CN117204398A - Method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model - Google Patents
Method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model Download PDFInfo
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Abstract
The invention relates to the field of raw materials of foods and cosmetics, and discloses a method for evaluating the antioxidation and anti-aging activity of an egg white protein polypeptide based on a caenorhabditis elegans model, wherein the method for evaluating the antioxidation and anti-aging activity of the egg white protein polypeptide comprises the following steps: preparing a culture medium, culturing and synchronizing nematodes, measuring the oxidative stress life of paraquat, measuring the oxidative stress life of H2O2, measuring the activity of antioxidant enzyme, measuring the normal life, measuring the lipofuscin content, measuring the spawning quantity and the like. The method for evaluating the oxidation resistance and the aging resistance of the egg white protein polypeptide based on the caenorhabditis elegans model has the characteristics of convenient operation, low cost, short period and the like, can evaluate the effect of the egg white protein polypeptide in organisms with high efficiency, and reflects biological significance.
Description
Technical Field
The invention relates to the field of raw materials of foods and cosmetics, in particular to a method for evaluating oxidation resistance and aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model.
Background
Caenorhabditis elegans (Caenorhabditis elegans), which are nematodes (Phyumnematoda), paraconchoides (Secernentetea), aphanotherhabdus (Rhabditida), aphanotheidae (Rhabditidae), and Aphanothedes (Caenochabditis), are abbreviated. Elegans includes oral, pharyngeal, intestinal, gonadal and collagen cuticle. There are two sexes, hermaphrodite and male, most individuals are hermaphrodite, and the only experimentally studied germ line for which males account for 0.05% is usually hermaphrodite N2 wild caenorhabditis elegans (C.elegans, the Bristol strainN 2). About 60% -80% of the human homologous genes of caenorhabditis elegans, including at least 42% of the human disease-related genes (Sugi et al 2016) and c.elegans do not require approval by the animal ethics committee. These advantages make c.elegans a widely used animal model, often used to study the direction of aging.
The caenorhabditis elegans has a short growth cycle, only 3-4 days are needed from egg development to adult, the average life span is 2-3 weeks under the culture condition of 20 ℃, the life cycle of C.elegans which is about 300 eggs of a hermaphrodite can be produced by the adult, the life cycle comprises four larval stages (L1-L4) and adult stages, the nematode eggs can enter the L1 stage of the larvae after being cultured for 15 hours at 20 ℃, and the nematode body type is only 250um at the moment, but the structure is not different from that of the adult. If the culture environment is proper and the nutrition is sufficient, the nematodes grow from the L1 stage to the L4 stage within about 3 days and form adults, and the nematode body size is 1-1.5mm. If there is insufficient growth conditions in stage L1, nematode growth will go to arrest and go to dormancy, a period known as the dauer period. The service life of the nematodes in the period can be as long as 6 months, and if the conditions suitable for the growth of the nematodes are reached again, the nematodes can directly skip the L3 phase to reach the L4 phase, and develop into adults. By utilizing the characteristics, the larvae in the L1 stage can be stored at the temperature of minus 80 ℃ for a long time, and then recovered when in use.
The caenorhabditis elegans is taken as a model to research the effect of the antioxidation stress. Oxidative stress in general means that in certain extreme cases, unbalance of the body's tissue and organs occurs. Studies have shown that the increase in oxygen radicals can lead to abnormal protein and accumulation of lipids, and cause oxidative damage to macromolecules such as DNA, and that the antioxidant activity in nematodes is closely related to oxidative stress. Research has demonstrated that Cirsium japonicum from different processing regimes can improve the oxidative stress of caenorhabditis elegans to varying degrees. Plant drinks rich in sterols are fed to nematodes and can improve the oxidative stress of nematodes under extreme conditions.
Nematodes are model organisms widely used in many fields such as genetic biology, gene function, aging, drug screening, etc. Caenorhabditis elegans has several advantages as a major model organism for exploring various biological processes:
(1) The structure is simple, the body is transparent, and the observation under a microscope is convenient; and the OP50 bacteria are taken as food, so that the culture is simple, and the storage and recovery are convenient.
(2) The life cycle is short, and the experimental operation is convenient; the synchronization is easy, and the interference of external factors to experiments is reduced.
(3) Having complex behaviours including mechanical and chemical perceptions; meanwhile, the human body oxidation product has similarity with human body behaviors and physiology, and the movement is slowed down and the oxidation product in the human body is increased along with the aging.
(4) 60-80% of the genes are similar to human genes, so that the research is more significant.
Disclosure of Invention
Based on the method, the invention discloses a method for evaluating the antioxidation and the anti-aging of an egg white protein polypeptide based on a caenorhabditis elegans model, which comprises the steps of culture medium preparation, nematode culture and synchronization, paraquat oxidation stress life measurement, H2O2 oxidation stress life measurement, antioxidase activity measurement, normal life measurement, lipofuscin content measurement, spawning amount measurement and the like.
Compared with the prior art, the evaluation method has the characteristics of convenient operation, low cost, short period and the like, can efficiently evaluate the function of the egg white protein polypeptide in organisms, and reflects biological significance.
In order to achieve the above experimental purposes, the invention adopts the following technical scheme
A method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model mainly comprises the following steps:
s1, preparing a culture medium: preparing nematode growth solid culture medium (NGM), LB liquid culture medium, LB solid culture medium, paraquat culture medium, and sterilizing for use.
S2, nematode culture and synchronization: and (3) marking the strain of E.coli OP50 on an LB plate, picking a single colony, culturing the single colony in an LB liquid culture medium to a certain bacterial liquid concentration, and coating the single colony on an NGM plate. Resuscitates frozen nematodes, inoculates the nematodes in NGM plates, washes and lyses the nematodes into adults with M9 buffer solution and lysis solution after oviposition, and then sucks proper amount of eggs of the nematodes on an ultra-clean workbench, drops the eggs on the NGM plates coated with E.coli OP50 with a pipettor, and places the eggs in a 20 ℃ incubator for 3 days to complete contemporaneous culture.
S3, measuring the oxidative stress life of paraquat: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, transferring the nematodes into a paraquat culture medium at 5-30mmol/L, recording the death condition and the number of residual nematodes of each group of flat nematodes every day, and picking out dead nematodes until all nematodes die.
S4, H2O2 oxidative stress life measurement: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, diluting 10-60% H2O2 for 1000 times, uniformly coating the solution, transferring the nematodes onto an NGM culture medium containing H2O2, recording the death condition of the nematodes and the quantity of the remaining nematodes every 30 minutes, and picking out the dead nematodes until all the nematodes die.
S5, measuring the activity of antioxidant enzyme: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 worms in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, preparing a nematode homogenate solution with the volume ratio of 5-30% by using M9 buffer, extracting nematode tissue proteins, and measuring the protein content, the enzyme activity of Catalase (CAT), the enzyme activity of superoxide dismutase (Superoxide dismutase, SOD), the enzyme activity of reduced Glutathione (GSH) and the content of Malondialdehyde (MDA) by using a kit.
S6, normal life measurement: picking the nematodes obtained in the step S2 into experimental group plates and control group plates, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, regarding the day as day 0, transferring the nematodes into new experimental group plates and control group plates every day, recording the death condition of the nematodes and the quantity of the residual nematodes every day, and picking out the dead nematodes until all the nematodes die.
S7, lipofuscin content determination: picking the nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 worms in each group in an incubator at 20-25 ℃, culturing for 72-168 hours, anesthetizing the nematodes with 1-20% NaN3, and placing the nematodes on a glass slide dripped with 3-20% agarose pads. The fluorescence intensity was measured by Image J software, which was observed and photographed with an inverted fluorescence microscope.
S8, measuring spawning amount: the nematodes obtained in S2 were picked up in experimental and control plates, 10-30 worms per group, placed in an incubator at 20-25℃and, from day 0, picked up into new experimental and control plates after 24h intervals until the nematodes would cease to spawn. The spawning plate is placed in an incubator at 20-25 ℃ for culturing for 24-72 hours, and then the number of nematodes is recorded.
According to the above scheme, the culture medium used for nematode culture is described in S1.
According to the scheme, the concentration of the E.coli OP50 bacterial liquid inoculated in the S2 is between 0.1 and 0.8, and the egg drop of the nematodes coated on the E.coli OP50 NGM plate is between 50uL and 300uL.
According to the scheme, the number of nematodes in each group of nematodes S3, S4, S5, S6, S7 and S8 is evenly distributed on 3-6 plates.
According to the scheme, the nematode death judgment standards of S3, S4 and S6 are that nematode individuals are regarded as death if the nematode individuals do not respond to slight mechanical stimulus; nematode counts exclude hatching and escaping nematodes in vivo.
According to the above scheme, the inverted fluorescence microscope observation in S7 is performed with a 10-30-fold objective lens at an excitation wavelength of 485nm and an emission wavelength of 530 nm.
In summary, the invention has the following advantages:
the method has the characteristics of convenient operation, low cost, short period and the like, can efficiently evaluate the function of the egg white protein polypeptide in organisms, and reflects biological significance.
Drawings
FIG. 1 is a graph comparing the evaluation results of the nematode of the present invention on paraquat stress life;
FIG. 2 is a graph comparing the evaluation results of hydrogen peroxide stress life of nematodes according to the present invention;
FIG. 3 is a graph comparing CAT antioxidant enzyme activity results in nematodes according to the present invention;
FIG. 4 is a graph showing the comparison of the results of the activity of SOD antioxidant enzyme in nematodes according to the present invention;
FIG. 5 is a graph comparing GSH-Px antioxidant enzyme activity results in nematodes according to the invention;
FIG. 6 is a graph showing comparison of MDA content results in nematodes according to the present invention;
FIG. 7 is a graph comparing the results of evaluation of the normal life of nematodes according to the present invention;
FIG. 8 is a graph showing the comparison of the evaluation results of the lipofuscin content in nematodes according to the present invention;
FIG. 9 is a graph showing the results of evaluating the egg laying amount of the present invention.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
The reagents and materials used in the present invention are commercially available unless otherwise specified.
Example 1:
preparation of a culture medium: preparing nematode growth solid culture medium (NGM), LB liquid culture medium, LB solid culture medium, paraquat culture medium, and sterilizing for use.
Nematode culture and synchronization: and (3) marking the strain of E.coli OP50 on an LB plate, picking a single colony, culturing the single colony in an LB liquid culture medium to a certain bacterial liquid concentration, and coating the single colony on an NGM plate. Resuscitates frozen nematodes, inoculates the nematodes in NGM plates, washes and lyses the nematodes into adults with M9 buffer solution and lysis solution after oviposition, and then sucks proper amount of eggs of the nematodes on an ultra-clean workbench, drops the eggs on the NGM plates coated with E.coli OP50 with a pipettor, and places the eggs in a 20 ℃ incubator for 3 days to complete contemporaneous culture.
Paraquat oxidative stress life measurement: picking the nematodes obtained in the step (2) into experimental groups and control groups, placing 180 nematodes in an incubator at 23 ℃, culturing for 96 hours, transferring the nematodes into 20mmol/L paraquat culture medium, recording the death condition of the nematodes in each group and the quantity of the remaining nematodes every day, and picking out the dead nematodes until all the nematodes die.
H2O2 oxidative stress life measurement: picking the nematodes obtained in the step (2) into experimental groups and control groups, placing 180 nematodes in each group in an incubator at 23 ℃, culturing for 96 hours, diluting 50% H2O2 for 1000 times, uniformly coating the solution on the solution, transferring the nematodes to an NGM culture medium containing H2O2, recording the death condition of the nematodes and the quantity of the residual nematodes every 30 minutes, and picking out the dead nematodes until all the nematodes die.
Antioxidant enzyme activity assay: picking nematodes obtained in the step (2) into experimental groups and control groups of plates, placing 180 worms in an incubator at 23 ℃, culturing for 96 hours, preparing a nematode homogenate solution with a volume ratio of 20% by using an M9 buffer solution, extracting nematode tissue proteins, and measuring protein content, catalase (CAT) enzyme activity, superoxide dismutase (Superoxide dismutase, SOD) enzyme activity, reduced Glutathione (GSH) enzyme activity and Malondialdehyde (MDA) content by using a kit.
Normal life measurement: picking the nematodes obtained in the step (2) into experimental group and control group plates, placing 180 nematodes in an incubator at 23 ℃, regarding the day as day 0, transferring the nematodes into new experimental group and control group plates every day, recording the death condition of the nematodes and the quantity of the residual nematodes every day, and picking out dead nematodes until all the nematodes die.
Lipofuscin content determination: picking the nematodes obtained in the step (2) into experimental group and control group plates, placing 180 worms in each group in an incubator at 23 ℃, culturing for 96 hours, anesthetizing the nematodes with 10% NaN3, and placing the nematodes on a glass slide with a 10% agarose pad. The fluorescence intensity was measured by Image J software, which was observed and photographed with an inverted fluorescence microscope.
Spawning amount measurement: picking the nematodes obtained in the step (2) into the experimental group and the control group plates, placing 20 worms in each group in an incubator at 23 ℃, picking the nematodes into the new experimental group and the control group plates after 24 hours every interval from day to day considered as day 0 until the nematodes are no longer spawned to stop. The spawning plates were placed in an incubator at 23℃and the number of nematodes was recorded after incubation for 24 hours.
Example 2:
(1) Preparation of a culture medium: preparing nematode growth solid culture medium (NGM), LB liquid culture medium, LB solid culture medium, paraquat culture medium, and sterilizing for use.
(2) Nematode culture and synchronization: and (3) marking the strain of E.coli OP50 on an LB plate, picking a single colony, culturing the single colony in an LB liquid culture medium to a certain bacterial liquid concentration, and coating the single colony on an NGM plate. Resuscitates frozen nematodes, inoculates the nematodes in NGM plates, washes and lyses the nematodes into adults with M9 buffer solution and lysis solution after oviposition, and then sucks proper amount of eggs of the nematodes on an ultra-clean workbench, drops the eggs on the NGM plates coated with E.coli OP50 with a pipettor, and places the eggs in a 20 ℃ incubator for 3 days to complete contemporaneous culture.
(3) Paraquat oxidative stress life measurement: picking the nematodes obtained in the step (2) into experimental groups and control groups, placing 180 nematodes in an incubator at 23 ℃, culturing for 96 hours, transferring the nematodes into 20mmol/L paraquat culture medium, recording the death condition of the nematodes in each group and the quantity of the remaining nematodes every day, and picking out the dead nematodes until all the nematodes die.
(4) H2O2 oxidative stress life measurement: picking the nematodes obtained in the step (2) into experimental groups and control groups, placing 180 nematodes in each group in an incubator at 23 ℃, culturing for 96 hours, diluting 50% H2O2 for 1000 times, uniformly coating the solution on the solution, transferring the nematodes to an NGM culture medium containing H2O2, recording the death condition of the nematodes and the quantity of the residual nematodes every 30 minutes, and picking out the dead nematodes until all the nematodes die.
(5) Antioxidant enzyme activity assay: picking nematodes obtained in the step (2) into experimental groups and control groups of plates, placing 180 worms in an incubator at 23 ℃, culturing for 96 hours, preparing a nematode homogenate solution with a volume ratio of 20% by using an M9 buffer solution, extracting nematode tissue proteins, and measuring protein content, catalase (CAT) enzyme activity, superoxide dismutase (Superoxide dismutase, SOD) enzyme activity, reduced Glutathione (GSH) enzyme activity and Malondialdehyde (MDA) content by using a kit.
(6) Normal life measurement: picking the nematodes obtained in the step (2) into experimental group and control group plates, placing 180 nematodes in an incubator at 23 ℃, regarding the day as day 0, transferring the nematodes into new experimental group and control group plates every day, recording the death condition of the nematodes and the quantity of the residual nematodes every day, and picking out dead nematodes until all the nematodes die.
(7) Lipofuscin content determination: picking the nematodes obtained in the step (2) into experimental group and control group plates, placing 180 worms in each group in an incubator at 23 ℃, culturing for 96 hours, anesthetizing the nematodes with 10% NaN3, and placing the nematodes on a glass slide with a 10% agarose pad. The fluorescence intensity was measured by Image J software, which was observed and photographed with an inverted fluorescence microscope.
(8) Spawning amount measurement: picking the nematodes obtained in the step (2) into the experimental group and the control group plates, placing 20 worms in each group in an incubator at 23 ℃, picking the nematodes into the new experimental group and the control group plates after 24 hours every interval from day to day considered as day 0 until the nematodes are no longer spawned to stop. The spawning plates were placed in an incubator at 23℃and the number of nematodes was recorded after incubation for 24 hours.
Results of activity evaluation:
as shown in fig. 1, fig. 2 and table 1, in the experiments of two antioxidant stresses, the growth curve of the nematode under the action of the egg white protein polypeptide is obviously shifted to the right (p is less than 0.01), and the average life, the median life and the maximum life are all increased, which indicates that the egg white protein polypeptide can relieve the antioxidant stress and has antioxidant activity in the nematode body. As shown in fig. 3, 4, 5 and 6, the enzyme activities of SOD, CAT, GSH-Px in caenorhabditis elegans are obviously increased (p < 0.01) by 0.4 times, 1.25 times and 2 times respectively compared with the control group, and the MDA in the caenorhabditis elegans is obviously reduced (p < 0.05) by 28% compared with the control group. The egg white protein polypeptide can activate the activity of antioxidant enzyme in vivo, relieve oxidative damage in vivo and has antioxidant effect. As shown in fig. 7 and table 2, the growth curve of nematodes under the action of egg white protein polypeptides is obviously shifted to the right within 0-15 days; the average life, the median life and the maximum life are all increased. The egg white protein polypeptide has the functions of resisting aging and prolonging the service life. As shown in FIG. 8, lipofuscin fluorescence in nematode after the action of egg white protein polypeptide is obviously reduced, and the fluorescence intensity is reduced by 1 time (p < 0.01). The egg white protein polypeptide can reduce accumulation of lipofuscin in vivo, and has effects of resisting oxidation and relieving aging. As shown in FIG. 9, the egg laying amount of the nematodes after the egg white protein polypeptide is not changed, and the egg white protein polypeptide has no influence on reproductive ability and is safe and harmless.
TABLE 1 evaluation results of the nematodes of the invention on paraquat stress life and hydrogen peroxide stress life
TABLE 2 evaluation results of the normal life of nematodes according to the invention
Although embodiments of the invention have been shown and described, the detailed description is to be construed as exemplary only and is not limiting of the invention as the particular features, structures, materials, or characteristics may be combined in any suitable manner in any one or more embodiments or examples, and modifications, substitutions, variations, etc. may be made in the embodiments as desired by those skilled in the art without departing from the principles and spirit of the invention, provided that such modifications are within the scope of the appended claims.
Claims (6)
1. A method for evaluating oxidation resistance and aging resistance of egg white protein polypeptide based on caenorhabditis elegans model mainly comprises the following steps:
s1, preparing a culture medium: preparing a nematode growth solid culture medium, an LB liquid culture medium, an LB solid culture medium, a paraquat culture medium and sterilizing for later use;
s2, nematode culture and synchronization: and (3) marking the strain of E.coli OP50 on an LB plate, picking a single colony, culturing the single colony in an LB liquid culture medium to a certain bacterial liquid concentration, and coating the single colony on an NGM plate. Resuscitating frozen nematodes, inoculating the recovered nematodes to an NGM plate, cleaning and lysing the nematodes to form adults by using an M9 buffer solution and a lysis solution after the nematodes lay eggs, sucking eggs of a proper amount of nematodes by using a pipette on an ultra-clean workbench, dripping the eggs on the NGM plate coated with E.coli OP50, and placing the NGM plate in a 20 ℃ incubator for culturing for 3 days to finish synchronous culture;
s3, measuring the oxidative stress life of paraquat: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, transferring the nematodes into a paraquat culture medium at 5-30mmol/L, recording the death condition and the number of residual nematodes of each group of flat nematodes every day, and picking out dead nematodes until all nematodes die;
s4, H2O2 oxidative stress life measurement: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, diluting 10-60% H2O2 for 1000 times, uniformly coating the solution, transferring the nematodes onto an NGM culture medium containing H2O2, recording the death condition of the nematodes and the quantity of the remaining nematodes every 30 minutes, and picking out the dead nematodes until all the nematodes die;
s5, measuring the activity of antioxidant enzyme: picking nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 worms in each group in an incubator at 20-25 ℃, culturing for 72-120 hours, preparing a nematode homogenate solution with the volume ratio of 5-30% by using M9 buffer solution, extracting nematode tissue proteins, and measuring the protein content, the catalase activity, the superoxide dismutase activity, the reduced glutathione enzyme activity and the malondialdehyde content by using a kit;
s6, normal life measurement: picking nematodes obtained in the step S2 into experimental group plates and control group plates, placing 150-300 nematodes in each group in an incubator at 20-25 ℃, regarding the day as day 0, transferring the nematodes into new experimental group plates and control group plates every day, recording the death condition of the nematodes and the quantity of the residual nematodes every day, and picking out dead nematodes until all the nematodes die;
s7, lipofuscin content determination: picking the nematodes obtained in the step S2 into experimental groups and control groups, placing 150-300 worms in each group in an incubator at 20-25 ℃, culturing for 72-168 hours, anesthetizing the nematodes with 1-20% NaN3, and placing the nematodes on a glass slide dripped with 3-20% agarose pads. Observing and taking a photo by using an inverted fluorescence microscope, and measuring fluorescence intensity by using imageJ software;
s8, measuring spawning amount: the nematodes obtained in S2 were picked up in experimental and control plates, 10-30 worms per group, placed in an incubator at 20-25℃and, from day 0, picked up into new experimental and control plates after 24h intervals until the nematodes would cease to spawn. The spawning plate is placed in an incubator at 20-25 ℃ for culturing for 24-72 hours, and then the number of nematodes is recorded.
2. The method for evaluating the oxidation resistance and the aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model according to claim 1, which is characterized in that: the culture medium used for nematode culture is described in S1.
3. The method for evaluating the oxidation resistance and the aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model according to claim 1, which is characterized in that: the concentration of the inoculated E.coli OP50 bacterial liquid in the S2 is between 0.1 and 0.8, and the egg drop of the nematode coated on the E.coli OP50 NGM plate is 50uL-300uL.
4. The method for evaluating the oxidation resistance and the aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model according to claim 1, which is characterized in that: the number of nematodes in each group in S3, S4, S5, S6, S7 and S8 is evenly distributed on 3-6 plates.
5. The method for evaluating the oxidation resistance and the aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model according to claim 1, which is characterized in that: the nematode death judgment standards in S3, S4 and S6 are that nematode individuals are regarded as dead if the nematode individuals do not respond to slight mechanical stimulus; nematode counts exclude hatching and escaping nematodes in vivo.
6. The method for evaluating the oxidation resistance and the aging resistance of an egg white protein polypeptide based on a caenorhabditis elegans model according to claim 1, which is characterized in that: the inverted fluorescence microscope observation described in S7 was performed with a 10-30-fold objective lens at an excitation wavelength of 485nm and an emission wavelength of 530 nm.
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