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Nucl. Acids Res. 34, 6215-6224. Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51 2006

Desfarages, S., San Filippo, J., Fournier, M. Calmels, C., Caumont-Sarcos, A., Litvak, S., Sung, P., Parissi, V.

Notes: In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase. (3704)

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Appl. Environ. Microbiol. 72, 2539-2546. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. 2006

Mohan Nair, M.K. and Venkitanarayanan, K.S.

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconsituted infant formula. (3464)

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Genetics 173, 2143–2149. Functional Implication of an ARG307GLY Substitution in Corticosteroid Binding Globulin, a Candidate Gene for a QTL Associated with Cortisol Variability and Obesity. 2006

Guyonnet-Duperat, V., Geverink, N., Plastow, G.S., Evans, G., Ousova, O., Croisetiere, C., Foury, A., Richard, E., Mormede, P. and Moisan, M.P.

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

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Nucl. Acids Res. 34, e67. GREM, a technique for genome-wide isolation and quantitative analysis of promoter active repeats. 2006

Buzdin, A., Kovalskaya-Alexandrova, E., Gogvadze, E. and Sverdlov, E.

Notes: The authors selected repetitive elements in the human genome using a novel technique: GREM. T4 DNA Ligase was used to ligate adapters to digested genomic DNA prior to PCR, and exonuclease III was used to generate the necessary 5´ termini. After the final amplification, the PCR products were cloned into the pGEM®-T Vector, then sequenced. (3550)

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Infect. Immun. 74, 3825-3833. Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. 2006

Shin, S.J., Wu, C-W., Steinberg, H. and Talaat, A.M.

Notes: In this study, insertional mutagenesis with the transposon TN5367 was used to generate a library of M. paratuberculosis mutants. Sequences containing transposons were then amplified, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and cloned into the pGEM®-T Easy Vector prior to sequencing. Bioinformatic screening was then used to identify potential virulence determinants for further study in a mouse model of M. paratuberculosis infection. (3534)

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Cancer Res. 66, 9090-9098. MicroRNA regulates the expression of human cytochrome P450 1B1. 2006

Tsuchiya, Y., Nakajima, M., Takagi, S., Taniya, T., and Yokoi, T.

Notes: These authors identified a region complementary to the microRNA miR-27b in the 3´ UTR of the cytochrome p450 CYP1B1 mRNA, and showed that miR-27b was involved in regulation of CYP1B1 expression. The 3´ UTR containing the miRNA target site was cloned downstream of the luciferase gene in the pGL3 Promoter Vector and cotransfected into the miR-27b-positive breast cancer cell line MCF-7 and into miR-27b-negative Jurkat cells. Luciferase expression levels from the reporter vector containing the CYP1B1 3´ UTR sequence were reduced in miR-27b-positive cells, but not in the Jurkat cell controls. Delivery of an antisense oligoribonucleotide directed against miR-27b to MCF-7 cells containing the reporter construct resulted in restoration of luciferase activity. The effects of inhibition of miR-27b on protein levels and enzymatic activity of CYP1B1 were then investigated in MCF-7 cells. CYP1B1 protein levels and enzymatic activity increased significantly in cells transfected with the antisense oligo; the enzymatic activity was measured using a p450-Glo™ Assay. The coding region and 3´ UTR of the CYP1B1 gene were also PCR-amplified, subcloned the into the pTargeT™ Mammalian Expression Vector, and transfected into HEK293 cells. The effect of overexpression of miR-27b on protein levels and enzymatic activity of CYP1B1 was then evaluated in these cells. (3622)

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J. Biol. Chem. 281, 8254–8263. Molecular cloning and characterization of UDP-glucose dehydrogenase from the amphibian Xenopus laevis and its involvement in hyaluronan synthesis. 2006

Vigetti, D., Ori, M., Viola, M., Genasetti, A., Karousou, E., Rizzi, M., Pallotti, F., Nardi, I., Hascall, V.C., De Luca, G. and Passi, A.

Notes: To test the effect that Xenopus laevis UDP-glucose dehydrogenase (xUGDH) expression has in mammalian cells, the xUGDH ORF was amplified, purified, A-tailed and cloned into the pTARGET™ Mammalian Expression Vector. Two clones were selected: one in the sense orientation and one in the antisense orientation. The constructs were confirmed by DNA sequencing and expression in a TNT® T7 in vitro transcription/translation system. Five micrograms of the plasmids were transfected into AoSMCs and human aortic smooth muscle cells, and UGDH activities werw measured 48 hours post-transfection. (3497)

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J. Physiol. 570, 283-294. Motor neurone targeting of IGF-1 prevents specific force decline in ageing mouse muscle 2006

Payne, A.M., Zheng, Z., Messi, M.L., Milligan, C.E., González, E. and Delbono, O.

Notes: Overexpression of IGF-1 can delay or prevent aging problems in motor neurons and skeletal muscle. The authors of this paper were able to target IGF-1 to motor neurons using a fusion protein containing tetanus toxin fragment C (TTC). Motor neurons will bind, take up and transport the TTC fragment with no toxicity to the neurons. Full-length human IGF-1 cDNA was generated by PCR and inserted into the pGEM®-T Easy Vector. TTC amplified from Clostridium tetani CN655 genomic DNA was inserted into the vector. The new IFG-1-TTC insert was used for PCR to eventually produce the fusion protein for the studies. (3635)

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Appl. Environ. Microbiol. 72, 2691–2706. Oligonucleotide array for identification and detection of pythium species. 2006

Tambong, J.T., de Cock, A.W., Tinker, N.A. and Levesque, C.A.

Notes: This study compared detection of Pythium species in soil samples by DNA array hybridization and PCR cloning. Three Pythium species were amplified from soil samples, a single 3´ A was added to the resulting PCR product, and the DNA was ligated into the pGEM®-T Easy Vector at 4°C overnight. After the ligation was transformed into JM109 Competent Cells, and 100 colonies were chosen and grown overnight in LB broth. The plasmid DNA was isolated using the Wizard® SV 96 Plasmid DNA Purification System and then sequenced. (3437)

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Clin. Can. Res. 12, 2032-37. Reversal of the malignant phenotype of cervical cancer CaSki cells through adeno-associated virus-mediated delivery of HPV16 E7 antisense RNA. 2006

Wu, S., Wang, S., Wang, W., Xi, L., Tian, X., Chen, G., Wu, Y., Zhou, J., Xu, G., Lu, Y. and Ma, D.

Notes: The coding sequence of the Human Papilloma Virus (HPV16) E7 oncogene was isolated following total RNA purification from CaSki cells, RT-PCR and PCR and then cloning into the pGEM®-T Easy vector. In order to test the effectiveness of antisense HPV16 E7 therapy against cervical cancer, an adeno-associated virus vector was used to transfer the antisense construct of the E7 coding sequence into CaSki cervical cancer cells. (3395)

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Am. J. Pathol. 168, 261-269. Role of apoptosis signal-regulating kinase 1 in stress-induced neural cell apoptosis in vivo. 2006

Harada, C., Nakamura, K., Namekata, K., Okumura, A., Mitamura, Y., Iizuka, Y., Kashiwagi, K., Yoshida, K., Ohno, S., Matsuzawa, A., Tanaka, K., Ichijo, H. and Harada, T.

Notes: The authors of this study investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in neural cell apoptosis during retinal development and ischemic injury. Nucleotides 283 to 713 of the ASK1 cDNA were amplified by PCR and cloned into the pGEM®-T Easy Vector, and sense and antisense probes for in situ hybridization experiments were generated. Anti-ACTIVE® p38 polyclonal antibody was used for immunohistochemistry analyses to investigate the localization of phosphorylated p38 in mouse retina. (3530)

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Appl. Environ. Microbiol. 72, 3274–3283. Stolbur phytoplasma genome survey achieved using a suppression subtractive hybridization approach with high specificity. 2006

Cimerman, A., Arnaud, G. and Foissac, X.

Notes: To purify stolbur phytoplasma DNA from total DNA of infected periwinkle plants, two rounds of suppression subtractive hybridization (SSH) were performed, followed by amplification with Taq DNA polymerase. The resultant PCR products (1µl) were ligated into 50ng of pGEM®-T Easy Vector using 3 units T4 DNA Ligase. After transformation of DH10B cells, ampicillin-resistant colonies were grown and the plasmids purified using the Wizard® Plus SV Minipreps DNA Purification System. The insert lengths were estimated after EcoR I digestion and agarose gel electrophoresis prior to amplification and labeling with digoxigenin. These probes were used for dot hybridization with denatured healthy or infected plant DNA (10µg) and the corresponding plasmid as a positive control (100 ng). (3436)

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Mol. Cell. Biol. 17, 1331-1343. The Arabidopsis thaliana PARTING DANCERS gene encoding a novel protein is required for normal meiotic homologous recombination. 2006

Wijeratne, A.J., Chen, C., Zhang, W., Timofejeva, L. and Ma, H.

Notes: The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM®-T Vector, then labeled with digoxygenin through in vitro transcription. (3468)

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J. Biol. Chem. 281, 9030-9037. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1alpha-dependent mechanism. 2006

Ullah, M.S., Davies, A.J. and Halestrap, A.P.

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

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Nucl. Acids Res. 34, 584-592. Unbiased in vitro selection reveals the unique character of the self-cleaving antigenomic HDV RNA sequence. 2006

Nehdi, A. and Perreault, J.P.

Notes: To characterize sequence variability of the catalytic center of the hepatitis delta virus (HDV) ribozyme, which includes a self-cleaving motif, the authors developed an in vitro selection process that allowed them to select self-cleaving sequence variants from a pool of HDV ribozymes. The selection process started with a library of DNA oligonucleotides corresponding to the HDV ribozyme sequence that had been randomized at a 25-nucleotide sequence. The oligos contained known sequences at the 5´ and 3´ ends to allow amplification. The library was amplified by PCR, then transcribed. The resulting RNA was separated by polyacylamide gel electrophoresis, and the self-cleaved transcripts were excised, purified and reverse transcribed. A 3´ extension using terminal deoxynucleotidyl transferase added a known sequence to the 3´ end so that the resulting cDNA could by amplified by PCR. Thus another cycle of selection could begin. The PCR products generated at the start of the process and at each cycle were cloned into the pGEM®-T Vector. (3467)

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J. Biol. Chem. 280, 19977-19985. A novel Myc-target gene, mimitin, that is involved in cell proliferation of esophageal squamous cell carcinoma. 2005

Tsuneoka, M., Teye, K., Arima, N., Soejima, M., Otera, H., Ohashi, K., Koga, Y., Fujita, H., Shirouzu, K., Kimura, H. and Koda, Y.

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

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Mol. Cell. Biol. 25, 2861-2870. Assembly of the mariner Mos1 synaptic complex. 2005

Auge-Gouillou, C., Brillet, B., Hamelin, M.H., and Bigot, Y.

Notes: A transposase protein (Tnp) open reading frame (amino acids 1 to 345) was amplified from the Drosophila mauritiana Mos1 mariner transposable element using GoTaq® DNA polymerase. The Tnp open reading frame was then cloned with into the pGEM®-T Easy Vector and sequenced before further subloning. Tnp protein was purified and used in gel-shift assays to demonstrate Tnp binding regions in the Mos1 transposable element. (3344)

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Plant Physiol. 137(2), 557–66. Beyond complementation. Map-based cloning in Chlamydomonas reinhardtii. 2005

Rymarquis, L.A., Handley, J.M., Thomas, M. and Stern, D.B.

Notes: These authors describe map-based cloning tools for Chlamydomonas reinhardtii, (green yeast). In this case-based study of existing and projected resources for C. reinhardtii mapping, mcd4 and mcd5 mutants were mapped by crossing to the infertile strain known as Chlamydomonas grossii, S1-D2, with several known marker sites. To reduce the time and expense of mapping, bulked segregant analysis (BSA) and marker duplexing were evaluated. In BSA, DNAs from multiple segregating progeny (up to six in this study) are combined, and results from PCR-based markers are examined for significant bias from a roughly equal contribution from each parent. This case study used 57–72 markers to span the 1,107-cM genome using approximately 3,350 PCR amplifications. The researchers picked single colonies from a plate and placed each colony in a hypotonic EDTA solution. After boiling for 5 minutes, the supernatant was collected by centrifugation and the DNA quantitated. Twenty nanograms of DNA thus isolated was used as the template for PCR. The reaction included GoTaq® DNA polymerase with the enhancers 8.5%glycerol and 0.83% formamide in a 30µl reaction. Forty cycles of amplification were performed and the results were analyzed by agarose gel electrophoresis. The various amplification reactions included the use of BSA and both monoplex and duplex reactions with amplicons of 90bp–625bp. (3279)

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Nucl. Acids Res. 33, 4762-4774. Characterization of SpPol4, a unique X-family DNA polymerase in Schizosaccharomyces pombe. 2005

Gonzalez-Barrera, S., Sanchez, A., Ruiz, J.F., Juarez, R., Picher, A.J., Terrados, G., Andrade, P. and Blanco, L.

Notes: An open reading frame encoding a putative DNA polymerase, SpPol4, was amplified from S. pombe genomic DNA by PCR. The resulting PCR product was cloned into the pGEM®-T Easy Vector, and the sequence was verified. Based on sequence analysis, the authors hypothesize that SpPol4 has deoxyribose phosphate (dRP) lyase activity, suggesting that the enzyme plays a role in base excision repair. The authors perform a dRP lyase activity assay with an oligonucleotide substrate labeled using [32P]ddATP and terminal deoxynucleotidyl transferase. (3465)

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J. Biol. Chem. 280, 28215-28220. Determination of the functionality of common APOA5 polymorphisms. 2005

Talmud, P.J., Palmen, J., Putt, W., Lins, L., and Humphries, S.E.

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM®-7Zf Vector. Transcription/translation experiments were performed using the TNT® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ GreenLys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe® System –T7 and the Ribo m7G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

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Infect. Immun. 73, 2611-2620. Molecular cloning and characterization of three beta-defensins from canine testes. 2005

Sang, Y., Ortega, M.T., Blecha, F., Prakash, O. and Melgarejo, T.

Notes: The investigators cloned three β-defensins, which are antimicrobial peptides, from canine testes. A canine expressed sequence tag (EST) was identified based on similarity to human β-defensins. Full-length cDNAs were obtained using 5´- and 3´RACE, then amplified by PCR and cloned into the pGEM®-T Easy Vector. cDNA sequences were confirmed using the SP6 and T7 Promoter Primers. The tissue-specific expression of canine β-defensins (cBDs) was characterized using the AccessQuick™ RT-PCR System to amplify β-defensin RNA from a variety of tissues. RNA was treated with RQ1 RNase-Free DNase prior to RT-PCR. The identity of the RT-PCR products was confirmed by electrophoresis, transfer to nylon membranes and hybridization to probes derived from sequence-confirmed β-defensin clones; the probes were synthesized using the Prime-a-Gene® Labeling System. To localize expression of the three β-defensin isoforms in canine testes, in situ hybridization (ISH) was performed. The 3´-RACE products were cloned into the pGEM®-T Vector, which was then linearized and treated with exonuclease III (Erase-a-Base® System) to delete an approximately 80-bp region shared by the three cBD isoforms. The resulting product was used to synthesize sense and antisense ISH probes. (3453)

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J. Biol. Chem. 280, 36802-36808. Structural and genetic analyses reveal a key role in prophage excision for the torl response regulator inhibitor. 2005

ElAntak, L., Ansaldi, M., Guerlesquin, F., Méjean, V. and Morelli, X.

Notes: GoTaq® Polymerase was used in this study investigating the role of TorI (Tor inhibition protein) in prophage DNA excision. PCR products were amplified from E. coli strains carrying a plasmid-encoded torI gene, and were analyzed by gel electrophoresis. (3350)

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J. Bacteriol. 187, 3762-3778. Sulfur amino acid metabolism and its control in Lactococcus lactis IL1403. 2005

Sperandio, B., Polard, P., Ehrlich, D.S., Renault, P. and Guédon, E.

Notes: To study the transcription levels of several genes involved in cysteine and methionine metabolism, Lactococcus lactis strains deficient in these genes were created by double-crossover homologous integration. To do so, PCR was performed to amplify sequences upstream and downstream of the target gene, and the PCR products were ligated and cloned into the pGEM®-T Easy Vector. The resulting vectors were fused to pGhost9, a vector designed for homologous recombination in L. lactis. Once the gene deletions were verified, quantitative real-time RT-PCR was performed to quantitate expression levels of a variety of genes involved in Cys and Met metabolism. (3461)

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J. Biol. Chem. 279(14), 13849-58. Cbfa1/RUNX2 directs specific expression of the sclerosteosis gene (SOST). 2004

Sevetson, B., Taylor, S. and Pan, Y.

Notes: A 1.8 kb region of the sclerosteosis (SOST) promoter was amplified from Human Genomic DNA. Various deletion mutations were created in the amplified SOST promoter and then cloned into the pGL2-Basic Vector. Using a 50:1 ratio of luciferase construct to SEAP (secreted alkaline phosphatase) normalization control, SAOS-2 and MG-63 human osteosarcoma cell lines were transfected and then assayed for luciferase activity using the Bright-Glo™ Luciferase Assay System.  Nuclear extracts prepared from SAOS-2 and MG-63 cells were incubated with four regions of the SOST promoter in the presence of Gel Shift Binding 5X Buffer for EMSA experiments.  Supershifting was performed by adding antibodies to the DNA binding reaction. (3115)

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Biochem. Biophys. Res. Commun. 321(1), 259-265. Cloning of hOST-PTP: the only example of a protein-tyrosine-phosphatase the function of which has been lost between rodent and human 2004

Cousin, W., Courseaux, A., Ladoux, A., Dani, C., and Peraldi, P.

Notes: Researchers used GoTaq® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard® SV Gel and PCR Clean-Up System prior to cloning into the pGEM®-T Easy Vector.  The Prime-a-Gene® Labeling System was used to make 32P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

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