Biomarker for diagnosing drowning and uses thereof

Abstract

The ERK2 and / or p-ERK2 of the present invention can distinguish drowning from other causes of death such as hypoxia and post-mortem drowning by confirming their expression levels, and additionally can distinguish between freshwater drowning and seawater drowning. As a biomarker, it can be effectively used for diagnosing drowning or distinguishing drowning environments.

Description

Technology Field

[0001] The present invention relates to a biomarker for diagnosing drowning that can distinguish drowning from other causes of death and additionally distinguish between freshwater drowning and seawater drowning, and to the use thereof. Background Technology

[0003] Drowning is the third leading cause of unintended (accidental) death worldwide, claiming approximately 200,000 to 500,000 lives annually. Consequently, numerous epidemiological studies have been conducted on drowning cases. Despite these research efforts, global and regional estimates of drowning deaths remain uncertain. One reason for this uncertainty in death figures is the difficulty in diagnosing drowning. According to the World Health Organization (WHO) definition, “drowning is the process of experiencing respiratory distress while submerged or immersed in liquid.” Currently, the diagnosis of drowning is based on autopsy findings, such as foaming around the mouth and nose, foaming in the airways, lung distension, subpleural petechiae, pleural effusion, and the detection of diatoms in the bone marrow, spleen, and brain. However, while these autopsy findings and diatom examinations support the diagnosis of drowning, they do not constitute conclusive evidence. Furthermore, since submerged bodies may not always have drowned, careful diagnosis is required during forensic autopsies regarding various causes of death, such as homicide followed by submersion. Furthermore, the diagnosis of drowning is not only essential for legal death investigations, but determining the attributes of the drowning environment and the postmortem interval (PMI) is also crucial. Therefore, it is necessary to estimate the PMI and distinguish the drowning medium. Traditionally, specific morphological differences have been described between freshwater and seawater drownings. However, some studies have not observed significant differences between freshwater and seawater drownings, and reliable techniques applicable to forensic practice have not yet been validated. This suggests the need for biomarkers to distinguish between drowning and postmortem drowning and to identify attributes of the drowning environment, such as freshwater versus seawater.

[0004] Markers responding to various mechanisms associated with drowning are recently being studied as biomarkers for the diagnosis of drowning. However, there is no technology to identify the expression patterns of ERK (extracellular signal-regulated kinase) in relation to drowning. Prior art literature

[0006] KR 10-2019-0061601 (2019-05-27) The problem to be solved

[0007] As a result of diligent efforts to provide a biomarker capable of specifically diagnosing, determining, or distinguishing drowning, the inventors confirmed that an extracellular signal-regulating kinase (ERK) belonging to a multiple MAPK (mitogen-activated protein kinase) pathway can effectively diagnose, determine, or distinguish drowning, and thus completed the present invention.

[0009] Accordingly, the objective of the present invention is to provide a biomarker for diagnosing drowning that can distinguish drowning from other causes of death and additionally distinguish between freshwater drowning and seawater drowning, and its uses. means of solving the problem

[0011] The present invention provides a biomarker composition for diagnosing drowning comprising ERK2 (Extracellular Signal-Regulated Kinase 2) and / or p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2).

[0012] In addition, the present invention provides a composition for diagnosing drowning comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene.

[0013] According to a preferred embodiment of the present invention, the protein is p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2).

[0014] According to a preferred embodiment of the present invention, a preparation for measuring the mRNA level of the gene comprises sense and antisense primers or probes that bind complementarily to the mRNA of the gene.

[0015] According to a preferred embodiment of the present invention, the agent for measuring the protein level comprises an antibody or an antigen-binding fragment thereof, or an aptamer, that specifically binds to a protein or a fragment thereof encoded by the gene.

[0016] In addition, the present invention provides a drowning diagnosis kit comprising the above composition.

[0017] In addition, the present invention provides a composition for distinguishing drowning environments comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene.

[0018] According to a preferred embodiment of the present invention, the drowning is fresh water drowning or sea water drowning.

[0019] In addition, the present invention provides a kit for distinguishing drowning environments comprising the above composition.

[0020] In addition, the present invention comprises the step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample;

[0021] A method for providing information for diagnosing drowning, including

[0022] According to a preferred embodiment of the present invention, the information providing method further comprises the step of a) classifying as drowning if the level of ERK2 increases compared to the control group when the postmortem interval of the sample is immediately after death but less than 48 hours, or b) classifying as drowning if the level of ERK2 decreases compared to the control group when the postmortem interval of the sample is 48 hours or more.

[0023] In addition, the present invention comprises the step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample;

[0024] A method for distinguishing drowning environments including is provided.

[0025] According to a preferred embodiment of the present invention, the sample has a postmortem interval of 48 hours or more.

[0026] According to a preferred embodiment of the present invention, the drowning is fresh water drowning or sea water drowning.

[0027] According to a preferred embodiment of the present invention, the distinguishing method further includes the step of classifying as fresh water drowning if the level of ERK2 decreases compared to the control group. Effects of the invention

[0029] The ERK2 and / or p-ERK2 of the present invention can distinguish drowning from other causes of death such as hypoxia and post-mortem drowning by confirming their expression levels, and additionally can distinguish between freshwater drowning and seawater drowning. As a biomarker, it can be effectively used for diagnosing drowning or distinguishing drowning environments. Brief explanation of the drawing

[0031] Figure 1 shows the results of the relative expression analysis of ERK1(a) and ERK2(b) using quantitative real-time PCR. Means were normalized to GAPDH and expressed as ratios. Control (CON), hypoxia (HY), freshwater postmortem infiltration (FP), seawater postmortem infiltration (SP), freshwater drowning (FD), and seawater drowning (SD). ***p < 0.001 vs. CON. Figure 2 shows the results of Western blotting analysis of ERK1 / 2 and phosphorylated ERK1 / 2 (p-ERK1 / 2) in the lungs. (a) There was no difference in ERK1 / 2 band intensity between groups, but 42 kDa p-ERK2 was higher in drowning cases than in other groups. GAPDH was used as an internal control to normalize ERK1 / 2 and p-ERK1 / 2. Relative expression results of ERK1 / 2 (b) and p-ERK1 / 2 (c) using an image analysis system. Control (CON), hypoxia (HY), freshwater post-drowning (FP), seawater post-drowning (SP), freshwater drowning (FD), and seawater drowning (SD). **p < 0.01 vs. CON. Figure 3 shows the results of immunohistochemical (IHC) analysis of p-ERK1 / 2 in the control group (a), hypoxia (b), freshwater post-mortem drowning (c), seawater post-mortem drowning (d), freshwater drowning (e), and seawater drowning groups (f). IHC staining revealed that granular patterns were found more frequently in drowning cases than in other groups. Magnification: ×200, Inset magnification: ×400. Figure 4 shows the results of the relative expression analysis of ERK2 in 2-hour drowning cases (a) and 48-hour drowning cases (b) using quantitative real-time PCR. Means were normalized to the GAPDH standard and expressed as ratios. Control group (CON), freshwater drowning (FD), and seawater drowning (SD). ***p < 0.001 vs. CON. #p < 0.001 vs. SD. Specific details for implementing the invention

[0032] The present invention will be described in more detail below.

[0034] The 'drowning diagnosis' of the present invention may mean identifying, determining, or distinguishing as drowning among various causes of death, such as hypoxia and post-mortem drowning.

[0035] The 'drowning environment distinction' of the present invention may mean confirming, determining, or distinguishing whether the medium of drowning is freshwater or seawater.

[0036] The 'freshwater' of the present invention refers to ordinary freshwater with a low salt content, such as groundwater or river water, and the 'seawater' of the present invention refers to seawater.

[0038] The 'biomarker' of the present invention refers to a molecule quantitatively or qualitatively associated with the presence of a biological phenomenon, and the marker of the present invention refers to a gene that serves as a criterion for diagnosing drowning. The marker may be derived from a genomic nucleotide sequence or from an expressed nucleotide sequence (e.g., from RNA, nRNA, mRNA, cDNA, etc.), or from a coded polypeptide. The term includes a nucleic acid sequence complementary to or flanked thereto by the marker sequence, such as a nucleic acid used as a probe or primer pair capable of amplifying the marker sequence.

[0039] In the present invention, 'expression' means the production of a protein or nucleic acid. 'Protein' is used interchangeably with 'polypeptide' or 'peptide' and refers to a polymer of amino acid residues, for example, as generally found in proteins in their natural state. 'Polynucleotide' or 'nucleic acid' refers to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) in the form of a single or double strand. Unless otherwise limited, it also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' refers to RNA that transmits genetic information (gene-specific base sequences) to ribosomes that determine the amino acid sequence from specific genes during the process of protein synthesis.

[0041] Diagnosing drowning is a challenging task in forensic investigations because it is difficult to verify actual drowning using currently available autopsy techniques. To evaluate the expression patterns of ERK1 / 2 (extracellular signal-regulated kinase 1 / 2), the inventors divided experimental animals into four groups: drowning, postmortem submersion, hypoxia, and control. Gene expression analysis revealed that only ERK2 was significantly increased in the drowning group compared to the other groups. This may be attributed to the genetic size difference between the two isoforms, ERK1 and ERK2. For example, in mammals, the ERK2 gene is on average 15 times larger than the ERK1 gene, while in mice it is approximately 6 times larger. This difference suggests that ERK2 may be regulated more precisely than ERK1. Immunoblot analysis showed that phosphorylated ERK2 (p-ERK2) was upregulated in the drowning group. Immunochemical staining also showed that p-ERK in alveolar cells exhibited a granular pattern in the drowning group. However, the expression pattern of ERK2 over time after drowning differed between the freshwater and seawater drowning groups. Taken together, these results suggest that ERK2 may be useful in distinguishing between drowning and postmortem drowning when the postmortem interval (PMI) is short. Conversely, if the PMI is long, it may be more helpful in distinguishing between freshwater and seawater drowning.

[0043] Accordingly, the present invention provides a biomarker composition for diagnosing drowning comprising ERK2 (Extracellular Signal-Regulated Kinase 2) and / or p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2).

[0045] In addition, the present invention may provide a composition for diagnosing drowning comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene.

[0046] According to a preferred embodiment of the present invention, the protein may be p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2).

[0047] For the sample in which the above ERK2 level was measured, a) if the postmortem interval is immediately after death but less than 48 hours, and the ERK2 level increases compared to the control group, it can be diagnosed as drowning, or b) if the postmortem interval of the sample is 48 hours or more, and the ERK2 level decreases compared to the control group, it can be diagnosed as drowning. The above control group may be a sample from an individual that died from a cause other than drowning.

[0048] More preferably, when the postmortem interval of the sample in which the ERK2 level was measured is within 2 hours immediately after death, the ERK2 level may be diagnosed as drowning if it increases compared to the control group, or when the postmortem interval of the sample is 48 hours or more and 72 hours or less, the ERK2 level may be diagnosed as drowning if it decreases compared to the control group.

[0049] According to a preferred embodiment of the present invention, a preparation for measuring the mRNA level of the gene may include sense and antisense primers or probes that bind complementarily to the mRNA of the gene.

[0050] The above "primer" refers to a short nucleic acid sequence having a short free 3' hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template replication. In the present invention, intermediate precursor cells can be identified by determining whether a desired product is produced by performing PCR amplification using the sense and antisense primers of the aforementioned biomarker polynucleotide. The PCR conditions and the lengths of the sense and antisense primers can be modified based on those known in the art.

[0051] The term "probe" above refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few bases to hundreds of bases in length, capable of forming a specific binding with mRNA; it is labeled to confirm the presence or absence of a specific mRNA. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc. In the present invention, hybridization is performed using a probe complementary to the aforementioned biomarker polynucleotide, and intermediate precursor cells can be identified based on whether hybridization occurs. The selection of a suitable probe and hybridization conditions can be modified based on those known in the art.

[0052] According to a preferred embodiment of the present invention, the agent for measuring the protein level may be a agent for measuring the phosphorylation level of the protein, and may include a protein encoded by the gene, an antibody or an antigen-binding fragment thereof that specifically binds to a protein or a fragment thereof to which the protein encoded by the gene is phosphorylated, or an aptamer.

[0053] The above “preparation for measuring the phosphorylation level of a protein” may include, without limitation, preparations used in conventional phosphorylation measurement methods, such as antibodies that recognize phosphorylation groups, chemicals that recognize phosphorylation groups, preparations used in luminescence methods, etc.

[0054] The term "antibody" above refers to a specific protein molecule directed toward an antigenic site, as is a term known in the art. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to the aforementioned biomarker. Such an antibody can be produced by cloning each gene into an expression vector according to conventional methods to obtain a protein encoded by the biomarker gene, and then producing the obtained protein by conventional methods. This includes partial peptides that can be produced from the protein. The form of the antibody of the present invention is not particularly limited; polyclonal antibodies, monoclonal antibodies, or parts thereof having antigen-binding properties are included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibody of the present invention includes special antibodies such as humanized antibodies. The antibody against the protein encoded by the biomarker gene of the present invention may be any antibody that can be produced by methods known in the art.

[0055] The term "aptamer" above refers to a single-stranded oligonucleotide, which is also called a chemical antibody because it has a function similar to an antibody, and is a nucleic acid molecule that has binding activity to a specific target molecule. The aptamer can have various three-dimensional structures depending on its base sequence and can have a high affinity for a specific substance, such as in an antigen-antibody reaction. The aptamer can inhibit the activity of a specific target molecule by binding to it.

[0056] The aptamers of the present invention may be RNA, DNA, modified nucleic acids, or mixtures thereof, and may be in a linear or cyclic form, but are not limited thereto. Aptamers having binding activity to each of the three types of biomarker proteins may be prepared by a person skilled in the art according to known methods by referring to each base sequence.

[0057] The nucleotide sequence of the preparation used in the present invention for measuring the expression level of a biomarker gene is interpreted to include a sequence that exhibits substantial identity with a sequence that specifically binds to the biomarker gene, provided that variations having biologically equivalent activity are taken into account. The "substantial identity" refers to a sequence that exhibits at least 60% identity, more specifically 70% identity, even more specifically 80% identity, and most specifically 90% identity when a specific sequence is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0059] In addition, the present invention provides a drowning diagnosis kit comprising the above composition.

[0060] The kit of the present invention may be composed of one or more other component compositions, solutions, or devices suitable for commonly used expression level analysis methods. For example, a kit for measuring protein expression levels may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, etc., for the immunological detection of antibodies.

[0061] The kit of the present invention may include a sample extraction means for obtaining a sample from an evaluation subject. The sample extraction means may include a needle or a syringe, etc. The kit may include a sample collection container for receiving the extracted sample, which may be a liquid, gas, or semi-solid. The kit may further include instructions for use. The sample may be any body sample in which a biomarker may be present or secreted. For example, the sample may be lung cells and / or lung tissue. Measurement of the biomarker in the sample may be performed on the whole sample or on the processed sample. The kit of the present invention may be manufactured in a number of separate packages or compartments.

[0063] In addition, the present invention provides a composition for distinguishing drowning environments comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene.

[0064] Since the preparation for measuring the level of the mRNA of the ERK2 gene or the protein encoded by the gene is identical to the concept used in the composition for diagnosing drowning, the description is replaced by the description thereof.

[0065] According to a preferred embodiment of the present invention, the drowning is fresh water drowning or sea water drowning.

[0066] If the postmortem interval of the sample in which the level of ERK2 was measured is 48 hours or longer, it can be classified as fresh water if the level of ERK2 is reduced compared to the control group. Preferably, if the postmortem interval of the sample in which the level of ERK2 was measured is 48 hours or longer and 72 hours or less, it can be classified as fresh water if the level of ERK2 is reduced compared to the control group.

[0068] In addition, the present invention can provide a kit for distinguishing drowning environments comprising the above composition.

[0069] Since the above kit is identical in concept to the drowning diagnosis kit mentioned above, the description is replaced by the description thereof.

[0071] In addition, the present invention comprises the step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample;

[0072] A method for providing information for diagnosing drowning, including

[0073] The above sample may be lung cells and / or lung tissue.

[0074] Since the preparation for measuring the level of the mRNA of the ERK2 gene or the protein encoded by the gene is identical to the concept used in the composition for diagnosing drowning, the description is replaced by the description thereof.

[0075] According to a preferred embodiment of the present invention, the information providing method may additionally include the step of a) classifying the case as drowning if the level of ERK2 increases compared to the control group when the postmortem interval of the sample is immediately after death but less than 48 hours, or b) classifying the case as drowning if the level of ERK2 decreases compared to the control group when the postmortem interval of the sample is 48 hours or more. The control group may be a sample of an individual that died from a cause other than drowning.

[0076] More preferably, the information-providing method may additionally include the step of a) classifying as drowning if the level of ERK2 increases compared to the control group when the postmortem interval of the sample is within 2 hours immediately after death, or b) classifying as drowning if the level of ERK2 decreases compared to the control group when the postmortem interval of the sample is 48 hours or more and 72 hours or less.

[0077] If the postmortem interval of the above sample is 48 hours or longer, and the expression level of ERK2 decreases compared to the control group, it can be classified as freshwater drowning, and preferably, if the postmortem interval of the sample is 48 hours or longer and 72 hours or shorter, and the expression level of ERK2 decreases compared to the control group, it can be classified as freshwater drowning.

[0079] In addition, the present invention comprises the step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample;

[0080] A method for distinguishing drowning environments including can be provided.

[0081] The above sample may be lung cells and / or lung tissue.

[0082] According to a preferred embodiment of the present invention, the sample may have a postmortem interval of 48 hours or more, and more preferably 48 hours or more and 72 hours or less.

[0083] Since the preparation for measuring the level of the mRNA of the ERK2 gene or the protein encoded by the gene is identical to the concept used in the composition for diagnosing drowning, the description is replaced by the description thereof.

[0084] According to a preferred embodiment of the present invention, the drowning may be fresh water drowning or sea water drowning.

[0085] According to a preferred embodiment of the present invention, the distinguishing method may additionally include the step of classifying as fresh water drowning if the level of ERK2 decreases compared to the control group.

[0087] The present invention will be described in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0089] Preparation of the experimental group

[0090] <1-1> Animals

[0091] Five-week-old Sprague-Dawley rats purchased from DBL (Korea) were used as experimental animals. All rat experiments were approved by the Soonchunhyang University Animal Care and Use Committee (IACUC Approval No.: SCH17-0003).

[0093] <1-2> Fauna

[0094] Experimental animals were randomly divided into four groups: hypoxia (n=10), post-mortem watering (n=10; freshwater n=5; seawater n=5), drowning (n=20; freshwater n=10; seawater n=10), and control group (n=5). Alfaxan (1.5 mL / kg, iv) was injected into all groups for anesthesia. After anesthesia, the control group was sacrificed by cervical dislocation. The anesthetized hypoxia group was exposed to a hypoxic environment chamber filled with 5% oxygen and 95% nitrogen for 8 minutes, followed by a 2-minute rest. This process was repeated until the animals were sacrificed. Post-mortem watering and drowning conditions were simulated using sterile plastic cages (0.09 × 0.19 × 0.12 m) filled with 11 L of water. The freshwater and seawater used in the experiment were collected from Sapgyo Lake (36°87'N, 126°84'E) and Janggo Port (37°03'N, 126°56'E), respectively. The collected specimens were placed in sterile bottles and refrigerated at 4°C. They were warmed to room temperature one day prior to use, and the water temperature was maintained at 15–20°C throughout the experiment. The post-mortem drowning group was sacrificed by cervical dislocation after anesthesia and placed at room temperature in freshwater and seawater cages, respectively, for 2 hours. The drowning group was divided into a 2-hour drowning model and a 48-hour drowning model to observe molecular changes over time. After anesthesia, the drowning group was subjected to repeated cycles of 1 minute of immersion and 1 minute of breathing in water-filled cages at room temperature for 2 or 48 hours to simulate actual drowning conditions. Mouse lung tissues from each group were fixed in 10% formalin for immunohistochemical staining and frozen in liquid nitrogen for Western blotting and RNA isolation.

[0096] Gene expression of ERK1 and ERK2

[0097] The inventors confirmed the gene expression of ERK1 and ERK2 from mRNA of mouse lung tissue, including cases of 2-hour drowning and post-mortem drowning. The target genes were quantified by comparing the expression levels of the housekeeping gene (GAPDH) in each group and expressed as a ratio relative to the control group.

[0098] Specifically, the mouse lung tissue of Example <1-2> above was homogenized in a mortar with liquid nitrogen, and then total RNA was extracted from the frozen lung tissue using the NucleoSpin®RNA Mini Kit (MAHEREY-NAGEL, Duren, Germany) according to the manufacturer's instructions. The RNA was quantified by BioDrop μLlTE (BioDrop Ltd., Cambridge, UK). Then, cDNA was synthesized from 2 μg of the RNA sample using ReverTra Ace®qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) according to the manufacturer's instructions.

[0099] cDNA samples were amplified by qRT-PCR on a StepOnePlus™ Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) using TOPreal™ qPCR 2X PreMIX (Enzynomics, Daejeon, Korea) and primers (Table 1). The PCR reaction mixture, with a final volume of 20 μL, contained 10 μL of master mix, 1 μL of each primer (0.5 pmol / μL), and 1 μL of cDNA template. The thermal cycling program was set to 40 cycles consisting of an initial denaturation step at 95°C for 10 minutes, a denaturation step at 95°C for 30 seconds, a primer annealing step at each temperature for 30 seconds, and an extension step at 72°C for 30 seconds. The threshold cycle (Ct) was calculated by the instrument software (StepOne Software v2.3). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. -ΔΔCt The relative quantification of gene expression levels was determined using a method.

[0100] gene Registration number Primer sequence (5'→3') Annealing temperature (°C) Amplification product (bp) Sequence number GAPDH NM_017008.4 F: ATG ACT CTA CCC ACG GCA AGR: GAT CTC GCT CCT GGA AGA TG 57 99 12 ERK1 ( MAPK3 ) NM_017347.3 F: CTG GAA GCC ATG GAG AGA TGT TR: TCG CAG GTG GTG TTG ATA AG 57 204 34 ERK2 ( MAPK1 ) NM_053842.2 F: TGT TGC AGA TGG AGA CCA TGR: CAG CCC ACA GAC CAA ATA TCA 56 132 56

[0102] As a result, there was almost no difference in the expression levels of ERK1 between the groups (Fig. 1a). On the other hand, in the drowning group, the expression of ERK2 was statistically significantly increased compared to the control group, hypoxia group, and post-mortem water group (p<0.001) (Fig. 1).

[0104] Immunoblot analysis

[0105] Western blot analysis was performed using antibodies specific to the total and phosphorylated (activated) forms of ERK.

[0106] Specifically, for immunoblotting, lung tissue was dissolved in RIPA buffer (500 mM Tris-HCl, 10% NP-40, 10% sodium deoxycholate, 1.5 M NaCl, 100 mM EDTA, 100 mM PMSF, 100 mM Na3VO4, 1 μg / μL aprotinin, 1 μg / μL leupeptin, and DdH2O). The lysate was centrifuged at 18,500 g for 20 minutes at room temperature, and the supernatant was separated. Protein concentration was determined using serially diluted bovine serum albumin (BSA) protein as a standard. After boiling the protein in loading buffer at 102 °C for 5 minutes, the prepared samples were electrophoresed on 15% SDS-PAGE. The isolated proteins were then transferred to an Immobilon®Transfer Membrane (MILLIPORE, Billerica, MA, USA) and blocked with 5% skim milk for 2 hours at room temperature. The membrane was then incubated overnight at 4°C with primary antibodies containing anti-ERK1 / 2 (1:10,000, Abcam, Cambridge, USA) and antiphosphorylated-ERK1 / 2 (1:2,000, Abcam, Cambridge, USA). After washing, the membrane was incubated at room temperature for 1 hour with a goat anti-rabbit IgG secondary antibody (1:2,000, Abcam, Cambridge, USA) and finally visualized using a Miracle-Star™ Western Blot Detection System (Intron Biotechnology, Gyeonggi-do, Korea). GAPDH was used as an internal control to normalize the levels of the target protein.

[0107] As a result, no differences were observed between groups regarding the total morphology of ERK1 / 2 (Figs. 2a and b). In contrast, compared to the control, hypoxic, and post-infarction groups, the phosphorylated form of ERK1 / 2 (p-ERK1 / 2) increased in a statistically significant manner in the drowning group (p < 0.01) (Fig. 2c). In particular, the band intensity of p-ERK2 at 42 kDa was significantly higher in the drowning group than in the other groups. Meanwhile, p-ERK1 at 44 kDa was hardly observed in any group (Fig. 2a).

[0109] Immunohistochemical analysis

[0110] Based on the results of immunoblot analysis, activated ERK was visualized using histological methods.

[0111] Specifically, lung tissue was fixed with 10% formalin and then embedded in paraffin. The embedded paraffin blocks were sliced ​​to a thickness of approximately 4 μm. The tissue slides were deparaffinized with xylene and rehydrated, after which the tissue sections were used for IHC staining. The tissue section slides were subjected to antigen retrieval in 0.1 M sodium citrate buffer in a microwave for 20 minutes. To reduce non-specific background staining, the samples were immersed in a horseradish peroxidase (HRP) blocking solution for 15 minutes and in 2% normal goat serum for 30 minutes. The treated samples were then incubated with rabbit anti-phospho-ERK1 / 2 polyclonal antibody (1:100, Abcam, Cambridge, USA) in a humidified chamber at 4°C for one day. Subsequently, the immunoreaction was visualized using HRP-conjugated secondary goat antibody and 3,3'-diaminobenzidine (DAB) solution from the HRP / DAB detection IHC kit (Abcam, Cambridge, USA) according to the manufacturer's instructions. Finally, after washing with phosphate-buffered saline (PBS), the sample was counterstained using Mayer's hematoxylin.

[0112] As a result, immunohistochemical expression of p-ERK was increased in the drowning group compared to other cases (Fig. 3). Specifically, p-ERK was observed as a granular pattern in alveolar cells of freshwater and seawater drowning cases. In contrast, p-ERK was hardly expressed in the control, hypoxic, and post-incident drowning groups.

[0114] Gene expression according to PMI of drowning

[0115] The inventors confirmed mRNA expression in lung tissue of the drowning group at different PMIs (2 hours and 48 hours) using quantitative real-time PCR.

[0116] As a result, there was no difference in gene expression according to water composition, but ERK2 expression increased in the drowning group compared to the control group under the 2-hour drowning condition (Fig. 4a). However, under the 48-hour drowning condition, expression significantly decreased in the freshwater drowning group compared to the control group (p<0.001). In addition, a statistically significant difference in expression was observed between seawater and freshwater drowning (p <0.001) (Fig. 4b).

Claims

Claim 1 A biomarker composition for diagnosing drowning comprising ERK2 (Extracellular Signal-Regulated Kinase 2) and / or p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2), wherein the drowning is fresh water drowning or seawater drowning, and the composition is characterized as being applicable to the diagnosis of drowning applicable to a) a sample with a postmortem interval of immediately after death to within 2 hours or b) a sample with a postmortem interval of 48 hours or more. Claim 2 A composition for diagnosing drowning comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or a protein encoded by said gene, wherein the drowning is fresh water drowning or sea water drowning, and the composition is characterized as being applicable to a) a sample with a postmortem interval of immediately after death to within 2 hours or b) a sample with a postmortem interval of 48 hours or more. Claim 3 A composition for diagnosing drowning according to claim 2, characterized in that the protein is p-ERK2 (phosphorylated form of Extracellular Signal-Regulated Kinase 2). Claim 4 A composition according to paragraph 2, wherein the preparation for measuring the mRNA level of the gene comprises sense and antisense primers or probes that bind complementarily to the mRNA of the gene. Claim 5 A composition according to paragraph 2, wherein the preparation for measuring the protein level comprises an antibody or an antigen-binding fragment thereof, or an aptamer, that specifically binds to a protein or a fragment thereof encoded by the gene. Claim 6 A kit for diagnosing drowning comprising the composition of claim 2, wherein the composition is applicable to a) a sample with a postmortem interval of immediately after death up to 2 hours or b) a sample with a postmortem interval of 48 hours or more, and wherein the drowning is fresh water drowning or sea water drowning. Claim 7 A composition for distinguishing drowning environments comprising a preparation for measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or a protein encoded by said gene, wherein the composition is applicable to a) a sample with a postmortem interval of immediately after death to within 2 hours or b) a sample with a postmortem interval of 48 hours or more, and wherein the drowning environment is a fresh water drowning environment or a sea water drowning environment. Claim 8 delete Claim 9 A kit for distinguishing drowning environments comprising the composition of claim 7, wherein the composition is applicable to a) a sample with a postmortem interval of 2 hours immediately after death or b) a sample with a postmortem interval of 48 hours or more for distinguishing drowning environments, and wherein the drowning environment is a fresh water drowning environment or a sea water drowning environment. Claim 10 i) a step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample; and ii) a step of classifying it as drowning if the measured level of ERK2 increases compared to a control group when the postmortem interval of the sample is within 2 hours immediately after death, or classifying it as fresh water drowning if the measured level of ERK2 decreases compared to a control group when the postmortem interval of the sample is 48 hours or more. Claim 11 delete Claim 12 i) a step of measuring the level of mRNA of the ERK2 (Extracellular Signal-Regulated Kinase 2) gene or the protein encoded by said gene in a sample with a postmortem interval of 48 hours or more; and ii) a step of classifying as fresh water drowning if the measured level of ERK2 decreases compared to a control group; wherein the drowning environment is characterized as being a fresh water drowning environment or a sea water drowning environment. Claim 13 delete Claim 14 delete Claim 15 delete

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