KR102242465B1 - Solid-state nuclear magnetic resonance probe for analyzing material and biomembrane containing phosphorus - Google Patents

Abstract

Provided is a solid-state nuclear magnetic resonance probe for analyzing a phosphorus-containing material and a biological membrane. According to an embodiment of the present invention, the solid-state nuclear magnetic resonance probe for analyzing a phosphorus-containing material and a biological membrane comprises: a coil module where a coil is wound to surround a phosphorus-containing sample to apply a magnetic field to the sample; a sample plate where the coil module is installed; and an RF unit composed of a 1-H-31-P double resonance circuit for observing phosphorus contained in the sample. Herein, the coil is provided to surround the sample in a scroll shape.

Description

Solid-state nuclear magnetic resonance probe for analyzing material and biomembrane containing phosphorus

The present invention relates to a solid-phase nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological membrane.

In general, Phosphorus is one of the most important components that make up a human biofilm. In particular, phosphorus is important for lipid bilayer packing, phase transition (gel phase, physiological liquid crystal phase, ripple phase, non-double phase), lipid head group orientation/dynamics, and the elastic properties of pure lipid bilayers and binding of proteins and other biomolecules. Plays a role.

In addition, the observation of phosphorus constituting the biological membrane plays a very important role in studying the interaction of the biological membrane with many kinds of proteins in the human body. Therefore, there is a need for a way to observe this.

US 9995801 B2

In order to solve the problems of the prior art as described above, an embodiment of the present invention is a material containing phosphorus suitable for experiments in various nuclear magnetic resonance spectroscopy observing ¹ H or ^{31 P, and solid-phase nuclear magnetic resonance for biofilm analysis.} I want to provide a probe.

However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

According to an aspect of the present invention for solving the above problems, a coil module is wound around a sample containing phosphorus to apply an electric field to the sample; A sample plate on which the coil module is installed; ^{And an RF unit consisting of a 1} H- ³¹ P double resonance circuit for observing phosphorus contained in the sample, wherein the coil is a material including phosphorus provided to surround the sample in a scroll shape, and A solid-phase nuclear magnetic resonance probe for biomembrane analysis is provided.

In one embodiment, the coil may be disposed in the center of the sample plate and may have a circular side surface.

In an embodiment, the solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm may further include a fixing member for fixing the coil to the specimen plate so as to maintain the shape of the coil. Here, the fixing member may be made of a phosphorus-free material.

In one embodiment, the ¹ H- ³¹ P double resonance circuit includes a first capacitor connected to one end of the coil, and a first capacitor ^{disposed between the first capacitor and the 1} H terminal, and between the first capacitor and the ground. variable and a capacitor, ¹ H channel tuning unit for performing frequency tuning and frequency matching of the proton nuclide ⁽¹ H); 3 is disposed between the second capacitor and the second capacitor and the ³¹ P terminal and between the second capacitor and a second variable capacitor that is disposed between the ground and the second capacitor and the ground connected to the other end of the coil ³¹ P channel tuning part comprising a capacitor, and perform frequency tuning and frequency matching of the nuclide ⁽³¹ P); And a coaxial cable connected between a connection point of the first capacitor and the first variable capacitor and a ground.

In one embodiment, the channel tuning unit ¹ H tuning range is 398 ~ 402 ㎒, the ³¹ P-channel tuning unit tuning range may be 157 ~ 166 ㎒.

In one embodiment, the coaxial cable may have a length of 1/4 of the resonance wavelength.

In an embodiment, the solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological film may include a temperature control module installed under the RF unit; And a cylindrical non-magnetic case surrounding the sample plate, the RF unit, and the temperature control module. Here, the temperature control module is a thermocouple temperature sensor for measuring the temperature of the coil module; A ceramic heater for increasing a temperature according to the measured temperature; And a dewar for lowering the temperature according to the measured temperature.

In one embodiment, the frequency of the current pulse supplied to the coil may be 400 MHz.

In one embodiment, the solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm may further include a coil cap that is detachably coupled to surround the coil module. Here, the coil cap may be made of polyethylene.

The solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention has a ¹ H- ³¹ P double resonance circuit, so it is suitable for observation of ¹ H or ^{31 P.} The properties of materials and biofilms can be easily analyzed.

In addition, the solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention has a scroll-shaped coil, so that a signal with much better pulse efficiency and better sensitivity than a solenoid coil can be obtained. Therefore, the reliability of observation can be improved.

1 is a perspective view of a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention,
2A to 2F are photographs of a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological membrane according to an embodiment of the present invention,
3 is a circuit diagram of the RF unit of FIG. 1,
4 is a graph showing the ³¹ P chemical shift magnetic resonance spectrum obtained using a solid NMR probe for material analysis and biological membranes, including in accordance with one embodiment of the present invention,
Figure 5 is a graph showing the ³¹ P NMR spectra in accordance with the obtained temperature using a solid state NMR probe for material analysis and biological membranes, including in accordance with one embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art may easily implement the present invention. The present invention may be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and the same reference numerals are assigned to the same or similar components throughout the specification.

The embodiments of the present invention are provided to more fully describe the present invention to those of ordinary skill in the art, and the embodiments described below may be modified in various other forms, and The scope is not limited to the following examples. Rather, these examples are provided to make the present invention more faithful and complete, and to fully convey the spirit of the present invention to those skilled in the art.

In the drawings, for example, depending on manufacturing techniques and/or tolerances, variations of the illustrated shape can be expected. Accordingly, the embodiments of the present invention should not be construed as being limited to the specific shape of the region shown in the present specification, but should include, for example, a change in shape caused by manufacturing.

1 is a perspective view of a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention, and FIGS. 2A to 2F are a material containing phosphorus according to an embodiment of the present invention, and A photograph of a solid-state nuclear magnetic resonance probe for biofilm analysis, and FIG. 3 is a circuit diagram of the RF unit of FIG. 1.

1 and 2A to 2F, a solid-state nuclear magnetic resonance probe 100 for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention includes a coil module 110 and a sample plate 120. , RF unit 130, temperature control module 140, non-magnetic case 150, and includes a port 160 and a coil cap 170.

Here, FIG. 2A is a photograph of the solid-state nuclear magnetic resonance probe 100. 2B is a photograph of the head of the solid-state nuclear magnetic resonance probe 100 with the case cover 150a removed. 2C is a photograph of a state in which the coil cap 170 is covered for an efficient temperature experiment. 2D to 2F are photographs of the fabrication of the coil 112 and mounting on the solid-state nuclear magnetic resonance probe 100.

The solid-state nuclear magnetic resonance probe 100 for analyzing phosphorus-containing materials and biological membranes is a dual resonance solid-state nuclear magnetic resonance probe for 400MHz narrow-bore magnets for analyzing phosphorus-containing biological membranes and various materials. .

Here, all signal transduction processes related to human sensory organs are the result of complex interactions between proteins and cells, and all these processes occur on the outer wall of cells. At this time, complex interactions mostly occur on the outer walls of cells made of phospholipids, and structural or morphological modifications of the outer walls can be an important indicator to confirm whether protein-cell interactions are well performed.

In addition, receptors that can accept signaling substances or proteins that play the most important role in maintaining homeostasis are also present on the outer wall of cells, and structural modifications of signaling substances or protein receptors also have a close correlation with structural modifications of phospholipids. .

Therefore, observing and analyzing the phosphorus of various phospholipids constituting the cell membrane plays a very important part in the pathological analysis of the correlation in which the onset of disease occurs when the human homeostasis or homeostasis is broken due to external factors.

On the other hand, phosphorus is widely used not only in biological membranes, but also in various materials such as plastics, glass, various organic and inorganic synthetic materials, synthetic catalysts, and pharmaceuticals. In particular, since it is also an essential element for human body, it is widely used for making bio-friendly materials.

For the analysis of various materials and biofilms including phosphorus as described above, the present invention uses a ¹ H- ³¹ P double resonance circuit and a scroll-shaped coil.

Here, in the solid-state nuclear magnetic resonance probe 100 according to the present invention, all materials may be made of a non-magnetic material.

The coil module 110 may apply an electric field to the sample by winding the coil 112 around the sample containing phosphorus. In this case, the coil 112 may be provided to surround the sample in a scroll shape. 2E and 2F, the coil 112 may be positioned at the center of the specimen plate 120 so as to be disposed under a uniform magnetic field. In addition, the coil 112 may be arranged to maintain a circular shape that is not crushed as a whole when viewed from the side.

As shown in FIG. 2D, the coil 112 is formed by winding a copper plate having a constant width in a cylindrical shape. Accordingly, when the copper plate is bent and rolled by coating the copper plate with Teflon tape, the coil 112 may directly contact each other, thereby preventing an arcing problem that occurs when a pulse is applied.

Here, the fixing member 114 may fix the coil 112 to the specimen plate 120 to maintain the shape of the coil 112. 2B and 2E, the fixing member 114 may fix the coil 112 so as to maintain the coil 112 in a cylindrical shape. In addition, the fixing member 114 may fix the coil 112 at the center of the coil 112. Accordingly, since the coil 112 can maintain the cylindrical shape of the sample from the outside, when a pulse is applied, a uniform magnetic field can be formed.

In this case, the fixing member 114 may be made of a phosphorus-free material. Accordingly, the fixing member 114 can exclude the influence of the magnetic field on the sample containing phosphorus.

In this way, when a current pulse is applied to the coil 112 wound on the sample containing phosphorus, a magnetic field is generated inside the coil in a direction perpendicular to the winding direction of the coil 112. An electric field is induced in the vertical direction. Here, the frequency of the current pulse supplied to the coil 112 may be 400 MHz.

Meanwhile, since a nuclear magnetic resonance experiment is generally performed by applying a pulse to a coil, in the case of repeated experiments, the temperature of the coil increases, which may cause a problem that the temperature of the sample inside the coil increases unintentionally.

However, in the solid-state nuclear magnetic resonance probe 100 according to an embodiment of the present invention, a relatively small electric field is generated inside the sample in the coil 112. Due to this, the problem of increasing the temperature of the sample may be reduced.

In addition, when measuring various types of samples having different dielectric properties, there is an advantage that the tuning range does not change significantly.

In addition, when a 2D or 3D nuclear magnetic resonance experiment is performed, a higher pulse can be applied to decoupling, so that a result value with good resolution can be obtained.

In addition, it is possible to minimize the cycle delay (waiting time until the coil and circuit are stabilized until the next pulse after applying a pulse once) due to an increase in the temperature of the sample. Therefore, it is possible to carry out many accumulation experiments within a given time.

The sample plate 120 may have a coil module 110 installed thereon. As shown in FIGS. 2B and 2E, the sample plate 120 may be provided in a circular shape to be provided in the non-magnetic case 150. In addition, the sample plate 120 may be made of polyethylene.

The RF unit 130 may be formed of ^{a 1} H- ³¹ P double resonance circuit for observing phosphorus contained in the sample. That is, the RF unit 130 ^{may be implemented as a 1} H- ³¹ P double resonance circuit in a 400 MHz narrow bore magnet. The RF unit 130 may be provided under the sample plate 120.

Referring to FIG. 3, the ¹ H- ³¹ P double resonance circuit may include a ¹ H channel tuning unit 132, a ³¹ P channel tuning unit 134, and a coaxial cable 136.

¹ H channel tuning unit 132 may perform the frequency tuning and frequency matching of the proton nuclide ⁽¹ H). In other words, ¹ H channel tuning unit 132 may perform the frequency tuning (tuning) the frequency matching (matching) for the high frequency channel ⁽¹ H terminal) of the 400㎒.

Here, ¹ H channel tuning unit 132 may comprise a first capacitor (C5) and a first variable capacitor (C1, C2).

The first capacitor C5 may be connected to one end of the coil L1. A first variable capacitor (C1) may be disposed between the first capacitor (C5) ¹ H terminal. The first variable capacitor C2 may be disposed between the first capacitor C5 and the ground.

^{The 31} P channel tuning unit 134 may perform frequency tuning and frequency matching of the ^{phosphorus nuclide 31 P.} That is, the ³¹ P channel tuning unit 134 may perform frequency tuning and frequency matching for a ^{161 MHz low frequency channel (31 P terminal).}

Here, ³¹ P-channel tuning unit 134 may include a second capacitor (C6), the first variable capacitor (C3, C4) and a third capacitor (C7). The second capacitor C6 may be connected to the other end of the coil 112. A second variable capacitor (C3) may be disposed between the second capacitor (C6) and ³¹ P terminal. The second variable capacitor C4 may be disposed between the second capacitor C6 and the ground. The third capacitor C7 may be disposed between the second capacitor C6 and the ground. That is, the third capacitor C7 may be connected in parallel with the second variable capacitor C4.

In this case, the capacitors include a quartz dielectric. For example, the first variable capacitors C1 and C2 and the second variable capacitors C3 and C4 have a variable capacitance of 1 to 10 ㎊, and the first capacitor C5, the second capacitor C6, and The third capacitor C7 may have capacitances of 10 ㎊, 5.6 ㎊, and 3.9 ㎊, respectively.

The coaxial cable 136 may be connected between the connection point of the first capacitor C5 and the first variable capacitors C1 and C2 and the ground. In addition, the coaxial cable 136 may have a length of 1/4 of the resonance wavelength λ. At this time, the length of the coaxial cable 136 (Length of λ/4 cable) is calculated as follows.

Here, C is the velocity of light, ε is the dielectric constant of the coaxial cable, and υ is the resonance frequency of the ^{1 H nuclide.}

Meanwhile, all RF components forming the RF unit 130 are soldered to a circuit connected to the ground. In particular, for reliable grounding between the RF component and the case cover 150a, brass fingers may be used. In addition, ^{it is preferable that the impedance of the 1} H- ³¹ P double resonance circuit is 50 Ω.

^{Electrical measurements of the 1} H- ³¹ P double resonance circuit can be made through a network analyzer (eg, Hewlett Packard 85046A). The final tuning range is a high frequency channel, that is, the tuning range of the ¹ H channel tuning unit 132 is 398 to 402 MHz, and the low frequency channel, that is, ^{the tuning range of the 31} P channel tuning unit 134 is 157 to 166 MHz. Can have a wide range of.

Accordingly, the solid-state nuclear magnetic resonance probe 100 according to an embodiment of the present invention can test various types of samples containing phosphorus.

Referring back to FIG. 1, the temperature control module 140 may adjust the temperature of the solid-state nuclear magnetic resonance probe 100 for various temperature change experiments. In addition, the temperature control module 140 may be installed under the RF unit 130.

In this case, the temperature control module 140 may include a ceramic heater, a thermocouple temperature sensor, and a dewar. The thermocouple temperature sensor may measure the temperature of the coil module 110. The ceramic heater may increase the temperature according to the temperature measured by the thermocouple temperature sensor. The Dewar may lower the temperature according to the temperature measured by the thermocouple temperature sensor.

In addition, after the solid-state nuclear magnetic resonance probe 100 enters the magnet, ^{resonance measurement by the 1} H- ³¹ P double resonance circuit is performed by software built into the nuclear magnetic resonance analyzer (for example, Bruker's spectroscopy software (Top spin spectrometer software)).

The non-magnetic case 150 may surround the sample plate 120, the RF unit 130, and the temperature control module 140. The non-magnetic case 150 may have a cylindrical shape as a whole. In addition, the non-magnetic case 150 may include a case cover 150a and a case body 150b.

For example, the non-magnetic case 150 may have a cylindrical shape having an inner diameter of 39.1 mm and an outer diameter of 39.5 mm. In addition, the non-magnetic case 150 may be made of aluminum 6061 pipe.

The port 160 may be a signal measurement terminal connected to a terminal of ^{the 1} H- ^{31 P double resonance circuit of the RF unit 130.} I.e., port 160 may be connected to each terminal ¹ H and ³¹ P terminal.

Referring to FIG. 2A, the port 160 may be disposed to be exposed from the lower portion of the non-magnetic case 150 to the front side. Here, when the solid-state nuclear magnetic resonance probe 100 is inserted into an external unit generating a magnetic field, a fixing plate 152 for fixing the coil module 110 to be located at the center of the magnetic field may be provided.

The fixing plate 152 may have a hole in the center so as to be inserted into the non-magnetic case 150. In addition, the fixing plate 152 may be provided with coupling holes along its edge. That is, the fixing plate 152 may be coupled to an external unit that generates a magnetic field through coupling holes at the edges.

The coil cap 170 may be coupled to an upper portion of the coil module 110 so as to surround the coil module 110. As shown in FIG. 2C, the coil cap 170 may be attached to the specimen plate 120 in a detachable form. In addition, the coil cap 170 may be made of polyethylene.

With this configuration, the solid-state nuclear magnetic resonance probe 100 for analyzing a material containing phosphorus and a biological membrane according to an embodiment of the present invention is suitable for observation of ¹ H or ^{31 P.} The characteristics can be easily analyzed, and since the pulse efficiency is much better than that of a solenoid coil and a signal with good sensitivity can be obtained, the reliability of observation can be improved.

Hereinafter, an example of a solid-state nuclear magnetic resonance spectroscopy experiment performed using the solid-state nuclear magnetic resonance probe 100 for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention will be described. ^{Here, a 400 MHz home-built solid-state nuclear magnetic resonance probe having a 1} H- ³¹ P double resonance channel equipped with a 5 mm scroll-shaped coil 112 was fabricated, and a biological membrane sample analysis experiment was performed according to temperature.

1. Preparation of the sample to be analyzed

^In order to observe the signal of the biomembrane sample containing _{31P, a reference sample of 85% H 3} PO ₄ and a similar biomembrane sample of bicelle were used. Both samples were put into each 5 mm OD round bottom tube, and the tube was closed with a rubber stopper, and then sealed with parafilm and Teflon tape. Thereafter, the tube was put into the scroll coil 112 of the solid-state nuclear magnetic resonance probe to prepare an experiment.

2. Solid phase nuclear magnetic resonance test process

Solid phase nuclear magnetic resonance experiments were performed using a 400 MHz (9.4 [Tesla]) narrow-bore magnet, a Bruker Avance III HD Spectrometer, and a solid phase nuclear magnetic resonance probe 100 (home-built probe) of the present invention. The wobble signal optimizes the tuning and matching of the probe circuit located inside the magnet, ^{and the experiment to obtain 1} H and ³¹ P signals is a one pulse experiment without decoupling and HPDEC (High-Power DECoupling ^{) with 1 H decoupling.} ) Proceed as a pulse experiment.

3. Solid phase nuclear magnetic resonance test results

Experimental results obtained using a 400 MHz home-built probe with ¹ H- ^{31 P channel are shown in FIGS. 4 and 5.}

^{FIG. 4 is a graph showing a 31} P chemical shift nuclear magnetic resonance spectrum obtained using a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm according to an embodiment of the present invention, and FIG. 5 is a diagram of the present invention. one embodiment the solid phase is a graph showing the ³¹ P NMR spectra in accordance with the obtained temperature using the NMR probe for material analysis and biological membranes, including in accordance with the.

4, only one ³¹ P chemical shifts without the background (back ground) signal ⁽³¹ P chemical shift) represents the nuclear magnetic resonance spectrum. Signal of ³¹ P occurring in 85% H ₃ PO ₄ may be divided out from the received interference signal ¹ H, but the form is only one signal is better the efficiency of the pulse application is well decoupled from 0 ppm occurred.

5, shows a spectrum observed in accordance with the ³¹ P signal of the phospholipids that form the similar biological membranes (bicelle) to the temperature change. Here, (a) is 25°C, (b) is 30°C, (c) is 35°C, (d) is 40°C, and (e) is 45°C.

The similar biomembrane consists of three types of phospholipids: 14-O-PC/DMPG/6-O-PC. It is known that the pseudobiomembrane has the best shape at 40℃~45℃ and has the best susceptibility within a magnet.

^{In this way, it can be seen that the 31} P spectrum is divided into three segments and observed at 40°C to 45°C. This was observed in each of the three types of phospholipids mentioned above, and it can be seen that the temperature control system of the home-built probe, the efficiency of the pulse applied to the scroll coil, and the uniformity of the magnetic field are very excellent.

Although an embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiments presented in the present specification, and those skilled in the art who understand the spirit of the present invention can add components within the scope of the same idea. It will be possible to easily propose other embodiments by changing, deleting, adding, etc., but it will be said that this is also within the spirit scope of the present invention.

100: solid-state nuclear magnetic resonance probe for analyzing phosphorus-containing substances and biological membranes
110: coil module 112: coil
114: fixing member 120: sample plate
130: RF unit 132: ¹ H channel tuning unit
134: ³¹ P channel tuning unit 136: Coaxial cable
140: temperature control module 150: non-magnetic case
150a: case cover 150b: case body
160: port 170: coil cap

Claims (9)

A coil module having a coil wound around a sample containing phosphorus to apply an electric field to the sample;
A fixing member made of a phosphorus-free material and configured to fix the coil to the specimen plate so as to maintain the shape of the coil in a circular shape that is not crushed;
A sample plate on which the coil module is installed; And
^{An RF unit comprising a 1} H- ³¹ P double resonance circuit for observing phosphorus contained in the sample;
Including,
The coil is provided to surround the sample in a scroll shape,
The ¹ H- ³¹ P double resonance circuit,
A first capacitor connected to one end of the coil, and ^{a first variable capacitor disposed between the first capacitor and the 1} H terminal and between the first capacitor and the ground, respectively, and frequency tuning of the ^{proton nuclide (1 H) 1} H and channel tuning unit for performing the frequency matching; And
3 is disposed between the second capacitor and the second capacitor and the ³¹ P terminal and between the second capacitor and a second variable capacitor that is disposed between the ground and the second capacitor and the ground connected to the other end of the coil ³¹ P channel tuning part comprising a capacitor, and perform frequency tuning and frequency matching of the nuclide ⁽³¹ P); And
Including; a coaxial cable connected between the connection point and the ground of the first capacitor and the first variable capacitor,
The ¹ H channel tuning unit tuning range is 398 ~ 402 ㎒,
The ^{tuning range of the 31} P channel tuning unit is 157 to 166 MHz,
The coil is a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological film, which is formed by winding a copper plate having a constant width in a cylindrical shape. The method of claim 1,
The coil is disposed in the center of the sample plate and a solid-state nuclear magnetic resonance probe for analyzing a material and a biological film containing phosphorus having a circular side. delete delete delete The method of claim 1,
The coaxial cable is a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus having a length of 1/4 of a resonance wavelength and a biological membrane. The method of claim 1,
A temperature control module installed under the RF unit; And
The sample plate, the RF unit, and a cylindrical non-magnetic case surrounding the temperature control module; further includes,
The temperature control module,
A thermocouple temperature sensor measuring the temperature of the coil module;
A ceramic heater for increasing a temperature according to the measured temperature; And
A solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological membrane including a dewar for lowering the temperature according to the measured temperature. The method of claim 1,
A solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biological membrane having a frequency of 400 MHz of a current pulse supplied to the coil. The method of claim 1,
Further comprising a coil cap coupled in a detachable form to surround the coil module,
The coil cap is a solid-state nuclear magnetic resonance probe for analyzing a material containing phosphorus and a biofilm made of polyethylene.

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