Patent Description:
Fine dust, also called particle matter, is a particulate material that floats in the air and is known to be typically generated from exhaust gases of automobiles, combustion of fossil fuels, road dusts, etc. Particle matter is a Group <NUM> carcinogen that can cause cancer, designated by the International Agency for Research on Cancer (IARC), and fine dust particles having a diameter of less than <NUM> may be induced into the lung and blood, consequently posing a major threat. It is known that the particulate matter increases the incidence of lung cancer by causing coughing or phlegm due to accumulation of dust in the bronchus, and inducing an inflammation reaction due to the dust accumulating in the pulmonary alveolus, thereby generating active oxygen, resulting in necrosis of tissues and degrading lung functions.

Therefore, since the particulate matter causes an inflammatory disease or leads to a potential risk of lung cancer in a serious case, there are constant demands for discovery of a biomarker capable of predicting a change in the lung due to the particulate matter and quickly responding to a change in risk factors.

The provided invention is according to the claims.

An aspect of the disclosure, which is not claimed, is to provide a composition for identifying exposure to particulate matter in a lung, comprising an agent for measuring an expression level of a Testican-<NUM> gene or an expression level of a Testican-<NUM> protein or a fragment thereof.

The term "particulate matter" refers to fine dust particulate matter (PM) floating in the air. The particulate matter may include ionic components, such as nitrates (NO<NUM>-), ammonium (NH<NUM>+), or sulfate (SO<NUM><NUM>-), carbon compounds, metal compounds, polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, or a combination thereof. The particulate matter may be categorized according to its particle size: particulate matter (PM<NUM>) with a diameter of not greater than <NUM> and ultrafine particulate matter (PM<NUM>) with a diameter of not greater than <NUM>. PM<NUM> may include PM<NUM>. The particulate matter may have an average diameter of about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, about <NUM> or less, or a combination thereof.

The "lung" may be a lung tissue or a lung cell. The lung cell may be a lung fibroblast.

The "exposure to particulate matter in a lung" may refer to a case in which particulate matter passes through the respiratory organ of a subject to come into contact with lung tissues or lung cells of the subject.

Testican-<NUM> may also be called SPOCK or SPOCK1. Testican-<NUM> may be a protein encoded by SPOCK1 gene in humans. Testican-<NUM> may be proteoglycan including chondroitin sulfate and heparan sulfate. Testican-<NUM> may include an amino acid sequence of Uniprot ID NO: Q08629 in humans. Testican-<NUM> may include an amino acid sequence of Uniprot ID NO: Q62288 in mice.

The fragment may refer to a part of Testican-<NUM> protein, and may be immunogenic polypeptide.

The Testican-<NUM> gene refers to a nucleic acid encoding Testican-<NUM> protein. The Testican-<NUM> gene may be a SPOCK1 gene.

The expression level of Testican-<NUM> gene may be an expression level of mRNA encoding Testican-<NUM> protein. The expression level of mRNA may be a relative or absolute amount of mRNA. The measuring of the expression level of Testican-<NUM> gene may be measuring an amount of mRNA.

The expression level of Testican-<NUM> protein or a fragment thereof may be a relative or absolute amount of Testican-<NUM> protein or the fragment thereof. The measuring of the expression level of the Testican-<NUM> protein or the fragment thereof may be measuring an amount of Testican-<NUM> protein or the fragment thereof.

The agent may be an antibody or an antigen-binding fragment, or aptamer, specifically binding to Testican-<NUM> protein or the fragment thereof. The antibody may be a polyclonal antibody or a monoclonal antibody. The term "antibody" may be used interchangeably with the term "immunoglobulin". The antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide including an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab', or scFv. The antibody or the antigen-binding fragment may be adhered to a solid support material. The solid support material may be, for example, a surface of a metal chip, plate, or well. The antibody or the antigen-binding fragment may be an anti-Testican-<NUM> antibody or an antigen-binding fragment. The aptamer may be a single stranded nucleic acid (DNA, RNA or modified nucleic acid) or peptide specifically binding to Testican-<NUM> protein or a fragment thereof.

The agent may be a nucleic acid including a polynucleotide identical to or complementary to a polynucleotide encoding Testican-<NUM> protein or a fragment thereof. The nucleic acid may be a primer, a probe, or an anti-sense oligonucleotide. The primer, the probe, or the anti-sense oligonucleotide may be labeled with a fluorescent substance, a chemiluminescent substance, or a radioactive isotope, at an end or inside thereof.

The composition may be a composition for in vitro measuring an expression level of Testican-<NUM> gene or an expression level of Testican-<NUM> protein or a fragment thereof in a lung tissue or a lung cell.

Described herein is a kit for identifying exposure to particulate matter in a lung, comprising an agent for measuring an expression level of a Testican-<NUM> gene or an expression level of a Testican-<NUM> protein or a fragment thereof.

The Testican-<NUM>, Testican-<NUM> gene, fragment, expression level, lung, and exposure to particulate matter, are the same as described above.

The kit may further include a sample necessary for identifying exposure to particulate matter. For immunological detection of the antibody or the antigen-binding fragment thereof, the kit may include a substrate, a suitable buffer solution, a chromogenic enzyme, a secondary antibody labeled with a fluorescent substance, or a chromogenic substrate. For detection of nucleic acids, the kit may include a polymerase, a buffering agent, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof. The polymerase may be, for example, a Tag polymerase.

The kit may be a kit for in vitro measuring an expression level of Testican-<NUM> gene or an expression level of Testican-<NUM> protein or a fragment thereof in a lung tissue or a lung cell.

Another aspect is to provide a method for detecting Testican-<NUM> for providing information required in identifying exposure to particulate matter in a lung, the method comprising: measuring an expression level of Testican-<NUM> gene or an expression level of Testican-<NUM> protein or a fragment thereof in a biological sample isolated from a subject with suspected exposure to particulate matter; comparing the measured expression level with that in a normal control group; and identifying whether the Testican-<NUM> expression level has increased as compared with that in the normal control group, wherein an increase in expression level indicates a high probability of exposure to particulate matter in the lung.

The method comprises measuring an expression level of Testican-<NUM> gene or an expression level of Testican-<NUM> protein or a fragment thereof in a biological sample isolated from a subject with suspected exposure to particulate matter.

The subject may be a mammal, for example, such as a human being, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The subject may be a subject suspected to be exposed or have been exposed to particulate matter.

The biological sample refers to a sample obtained from the subject. The biological sample may include a lung tissue, a lung cell, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof. The lung cell may be a lung fibroblast. The biological sample include a protein sample or a nucleic acid obtained from the sample.

The measuring may include incubating the biological sample with an antibody or an antigen-binding fragment thereof, or an aptamer that specifically binds to Testican-<NUM> protein or a fragment thereof. The measuring may be performed by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), protein chip, immunoprecipitation, microarray, electron microscopy, or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, 2D electrophoresis, or a combination thereof.

The measuring may include incubating the biological sample with a polynucleotide identical to or complementary to a polynucleotide encoding Testican-<NUM> protein or a fragment thereof. The measuring may be performed by northern blotting or polymerase chain reaction (PCR). The PCR may be real time PCR or reverse transcription PCR.

The method includes comparing the measured expression level with that in a normal control group. The method includes identifying whether the Testican-<NUM> expression level has increased as compared with that in the normal control group. The normal control group refers to a group without exposure to particulate matter, and is referred to as a negative control group. In the method, the amount of the measured expression level of Testican-<NUM> from sample exposed to particulate matter may be increased compared to the expression level of Testican-<NUM> derived from the sample of the normal control group. For example, when the amount of detected Testican-<NUM> protein is greater than that in the normal control group, the subject may be diagnosed as having a high probability of being exposed or being exposed to particulate matter. The subject identified or diagnosed with exposure to particulate matter may be determined to suffer from or have a high probability of a respiratory disease, a vascular disease, a brain disease, a cancer, or a combination thereof. The respiratory disease may be cold, nasal inflammation, asthma, sinusitis, or pneumonia. The vascular disease may be arteriosclerosis, arrhythmia, angina, myocardial infarction, heart failure, heart attack, or a combination thereof. The brain disease may be stroke, ischemic stroke, cognitive disorder, dementia, depression, or a combination thereof. The cancer may be a lung cancer or a bladder cancer.

Hereinafter, the present disclosure will be described in greater detail through examples. However, the following examples are provided for illustrating one or more specific examples, and the present disclosure is not limited thereto.

CCD-8Lu (ATCC) as a lung normal fibroblast cell line of a human lung was culturedin the presence of a medium with <NUM>% (v/v) fetal bovine serum (Gibco®, Thermo Fisher Scientific Inc. ) and <NUM>% (w/v) antibiotic-antimycotic (Gibco®, Thermo Fisher Scientific Inc. ) added to an Eagle's Minimum Essential Medium (EMEM, BD Bioscience) in a <NUM>, <NUM>% CO<NUM> incubator.

A particulate matter powder (SRM2975, National Institute of Standards & Technology) was dissolved in a phosphate buffered saline (PBS) to prepare <NUM>/mL of a particulate matter solution. The prepared particulate matter solution was diluted with an EMEM medium to prepare concentrations of <NUM>µg/mL, <NUM>µg/mL, <NUM>µg/mL, and <NUM>µg/mL. The prepared particulate matter solutions were added to CCD-8Lu cell line and incubated at <NUM> for <NUM> hours or <NUM> hours.

Proteins that are differentially expressed in CCD-8Lu cells treated with particulate matter were analyzed.

Specifically, lysis buffer (<NUM> Tris-HCl (pH <NUM>), <NUM> urea, <NUM> thiourea, <NUM> EDTA, <NUM> NaCl, a protease inhibitor cocktail, and phosphatase inhibitor cocktail (Roche) were added to CCD-8Lu cells treated with particulate matter in the same manner as in Example <NUM>, and then the CCD-8Lu cells harvested using a scraper. The harvested cells were transferred to a <NUM> Lo-bind tube and lysed using ultrasonic processor (Sonics & Materials, Inc. ) to extract proteins from the CCD-8Lu cells. The extracted proteins were quantified using a Bradford assay kit.

Urea was added to the extracted proteins so as to have a final urea concentration of <NUM>, and <NUM>µmοl of TCEP (tris(<NUM>-carboxyethyl)phosphine) was added thereto and incubated for <NUM> hour at room temperature to reduce disulfide bonds. Afterward, <NUM>µmοl of IAA (<NUM>-lodoacetamide) was added and then incubated at room temperature under a dark condition for about <NUM> hour for protein alkylation. <NUM> of an ammonium bicarbonate solution was added to the protein reaction product to reduce the concentration of urea in the protein samples to <NUM> or less. The proteins were incubated at <NUM> for <NUM> hours in the presence of <NUM> mAU of endoproteinase LysC and then incubated at <NUM> overnight in the presence of trypsin. The obtained peptide sample was purified using a SepPak® tC18 cartridge, followed by vacuum drying. The dried peptide sample was suspended in <NUM> of tetraethylammonium bromide (TEAB), and then the peptide was quantified.

TMT labeling reagents (Thermo Fisher Scientific) were dissolved in <NUM>µl of acetonitrile and then added to <NUM>µg of a peptide sample prepared in the same manner as in Example <NUM>. As a negative control group, a peptide sample derived from CCD-8Lu cells incubated for <NUM> hours or <NUM> hours without treatment with particulate matter was used. After the labeling reaction was made by incubation at <NUM> for <NUM> hour, <NUM>µl of <NUM>% (w/v) hydroxylamine was added and incubated at <NUM> for <NUM> hour to stop the labeling process. The TMT-labeled peptide samples were combined using a single tube, and then purified using a SepPak® tC18 cartridge, followed by vacuum drying.

The dried peptide sample was suspended in <NUM> of ammonium formate (pH <NUM>), injected into a C18 column (<NUM> × <NUM>, <NUM>, <NUM>Å; Waters) and fractionated on an Agilent <NUM> ultrahigh performance liquid chromatography (UHPLC) system at a flow rate of <NUM>/min. <NUM> fractions were combined to give <NUM> fractions, and the fractions were then dried (<FIG>).

Each <NUM>µl of the samples obtained in the same manner as Example <NUM> was injected into a reverse-phase PepMap RSLC C18 column (<NUM> × <NUM>µl ) on Ultimate <NUM> system. The column was equilibrated with <NUM>% buffer A (<NUM>% water containing <NUM>% (v/v) formic acid). <NUM>% acetonitrile containing <NUM>% (v/v) formic acid was used as a buffer B. The flow rate was <NUM> nL/min under the following gradient conditions:.

A nano high-performance liquid chromatography (HPLC) system was connected to Orbitrap Fusion Lumos Tribrid mass spectrometer. Survey full-scan MS spectra (<NUM> to <NUM>,<NUM>/z) were obtained with <NUM> microscan and <NUM>,<NUM> resolution, precursors were screened, and a preview mode for measuring a charge state was set. Through preview survey scanning, MS/MS spectra for <NUM> most intense ions were obtained from full-scanning ion traps with the following options: isolation width of <NUM>/z; normalized collision energy of <NUM>%; and a dynamic exclusion duration of <NUM> sec.

The respective LC-MS/MS files were analyzed using the SEQUEST algorithm within Proteome Discoverer <NUM>. MS and MS/MS data were searched by comparison with SwissProt human database. The searches were limited such that two missed cleavages were allowed for tryptic peptides. The searches were performed with fixed modification on carboamidomethylation of cysteines (+<NUM> Da) and TMT labeling of lysine and peptide N-terminal (+<NUM> Da), and variable modification on oxidation of methionine (+<NUM> Da). Mass tolerances for MS/MS data and MS data were set to <NUM> Da and <NUM> ppm, respectively. For decoy database searches, a total of two target values, that is, <NUM> as a strict false discovery rate (FDR) and <NUM> as an eased FDR, were employed.

Relative amounts of proteins existing in cells treated with particulate matter in concentrations of <NUM>, <NUM>, <NUM>, or <NUM>µg/mL for <NUM> hours or <NUM> hours were calculated based on the signal to noise values of reporter ions from the labeled peptides. The signal to noise values of reporter ions of the respective peptides were obtained using Proteome Discoverer <NUM>. To obtain statistically significant proteins based on the signal to noise values for the respective proteins, Perseus software was used. Even when no protein exists or proteins detected, if any, fall short of a detection limit, for compensating for null values and calculating a difference in the fold change among the respective samples, missing values were complemented by downshifting <NUM> and <NUM> in width from a normal distribution.

With regard to statistical significance, ANOVA test was performed on signal to noise values obtained three technical replicates of LC-MS/MS runs, FDR-adjusted p-values were obtained using a Benjamini-Hochberg correction, and, when the FDR-adjusted p-values were less than <NUM>, the protein samples were considered to be statistically significant.

From the results of protein identification performed in the same manner as described in Example <NUM>, it was confirmed that a total of <NUM> proteins were identified in all of the cell samples exposed to particulate matter according to particulate matter concentrations. Out of the identified proteins, <NUM> proteins had signal to noise values suitable for quantitative analysis. When ANOVA statistical analyses were performed on the signal-to-noise based abundance values in the <NUM> proteins, <NUM> proteins having an FDR-adjusted p-value of less than <NUM> were obtained. ,Some of the statistically significant proteins are listed in Table <NUM>.

As shown in Table <NUM>, it was confirmed that the expression levels of Testican-<NUM> in statistically significant proteins were increased as the particulate matter concentration increased. A graph showing signal to noise values of Testican-<NUM> according to particulate matter concentrations is shown in <FIG>.

Accordingly, the Testican-<NUM> was identified as a biomarker for identifying exposure to particulate matter in a lung.

Claim 1:
In vitro use of a composition for identifying exposure to particulate matter in a lung,
wherein the composition comprises an agent for measuring an expression level of Testican-<NUM> gene or an expression level of Testican-<NUM> protein or a fragment thereof; and
wherein identifying exposure to particulate matter in a lung is by identifying an increase in expression level of Testican-<NUM> gene or an increase in expression level of Testican-<NUM> protein or a fragment thereof.