• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    Composition to promote cardiac recovery after a myocardial infarction (im). The present invention offers a solution to the lack of compounds capable of efficiently avoiding deterioration of the heart muscle after mi, and of improving the long-term prognosis of patients who have suffered an mi. The present invention claims compounds capable of inhibiting the expression of the transcription factor yy1 in cardiac cells, thus reducing the expression of the st2s gene and allowing the activation of the cardioprotective signaling chain il33/st2l. The present invention also describes a screening method that allows to identify compounds capable of improving the long-term prognosis of patients who have suffered an mi. This method is based on identifying compounds capable of inhibiting the expression of yy1 and/or activating the cardioprotective signaling chain il33/st2l in cardiac cells. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2637032A1
    申请号:ES201700154
    申请日:2017-02-06
    公开日:2017-10-10
    发明作者:Domingo Andrés PASCUAL FIGAL；María Del Carmen ASENSIO LÓPEZ；Antonio Manuel LAX PÉREZ
    申请人:Universidad de Murcia；
    IPC主号:
    专利说明:

    ~~
    ENTRY
    COMPOSITION TO PROMOTE CARDIAC RECOVERY AFTER A MIOCARD INFRATEMENT 11M).
    Field of the m'cndón
    The present invention belongs to the field of compounds. and methods to identify compounds. able to improve the cardiac recovery of patients who have suffered a myocardial infarction
    (1M).
    Background of the introduction and state of the art
    Cardiovascular diseases cause more deaths than any other condition in the West,
    10 and 1M is still a major health problem. It is caused by the sudden blockage of the coronary blood supply. which leads to the irreversible death of cardiomyocytes. Although the survival levels of patients who have suffered 1M have increased in recent years. The long-term prognosis is still bad due to the structural and functional effects caused by cardiomyocyte necrosis. After 1M, a series of mechanisms of
    15 local repair in the infarcted tissue. mainly inflammation and fibrosis, which carry a risk of ventricular arrhythmia and deterioration of systolic and diastolic myocardial function. That is why 1M carries a high risk of death and evolution to heart failure.
    The treatment of patients who have suffered 1M depends on the size of the infarction. but broadly it is usually based on the following types of drugs:
    20-Acetylsalicylic acid. Heparin Clopidogrel, prasugrel or ticagrelor to inhibit platelet aggregation.
    - Betahloquentes to inhibit the stimulating effect of adrena lina in the heart, thus regulating the heart rate. thus reducing the need for oxygen by the heart muscle.
    - Cholesterol lowering medications (statins).
    25-Drugs to block the harmful effects of angiotensin and aldosterone (EeA inhibitors or angiotensin ARA 11 receptor antagonists, and aldosterone mineralocorticoid receptor antagonists).
    These treatments are mainly focused on avoiding the risk of having a new heart attack, and avoiding the occurrence of complications. ventricular arrhythmias and heart failure. But nevertheless. 30 as mentioned above. today the long-term forecast is still bad due to
    ",,,,., I ', tJbI! M P201700lS4 06/02/2017 27/0212017
    progressive deterioration of cardiac function. For tanlo. new and new treatments are necessary
    strategies to identify new drugs and / or administration doses more effective and capable
    to avoid the deterioration of the heart muscle, you would have a heart attack and thus prolong the life of these patients.
    The Suppression of Tumorigenicity-2 (ST2) protein belongs to the family of receptors of the
    5 interleukin 1 (lU). There is the transmembrane isofonna ST2-1igand (ST2L) and the soluble safanna
    ST2s Recently, Interleukin 33 (IL33) has been identified as a specific ligand of
    ST2L The interaction of 1L33 with ST2L activates a cardioprotective response. The ST2s isoform
    You can also join ILJ3, but the ST2s1ILJ3 interaction causes inhibition of the interaction of
    ST2UILJ3 (Pascual-Figal DA et al., Clinical relevance of ST2s in cardiac diseases. Clin Chem Lab
    10 Med CCLM FESCC 2015; Kakkar R, Lee RT, The IL-33 / ST2 pathway: therapeutic target and novel
    biomarker Nat Re 'Drllg Disco !. 2008). In vitro studies have shown that ST2s expression
    It is related to cardiac hypertrophy. fibrosis and ventricular dysfunction (Sanada S el al., IL ·)) and
    ST2 comprise a critical biomechanically induced and cardioprotective signaling system. .J eli "Im'esl.
    2007). It has also been proposed that both IL3) and ST2s may be possible targets in
    lS Adverse remodeling treatments You will be 1M (Sanchez More. J. et al., Modulation of IL-33 / ST2
    system in postinfarction heart fai lure: correlation with cardiac remodeling markers. European
    JOl / mal ofClinicallnwstigalio ", 2014).
    The mechanisms for regulating the expression of the two ST2 isofermas are unknown. For now
    it seems that they have only been shown to be regulated by two different promoter regions that
    twenty They contain consensus zones for various transcription factors.
    The transcription factor Vin Vang 1 (Yyl) is a very multifunctional "zinc finger" protein that
    It belongs to the GLI-Kruppel family of nuclear proteins. Yyl can start. activate or repress the
    gene transcription depending on its interaction with other factors, it has been observed that it is
    over-expressed in patients with cardiac hypertrophy and heart failure (Sucharov c.e. et al.,
    25 Yin Vang I is increased in human heart failure and represses the activity of the human a-myosin
    heavy chain promoter, Thejollmal ofhiological cJ¡emislry, 2003; Mariner P.D .. Vin yang 1 represses
    a-myosin hea ')' chain gene expression in pathologic cardiac hypertrophy, Biochemical and
    Biophysical Research Cmmmmications, 2004; Glenn OJ. el al., Endothelin-stimulated human b-type
    natriuretic peptide gene expression is mediated by Yin yang I in association with histone deacetylase
    30 2, Hyperfem¡; On, 2009). However, we have not found papers that indicate a relationship between
    Yyl with myocardial infarction or with the regulation of ST2s.
    In the present invention, the inventors show how Vyl regulates the transcription of ST2s and uses
    this discovery to deal with the ca-diacos problems that arise after a 1M. They propose utili7..ar
    compounds that reduce the amount of Vyl protein in cells to reduce the expression of ST2s
    35 and thus improve the recovery of patients who have suffered 1M. avoiding progressive deterioration
    - ~~ -.... t.lectiyll.- ~~ __... (, Jbt'M
     06/0212017 27/0212017
    of the cardiac muscle. This strategy allows treating, and identifying compounds capable of treating, the
    Heart deterioration You will be 1M.
    Bren de! Iicripción de la nnnción
    In the present invention. the inventors show how Yyl inhibition results in an inhibition
    S of the expression of ST25. Since ST25 competes with ST2L to interact with the IU3, the
    ST25 inhibition is associated with an activation of the ST2L11L33 cardioprotective cascade.
    Indeed, after 1M, an increase in Yyl and ST25 expression is observed. But inhibition
    of Yyl by siRNAs in H9C2 cells exposed to biomechanical stress. results in a reduction
    of the levels of the mRNA encoding ST2s and the protein ST2s.
    10 The inventors also show how metformin. known for its therapeutic ability after a
    1M (Yin M et al. Metfonnin improves cardiae function in a nondiabetie rat model of post-M I heart
    failure. Am J Phy. ~ Iol Hear / Cire PhysioJ. 2011; Bhamra GS el a l., Melfonnin prolecls the ischemic
    heart by the Akt-mediated inhibition of mitochondrial penneability transition pare opening. Bosic Res
    Cordiol 2008: Calvert JW el al., Acute metfonnin therapy confers cardioprotection against
    fifteen myocardial infarelion via AMPK-eNOS-mediated signaling. Diaheles 2008.Paiva M et al ..
    Metformin prevents myocardial reperfusion injury by aetivating the adenosine receptor. J Cardiorasc
    Pharmacul 2009; Gundewar S. et al .. Activation of AMP-aclivated protein kinase by metfonnin
    improves left ventricular fun ction and survival in heart failure. Cire Res. 2009) has an effect
    similar to Yyl inhibition by siRNAs in the IL33 / ST2L cardioprotective cascade. In
    twenty effect. Metfonnine reduces Yyl and ST2s expression levels. But nevertheless. in this case the
    Effect seems to be mediated. at least in part, by an increase in the levels of expression of
    HDAC4 and by a decrease in phosphorylation levels of HDAC4, resulting in a
    increased HDAC4 in the heart cell nucleus. An increase in HDAC4 levels in the
    nucleus are associated with an inhibition of Yyl regulated gene expression (Han S el al .;
    25 Rccruitment of histone deacetylase 4 by transcription factors represses inlerleukin-5 Iranscription.
    Biochem J. 2006; Mathiyalagan P. el al .. Chromatin modifications remodel eardíae gene expression.
    Cardio 'Ose Res. 2014).
    In summary. the inventors have developed a new method to treat patients who have suffered
    1M based on the reduction of Yyl's expression. which results in a reduction of the
    30 ST2s expression after 1M. This invention also allows to detect compounds with properties
    cardioprotective Indeed. compounds with effects similar to those described for metformin in
    the waterfall 1L33 / ST2L. preferably that result in the inhibition of Yyl expression, they are
    potentially interesting for the treatment of patients who have suffered 1M.
    P2017001 54 06/021201702/27/20 17
    8reH description of the figures
    S To complement the description that is being made and in order to help a better understanding of the characteristics of the invention, according to preferred examples of practical embodiments thereof, it is accompanied as an integral part of said description. . a set of figures where, for illustrative and non-limiting purposes, the following has been represented:
    10 Figure 1: Figure 1 shows how metform ina modulates the IL · .JJ / ST2 system in myocardial infarction. (A-C), expression levels of mRNA molecules involved in IL · 33 / ST2 signaling: 'L-33, ST2L and ST2s. Westem-blot representative of IL-33 (O) and protein levels for ST2s (E) in rat-fed tissue and give them from densitometric analysis. The dalas were expressed as control times. ··· p <O.OOI, ·· p <O.OI compared to the control group. ## p <O, OI comparing with the 1M group. MET = metfonnine; IM = myocardial infarction; IL "" Interleukin; ST2L = ST2 linked; ST2s = ST2 solbleble; GA POH = glyceraldehyde 3-phosphate dehydrogenase.
    15 20 Figure 2: Figure 2 shows how metformin regulates ST2s nhleles through the modulation of transcription factor Yy1. (A) Analysis of Yyl mRNA expression by RT-PCR. (8) Representative Westem-blot of the Yyl protein in nucleus and cytoplasm of infarcted tissue. and densitométnco analysis calculated as Yyl core / eitosol ratio. ··· p <O, OOI comparing with the control group. ### p <O, OO I comparing with 1M. Yyl = transcription factor Yin-yang 1; Cyl-cytosol; other abbreviations as in figure 1.
    25 30 35 Figure 3: Figure 3 shows how rnetformin regulates the activity of the Vyl transcription faclOr through modulation of the HDAC4 factor. (A) mRNA levels for HOAC4. (B) Weslern-blol representative of protein levels for HDAC4 in rat infarcted tissue and itometric ns analysis. (e) Representative Westem-blot of the phosphorylated protein Scr-632-HDAC4 in nuclear and cytosalic fractions in infarcted tissue and densitometry analysis calculated as Ser-632-J-IDAC4 nucleus / cytosus ratio !. ··· p <O, OOI comparing with the control group. ## p <O, O 1; ### p <O, OO l comparing with 1M. HOAC4 = histone deacetylase 4; Hi st H3 = histone 3 protein; ot abbreviations as in previous figures. Figure 4: Figure 4 shows that, under biomechanical stress, the preformed metformin of the ST2s increase induced by Y and t. (A-O). RT · PCR analysis of IL-33, ST2s, Y and l and HOAC4 using a biomechanical stress model. (E) Westem-blot representative of ST2s levels in infarcted tissue and normal densitropic analysis raised to levels of GAPD H. (F) Western-blot representative of phosphoryl protein ada Ser-632-HDAC4 cytosoliea in Infarcted tissue and analysis densitometric calculated as
    P201 700154 02/02/201 7 27/021201 7
    Ser-632-HDAC4 / GAPDH ratio. "· P <O, OOl comparing with the control group. ## p <O, OI, ### p <O.OOI comparing with 1M. PMA = forOO acetate! Myristate; A = anophora A23187; other abbreviations as in previous figures.
    5 10 Figure 5: Figure 5 indicates the inhibitory efficacy of siYy l and its effect on the expression of ST2s. (A. B AND O) Quantitative RT-PCR analysis of Yyl mRNA expression levels, ST2s and ST2L using a cellular model of biomechanical stress. (C) Westem-blot representative of protein levels of ST2s and densitometric analysis. (E) Confocal microscopy images to characterize the expression of Yy I (red) and y-tubulin (green) by focal microscopy in H9c2 cells. The cores in blue (DAPI). Bar scale: 50 ~ m. "· P <O, OOt comparing with sham. ### p <O, OOI comparing with [M. siYyl = small interfering RNA; src = scramble as a negative control of whether or not lecture using siRNA: other abbreviations as in previous figures .
    lS 20 Figure 6: Figure 6 shows how the effect of each Y and l siRNA separately has the same erythro as the 4 siRNAs together. H9c2 cells were preincubated.r; with scrambJe siRNA (ser), a combination of four if RNAs for Yy [(Yyl siRNA a + b + c + d) or with a single siRNA before the induction of biomechanical stress, lasts 6 hours. Yyl (A) and sST2 (B) mRNA levels were analyzed by RT-PCR. .. · p <O.OO I vs ser; ### p <O, OO I vs biomechanical stress. Detailed description of the in <en tion
    Definitions
    2S -In the context of the present invention, the term "gene expression" refers to the process by which the nucleic acids of a gene are used to direct transcription resulting in [a protein synthesis. The gene expression process comprises 2 main steps:
    1) Transcription: the production of messenger RNA (mRNA) by an RNA polymerase using the AON sequence as a template. This stage continues with the processing or maturation of the resulting mRNA molecule.
    30 2) Translation: the use of mRNA to direct protein synthesis. After this second stage, post-translational modifications of the protein can occur.
    In addition, in some cases, the expression génü; a entails the production of other forms of RNA that are not translated into proteins. as transfer RNA (tRNA). Ribosomal RNAs (rRNAs), long non-coding RNAs (RNA cnc), or interference RNAs (RNAi).
    ¡¡
     02/06/2017 02/27/2017
    s
    lS
    2S
    . In the context of the present invention, "regulatory sequence" refers to a segment of a
    nucleic acid molecule that is capable of increasing or reducing the expression of specific genes in
    an organism Some examples of regulatory sequences would be promoters, enhancers, signals
    polyadenylation, splicing sites etc.
    - In the context of the present invention. "RNA interference (RNAi)" is understood as an RNA molecule that suppresses the expression of specific genes by mechanisms known globally as ribointerference or RNA inference. The interfering RNAs are small molecules (from 20 to 25 nucleotides) that are generated by fragmentation of longer precursors. They can be classified into three large groups of molecules:
    - Small interfering RNA (or small interfering RNA siRNA). They are perfectly complementary double-stranded RNA molecules of approximately 20 or 21 nucleotides (ni) with 2 nucleotides unpaired at each end) '. Each strand of RNA has a 5 'phosphate group and a hydroxyl (-OH))' group. This structure comes from the processing carried out by Dicer. an enzyme that cuts long molecules of double stranded RNA (dsRNA. double stranded RNA) into several siRNAs. One of the strands of siRNA (the 'antisense' strand) is assembled in a protein complex called RISC (RNA-induced silencing complex). which uses the siRNA strand as a guide to identify the complementary messenger RNA. The RISC complex catalyzes the cutting of the complementary mRNA into two halves. which are degraded by cellular machinery, thereby blocking gene expression. The siRNAs can also be introduced from exogenous fonna into cells using transfection methods based on the complementary sequence of a particular gene, in order to significantly reduce their expression.
    - miRNA (or miRNA of the English micro-RNA or miRNA) are small interfering RNAs that are generated from specific precursors encoded in the genome. which, when transcribed, fold into inlramolecular hairpins containing segments of imperfect complementarity. Precursor processing generally occurs in two stages. catalyzed by two enzymes. Drosha in the nucleus and Dicer in the cytoplasm. One of the strands of miRNA (the 'antisenlido' strand). As OCUITe with 105 siRNA, it is incorporated into a complex similar to the RISC (RNA-induced silencing complex). Depending on the degree of complementarity of the miRNA with the mRNA, the miRNAs can either inhibit the translation of the mRNA or induce its degradation. However, unlike with the siRNA pathway. miRNA-mediated mRNA degradation begins with the enzymatic removal of the poly (A) tail of the mRNA.
    P201 700154 02/06/201702/27/201 7
    -RNAs associated with Piwi (piRNAs): they are generated from long single-chain precursors, in a process that is independent of Orosha and Dicer. These small RNAs are associated with a subfamily of the prolines 'Argonauta' called Piwi proteins.
    5 10 -In the context of the present invention, the term "RNA antisense oligonucleotide" refers to a single strand of RNA that is complementary to a specific sequence. This sequence of hybrid antisense RNA with a specific mRNA thus inhibiting its translation. since mRNA translation requires a simple strand of RNA. Thus, the antisense RNA oligonucleotide in hibe the synthesis of a particular protein. On the other hand, RNase H degrades RNA-DNA complexes, so qUf: hybridization of the antisense RNA oligonuc1eotide to a specific DNA sequence will also cause degradation of said DNA sequence.
    lS -In the context of the present invention, the term "plasmid" is understood as the circular extrachromosomal DNA molecule that is replicated and transmitted independently of chromosomal DNA. Plasmids are normally present in bacteria. and sometimes in eukaryotic organisms such as yeasts. The pBasmids are used as cloning veclores in genetic engineering because of their ability to reproduce independently of chromosomal DNA as well as because it is relatively easy to manipulate and insert new genetic sequences into them. They usually include regulatory sequences. as promoters that are used to synthesize the RNA of interest, which in most cases ends up being translated into a arrested protein.
    20 25 -In the context of the present invention, the term "vector" refers to a small DNA fragment, obtained from a virus, from a plasmid. or from! a cell of a higher organism into which heterologous genetic fragments can be inserted and which are usually used to artificially transport this heterologous genetic material to another cell. where it can be replicated, expressed and / or inserted into the genome of the host cell. In general, the vector itself is a DNA sequence that consists of a transgene and a longer sequence that serves as a "skeleton" of the vector. It can be single or double chain. Option. it may be formed, or comprise, modified nucleic acids, such as, for example, methyl phosphate or phosphorothioate main chains.
    30 -In the context of the present invention, the term "cardiac cell" refers to a cell that forms part of the heart. Among the different cardiac cells are mainly cardiomyocytes, but also others such as fibroblasts or endothelial cells of the heart. In the context of the present invention, the term "cardiomyocyte" refers to a myocardial cell or cardiac muscle capable of contracting spontaneously and individually.
    -In the context of the present invention, the term "increase" or "increase" refers to increases above a reference or control level. For example, the reference or control levels are the
    8  02/02/2017 02/27/201 7
    ,,
    that are observed before, or in the absence of. the addition of an activating compound or the activating action. The increase may be assessed by a person skilled in the specific technique. but in the aspects and embodiments described in this invention. refers to increases of at least 8. 8. 10,
    11, 12.13,14, 15, 16.1 7, 18,19,20,21,22,23,24,25,26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36 , 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61 , 62, 63, 64, 65.66.67.68.69,70,71,72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82. 83, 84, 85, 86. 87, 88 , 89.90.91, 92.93.94.95.96.97.98.99, or 100%, or del.5, 2.3.4, S, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20,25, 30,
    35,40,45,50, or more times with respect to the control levels.
    . In the context of the present invention, the term "reduction", "reduce", "inhibit" or "inhibition" refers to reductions below a reference or control level. For example. the reference or control levels are those observed annually. or in the absence of. the addition of an inhibitor compound. The decrease can be evaluated by a person skilled in the art. but in the aspects and embodiments described in this invention, it refers to decreases of at least 8. 9, 10, 11, 12,
    13, 14, 15, 16, 17, 18. 19,20,21, 22,23,24,25,26.27,28,29,30.3 1,32,33,34, 35, 36. 37, 38. 39 , 40.41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56. 57, 58. 59, 60. 61, 62, 63, 64 , 65, 66. 67.68.69.70.71.72.73.74.75.76.77.78.79.80.81, 82.83. 84, 85, 86. 87, 88. 89. 90, 91, 92, 93, 94.95.96.97.98.99, or 100%, or 1.5,2,3,4, S, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,20,25,30,35,40,
    45, 50, or more times with respect to control levels.
    - In the context of the present invention, the term "polynucleotide" refers to a sequence of several nucleotides. formed by at least 20 nucleotides. It can be formed by deoxyribonucleotides or by ribonucleotides and therefore constitute DNA or RNA molecules. In the present invention they can also be formed by synthetic or modified nucleotides. They can include, for example, phosphorothioate main chains, or the addition of acridine. For the purposes of the present invention, it should be understood that the polynucleotides described herein can be modified by any method available in the art.
    - In the context of the present invention, the term "screening method" is understood as a method that allows the reading of compounds capable of promoting a certain change measurable by in vi / ro methods known in the art. in a biological sample or in a non-human model animal to which said compound has been administered. compared to a similar and comparable non-human biological or animal sample, to which the compound has not been administered. Said screening method may also include an additional phase of isolation of the selected compound or of the selected compounds.
    3S -In the context of the present invention, the term "pharmaceutically acceptable carrier" refers to the compound that is mixed in some way with active ingredients to stabilize them and
    P201 700154 02/02/201 7 02/27/201 7
    5 solubilize and administer them to live animals, including humans. This includes any and all solvents, means of di: .persion. coatings, antibacterial and antifungal agendas, isotonic and absorption retarding agendas. and similar. compatible with the pharmaceutical administration. Except to the extent that any conventional media or agent is incompatible with the active compound. The use in the compositions is contemplated.
    10 15 20 The inventors identify a new strategy to promote recovery after 1M. This strategy is based on reducing the expression levels of the Yyl transcription factor. which will result in a reduction in the expression of the ST2s protein. The interaction between the isofonna ST2L and the ILJ3 activates a cascade of reactions that promotes. cardiac recovery after 1M (Sanchez More. J. et al., Modulation of IL-33 / ST2 system in postinfarction heart failure: correlation with cardiac remodeling markers. European JUllrnal ojClinicallnvesligation. 20 I 4). Since ST2s competes with ST2L to interact with 1L33, inhibition of ST2s is associated with a positive effect on cascade activation mediated by the ILJJ / ST2L interaction. A first aspect of the present invention. it refers to the use of a composition comprising a compound capable of reducing the expression of the Yin yang I gene (Yy 1). in cardiac cells, preferably in cardiac cells that have suffered identical or similar biomechanical stress as a myocardial infarction (1M). of a human or animal subject with respect to the expression observed in the absence of the compound in said cells. selected from the list consisting of:
    -RNA interference (RNAi) of the Yyl gene,
    - an antisense RNA oligonucleotide corresponding to the Yyl gene.
    -a nucleic acid sequence that is introduced into the gene or regulatory sequences of the Yyl gene inhibiting its expression;
    2S To develop a drug that inhibits the e "pressure of the gene that encodes the soluble protein suppression of tumorigenicity-2 (ST2s) to promote cardiac recovery, you would have a heart attack (1M).
    3D As just indicated, inhibition of Yyl gene expression can be achieved using RNA interference (RNAi). This mRNA comprises a unique sequence and complementary to the messenger RNA (mRNA) of the Yyl gene to inhibit the translation of said mRNA. Alternatively, an antisense RNA oligonucleotide encoded by a unique sequence of the Yyl gene complementary to the Yyl mRNA that can bind said mRNA and also inhibit its translation can be used. This methodology is known in the art. These RNA constructs must be able to hybridize with a Yyl target mRNA sequence selectively and specifically, so that they are identical
    P201 7001 54 06/02/201 7 02/27/201 7
    at a complementary sequence of mRNA Y and 1 I ~ n at least 90% of its nucleotides. 92%, 94%. 96%, 98% .99% or 99.5%. The Yyl mRNA sequence is identical to the SEQlde Yyl sequence of the human genome (or to the homologous sequence encoding a protein with homologous functions in other animal genomes) in at least 90% of its nucleotides. 92%, 94%. 96%, 98%, 99% or a
    99.5% Alternatively. nucleotide sequences can be introduced into the genome of the cell of interest in the gene or regulatory sequences of the genYyl to inhibit its expression. The methods for introducing these sequences into the genome of the cell are known in the art. For example. You can use the CRI $ PR-Cas technique.
    To introduce RNA molecules described just above into the target cells. Different strategies can be used. For example. one can simply inject the siRNAs or antisense oligonucleotides into the environment of the cells so that they come into contact with the cells. The medium in which these RNAs are found can be any pharmaceutical vehicle compatible with the stability of the RNAs and compatible with the cells and, in case of administration to a subject, with the physical health of the subject. This vehicle is known by the technique. such as a saline solution. Biodegradable nanoparticles can also be used. as liposomes Jiposomes are vesicles formed by lipids that contain molecules of interest inside them (in this case [os siRNAs or antisense oligonucleotides) and that can fuse with a cell membrane and release their content in that cell. To increase the. specificity of the cells in which the liposome content is released, proteins that improve cell direction can be added. as specific markers of the target cells. The cells can also be electroporated.
    Another option is to introduce the AON sequence corresponding to the RNA molecule into the cells "as described above. This DNA sequence can be transcribed into the target cells. To do this, vectors or plasmids capable of being introduced into the cells can be used. A number of strategies can be used to enter these cells, which are known in the art.As well as to introduce RNA, liposomes can be used, methods based on electroporation or biobalysis can also be used. viral vectors, such as adenovirus, adenoassociative virus, two, herpes viruses, vaccination virus, polio virus, AIDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including among them, viruses with the structure of HIV. Viral families that share the properties of these viruses that make them suitable for use as vectors are also preferred. they are Maloney's murine leukemia virus, MML V, and retroviru :) that express the desirable properties of MMLV as a vector. Retroviral vectors are capable of carrying a larger genetic payload than other viral vectors, that is. a gene and / or transgene marker, and for this reason they are a commonly used vector. But nevertheless. They are not as useful in nonproliferative cells. Adellovirus vectors are relatively stable and easy to use (and can be transfected into non-dividing cells. Smallpox viral vectors are large and have several sites for the insertion of
     06/0212017 02/27/2017
    5 genes are lennoslables and can be stored at room temperature. Lentiviruses are capable of infecting all types of cells, both quiescent and dividing, penniten medium sized inserts and integrate into the genome of the host cell. leading to a long term expression. A preferred embodiment is a viral vector that has been designed to suppress the host organism's immune response, caused by viral antigens.
    10 Therefore, another preferred embodiment of the first aspect of the invention relates to the use of the first aspect of the invention, wherein said compound is comprised in a vector or plasmid capable of transporting or delivering said compound to cardiac cells. Preferably. said vector is a viral vector. More preferably. said viral vector is selected from the list consisting of adenoviral, lentiviral, retroviral and associated vectors.
    lS Once inside the cells, transcription of the nucleic acid sequences encoding the RNAs of interest capable of silencing the expression of the Yyl gene will occur within the target cells. under the control of prc-dctcnninated promoters and present in the vectors or plasmids used. In another preferred embodiment of the invention, the sequence of nude acids encoding the RNAs of interest capable of silencing the expression of the Yyl gene is inserted into the genome of the cardiac cell into which it was introduced.
    twenty For any of the preferred embodiments described so far. Modified synthetic nucleotides of the invention can be included in the polynucleotides of the invention. A number of different types of polynucJeotide modification are known (: n the art. These include, for example, phosphorotium ioate main chains, or the addition of acridine.
    The use of the composition according to any of the preferred embodiments described so far is directed to reduce the size of myocardial infarction and / or prevent adverse cardiac remodeling after 1M.
    2S 30 The compounds can be administered locally: in the inert cells. cardiomyocytes, or parenteral fonnar. intravenous or intramuscular. It can also be administered orally, provided that any of these pennite administration sources transport and deliver the compound described in any of the above embodiments to the cardiac cells of interest. Therefore, in a particular embodiment of the first aspect of the invention. The composition is administered locally in the heart cells. In another embodiment of the invention, the composition is administered intravenously.
    The composition used in any of the above embodiments may further comprise an acceptable pharmaceutical carrier and optionally an additional active ingredient. In this sense. The compound can be administered in vivo together with an acceptable pharmaceutical carrier. The vehicle,
     02/06/2017 02/27/2017
    naturally, it is selected to minimize any degradation of the active ingredient (or active ingredients. if it contains an additional active ingredient) and to minimize any adverse side effects on the subject, as is well known to one skilled in the art. and as indicated in the definitions. Sorry. In one embodiment of the invention it refers to the use according to any of the previous embodiments wherein said composition further comprises an acceptable pharmaceutical vehicle and optionally an additional active ingredient. A second aspect of the invention relates to a screening method to identify compounds capable of reducing the expression of the gene encoding the ST2s protein comprising:
    a) Obtain a library of compounds.
    b) Determine whether an administration to cardiac cells. preferably subjected to similar stress
    or identical to that produced by 1M. of one or more of the compounds of step a) results in a reduction of the intracellular levels of the transcription factor Yyl and / or the expression of the gene encoding the transcription factor Yy 1 with respect to the cardiac cells, preferably subjected to a Biomechanical stress similar or identical to that produced by 1M, which is not given said compound (s).
    c) Select those compounds that promote the reduction described in step b).
    Said screening method can be performed with in vitro biological samples. or with nonhuman model animals.
    In example 3, it is observed how an increase in the expression of the transcription factor Yyl is related to an increase in the expression of ST2s and how the silencing of Yyl with small interference RNAs (siRNAs) translates into a reduction of the expression of ST2s. So. it is expected that compounds capable of inhibiting Yyl cxpression, similar to siRNAs. they also end up inhibiting the expression of ST2s. As commented above. the expression of ST2s translates into an inhibition of the interaction between lL33 and ST2L, thus inhibiting the cardio-protective cascade IL33 / ST2L and its beneficial effects on recovery after a myocardial function. Therefore, compounds capable of reducing Yyl expression, and consequently of ST2s, seem to trigger molecular mechanisms capable of promoting cardiac recovery. Therefore, these compounds are selected in step e) of the screening method described above. To measure the effect that a compound has on the expression of Yy 1, cells expressing the transcription factor Yyl are grown, preferably cardiac cells comprising cardiomyocytes. that even more preferably they have suffered a biomechanical stress identical or similar to that of a myocardial infarction (M I) and they are administered the compound. Biomechanical stress is known in the art and preferably consists of that described in Neshali Z al. (Neshati Z the aL,
    yt <.. l
    P201 700154 02/02/201 7 02/27/201 7
    Development of a new madel of cardiac hypertroph) 'wilh pro-arrh) 1hmic features lo study the role of
    hypcrtrophy in arrhythmogenesis. ElIr Hearl J. 20 t3).
    Alternatively, to measure the effect that a compound has on Yyl expression, it is administered
    the compound fI nonhuman model animals. preferably non-human mammals. and more
    5 preferably. whose cardiac cells have undergone stress whether milled or identical to that of a 1M. and
    It measures the level of Yyl expression in the cardiac cells of interest. preferably in
    cardiomyocytes The route of administration in both cultured cells and model animals,
    It will depend on the type of compound. In case of interference RNAs or oligonucleotides
    antisense they can be found in the same fonts that have been described in the first aspect of the
    10 invention. and introduce them into the cardiac cells as described in the first aspect of the
    invention. If it is another type of molecule (such as metformin). the
    method of administration to the decite in the art. To check if the compound promotes a
    decreased levels of Yy l protein, in cultured cells or in cardiac cells of animals
    model described above, these levels will be measured in the cardiac cells that have been
    lS administered the compound (or of model animals to which the compound has been administered) and in
    heart cells that have not been given the compound (or animals that have not been given
    has administered the compound). In case there is a lower level of Yyl in the cells
    cardiac that if they have received the compound (or d.e animals to which if the
    compound). it will be concluded that the compound promotes a reduction in intracellular levels of
    twenty Yyl transcription factor. This reduction will be evaluated by a person skilled in the art, and
    understands that to be considered a decrease must be as described in the definitions more s
    alTiba. To measure Yy l intracellular protein levels. different methods may be used
    known in the art. comparing a control sample (heart cells that have not been
    administered the compound or of animals to which the compound has not been administered) with the
    2S cell sample to which the compound (or of animals that have been given
    administered the compound). As an example you can use the westem blot technique,
    immunohistochemistry or the "f1uorescence-activated ceH sorting" (FACS) technique. Other indicative. or
    confirmation. that there is a reduction in intracellular Yyl protein levels is that it occurs
    a reduction in the levels of gene expression. This reduction will be evaluated by an expert
    30 in the art, reduction is understood to be the same as indicated for the case in which the
    protein levels and as indicated in the definitions. To measure genie expression. be
    they may use any of the methodologies known in the art. such as the "Real
    Time-Polymerase Chain reaction (RT-PCR), the "Real Time-Quantitative Polymerase Chain"
    reaetion "(RT-QPC R) or · 'micro-alTays".
    3S In examples 2, 3 and 4. a collation between HDAC4 expression, phosphorylation, Yyl expression and ST2s expression is observed. More specifically. it is observed how after inducing a 1M or 14
    ¡¡
    P201 700154 02/02/201 7 02/27/201 7
    a biomechanical stress to HQC2 heart cells. phosphorylation of HDAC4 increases. When administering Melfonnine. there is an increase in the expression of HDAC4. and a decrease in its phosphorylation level (fig 3,4). Figures 2 and 4 show how metformin reduces levels of Yyl and ST2s that had increased due to 1M or biomechanical stress in H9C2 cells. 5 Given that Yyl activates the transcription of ST25 You will be 1M or biomechanical stress, the results suggest that HOAC4, which is capable of interacting with Yyl (Han S el al .. recruitment of histone deacetylase 4 by transcription factors reprcsses interleukin-5 tran! '; cription. Biochem 1. 200615; Malhiyalagan P. el aL, Chromatin modifications remodel cardiac gene expression. Cardiovasc Res. 2014). prevents Yyl from activating the transcription of ST2s. Phosphorylation of HDAC4 10 prevents its importation into the cell nucleus (Backs J el., CaM kinase 11 selectively signals lO histone deacetylase 4 during cardiomyocyte hypertrophy. J CJin JI / ves /. 2006: Walkinshaw DR el al., The tumor suppressor kinase LKBI activators Ihe downstream kinases SIK2 and SIK3 to stimulate nuclear export of class lIa histone deacetylases. J ~ iol Chem. 2013; Wang X. el al., YYI reslrained cell senescence Ihrough repressing the transcription to 'p16. Biochim Biophys Acta BBA. Mol Cell Res. 15 2008). Therefore, its dephosphorylation favors its importation into the nucleus, where it can interact with Yy I, resulting in repression of the transcription of ST2s. In summary, a compound that promotes an increase in HOAC4 expression, and / or reduces its phosphorylation, also results in a reduction in the expression of ST2s by the inhibition of Yyl activity. Therefore, in a preferred embodiment of the invention, in the screening method described in the second aspect of the
    The invention also includes, in step b) to determine whether the administration of said compound (s) compared to cardiac cells, preferably subject to biomechanical stress similar or identical to that produced by 1M, to which no they are administered / n results in:
    - an increase in histone protein levels. deacetylase 4 (HDAC4) or [a expression of the gene encoding the HDAC4 protein and / or
    25 -a decrease in phosphorylation levels of the HDAC4 protein and / or
    - an increase in the amount of Yy 1 protein that is bound to HDAC4 and / or
    - an increase in [the expression of Interleukin 33 (lL33) or the expression of the gene encoding the IL33 protein, and / or
    - a decrease in ST2s or e levels) (pressure of the gene encoding the ST2s protein, and
    C) select those compounds that produce one or more of the effects described in b).
    Methods to determine the increase or decrease of protein levels, or expression
    gene of the different proteins mentioned in this preferred embodiment of the invention,
    They will perform similarly to the one described above in the second aspect of the invention. In
    P201 700154 02/02/2017 02/27/201 7
    As for the measurement of phosphorylation levels of HDAC4, the methods known in the art will again be used. such as the wcstcm blot. immunohistochemistry or the use of FACS. To compare the amount of Yyl protein bound to HDAC4 in the presence and absence of the compound, methodologies known in the art may be used, such as Yyl Y immunoprecipitation followed by a westem blo! to delegate YAC-bound HDAC4 protein.
    To evaluate or confirm the therapeutic effect of the compounds that produce the changes described in the preceding claims, the compound is administered to myocardial infarction model animals, and the size of the infarction is evaluated and as observed for example in Figure 6b of work of Yin M el al .. Metfonnin improves cardiac funclion in a nondiabetic rat model of post-M I heart fa ilure. Am J Physiol Hearl Ore Phy. ~; Ol. 2011. The evaluation of the size of the infarction can be done using the methodology known in the art, such as magnetic resonance imaging or especially when using model animals by surgery. The model animals may be laboratory model animals, preferably rats, mice or pigs, to which a descending coronary artery ligation is performed. Therefore, a third aspect of the invention consists of a method where in addition to the steps described in any of the embodiments of the second aspect of the invention, a step d) is included in which it is evaluated in non-myocardial infarction model animals. humans. preferably in non-human mammals, if the administration of V of the compound / s evaluated in steps a) and b). or selected / s in step c), reduce the size of myocardial infarction.
    The screening method can also be performed to determine the most appropriate ranges of administration to achieve the highest efficacy of the therapeutic compound. Therefore, in another embodiment of the invention. a range of the amount of V of the administered compounds / s which causes the effects described in any of the claims of aspects 2 and 3, preferably the effect described in the second aspect of the invention that relates to the levels of the invention, is determined more intensely Yyl transcription factor, in biological samples in vi / ro, or in nonhuman model animals.
    In any case, the dosage ranges for administration of the compositions are those large enough to produce the desired cardiac recovery effect after 1M. The dosage should not be large enough to cause adverse side effects, such as undesired screening reactions. anaphylactic reactions, or the like. Generally, the dosage will vary with age, 1M aggressiveness, route of administration, or if other additional drugs are being administered. The dose may vary. and it can be administered in one or more administrations, they can be daily doses. for one or several days. Guidance can be found in the literature for appropriate dosages for the given classes of products.
    Fannaceuticals
    SOIlCIlUg. (!
    P201 7001S4 06/02/201 7 02/27/2017 06/0212017 02/27/2017 06/0212017 02/27/2017
    The composition comprising the compound of any of the previous embodiments of the
    second and third aspect of the invention may be marked by the compound being tested
    in the screening method. and an acceptable pharmaceutical vehicle. In this sense, the composition
    The resulting fan can be administered in vivo to a non-human model animal. The vehicle,
    S naturally. is selected to minimize any degradation of the active ingredient and to
    minimize any adverse side effects on the subject, as is well known to an expert in
    the technique and as indicated in the definitions .. The proportion of compound and vehicle
    Suitable pharmacist may be arrested using the same screening method described
    previously. So. In a particular embodiment of the invention, a range of
    10 amount of compound tested by the screening method and vehicle that causes most
    intensity of any of the effects of aspects 2 and 3 of the invention, preferably the effect
    described in the second aspect of the invention which refers to the levels of the transcription factor
    Yy1. In another preferred embodiment of the invention, the mode of administration of the
    compound that causes with greater intensity any of the effects of aspects 2 and 3 of the
    fifteen invention in nonhuman model animals, preferably the effect described in the second aspect
    of the invention that relates to the levels of the transcription factor Yyl. In one embodiment
    preferred. in the method of any of the embodiments of the second and third aspects of the
    invention. The compound library consists of biguanides. Since [a metfonnine is a
    compound well known for its ability to treat recovery after 1M (Yin M el al ..
    twenty Metfonnin improves cardiac function in a nondiabetic rat model of post-MI hean failure. Am J
    Physiol Hearl Cire Physiol. 201 1; Bhamra GS et al., Metfonnin prolecls the ischemic heart by the
    Akt-mediated inhibition of mitochondrial penneability transition pore opening. Basie Res Cordial.
    2008; Calvert JW el al., Acute metformin therapy confers cardioprotection against myocardial
    infarction via AMPK-eNOS-mediated signaling. Diahele. ~. 2008.Paiva M et al .. Metformin prevents
    2S myocardial repe: rfusion injury by activating Ihe adenosine receptor. J Cardü) 'afC Pharmaco /. 2009;
    Gundewar S. el al., Activation of AMP-activated protein kinase by metformin improves left
    ventricular function and survival in heart failure. Circ Res. 2009 »and as seen in examples 1
    to 4, reduces the expression and levels of protein Yy 1, increases those of HDAC4, reduces phosphorylation
    of HDAC4 and reduces the expression of 5T2s and finally. reduce the size of myocardial infano
    30 (Yin M et al., Metfonnin improves cardiac function in a nondiabetic rat model of post-MI heart
    failure. Am J Physiol Hearl Circ Physiol. 2011), this compound can be used as a compound of
    reference. or as a positive control, in the screening methods described in the second and third
    aspect of the invention. Pair so much. in another particular embodiment of the invention, in the method of
    Any of the embodiments of the second and third aspects of the invention, merfllnnine is used
    3S as a positive control
    A fourth aspect of the invention makes reference to a product that contains the information of the results obtained by the screening method described in any of the above embodiments.
    5 A fifth aspect of the invention refers to a method of obtaining one compound comprising the identification of the compound / s according to any embodiments of the second and third aspects of the invention and their isolation and / or purification.or more of the
    DI! Licripción of a main embodiment of the invention
    Materials and methods
    A. Animal model.
    Animals
    10 Male Wistar rats were used (with a pc ~ s between 220-250 g, and with 6-8 weeks of age). All protocols were approved by the University ethics committee (A13150105). Lus animals were kept in standard laboratory conditions, in pathogen-free facilities and under strict veterinary surveillance. Maintained in controlled rooms with 12h light-dark cycles, the animals received a commercial diet of yagua ad Iibitllnl rats.
    fifteen Induction of experimental 1M in r.lltas.
    Acute 1M was induced by pemlanent ligation of the left anterior descending coronary artery (LA O). To demonstrate the elevation of the ST and. therefore, to confine the success of the surgery, electrocardiography was used. The control group was subjected to the same procedure as the other groups except for LAD ligation.
    twenty Experimental groups and design.
    The animals were divided into three groups:
    2S Rats operated without LAD (sham group, n "" '10) and with placebo (0.9% NaCI in gelatin). Rats operated if n LAD (sham + MET group, n = IO) and treatment with metfonnine: 250 mglkg of daily oral dose, adm or strada in gelatin. Rats operated with LAD and placebo (group MI, n = 1 5). Rats operated with LAD and metfonnine treatment: 250 mglkg of daily oral dose, administered in gelatin (IM + MET group. N = 20).
    30 The administration of metfonnin began three days before the induction of 1M and was maintained until sacrifice, which was performed between 1 and 4 weeks after the induction of 1M. The dose of
    Melfonnine was adjusted weekly according to the body weight of each animal. Metfonnine used was purchased from Sigma-Aldrich Corp. (St. Louis. MO).
    On the day of sacrifice, the infarcted area of the left ventricle (VI) was carefully removed and processed for analysis. The variables of LV function were assessed using a transthoracic ultrasound3. In the region of sacrifice, the concentration of methotonin in plasma was quantified with HPLC (High-performance liquid chromatography).
    E: RNA traction and RT-PCR cuanlitalin.
    Fresh tissue from the VI was washed using cold PBS and crushed in an ice bath manually using a scalpel. RNA isolation and quantitative RT · PCR were carried out following the protocols previously used by the group (Lax A et al., Mineraloconicoid Receptor Antagonists Modulate Galectin · 3 and Interle ukin-33 / ST2 Signaling in Left Ventricular Systolic Dysfunction After Acute Myocardial Infarction. IACC Hean Fail. 2015), without any significant modification.
    Protein fraction and We. ,, 'ern blolling.
    Approximately 30 mg of myocardial infarction were homogenized by sonication and centrifuged at 20,000 xg for 20 min at 4 oC. The supernatant was aliquoted and frozen at · 80 "C. The cytosolic and nuclear fractions were separated using the method described by Dimaurus 1. et al. (Dimauro L. et al., A simple protocol for the subcellular fractionation of skeletal muscle cells and ti ss ue. BMe Res Notes. 20/2). Protein concentration was determined by the bicinconinic acid method (Smith PK, et al., Measurement of protein using bicinchoninic acid. Anal Biochem. 1985) .The Weslern blotting was performed with an electrophoresis system with SOS-PAGE.
    Immunoreactive bands were detected using ECL and by using the ChemiDocTM XR system, ') + with the BioRad LahoralOr imaging software; it is ne. (Berekley. CA). A quantitative analysis was performed with the Ge / -Pro Ana / yzer 3. J program by Sigma.
    B. Cellular model.
    Biomechanical stress model.
    H9c2 cells were maintained in Dulbecco-modified Eagle's medium (DMEM), supplemented with 2 mM glulamine and 10% fBS. For the performance of the assays, 3S cells were deprived of serum for 24 h. Stress. Biomechanical was carried out following the design
    } t.
     02/06/2017 02/27/2017
    Experimental by Neshati Z el al. (Neshati Z (~ t al .. Developmenl of a new medel of card iac hypertrophy with pro-arrhythmic features lo sludy the role of hypertrophy in arrhythmogenesis. ElIr HeartJ.20 13).
    ID munOnU Or esCl'ncia.
    S 10 Initially, the cells were fixed with 4% (v / v) formaldehyde, penneabilized with Trilon X · 100 and treated with the lmage-iPM FX kit signal enhancer following the experimental protocol described by Thermo Fisher Scientific (Molecula.r Probes, (36933) The cells were then incubated at 4 OC throughout the entire night with the anti-Yyl antibodies (dilution 1: 1000) and y-Tub ul ina (dilution l: 1000) and subsequently at 37 OC for 1 hour with anti-mouse IgG conjugated with Alexa Fluor® 488 (1: 200 dilution) or rabbit anti-IgG conjugated to Alexa Fl uor 593 (1: 200 dilution), the fluorescence of the cells was analyzed With a confocal team from Leica Mycrosystems, selecting the emission windows at 508-550 nm and 61 0--65 nm respectively, in addition, the fUt: rum nuclei stained using DAPI.
    15 20 Silendamit'nlo for! I "nwll in / t rfering RNA (siRNA) specific Yyl siRNA or control siRNA (Dhannacon, Lafayelte, COl) were used to transfect cells using Lipojectamine 2000 according to the protocol provided by the commercial house. To prevent off-target effects, a commercial mixture of four specific siRNAs (ON-TARGETplus ™ Yyl) was used, described previously ~ (liu Q. el al., Prostaglandin F2alpha suppresses ral steroidogenic acule regulatory protein expr (~ session via induction of Yin Yang I prolein and recruitment of histone deacetylasc 1 prolein. Endocrin% gy. 2007) .The gene studied was Yy , while GAPDH was used as an internal control gene. The target sequences were:
    ON-TARGET plus SMARTpool siRNA Yy l J-091624-05 J-091624-06 J-091624-07} -091624-08 IF! Target count (5 "-3") CAUCUUAACACACGCUAAA AGGCiAUAACUCUGCUAUGA GGGCGACACCCUCUACAUU GCGCCGACCCGGGUAAUAA
    t; (! C 06/02/2017
    H9c2 cells were incubated in six-well plates in complete medium. The next day. These cells were transfused using 25nM of each. one of the siRNA for eight hours. followed by a recovery period in serum medium. After 24h of transfection the cell culture was washed
    twice with PBS tempered at 37 oC. before starting the treatments.
    5 RNA extraction and RT · PC R quantitalha. the cells were sedimented by centrifugation at 480 xg. for 10 min at 4 oc. For the rest of the procedure see animal model.
    Protein fraction and Weslerll hlotting. 10
    See animal model.
    Statistic analysis
    15 Data are expressed as mean ± esl error: walking or standard deviation as indicated. Analyzes were performed for independent samples. and paired, or analysis of variance, as appropriate in each case. Data were analyzed using the statistical program SPSS statistics v. 19 (SPSS Inc., USA), and graphics were constructed using the SigmaPlot program
    v. 11 .0. A value of p <0.05 was considered statistically significant.
    20 H. Results
    1-Protective effects of metformiine therapy.
    In order to find new therapeutic strategies to treat patients with 1M, a regulatory mechanism of the IL-33 / ST2 signaling cascade was sought. For this purpose, a compound 25 capable of treating patients with 1M and acting on said signaling cascade was searched for use as a reference condition. Metformin is widely known for its therapeutic capacity after 1M (Vin M et al., Mctformin improves cardiac function in a nondiabetic rat model of post-MI heart failu re. Am J Physiol Hear! Cire Physiol. 20 11; Bhamra GS et al., Metformin protects the ischemic heart by the Akt-medialed inhibilion of mitochondrial penneahility transition pare opening. 30 Basie Res Cardiol. 2008; Calvert JW el al .. Acute metformin therapy confers cardioprotection against myocardial infarclion via AMPK-eNOS-mediated signaling. Diabetes 200R, Paiva M el al., Metformin prevenls myocardial reperfusion injury by activating Ihe adenosine receptor. J Cardiovasc PharmacoJ. 2009; Gundewar S. et aL, Activation of AMP-activated protein kinase by metformin improves len ventricular function and survival in hear1 failure, Circ Res. 2009). Indeed, Yin M. on 35 al. show how treatment with metfonnin reduces the size of 1M in rats treated with metformin (Figure 68 in Yin M et al., Metforrnin improves cardiac function in a nondiabetic ral
    02/27/201 7
    IY · SO "ISIC. ____ .. b l« n (II ._____
    .. jl..Ul!. ~ I 1
     02/06/2017 02/27/2017
    model of post-MI heart fai lure. Am J Physiol Hearl Cire Phys; ol. 2011). So. it was examined if theThe therapeutic effect of metformin after 1M is related to the IL-33 / ST2 system. For itwe analyze the spatio-temporal modulation dl ~ the signali7..3ci IL-33 / ST2 by RT-PCRquantitative and Westem Blot with (+) and (-) metfonnine and (+) and (-) 1M. Expression levels of IL5 33, ST2L and ST2s increased one week after causing 1M and remained elevated. 4weeks later compared to the sham group (p <O. OO 1) (Figure I A-C). Therapymetfonnine was associated with an increase in IL-J3 and a reduction in ST2L and ST2s at the level ofMRNA (Figure I A-C). Results were obtained; similar when evaluating levels of IL-33 protein andST2s by WB (Figure 1, panels O and E). In the absence of 1M. metformin therapy did not change the
    10 levels of mRNA expression or protein levels. Therefore, it seems that after a 1M. Metfonnin increases IL33 levels and reduces ST2L and sST2.
    2-Response of the factors that act in trans Yyl and HDAC4 at 1M and to the therapy ofmetformin
    Yyl transcription factor expression levels showed a considerable increase after I and
    15 4 weeks of having caused a 1M. similar to changes in ST2s (Figure 2A). Indeed. Yyl expression levels were positively correlated with the expression of ST2s (rs "" 0.362, p = 0.018). Metformin therapy resulted in a reduction of Yyl expression levels. after 4 weeks of having induced 1M and after prolonged treatment with metfonnine until week 4 (p <O.OO 1) (Figure 2A). The analysis of the cytosogenic protein extract and
    20 nuclear infarcted area also showed an increase in the accumulation of Yyl protein in the nucleus. compared to the sham group (p <O.OOI. Figure 28). The administration of metformin also significantly reduced Yyl protein levels in the nucleus (p <O.OOI). In the absence of 1M, metformin therapy did not modify the Yyl distribution compared to the sham group. So. The therapeutic effect of metfonnine seems to be related to a regulation of
    25 expression of Yyl transcription factor after 1M.
    As shown in Figure 3A, there was no significant change in HOAC4 at the mRNA level after 1M compared to the sham group. However, metformin therapy induced a significant increase in HOAC4 mRNA after I week. This effect was greater when the treatment time lasted up to 4 weeks (p <O.OOI) (Figure 3A). Rats treated with metformin in the absence of 1M also showed elevated levels of HDAC4 (p <O.OO J) compared to the sham group. When HDAC4 protein levels were evaluated after 4 weeks, the results were similar (Figure 3B). To determine the role of HDAC4 in the regulation of Yyl activity, we study phosphorylation levels and distribution of HDAC4 in infarcted tissues (Figure 3C). WB analysis of cytosolic and nuclear protein extracts showed an increase in the accumulation of phosphorylated HOAC4 protein in infarcted rats
    - ¡¡
    P201 7001S4 06/02/201 7 02/27/2017 P201 7001S4 06/02/201 7 02/27/2017
    in the citoso! (p <O.OOI). Melfonnine treatment significantly reduced these levels
    (p <O.OOI). In August: nt dt: 1M. metfonnin treatment did not alter the distribution of HOAC4
    phosphorylated (Figure 3C). In conclusion, the reduction of HOAC4 phosphorylation ion results from the
    Metformin administration induces the translocac: ion of HDAC4 to the nucleus. where by acting as co-
    S Yyl temlina transcription factor repressor by blocking the expression of the gene encoding ST2s.
    3-Yyl modulates the expression of ST25.
    To assess whether Yyl participates in the modulation of the expression of the gene encoding ST2s. and to
    To evaluate the implication of HDAC4, we use a biochemical model of cellular stress. The bosses
    The resulting gene expression were analyzed by quantitative RT-PCR and by WB. As shown
    10 in Figure 4, and similar to the results obtained with infarcted LV tissues. stress
    biomechanical was associated with an increase in IL-3J (p <O.OO I "ST2s (p <O.OO]), Yyl (p <O.O 1) and did not affect
    to the expression of the mRNA HDAC4. Metformin therapy did not affect IL-33 but reduced
    significantly expression levels of ST2s (p <O.OO 1), Yyl (p <O.OO 1) and increased the
    expression levels of! MRNA HDAC4 (p <O.OOI) (Figure 4 A-O). Results were obtained
    fifteen similar when evaluating ST2s protein levels by WB (Figure 4E). Further. when we study the
    Cellular distribution of phosphorylated HOAC4. the results were similar to those obtained in the
    animal model (Figure 4F). In the absence of biomechanical stress. metformin therapy did not change
    the cytosolic levels of the phospho-HOAC4 protein.
    To assess whether Yyl is involved in modulating the expression of the gene encoding ST2s. be
    twenty they used small interfering RNAs (in English small interfering RNA, "siRNA") in order to read
    Yyl The inhibitory efficiency of siYyl on endogenous Yyl expression was verified by RT-PCR
    quantitative, WB and immunonuorescence by seal microscopy] (Figure 5). As the
    Figure 5A the increase in Yy mRNA expression] induced by biomechanical stress (p <O.OOI)
    completely blocked by silencing Yyl expression using a group of 4 siRNAs
    25 specific (p <O.OOI). Yyl's silencing was not erect in the absence of biological stress.
    In a parallel trial. We rule out the pos: ibility of an "off-jarget" effect, when studying
    individually the effect of each siRNA on the expression levels of Yyl and ST2s mRNAs.
    As shown in figure 6. the results were similar to those obtained when we used a
    group of 4 siRNAs (Figure 5A). As Figure 58 shows. Biomechanical stress increased the
    30 ST2s expression (p <O.OOI); however, in cells where Yyl's expression was silenced. he
    Biomechanical stress did not result in an increase in intracellular ST2s; and finally. the silencing of
    Yyl in the absence of damage had no effect on the expression of ST2s. compared to the respective
    control. Similar results were obtained when the level of 5T2s prolein was evaluated by WB
    (Figure 5C). ST2L gene expression no: was altered when endogenous Yyl was silenced
    35 (Figure 50). Using these cell stress models, we tested the activation of Yyl, and the
    effectiveness of gene silencing by immunonuorescence (Figure 5E). An increase in
    ; n
    Yyl nuclear staining in cells under biomechanical stress. Compared to the control conditions, he confirmed that Yyl is activated in response to biomechanical stress. Representative images are shown in Figure 5E; In this case, the violet color in overlapping images is consistent with Yyl's nuclear location. A co-location analysis provided a Pearson coefficient of
    S 0.96 ± 0.04 and a Manders coefficient of 0.98 :: 1: 0.02. Similarly, the silencing of endogenous Yy 1 prevented this effect mediated by biomechanical stress.
    In conclusion. the blockage of the expression of ST2s after the transposition factor YY 1 is detected, shows that Yy1 is involved in the modulation of the expression of the ST2s isofonna.
    10 SEQ ID I
    YYl (Furnace sooiens. Humans)
    > NC 000014. 9: 100238765-100279034 Oven sapiens chrom050me 14, GRCh38. p7
    gene- "YY1"
    gene_synonym = "DELTA; IN080S; NF-El; UCRBP; YIN-YANG-1"
    lS rnRNA join (1.. 1159, 2354 O. 23702, 35934.. 35994, 3772 6 .. 3788 <1,
    38654 .. 40270)
    product "YYl transcription factor"
    twenty 5 '-AGGGCGAACGGGCGAGTGGCAGCGAGGCGGGGCGGGCTGAGGCCAGCGCGGAAGTCTCGCGAGGCCGGGC
    CCGAGCAGAGTGTGGCGGCGGCGGCGAGATCTGGGCTCGGGTTGAGGAGTTGGTATTTGTGTGGAAGGAG
    GCGGAGGCGCAGGAGGAAGGGGGAAGCGGAGCGCCGGCCCGGAGGGCGGGAGGAGGCGCGGCCAGGGCGG
    GCGGTTGCGGCGAGGCGAGGCGAGGCGGGGAGCCGAGACGAGCAGCGGCCGAGCGAGCGCGGGCGCGGGC
    GCACCGAGGCGAGGGAGGCGGGGAAGCCCCGCCGCCGCCGCGGCGCCCGCCCCTTCCCCCGCCGCCCGCC
    2S CCCTCTCCCCCCGCCCGCTCGCCGCCTTCCTCCCTCTGCCTTCCTTCCCCACGGCCGGCCGCCTCCTCGC
    CCGCCCGCCCGCAGCCGAGGAGCCGAGGCCGCCGCGGCCGTGGCGGCGGAGCCCTCAGCCATGGCCTCGG
    GCGACACCCTCTACATCGCCACGGACGGCTCGGAGATGCCGGCCGAGATCGTGGAGCTGCACGAGATCGA
    GGTGGAGACCATCCCGGTGGAGACCATCGAGACCACAGTGGTGGGCGAGGAGGAGGAGGAGGACGACGAC
    GACGAGGACGGCGGCGGTGGCGACCACGGCGGCGGGGGCGGCCACGGGCACGCCGGCCACCACCACCACC
    30 ACCATCACCACCACCACCACCCGCCCATGATCGCTCTGCAGCCGCTGGTCACCGACGACCCGACCCAGGT
    GCACCACCACCAGGAGGTGATCCTGGTGCAGACGCGCGAGGAGGTGGTGGGCGGCGACGACTCGGACGGG
    CTGCGCGCCGAGGACGGCTTCGAGGATCAGATTCTCATCCCGGTGCCCGCGCCGGCCGGCGGCGACGACG
    ACTACATTGAACAAACGCTGGTCACCGTGGCGGCGGCCGGCAAGAGCGGCGGCGGCGGCTCGTCGTCGTC
    GGGAGGCGGCCGCGTCAAGAAGGGCGGCGGCAAGAAGAGCGGCAAGAAGAGTTACCTCAGCGGCGGGGCC
    35 GGCGCGGCGGGCGGCGGCGGCGCCGACCCGGGCAACAAGAAGTGGGAGCAGAAGCAGGTGCAGATCAAGA
    CCCTGGAGGGCGAGTTCTCGGTCACCATGTGGTCCTCAGGTGAGCGCCGGCCGCGCGCCCCGGCCCCGGG
    ATGTTTTTGTTTTGAAGGGAAGCCATGTTTTATGTGTGTGATGGGCGCCGCCATCTTCTTCGCCAGGGCA
    AGTATGGCGGCCCCAAAAGATGGCGGGCCGTGCGGCGGCGGGGGCGCGGCGGCGGCGGCGGGCGGCGGGC
    CGCGAAGATGGCGGCAGGCCGCGGGGGGGAGGGGCCGGCGCCGCCCGGCTAGGCCGCAATGGCGTAGTTT
    40 CTCCGAGAGGGGGAAGCGCCGCGCGGGGCGGAGACGGCCGGGGGAGGGGCCGGCGCGCTCGCTCCCCGAC
    GCCCTCGCTCCCCGCGCCCGCATCAACGGGCGCGCCCGCCGGCCCGTTGTGGCGGCAGCTGCGCGCCCCC
    GGCCCGGGCGGGACGCGGGAGGTCGTTGGCGGGGCTGCGCCGCGGCCTCGCGGGCCAGGGATTGCTGCCT
    CCGGGACGCGCGCCCCCGCCCCCGCCCGGCGC'fGCCGTTACCCCATCGGCCGCTGGAGGGCGCCGCGGGC
    GCTTGCCCCGCCCGCCCCCAACTTCCCCGGACTCCCAACTGCTCCCCGGTGGCACCTTCCAGAAGGAGGG
    Four. Five GGCTTGGCACTCGTGGGCGCCCTCCGGCCTCCTTGCTCCCCGCCCACCCAGCGCCGGGCTGGACCCTGCC
    CCGGCGGTCACGCTCGCCCAAGTCGTCGTTTGCTGCGGGGCGGGACTTGGGGCTGCACGTAGGGCTCGCG
    TGTGCGGGCTCCGAGCGTCAGTCGGAGCCTGTCACCGCCGTTGCCAGCGAATTCCTGGCCTTTTCGCCTT
    CCTGCGGTACCTAGTGGAAAGGGCCTGTGAATGTTAGCGAATAATTCTTAGTCACTTACGTTTTGGGAAA
    GCTTTTTTTTTTTCCCTCAAGTATTTAGCTCTTCTGAATAGGTTGGGTTAGTTTACTCAACTGTGGACAT
    fifty TTGCCAGTAAATAAAATCGGGTAGTTTAATTTTAAGCCTTTATGGTTTTTACTTTGTAGAAAGGTATCTA
    .._---
     06/0212017 27/0212017 P2017001S4 06/02/2017 02/27/2017
    TTTTCCCCATGAAAAGAACGATAATAGAAAGCGGGTT'TTTTTGAGTTTTGTTTTACTGTTTTAATACTAC
    TTTTAACGAAGGCCTGAAAACCATCTAGAGCAAAAGG. ~ TCTTATCTGCCTGTGTAAAATCTGTTTTTAer
    TCAAAGGCMTTATTAATTCAATGCTGGTTTTTTAGGTCAGTATCCTATTAGTATATTCATATGCTACCT
    AGCTAAATTGCTGAGATACTAAATAGACTGAGAATAATCCTGCCATCGTAGTTMTCTAATGCCTGTGAT
    5 GATTACGTTTAGTACTCCATATMGCGGTGGAACCTC'TTCCCTATAAGTACTTTAAAGCTTCATAAATTC
    TCAGAACTGMTGGAGACTCAAAATATGGTGACCTTTI3AGTATTAAAAGATCGTTAAAATTCGGAGGAAT
    GAATGAAACAGAAATGAAAAGGCTGGACTGAAAAAGC'TAGTTTGTATCTGGTGAAAGCCTCAAGATGAGA
    CMCTACAGCTGGGTGGATCTGTGATTGAAAGAGGCC'TGAGCAAAGGGATCTAGGAGCTGTCAAAATCTA
    ATAGGAAGGTGCTAACAGGCGCTGTCCTGCATCCAGGGTGCCTTGTAGAATTCATAGCAGTTTGAGTTTA
    10 GCAGAAACTATGTTTAAATGAAACATGAATACTTTAG'TACTTAATGGTTATCAAAAGATAATTTCCATGT
    ATTTTGGTAGCTCAAGAGATTACATACTGATCATGTTIGGAACTAAAAGTCATGGTTTTGGCCGGGCGCGG
    TGGCTCACGCCTGTAATCCCAGCAGTTTGGGAGGCTG, ~ GGCCGGTGGATCACTTGAAGTCTGGAGTTCGA
    GACCAGCTGGCCATCATAGTGAAACCCCATCTCTACT, ~ TATAAAAATTAGCCAGGCGTGATGGCGG
    GAGCCCGTAATCCCAGCTACTCGGGAGGCTGAGGCAGIGAGAATCGCTTGAACCCAGGAGGCAGAGGTTAA
    fifteen CAGTGAGCCGAGATTGCACCACTACACTCCAGCCTGGGTGACAGAGCGAGACTCTGTCTCAAAAGGGGGG
    GGGGGGCATTTTTTTTTGTTAATACCAATGAGTAAGGIGATGGGGGTTTTAGTTATTGATGAATTTGCAGA
    ATTCAGAATACGATTTGAAATAAGATATTTAGGTAAAIGATACATTAACCACAAAAGTAAATTTACTTTTT
    CTCATAGTGTGTGGAATTAGAGGAATTTAGAAAACCG, ~ CATATTTTTACATGATCTGCATTTTATCTTAA
    CAAATTGCTGGTCCACTTTTCCACATCCAGAGAAGCTGGTAGTGTACGGCTTTGCTTGCTGAAGTGGTAT
    twenty GTATGGAGTCAGCCAGTTAGGGGAGAAAGTCTGAGGGGCTTGACTGATGGA1 GTCAATTTACAATTAAGT
    GAGCATTTACAAGTGTGCAAGTGGCTGTTTTGTTTTG'TTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTT
    TTGAGATGGAGTCTCGCTCTGTCGCCCAGGCTGGTGTGCAGTGGCATGATCTCGGCTCACGGCAAGCTCC
    GCCTCCCGAGTTTACGCCATTCTCCTGCCTCACCCTCCCGAGTAGCTGGGACTACAGGCGCCCACCACCA
    CGCCCGGCTAATTTTTTGTGTTTTTAGTAGAGACAGGGTTTCACCATGTTAGCCAGGATGGTCTTGATCT
    25 CCTAACCTCGTGATCCGCCTGCCTCGGCCTCCCAAAG'TGCTGGGATTACAGGTGTGAGCCACCGCGCCCA
    GCCMGTGGCTGCTTTTAAGGTGGCACAGTTTGGGGT'TGTTTTCACGTCCGTTTCTTTTCTTCTCTTTTC
    TTTTTTTAATTTGAGACAGAGTCTTGCTCTGTTGCCCAGGCTGGAGTGCAGTGTCACGATCTTGGCTCAC
    TGCAACCTTCACCTCCCGGATTCAAGGGATTCTCATG'TCTCAGCCTCCCAAGTAGCTGGGATTGCAGGGA
    TCCACCACCACCCCTGGCTATAATGTCCATTTCTTTA ~ GCGCTGGTTGTCTAGCTAGTGTGGTAGAGTA
    30 CGCTGTGCATMGGGTAGTTATAAATATTTGATGGCAGTAACATAGGATGGCCTGTTTCTCGGACATGTG
    TTTTGTGGTAAACTATCCCCCTTACTTTTCCTTGTGA'TTTGGAATTATTACTAAAGAGAAGCTAAAGAGC
    TTATGGATAACTCTGCATTTCCCMGCTGAAATTCTGAAGGTGATTTCATCTTCAGAACTGAAATGACGT
    GATATACTCAGTATAATTCCATAACAAGTGGAGAAATGAAAGTGGGTTAATTGTTGGGTAAATCTGTATG
    TATGTGTACATATGGGTTGAGCATGGTGGGCAGCATTACtGCTTTCGTGTACTTGATCTCATTTGATCCT
    35 AGGCATGAAGGTTTTAACTACACTTTAGTTTCACTAAAATAGCCCCTTAGTTTGGAATGAACTGTGAAGT
    AACAGTTTTGCATAAGCCCCAAGCTGAGTTACTTGAAIGTCCTTATAAAAATAGTTGTACATACAGGGTAT
    TCCCCGAGGGTCTAGTTACTTCTTAGACCCTCTTGGTTTAAAAAGCAAAAAGATCAGGCTGGGTGCAGTG
    GCTCACACCTGTAGTCCCAGA1 CTTTGGGAGGCCAAGGCAGGAGGATCGCTTAAGCCCAGGAGTTCGAGA
    TGAACCTTGGCAATATAGTGAGACCCCGTCTCTGC ~~ CAAAAAACCCCACAAAAATTAGCCAGTC
    40 ATGGTAGCACATGCCTGTAGTCACTCTCAGCTACTTGGTAGATTGAGGTGGGAGGATCTCCTGAGCCTGG
    GAA1 TCGAGGCTGCAAGTCAGCGGTGATTACACCACTGTACTCCAGCCTAGGTGACAGTGAGACACTGTC
    AAAAAAAAAAAAAAATTGGCCGGGCGCGGTGGCTGACGCCTGTAATCCCAGCACTTTGGGAGGCCGAG
    GCGGGTGGGTCACGAGCTCAGGAGATCGAGACCATCC'TGCCTAACACGGTAAAACCCTGTCTCTACTAAA
    AATACAAAAAGAAATTAGCCGGGCGCTGTGGCGGGTGCCTGTAGTGCCAGCTACTCAGGAGGCTGAGGCA
    Four. Five GGAGAATGGCGTGAACACGGGAGGCGGAGCTTGCCGTGAGCCGAGATAGCGCCACTGCACTCCAGCCTGG
    GCGACAGAGCAAGACTCCGTCTCC ~~ TTAGTGGGAGTCATCATATTTCTGGAAACC
    AAATGGGTTTCTTTTGCGCACATGCCATGTTCCTTTA ~ TTTCCCACAGTAAGAGCACTCTGGTGGGCATC
    TTTTGAATGTAATATCAGTATAGGMTCTAATAGTTT'TTTAGTAGTTTTAGGCCTTATAGGATCCTTAGA
    GATTGGCTTTTTTTTTCCTTTTTTACTTTTTTTTTTT'TTTTTTTTTTTTGAGACAGGGGTCTCACTCTGG
    SW CACCCAGGTTTGAGTGCAGTGTGAGATCTCGGCTCAC'TGCAACCTCCACCTTCCGAGTAGCACGTGCCAC
    CATGCCTGGCTAATTTTGTTGTATTTTTGGTAGAGAC'GAAGTTTCGCCATGTTGCCCAGGCTTGTCTTGA
    ACTCCTGATCTCAGGTGATTCACCCACCTCGGCCTCCCAAAGTGTTGGGATTACCAACCTGCCCGCCCCA
    CTCCCCACCCCTTACTTTTTTTTTTTGAGGCAGGGTC'TCATTCTGTCACCCAGGCTAGAGTGCAGTGAGA
    CAGTCACGGCTCACGGCAGCCTCAACCTCCCGGGCTG, MGCGATCCTCCCACCTCAGCCTTCCAAGCAGC
    55 TGGGACCACAGGCTAATGTTTTAATTTTTTAGGCATGGGGTCTCCTTATGTTGCCCAGGCTGGTGTCAAA
    CTCCTGGGCTCCAGCAATCCTTCCTCAGCCTTCCAGAGTGATGGGATTATGGGTGTGAGCCACTGTGCCT
    GACCTCTTTATTTTTTCCTAAATTTTTAATTGTCATACACATGTTTAGTTTCTTCTGTATTTA7TTTTAT
    TTATTTATTTTTTATTTCTTATTTTTTTTTGAGACAGAATCTCGCTCTGTCACCCAGGCTAGAGTGCAGT
    GGCACGATCTCGGCTCACTGCAAGCTCCACCTCCCGGGTTCACACCATTCTCCTGCCTCAGCCTCCAGAG
    60 TAGCTGGAACTACAGGCGCCCGCTGCCACACCCGGCT, ~ TTTTTTGTATTTTTAGTAGAGATGGGGTTTC
    ACCATGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCTGCCTCGGCCTCCCAAAGTGCTG
    GGATTACAGGCGTGAGCCACTGCGCCTGTCCTGTTTCTTCTGTATTTAACCTGAATATACTTAACTGTTT AGTGTGTCAGAGAACTCTGTGCATCTGGAGTTTTTTAGTTGTTGCTTTTTAAATTAGAGATGGGGTCTTG CCTTGTTGCCTAGGCTGGCCTTGAATTCCTGGGTTCAGAGTATCTTCCTGCCTCAAATTCCTGAGTAACT GGGACTACAGACACCCAGCTTCAACTATGTTTTGAATAATCTTAATCAATTGCTTTAATGCCAGTGTTTA CTTTGGCCTTTTTCACTATATATGATTAATAAACATTTCAGTGTTGATAGATTGGGGCACAGTACTATAT ATTTTTTCCCTAGATACTAATGCTGTTACATAGGAGTAACAGTTGTTGTGGGGTTTTTTGGGTTTTTTTT GAAACGGAGTCTCGCTTTGTTGCCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACTGCAAGCTCCAC CTCCCGGGTTCACGACATTCTCCTGCCTCAGCCTCCCGAGTAACTGGGATTACAGACGTGTGCCACCATG CCCGGCTAATTTTTGTATTTTTAATAGAGTCTGGGTTTCTCCATGTTGGCCAGGCTGGTCTCAAACTCCT GACTTCAGGTGATCCACCCACCCCAGCCTCCCAAAGTGCTGGGATTACAGACGTGAGCCACCGCGCCCAG CCAGGl GTAACTGTTTTATAACTTl GTTAATTl TAAACTAAGCCACTTAACCATTGCATTTAATl GGGAA GATAATGATAAAAACTGAGMTTTAAAACATTTAGTTCAGl GAGAAATTTl GGTGATTTTAAAATCTTGA GATTTCAGGTGGTGACAAACTGTGACCCTCTCTAAGGCTGTTGATATTTTAATTCGGAGCAGAAGTTGCA TTGCCATGTGCATAAACTGCCGCl TI GATGCCTGGAACCCTGGAAACAAGCCAGCATGTCCATGGTCACC CCCACCCCGTTTCCATTTCCATACATGGGTTTTGTGAGGTGAGGCCATCATGTGCATACTGCCTGCCCAT GGCTAATGAGGATAGCl Cl GGTGCGGGGCCl GTGTCCCCCCCATTCTGTCCCGTGCCAGTCAGCACAGCT TGGGTTGTGGGGGACATGTGCCTTCTCAAAGTGCTGGCAGCTGl GTGGCACCTCCTTCl CGCCTAGGAGT CGl TGGTCCCTCCCCTGGTGTGCCCl GGTTGGCCTCATGCTCCGGGTAGAGAACCCTtGTTTGGACCtTG TGGGGATGTGGGGTGGTGAGACCCCACCtGAGCTGTGl CCTTCTGl GAGGAGACAAATTAAACTGCTGAA GGAGAGGCAAAAATAATAATCATTATTCTGTTACACTGAATAGAGAAGCGGCTTGTGCTGGATTAAGCAA AACCTGTAAGACCAGAAAACCATTGATACAAAATCAG'TAAATGCAAGGAAGTTGAAATGCATTGAATTTT TCCCAATTTTATAAGTTCl GAAGATGCl GGTTTGCCAAAAATCACTAGTCATGACCGTTTGCCATTCCTC TGTGGGTTTTTCACACCCTCCCTCCCCl GTAGAAACTGCGCTGTTTATCACTAAGATGCTATAAACATGA l TTTCCATGGAGCCAAGCTCAAAAAGCCCTCCAAAGT, AATCACTTCACCCCCATGl CCTL Cl TAGGGTl G CCTAAGTCATAGATCTCGAATCCTGATCTGGTl GCTCTGAAAGGTTTAGCTTCCTAATAAAGCCTCTTGT GCTGtCAGACTCTCCCCTCCAGGTTl TTAACCTTGCCTTTTCCCTCCCACTATCAACAACTGl CTCCCCC TAGTGGGAAAAGAAAAATCCGTl TCAGGAAAAGGTCAGGGTTCCTGTCCTATCTACAGTTAGTATTTTAT AGGCTTTTGGAAGl GGGTAAATTTTTTAAAACATCCATGTTGGACTCCTl G GTGGl Tl TTTACAAAAGGC ATTTCTl TTTTGTTTTTGAMCCTGCTTAACAAGGTCAGTCTGTTTTAAGGCTGGGAAACTTAGGTTATl TTAGCAAAGCAGTTATTGCAATGAATGTTACTTAGTA7TTGCATTCTTTTATTTAACTCTTGATCTGCTC AGGTGACCATTTTTl TTCTCTCTGTGCTCTTACTl CTTCCAATAGGAAGAGTAAGCAAAAATGAATTTTT CTTTTTTTCTTTTTTTTTTTTTTTCGAGl CAGGGTCTCACTTTGTCl CCCl GTCGGGAGTACl GTGGCAC AATCTCAGCTCATTGCl GCCTCGACCTCCTGGGCTCAAGCAGTCCTCCTGCCTCAGTGCCCCAAGAGCTG GGACTACAGGCACGCACCACCl GGCl TGGCTAATTTT'TTGTAGl GACAGGGTTATCAGAATCATGACTTG GATGCAGAAGATAACATTCATGTAATTACTGCAGl GT.ATTAGGAAGAGCCAAGGGAGl TGTTAGAAGTTA GCTGATTGTTGGGGGGCTTGCACCTTTTCATGTGTGATGGACTTTCATAACAGCACGTAl GGGGAGCCAl GCCATTTCCTCGTTCTAAGTATCTTCTCAGTAGGTTCCATTCTGTTTAACTTCATGAATAATTATCAAGA ACAGAAAGCCAGCAGCATCATATTTCCTCTCTAGAGGGGACTGACCTTGAGATCTTTCTAAAATGl GTTG GGGTAGCGGTl TCCTTAAAGTCACTTTGAATCTTTATl GCAGGAAATTCTATTCCTTTACAAACAGAACA ATATACTCAATTTCTTTCTTTCTTTCTTTTTTACl GAGTCTTACTCTGTTGCCCl GGCTGGAGTGCAGTG GTTTGATCTGGCTCACTGCAACCTCTGCCTCCCGAGTTCAAACAATTTTCCTGCCTCAGCCTCCTGAGTA GCTGGGl TTl CAGGTGTGCACCACCACGCCC GGCTAATTTTTTTTTCTTTTTTTTTTTTTTGAGACAGTC TCGCTCTGTCl CCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACTGCAl GCTCCGCTTCCCGGGTTC ATACCATTCTCCTGCCTCAGCCTCCCAGGTAGCTGGGACTl CAGGCGCCCACCACCACGCCTGGCTAATT TTTTTTCTTTGTl TTTTTTAGTAGAGATGGGGTTTCACCGTATTAACCA.GGA.TGGTCTCAATCTCCTGl C CTCGTGATCCGCCCGCCTTGGCCTCCCAAAGTGCTGGGATTATl GGCATGAGCTACTGTACCCAGCCTAC TCAATTTCTTAAAGCAAGAGTCTtCCATTTTAACATGACCTGGCAAATGAGATATTAGGAACTCTTGAGA AAAAAAAATTTTTTTGAGACGGGTTTTTGCTATGTTGTTGAAACTGGAGTGCACTGGCTATTCACAGGTG TGATCATTGTGCGCTACA.GCCTTGAACTCCTGGGCTCAAGCGGTTCTCTGGCCTTAGCCTCCCTAGTAGC TGGGACTAGAGGTGTGTGCCATTCCACCCAGCTTGAGTAAAATTCTTTTTTTTTTTTTTTTTTTTTTTI T TGAAACGGTGTCTCGCTCTGTCACCCl GGCTGGAGTGCACTGGCGCGATCTCGGCTCl CTGCAAGCTCTG CCTCCCGGGTTCACACCl TTCTCCTTCCTCl GCCTCCCGAGTAGCTGGGACTACAGGCGCCCACCACCAT ACCTGGCTAATTTTTTTGTl TTTTTGGTl Gagl CGGGTTTCACCGTGTTAGCCAGGATGGTCTTGATCTC CTGA.CCTCATGATCGGCCCGCCTTGGTCTCCCAAAGTGCTAGGATTACAGGCGTTAGCCATCACGCCCAG CCAAGTAAAATTCTTAATAGCCATTCAGTTACTTTGGTTTTACl CAGTGGTTtCAAATGTGGTTTAGCAT TAAAACAATATAAATGGT TGTTTGTGATGGACTCTTTcrCGTGTAATTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTGGGAGACAGAGTTTTGCTCTGTTGCCCAGGCTGGAGTTCAGTGGCACGATCTCGGCTCA CCTGCAACCTTCACCTCCTGGGTTCAAGCGl TTCTCC'TGCCTCAGCCTCCTGAGTAACTGGGATTACAGG CTTGCACTACTACACCCAGCTAATTTTTGTACTTTTAGTAGAGATGGGGTTGCACCATGTTGGCCAGGCT GGTCTTGAACTCCTGACCTCAAGTGl TCTGCCTGCCTCAGCCTCCAAAGTTCTGGGATTl CAGGCGTGAG l CACCGTGCCTGGCTTTCTTGTGTAAATTAATTCCCC'TTCTCAAATAGAAGATCTGTGAGTCAGGGAAAG
     02/06/2017 02/27/2017
    GGGCTGCCATTGCTGTGCAAGAAGCTGTTCTGTTGGTGTCAGGACCTGTCTTTTCAGGAGAGTATTGAACTTTGAATATTGAGTGAATGAATGGCTGGAATTTGTGCTGTAAGGCAGAATCAGGGCTTATACCAAAATACTATTTTGGTCTATTCCTGTATAGGTGGAAAAGAAGTATTAGGGATAGAGAGAAGGTGCAGAAGGTTCTGTTTTAGAAGCAGCATGGGCTTTTAGAGTCAATTCTCAATTCAAATCTCAGATTTGCTACTTACeGTT! TTTTTTTTGTTTGTTTTTGTTTTTGTTTTTGTTTTTTTTTGAGACAGAGTCTTGCTCTGTCGCCCAGACTGGAGTGCAGTGGCGTGATCTCAGCTCACTGCAACCtCCGCCTCCCAGGTTCAAGCAATTCTCCTACTTCAGGTTCCTGAGTAGCTGAGACTGCAGGTGCCTGCCACCACACCTAGCTAATTTTGTATTTTTAGTAGAGATGGGGTTTCTGCATGTTGGTCAGGCTGGTCTCGAACTCCCGACCTCAGGTGATCTGTCCGCTTCAGCCTTCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCACCCGGCCTACTTACTGCTCTTTAGATACATTGCTTGTCATTTCTGAGGTCAGTTTCTTCAGCCAAAAACATAGGGTTTAATACCTTAAAGATATTGTTTGGATTGAGAAATGTGACAAGTTGCCAGAATGAGACAGGTACTTAATAAAGGATAGTATCCTTTCTTAAGGTCTTTATTAGTGGGAAAAACTGGGAAACAACCTATGTCTGATTTTTGCCTGTTGCTAAGCAGTGAATCGCAAATGTTTAGCTCTCCCCTGTTTCAGTGAAAGGTTGGGTGATGGCAGCGGTGACTCTTTTTTGTCTTTTTTTGCCAGAAGTAACCTGGAGTGGCCTAAGATAGAAGGTGTGGAGAAACAAAATCTAGCCAAACATATACATGCATACATACATAGGTAAATATCTCAGTTGGTGAAGGTGAATATAGAAAATCGGGATCACAGGAAAAATTACCATCAACCCTAATGAGGTTACTGGCAGTCAAAATTAAAAAAAAAGGAACTTATACTACCTTCATCTGATATTAATTCTGAGGAAATAAAATGTTTCTTAGGAATGAATTCCTTTGTGTATGAAGGGGACCACTGAGTGGTAGTACTGGCATTTTTCAGCCAATGTGACAAATTTCACAGTGCTGTCTTAGAGATGCCTAAAAAATGTATTAAGGAACGTGGCTGGGTATGGTGCACTTTTGGAGACTGAGGTGGGAGGATCTGAGGCCAGGAGTTTGAGACCAACCTGGGCAACATAGTGAGACCTTGTCTCCACAGAAAATTAAAAAAAAAAAAAAAAGGCCAGGCATGGTGGCATGTGCTTTTGTAGCCCCAGCTACCCAGGAGGCTGACGTGGGAGAATCACCTGAGCCCAGGAGGTCAAGGCTGCAGTGAGCTCCACTCCAGCCTGGATGACATAATGAGATTCTGTCTCAAATGAAAAGAGAGAGAGAGAAATAATATATAAGGCCTTTGATTTTTCTGATTAACATATTGACTAACTGGTTGACTGGTTGACTGAATAATGACTCCCAATATAGCCAGATCCTAATTCTTGGAACCTGTGGGTGTTAACCTTATTTGCAAGTGTGATTAAACCAGGGCTCTTGGAATAGGGAGAGTATCCTGGTTTATCCAGGTGGGTACCAAATGTAATCACACAAGTGTCCTTGTATTCACAGGAAGGCAGTGGGAGATTGACTGCCAAAGAGGAAGTAGGAGAGGTGTGAGAGGTTGGAGTCCAGTGAAGAGAAGGGGTCAAGGAATGTAGGCAGCCTTCAGAAGCTAGAATATGTGAGGGACCAGATTCTCCACTAAAGCTGCCAGAAGGAACCCACACAACAACTTTATAGCTCTGAAACTGATTCTCACTGCTGGCCTCTAGAACTATAAGAGAATAAATTCCTGTTGTTTCAAACCACCAAATTGGTGGTAGTTTGTTACAGTGGTAATAGGAAACTAATACATGTGCTAGCATCCCCTGTATAGCTTCATTCTGCAGGAACTTGGCAAGTCCCTGTTATGTGCAAGGCACAAGACCTACTGTTCCCCCTTAAACCTAGGAGAGGGGGGAAGTGGAATAAAGGGGCTTTTTTTGCACACTACTCAATTTCCCAGACATGATGATTGGCATTTAACATGTTACCTTGTTTTAGCCTTATAAGATCAGTGGTATCCTTCCTCCCTCCCTCCCTTTGTCCCTTCCTCCCCTCCCCTCCCTTTGCTTCTCTTTCTTCTCTTTATTTCTTTGTAGAGGCAGGGTTTTCTCTTTGTTGCTCAGGCTGGTCTCTAATTCCTGGCCTCAAGAGATCCTCCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAACTCCTGTGCCTGGCCTCATTGTTTAATAAATGAGGAAACCAGGCGAGGTTAAGAGTAGAGGCTGGAGTGACAGGAATGAATGGCTTCTTGAAAGGAAGAGATCTTAATTTGGGTTTTGGAGAGTGGACTGGGCTTAGGTAGGTCATGAGAGGAAGGCAAGAGGGTAAATTGTATTGAGTACCTTTTACT.AGACACTGAGAAAACCACTTTGCTTGGAGCGTGGAGGGCAGGTGAGACTCCATCTTTTTACTTCTCTGGACTTGTAGAGTGCTTCTAGCCTTTCCATTTATTTAGTTCCTTAATTATATCTTTTTGACTGGAAATTGGAGTCTACAAACTCAGTCTTATGCTTTGCTCCTCAAACTAGACTTGGAGTGATTATTGCTTTGGTAACATAGAAGAACACCGAGCAGCTGGGAGATTGGGATGGGCCAGCGGCTTATTTATACAATACCTTTAACCTTCTGAATAGTACCATATGCAGAAATAGAAACTTTTAAATGTGAGTTAATTGTCCTTCCACATATATGACTAGTGTTGGCTAGATTTTGTTTTTCCACACATTCTATCTTAGGCCAAGTTAGTCTTGGTCTAGTTGTTTTAGGTCATTGCATTTAGCCAGAGTGTGAGTGAGCACTGTAGTCACCTAATCGACAGTTTTGGGTAGAAGTTCCTAACATGATGGTTGCCCACATCACCTTACCCCCTTTGAATCAATGACTGTATTCTCAGCTTTTTCCAAATAGGAATTCTGAGATTTGGCTTTAATTGGCCTTGCCCACTTGTGTTCTGGTAATTAGCAATTGTAAACTTAAGTTATATTGGTTAAAGAAACCCATTGCAGGCCAGGCATGGTGGCTCACACCTCTAACCCCAGGGATTTGGGAGGCCAAGGCAGGAGGATCACTTGAGCCCAGGAGTTCAAGACTAGCCTGGGCAACATAGTGAGACCCCATCTCTACCAGGAAAAAAAAAATCAACTGGGTGTGGTGGTGTGGTGCACCTGTGGTCTCAACTACTGGTGGAGTGGGGTTGCTGGAGGATCACTTGAGCTAGGGAGGTTGAGGCTGCATTGAGCAGTGATAGTGCCACTGCACTCCAGCCTGGACAACAGAGTGAGACCCTGTCTCAAAGGAAAAGAAAGAAAGAAACCCATAGAAACTTCTAATGGGGTTAACTATGTCCTGTGAAACAATAAAAGATCCTGTCCTCAAGGGAAACTCCAGCAGGGAATAGACATACAACAATTTTTTAATGACCTATAGTGGGTCCAGACAATTTTTAAAGCAGATTTAGATACTTTGCTTCCTGAACATGTTTTACCTAGATGTACATTTGATATTACCCCATCGCTAGTCAATAGAAATCAGTAACATTCATAGCATAATTTGACATTCAAAGTGGATCAGTAAACACTCTCCTCTATGTAGCAAATCTTTTTCAGTAGTAATTATGAAGGGAATAGTATTTTTAGTAGCAATGGAGGTTTGCCATGTTAGCCAGGCTAGTCTTTTTTATTTTTTGAGACAGAGTCTCGCTCTGTTGCCCAGCCTGGAGTGCAGTGGCGTGATCTCGGCTCACTGCAACCTCTGCGTCTCAGGTTCAAGCTATTCTCCCACCTCAGCCACCCGAATAGCAGGGATTACAGGTATGTGCCACCATGCCCAGCTAATTTTTGTATTTTTAGTATTGATGGAGTTTCACCTTAACCTCAGGTGACCAGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTATAGGCGTGAGCCAGTGTACCCAGCCAACCAGGGTAATCTTGAACTCCTGGCCTCGGGTGATCCGCCCACCTCAGCCTC
    "';" !:P201 700154 02/02/2017 02/27/2017
    CCAAAGTACTGGGATTCAGGCATGAGCCACTGCGCTGGGCCAGGGTTAATACTTTTTAACAAAGATTTTT ATTATTATTATTTTTTATAATTTTCTTTTTTTGAGATGGAGTCTCACTGTTGTTGGCTCAGGGTGGAGTG CAATGGCATGATCTCAGCTCACTGCAACCTCTGCCTCCTGGGTTCTAGCAGTTCTCCTGCCTTGCCTTCC CAGTAGCTGAGATTACAGGTGCCCGCTACCACACCTA'TAATCTTTTTTTTTATATATTTTTGCTAGAGGT 5 GGGGTTTCACCATGTTGGCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCCGCCTCGGCCTCT CAAAGTGCTGGGATTATAGGCGTGAGCCACCACACATGGCCAAGATATTTTTTTGAACAAAGAAAGAAAC ATATGTACATATGGCCTGTAATGTAATAAATAAAAAT'TACTAGCTTCTTTTACAGCATCATAAATTTeTA
    TTCTATTTGGTAAAAACTTCTGATTTTCTTTTTGCAA ~ TTAGCAAAATGGCCAAATGATAGCAAATAT
    TGACATTATTTAATGTATTGCTTTATTGTGTGTCCAGCTTGTGAAAATGAGGCAGGTCAAATATTGTCCT
    10 TTTGATTTCTTCTGGCAACAAAAGCCAGTGTCTTTTGTCATTTCTCTAGAAGTCTTTTTGCAGCTTTTTC CTATATGTTTCTTTTCTCTTTTCTTTTCTTTTCGACGGAGTCTTACTCTGTCGCCCAGGCTGGAGTGCAG TGGTGCAATTTCAGCTTACTGCAAACTCTACCTCCCAI:; GTTCAAGCGGTTCTTTTGCCTCAGTCTCCCGA GTGGCTGGGATTAAAGGCACACGCCACCACACCTGCT, ~ TTTTGTATTTTTAGTCAAGATGGATTTTTG CCATGTTGGCCGGGCTGGTCTCGAACTCCTGACCTTAI3GTGATCAGCCCACCTTGGCCTCCCAAAATGCT 15 AAGATTACAGGTGCGAGCCACCATGCCTAGCCTATTT'TATTTATTTATTTATTTTTTTTTTTTTTTGAGT CGAAGTCTCACAGTGTCGCCCGGGCTAGAGTGCAGTGGTGTGATCTTGGCTCACTGCAACCTCCGCCTCC TGGGT TCAAGCAATTCTCeTGeCTeAGCeTeeeAAGTTAGeTGGGATTAeAAGeGeCeGCeACCAeGCCC AGCTAATTTTTTTTTTTTTTTTTTTTTTTTTTTTTGG'TATTTTTAGTAGTGACGGGATTTeACTATGTTG GCCAGGCTGGTCTeAAACTACTGACeTeGTGATCCAecCACCTTGGeCTCCCAAAGTGCTAGGATTACAG 20 ATGTGAGCCACTGTGCeeAGCCAGeeTATTTTCTTAT, ~ TTTTCTTGTTCACAATTTCTGTGCAGTGGTeT TTGGAGAGTTCTAGTTATCTTCCTTGTAGGAAACACTGTTTTTTAAGGAATAAGTCATAAATGTGATAAe ATGTGTeTTAAATCTGTATeTTACGAGAATGTACAAT, ~ CCACAAAAATTATTTCAAACTTAGACCAACA AAATATGTTTTATTTTAAGGCAAGAAACTGATATTAT'TTGAAAGTGTGGGAGGGCCGGGCA CGGTGGCTC ATGCCTGTAATCCCAGCAGTTTGGGAGGeTGAGGTGGGCGGATCACCTGAGATCAGGAGTTAGAGACCAG 25 CCTGGeCAACATGGTGAAACCCCATCTCTGCTAAAAA'TACAAAAAATAGeTGAGCGTGGTGGTGTGeeee TGTGATeCCAGCTACTTGGAAGGCTGAGGCAGGAGAATCACTTGAACCAGGGAGGCAGAGGTTGCGGTGA GCeAACATTGCACCACTGCACTeCAAeCTGGGGGGGACAGAGCGAGACTCCATCTCAGGAAAAATAAATA AATAAATAAGTGTGGAATGTGGTAATGATCAAGAGTACACTTATTTTCAGCCAGTTTTATTATGGTTTTA AAATTATGTACAGACCATGCAGeCTCTCTTGTCTGTGCTTAGGGCAAACTGCTTTTGCTGTATGCCACTT 30 GGCACTTGATACGTCACTGAGTACAGTTTTCTTTAGACAGAGCTAGTTGTCTCCCTACTTGTGGACATCT CTCTTCAGTCTCCTTTGCAAAGTCTTCCTCATTCTCTTAATATTAGAATTGTTCAGGTTTTGATTATAAT CCTTCTCCTCTCTGACTGTATTCTTTTTCAGTAGAT ~~ TCTTACAATGTCTTTCTGGGGCTTTCAATGCA GTCTGAGTCAGGCATGGTATTGTGGGCCTGTAGTCCC.Pr.CCTACTTGGGAGGTTGCAGCAGGAGGGTCAeT TGAGTCCAGGAGTTTGAGGCTGCAATGAGCTATGATC, ~ TGeAGACTATGATCACTCTAGTCTGGTCAACA 35 GAGCCAGACCCTGTCACAAAAAAAAAAAAACATCTAT, TGCCAGTGATTeTeACAGATTACGATATAATT CTCTCTGACCTAGGTTTCTTATCCCCTATACATGCAT ~, ~ ~ TTTAGTTGCCTTTTTGAGGTCTGCCCATTAGA TTTTGCACAGATACCTCAAACCAAAGAGGAAeTCTAA TCTTCTGCACATC ATCTCTTTCACACTCTTGT CCTCCATCTCATCAAATGACACCACCATCTTCT ~~ TTAGAAGCAAGAAACTCAGGTGGCATACTTGG
    CATCCCTGTTCCTCCCCAACCCATCTGATTAATTATC'TAGCCATGTTTATTTCTCCATCCATTAACAGAT 40 GGACAAAATGGTATGTATGTACAGAATATTATTCAGCCATGAAGAGGAATGGAATGTTGTTATATGTTAC
    AACAGGTGGGTCTTAAAAACATTATGeTTAGTGAAAT, ~ CCAGGCATGGATGGCAAATACTGTATGATT
    CAACTGGTATGAGGTACeTGAAATAGGCAAATTCATAGAGACAGAAAGTAGAATTGAGGTTAGCAGGTGC TGAGGGGAAGTGAAAGTTCTTGCATAATGGGTATAGAGTTTTTTGCGGGAATGAAGAAAATATTTTGGGT
    ATAGATAGTGGTGATGGTTACACATTCTGAATGTATT, ~ TGGCACTGAACTGTACACTTACATATCGTT
    45 AAAATGAGAAGTATTATGGTAATGTATATTTTACCAC, ~ TTTTTTTTTTTTTTACGAAAACCGTGGAAAT
    ATAGAGGTTGGATTTGGeCAACTGGCTGTACTAGTTG, ~ TTCCTGATATAGGTCATAAAGTCTTGTCAGCG
    TCTACTTTAGGCTTCTTTCATATATTTTTTTGGTTTC'TGATTCTGAATTATTTTAGTGACeATCCTGAGG GTTCTTGATATTCTTCAGTGTTGGTAGTGTATGCCACTTGCACTTGATATGTCGCTAAGTTCTCTTTAGA CAGAGCTAGTTGTCTCCCTACCTGTGGATATACCTCT'TCAGTCTCCTTTGCAAAGTATTCCTCAGTCTTT 50 eAATATTAGAATTGCTCAGATTTGGGTTCTGAGCCCC, Pr.CTCAAGAAGTATTTecTcATTTGACTTCCCTC ACTAGA.CTGTAAGTTCCTTGAGAGCAGATACTTTGTT '! CTAGCTTTTAT1CCCAGTGCCCAGAACAATGC CAGGCACATAGGAGGCeeTCAATATGTGTAGAACTAATGATAATAATCATTTAGCCTGTGATTCCTTTAT GCCATGAGTTCAGTCTTCTGTCTCAGCACACTTGAGACATGCCCTCTGGGGTAGACTGCATGTTTGTGTC
    TCTCCCAGATTCATGTGTTGAAATCTTAACCCCCAAT '~ TGATGGTATTAGAGGGTAGGTCCTTTGGAAGG
    55 TAATTTAGGTCTGAGGGTAGAACCCTAATGCATGGGA'TTAGTGCCCTATAAAAGAGACTCCAGAGTGCTA GCCCTCTGGTTCTCTCTATGeTATGTGACAAGACAGTGTGAAGATGGCTATCTGCAAACCAGGGAGTAGG TCCTCACCAAGAACACCACCAfGCfGGCACCfTGATCTTGGACTTCTAGCTTeCGAACTGTGGGAAATAA ATGTTGCCTTAAGCeACCTAATCTATATTTTTGTTGT, Pr.GCAGCCCAAATGGATTAAGACACTTTCATCAG TAGCTTATTCAGAGTAGCACTGATTCTCAACATTGCTATCATTCTGTATCAGAAATATAAAACATGGCCC
    60 AeACeTGTAATCCCAGCACTTTGGTAGGCCAAGGCGG, Pr.TTGCTTCAGCCCAGGAGTTCGAAACCAGCCTA
    GGTAACATAGTGAGACCACATCTCTACAAAAAAT ~~ TTAGCTGGGCATGGTGGTGCCCGTCTGTGGTC
    28
    P201 700154 06/021201 7 27/021201 7
    CCAGCTACATGGGAGGCTGAGGTGGGAGGATTGCTTGAACCtTGGAGGTCAAGGCTGCAGTGAGCCATGA TTGCTCCACTGCACTCCAGCCTGGACAATAGAGGGAG.ACTGTGTCAAAAAAAAAAGAAAAGAAAGAAATA TAAAACATGAGGACAAATAGCTCCCTTGGCCACCAAAGTATTTCTGTACCCCGTGAAAGTTTTGATGTAG AAGTAGATGTGCCAGGCTAGAGGGATGGGCACGTGCAGGGGGTGTCAGTAGGAGCAGGCAGCCCTGGGAA S CAGGAATGAAATTAAAGAATTTGAGTCCTGGGTTTGTGTCTTAGGGTCAGTAACCATATTTeCTGteTeT TTTTCGGAATACATCCAGCTATTTTTTTCCTAGGCCCCACCTAGGAATAGATAGTCTTCATCTGTGAATG
    GATAACCTTGGGTATATTATCACTAGTGTTCTTTCTG, ~ CAAGAGGCAGCAGAGr.TTAACAGTTAAGCAT
    ATAAATCTTAGGGGTAGACTTCATGAGTTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGGTCTCG ATCATCCTGACCTCGTGATCCGCCCGCCTCGGCTTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCAC GCCCGGCCCTCATGAGTTTACATCAGCCACTTCCTAGCCAGTTAACCCTGAGCAGGTTACTTAACCTCTC TGTGCCTGCGCATTCTCCTCAGTAAAATGAAAAAAATCTGTGATTCCTTGTAATGATGTATAATTATATT TCCTAGAATGAAGAGTGGTGGAATTAGAGTCATGTAAAGTTTACAATTGTATTCAGTGCTTTTCATTGCT GAGAGTGATTGAACTTGATAATGACCACTTGAATCAAATGGAACCTACGTTCTATCTTGATCTTCCTCTC ATACTCCAGCTTTGCTCTGAGGTCAGAAAGCTGTGTGCTAAGCAGTTATATGTAGTTTTTGGTGGTTGTA 15 GATTTCGTTTTGGTTTGGTTGTTGGTCTTGATTTTTAACACTGGTGGAAAGGTAATTCAAGGTAAAAGTT GAAGCCAGTTTCACTTCTGAAATATTTTATTTTACTACTTTTGGTGAGACAGACAATTTGAGAAGCAAAA ACTTGCAGTTAGCCTTGAAAATTTCGGAGTACCTATTTCTAATTTAGGCATCAAAGGTAGAAGAAGACTT GTTTTTGGCCTCTGGGCACTTCTGCTCTACCTTAAGAGGCAAGACCCGCCAGGCACTGTGGCTCCCAGCA CTTTGGGAGGCCAAGGCGGGTGGATCACGAGGTCAAG, AGATCGAGACCATCCTGGCCAACGTGGTGAAAC CCTGTTTCTACTAAAAATACAAACGATTAGCTGGGCATGGTGGTGTGTGCCTGTAGTCCCAGCTAATCGG AC GAGGTGGAGGCAGGAGAATCACTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCTGAGATTGTGCCACTGC TCTAGCCTGGGTGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAGAGGCAAGACCAACAAAAATTCA AACTGGAATGATTGAGTCTGTCAGGGAATAATCTAGGAGCTCAAAGAAAAGGTAGGAATCAAGACTGAAC TGATAAAATGAACATGCCCTCAAATTCTCTAGAATTTGAGCTAGACTTTGGAGGGGAGGCTGGGTTGAGA 25 AGAGAGGTGGTAAGAGAGAGCAAAACTCACACTTACTGAAACCTCCTGCCATGCACTTGTGAATTTAACC CATGAGAAAAGCTGGTTAGAATATAGAAGGCATGTGC, AAAGGAGAAATAGAAGGTAAGACCAGGTATGTT GTTAAAGTGGGACCAGACAGCATTGGACCCAGAATGCCAGATTGAAATAATTTACAGCAGCACTGAAGGG ATTATACCATGGGTGAGAATATATCAGCTATATTTTCTGTGCCAGTTTATCCTTTGCAGTATCACTAGAG AAAAtAATTGTGAAAATCAATTAAGAAACTACTACAGTCATTTAGGTTTGAGATAATAGGAAATAAGTTG ATGGCAGTGGATCAAAGAGGGAAGACTGGTGTTTTTTGTTTGTTTGTTTGTTTGTTTTGAGACGGGGTCT TGCTCTGTTGCCCAGGGTGGAGTGCAATCTCGGCTCACTGCAACCTCCACTTCCCGGGTTTAAGCAATTC TCTTGCCGCAGCCTCCCGAGTAGCTGGGCTTACAGGCGCCCGCCACCACACCTGGCTAATTTTTGTATTT TTAGTAGAGTTGGGGTTTCATCATGTTGGCCAGGCTGGTCTTGAACTCCCAAAATGCTGGGATTACAGGC ATGAGCCACTGTGGCCTGGCCGAGAGATCTTTTAAAGGGGATCAATAGGATTTGGTGTCTGAGATATGGG 35 AAATGAATTATATGGGGTCTGAATATATGAATATATATATATATATATATATACACACACACACACATAT A TGTATTTTTTTTTTTTTTCTTTTTTGAGACAGAGTCTCGCTCTGTCACCCAGGCTGGAGTACAGTGGCA CAATCTCTGCTCACTGCAAGCTCTGCCTCCCAGGTTC, AAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGC CAGGACTACAGGTGCCCGCCACCACGCCTGGCtAATTTTTTGTATTTTAGTAGAGACAGGGTATCATTGT ATTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCACCTGCCTCGGCCTCCCAAAGTGCTGGGATT ACAGGTGTGAGCCACCGTGCCTGGTCCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGAG GCAGAGTCTTGCTCTAGACTGAAGTTGCAGTGACGCAGTGACGTGGTCACGGCTCACTACAGCCTCGACC TCCTGGGCTCAAGTGATTGTCTTGCCTCAGCTTCCCAAGTAGCTGGGACTACAGGCATGCACCCCATGCC TGGCTAATTTTAATTTTTTTGTGGAGATGGGGTCTCTCCCTTTATTGCCCAGGCTGGTCTAGGAACTTCT GGGCTTAAGTGATCCTCCTGTCTTGATCTCCCAGAGTGCTGGGATTACAGGCATGAGCCACTGCACCTGG 45 CCTATTTTTTGTAACCTGTTGGATATGAGATGATAGT.AAGACATACAAATAGAGATGTCGTACATTTCTT TGAACATATGCTTATTTATTTTTATTTTTTGCTTTGTTTTTTTGAGACAGACTCTCACTCTGTCACTCAG GCTGGAGGGCATTGGCATGATCTCAGCTCACTGCAAACTCTGCCTCCCAGTTCAAGCAATTCTCCTGCCT CAGCCTCCTGAGTAGCTGGTATTACAGGCATGCACCACCATGCCTGGCTAATTTTTGTATTTTTAAGTAG AGATGGGGTTTCGCCGTGTTGGCCAGGCTGGTCTCAAACTCCTGATTGCAAGCAATCCACCGCCTCGGCC TT CCAAAGTGCTGGGATTATAGGCATGAACCACTGGTGCTGGGCCGAACATATATTTGTTGAGGGCCAGC TATGTGCAGATTTTGAACAAGGCTCTGTAAATGCACGGGATCAAAACTGACCAACCTCCATGTCCATATG CAGCTTACAGTCTAGTGGGAGGATGACAAAAATCACACAGCTATAATTACAAATTTTAGTCAATGGAAAT TGCATGAATAAACACCTTGAGGTTAGGGTAGATGGGCCATGATACAACTGGGGAGCTAAAGGGCCAAAAT AGAGGAGTGAAGAGGGAGGAGGCTGGAGAGGTAGCAAGAGACTAGCTAAGAGGTGAAGGCTATGAAATCA
    55 GATTTGAATCAGCACCACTGAATTGGTAGTTGCCAGG.AAATGTGTGTGGTGTGAGTGAAAAGCCGAAATC TAGGCTGGGTGTGGTGGCTCATGCCTGTAATACGAGG, ACTTTAGGAGGCCAAGGCAGGAGGATCGCTTGA GTGCATGTGTTCCAGACCAGCCTGGGTAACATTGTGAGACCCTGTCTCTACAATAAATAAAAATAATTAG CTGGGTGTGGTGGCACCTGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGTGGGGGGATTGCTTGAGCCC AGGAGTTTGAGGCTGAATGAGCCGTGATTGTGATTGGGCCACTGCACCACTGCACTCCAGCCTGGGTGAC AGAGTGAGACCGTTCTCCAAAAAAAAAAAAGGGAGAGGGGAGAACAAATTGAGCTGGTGGCTATAAATCA CCCCATtTAAGAAGTtACTGGtGAAAGCAGTTTTTGTAGTTTTTAAAATCTATTTACTCAGCTAAAGATA
     02/02/2017 02/27/2017 P20 1700 154 06/02/2017 02/27/2017
    ATCTCATGATGTGTTTCAGATGAAAAAAAAGATATTGACCATGAGACAGTGGTTGAAGAACAGATCArTG
    GAGAGAACTCACCTCCTGATTATTCAGAATATATGACAGGAAAGAAACTTCCTCCTGGAGGAATACCTGG
    CATTGACCTCTCAGATCCCAAACAACTGGCAGAATTTGCTAGGTAAGTTATAAGAACTTTCerrreTrr1
    AATTATAGAAAGTGCTTGAGTCTTGCCAGCTATGTTTTCCTGTGCCTAAGTCAAAATGGTGGrTTGrATT
    5 CTTTCTCTAGGGAAATTCCTGAAAACAAAAGCAGTCAGTTTTATTTGTTTGTTTTTTGTTTCCTTTTTTT
    GAGATGGAGCCTCGCTCTGTTGCCCAGACTGGAGTGC.AGTGGTGCGATCTCGACTCACTGCAGCCTCTGC
    CTCCCGGGTTCAAGCAATTCTGACAGTCAGTTTTATTTTTAGTAGTAACTATGCTATTAATGTGAGTGGC
    AGCATTTTAGATCCTTGCAGTAAGATGAAGCAAATACCCTTAAATAGGCATTGCATTTGAGTAAATAAAA
    TTGCACGTAATTTGTTTTGGAATAGATATCTTCCACACTGCCTTATGGCTTCTTGGTACACATTTTATTT
    10 TTTATTTTTTTGAGACAAGAGTCTTGCTTGTTGCCCAGGCTGGAGTGCAGTGGTGCAATCTCAGCTCACT
    GCAACCTCCACCTCCCTGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACATGCGT
    GTGCCACCACGCCCAGCCAATTTTTGTGTGTTTAGTAGAGACAGGGTTTCATCCTGTTGGCCAGTCTGGT
    CTCGAACCAGTCCTGGCCTCAAGTGATCCGCCTGCCTTGGCCTCCCAAAGTGCTAGGATACAGGCATGAG
    CCACTGTGCCCAGCCTGTAGTACACATTTTAAATTTGGGTGTGTTGTATTGGTCTATGCTGCTGGAGACC
    fifteen TGGGTCTGTACTTGACTCATCCAGCTGACCACTGGAAAGGATTTCATAAATTCGTGTTTGTCTTGTCATC
    AGTTAAGCTCCTGGTATGTTAATTTAAGCTGGCATGACAGGAGGAGATCAAGTATTTATGGCTTGGACAT
    GGCGAGCAGTGGAGAAACTCTGAACCCATTGGTTGGGTGTGTTTGTTTTGCCTAAAGTGGGCTGGGTTCT
    TATAGAAGCCTACTTTTTGTGTTACTTTCTAGTACTGTTTGGCAGTTTGTACAAGGAGCTGAAAATACCA
    CAAAGATGTAAACTTTTAAGTTAAATGAACTAATAAACtCTGGAGCAGAAAGCCTAATGCCTTTCCATAC
    twenty CTGAGGTTGGTTGGAACCTGGAATAGCTCAGGAAATTTCGGCAAGGGCAGGATCAAGAAGAAAGAAATCC
    TTTTGATCTTGCACATTCTAAGAACAGAGTGGGTTGCTTTTTTTTTTTCTTTTGAGAGCAGAGTGGTAGA
    GGTGGAGACCGTTCAGTGTCCTGGCTTCCTTCTTGGCCCTGCCGAAAACAAGGTGGGCAGTTCCTGCCAC
    CTGGAGCTCGTTTCCCCACCTGTCAAATGGGCGTTAAGAATTCCATTGTGGTTTGATACATGGAGGTTCT
    CACTGTGTTGCCCAGGCTGGAGTGCAGTAGCATGATGATAGCTCACTACAGCCTCAAGCAATCCTCCTGC
    25 CACACCTCCCGAGTAGCTGGAACTACAGGTGTGTGCCACCATGCCTGGCTAATTTTTTTATTTTATTTTA
    TTTTATTTTTTGTAGAGATGGAGTCTCACTATGTTGCCCAGGCTGGCCTCAAACTCCTGGCCTCAAGTGA
    TCCTCCTGCCTCGGTCTCTCAAATTGTTGGGATGACAGGTATGAGCCACTGCACATGGCCCAAGAGTTTT
    TGTTTGTTAGTTTTGTTTTTTGAGATGGATTTTCACTCATCTCCCAGGCTGGAGTGCAGTGGTATGACCT
    CGGCTCACTGCAACCCCCACCTCCTGGGTTCAAGCGATTCTCCTACCTCAGCCCCCCAAGTAGCTGGGAT
    30 TACAGGTGCATGCCACCATGCCTGGCTGATTTTTGTGTTTTTAGTAGAGACGGAGTTTCACCATGTTGGC
    CAGCCTGGTCTCGAACTCCTGACCTAAAGTGATCCACCCACCTTGGCTTCCCAAAATGATTATAGACGTG
    AGCCACTGCACCCAGCTGGCCCTAGAGTTTTGATAGAGATGGAGGGTTTACTGGGAATTAGGGCGCTTCC
    AGGGAAATGACTGGAGTTGAGGTAAACTGGAACCAGATCATTAAGGGCGATGAATCCTAGGCCAGGCTGT
    GCAGACATTTCTCATATGCAGTGGGCAGCTACTGCAGACTTTGAGCTGGGGAGAAGGGGCTGTGTGAGTA
    35 GAGCGTTAACTTGGGAGAAGTCCTTTGCAGCTGTCTGTGCAGGCTAGATTCATACAGGCACAGGAAATGA
    GAGAGGTCAGTTGAACAAGAGTTAATGAAGGCCTGAGTTAGAATTGTGGCAGTCAGATAACAAAGGAATA
    AATGTGATAGAAATTACAAGAGACCAGCTGGGCTCTGTGGCTCAAGCCTGTAACCTCAACACTTTGGGAA
    GCTGAGGTGGGATCGCTTGAGGCCAGGAGTTTGAGACCAGCCTGGGCAAAATGATGAGACTCCATCTCTA
    AAAGCTCAGTTTGGTGATATGCAGCTAGCTTCTTGGGAGGCTGAGTGGGAGGATCGTTTGAACATGGGAG
    40 GTCAAAGCTGCAGTGAGCTCATGCCACTGCCCTCCAGCTTGGGTGACAGAGCAAAATTGTCTCTAAAAAA
    GAAGGAAACTGAACCAGGCATGGTGGCTCACGCCTGT.AATCCCAGCACTTTGGGAGGCTGAGGTGGGTGG
    ATCGCCTGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATAGTGAAACCCCATCTCTACTAAAATACAA
    AAACGTAGCTGGGCT-TGATGGCAGGCGCCTGTAATCCCAGCTACTAGGGAGGCTGAGGCAGGAGAATTGC
    TTGAACCCAGGAGGGGGAGGTTGCAGTGAGCTGAGATCACGTCATCGCACTCCAGCCTGGGCAACAAGAG
    Four. Five GGAAACTCCTTCCCCGCCTCCCCCACCAAAAAAAAAG.AGAAAGAAAAAAACTACAAAAGATAATGGAAAA
    GATTTAGAAACAGACTGTGTGCTCAGTTGGGGGCAGGGGGGAGGAGTAAGAGCATGCTTACCGTTTTGAT
    GCTTTTAGCCACTGCTCATTAGATGACTCCAGCCCCAACTTACTTCCTAAAAACCACCTCAAGTCCAACA
    TGGATGTTTTATTATTAACAGCATCTCAAACCAAATACTGACTACTCTGAGCCCCAAACCAGAACACTGA
    AGCTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAGTGGCACGA
    SW CCTCGGCTCACTGCAGCCTCTTCCACCCGGGTTCAAGTGATCTCATGCCTCAGCCTCCCAAGTAGCTGGG
    ATTACAGGCGCCTGCCACCGTACCCGGCTAATTTTTGTATTTTTCATAGAGACAAGGTTTCACCATCTTG
    GCCAGGCTGGTCTTGAACTCCTAACCTCATGATCCACCCGCCTCGGCCTCCCAAAGGGCTGGGATTACAG
    GTGTGAGCCACCGTGCCTGGCCTTTTGAAACTTTAAATTACAATTGTGATTCTCCCGTGTCCACATGGCT
    GGGGAGGCCTCAGAATCATGGCAGGAGGCAAAAGGCATTTCTTACTTGGTGGCGGCAAGAGAAAAATGAG
    55 GTAGAAGCAAAAGTGGAAACCCCTGATAAACCCATCTCAGATCTCGTGAGACTTACTATCATGAGAATAA
    CATGGGAAAGATTGGTCCCCATGATTCAGTTACCTCCCCCCGGGTCCCACCTACAACATGTGGGAATCCT
    GGGAGATAGAATTCAAGTTGAGATTTGGGTGAGGACGCAGCGAAACCATGTCAGAGGGTAAGAGGTCCCA
    GTGGCAGAGTAAAAGCATCATTCAGACAAATAGAAGGAGACTCAGGAGGTACGAGAGAGAGGAATAAAAC
    CTCTTTGGATTATACATTGTGAAGGAGTTGCAGTGGAGGGGCGGAGGGCTGAAGGI AAGGTTTTGAAGAG
    60 AGAGGGAATCTATGGGTGGTGAGCAGTAAAGCCATTAGGTGTTGGTGACCCTTGGCACTGCTCACCCTCC
    TTGGAGCATTGGAGCAGCTGATCTAAAAGTTTGTCCCCAGTCTTCTGAGCAGAAAATCCAGGGACACATG
    TTACTGTTGGGAAGGTAACATGGCTACCTTAGACCATACTCCTTAAGAAAGGGACCACCCACAGAGACTG GCACCCGGGTACCAGATAGCCATCCAAAACACCAGGGCTGAAGAACCCAAAAGTTTCAGTAAGATAGGCC ATCAAGTGGAAAGAATGATAGACCTGAATGAGACTTTTCTTCAGTAGCAGGGGATAATTAGAGACAATAA AACAATGCTTTGAGAGGAGAAATTACTGTAAACATAGAGTTATTGATCCAGCAAAGCTATCAAGTCAGAT S ACAAGGTCAAGAGATGACAATTTTAGAGACCCAAGGATTCATGTTGCATGCACCCTTTCTGAGGAGGTAA TTTGAAGATATAATACAGCGAAATGAGGATGAACATCAGAAGGAAGATTTCCTAGTGCACGACAACCAAG AGGACAGCTGTGTAGCAGGCTGGAAAATGTATTCCATTTAGAATAGAAAGTTGGCTGGCTGTAAGAATTT CTTAAGGAAAAGTCAGTTTCATTCAGTAGTGGGTAGAGTGAGTAAGAAGCTTGGTGGTATGGAAAAGGGT CTTCTGAAAAAGAAAAAGAAAAGGCAATCGGAAACTCCATGAAAAGGGGTGAGGGAGTAGGGCAAAGAAA GCTGTAGCCTGAAAAATGGGGCCTAATTTTGACAGTATTGAAAGAGAGAGCTTCTTTGACCTGACTCTGG GAAGATTTTCTCCTTGCAGCTCCTATGTTGTATACTTGTAGAGCATTAAGTGTGATCCTAGCACACTGTT GAGCCCCACAGTAAATAATATTTGCATAATCACAATATGAATGTAAATAGCATTTCTTTTATTAGCCTGT TAAAAGATTTCATTTGTGTATAGAAAAGAATGAAATCTTGCTCGCCTTGACAGTGTAAAAAGTAAAGTTA CAGAGTTTGGGCCCTGTGAGGTGGGTCAAGGGTTGGGCTCCCTCTCATCCACATTCTACATAGTAGcTTT 15 T TTTTTTTTAAATGGAGTCTTGCTCTGTCGCCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTGCAG CCTCTGCCTCCTGGGTTCAAGCAATTCTTCTGCCTCAGCCTTCCGAGTAGCTGGGACTACAGGTGCCCGC CACTACGCCCGGCTAATTTTTGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGGCCAAGATGGTCTCG ATCTCTCGACCTCGTGATCCACCCACCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCG CCCGGCCTCTAACATAGTAGCTCTTGAAGCTCATGTTCATCTGAACAAATATCTGTAATTGGACAGTGGT GTTGGTGAGAACTGTTTGCACCTAGGTGCCCTGTTTTGTTAGATCTTCACGTTTTGAGGATGTGTGTAAC AGTTGAGTGGGATTTTCTGGAGGCTCAGGAATTGATGCCCGGGACTCTGAGAGCAGAAGATACAGAGGCA CCAAGAGTTTTAAACTCTTGGGGGCCAGACCACATCTTCGCTAATTACGGCCCATAGAACTTGTCCTGCT GGCCTCCAGTCTTCGCCCCCAGCACTCACAGTGGCATTAGAGCCGCAGCTGTTGGCTTAAGGTAGAATGT AAGCTGCTTTTCTGTCTCCTCTGTGCACTGGGGTGATGGGGTGCTGGACTGGACTTGAAACTGTCCAAAG 25 AGTATTTCAGGCTCATGCTGCACTCAGCCTAGACTGCCTAGTAAATCAGTTACATACGCACATG ~ GCAAA ATATAAGCCATAATGACTTTAATTGGAAAGTCTGACATTGGGAAGGTGGACAGTCTATGGATGACTTTTT TTCCCGAAATGTCTGTTCAGTAAACATTTTGAAAAGTGGTCAGTGAGTGGTCCAGTGAGTTCTGTTTCCT GTAAAGTGACCACCTTTGTATGTGTTTCACTAAATAAAGTTAGACCTTTGAAAACCTTTGGTCTAGAAAG ATTA GAATGGTTAGGCCAATCCCAAATGGGTATTTTTGTTGCTGTTCACTACTTACCCTGAGACTTTCAC AATATTAATGTATAGATGAAATGTGATAATGGAGATGAAAATACCTCACAACTCTTCAGTATCATCACAT GAATATTAATATTTAAGAGATAATTCATTGAACAAACTTTATTTGTCTAAGGCAATATTATCTTTTCACA AAGAAGGGAGGTGAATCATGCTTGCAAATGACAGCCGTATTACGCAGGAACTTCTGTCCCAATAGCACAG ATCAGAGGATGGTTCTGGTCTGGAAGTCAGCTAACCTTGTTTTGTATGGAAGAGGCTTATTTTCATTAAA GAGTCACTTGTCCAAAAATGTTTCATCTCCCTTTCTGGGATATTTGCAGATCTGTTGGTTAGAACTTCGG 35 TTTACACAATGCTCTGCTGATACTGTGCCAGAAGTTTGGGTTCTGAAGTCCACAATCTTTACAACGTTCT TCCCACATATGCCAGAATGAGCTATTATAAATCGGGCAGTCATGTCATTGGGAGTGTATTCTCCCTCTCT ATTTGACAGGAAACTTGAGTTGTTGGGTGTCTGACTCCTCCATATAACATGTACTGCGTTGAGGGGTGTG TTCCTCCTAGTGCCATGCTTTCTCCAGTTTCTCAGTCCTTTATCGAAGGGTGGTTAAGAATGAACTCATT TTTAGCTAGACCCATTTCACACATAGCCAATGGTAATAAGAGCTATCATTTCCCAGGTGCTTAACATACA GCTCTTAAGTAGGTAGCATTATACCTTTTTACAGAATAAGAAACCGAGGCCGAGAAGTTACAGTTTGCTC AAGGTCATGCAGCTAGTAGAAGTCACCTCCAAGTCACACATCCCATACTGCCTCATTCATAATGTCTATG CTGTTGTCATCATTGTCTCCAGGTTCAAAAGGACTCTCTGGGTTCAAAAGTGTCCGTTGGTGAATGGAAG ACTTAAA TCTTAGTTACTTCTGAATAGTTGAGGAGGGTGAGAAGATGTTCTCAGTGGTAGTATATGATTT TTACTGTTTACTCGCAAAATGAAACACTGTTTTAACCTAAGTATATTTTTGAAAAATGACTTTTTTCATT 45 GTTGGCATTCAAATAAAATCTTAAAAGTACCAACGTATACTGCTGCATTTGCCACTCTGTGTGAAGGAGG ATGATTTGCCAAGGAGCTTAAGAGTGGCTGCATTTAAATCTTCCACTTACTCCTTAAGTCCAAGGGTATT TGTCCAAATGCATTTTAAACTCTCATTCTAAAATTAAATATATTTCTAAATACTAGCACACATTTTGAAA ATGACATTTTAAGAAAAAATAATACCACTTATAATAACAAACCAAGAGTTGGGTAAATGGTACCTGAGAC TGTTCTCTTTACTTTGGGGGCGTATTTGAAAATTTCCATGATAAAATATCTTTTAAAAAATTAAGTGGCC AGGCGCGGTGGCTCATGCCTGTAATCCCAGCACTTTGCGAGGCTGAGGCAGGTGGATCACGAGGTCAGGA GATCGAGACCATCCTGGCTAGCACGGTAAAACCCCGTCTCTACTAAAAATACAAAAAAATTAGCCTGGTG TGGTGGCAGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATGGCCTGAACCTGGGAGGC GGAGCTTGCAGTGAGCCAAGATTGCACCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCTGTCTCAA
    AAAAAA ~~~ VVUVUV ~~ AAATTAAGTTACTAGGAATAACTCTAGCAAAATGTATGGGAGGCCGG
    55 GTGCCGTGGCTCATGCCTGTAATCCTAGCACTTTGGGAGGCCGAGGTAGGTGGATCACCTGAGGTCAAGA
    GTTCCAGACCAGCCTGACCAACATGGTGAAACCCCGTCTCTACTAAAAATAC ~
    TAGCCAGGCATGGTGGCACGCGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAA CCCAGGAGGCGGAGGTTGCAGTGAGCCAAGATCAAACCACTTCACTCCAGCCTGGGCAAAAGAGTGAAAC TCCATCTCAAAAAAGAAAAGAAAAGAAAATGTATGAGAGAGGACCTTACTAAGCCATGTTTACTAAGATT TAATTTGCATATGGTAAAGTCTACCCCCCTTTTAGGTATGTATGTACTATTCTGTGAATTTGGACAAACA GTCATGTAGCCATCTTCACACTCAACACAGAATGTTTCCAAGGAGTCCTAGGCAGGTGGATTGCTTGAGC
    .._---
     06/0212017 27/0212017 06/0212017 27/0212017
    TCAGGAGTTGAGAATAGTCTGGGCAACATGGTGAAACCACATCTCTACAAAAACAAAATAAAACAATGTT
    TCAGTAGAATTTTCAAAGAATAAGTGTTCACTCTTAATTGGGAACATTAAAACTT ~ TAG
    GCTGGGCGCGGTGGCTCACGCCTGTAATCCCCGCACTTTGGGAGGCCAAGGCAGGCGGATCACGAGGTCA
    GGAGATCGAGACCATCCTGGCTAACACGGTAAGAAACCCCGTCTCTGCTAAAAATACAAAATATTAGCCG
    S GGCATAGTGGCGGACACCTGTAGTCCCAACTACTCGGGAGGCTGAGGCAGGAGAATGGCATGAACCTGGG
    AGGCAGAGCTTGCAGTGAGCTGAGATTGCGCCACTGC, ACTCCAGCCTGGGCGACAGAGCGAGACTCTGCC
    TCAAAAAATAAATAAATTAATTAAATTAAATTAAAAATAAATAATGTTTGAGTGGAATAGAGACCTTTCA
    TATCAATCACTGTTATTTTCCAAACTGATGTAACCTC, AAGGTGGCGCAGTGGCACCAAACTTTTGTTCTA
    CCTGATTTTGTGAACTCAGGCCAGAACTCCAGGATTT.ATTTGTATTTAAGATAAGAAAGGCTAGTTCAGA
    10 GTCCTGTTTACAACAGGCACTTAAAAAAACTTGCTAACTGGTATTACAGGTTGAGCATCCTTTATTCAAA
    ATGCTTAGGAACAGTAGCACTTTGGATTTTGGGGTTTTGCATATGCATAATGAGTTATCTTAAGGCTAGG
    ACTCGGGTTTAAATACGAAATTTGAAATTCATTTGTGTTTCATTACACCTTATACACATAGCCTGAAGGC
    AATTCTGTACTTTTTTTTTTTTGAAACAAGGTCTCGTTCTGTCACCGAAGTTGGATCGCAGCCTCAAATT
    CCTGGGCTGAAAGGATTCTACCTCCTCAGCCTCCCAAGTAGCTGGGACTGTAGGCATGTGCCACCACGCC
    fifteen TGGCTAACTTTTTATTTTTTTTAGAGACGGGGTGTTGCTTTCTTGCCCAGGCTGTTATACAGTATTTTAA
    ATAATTTTGTACACGAAACAAAGTTTTCACTGTGACCCTATCACACGAGTGCATCATGTTAGCACTCAGA
    TAGTTTCAGATTTTGGATTTTGGATGTTAGGGATGCTCAATCTATAATCCATTTACGAGAAAGGACGAAT
    ATCTACAGGGTTGGGAAGGGGGAGGACACTGAATTGAAAATGTTCTCGTGGGTGGGCGGATCACGAGGTC
    AGGAGATCAAGACCATCCTGGCTAACATGGTGAAACCCCATCTCTACAAAAATACAAAAAAATTAGCCGG
    twenty GCATGGTGGCGGGCGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGACAGGAGAATGGCATGAACCTGGGA
    GGCGGAGCTTGCAGTGAGCCGAGATAGCGCCACTGCACTCCAGCCTGGGTGACAGAGTGAGACTCTGTCT
    CAAAAAAAAAAAAAAGAAAGAAAAGGAAAAGAGAAAATGTTCTCGTAGAATTTAAAATAACAAATATGTA
    TAGTATCGGACCAACTCCATGGAGATAAGTGATAGTT.ACCAGATCTGTTGTTCCTCATATACTCCTAGGG
    CTGGAAAGTTTAACCAATGATTTCATGTGTTCAGGTT.ACAGATCAGGTCATCCTTTTCCCAGATTATAAG
    25 TGAAGAAGGACATTGTTAGGGCCTTCACAAACTTAAAAGATCACAGAATTAATTTTTTTTTAAGGAGTAT
    TTTTTTTGTAACAAAAGAGTGGTGGTAAATTAGCTCTTGCTTTTAAGGATTCACTTAGGTGGGCTTTTTT
    TTTTTCGACTTTGACTTTTTAAAAAATTTCAATAGCTTTTGGTGTATAAGTGGTTTTTGGTTACATGGAT
    GAATTCTATATTGATGAATTCTGAGGTCCGAGTGCACCCCTCGCCAGAGCAGTGTACACTGTATCCGGTA
    TGTAGTCTTTTATCCCACACCATTCTTTCCCTACTAAGTCCCCAAAGTCCATTATATCACTTGTATCCTC
    ~ O ATAGCTTAGTTCCCACTTATAAGTGAGGACATACCGCGTTTGATTTTCCATTCCTGAGTTACTTCACTCA
    GAGTAGTGGCCCCCAGCTAGTTTTTTGTTGTTTTTTTTTTGTTTTTTTTTTTTTGAGACGGTATTTCCTT
    CTTGCCGCCTAGGCTGGAGTGCAGTGGCACGATCCCGGCTCACGGCAACCTCCGCCTCTCTGGCTGAAGC
    AATTCTCCTCCCTCAGCCTCCTCCTAAGTAGCTGGGATTACAGGCATCTGCCACCACACCCGGCTAATTT
    TTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAACATAGTGTCAAACTCCTGGCCTCAAGTGA
    35 TCCATCTACCTCGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTACGCCCAGCTATGGTTTTTT
    AAATTATTATTTTATTTATTTTTTTAAATCCAATACAGAGTTTCGTTCTGTCACTTAGGCTGGAATGCAG
    TGACACAGTCATGGCTTACTGCAGCCTTGACCTTCTGGGCTCAAGTGATCTTACTGCCTTCGGCTTCCCG
    AGTGGTTGGGACTATCGACACACACCACCATGCCTGGCTGATTTTGGGGGATTTTTTGTTTGTTTGTTCA
    TTTGTTTTTGATAGAGACAGGGTTTTGTCCATGTTGCCCAGGCTTAATTTTTTTATTTTTTGTAGAGACA
    40 GAGTCTCACTGTGTTTCCCAGGCTGGTCTTGAACTCCTGGGCTCAAGCAATCCTCCCAGTTTGGCCTCCC
    AAAGTGCTGGGATTACAGGCGTGACCCACCACACCCGGCTGATTTATTTATTTATTAAATATGAACCATC
    ATGGAGGGGTGTGAGAGGGGTAAAACTAGGGACAAAAAGAAAATCTGTTTCCGAGATTTAAATTTATTCT
    CTAGTTTTTTTAGGMCGCTTMTTTCTCACCCCCCT, ACCACACACAGATCTATAGGGAATATTTGGAGA
    GAAAGTTTTGACAAAAGTCCAAAGCTTGATTCAGTGGAGAAGCAGGTAGGTAAGTTACCAAACCAGGAGG
    Four. Five CTCTCTGGACAGTAATTTCACCTGGGGCTCTGCCAGACGGTTACCACATTACCAGGACTCTTGGTACAAA
    TGAGAACTTCCAAGCCCCCATCCAGAAGTAGGGTAACAGTATGAGAGTATGTGGTGTTCCCATCTGTCCA
    CCAGGCCCTAGGAGCTCTCTCCCCAAAGTAACTGGTGCTAATGGTTTGTCGTGTGTCCTTCCAGAGATAT
    TTGATATTTAATATCTATAAATCTTGATTTAAAAGGACTCTTCTGTACCCTTTAAAATGATATATTTTAA
    AGATCATTCGTGTCAGTACTAATTGGACCCATTTCGTGTATAGAGATGGATATTTTAGGACACTCCTGTG
    SW TTCACCGCTTCTTTTTCATAAGGCACCTTCATCATCGCAGCAGCTGCCTTTGGAAATCTTCCCTTGCCTC
    TCACAAGGAGATTCATCTGATTGTCTGAGTTGATTTTTTGTGTTTTTTTAAAGGGTTTGGAGTTAAGAAC
    AGGTATGrGAATCACACAGATCATAGTGCCATTCTAAGCTGCACCAGCTTATTAGCTATGAGACTGTAGG
    CGGCAACTCGATGTTTCTAGCCTGAGCATCCTTATCTGTAAGATGGGAAGAGTGCTTCACAGTCATTGGT
    ACTTAGTGAATGCAAGT'l'GTCTCATCC'fTTCTGCTTC.ATGGAAATGTATCATAATTTTAGTTTGGGTACC
    55 TATAAGGAAAGCATTTTAGGGACTATATGATTTATGCCAGTTGAGTCTACCCACTCCTACAAATCTGTCT
    GTCTCTCTTTTTCTTTTGATAAGAATGAAGCCAAGAAAAATTAAAGAAGATGATGCTCCAAGAACAATAG
    CTTGCCCTCATAAAGTAAGTAATGCTAGAGGGAGAGTGTTCATTAAACTGTTGATGAAGTTTTAGAGAGT
    TATAAGAAATTTGCACTTTTGTTTCTGCTTGTGATAATTTAAATGAAGCAAGGAATTAGAATCTTCGGTT
    TCTAAATCCAAGAGATGAAGTTTTGTTATTTAGTAAAGGCAACTTTTTAAATACTGTTAAATGGGATGAC
    60 TGCAGGGAGCCGTTGCTTGATTACTGTGTGTTGACTCTACTGTTAGCGTTTCATTTCTTTTTGGATTATA
    TTMCAAGTCCGTCTTAAGATCCTGGGTTGTGTC1CACCTATTGACCTTTATGGGAATGTAGGCTTGTAA
    CTGAAATTTGAAAATTATTtTGGATGTTGCTAAAATAAGCCAAATAAAGACTTAATAATTGTGTGTGATT AGAGAGGCACTTAATTGTTTTCAAATGTAGTTAGTCTTTAAAATAAAAGGATAACCTGAAAAGTACTTTT GAGATATTTGTTGGGGGCTATTTTCAAACTTAATTGGATATGAGACTAACAGAAAAATCAAGTATCACAT AGGTACAGAGAACAAAGTAGGAGAGATTGAGTGGAATAAAGAATGTTAAAAATGAACAATCGGAGCAGGC
    5 TACATTTAGTTTCACGGTCTCTTCCCTGTAATGACTGTGCACTAGCAAGTCCTTCCTGTTCTTTGTCAGC GTTCTTGATGTGGCGATTTGAAATGTAAACGTATCTCCATTAGGGCCGAAATAACAAGCATTTGAAAACA r, TTAGGCTGGAAGTGCCTATGCCATCCCTTTAGCTTCTCTGAAGTTTTCTTTATAATGGACACATAATAC TTGTACATATTTATGGGGTACCGTGTGATGTTTCAATGCATGTATACATTGTGTACCAGTTAAATTTTTT AAATTTCCATTTTCTAAATGGAAATTCAACCTAGGGGTAAAGGCGGAAAAACTGGGGCTTCCCTCTCCCT TCTGCCTGTGAGCTGTCTAGTGACTTGTGCTGTCCTTCTGTCATGCTGTCTTATCATTTCCCCATTCACT I CCATTACTGGGCAACAACTGCAGAAGAAATTGTTGAGGACATGCTTCTCGAGCGCCACACATCCATCTGC CTTTCCCGCCCTGCACTGGTATCAATTTGCCTGCTTCTTCCTTTTCCTAGTGTCTCACAAAATAAATTTC TTCTGTAGTTGCCATTCCTTGGAGACCCTCTTGCTTCTTTCTCTGTCAAGAAGGGGAAAGAGGCAAAGAG AGAAGAGGGAACAGTAGGCGATGGAGAATGACAGTCCATTGTTGCAGCACAGAATATGGGGCCACCAAAG
    15 GTAGTGGTGGACTACCGTCATTTTAACTCCATTTGCTTAGCAGTGCTTCTGTTACTTCATTTTGGGGGAC ATAGTCGGGTGAGGTGGCTGTGGTAGAGCTGGAAGCCAGGGTGTTGGGTTCTATTCCCAGTTTGGCTACT ACTAGTAGTGTGACCTTGGGCAAGTCTACTTAAAGACATCTCTAAGCCTCAGTTTCCTCATCTGTAAAAC TAGGGTTTGCATTGAATGATGACTTTTTCAGCTGTCTGCTTTTTGTCTTAAATGATATTAATGTTCTACC GTAATACTAAGTAAAATTAAAATGGGGGGTTGGGGAGGTGGTTTTGTTTTAATATGTCAGTAAAGGCTGT TAAATGGTTGAATCCTTTCTAACAGTTTGCAATGTGAACTTCTAAGCTGCTTTCTCTGTTTTAAGGGCTG CACAAAGATGTTCAGGGATAACTCGGCCATGAGAAAACATCTGCACACCCACGGTCCCAGAGTCCACGTC TGTGCAGAATGTGGCAAAGCTTTTGTTGAGAGTTCAAAACTAAAACGACACCAACTGGTTCATACTGGAG AGAAGCCCTTTCAGGTAGAGCCAGTTCCCTCTCTTCCCCACACTGCCTTGCCTGTCTGAACACTGCAAGT GTAGGTGGTGTGGTGATGAGGCAGGAGGCGCCAGCCCAGAGACTCAGGGTCTTATTACTCCTTGGTGAGA
    25 AACAAAACTTCTCCTGGGGAGTCGCTTAGAAGGGTTGCCGGGGCTCTGGACATCCTTGTCTATACTCTGT GGTACTGGGTACGCAATGTATTGGTGTTGATGGAGTACACTTTATGGCAGGAGGAGAACAGGATTGAAGA GCATACCTAAAACTCAATTTCTACTTCATTCATTACAGGATTGAAGTAATTTATTTTTGGTAGTTAATAG TATAATTACTAACTGATAAAGTTTCTAGAGTGTAAGTATAGGTAATACTTTAGCCCATAAATAAGTGAAA GAACTAGCAGTGCAGCTAGTAAATCTAACGTGGTTCTTTTTTGACAACTGACACCAGAACCCTTAATCAT TTGTATTTTACTGTTGGAAATGTTTACAGCAGCACTAGGACTCTAGCCTGCATTTAGGAAGACTTGCCAT TTTGCCAAGTGTTTTAAACAGTTCGTGAGGGCTCTCCTTGGGTTTTCGGGGTTGTCCAACCTGCCTGCTG ATTCTAGACTATATCCACAAAGTTCACCCAGGGCAGGAATGAAAAGTGTTTGGTAAAATAAATAGGCTGT TCTCTAGCAAAGCAGTGAGCTCCTGACTCCCAGGGTCTGGTCAGAGTTGCTGAGTGGGTTGATCTCTGGT CTTTCCTTGACAGTGCACGTTCGAAGGCTGTGGGAAACGCTTTTCACTGGACTTCAATTTGCGCACACAT
    35 GTGCGAATCCATACCGGAGACAGGCCCTATGTGTGCCCCTTCGATGGTTGTAATAAGAAGTTTGCTCAGT CAACTAACCTGAAATCTCACATCTTAACACATGCTAAGGCCAAAAACAACCAGTGAAAAGAAGAGAGAAG ACCCTTCTCGACCACGGGAAGCATCTTCCAGAAGTGTGATTGGGAATAAATATGCCTCTCCTTTGTATAT TATTTCTAGGAAGAATTTTAAAAATGAATCCTACACACCTAAGGGACATGTTTTGATAAAGTAGTAAAAA TT ~ CTTTACTAAGATGACATTGCTAAGATGCTCTATCTTGCTCTGTAATCTcGTTTCAA
    AAACACAGTGTTTTTGTAAAGTGTGGTCCCAACAGGAGGACAATTCATGAACTTCGCATCAAAAGACAAT TCTTTATACAACAGTGCTAAAAATGGGACTTCTTTTCACATTCTTATAAATATGAAGCTCACCTGTTGCT TACAATTTTTTTAATTTTGTATTTTCCAAGTGTGCATATTGTACACTTTTTTGGGGATATGCTTAGTAAT GCTACGTGTGATTTTTCTGGAGGTTGATAACTTTGCTTGCAGTAGATTTTCTTTAAAAGAATGGGCAGTT ACATGCATACTTCAAAAGTATTTTCCTGTAAAAAAAAAAAAGTTATATAGGTTTTGTTTGCTATCTTAAT 4S TTTGGTTGTATTCTTTGATGTTAACACATTTTGTATAATTGTATCGTATAGCTGTATTGAATCATGTAGT ATCAAATATTAGATGTGATTTAATAGTGTTAATCAATTTAAACCCATTTTAGTCACTTTTTTTTTCCAAA AAAATACTGCCAGATGCTGATGTTCAGTGTAATTTCTTTGCCTGTTCAGTTACAGAAAGTGGTGCTCAGT TGTAGAATGTATTGTACCTTTTAACACCTGATGTGTACATCCCATGTAACAGAAAGGGCAACAATAAAAT AGCAATCCTAAAGCAAGAATATGGCAGAACAAGATCTGTAAGCACAGTCTTATTTTCTTTTGTTGTCCAG AATACTTATAATTCTTGAGCCTCCCAGAAATTGGAAGCTAAATAAAGCAACTCAAGTTTCCTTTATTTTG CACTCAATTACAGTGATTATTGATGAAAGCGATGCATGGATATTTTAATACTTCCTACATGTCCTGACTT CGC CTGAAAGAGAGTAGGTAACAGGCATCCCGAGTTCAGGAACTACCTCAGAACACCCCAGGCCAGGTTGGTC ATAGGCTGTGATTTTAGCCCCCGGCAAGTGTGAGTGAAGCATCTGTACCACCGCGCAGGCTGAGCGCCTG AGGGTAAGGTGCCACCTGGCAGTGGGGCACACAGAGGGAAGACCAGGCCTGTCCATCAGCCGGCTGC
    SS CTTCAGAGGCAGCTCCAGCAGGACCTTGGCTTGTCTGACAGGAAATGCTTGTGGTCGTTGGTTATTTGGT TTGAGAGCCCTTGTTCCTCCATCTAGTGGAGTCCTTATTAAATGCTAGCAATGTGGATATTGAGTGCCAGGGTTTCC
    权利要求:
    Claims (1)
    [1]
    1-Use of a composition comprising a compound capable of reducing the expression of the Yin yang l (Yyl) gene, in cardiac cells of a human or animal subject with respect to the expression observed in the absence of the compound in dic have cells, for develop a drug that inhibits the expression of the gene that encodes the soluble protein suppression of tumorigenicity-2 (ST2s) to promote
    10 cardiac recovery after myocardial infarction (1M).
    2-The use according to claim 1, wherein said medicament is used to reduce the size of myocardial infarction and / or prevent adverse cardiac remodeling after 1M.
    3-The use of claim 1 to 2 wherein the composition is administered locally in the heart cells
    The use of claim I to 2 wherein the composition is administered from intravenous fOffila
    5-A screening method to identify compounds capable of reducing the expression of the gene encoding the pro (eina ST2s comprising:
    a) Obtain a library of accounts
    b) Determine whether an administration to cardiac cells of one or more of the compounds of the
    Step a) results in a reduction in the intracellular levels of the Yyl transcription factor and / or the expression of the gene encoding the Yyl transcription factor with respect to cardiac cells to which no sc is administered / n said computer / s / s
    c) Select those compounds that promote the reduction described in step b)
    6-The method of claim 5, which also includes, in step b) administration of said compound / s in comparison with cardiac cells to admin istra / n results in:
    - an increase in histone deacetylase 4 (HDAC4) protein levels of the gene encoding the H.DAC4 protein and / or
    - a decrease in phosphorylation levels of the HDAC4 protein and / or
    30-an increase in the amount of Yy l protein that is bound to HDAC4 and / or
    determine if the ones that are not
    or of the expression
    - an increase in the levels of Interleukin 33 (IL33) or the expression of the gene encoding the ILJ3 protein, and / or

    5 -a decrease of the ST2s dc levels or the ccnprcsion of the gene encoding the ST2s protein, and
    c) select those computers that produce one or more dc the signed documents cn b)
    10 7-A method where, in addition to the steps described in any of claims 5 to 6, a step d) is inelucted in which myocardial infarction models are evaluated in animals if the administration of the computer / s evaluated / s in steps a) and b), or selected in step e), reduce the size of the myocardial infarction.
    8 - The method of using the claims 5 to 7 where a range of the amount of the compound / s administered / s which causes the effects described in claims 10 to 12, preferably the described effect, is more intense in claim 10.
    lS 9-The method of any of claims 5 to 8, wherein the compound is formed by an active ingredient and an acceptable pharmaceutical vehicle.
    10-The method of claim 9 wherein a range of amount of active principle and vehicle is determined which causes the effects of claims 10 to 12 more intensely than the effect of claim 10, preferably the effect described in claim 10.
    twenty 11 The method of any one of claims 12 through which the mode of administration of the compound that most intimately causes any of the effects of claims 5 to 7 is determined, preferably the effect described in claim 5.
    12-The method of any of claims 5 a formed by biguanides. 11 where the compound library is
    2S 13-The method of any one of claims 5 to 12 wherein the biguanide Metfonnine is used as a positive control.
    14-A product containing the information on the results obtained by the screening method described in any of claims 5 to 13.
    30 15-A method of obtaining one or more compounds comprising the identification of the compound / s according to any of claims 5 to 13 and their isolation.
    类似技术:
    公开号 | 公开日 | 专利标题
    JP6793649B2|2020-12-02|RNA agent for P21 gene regulation
    Luo et al.2013|MicroRNA-26 governs profibrillatory inward-rectifier potassium current changes in atrial fibrillation
    Tian et al.2018|MiR-320 regulates cardiomyocyte apoptosis induced by ischemia–reperfusion injury by targeting AKIP1
    ES2735334T3|2019-12-18|Signaling of hippo and dystrophin complex in cardiomyocyte renewal
    Wei et al.2014|NF-κB-mediated miR-30b regulation in cardiomyocytes cell death by targeting Bcl-2
    JP2017511694A|2017-04-27|Compositions and methods for using microRNA inhibitors
    Gong et al.2019|Enhanced ROBO4 is mediated by up‐regulation of HIF‐1α/SP1 or reduction in miR‐125b‐5p/miR‐146a‐5p in diabetic retinopathy
    Huang et al.2017|MicroRNA-133b negatively regulates zebrafish single Mauthner-cell axon regeneration through targeting TPPP3 in vivo
    ES2393054T3|2012-12-18|Means and methods to counteract, delay and / or prevent heart disease
    ES2637032A1|2017-10-10|Composition to promote cardiac recovery after myocardial infarction | |
    ES2855577T3|2021-09-23|Functional nucleic acid molecule and its use
    AU2009289239B2|2015-11-12|Treatment of scleroderma
    US9493772B2|2016-11-15|Method for reducing expression of downregulated in renal cell carcinoma in malignant gliomas
    US10308936B2|2019-06-04|miR-96-5p inhibitor and a screening method for the inhibitor
    KR101736025B1|2017-05-15|Reverse-aging induced method using a circulating aging marker
    Ahn et al.2012|Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin
    US11225660B2|2022-01-18|Methods and compositions for managing vascular conditions using miR-483 mimics and HIF1alpha pathway inhibitors
    AU2015262889A1|2016-12-01|Small interfering RNA | for the therapy of type 2 | autosomal dominant osteopetrosis caused by CLCN7 | gene mutation
    TWI585204B|2017-06-01|Short interfering rna for treating cancer
    JP6751185B2|2020-09-02|RNA interference agents for regulating the GST-π gene
    Zhu2022|RNA pull-down-confocal nanoscanning |, a novel method for studying RNA/protein interactions in cell extracts that detected potential drugs for Parkinson’s disease targeting RNA/HuR complexes
    WO2012020839A1|2012-02-16|Pharmaceutical composition for cancer therapy
    Traeger et al.2022|UBA6 and NDFIP1 regulate the degradation of ferroportin
    CN113930423A|2022-01-14|SaRNA for protecting myocardial cells from stress injury and application thereof
    Allen2016|Post-transcriptional regulation of BDNF and the Cdk5 pathway by the neuronal RNA binding protein HuD
    同族专利:
    公开号 | 公开日
    ES2637032B1|2018-07-17|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP3922720A1|2020-06-09|2021-12-15|Universidad de Murcia|Therapy to prevent adverse cardiac remodeling following an acute myocardial infarction|
    法律状态:
    2018-07-17| FG2A| Definitive protection|Ref document number: 2637032 Country of ref document: ES Kind code of ref document: B1 Effective date: 20180717 |
    优先权:
    申请号 | 申请日 | 专利标题
    ES201700154A|ES2637032B1|2017-02-06|2017-02-06|Composition to promote cardiac recovery after myocardial infarction |ES201700154A| ES2637032B1|2017-02-06|2017-02-06|Composition to promote cardiac recovery after myocardial infarction |
    [返回顶部]