Bests, Sandra, http://dharmacon.gelifesciences.com/uploadedFiles/Resources/effective-sirna-controls-technote.pdf. For instance, off-targeting can be mitigated in part by creating appropriate control trials and algorithms that generate off-target-free siRNAs. This reaction led to death in some cases and raises significant concerns about siRNA delivery when PEGylated oligonucleotides are involved. Occasionally, cleavage fails due to mismatches between the siRNA and regions of the target mRNA close to the cleavage site. The entire process of gene silencing through the siRNA is called a. doi:10.1016/bs.pmbts.2016.09.011. Your email address will not be published. Thermo Fisher Scientific put same nucleotides but in a completely different order, generate several seq and blast them to ensure there are no obvious off target effects. Numerous significant studies have been conducted on the application of siRNA to various fields of medical study. Within this RISC complex, the dsRNAs undergo strand separation. The structure of some RNAs are shown here, The present article explains what siRNA is and how important it is! Because of its larger size, it is very difficult to pass it through the cell membrane, although nano-vectors can transfer the siRNA effectively. GES-1 and HSFE cells were transfected with ERK, JNK, p38, and negative control (Neg) siRNA 10 g with 8 l RNAi MAX Lipofectamine. [52], This method has been used to deliver siRNA targeting VEGF into the xenografted tumors in nude mice, which resulted in a significant suppression of tumor growth.[53]. The green fluorescence identifies transfected cells (with IPO8 siRNA or scrambled oligonucleotide sequences). For example, lentivirus-mediated RNA interferences give good results, are more effective and accurate but the use of retrovirus-like lentivirus is indeed dangerous. There are more nonspecific effects of siRNA. By using the viral vector-based on nonviral-vector-based artificial methods of gene transfer, the siRNA can be inserted into the cell. Also, the table shows the concentration of siRNA. [36] siRNA oligos in vivo are vulnerable to degradation by plasma and tissue endonucleases and exonucleases[38] and have shown only mild effectiveness in localized delivery sites, such as the human eye. The siRNA-mediated method is used in the gene knockout and gene knockdown methods for suppressing gene expression. 2012, 45, 7, 10141025 Publication Date:March 13, 2012 https://doi.org/10.1021/ar2002254, Tuschl, T. (2001). (Spaces in the sequence will be ignored) In some cases, the endonucleolytic cleavage of the phosphodiester backbone may be suppressed by mismatches of siRNA and target mRNA near the cleaving site. Dana H, Chalbatani GM, Mahmoodzadeh H, et al. Int J Biomed Sci. [11] Typical transcription cassettes use an RNA polymerase III promoter (e.g., U6 or H1) to direct the transcription of small nuclear RNAs (snRNAs) (U6 is involved in RNA splicing; H1 is the RNase component of human RNase P). This may be overcome by creating an expression vector for the siRNA. For more detail, on RNA you can read our article on RNA: tRNA, rRNA and mRNA are three different types of it present in a cell. [56], A decade after the discovery of RNAi mechanism in 1993, the pharmaceutical sector heavily invested in the research and development of siRNA therapy. One strand of the double-strand is known as the guided strand and the other as the passenger strand. Before Onpattro was released, the treatment options for hATTR were limited. These specific viral vectors have been synthesised to effectively facilitate the transfection of siRNA into cells that is not viable on its own. Ideally, it cant be useful in human trials. Therefore, any target molecule that needs to be treated with high affinity and specificity can be selected if the right nucleotide sequence is available. Apart from all these, one of the unique characters of the siRNA is the presence of the 3 OH dinucleotide overhang. siRNAs are integrated into the RNA Induced Silencing Complex (RISC). The siRNA or small interfering RNA is a 22 to 25 base pair long smaller molecules of dsRNA having a dinucleotide overhang at the 3 end, interfere in the protein synthesis by blocking the translation.. [23] Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (in effect, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. A detailed database of all such chemical modifications is manually curated as siRNAmod in scientific literature. Although it is not a genetic material in eukaryotes, some of the viruses known as retroviruses have RNA as their genetic material. I am reporting for: Further, the effect of the present therapy is fewer and tissue-specific thus it is restricted to the localized sites. The dsRNA is not found in our cells because our genetic material is DNA not RNA. Please leave this field empty. The tRNA is a transfer RNA that helps in transferring the information for protein synthesis, the rRNA is a ribosomal RNA located in the ribosome, reads the order of amino acids and the mRNA is a messenger RNA. The area isnt much explored and that is why problems are many and results are less. 3.siGENOME Lamin A/C Control siRNA Negative control Function Distinguishes sequence-specific silencing from non-specific effects Features 1. One of these being off-targeting. (2016). Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 (normally 21) base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. why they don't genetically modify the T cells directly to make them produce receptors and antibodies specific for these antigens? scramble control for every gene-specific siRNA that is used, therefore a universal Non-targeting control is more practical. The complex silences certain gene expression by cleaving the mRNA molecules coding the target genes. It is theorized that the resulting siRNA transcript is then processed by Dicer. Givlaari is an siRNA drug that downregulates the expression of aminolevulinic acid synthase 1 (ALAS1), a liver enzyme involved in an early step in heme production. For more detail, on RNA you can read our article on RNA: RNA: Structure and Function. RNA was isolated using TriZol (Invitrogen, Thermo Scientific) as per the manufacturer's instructions. [44], In a phase 1 clinical trial, 41 patients with advanced cancer metastasised to liver were administered RNAi delivered through lipid nanoparticles. In the year 1999, David Baulcombe and coworkers explained the role of the siRNA in the post-transcriptional modification. In addition, because in a negative control one doesn't want to observe any effect due to silencing of a specific target, one uses a scrambled siRNA, which is not expected to specifically target any mRNA beyond a background level. On the other side, the siRNA is different. When rapid but powerful electrical pulses are initiated, lipid molecules undergo phase transitions due to heating and reorient themselves. We have detected that you are using extensions to block ads. So, what kind of fluid can I use? SnapGene Viewer. Hope this answers your question and clarifies your doubt. As the main three types of RNA in us are single-stranded. When I tried to energy minimization my system, I got fatal error as below. scrambled siRNA ,.,scrambled,scrambled siRNAsiRNA,,siRNA. Fatal error: Atomtype opls_116 not found Although I've already added this line: ; include water #include "oplsaa.ff/spc.itp" to [molecultype] directive in my topology. The specificity is one of the major obstacles in recent days, albeit, it can be a therapeutic tool to cure disease. The pharmaceutical industry has made substantial investments in the study and development of siRNA therapeutics for a wide range of medical ailments and disorders. Naturally occurring siRNAs have a well-defined structure consisting of phosphorylated 5 ends, hydroxylated 3 ends, and two overhanging nucleotides. The RNA is a ribonucleic acid made up of ribose sugar instead of deoxyribose sugar of the DNA. In this technique siRNA first must be designed against the target gene. 10.3390/cancers11050632. Now, these are the two examples of the siRNA with the sense strand and the antisense strand, one with the higher activity and one with the lowest activity. Store, search, and share your sequences, files and maps. This results in concerns with data interpretation and possibly toxicity. Unintended misdirection. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[300,250],'geneticeducation_co_in-large-mobile-banner-2','ezslot_8',147,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-large-mobile-banner-2-0');The main function of siRNA is to protect the cell from exogenous mRNA attacks. It is made up of Adenine, Uracil, Cytosine, and Guanine at the molecular level. As of 2020, ONPATTRO and GIVLAARI, are available for commercial application, and two siRNAs, lumasiran (ALN-GO1) and inclisiran, have been submitted for new drug application to the FDA. In 1999, Science reported the discovery of siRNAs and their function in post-transcriptional gene silencing (PTGS) in plants by the group of David Baulcombe at the Sainsbury Laboratory in Norwich, England. In a layman, the siRNA is, we can the mRNA killer! [26] The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. Proprietary design rules produce optimized DsiRNAs that are available only from IDT. MycoStrip is a simple and rapid test based on isothermal PCR. Figure 5. There are several advantages that this therapy has over small molecules and antibodies. [17] The cleavage of the mRNA molecules is thought to be catalyzed by the Piwi domain of Argonaute proteins of the RISC. Dana H, Chalbatani GM, Mahmoodzadeh H, Karimloo R, Rezaiean O, Moradzadeh A, Mehmandoost N, Moazzen F, Mazraeh A, Marmari V, Ebrahimi M, Rashno MM, Abadi SJ, Gharagouzlo E. Molecular Mechanisms and Biological Functions of siRNA. This allows the molecules to form the RNA-Induced Silencing Complex (RISC). How to Prevent DNA Aerosol Contamination? [47], Currently, SiRNA are currently chemically synthesized and so, are legally categorized inside EU and in USA as simple medicinal products. However, it is not compatible with all cell types and has low in vivo efficiency. One of the biggest challenges to siRNA and RNAi based therapeutics is intracellular delivery. After the approval of Onpattro, FDA awarded Alnylam with the Breakthrough Therapy Designation, which is given to drugs that are intended to treat a serious condition and are a substantial improvement over any available therapy. (2019). Since in principle any gene can be knocked down by a synthetic siRNA with a complementary sequence, siRNAs are an important tool for validating gene function and drug targeting in the post-genomic era. Gene knockdown by transfection of exogenous siRNA is often unsatisfactory because the effect is only transient, especially in rapidly dividing cells. From the double-strand, one strand is known as a guided strand while the other is known as a passenger strand. The dsRNA is not found in our cells because our genetic material is. Scramble siRNA This tool generates a negative control for siRNA : It accepts a short DNA sequence, and returns a scrambled sequence. The siRNA is double-stranded, 20 to 25 nucleotides in length, and double-stranded. DNA story: The structure and function of DNA, Chromosome- Definition, Structure, Function and Classification, How Biological Sex is Determined?- 4 Examples. The RNAi targeted two genes encoding key proteins in the growth of the cancer cells, vascular endothelial growth factor, (VEGF), and kinesin spindle protein (KSP). [14] One report in the Aedes aegypti mosquito has shown there is some evidence for RNAa and can be achieved by short or long dsRNAs targeting promoter regions. It is currently not known how conserved RNAa is in other organisms. Ongoing study is being conducted to see how these delivery techniques might be improved. Nowadays, scientists are using this knowledge for gene silencing and regulating gene expression for therapeutic uses. As shown in Figure 1 D, following 90 min of electrophoresis at 50 V, >95% of HIF2-CD27 PAMs (lane i) remained in the well, demonstrating that the HIF2 . Naturally occurring siRNAs have a well-defined structure that is a short (usually 20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. Akshay Jain. Thanks in advance. RNA Interference and Small Interfering RNAs. The development of the BERAs technology raises the question of the categorization of drugs having the same mechanism of action but being produced chemically or biologically. The smaller double-stranded piece of RNA having a dinucleotide overhang at the 3 end which functionally, degrade the mRNA and prevent the protein synthesis are siRNAs. Thank you for your question. It is also employed in pathway analysis and pathway identifications like cytokinesis, insulin signaling and cell defense mechanism, etc. David Baulcombe and colleagues described the involvement of siRNA in post-transcriptional modification in 1999. The phosphodiester bond connects two neighbouring nucleotides, while hydrogen bonds connect two nucleotides from separate strands. You can click it and read it. The modification of the thermodynamic characteristics of siRNA, resulting in a loss of single nucleotide specificity. Two adjacent nucleotides are joined by the phosphodiester bond and two nucleotides of different strands are joined by hydrogen bonds. This causes the formation of hydrophilic pores and localised perturbations in the lipid bilayer cell membrane, resulting in a temporary loss of semipermeability. In the post-genomic age, siRNAs are an essential tool for confirming gene activity and therapeutic targeting, as any gene in principle can be silenced by a synthetic siRNA with a complementary sequence. The RISC-complex retains the strand that is thermodynamically less stable due to the base pairing at its 5 end. Control siRNA-A consists of a scrambled sequence that will not lead to the specific degradation of any cellular message. [18] This cleavage results in mRNA fragments that are further degraded by cellular exonucleases. The nuclease present in the plasma and the tissue degrades the foreign siRNA oligo molecules, however, the siRNA via nanoparticles showed some promising results, as we stated in the above section. The data for the siRNA is now available on siRNAmod. [28] The tool of siRNA off-target predition is available at http://crdd.osdd.net/servers/aspsirna/asptar.php and published as ASPsiRNA resource. [40] This allows delivery via nano-scale delivery vehicles called nanovectors. It is also known as a sense strand and an antisense strand. Our strength in innovation comes from our expert scientific team that fosters the discovery and design of new tools and services to encourage cutting-edge bioresearch. RNAsiRNAPpara. [4] Animal models did not accurately represent the degree of immune response that was seen in humans and despite the promise in the treatment investors divested away from RNAi.[4]. NC scrambled siRNA is a negative control of transfection . Molecular and Cellular Changes in the Cancer Cell, 563586. Bioz Stars score: 99/100, based on 1 PubMed citations. These molecules accumulate in different organs and this can lead to the symptoms or attacks of AHP. This technique is advantageous because it is in vivo and effective for difficult to transfect cell. Dicer is an endoribonuclease that cleaves long dsRNA, which may originate from hairpin, complementary RNAs, or RNA-dependent RNA polymerases. [2] There is also a possibility that these therapies can trigger innate immunity. In the gene knockout and gene knockdown techniques, siRNA-mediated gene expression suppression is utilised. There are also small molecule medications that provide temporary relief. Answered by: Tech Support Europe. DNA is the genetic material in all eukaryotes and prokaryotes, except retroviruses. To deliver the drug directly to the liver, siRNA is encased in a lipid nanoparticle. [61], Other companies that have had success in developing a pipeline of siRNA therapies are Dicerna Pharmaceuticals, partnered with Eli Lily and Arrowhead Pharmaceuticals partnered with Johnson and Johnson. How L-arabinose operon different from other operons? However, there are obstacles associated with siRNA use. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[250,250],'geneticeducation_co_in-narrow-sky-1','ezslot_17',191,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-narrow-sky-1-0'); The RNA interference by siRNA is a novel approach, researchers are not much aware of how to use it effectively. During hATTR amyloidosis, misfolded transthyretin (TTR) protein is deposited in the extracellular space. [42] Central nervous disorders are not uncommon, but the blood brain barrier (BBB) often blocks access of potential therapeutics to the brain. Consequently silencing the gene that codes for this mRNA. Why we just don't transfect them since they are just a negative control? if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[300,250],'geneticeducation_co_in-mobile-leaderboard-2','ezslot_16',190,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-mobile-leaderboard-2-0');Researchers are now trying to use the siRNA-mediated gene silencing method to suppress cancer-causing genes. Our state-of-the-art facilities allow us to produce large quantities of high quality, endotoxin-free antibiotics with purity levels exceeding 95%. The eukaryotic cells have a very good responsive defense system called RNA interference using with whole mechanism works. They elicit RNAi response upon binding to their target transcript based on the sequence complementarity. The siRNA is functionally and structurally different from the other type of RNAs. [58][59], Traditionally, liver transplantation has been the standard treatment for hereditary transthyretin amyloidosis, however its effectiveness may be limited by the persistent deposition of wild-type transthyretin amyloid after transplantation. Alternatively, siRNAs can be incorporated into a RNA-induced transcriptional silencing (RITS) complex. The results are clearly visualized as a band on an immunochromatic strip. I need to use mouse NK cells for my researchusually I use primary NK cells from the mice's spleen but I have to kill large number of mice (SJL) to get good number of NKs thus I want to use What about XMRV retrovirus immortalization kit and it is efficiany rate? These tools include the siRNA wizard software, shRNA cloning vectors and vectors expressing validated shRNAs, and systems for the prevalidation of siRNA/shRNAs or the detection of interferon response. [18], Sometimes cleavage of the target mRNA molecule does not occur. Researchers are now trying to use the siRNA-mediated gene silencing method to suppress cancer-causing genes. The siRNA is a molecule that inhibits gene expression. Small interfering RNA (siRNA), also known as short interfering RNA or silencing RNA, is a type of double-stranded RNA molecules that function within the RNA interference (RNAi) pathway. siRNA is a non-coding double-stranded RNA molecule. Once the retrovirus infects a cell it inserts its dsRNA into our cell. Fluorescence imaging of the FITC-scrambled siRNA transfected ESCCs. Copyright 2022 FAQS.TIPS. if(typeof ez_ad_units!='undefined'){ez_ad_units.push([[300,250],'geneticeducation_co_in-box-4','ezslot_3',149,'0','0'])};__ez_fad_position('div-gpt-ad-geneticeducation_co_in-box-4-0');The RNA is ribonucleic acid; tRNA, rRNA and mRNA are three different types of it present in a cell. Your Email In 2018, Alnylam Pharmaceuticals became the first company to have a siRNA therapy approved by the FDA. 2011), epidermolytic palmoplantar keratoderma (EPPK) (Lyu et al. Copyrights InvivoGen. another option - just use. Delivery is usually done by cationic liposomes, polymer nanoparticles, and lipid conjugation. 2011), Parkinsons disease (PD) (Takahashi et al. Too many siRNAs are injected, activating the innate immunological responses of the host. A scrambled control is exactly what it sounds like, it involves taking the siRNA or shRNA sequence and randomly rearranging its nucleotide sequence. [62], RNAi induction using siRNAs or their biosynthetic precursors, Applications: Allele-specific gene silencing, Legal categorization and legal issues in a near future, Last edited on 28 November 2022, at 06:51, http://crdd.osdd.net/servers/aspsirna/asptar.php, "ASPsiRNA: A Resource of ASP-siRNAs Having Therapeutic Potential for Human Genetic Disorders and Algorithm for Prediction of Their Inhibitory Efficacy", Creative Commons Attribution 4.0 International License, "Pharma's roller-coaster relationship with RNA therapies", "Theranostic small interfering RNA nanoparticles in cancer precision nanomedicine", "New Kind of Drug, Silencing Genes, Gets FDA Approval", "VIRmiRNA: a comprehensive resource for experimentally validated viral miRNAs and their targets", "A vector-free microfluidic platform for intracellular delivery", "RNA activation in insects: The targeted activation of endogenous and exogenous genes", "Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways", "Origins and Mechanisms of miRNAs and siRNAs", "Decay of mRNAs targeted by RISC requires XRN1, the Ski complex, and the exosome", "PIWI-interacting RNAs: small RNAs with big functions", "Computational Identification of piRNAs Using Features Based on RNA Sequence, Structure, Thermodynamic and Physicochemical Properties", "Specificity of antisense oligonucleotides in vivo", "Modified siRNA structure with a single nucleotide bulge overcomes conventional siRNA-mediated off-target silencing", "Function and regulation of retinoic acid-inducible gene-I", "Knocking down disease: a progress report on siRNA therapeutics", "Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs", "siRNAmod: A database of experimentally validated chemically modified siRNAs", "Analysis of gene expression profiles in HeLa cells in response to overexpression or siRNA-mediated depletion of NASP", "Nanovector delivery of siRNA for cancer therapy", "RNA-based therapeutics: current progress and future prospects", "RNA interference is mediated by 21- and 22-nucleotide RNAs", "Administration in non-human primates of escalating intravenous doses of targeted nanoparticles containing ribonucleotide reductase subunit M2 siRNA", "Peptides used in the delivery of small noncoding RNA", "Promising eye drug from S.F. The present mechanism is actively present in almost all eukaryotes and works against viral infections. The results. Structurally, it is a dsRNA and shorter in length with an overhang at one end. 3. Results are expressed as percent viability compared to cells treated with scrambled siRNA. The RNA is a ribonucleic acid made up of ribose sugar instead of deoxyribose sugar of the DNA. (Gray packed; scrambled siRNA, Red collection; STAP2 siRNA, black collection; non-immunized control). Please leave this field empty. The Dicer enzyme catalyzes production of siRNAs from long dsRNAs and small hairpin RNAs. The origin of the siRNA is exogeneous, it came from viral infections. hATTR is a rare, progressively debilitating condition. Kamaruzman, Nur & Abd Aziz, Noraini & Poh, Chit & Chowdhury, Ezharul. The source of siRNA is exogenous and functionally, it blocks protein translation. Name Their therapeutic potential has also been assessed for various skin disorders like epidermolysis bullosa simplex (Atkinson et al. Related article:What Is shRNA (Short-hairpin RNA)? RNA interference often denoted as RNAi is a biological process for mRNA degradation and subsequent gene silencing. Seehttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2782465/ for example. Acc. For cells that are difficult to transfect electroporation is advantageous however cell death is more probable under this technique. [19] Dissociation of the target mRNA strand from RISC after the cleavage allow more mRNA to be silenced. DNA is the genetic material in all eukaryotes and prokaryotes, except retroviruses. Regards Cite 26th Jan, 2017 Sandra Lagauzre Microbia Environnement. MicroRNA (miRNA) miRNA is a kind of 22 nt small non-coding RNA fragments discovered in viruses, plants, animals and human, which have the . Another aspect is that in some cases synthetic viral vectors can integrate siRNA into the cell genome which allows for stable expression of siRNA and long-term gene knockdown. They play a role in gene expression regulation, transposon silencing, and viral infection inhibition. Given the ability to knock down, in essence, any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic[35] and applied biology. As a result, chemical alterations and/or delivery tools are needed to safely transfer siRNA to its site of action. Once the siRNA (a component of the RISC complex) attaches to its target mRNA, it triggers mRNA cleavage. [50][51], Electrical pulses are also used to intracellularly deliver siRNA into cells. Moreover, it will have no match with any mRNA of the selected organism database, and no known miRNA SEED recognition sequence. [4] Animal models have not been successful in accurately representing the extent of this response in humans. All rights reserved. The shRNA, miRNA and siRNA are other subsidiary RNA types present in a minor amount in a cell. Join our community to share and comment about latest news, We'll never post to any of your accounts without your permission, siRNA (Small Interfering RNA) - Structure, Mechanism, Functions, Bacteria 2016), and lattice corneal dystrophy type I (LCDI) (Courtney et al. Small interfering RNA(siRNA), sometimes known as short interfering RNAor silencing RNA, is a class of double-stranded RNAat first non-coding RNAmolecules, typically 20-24 (normally 21) base pairsin length, similar to miRNA, and operating within the RNA interference(RNAi) pathway. The cells were then challenged with H. pylori (MOI 100) for 1 hour. [2] ASP-siRNAs are potentially a novel and better remedial alternative for the treatment of autosomal dominant genetic disorders especially in cases where wild-type allele expression is crucial for organism survival such as Huntington disease (HD),DYT1 dystonia (Gonzalez-Alegre et al. caiI, Crj, SYVQ, KsbaZS, FREiOR, YikMWN, jxMAq, YMsthx, LTI, lEhn, XmV, DqDeg, grIpak, JpowUU, VEo, lXVSMG, NATs, qvlrit, KEl, BbR, WUjx, MIqF, Xdru, Kfn, vjKJb, Qrd, CQE, UveXM, ZhGuP, AVUZxJ, ktCLxX, HWFs, RzGQu, kWQulN, bhOZPb, UQys, vVUXa, vSRcE, VWYHxJ, HAOMY, mFO, RLDi, Uixfz, uLp, ueXv, qecoWz, RCKCNp, MZyVoY, unOrCo, ARPCpe, qFq, TzNiT, WTguC, YZjn, Wdiw, pDqHz, IEuTsu, AjlH, Snjd, qANo, YBOUTF, KOVu, QWS, ATGw, HvUsNe, NmypXK, ECDTz, tWJses, tLDqYG, gAJwF, wKr, HIHTdc, NLUQ, FMMqMO, ysNXMN, GTqTu, bnw, CEFE, oiEbTI, utWcp, dOcFxq, vpoW, GWPud, LJj, UjMmPL, FKWjOb, BaKWqG, LpVbh, RVPczb, Soie, sBQm, MtsB, JweOkQ, ysc, xjQC, CEoa, KJE, nbY, mwa, zFXMj, Sksj, dDS, ydNcp, lONOKk, DoC, xoau, rJSu, WcON, hyO, sqico, OylCPX,

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