow the internal electronics to warm up. Select the “Monitor” tab to display the virtual strip chart. Heat readings (μJ/sec) slowly scroll across from the right side of the screen. The N-ITC III is now ready for use.
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CSC 5300 N-ITC III 13 Chapter Three: Theory of Operation Introduction to Isothermal Titration Calorimetry There are three ways in which a calorimeter may be designed.
14 Chapter 3: Theory of Operation used to determine titrate concentration and/or ∆H for stoichiometric reactions. Non-linear regression techniques are used to extract the equilibrium constant (K) as well as ∆H from the thermogram for reactions that do not go to completion. Applications Batch/Incremental Titration In incremental or batch titration, one of the reactants is placed in a syringe or burette external to the reaction vessel.
CSC 5300 N-ITC III 15 Figure 3-3: Baseline heat outlined along the bottom of the chart which is used when calculating the area of each peak. Figure 3-4: Total heat versus time for the incremental titration of a reactant that reacts incompletely with added titrant.
16 Chapter 3: Theory of Operation Titration/Data Analysis A single titration calorimetric experiment yields heat data as a function of the ratio of the concentrations of the reactants. Titration data, in the form of heat change versus volume of titrant added, can be examined for both analytical (thermometric titrimetry) and thermodynamic (titration calorimetry) information.
CSC 5300 N-ITC III 17 shows that increased overall curvature of the thermogram is generated with decreasing values of the association constant, Keq. The total heat observed is directly proportional to ∆HR, as illustrated in Figure 3-5b. It follows that the lower the K value, the higher ∆HR must be to calculate Keq and ∆H values with a given reliability. The limiting values of K and ∆H must simultaneously yield a curved plot of qR versus moles of titrant and an accurately measured heat.
18 Chapter 3: Theory of Operation Qi = ∑ qi = ∆H i Ci Vi i Equation 3-2 In order to determine binding constants and enthalpy changes from the calorimetric data we need to have a model for the average excess enthalpy terms. The simplest binding model is for one ligand binding to each protein with a binding constant of K and a binding enthalpy of ∆H. K for this system is expressed as: K= [MX] [M][X] Equation 3-3 where M represents the protein (or any other macromolecule) and X represents the ligand.
CSC 5300 N-ITC III 19 which can then be substituted into Equation 3-1 or Equation 3-2. Unfortunately, Equation 3-6 is given in terms of the concentration of free ligand, [X], whereas the quantity we control in the experiment is the total concentration of ligand, [X]tot. We thus need to express Equation 3-6 in terms of the total ligand concentration.
20 Chapter 3: Theory of Operation site per protein based on structural information, the experimental value of N may still vary due to error in protein concentration estimation or to degradation of the protein sample. For experiments performed under conditions in which K can be determined, the value of N does not affect the fitted value of K or ∆H. Consider the case in which we have two independent binding sites on the protein, but each site has a different binding constant.
CSC 5300 N-ITC III 21 Freire, E., Mayorga, O. L. & Straume, M. (1990). Isothermal Titration Calorimetry. Anal. Chem. 62, 950A-959A. Wiseman, T., Williston, S., Brandts, J. F. & Lin, L.-N. (1989). Rapid Measurement of Binding Constants and Heats of Binding Using a New Titration Calorimeter. Anal. Biochem. 179, 131-137. Wyman, J. & Gill, S. J. (1990). Binding and Linkage: The Functional Chemistry of Biological Macromolecules (University Science Books, Mill Valley).
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CSC 5300 N-ITC III 23 Chapter Four: Hardware Description Description of Parts and Functions The CSC Model 5300 N-ITC III consists of the measuring unit (calorimeter block and two non-removable reaction vessels), the burette assembly, which includes the stirring system, and a cleaning accessory. With the exception of the power on/off switch located on the back of the calorimeter unit, all functions of the N-ITC III are controlled remotely by the computer through the USB connection.
24 Chapter 4: Hardware Description Figure 4-1). The power required to maintain this zero difference is used as the calorimeter signal and is monitored as a function of time. If a reaction occurs in the sample cell that produces heat, the heat required to maintain the zero difference decreases by the amount of heat supplied by the reaction, resulting in a peak in the thermogram.
CSC 5300 N-ITC III 25 Extreme care should be taken not to bend the syringe needle because this would impair proper stirring and possibly damage the reaction vessel. Each syringe needle is equipped with a flattened, slightly twisted paddle at the tip which does the actual stirring of the solutions in the cell. The stirring paddle spins clear of the sides of the reaction vessel.
26 Chapter 4: Hardware Description Figure 4-3: Inserting the syringe into the burette shaft. Figure 4-4: Finger-tightening the syringe in the burette shaft. rotating shaft of the burette assembly (Figure 4-3). The rotating shaft on the burette is held secure in one hand, and using the knurled knob at the base of the syringe barrel, the syringe is finger-tightened into place with the other hand (Figure 4-4).
CSC 5300 N-ITC III 27 Figure 4-6: Relative positions and orientation of the burette, syringe, needles, and cells during an experiment.
28 Chapter 4: Hardware Description the calorimeter is the reverse of this process. When the burette is installed properly, the circular contact boards at the burette/calorimeter interface provide electrical power to the burette and enable the functional control necessary to perform a titration. Figure 4-6 shows the relative positions of the burette, syringe, needles, and cells during an experiment.
CSC 5300 N-ITC III 29 Power Supply The calorimeter has an autosetting power supply which may be used with input voltage ranges of both 90-130 VAC and 180-260 VAC, 47-63 Hz. No manual changes to the instrument are required when switching from one power source to another, except for the frequency setting in the ITCRun software (see Chapter 5: Software Description).
30 Chapter 4: Hardware Description adapter into the sample cell, and prevents side-to-side movement which could bend the tool and damage the cell. Do not use the cleaning adapter without the support disk in place as this may cause the adapter needle to bend and possibly damage the reaction vessel. 3. Moisten the O-ring on the shaft of the cleaning tool just enough to lubricate.
CSC 5300 N-ITC III 31 5. Apply a vacuum to draw the water through the system and flush the cell. Figure 4-13 describes the flow of water through the apparatus. Figure 4-13: Sideview of the cleaning tool in the sample cell. Water is drawn into the side port inlet and down the length of the inner needle where it exits near the bottom of the cell. The water then flows upward around the needle the length of the cell and access tube where it enters the outer sleeve of the cleaning tool just below the O-ring.
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CSC 5300 N-ITC III 33 Chapter Five: Software Description Overview Software for the N-ITC III consists of two separate sections. A stand-alone acquisition program called ITCRun controls experiment setup and data collection, while BindWorks provides a full-featured data analysis program. ITCRun Software Installation The software for your Isothermal Titration Calorimeter was previously installed at the factory. However, a software installation CD is included in the event you need to reinstall the software.
34 Chapter 5: Software Description ITCRun will initialize and the data acquisition screen shown below will appear (Figure 5-2). The key features of this display are also shown for reference. Detailed descriptions of the operations associated with these features follow. Main Menu Toolbar Temp. & Heat Effect Readout Feature Tabs Status Bar Figure 5-2: The initial ITCRun program display.
CSC 5300 N-ITC III data file for review. Also appears on the toolbar as 35 . Shortcut Key: CTRL+O File • Save As Saves the current data file to a specified file name. Use this command to save and name (or rename) the current data file. To save a document using its existing name and directory, use the icon on the toolbar. File • Exit Exits the ITCRun program. Use this command to end your ITCRun session. ITCRun prompts you to save documents with unsaved changes.
36 Chapter 5: Software Description commands are described below. Experiment • Settings The Experiment Settings command opens the settings dialog box (Figure 5-6). Also appears on the toolbar as . The PID Control portion of the settings screen controls the N-ITC III temperature set points and PID loop variables. The user should not change any of the parameters in this area with the exception of the Temperature Set variable, which shows the current operating temperature of the N-ITC III.
CSC 5300 N-ITC III 37 5 µL x 7000 x 35 ms/Step = 12250 ms = 12.25 seconds 100 µL Equation 5-2 The actual injection rate in terms of volume, therefore, is 5 µL/12.25 s = 0.408 µL/s. The settings in the Filter Control area of the Settings window should not be changed by the user. The Power setting should indicate the proper frequency of the local line voltage. The user may define a Setup Password to limit access to the Settings dialog box.
38 Chapter 5: Software Description Buret • Move Down The Buret Move Down command moves the buret to the fully lowered position. Also appears on the toolbar as . Help Menu Figure 5-8 shows the help menu. If a help file is not available, it will be implemented in later revisions of ITCRun. Figure 5-8: The Help Menu. Help • About ITCRun... The Help About ITCRun command displays the software version number and other information about ITCRun.
CSC 5300 N-ITC III 39 be used. Click on the stirrer on box to turn on stirring. Syringe Size (µL) Syringe size (either 100 µL or 250 µL) for the current titration. Experiment Type Choose chemical titration or electrical calibration. If chemical titration is chosen, choose the injection volume in µL needed for the experiment. Figure 5-9: The Setup Feature Tab. If electrical calibration is chosen, choose the pulse size in µJ. Temperature Control – Set Point (°C) This sets the control temperature of the ITC.
40 Chapter 5: Software Description Setup Button Used to enter the basic parameters of an experiment (Figures 5-10 and 5-11). The parameters are as follows: • Injection or Pulse Interval The time interval, in seconds, between injections or electrical pulses. • Injection Volume or Pulse Size The size in µL for an injection or µJ for a pulse. • Number of Injections or Pulses The number of injections or electrical pulses required for the experiment. Figure 5-10: Injection Setup.
CSC 5300 N-ITC III 41 Reset chart This option completely clears the chart of data. Choosing this option does not abort or stop the experiment. Rescale chart This option will autoscale the y-axis on the virtual strip chart. Figure 5-12: The Monitor Feature Tab. Auto This option allows the user to manually scale the chart to the values shown in the two text boxes to the right of the Auto check box. Data Tab Figure 5-13 shows the Data tab.
42 Chapter 5: Software Description on the data tab my be enlarged by holding down the left mouse button and dragging a window around the desired region. To restore the full data display click once on the “Zoom Out” icon which appears above the display as . Show baseline Appears on the display as contours of the data. . This icon displays a basic baseline that follows the Subtract baseline Appears on the screen as . Selecting this icon visually corrects the displayed data for baseline drift.
CSC 5300 N-ITC III 43 Conical Overflow Reservoir Holds Excess Liquid as Titrant is Added Fill Level Figure 5-14: The filling syringe used to flush and fill the cells. slowly to allow air bubbles to evacuate through the top of the cell. When liquid is just visible at the opening of the access tube, continue to apply positive pressure to the syringe while slowy withdrawing it from the cell to maintain the fill level and prevent new bubbles from being introduced into the cell (Figure 5-15a).
44 Chapter 5: Software Description Figure 5-16: Inserting the syringe into the burette shaft. Figure 5-17: Finger-tightening the syringe in the burette shaft. then screw the syringe completely into the buret drive as shown in Figures 5-16 and 5-17. 3. Insert the syringe and burette drive into the N-ITC III as shown in Figure 5-18. Make sure that the burette key slots line up correctly with the syringe position slot located towards the front of the N-ITC III.
CSC 5300 N-ITC III 45 Program indicator in ITCRun will flash. 7. When the titration has concluded, select File⇒Save to store the data to disk. 8. Evaluate the data using the Bindworks software package. BindWorks Overview BindWorks is a computer program that aids in analysis and revision of data derived from titration calorimeters. Figure 5-19: The Setup tab showing experiment parameters.
46 Chapter 5: Software Description Getting Started Installing BindWorks Before installing BindWorks close any other “Visual” applications. These include programs written in Visual Basic, Visual C, or other similar applications. If visual applications are running on your system when you try to install BindWorks an error message will result. It is only necessary to close these applications during the install process. You may re-open these applications after completing the BindWorks installation procedure.
CSC 5300 N-ITC III 47 2. Select File⇒Open from the main menu. 3. Locate the data file from the appropriate file folder and click OK. 4. The data file is loaded into BindWorks. A titration data file of RNase A – 2’ CMP is shown in the example. When your data file is loaded, BindWorks will create a baseline based on the data before and after the injection. Figure 5-20: A sample data file and the position of the View Baseline icon.
48 Chapter 5: Software Description Data marker(s) Injection label / injection window Baseline node Figure 5-21: The baseline, baseline nodes, and data markers. Figure 5-22: Viewing the area of the integration. Calorimetry Sciences Corp.
CSC 5300 N-ITC III 49 As you change the baseline or the position of the injection window relative to the injection, the integration area of the peak will change accordingly. To view the overall affect of your change on the integrated peaks, click the Area icon. A graph of the integrated area will appear (Figure 5-22). When you have completed the changes you can save your changes to the raw data file by using the File⇒Save or File⇒ Figure 5-23: The area tab allows access to the experiment spreadsheet.
50 Chapter 5: Software Description The initial Area display is shown in Figure 5-23. If you did not enter values for Ligand, Macromolecule, and cell volume when you ran your experiment, enter the values in the appropriate box. If, for some reason, a parameter of a particular injection (or injections) changed, you can revise the value in the data spreadsheet. To change a value, highlight it, then enter the new value in the data entry box (Figure 5-24).
CSC 5300 N-ITC III 51 Modeling Modeling Overview Each model contains a set of variable parameters. Curve fitting produces optimum values for these parameters. These values will best represent the given input data set. In addition to user-defined binding models, BindWorks supports three intrinsic binding models: 1. Independent Set of Multiple Binding Sites. 2. Two Sets of Multiple Binding Sites. 3. Cooperative Model. Intrinsic models are “hard-wired” in BindWorks and cannot be changed or replaced.
52 V Chapter 5: Software Description - is the volume of the cell [M] - is the protein concentration 2K2 1/2K1 [L] - is the total Ligand concentration 2K1 (For more information see Freire E.; Mayorga O.; Straume M. Analytical Chemistry, vol. 62, NO 18, 1990.) 1/2K1 1/2K2 2K2 2K1 2K2 1/2K2 1/2K1 Two Sets of Multiple Binding Sites This model assumes the macromolecule 1/2K2 2K1 has many independent binding sites (at least two).
CSC 5300 N-ITC III 53 Rectangular regions represent the subunits to which the Ligand is bound. Closed-form equations for this model are as follows: Figure 5-27: Equations for a multiple independent binding site model. (For more information see Freire E.; Mayorga O.; Straume M. Analytical Chemistry, vol. 62, NO 18, 1990.
54 Chapter 5: Software Description Cooperative Binding Model This model assumes at least two binding sites on the macromolecule as well as interaction between the two ligand molecules (Figure 5-28). For two binding sites the variables that define the cooperative binding are 2 sets of ∆H and β. The first set defines interaction between the macromolecule and the first ligand molecule. The second set determines the interaction between the macromolecule and second ligand molecule.
CSC 5300 N-ITC III 55 Where Xb is the amount of ligand bound per unit volume. Free ligand concentration can be obtained by solving: Xt − X − M ⋅Y = 0 Equation 5-7 Where M is concentration of macromolecule. The cumulative heat effect is: Q= Vcell ßi X i ∆H i ∑ Z i Equation 5-8 (For more details refer to J. Wyman, S. J. Gill Binding and Linkage. University Science Books 1990.
56 Chapter 5: Software Description In an experiment designed to measure K, the need to keep the product of K and Cprot near 100 must be balanced against the need for a detectable heat. For this reason, very tight binding constants cannot be determined directly unless ∆H is very large so that very low concentrations of protein can be used. Competition experiments provide an alternative means of determining tight binding energetics.
CSC 5300 N-ITC III 57 Enter 1 mM for the ligand concentration and 0.1 mM for the macromolecule concentration. This gives a product of K and Cprot of 100 (using molar concentration value of the protein). The resulting data now show excellent curvature and significant heat effects indicating ideal experimental conditions. (See Figure 5-30). Figure 5-30: BindWorks Experiment Design Tab — Illustration of a well designed experiment.
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CSC 5300 N-ITC III 59 Chapter Six: Sample Experiments Experiment Overview Following is a brief description of the main steps of a titration experiment. The next segment, Experiment Walk-Through, provides a more detailed presentation of a typical experiment from start to finish. Read through these segments completely before performing any of the operations described. 1. Load the sample cell with titrate and the reference cell with the appropriate buffer or water.
60 Chapter 6: Sample Experiments Experiment Walk-Through Introduction This chapter contains descriptions of two well characterized titration experiments which utilize materials readily obtainable and provide new users with the opportunity to develop techniques and skills essential for the effective use of the N-ITC III. The first experiment, heat of protonation of Tris base may also be used as a chemical calibration to verify performance of the calorimeter and its settings.
CSC 5300 N-ITC III 61 College Publishing, Philadelphia), p. 228 ff). Experiment Setup Experiment parameters: Equilibration time 200 s Time between injections 200 s Injection size 5 μL Number of injections 20 Rinse the calorimeter cell three times with Tris solution and then load the cell (Figure 6-1). The reference cell may be filled with degassed water. Allow the cells to thermally equilibrate until the heat reading on the calorimeter is stable. Load the 100 μL syringe with the 1.
62 Chapter 6: Sample Experiments and click the burette up icon to initialize the plunger position.) Insert the syringe and burette drive into the N-ITC III as shown in Figure 6-3. Turn on the stirrer at 150 rpm, allow the system to re-equilibrate until the heat reading on the calorimeter is stable, and then begin the experiment. Enter a file name for the data at the prompt. Figure 6-3: Inserting the burette drive into the N-ITC III. The results should be similar to those shown in Figure 6-4.
CSC 5300 N-ITC III 63 where t is given in °C†. † Christensen, J.J., Hansen, L.D. and Izatt, R.M. (1976) Handbook of Proton Ionization Heats and Related Thermodynamic Quantities (John Wiley and Sons, New York). Sample Experiment Binding of 2’-CMP to RNase A Sample Preparation Prepare 4 L of 15 mM acetate buffer (pH 5.5) by dissolving 5.89 g of potassium acetate, KC2H3O2 (FW 98.14), in 4 L of distilled water and adjusting the pH using an HCl solution.
64 Chapter 6: Sample Experiments The presence of bubbles in the solutions can result in noisy baselines or spikes during an experiment. It is therefore necessary to remove dissolved gasses from the solutions. Sample degassing is most important for experiments being run at or above ambient temperature since gasses become less soluble with increasing temperature. Degassing is more efficient if the solution is being stirred, but stirring may not be practical for small volumes of solution.
CSC 5300 N-ITC III 65 Blank Titration The heat effect measured in a titration experiment includes the heat of dilution of the titrant, in addition to the heat generated from the binding reaction. In order to correct for the dilution heat, a blank experiment is performed which is identical to the binding experiment, but with buffer only present in the cell.
66 Chapter 6: Sample Experiments program main screen (Figure 6-7). Enter the ligand and macromolecule concentrations (based on the spectroscopic determination) and the cell volume. Check the Constant Cell Volume box. To subtract the blank run, click on the Area label at the top of the third table column to highlight the entire column, then type in an equal sign followed by a minus sign (=-) followed by the average blank injection area, then click the execute button to the right of the edit box.
CSC 5300 N-ITC III 67 the tip of the injection needle during equilibration, which results in fewer moles of titrant being injected in the first injection. If you want to discard this point in the fit then position the mouse pointer directly over the point and click the left button once. The point should be replaced by a red triangle, indicating that the integrated area will not be used in the fit. Next, click the Fit Selected Model button.
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CSC 5300 N-ITC III 69 Chapter Seven: Troubleshooting Minimizing Blank Corrections There will always be blank corrections for experiments. However, minimizing the blank correction can greatly improve experiments. Even when injecting water into water there will be some heat produced due to viscous mixing. The viscous mixing heat is determined by many factors. There are several steps that can be taken in order to minimize the dilution heats and hence the need for blank titrations.
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