Method for determining non-small cells lung cancer sensitivity to preparations reactivating protein p53. RU patent 2509808.

FIELD: medicine.

SUBSTANCE: invention refers to oncology and molecular biology. What is presented is a method for determining the non-small cells lung cancer sensitivity to the preparations reactivating protein p53, involving the recovery of RNA from samples, the synthesis of complementary DNA of the genes CDKN1A, BTG2 and E2F1 by reverse transcription and real-time polymerase chain reaction, and the determination of a relation of the amount of complementary DNA of the gene E2F1 to the amount of complementary DNA of the gene CDKN1A or the gene BTG2, wherein if observing the relation of E2F1/CDKN1A>3 or E2F1/BTG2>1.5, the non-small cells lung cancer cells are considered to be sensitive to the preparations reactivating protein p53.

EFFECT: invention may be used in treating oncological diseases.

3 dwg, 3 tbl, 1 ex

The present invention relates to the field of Oncology and molecular biology, and can be used as a method of additional examination of patients with non-small cell lung cancer to determine the sensitivity of tumor cells to the action of drugs, reactivating p53 protein.

Perspective direction of cancer treatment is targeted therapy, implying aimed pharmacological effects on the key for the tumor cells regulatory protein (called the target of the medicinal product). One such protein targets is a protein p53. P53 is a powerful tumor suppressor. Its activation in response to stress or violation of the integrity of genomic DNA leads to cell cycle arrest and apoptotic cell death. Loss of cell function of p53 protein is the Central event in the formation of malignant phenotype of cells, and also serves as one of the causes of drug resistance of tumor cells to the action of genotoxic chemicals. Thus, the restoration of normal activity of p53 in the cell is a promising approach to treatment of malignant diseases.

A key negative role in the regulation of p53 in tumor cells plays a protein Mdm2, representing an E3 ubiquitin ligase. In the cell Mdm2 is associated with the N-terminal domain of p53 protein, thus there is inactivation last and subsequent proteolytic ubiquitination degradation [1]. Structural homologs Mdm2 - Mdm4 (or Mdmx) also participates in the negative regulation of p53, forming heterocomplexes Mdm2 due to the interaction of RING domains [2]. In tumor cells, as a rule, there is an increase in gene expression MDM2 and (or) MDM4. As a result, the tumor cells rapid degradation of p53 protein, which allows them to avoid p53-dependent apoptosis. The destruction of the complex Mdm2-p53 contributes to the stabilization of p53 and restoration of its activity, which in turn causes it to stop proliferation and (or) destruction of tumor cells, as well as to increase the sensitivity of tumors towards radiation, and genotoxic chemotherapy. This strategy may be promising in the treatment of malignant disease, which remains the expression of a gene TR wild type.

As preparations reactivating p53, known connection, a displacement of protein p53 complex Mdm2-p53. These compounds include (but not limited to) Nutlins (tested nutlins) [3], BDAs (benzodiazephine) [4] and a series of Mdm2 inhibitors (Ml) derivative spirooxazines, including MI-63, MI-219 and MI-43 [5, 6]. All three series of compounds mimic amino acid residues F19, W23 and L26 protein p53 and with high affinity contact Mdm2 in the field of p53-specific pockets and thus displace p53 protein of Mdm2-p53 complex. Table 1 provides a list of the chemical compounds - antagonists Mdm2, which today are investigated as a therapeutic drugs. Information about ongoing clinical trials is obtained from the site .

Of all the compounds inhibiting the interaction of proteins p53, Mdm2, the most well-studied Nutlins (tested nutlins). Data preclinical trials connection Nutlin-3A for the treatment of acute myeloid leukemia [7, 8] confirm the ability of the Nutlin-3A to induce apoptosis of blast cells but not normal hematopoietic blood cells). The results of preclinical trials Nutlin-3A in the treatment of neuroblastoma [9] also confirm the ability Nutlin-3A to induce apoptosis in neuroblastoma cells resistant to chemotherapy. In both cases, the cytotoxic effect Nutlin-3 was observed only in the case of a tumour nematanthus genome TR.

To assign a target therapy based on the use of medicinal compounds, reactivating protein p53, you must define the criteria characterising a category of patients with a high probability of responding to treatment with drugs of the group under consideration. Therefore, the urgent task is the development of methods of determining the sensitivity of tumor cells, in particular, non-small cell lung cancer (NSCLC), to the treatment of drug reactivating p53 protein.

The most closest to the claimed method of the prototype is the way determining the sensitivity of cells lymphocytic leukemia to the drug (Nutlin-3)reactivating p53 protein [10], which includes the following stages: allocation of RNA from the blood cells using RNeasy Mini Kit (Qiagen, Germany); cDNA synthesis reaction reverse transcription using random hexanucleotide and oligo(dT) primer; amplification of cDNA fragment of the gene MDM2 through Poland to the detection results in real time, the amount of cDNA gene MDM2 and determination of the level EKSPRESSII gene MDM2 by regulation of the results obtained regarding the number of cDNA normalization of the gene. As normalization gene authors use gene household GAPDH.

The disadvantage of this method is considerable variability of the results of the analysis if the wrong choice of one of normalization of the gene. When choosing normalization of the gene it is assumed that the level of its expression in cells does not change depending on the type of sample. However, there is no perfect normalization of the gene whose expression level will remain constant regardless of the fabric and the state of the cells in the analyzed sample.

Object of the present invention to provide a more reliable and fast method of determining the sensitivity of cells of non-small cell lung cancer to the action of drugs reactivating p53 protein.

Technical result achieved when using the proposed method is increase the accuracy of the method by reducing errors calculations made by using one of normalization of the gene; in reducing the number of amplification by eliminating the need to analyze the level of expression of several genes normalization.

The task is achieved offer the method lies in the fact that as a criterion of the sensitivity of cells NSCL consider the ratio of cDNA gene E2F1 and the amount of cDNA gene CDKN1A or gene BTG2, while the relative amount of cDNA genes CDKN1A, BTG2 and E2F1 determine the method of reverse transcription and polymerase chain reaction detection results in real time.

The method of reverse transcription and polymerase chain reaction (RT-PCR) with registration of the results in real time allows to determine the relative or absolute number of mRNA molecules with a given nucleotide sequence in the sample. One of the applications of the method is the determination of the level of expression of genes encoding diagnostically significant protein factors in the cells of various tissues. Method RT-PCR includes three phases: synthesis of cDNA molecules (DNA complementary cellular RNA) by using the reverse transcription reaction (); amplification of cDNA sequence corresponding to the fragment of the analyzed gene; the analysis of the results and determination of the relative number and level of expression of cDNA analyzed gene in the sample. A number of specific cDNA in the sample on the one hand depends on the level of mRNA expression of the corresponding gene in the cells of the sample, on the other hand, it is determined by the volume of the cell material, degree of degradation of the analyzed RNA and presence in the sample inhibiting admixtures reaction FROM. To consider the influence of additional factors the level of expression of cDNA analyzed gene is calculated relative to the expression of cDNA normalization of the gene. As such normalization of genes used, as a rule, the genes of the ' household'. However, there is no perfect normalization of the gene whose expression level will remain constant regardless of the fabric and the state of the cells in the test specimen, therefore, in practice, the valuation is performed with the use of multiple genes normalization. Thus normalizing factor is calculated as the arithmetic average of the values of the relative amount of cDNA all genes normalization [11].

More specifically, the proposed method consists in definition of the relative amount of cDNA genes CDKNJA, BTG2 and E2F1 RT-PCR mode real time with the subsequent calculation of the amount of cDNA gene E2F1 to the amount of cDNA gene CDKN1A or gene BTG2 and when the value of the ratio E2F1/CDKN1A>3 or E2F1/BTG2>1,5 believe NSCLC cells sensitive to preparations of reactivating p53 protein.

The relative amount of cDNA calculated using the calibration curve was constructed at amplification standard DNA samples, taken 5 consistently decreasing known concentrations. The standard is a mixture of fragments of DNA that carry amplificarea sequence to DNA genes CDKN1A, BTG2 and E2F1 length of 500 base pairs. The relative amount of cDNA each gene determined on the basis of the equations of the calibration curve, thus take into account the efficiency of amplification of each sample. Next, calculate markers, taking into account the dynamics of the cell cycle (the balance of factors that regulate G1/S cell cycle arrest and the stage of replication).

Markers calculated by the formula:

where P S is the number of cDNA gene factor controlling replication;

P G1/S - the number to the DNA of a gene factor controlling G1/S cell cycle arrest;

E 1 and E 2 - effectiveness of gene amplification in Ps and PG]/S, respectively.

Ct1 and Ct2 - value threshold cycle PCR, automatically determined by the device the amplification of the gene fragments P S and P G1/S respectively.

The proposed method includes the following standard stage.

The allocation of RNA from samples.

As samples for analysis can be taken biopsies and(or) punctate tumor tissue, culture.

Method of extraction of total RNA from tissue samples mammals includes the following stages: crushing samples in the homogenizer, or in liquid nitrogen (refers to a dense, difficult homogenisierung fragments of tissue), lysis of cells, the allocation of RNA and its treatment, quality RNA electrophoresis in 1-1,5%agarose gel in the presence of dye ethidium bromide, and spectrophotometric determination of the number of RNA. Homogenization pieces of fabric carried out manually, rubbing with a pestle in a ceramic mortar, or by using mechanical homogenizers, for example TissueLyser (Qiagen). For lysis of cells and RNA extraction using strong atrophie agents in combination with a detergent, such as guanidinoproprionic and Sarkozy, then successively the extraction is carried out by phenol when rn 5.0 and chloroform for denaturation and remove protein and genomic DNA [Chomczynsci P., Sacci N. Single-step method of RNA isolation by Acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry. 1987/ Vol 162. P 156-159]. Next RNA precipitated in the presence of isopropanol or ethanol or adsorb on solid media, for example, Silica S-5631(Sigma) or spin-speakers (Qiagen; Promega). To prevent the erosion of RNA RNase can be used inhibitors of ribonuclease, such as recombinant RNAsin (Promega). For elimination of impurities genomic DNA can handle the drug RNA purified from the ribonuclease Nakasai. The allocation of RNA carried out using Trizol reagent®Reagent [Invitrogen] or by using commercially available kits, such as RNeasy kit (Qiagen), SV Total RNA Isolation System (Promega), etc.

The cDNA synthesis with the help of the reverse transcription reaction.

As a result of the reverse transcription to RNA-matrix is synthesized single-stranded DNA. The resulting cDNA molecules are more stable in comparison with unstable molecules of RNA and can be amplified by polymerase chain reaction to quantities required for detection.

Reaction reverse transcription is done using various commercial preparations reverse transcriptase inhibitors, for example, reverse transcriptase leukosis virus of mice, Malone (M-MLV), virus myeloblastosis birds (AMV), PowerScript (point mutation M-MLV-RT), .Therm Polymerase and other Can be used thermostable DNA polymerase Thermus thermophilus (Tth)with reverse transcriptase activity in the presence of ions 2 MP+ .

The reverse transcription reaction starts with the formation of double-stranded seed, for that use different types of primers. For example, oligo(dT)n primers (number n is usually 12-18)that communicates with endogenous the poly-a tail on the 3'-end of mRNA. These primers used for the full-size cDNA. To 3'-end of oligo(dT) sequences can be added nucleotides a, C, or G to "anchor" primer on the border of the transcript and poly-And tract.

Can also be used random hexanucleotide primers (statistical primers), hybridization in several areas throughout the RNA. In response reverse transcription using statistical primers are synthesized shorter (about 500 gel) cDNA fragments. The reverse transcription reaction using statistical primers, as a rule, is more efficient, especially when reverse transcription GC-rich areas and 5'-region of mRNA.

In addition to the review of the primers can be used longer 9-12-dimensional primers. You can also use statistical primers in combination with oligo(dT) primer or specific primers, complementary sequence analyzed gene.

Amplification of cDNA genes CDKN1A, BTG2 and E2F1 using PCR detection in real time.

Amplification of the transcript fragments of genes CDKN1A, BTG2 and E2F1 perform using pairs of primers complementary to the transcripts. When choosing primers for amplification of cDNA take into account the exon-intron structure of the gene and place the primers so that their sites hybridization were located in different exons. Thus, supressirovano possible impurities genomic DNA in the sample.

Reaction amplification is carried out using commercially available drug DNA polymerase Thermus aquaticus, for example HS Taq DNA polymerase, (Email) GoTaq© Hot Start Polymerase (Promega) and others as a matrix using cDNA is synthesized on the matrix total cellular RNA.

The accumulation of products of amplification of cDNA CDKN1A, BTG2 and E2F1 detects in real-time using one of the methods:

- or using inteilimouse double-stranded DNA fluorescent dye with a high index of discrimination fluorescence from related and unrelated forms, for example, SYBR GreenI (Invitrogen);

- or using oligonucleotide sample type TaqMan complementary Central part amplificarea fragment of the transcript. Sample length of 20-30 nucleotides bears at the 5'end of the fluorophore (FAM, R6G, ROX) and at the 3'end, not fluorescent cositel (BHQ, FTQ, and others). On each cycle amplification hibridizarea sample hydrolyzed by the 5'-end, while the fluorophore spatial disjoined with tusitala and, as consequence, increases fluorescence reaction mixture.

Determine number of cDNA genes CDKN1A, BTG2 and E2F1 carried out by means of the equations of the calibration curve according to the effectiveness of each amplification reaction. To construct the calibration curve simultaneously with the analyzed samples amplified standard DNA samples with a known sequence shrinking three times the concentration of DNA. Standard sample is a mixture of double-stranded DNA fragments length of 500 gel, bearing sequence amplificarea fragments transcripts CDKN1A, BTG2 and E2F1. Construct a standard curve depending on the values of Ct and Log the number of DNA in the standard sample. In accordance with the schedule of the calibration curve using regression analysis to determine the coefficients of the linear equation of the calibration curve. The equation used to determine the amount of cDNA CDKN1 A, BTG2 and E2F1 in the analyzed samples.

Below the invention in detail illustrated by concrete examples.

Example.

The research was NSCLC cell lines, carrying gene TR wild type. Just analyzed 8 cell lines: A, NCI-H292, A, COR-L23, DV-90, NCI-H1395, NCI-H1944, NCI-H2228.

Cultivation of NSCLC cell lines.

The sensitivity of cells to the connection Nutlin3 (+or -) (Cayman Chemical) was estimated by the value of the parameter IC50 (inhibitory concentration; concentration of the drug, which in culture there is a dividing 50% of the cells). For this cell A located in the exponential growth phase, were sown in the wells 24-hole tablet 60 Tyskie per well. The cells were cultured in DMEM/F12 containing 10% FBS, 0.03% of glutamine and kanamycin at a concentration of 100 mg/ml at 37 C and 5%concentration of CO 2 in the atmosphere. After 24 hours culture medium was replaced with a fresh containing Nutlin3 (+or -) in concentration from 50 microns to 1 micron (each point has been presented in the triple repetition). 48 hours after the addition of chemotherapy drugs was determined share of viable cells using test the viability with rezisoriem. Resazurin (resazurin sodium salt, Diaem) was added to the cells in a concentration of 50 mg/ml Amount culture medium was 500 ml. As a control (background) used 500 cells / mm culture medium, not containing cells NSCL. The length of time the incubation of cells with rezisoriem was 1 hour. Incubation was performed in sterile conditions, when 99% humidity and a temperature of 37 C and the concentration of 2 - 5%. The accumulation resorufin (restored form of resazurin) was estimated by using the fluorescence detection in the range 587/607 nm. Results of the test were constructed calibration curve changes fluorescence depending on the number of cells. IC50 values for each cell lines are given in table 3.

The allocation of RNA and cDNA synthesis reaction of reverse transcription.

Cells not exposed to Nutlin3 (+or -) and in the exponential growth phase, were sown in the hole 6-hole tablet and cultivated up to 70%density monolayer. After this culture medium was removed, and the cells were literally reagent Trisol ("Invitrogen"). Received the drugs were stored at -70°N Total cellular RNA was allocated according to the recommendations of Invitrogen. The quality of drugs RNA was evaluated according to the results of electrophoretic separation 1.3% agarose gel. The reverse transcription reaction was carried out at 42 degrees C for 45 min in 20 ml reaction mixture containing 10 mm Tris-HCl (pH 8.3), 5 mm MgC12, 10 mm DTT, 50 mm Kl, 0.2 mm dNTP, Stat-9 primer (10ng/ml), 100 edict. DNA-dependent RNA polymerase MoMLV (Biosan, Siberian branch of the SOR EN) and 500 ng of total cellular RNA.

Amplification of cDNA genes CDKN1A, BTG2 and E2F1 with detection in real time.

Reaction amplification was performed using thermal cycler with optical unit for the detection of fluorescence CFX96 (Bio-Rad). The possibility of registration of fluorescence in the real-time mode was achieved through hybridization sample type TaqMan.

Poland was carried out in the amount of 25 MKL, the reaction mixture contained 65 mm Tris-HCl (pH 8.9); 24 mm (NH 4 ) 2 SO 4 ; a 0.05% Tween 20; 2.5 mm MgCl 2 , 0,2mM dNTP, 300 nM primers (forward and reverse), 100 nM Taq-Man test, 0.5 ..Taq-DNA polymerase (Biosan, Siberian branch of the SB RAS) and 0.1-5 ng cDNA.

Protocol amplification:

- initial denaturation - 3 minutes at 96 Degrees;

- amplification cycle (x 40);

- denaturation - 10 seconds at 96 Degrees;

- annealed primers, elongation and eat fluorescent signal is 60 seconds at 60 C.

Specificity of amplification was confirmed by electrophoretic analysis in polyacrylamide gel.

For amplification used appropriate hybridization tests and forward and reverse primers. Patterns of oligonucleotide primers and hybridization of samples is presented in table 2.

Analysis of the results of RT-PCR.

Just 8 samples were analysed. For each sample using the software of the device identified the number of threshold cycle (Ct), where the amount of fluorescence reaction mixture has reached a certain threshold value (T) is the same for all compared samples. Determination of the relative amount of cDNA CDKN1A, BTG2 and E2F1 (X 0 ) in the analyzed samples was calculated by the formula:

Log(X 0 )=a+b·Ct,

where a=log(T); b=log(1+E); E - effectiveness PCR, the same for all the analyzed samples.

Among the eight NSCLC cell lines were allocated to three lines (NCI-H2228, NCI-H1944, A), resistant to Nutlin3(yo) for which the average value IC50 amounted to 24 microns±4,5 microns and five lines (NCI-H292, A, COR-L23, DV-90, NCI-H1395), sensitive to the action Nutlin3(yo) for which the average value IC50 was 7.9 microns±2,9 mm. IC50 values connections Nutlin3 (+or -) to eight NSCLC cell lines are given in table 3.

The ratio of the level of expression of E2F1/CDKN1A and E2F1/ BTG2 was significantly higher in the group is sensitive to the action Nutlin3 (+or -) lines NSCL compared with the group resistant to Nutlin3 (+or -) lines NSCLC (p=0.045 and 0.007 respectively). The values of the ratios of E2F1/CDKN1A and E2F1/ BTG2 for each cell line is shown in figure 1.

The dependency ratio E2F1/CDKN1 and the ratio of E2F1/BTG2 from the IC50 values connections Nutlin3 (+or -) to eight NSCLC cell lines presented in figure 2, 3. Obtained values of the ratios of the levels of expression of E2F1/CDKN1A and E2F1/ BTG2 significantly correlated with IC50 values with a correlation coefficient R=0.64 and R 2 =0,48. The use of the proposed method will allow us to more accurately predict the sensitivity of cells non-small cell lung cancer to the action of drugs reactivating p53 protein.

Sources of information

1. Haupt, Y., Maya, R., Kazaz, A. & Oren M. Mdm2 promotes the rapid degradation of p53. Nature (1997) 387, 296-299.

2. Wade, m., Wang, Y. V. & Wahl, G. M. The p53 orchestra: Mdm2 and Mdmx set the tone. Trends Cell Biol. (2010) 20, 299-309.

3. Vassilev LT, Vu W, B Graves, Carvajal D Podlaski F, Filipovic Z Kong N, Kammlott U, the Lukacs C, Klein C, et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science. (2004); 303(5659), 844-848.

4. Grasberger BL, Lu T, Schubert C, Parks DJ, Carver THOSE Koblish HK, Cummings MD, LaFrance LV, Milkiewicz KL, Calvo RR, et al. Discovery and cocrystal structure of benzodiazepinedione HDM2 antagonists that activate p53 in cells. J Med Chem (2005) 48: 909-912.

5. Shangary S, Qin D, McEachern D, Liu M, Miller RS, Qiu S, Nikolovska-Coleska Z, Ding K, Wang G, Chen J, et al. Temporal activation of p53 by a specific MDM2 inhibitor is selectively toxic to tumors and leads to complete tumor growth inhibition. Proc Natl Acad Sci USA. 2008; 105(10): 3933-3938.

6. Ding K, Lu Y, Nikolovska-Coleska Z, Wang G, Qiu S, Shangary S, Gao W, Qin D, Stuckey J, Krajewski K, et al. 2006. Structure-based design of spiro-oxindoles as potent, specific small-molecule inhibitors of the MDM2-p53 interaction. J Med Chem 49: 3432-3435.

7. Kojima, K. et al. MDM2 antagonists induce p53-dependent apoptosis in AML: implications for the leukemia therapy. Blood (2005) 106, 3150-3159.

8. Carter, B. Z. et al. Simultaneous activation of p53 and inhibition of XIAP enhance the activation of apoptosis signaling pathways in AML. Blood (2010) 115,306-314. 9. Van Maerken, T. et al. Antitumor activity of the selective MDM2 antagonist nutlin-3 against chemoresistant neuroblastoma with wild-type p53. J Natl Cancer Inst. (2009) 101, 1562-1574.

10. L Gu, N Zhu, H W Findley and M Zhou. MDM2 antagonist nutlin-3 is a potent inducer of apoptosis in pediatric acute lymphoblastic leukemia cells with wild-type p53 and overexpression of MDM2Nutlin-3 dosage apoptosis in ALL cells. The Leukemia (2008) 22, 730-739.

11. Tricarico, C, Pinzani, P., Bianchi, S., Paglierani, M., Distante, V., Pazzagli, M., Bustin, S.A., Orlando, C. Quantitative real-time reverse reduced polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies. // Anal Biochem. (2002) - N309. - P.293-300.

The method of determining the sensitivity of cells non-small cell lung cancer to the action of drugs reactivating p53 protein

Testing phase

Nutlin/RG7112 (Roche, Penzberg, Germany)

Clinical trials of phase-I NCT00623870 and NCT00559533 (hematological neoplasms)

MI-219/AT-219(Ascenta Therapeutics, Malvern, PA, USA)

Preclinical trials

RITA (Aprea, Solna, Sweden)

Preclinical trials

JNJ-26854165 (Johnson & Johnson, New Brunswick, NJ, USA)

The clinical trial stage-SST (hematological neoplasms)

PXN727 and PXN822 (Priaxon, Munich, Germany)

Preclinical trials

Designation primer

Patterns of oligonucleotide primers

CDKNIA F CDKNIA R CDKNIA PR

5' -TGTGG ACCTGTC ACTGTCTTGT-3' 5' -CGTTTGG AGTGGTAG A AATCTGT-3' 5'-FAM - CTTGTGCCTCGCTCAGGGGAGC-BHQ - 3'

E2F1-F E2F1-R E2F1-PR

5' -WITH ATCC AGCTC ATTGCC AAG AG A-3' 5 '-GCTGCGTAGTACAGATATTCATCA-3' 5'-FAM - CCACATCCAGTGGCTGGGCAGC-BHQ-3'

BTG2 F BTG2 R BTG2 PR

5' -TGGGCTT AGGG AACC ATCTCT-3' 5 '-TTCAGCC AAGGAATACATGCAA-3' 5'- FAM-CTGCTCTCCTTGGGATGATGGC-BHQ-3'

Designation cell lines

The average value IC50

Standard deviation

At 23.67

The method of determining the sensitivity of cells of non-small cell lung cancer (NSCLC) to the action of drugs reactivating p53 protein including the allocation of RNA of the samples, the cDNA synthesis using reaction reverse transcription and amplification of cDNA corresponding genes by means of PCR detection in real time with the subsequent analysis of the level of expression of cDNA selected genes, wherein determine the relative the amount of cDNA genes CDKN1A, BTG2 and E2F1 RT-PCR in real time, then calculate the ratio of cDNA gene E2F1 to the amount of cDNA gene CDKN1A or gene BTG2 and when the value of the ratio E2F1/CDKN1A>3 or E2F1/BTG2>1,5 believe NSCLC cells sensitive to drugs, reactivating p53 protein.