KR102070969B1 - A marker for the identification of pain - Google Patents

Abstract

The present invention relates to a marker for predicting pain, the present invention relates to a pain determination or prediction composition comprising an agent for measuring the expression level of a microRNA, or a kit comprising the composition, or an information providing method for pain determination or prediction. . The microRNA provided by the present invention can be used as a marker to objectively determine the degree of pain that can be neglected by subjective sensation and to determine or predict the severity thereof. Therefore, it can be widely used as a marker having an excellent effect.

Description

A marker for the identification of pain

The present invention relates to a marker for determining or predicting pain, the composition for determining or predicting pain comprising an agent for measuring the expression level of microRNA, or a kit comprising the composition, or an information providing method for determining or predicting pain. It is about.

Today's modern people spend a lot of time on desk work using computers, and suffer from chronic pain, such as pain and pain. However, pain is a problem that is not easily solved because it is easy to neglect it with a subjective sense. Therefore, rather than judging the severity of pain by subjective pain appeal, there is a need for a technique for determining objective pain.

MicroRNAs are short single-stranded ribonucleic acids (RNAs) with 19 to 25 nucleotides in length and are known to be expressed intracellularly to regulate the expression of many genes (Bartel DP, Cell, 116 (2): 281-297, 2004). To date, about 2000 species have been found in humans, and it is predicted that about 30% of human genes will be regulated by microRNAs, since one microRNA is capable of regulating several types of mRNAs (Bentwich, et al., Nat. Rev. Genet., 37 (7): 766-770, 2005).

MicroRNAs are made naturally in the cell and are mostly transcribed into primary microRNAs by RNA polymerase II, followed by the RNase III enzymes drosha and pasha in the nucleus. DGCR8) and the like are cleaved into precursor microRNA (precursor microRNA). Precursor microRNAs then migrate from the nucleus to the cytoplasm by Exportin-5 and are produced into short and incomplete double-stranded microRNAs by the RNase III enzyme dicer in the cytoplasm. The prepared microRNA is separated into a single strand by combining with a risk-risk RNA-induced silencing complex (RISC) protein complex, and the isolated single-stranded microRNA is complementary to the base sequence of the 3'-UTR (untranslated region) of the target mRNA. It is known to inhibit the expression of genes by binding to the ribosome to inhibit the translation of mRNA into protein or to cause the degradation of target mRNA (Lee, et al., Nature, 425 (6956): 415 -419, 2003).

It has been reported that microRNAs are very stable in serum and that expression levels in cardiovascular diseases and various cancers are significantly changed compared to normal subjects. Therefore, it is suggested as a very useful biomarker for the diagnosis of these diseases (Xi Chen et al., Cell Research, 2008). However, the use of microRNA as a marker for pain discrimination or prediction has not yet been identified.

Against this background, the present inventors have made intensive research efforts to develop a method for discriminating or predicting pain. As a result, the present inventors have identified microRNAs whose expression levels change in comparison with normal individuals in adult populations with persistent or recurrent pain. It was confirmed that the can be determined or predicted to complete the present invention.

One object of the present invention is to provide a composition for determining or predicting pain, including an agent for measuring the expression level of the microRNA consisting of SEQ ID NO: 1.

Another object of the present invention is to provide a pain determination or prediction kit comprising the composition for pain determination or prediction.

Another object of the present invention is to measure the expression level of the microRNA of SEQ ID NO: 1 or SEQ ID NO: 1 and 2 in a biological sample isolated from the individual; And (b) comparing the measured expression level of the microRNA with the expression level of the microRNA of the normal control sample, to provide an information providing method for pain determination or prediction.

One aspect of the present invention for achieving the above object provides a composition for determining or predicting pain, comprising an agent for measuring the expression level of hsa-mir-3135b.

As used herein, the term “microRNA” is mostly contained in an intron on a chromosome, and this means that the transcription is processed by Drosha and the like and converted into precursor-microRNA (precursor form). . The nucleus then exits the nucleus by exportin 5 and is converted into a mature microRNA of about 18 to 22 bp in length by Dicer. This mature microRNA is combined with the RNA induced silencing complex (RISC) to function (Nat Rev Mol Cell Biol 6, 376-785; 2005). In the present invention, microRNA may be used mixed with miRNA or microRNA.

For the purposes of the present invention, the microRNA is not limited as long as it can be used for the determination or prediction of pain. Specifically, the microRNA may be hsa-mir-3135b, and may further include has-mir-1285-3p. More specifically, it may be a polynucleotide having a nucleotide sequence of SEQ ID NO: 1, and may further include a nucleotide sequence of SEQ ID NO: 2.

Each microRNA has at least 80%, specifically at least 90%, more specifically at least 95%, and even more specifically at least 97% homology with the sequence, as well as the microRNA sequence described by each SEQ ID NO: microRNA sequences may be included. As the sequence having such homology, any sequence representing a microRNA exhibiting the same or corresponding efficacy as that of each microRNA is included without limitation. In addition, if the microRNA sequence having such homology, it is obvious that the microRNA sequence in which some sequences are deleted, modified, substituted or added is also included in the scope of the present invention.

As used herein, the term “pain” refers to an unpleasant sensory and emotional experience associated with or described as actual or potential tissue damage, and through the afferent cortical pathway consisting of nociceptors and nerve fibers. It also refers to pain and sensory impairment caused by stimulation of the area in contact with the marginal system region. This is a protective measure to protect the body and can be said to be a warning reaction that conveys abnormalities occurring inside or outside the body.

The causes of pain can be largely divided into perceptual cases caused by damage or inflammation of somatic or visceral tissues, or neuropathy occurring after nerve injury. In the case of perceptual pain, skin pain, visceral pain, somatic pain, perceptual neuralgia, neuromuscular pain, somatic pain, etc., and neuropathic pain due to peripheral or central nervous system dysfunction.

Specifically, the subject having a pain symptom in the present invention is 35 years or older, less than 45 years old, have a certain occupation, complains of neck pain that persists or repeats for more than 4 weeks, and the Numerical Rating Scale (NRS) pain questionnaire Excludes individuals with pain intensity of 3 or more and 14 points or more on the disease survey who have been diagnosed with traumatic or organic disease of the cervical spine or who are suspected to have pain symptoms due to placebo, paresthesia, pregnancy, or lactation. It was.

The term "discrimination or prediction" in the present invention means identifying the presence or characteristic of a pathological state. In the present invention, the determination or prediction may be interpreted as confirming the presence or degree of pain.

In the present invention, the microRNA may be a polynucleotide having a nucleotide sequence set forth in SEQ ID NO: 1, or a fragment thereof, and may further include a base sequence expected as set forth in SEQ ID NO: 2. The microRNA may be used as a discrimination or prediction marker capable of determining or predicting the presence or extent of pain, and the sequence may be modified to some extent in determining or predicting the presence or degree of pain. Those skilled in the art will appreciate that base sequences that maintain homology of at least 80%, preferably at least 90%, more preferably at least 95%, and most preferably 98% by such artificial modifications are desired in the present invention. As long as the expression level difference between the normal and the affected individuals is significantly compared as a marker for discriminating or predicting pain, it will be easily understood that the equivalent of the nucleotide sequence of the present invention is equivalent.

The term "agent for measuring the expression level of microRNA" in the present invention means an agent used in a method for confirming the expression of a microRNA or a fragment thereof contained in a sample. For example, RT-PCR, Competitive RT-PCR, Real-time RT-PCR, RNase protection assay (RPA), Northern blotting It may be a primer or a probe that can specifically bind to a target gene used in a method such as gene chip analysis, but is not particularly limited thereto.

As used herein, the term "primer" refers to a nucleic acid sequence having a short free 3 'hydroxyl group, capable of forming complementary templates and base pairs and starting for template strand copying. By a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (ie, DNA polymerase or reverse transcriptase) at appropriate buffers and temperatures.

As used herein, the term “probe” refers to a nucleic acid fragment such as RNA or DNA, which may correspond to a short number of bases to hundreds of bases, which is capable of specific binding with a gene or mRNA. An oligonucleotide probe , Single stranded DNA (single stranded DNA) probe, double stranded DNA (double stranded DNA) probe, RNA probes and the like can be produced in the form of, can be labeled for easier detection.

Primers or probes of the invention can be chemically synthesized using phosphoramidite solid support methods, or other well known methods. Such nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, eg, uncharged linkages such as methyl phosphonate, phosphoester, phosphoro Amidate, carbamate, etc.) or charged linkers (eg, phosphorothioate, phosphorodithioate, etc.).

Specifically, in one embodiment of the present invention, as a result of determining or predicting pain, it was confirmed that the microRNA expression amount of SEQ ID NO: 1 or 2 is down-regulated in women compared to normal people (Table 3).

Through these results, the microRNA was able to confirm that the expression level is significantly different from the normal group as the pain progresses, it can be seen that the pain can be effectively determined or predicted using the composition of the present invention.

Another aspect of the invention provides a pain determination or prediction kit comprising the composition for pain determination or prediction.

The determination or prediction kit of the present invention may be used to determine or predict a disease by measuring the expression level of the microRNA from a sample of a subject in question of pain, but is not particularly limited thereto, and expression level of the microRNA, or fragment thereof. As well as primers or probes for determining the activity, one or more other component compositions, solutions or devices suitable for analytical methods may be included.

As a specific example, the determination or prediction kit for measuring the expression level of the microRNA may be a kit including the necessary elements necessary to perform RT-PCR. The RT-PCR kit includes a test tube or other suitable container, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, in addition to each primer pair specific for the gene. Such as enzymes, DNase, RNAse inhibitors, DEPC-water (DEPC-water), may include sterile water and the like. It may also comprise primer pairs specific for the gene used as the quantitative control.

As another example, the kits of the present invention may include the necessary elements necessary to perform gene chip assays. The gene chip analysis kit may include a reagent, an agent, an enzyme, or the like for preparing a fluorescent label probe to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe.

Another aspect of the present invention provides a method of preparing a sac, comprising: (a) measuring the expression level of hsa-mir-3135b, or hsa-mir-3135b and has-mir-1285-3p, in a biological sample isolated from an individual; And (b) comparing the measured expression level of the microRNA with the expression level of the microRNA of the normal control sample, providing an information providing method for pain determination or prediction.

In addition, in step (b), the expression level of hsa-mir-3135b consisting of SEQ ID NO: 1, or hsa-mir-3135b consisting of SEQ ID NOs: 1 and 2, and has-mir-1285-3p was higher than that of the normal group. And (c) determining that there is pain when down regulated.

The "downregulation" may mean a case where the amount of microRNA expression in the experimental group is reduced by 1 to 20 times, specifically 2 to 15 times, compared with the normal group.

The term "individual" of the present invention may refer to any animal including a persistent pain group or a person suspected of suffering from constant pain. The animal may be a mammal such as, but not limited to, a human, a cow, a horse, a sheep, a pig, a goat, a camel, a antelope, a dog, a cat, and the like, which require treatment of similar symptoms.

The subject may be specifically a human, more specifically a human female.

The term "control" in the present invention refers to a normal subject having no pain, and for the purposes of the present invention, the subject having no symptoms of fatigue and pain and general health within 6 months based on the results of the questionnaire of Table 1 Can be.

The term "sample" in the present invention means blood separated from the pain group that feels persistent pain, and specifically, may be serum separated from the blood and RNA separated from the serum.

The microRNA provided by the present invention can be used as a marker to objectively determine the degree of pain that can be neglected by subjective sensation and to determine or predict the severity thereof. Therefore, it can be widely used as a marker having an excellent effect.

1 is a diagram illustrating the overall research method of the present invention.
FIG. 2 is a diagram of questionnaire content of a Numerical Rating Scale (NRS) for measuring a pain questionnaire score.
Figure 3 is a graph showing the results of RT-qPCR analysis of pain microRNA.
4 is a graph showing the results of ROC analysis of pain microRNA.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Selection of subjects

In order to discover markers for predicting pain, experiments were conducted as shown in FIG. 1. The subjects selected to conduct the experiment were 65 patients and women with a Numerical Rating Scale (NRS) pain questionnaire score (FIG. 2) and a disease free questionnaire who were admitted to the Oriental Hospital due to persistent or recurring pain symptoms. 108 patients were set as pain group, and 33 men and 44 women were set as health group (control group) based on the questionnaire about fatigue, pain, and general health. For the analysis of microRNA NGS (Next Generation Squencing), five men and women were selected from the pain group and the control group, and five men and women were selected as the control group. Selection and exclusion criteria are as follows.

(1) Criteria for selecting persistent / recurrent pain groups

-35 to 45 years old

-Complain of neck pain that lasts more than 4 weeks or recurs

-Patients with an average neck pain intensity of NRS 3 or more in the last week

-White collar workers who work more than 20 hours a week

-14 points or more in the disease survey

(2) criteria for excluding persistent / recurrent pain groups

-Those who have been diagnosed with traumatic or organic diseases of the cervical spine (severe spinal diseases (e.g. ankylosing spondylitis), infectious diseases, spinal fractures, spinal cord disorders, acute cervical disc herniation in the last 3 months, tumors, immune diseases, past 1 year) Whiplash injury during spinal surgery)

-Those with weakness or paresthesia in the upper limbs

-Those who have received medical or oriental medical treatment for neck pain within the last month

Pregnant women, lactating mothers, and those who plan to become pregnant within 6 months

Persons whose work environment is expected to change during clinical research

-If you are taking a drug that may affect your clinical research, or if other clinical research staff determines that it is not appropriate

(3) Pain group NGS subject selection

-The top 10 in the NRS questionnaire (sum of neck pain average intensity over the past week, neck pain average intensity over the last three months, plus the most severe intensity scores)

-Those with pain-related scores (sum of pain, discomfort, duration, recovery scores) among the top 10 in the disease survey

(4) RT-qPCR subject selection for pain group

(4-1) 1st RT-qPCR Verification

-60 males and 103 females (excluding NGS subjects) were selected from the top 20 males and 20 females with pain scores (NRS score + non-pain score).

(4-2) 2nd RT-qPCR Verification

-Top 30 men and 30 women with pain were selected from 40 men and 83 women, excluding NGS and the samples used for the primary test.

(5) Selection of control group (health group)

-35 to 45 years old

-Selecting people without discomfort through the questionnaire about fatigue and pain and general health (Table 1)

-NGS and RT-qPCR subjects were selected for 10 persons (5 men and women), 40 primary tests (20 men and women), and 40 secondary tests (20 men and women).

 Control group (health group) selection criteria ① What is the degree of tiredness?
-None (none, slightly, medium, severe)
② Does physical pain interfere with normal life?
-None or a few (no, some, some, many, very severe)
③ What is your overall quality of sleep?
-Very good or generally good (very good, mostly good, generally bad, very bad of items)
④ Is digestion good?
-Yes (Yes, No of Items)
⑤ How is your health?
-Best or Very Good (best, very good, good, little, bad)
⑥ Was your heart hurt and depressed?
-Rarely or not at all (always, most often, sometimes, rarely, not at all)
⑦ What is the level of difficulty in social activities (eg, visiting friends or relatives) due to physical or emotional problems?
-Rarely or not at all (almost always, most often, sometimes, rarely, not at all)

Experimental Example 1. Discovery of candidate microRNA associated with pain through NGS

1-1. Separation of microRNA in Serum

Total RNA was isolated from the serum using Mirvana Paris kit (Ambion) in 1.5ml serum of the subject, and then microRNA was isolated using Bioanalyzer (Agilent Technologies).

1-2. cDNA Library Fabrication

10ng small RNA per sample was prepared using the NEBNext® Multiplex Small RNA Library Prep Set.

1-3. Quality control

Analyzing cDNA library sequences produced using Illumina Nextseq 500 (Illumina) equipment, sequence information showing unnecessary information (adapter dimers generated during library creation) and low quality (read length less than 15nt) was added to the FASTQ Toolkit app ( Removed using Illumina.

1-4. RNA sequencing

BaseSpace® Small RNA v1.0 was used for qualitative analysis (read mapping using human genome and microRNA database) and quantitative analysis (normalized read count analysis).

1-5. Statistical analysis of difference in expression level of microRNA

The expression increase / decrease rate and statistical significance (P-value <0.05) of the microRNAs between the pain group and the control group were analyzed using the DESeq2 algorithm.

As a result of confirming the log2 magnification change value to the standardized mean number analyzed through the above steps, the read count was significantly increased (P <0.05) for 10 pain groups (5 men and women each) compared to 10 control groups. Or 14 microRNAs were found to be reduced (Table 2).

Pain Serum microRNA NGS Assay number microRNA gender Increase / decrease
(10 pain, 10 healthy people) P value One hsa-miR-122-5p female
male decrease
increase 2.7 times
3.5 times 0.0094
0.0046 2 hsa-mir-3135b female decrease 2.9 times 0.0071 3 hsa-let-7d-3p Female + Male increase 2.2x 0.0071 4 hsa-mir-2392 female increase 4.1x 0.0010 5 hsa-miR-193b-5p Female decrease
increase 2.2x
2.9 times 0.036
0.011 6 hsa-miR-1273h-5p Female decrease
increase 2.3x
2.5x 0.030
0.019 7 hsa-miR-148a-3p male decrease 2.1x 0.020 8 hsa-miR-501-3p female decrease 2.4x 0.022 9 hsa-let-7f-5p Female + Male increase 2.0 times 0.028 10 hsa-mir-1285-3p Female Female + Male decrease
decrease 2.7 times
2.0x 0.031
0.028 11 hsa-mir-885-3p male increase 2.7 times 0.035 12 hsa-miR-744-5p female increase 2.0 times 0.036 13 hsa-miR-22-3p Female + Male decrease 1.7x 0.041 14 hsa-miR-1307-3p Female + Male decrease 1.7x 0.042

Experimental Example 2. Verification of pain-associated microRNA

Of the 14 microRNAs discovered in Experimental Example 1, five microRNAs (hsa-mir-2392, hsa-miR-193b-5p, hsa-miR-501-3p, hsa-mir-885-3p, Nine microRNAs except hsa-miR-22-3p) were tested twice.

2-1. Separation of microRNA in Serum

After adding spike-in control RNA (Cel-miR-39) to 0.4 ml serum of the subject, total RNA was isolated from the serum using the Mirvana Paris kit (Ambion).

2-2. cDNA Library Fabrication

cDNA libraries were prepared using miScript II RT kit (Qiagen).

2-3. RT-qPCR Analysis

After pre-amplification (12 cycles) using a selectively recognizable primer and miScript PreAMP kit (Qiagen), microRNA using miScript SYBR Green PCR kit (Qiagen) and real-time PCR system (LifeScience, ABI) The quantitative analysis of these was carried out.

2-4. Statistical analysis of difference in expression level of microRNA

After correcting the experimental error with the spike-in control RNA (Cel-miR-39) Ct value of the microRNA, the increase or decrease rate and the statistical significance of the expression levels of the microRNAs between the pain group and the control group were measured. -value <0.05) were analyzed by Wilcoxon rank sum test method.

As a result of verifying the primary pain-associated microRNAs through the above steps, five microRNAs showed a significant result while agreeing with the NGS results (Table 3).

In addition, after verifying the secondary pain-associated microRNAs by increasing the experimental group and the control group by the same method, and comparing the NGS and the 1st-second verification results together, two microRNAs (hsa-mir-3135b and has-mir) were compared. -1285-3p) showed significant results with the same tendency (decrease) with NGS and the 1st test result for all women (Table 3 and FIG. 3).

Pain-associated microRNA RT-qPCR Validation Results Pain microRNA female male Increase / decrease P value Increase / decrease P value NGS (10 pains vs 10 healthy) hsa-mir-3135b decrease 2.9 7.1 × 10 ^-3 increase 1.6 0.20 hsa-mir-1285-3p decrease 2.7 3.1 × 10 ^-2 increase 1.7 N.A hsa-miR-122-5p decrease 2.7 9.4 × 10 ^-3 increase 1.8 4.6 × 10 ^-3 hsa-let-7f-5p increase 1.7 0.23 increase 1.2 0.07 hsa-miR-1307-3p decrease 1.5 0.28 decrease 1.9 0.076 First RT-qPCR Validation (40 Pain vs 40 Healthy) hsa-mir-3135b decrease 2.5 3.6 × 10 ^-3 increase 2.1 3.4 × 10 ^-3 hsa-mir-1285-3p decrease 3.3 3.1 × 10 ^-4 increase 1.5 4.0 × 10 ^-2 hsa-miR-122-5p decrease 2.1 1.3 × 10 ^-2 increase 2.4 1.0 × 10 ^-4 hsa-let-7f-5p - 1.0 0.44 increase 7.9 2.7 × 10 ^-7 hsa-miR-1307-3p increase 1.2 0.49 increase 3.9 1.0 × 10 ^-6 RT-qPCR Secondary Verification (60 Pain vs 40 Healthy) hsa-mir-3135b decrease 13.8 9.1 × 10 ^-9 decrease 5.3 3.4 × 10 ^-7 hsa-mir-1285-3p decrease 10.9 1.2 × 10 ^-8 decrease 8.7 2.2 × 10 ^-6 hsa-miR-122-5p increase 1.3 0.79 decrease 2.7 1.7 × 10 ^-5 hsa-let-7f-5p increase 1.5 4.1 × 10 ^-3 decrease 1.1 0.22 hsa-miR-1307-3p decrease 1.7 2.9 × 10 ^-2 decrease 2.5 5.9 × 10 ^-5

Experimental Example 3. Analysis of Receiver Operating Curve (ROC)

Using the SPSS statistical program, pain prediction probability values, sensitivity, and 1-specificity were calculated for each sample using a logistic regression model using age, BMI, and microRNA expression as variables. . Then, using the SPSS program based on the calculated result values, the ROC with the x-axis as the 1-specificity and the y-axis as the sensitivity, and the AUC (Area Under the Curve) value that means the discrimination accuracy and P value showing the significance Was calculated.

As a result, as shown in Figure 4, the pain was able to discriminate with 77% and 69% accuracy, respectively, using hsa-mir-3135b and has-mir-1285-3p expression levels in the pain women, simultaneously two microRNA When used, the accuracy was 78%. This result shows the possibility of using two microRNAs as objective biomarkers to distinguish persistent / recurring pain.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

<110> Korea Institute of Oriental Medicine <120> A marker for the identification of pain <130> KPA180422-KR <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> hsa-miR-3135b <400> 1 ggctggtccg agtgcagtgg 20 <210> 2 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> hsa-mir-1285-3p <400> 2 tctgggcaac aaagtgagac 20

Claims (9)

A composition for determining or predicting pain comprising an agent for measuring the expression level of the microRNA consisting of SEQ ID NO: 1 in women.
delete The composition of claim 1, wherein the composition further comprises an agent that measures the expression level of hsa-mir-1285-3p.
The composition of claim 3, wherein the hsa-mir-1285-3p consists of SEQ ID NO: 2.
The composition of claim 1, wherein the agent comprises a primer or probe that specifically binds to a microRNA.
Claim 1 and the pain determination or prediction kit comprising a composition according to any one of claims 3 to 5.
(a) measuring the expression level of a microRNA consisting of SEQ ID NO: 1, or a microRNA consisting of SEQ ID NO: 1 and hsa-mir-1285-3p in a biological sample isolated from a female individual; And (b) comparing the measured expression level of the microRNA with the expression level of the microRNA of the normal control sample.
The method of claim 7, wherein the hsa-mir-1285-3p consists of SEQ ID NO: 2.
The method according to claim 7, wherein the expression level of the microRNA consisting of SEQ ID NO: 1, or the microRNA consisting of SEQ ID NO: 1 and hsa-mir-1285-3p in step (b) is lower than the expression level of the microRNA of the normal group If controlled, further comprising the step (c) of determining pain.

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