CN110082444A - The construction method of the mouse model for screening particulate matter exposure early effect marker based on lipid composition analysis - Google Patents
The construction method of the mouse model for screening particulate matter exposure early effect marker based on lipid composition analysis Download PDFInfo
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Abstract
The invention discloses a kind of construction methods for being used to screen the mouse model that particulate matter exposes early effect marker based on lipid composition analysis, its tracheal instillation for including the following steps: S1, carrying out PM2.5 particle to test mice, after experimental period, the serum of test mice is collected；S2, the data that serum is extracted and obtains LC-MS；S3, the ion extraction identification is carried out with LC-MS data described in Commonpound Discover software, again with statistical method, data processing and pattern-recognition are carried out to result is extracted, searches out the biomarker of PM2.5 sample exposure mouse lung tissue metabolism group；S4, with metabolism group Relational database and analysis software, construct the metabolic pathway of biomarker and analyzed.The present invention is the screening of early effect marker, evaluation and application provide iipidomic method.
Description
Technical field
The invention belongs to lipid-metabolism analysis technical fields, specifically design it is a kind of based on lipid composition analysis for screen
The construction method of the mouse model of grain object exposure early effect marker.
Background technique
The health risk that atmosphere pollution is caused increasingly is paid close attention to by people, and the particulate matter of different-grain diameter can cause different
Health risk.The fine particle of aerodynamic diameter≤2.5 μm is referred to as PM2.5, pays close attention in recent years by people.Due to
PM2.5 partial size is small, large specific surface area, it is easier into alveolar and rest on deep lung and is not easy to be excluded, therefore is strong to people
Health influences bigger.Lung is one of the first target organs of PM2.5 exposure, will cause serious injury of lungs, increases asthma and respiratory tract
The disease incidence of inflammation, epidemiologic data show that PM2.5 exposure has stronger correlation with lung cancer.Such as Canada and beauty
State scientist has found that long-term PM2.5 exposure will increase the disease incidence of lung cancer, 10 μ g/m of the every increasing of concentration by long-term research3,
Lung cancer mortality increases by 8%.Therefore, the early screening of lung disease is particularly important, and explores and establish a kind of quick, sensibility
High diagnostic techniques there is an urgent need to.
Metabolism group is a kind of completely new omics technology to grow up after genomics, protein science, is that system is raw
The important component of object, the advanced instruments such as LC-MS limited using high separation rate, high sensitivity, low detection, by comparing not
With the metabolic profile information change of endogenous small molecule in organism under physiological status, by information extraction, mathematics dimensionality reduction (PCA,
The methods of OPLS-DA etc.), cause the key organism marker of pulmonary metabolism disorder after identification particulate matter exposure, is lung disease
Early diagnosis provides help.
Summary of the invention
In order to disclose the harmful organism effect that atmosphere pollution causes breathing and cardiovascular system, determines all kinds of neurovirulent phenotypes, visit
The toxic action mode of atmosphere pollution is begged for, the potential marker of atmosphere pollution early effect is screened.The present invention provides one kind to be based on
The construction method of the mouse model for screening environment reference PM2.5 sample exposure early effect marker of lipid composition analysis.
The present invention is achieved by the following technical solutions:
The present invention provides a kind of mouse based on metabolism group characterization particulate matter exposure lung metabolic disorder marker screening
Model building method comprising following steps:
S1, the tracheal instillation that PM2.5 particle is carried out to test mice, after experimental period, collect the blood of test mice
Clearly；
S2, the data that serum is extracted and obtains LC-MS；
S3, with LC-MS data described in Commonpound Discover software carry out the ion extraction identification, then with count
Method carries out data processing and pattern-recognition to result is extracted, and searches out PM2.5 sample exposure mouse lung tissue metabolism group
Biomarker；
S4, with metabolism group Relational database and analysis software, construct the metabolic pathway of biomarker and divided
Analysis.
Preferably, in step S1, mouse reconditioning are as follows: mouse tracheal instillation volume is 60 μ L, low dosage
Exposure group PM2.5 concentration is 25 μ g//times, and high dose exposure group PM2.5 concentration is 150 μ g//times.
Preferably, in step S1, mouse tracheal instillation uses medical Y-shaped remaining needle, model: 26G.
Preferably, in step S2, quality control sample sheet (QC) is interspersed in parallel in entire analytic process, is used for
Evaluate the repeatability of entire analytic process.
In preceding method, animal processing and sample collection method particularly includes: by 75 C57BL/6 mouse in SPF grades of rings
Adaptive feeding one week in border is randomly divided into 3 groups, i.e., blank control group, low dosage expose group, high dose exposes group, and every group 25
Mouse；Wherein blank group is isometric physiological saline that instils, and low dosage exposure group is 25 μ g//times, high dose exposure group
For 150 μ g//times, anesthesia tracheal instillation is carried out to C57BL/6 mouse；Each group mouse only carries out primary gas in entire exposure period
Pipe instils, and puts to death mouse after exposure 48h, carries out sample collection.
The preparation of blood serum sample, lipids in serum extract and LC-MS data acquisition method particularly includes: in acute exposure reality
After testing, mouse is directly put to death using head-breaking, general blood collection tube collects whole blood, after standing 2h at room temperature,
3000rpm under the conditions of 4 DEG C, is centrifuged 15min.Take supernatant liquid (as serum) in -80 DEG C of cryo-conservations.Carry out serum lipids
When extraction, serum is first placed on -20 DEG C, is then transferred to 4 DEG C, after all thawing postposition balances at room temperature after serum, takes 80 μ
L serum is added in 10 μ L SPLASH and is marked in centrifuge tube in 4mL glass centrifuge tube, and 600 μ L methanol are added in centrifuge tube
1mL chloroform vortex 30s is added in vortex 30s, and the 500 μ L eddies of waters are added and revolve 30s, stand.Mixed liquor is centrifuged under the conditions of 3000rpm
It 10 minutes (Eppendorf company, 5804R Germany), collects lower layer's organic phase (chloroform), and be added in remaining upper layer phase
600 μ L chloroforms, repeating above-mentioned centrifugation and extraction process extracts it sufficiently.By the organic phase extracted twice as vacuum concentration instrument
Middle drying (Thermo company, the U.S.).The redissolution of 100 μ L methanol/isopropanol (1:1) mixed liquors is added in drying sample, in equipped with interior
Sample introduction is used in vial in bushing pipe.Quality control sample sheet (QC) is interspersed in parallel in entire analytic process, for evaluating
The repeatability of entire analytic process.Mixing of the QC sample from all blood samples.The sample prepared according to the method described above carries out LC-
MS analysis.LC-MS condition: liquid-phase condition AccucoreTMC30 LC chromatographic column (150mm × 2.1mm, 2.6 μm, the U.S.
Thermo company), 2 μ L of sample volume, runs 40min by 45 DEG C of column temperature；Mobile phase A is acetonitrile/water (60:40), and Mobile phase B is different
Propyl alcohol/acetonitrile (90:10), while two-phase flow mutually all contains the ammonium formate of 10mmol/L and 0.1% formic acid；Flow velocity is
0.2ml/min, elution requirement are shown in Table 1；Mass Spectrometry Conditions are as follows: positive model scanning range is 250-1200Da, and impact energy CE is 25eV
With 30eV, spray voltage 3.5kV.Negative mode scanning range be 200-1200Da, impact energy CE be 25eV, 24eV and 28eV,
Spray voltage is 3.5kV.
Chromatography gradient condition is as shown in the table:
What LC-MS data processing and biomarker screened method particularly includes: using Lipid Search TM software (beauty
Thermo company, state) lipids extraction and identification are carried out to the raw mass spectrum data information that LC-MS is obtained, by data obtained above
It imports SIMCAP-14.0 software and carries out principal component analysis.Using the principal component analysis (PCA) of unsupervised pattern-recognition, it is orthogonal partially
Least square method (OPLS-DA) discriminant analysis compares the difference between each group sample group by shot chart (Scores Plot), with
The parameter values such as R2X, R2Y, Q2 carry out assessment models quality, and wherein R2X, R2Y indicate that model is more stable closer to 1, the table of Q2 > 0.5
Show prediction rate height；The variable weight value (VIP) obtained according to OPLS-DA model, the variable using VIP value greater than 1.5 is as candidate
Biomarker is tested to verify whether the candidate variables found in multidimensional statistics in unit statistically have marked difference
Middle to correct non-parametric test using FDR, wherein there is significant difference in p < 0.05；Possible biology mark is finally determined in conjunction with P, VIP
Will object；All data are handled using 4.0 Online statistics analysis method of MetaboAnalyst.
Further, 4 biomarkers of mice serum lipid group of determining environment reference PM2.5 sample exposure is new
Purposes, which is characterized in that cause mouse cardiovascular disease for Evaluation Environment reference PM2.5 exposure with this 4 biomarkers
Early effect diagnosis, 4 biomarkers be ChE (18:0), DG (18:0_18:2), PC (34:1e), SM (d43:
5)；The method that wherein 4 biomarkers are used to evaluate the early effect that environment reference PM2.5 sample exposes is as follows:
Blank group is obtained using the LC-MS analysis method of step 2), low dosage exposes group, 4 kinds in high dose exposure group
Biomarker content；If ChE (18:0), DG (18:0_18:2), PC (34:1e), SM (d43:5) four in model mice serum
Lipoids metabolite concentration is inversely proportional with PM2.5 exposure concentrations, then environment reference PM2.5 sample is to the success of C57BL/6 mouse model.
Further, in the serum lipids group that the environment reference PM2.5 sample that step 3) determines intervenes C57BL/6 mouse
Application of 4 kinds of biomarkers in the screening of early effect marker, which is characterized in that specific screening is as follows:
1) experimental animal is divided into blank control group, low dosage exposure group, high dose exposure group, collects blood after putting to death mouse
Final proof sheet；
2) using 4 in the LC-MS analysis method acquisition blank group of step 2), low dosage exposure group, high dose exposure group
Kind biomarker content；If ChE (18:0), DG (18:0_18:2), PC (34:1e), SM (d43:5) in model mice serum
Four lipoids metabolite concentrations are inversely proportional with PM2.5 exposure concentrations, and difference is that significantly, then this 4 kinds of lipids can provide early stage
The screening scope of effect biomarker.
Therefore, the invention has the following beneficial effects:
Test is analyzed using mouse lung tissue sample detection of the LC-MS spectrum to acquisition, utilizes PCA, OPLS-DA diagnostic model
Method, examination is simulated by variation of the biological effective biomarkers under various concentration generated to various concentration PM2.5 exposure
Test the biological response reaction that mouse is placed in environmental exposure.For PM2.5 exposure caused by effect biomarker screening, evaluation and
Using offer metabolism group method.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is techniqueflow chart of the invention.
Fig. 2 is serum lipids group mark object screening process figure.
Fig. 3 is the LC-MS Metabolic fingerprinting schematic diagram of lung tissue all samples (QC).
Fig. 4 is the case where Lipid Search identifies 21 1041 lipid-metabolism objects of lipoids schematic diagram.
Fig. 5 is low dose group, high dose group and control group mice volcano figure as a result, wherein A: control group-low dosage exposure
Group；B: control group-high dose exposure group.
Fig. 6 is the orthogonal ginsenoside (OPLS- of low dose group, high dose group and control group mice
DA) shot chart, wherein A: control group-low dosage exposure group；B: control group-high dose exposure group.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection scope.
Fig. 1 shows the imitating for screening PM2.5 exposure early stage based on lipid composition analysis provided by the invention about a kind of
Answer the experimental method process of the construction method of the mouse model of marker.For ease of description, it illustrate only and phase of the present invention
Close part.
Experimental animal is divided into blank control group, low dosage exposure group, high dose exposure group by [embodiment 1], carries out tracheae
It instils, collects serum sample after sacrificed by decapitation.
Animal processing and sample collection method particularly includes: the adaptability feeding in the SPF grade environment by 75 C57BL/6 mouse
It supports one week.Being randomly divided into 3 groups, i.e., blank control group, low dosage expose group, high dose exposes group, and every group 25.Blank control group
60 μ of instillation physiological saline L//times, low dosage exposure group are 25 μ g//times, and high dose exposes group for 150 μ g//times.
PM2.5 mixed liquor is configured according to the exposed amount of every mouse and physiological saline dosage, ultraviolet sterilization 2h after the completion of preparation,
It carries out being required to vibrate before tracheal instillation every time being uniformly mixed.Different treatments only carry out a tracheae drop to mouse
Note puts to death mouse using head-breaking, and collect associated sample after 48h is completed in exposure.
[embodiment 2] prepares mice serum sample, and serum lipids extract and obtain LC-MS modal data
The preparation of serum sample and serum lipids extract method particularly includes: place the Mouse whole blood being collected into quiet at room temperature
After setting 2h, it is centrifuged 10min under the conditions of 3000rpm, 4 DEG C, taking supernatant is blood serum sample, is stored under -80 DEG C of low temperature.It carries out
When serum lipids extract, serum is first placed on -20 DEG C, is then transferred to 4 DEG C, all melted postposition to serum and balance at room temperature
Afterwards, take 80 μ L serum in 4mL glass centrifuge tube, be added 10 μ L SPLASH in be marked in centrifuge tube, be added 600 μ L methanol in
1mL chloroform vortex 30s is added in centrifuge tube mesoscale eddies 30s, and the 500 μ L eddies of waters are added and revolve 30s, stand.Mixed liquor is in 3000rpm item
It is centrifuged 10 minutes (Eppendorf company, 5804R Germany), collects lower layer's organic phase (chloroform), and on remaining upper layer under part
600 μ L chloroforms are added in phase, repeating above-mentioned centrifugation and extraction process extracts it sufficiently.By the organic phase extracted twice as true
In empty concentrating instrument dry (Thermo company, the U.S.).The redissolution of 100 μ L methanol/isopropanol (1:1) mixed liquors is added in drying sample,
Sample introduction is used in equipped with the vial in internal lining pipe.Quality control sample sheet (QC) is interspersed in parallel in entire analytic process,
For evaluating the repeatability of entire analytic process.Mixing of the QC sample from all blood samples.The sample prepared according to the method described above
This progress LC-MS analysis.LC-MS condition: liquid-phase condition AccucoreTMC30 LC chromatographic column (150mm × 2.1mm, 2.6 μm,
Thermo company, the U.S.), 2 μ L of sample volume, runs 40min by 45 DEG C of column temperature；Mobile phase A is acetonitrile/water (60:40), Mobile phase B
For isopropanol/acetonitrile (90:10), while two-phase flow mutually all contains the ammonium formate of 10mmol/L and 0.1% formic acid；Stream
Speed is 0.2ml/min, and elution requirement is shown in Table 1；Mass Spectrometry Conditions are as follows: positive model scanning range is 250-1200Da, and impact energy CE is
25eV and 30eV, spray voltage 3.5kV.Negative mode scanning range be 200-1200Da, impact energy CE be 25eV, 24eV and
28eV, spray voltage 3.5kV.Lipid is extracted to 3 groups of mice serums using LC-MS technology to analyze, and obtains full scan inspection
The serum total ion current figure of survey.As shown in Fig. 2, the compound information of reaction blood serum sample, therefrom extracts Information in Mass Spectra, m/z value
And corresponding abundance etc..
[embodiment 3] uses statistical method, carries out processing and pattern-recognition to LC-MS data, finds environment reference
Early effect marker of the PM2.5 sample to the exposure of C57BL/6 mouse
This research uses the scan pattern of data dependence type, and second level spectrum is obtained while full scan obtains level-one spectrogram
Figure information, therefore more detailed lipid structure information can be quickly obtained.Then according to retention time, accurate mass number, two
Grade Fracture, and it is fixed using the original realization that Lipid Search TM software (Thermo company, the U.S.) obtains LC-MS
Property analysis.Identifying lipid species includes free fatty acid (FFA), Lysophosphatidylcholone (LPC), hemolytic phosphatidyl second
Hydramine (LPE), hemolytic phosphatidylinositols (LPI), phosphatidyl choline (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositols
(PI), phosphatidyl glycerol (PG), phosphatidic acid (PA) and sphingomyelins (SM), ceramide (Cer), glycosylated ceramide
(HexCer), cholesteryl ester (CE), diacylglycerol (DAG), triacylglycerol (TAG).This research detects 1041 lipid generations altogether
Thank to object.SIMCA14.0 software is imported to Lipid Search TM software treated data and carries out principal component analysis, using nothing
The principal component analysis (PCA) of supervised recognition, the orthogonal Partial Least Squares discriminant analysis (OPLS- for having supervised recognition
DA) discriminant analysis.Compare the difference between each group sample group by shot chart (Scores Plot), with parameters such as R2X, R2Y, Q2
Value carrys out assessment models quality, and wherein R2X, R2Y indicate that model is more stable closer to 1, and Q2 > 0.5 indicates that prediction rate is high；According to
The variable weight value (VIP) that OPLS-DA model obtains, the variable using VIP value greater than 1.5 as candidate biomarker, in order to
Whether the candidate variables that find in unit statistically have marked difference in verifying multidimensional statistics, are corrected in experiment using FDR non-
Parametric test, wherein there is significant difference in p < 0.05；Possible biomarker is finally determined in conjunction with P, VIP；All data are adopted
It is handled with 4.0 Online statistics analysis method of MetaboAnalyst.
It is analyzed by the statistical model of orthogonal Partial Least Squares (OPLS-DA), discovery low dosage exposure in this experiment
There are the Difference of Metabolism of conspicuousness with mice serum lipid-metabolism object under blank group comparative analysis with blank group, high dose exposure.
It is analyzed by OPLS-DA discrimination model we have found that apparent change has occurred in lipid-metabolism object.In being analyzed according to OPLS-DA
VIP histogram shows the VIP value of each variable, and general VIP value is bigger, indicates that its contribution function is bigger, usually by the change of VIP > 1
Amount, which is considered as, is of great significance to model.In this experiment in order to further screen important potential source biomolecule mechanism, we
Determine that it contributes maximum lipid-metabolism object for difference using the setting of VIP threshold value in OPLS-DA.Simultaneously in order to further
Illustrate its significant difference between the two groups, the non-parametric test that we are corrected by FDR simultaneously, and variation multiple (FC,
Fold Change) metabolin difference condition group is described.In conjunction with above-mentioned model, we by VIP>1.5, P-FDR<
0.05 and FC>2 or<0.5 lipid-metabolism object be set as difference metabolin.The positive negative mode one in low dosage and blank
184 difference metabolins are identified altogether, which includes a large amount of phosphatidyl choline (PC), sphingomyelins (SM) and triacylglycerol
(TAG) substance.Phosphatidyl choline (PC) substance is obviously lowered in exposure group, and part SM, DAG, TAG are bright in exposure group
Aobvious up-regulation.In high exposure and control group comparison process, we obtain result similar with upper one group of comparing result.As a result illustrate
Influence of the acute exposure of pollutant for mouse lipid-metabolism is all more significant under the conditions of various dose.Consistent with the above
Screening process after, we identify 38 lipid biomarkers.Pass through the integration of above-mentioned two groups of difference metabolins, Wo Menjian
Common lipid biomarkers under different reconditionings 21 are defined, and wherein have 4 biomarkers and pollutant
There are dose-effect relationship, lipid-metabolism concentration is inversely proportional with exposure concentrations for exposure.Which includes phosphatidyl choline, sheath phosphorus
Rouge, cholesteryl ester, four potential source biomolecule markers of Diglycerides.Also illustrate that the exposure of PM2.5 significantly affects mouse
Regulating Lipid Metabolism.Also the biomarker for PM2.5 exposure provides a good reference standard.Wherein 4 and pollution
Object exposure is shown in Table 1. there are the biomarker of dose-effect relationship
The potential biomarker of table 1
Note: 1.FC:Fold-change；
2.L-C: the ratio between low dosage exposure group and blank group is indicated；H-C: high dose exposure group and blank group are indicated
Between ratio
This experiment has determined that 4 may cause C57BL/6 mouse lipid-metabolism object to make with the exposure of environment reference PM2.5 sample
For the early effect marker of cardiovascular disease.Blank group, low dosage exposure group, high dose expose cholesteryl ester, glycerol in group
Diester, phosphatidyl choline, four lipoids metabolite concentration of sphingomyelins and PM2.5 exposure concentrations are inversely proportional, i.e., with the increasing of exposure concentrations
Greatly, these four lipid-metabolism objects are lowered, and difference is significantly, then this 4 kinds of lipids can provide early effect mark
The screening scope of object.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring substantive content of the invention.
Claims (4)
1. a kind of building side of the mouse model for screening particulate matter exposure early effect marker based on lipid composition analysis
Method, which comprises the steps of:
S1, the tracheal instillation that PM2.5 particle is carried out to test mice, after experimental period, collect the serum of test mice；
S2, the data that serum is extracted and obtains LC-MS；
S3, the ion extraction identification is carried out with LC-MS data described in CommonpoundDiscover software, then with statistical
Method carries out data processing and pattern-recognition to result is extracted, and searches out PM2.5 sample exposure mouse lung tissue metabolism group
Biomarker；
S4, with metabolism group Relational database and analysis software, construct the metabolic pathway of biomarker and analyzed.
2. as described in claim 1 based on the mouse for exposing early effect marker for screening particulate matter of lipid composition analysis
The construction method of model, which is characterized in that in step S1, mouse reconditioning are as follows: mouse tracheal instillation volume is 60 μ L, low
Dosage exposure group PM2.5 concentration is 25 μ g//times, and high dose exposure group PM2.5 concentration is 150 μ g//times.
3. as described in claim 1 based on the mouse for exposing early effect marker for screening particulate matter of lipid composition analysis
The construction method of model, which is characterized in that in step S1, mouse tracheal instillation uses medical Y-shaped remaining needle, model: 26G.
4. as described in claim 1 based on the mouse for exposing early effect marker for screening particulate matter of lipid composition analysis
The construction method of model, which is characterized in that in step S2, quality control sample sheet (QC) is interspersed in entire analytic process in parallel
In, for evaluating the repeatability of entire analytic process.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110646601A (en) * | 2019-10-15 | 2020-01-03 | 大连工业大学 | Method for detecting influence of heavy metal exposure on cell lipid metabolism |
| CN111710372A (en) * | 2020-05-21 | 2020-09-25 | 中国医学科学院生物医学工程研究所 | Exhaled air detection device and method for establishing exhaled air marker thereof |
| CN112630344A (en) * | 2020-12-08 | 2021-04-09 | 河北医科大学第二医院 | Use of metabolic markers in cerebral infarction |
| CN112986425A (en) * | 2021-02-09 | 2021-06-18 | 首都医科大学 | PM2.5Detection marker for influence on lipid metabolism and application |
| CN114544981A (en) * | 2022-02-22 | 2022-05-27 | 中国农业科学院农业质量标准与检测技术研究所 | Marker for determining PFOS (Perfluorooctane sulfonate) exposure obtained based on lipidomics technology and application thereof |
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2019
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110646601A (en) * | 2019-10-15 | 2020-01-03 | 大连工业大学 | Method for detecting influence of heavy metal exposure on cell lipid metabolism |
| CN111710372A (en) * | 2020-05-21 | 2020-09-25 | 中国医学科学院生物医学工程研究所 | Exhaled air detection device and method for establishing exhaled air marker thereof |
| CN111710372B (en) * | 2020-05-21 | 2023-11-28 | 万盈美(天津)健康科技有限公司 | Exhaled air detection device and method for establishing exhaled air marker thereof |
| CN112630344A (en) * | 2020-12-08 | 2021-04-09 | 河北医科大学第二医院 | Use of metabolic markers in cerebral infarction |
| CN112986425A (en) * | 2021-02-09 | 2021-06-18 | 首都医科大学 | PM2.5Detection marker for influence on lipid metabolism and application |
| CN114544981A (en) * | 2022-02-22 | 2022-05-27 | 中国农业科学院农业质量标准与检测技术研究所 | Marker for determining PFOS (Perfluorooctane sulfonate) exposure obtained based on lipidomics technology and application thereof |
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