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Appl. Environ. Microbiol. 73, 4234-4242. Rapid engineering of bacterial reporter gene fusions by using Red recombination. 2008

Gerlach, R.G., Hölzer, S.U., Jäckel, D., and Hensel, M.

Notes: These authors describe use of a red recombinase mediated method for generation of reporter constructs in Salmonella enterica setrovar typhimurium. Among the reporter constructs created was a HaloTag® reporter using the HaloTag® coding region from the pHT2 promoter. (3924)

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Nucl. Acids Res. 36, 5391–404. miR-16 family induces cell cycle arrest by regulating multiple cell cycle genes. 2008

Liu, Q., Fu, H., Sun, F., Zhang, H., Tie, Y., Zhu, J., Xing, R., Sun, Z. and Zheng, X.

Notes: To identify microRNA targets, the authors created a Drosha-knockdown cell line and confirmed depletion of Drosha and three randomly selected miRNAs in these cells by quantitative RT-PCR, using β-actin as a control. The reverse transcription step was performed using the ImProm-II™ Reverse Transcription System. The authors then performed microarray analysis to monitor expression of transcripts to determine which were upregulated as a result of Drosha depletion; cRNA used in these microarray experiments was synthesized using the T7 RiboMAX™ Express Large Scale RNA Production System. Cyclin D1 was identified as a potential miRNA target. To screen miRNAs that regulate cyclin D1, the authors cloned the cyclin D1 3´ untranslated region downstream of the firefly luciferase gene of the pGL3-Control Vector and measured luciferase levels in transfected cells using the Dual Luciferase Reporter Assay System. Renilla luciferase in the pRL-TK Vector was used as a normalization control. (3894)

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J. Clin. Microbiol. 46, 652–64. Multilocus sequence typing reveals that the population structure of Candida dubliniensis is significantly less divergent than that of Candida albicans. 2008

McManus, B.A., Coleman, D.C., Moran, G., Pinjon, E., Diogo, D., Bougnoux, M.E., Borecká-Melkusova, S., Bujdákova, H., Murphy, P., d'Enfert, C. and Sullivan, D.J.

Notes: To determine the usefulness of multilocus sequence typing (MLST) in differentiating Candida species during epidemiological studies, the authors investigated the population structure of C. dubliniensis by amplifying the same 10 MLST loci found to be useful in differentiating isolates of C. albicans, a closely related species. PCRs were performed using 1.25 units of GoTaq® Flexi DNA Polymerase and 1ng of DNA template in a 50µl reaction. (3880)

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Plant Physiol. 146, 1469–81. Deregulation of maize C4 photosynthetic development in a mesophyll cell-defective mutant. 2008

Covshoff, S., Majeran, W., Liu, P., Kolkman, J.M., van Wijk, K.J. and Brutnell, T.P.

Notes: The authors identified the maize homolog of hcf136 (Zmhcf136), a gene involved in photosynthesis, and used an RNA blot to determine if ZmHcf136 transcripts accumulate preferentially in mesophyll cells. DNA probes for Zmhcf136 and several cell-specific markers were generated by PCR using GoTaq® Green Master Mix, gel purified and radiolabeled prior to use in the RNA blots. To examine differences in protein accumulation and localization in wildtype and hcf136 mutants, proteins from subcellular fractions were subjected to two-dimensional gel electrophoresis, and spots of interest were excised, digested with Sequencing Grade Modified Trypsin, then analyzed by electrospray ionization-tandem mass spectrometry. (3883)

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Biol. Reprod. 79, 594–7. Can bovine in vitro-matured oocytes selectively process X- or Y-sorted sperm differentially? 2008

Bermejo-Alvarez, P., Rizos, D., Rath, D., Lonergan, P. and Gutiérrez-Adán, A.

Notes: To determine whether oocytes are able to select X-bearing or Y-bearing spermatozoa, the authors performed in vitro fertilization of bovine oocytes with X-sorted semen, Y-sorted semen, a mixture of X- and Y-sorted semen, and unsorted semen. The gender of the resulting embryos was determined by amplifying two DNA targets: a Y chromosome-specific target for gender assignment and a bovine-specific satellite sequence as a control. PCRs were performed using GoTaq® Flexi DNA Polymerase (1 unit per 25µl reaction), and amplified products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. (3881)

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Clin. Vaccine Immunol. 15, 418–424. Sequential analysis of Anaplasma phagocytophilum msp2 transcription in murine and equine models of human. 2008

Scorpio, D.G., Leutenegger, C., Berger, J., Barat, N., Madigan, J.E. and Dumler, J.S.

Notes: The authors examined the pattern of Anaplasma phagocytophilum msp2 expression, a gene that modulates with little immune pressure and has decreased virulence with prolonged in vitro passage. C57BL/6J mice were inoculated with HL-60 cells infected with low-passage (passage 5) or high-passage (passage 26). Blood samples were taken 2–21 days post-inoculation, and total RNA was isolated. The purified RNA was subjected to RT-PCR, cloned into the pGEM®-T Easy Vector, transformed and plated. Plasmids were purified using the Wizard® SV 96 Plasmid DNA Purification System, and the insert size analyzed after EcoRI digestion. The inserts were sequenced, aligned with A. phagocytophilum Webster strain msp2 references using ClustalX and the diversity of msp2 transcripts divided into low- or high-passage bacteria. (3975)

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J. Exp. Bot. 59, 2253–65. Interaction study of MADS-domain proteins in tomato. 2008

Leseberg, C.H., Eissler, C.L., Wang, X., Johns, M.A., Duvall, M.R. and Mao, L.

Notes: The authors characterized the network of protein-protein interactions for 22 MADS-domain proteins in tomato using yeast two-hybrid and three-hybrid assays. To construct bait and prey proteins, total RNA from various tissues was reverse transcribed using the Reverse Transcription System, then amplified using PCR primers containing restriction enzyme sites for cloning into the bait and prey vectors. (3886)

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Clin. Chem. 54, 1080–4. Rapid determination of monozygous twinning with a microfabricated capillary array electrophoresis genetic-analysis device. 2008

Yeung, S.H., Medintz, I.L., Greenspoon, S.A. and Mathies, R.A.

Notes: The authors used a microfabricated capillary electrophoresis instrument to rapidly assess the genetic relationship between same-sex twins and their parents and siblings. STR typing was performed to determine if the twins were monozyotic or dizygotic and to confirm familial relationships. The authors used the PowerPlex® 16 System to examine 15 STR loci in this study. (4045)

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J. Biomol. Scr. 13, 968–974. Mouse thymus targeted peptide isolated by in vivo phage display can inhibit bioactivity of thymus output in vivo. 2008

Yu, Y., Wang, Z. and Du, T.

Notes: In this study, in vivo selection of a phage display random peptide library was performed in mice, isolating specific peptides that homed to mouse thymus. A thymus-homing and kidney-recovered peptide were injected into BALB/c mice, and peripheral blood removed at intervals of 15 minutes, 1 hour, 3 hours, 6 hours and 24 hours. DNA was extracted using the ReadyAmp™ Genomic DNA Purification System. Thymus activity was assessed by using real-time PCR. (4020)

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J. Clin. Microbiol. 46, 1741–1746. High-throughput genotyping of Salmonella enterica serovar Typhi allowing geographical assignment of haplotypes and and pathotypes within an urban District of Jakarta, Indonesia. 2008

Baker, S., Holt, K., van de Vosse, E., Roumagnac, P., Whitehead, S., King, E., Ewels, P., Keniry, A., Weill, F.X., Lightfoot, D., van Dissel, J.T., Sanderson, K.E., Farrar, J., Achtman, M., Deloukas, P. and Dougan, G.

Notes: The authors examined strains of Salmonella enterica serovar Typhi isolated from typhoid cases originating in or around Indonesia or from travelers returning from Indonesia to examine if serovar Typhi from this area has a greater level of genetic diversity compared to other countries. Genomic DNA was isolated using the Wizard® Genomic DNA Purification Kit, diluted to 4 ng/µl and used in locus-specific PCR genotyping. (3980)

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Carcinogenesis 29, 1184-1191. Interaction of the cytochrome P4501A2, SULT1A1 and NAT gene polymorphisms with smoking and dietary mutagen intake in modification of the risk of pancreatic cancer. 2008

Suzuki, H., Morris, J.S., Li ,Y., Doll, M.A., Hein, D.W., Lium J., Jiao, L., Hassan, M.M., Day, R.S., Bondy, M.L., Abbruzzese, J.L., and Li, D.

Notes: In this study, the Maxwell® 16 Instrument was used to purify genomic DNA from blood samples. The extracted DNA was amplified by PCR for subsequent genotype analysis. (3902)

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Proc. Natl. Acad. Sci. USA 105, 12445-12450. Oncogenic bystander radiation effects in Patched heterozygous mouse cerebellum. 2008

Mancuso, M., Pasquali, E., Leonardi, S., Tanori, M., Rebessi, S., Di Majo, V., Pazzaglia, S., Toni, M.P., Pimpinellam M., Covelli, V. and Saran, A.

Notes: To examine radiation-bystander responses in neonatal mouse cerebellum, heterozygous radiosensitive Patched-1 (Ptch1) mice were exposed to either whole body (WB) x-rays or shielded head/rest of body (SH) irradiation. Genomic DNA was isolated from tumors and normal tissue using the Wizard® SV Genomic DNA Purification System. Loss of heterozygosity was tested using PCR and sequencing of exon 23 of the Ptch1 gene. (3940)

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Clin. Can. Res. 14, 3278-3282. Mutant epidermal growth factor receptor in benign, borderline, and malignant ovarian tumors. 2008

Dahl Steffensen, K., Waldstrøm, M., Olsen, D.A., Corydon, T., Lorentzen, K.A., Hans Jørgen Knudsen, H.J., Jeppesen, U., Brandslund, I., and Jakobsen, A.

Notes: These authors evaluated 225 tumor samples from various ovarian and peritoneal tumor types for expression of the epidermal growth factor receptor type III variant EGFRvIII. EGFvIII has not been observed in normal tissues and so was evaluated as a potential target for tumor-specific therapies. However, none of the 225 samples evaluated were positive for this marker, suggesting that the EGFRvIII mutation is rare in ovarian tissue and is not a good therapeutic target for this disease. The authors used the Maxwell 16 Total RNA Purification Kit and the Maxwell 16 Instrument to purify RNA from tissue samples that had been fresh-frozen and fixed in the stabilization reagent RNAlater (Qiagen). Complete details of the RNA purification procedure are provided in the paper. (3878)

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Cancer Res. 68, 4623–4630. Integrative genomics identifies RAB23 as an invasion mediator gene in diffuse-type gastric cancer. 2008

Hou, Q., Wu, Y.H., Grabsch, H., Zhu, Y., Leong, S.H., Ganesan, K., Cross, D., Tan, L.K., Tao, J., Gopalakrishnan, V., Tang, B.L., Kon, O.L. and Tan, P.

Notes: In this article, the researchers explored the role of RAB23 in gastric cancers. Twenty-four hours before transfection, AGS cells (gastric cancer cell line) that expressed RAB23 were seeded into a 24-well plate at a density of 1.8 × 105 cells/ml. To overexpress RAB23, a full-length RAB23 cDNA was cloned into the pCI-neo Mammalian Expression Vector and transfected into AGS cells. The effect of RAB23 overexpression on the cells was determined using a Matrigel invasion assay while protein expression levels were visualized with Western blotting. (3986)

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Malaria Journal Oct 29:7, 223. A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking. 2008

Daniels R, Volkman SK, Milner DA, Mahesh N, Neafsey DE, Park DJ, Rosen D, Angelino E, Sabeti PC, Wirth DF, Wiegand RC.

Notes: These authors used the Maxwell® 16 System to isolate DNA from frozen whole blood samples infected with Plasmodium falciparum. The isolated DNA was used in a qPCR-based SNP genotyping assay that sought to uniquely identify the parasites based on their SNP marker profile. (3962)

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J. Virol. 12, 5940–50. Sulfatide is required for efficient replication of influenza A virus. 2008

Takahashi, T., Murakami, K., Nagakura, M., Kishita, H., Watanabe, S., Honke, K., Ogura, K., Tai, T., Kawasaki, K., Miyamoto, D., Hidari, K.I., Guo, C.T., Suzuki, Y. and Suzuki, T.

Notes: Sulfatide is present in mammalian organs where influenza A replicates. Ceramide galactosyltransferase (CGT) and cerebroside (galactosylceramide) sulfotransferase (CST), which synthesize sulfatide, were cloned by PCR into the pTargeT™ Mammalian Expression Vector and the pGEM®-T Easy Vector, (CST with or without a three base insertion), respectively. The two genes were removed by restriction digestion and cloned into pIRES-neo to forma bicistronic construct. Arylsulfatase A (ASA), which degrades sulfatide was also amplified and cloned into the pGEM®-T Easy Vector, before being subcloned into a neomycin-resistant expression vector. The expression vectors were transfected into COS-7 cells and selected for stable expression using G418. (3990)

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Proc. Natl. Acad. Sci. USA 105, 7141-7146. Lack of aldose 1-epimerase in Hypocrea jecorina (anamorph Trichoderma reesei): A key to cellulase gene expression on lactose 2008

Fekete, E., Seiboth, B., Kubicek, C.P., Szentirmai, A., Karaffa, L.

Notes: To amplify yeast mutarotase, S. cerevisiae was used, and E. coli strain JM109 (Promega Cat.# L2001) was used for plasmid propagation. Fungal mycelia were harvested by filtration, washed, frozen and ground under liquid nitrogen. Genomic DNA was extracted using the Wizard Genomic DNA Purification System (Promega Cat.# A1120). RNA for hybridization and RT-PCR was extracted from mycelia using the SV Total RNA Isolation System (Promega Cat.# Z3101) and plasmid DNA isolated using the PureYield(TM) Plasmid Midiprep System (Cat.# A2492). (3919)

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Cancer Res. 68, 6803-6809. Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia 2008

Case, M., Matheson, E., Minto, L., Hassan, R., Harrison, C.J., Bown, N., Bailey, S., Vormoor, J., Hall, A.G. and Irving, J.A.E.

Notes: The authors of this study investigated the relationship of somatic mutations that deregulate the RAS-RAF-MEK-ERK pathway and Acute Lymphoblastic Leukemia (ALL) and its progression to relapse in some patients. They show that such mutations are common in ALL and its recurrence. Furthermore, lymphoblasts from patients with mutations that were associated with upregulation of ERK showed increased cytotoxicity over wild-type controls when treated with U0126, suggesting that specific ERK inhibitors may eventually yield useful therapeutics. Following drug exposure, cytotoxic effects were assessed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay. (3905)

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J. Biol. Chem. 283, 21579–87. ATP modulation of Ca2+ release by type-2 and type-3 inositol (1, 4, 5)-triphosphate receptors. Differing ATP sensitivities and molecular determinants of action. 2008

Betzenhauser, M.J., Wagner, L.E. 2nd, Iwai, M., Michikawa, T., Mikoshiba, K. and Yule, D.I.

Notes: The authors examined the different ATP sensitivities of inositol (1,4,5)-triphosphate receptor (InsP3R) isoforms InsP3R1, InsP3R2 and InsP3R3. To compare the ATP-binding properties of InsP3R2 and InsP3R3, nucleotide sequences encompassing the ATP-binding domains were amplified by PCR and cloned into the pFN2A (GST) Flexi® Vector. The ATP-binding sites were expressed as glutathione-S-transferase (GST) fusion proteins in BL21(DE3)pLysS cells. Fusion proteins were purified, and the GST tag removed by cleavage with tobacco etch virus (TEV) protease. Purified proteins were then used in a fluorescent ATP-binding assay. (3901)

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Br. J. Ophthalmol. 92, 848–851. Mutations in the quinolone resistance determining region in Staphylococcus epidermidis recovered from conjunctiva. 2008

Yamada, M., Yoshida, J., Hatou, S., Yoshida, T. and Minagawa, Y.

Notes: To study how mutations in the quinolone resistance determining region (QRDR) of Staphylococcus epidermidis may have a role in fluoroquinolone resistance, 138 samples of S. epidermidis were swabbed from the conjunctival sacs of 129 patients. These samples were cultured overnight in tryptic soy broth, and genomic DNA isolated using the Wizard® SV 96 Genomic DNA Purification System. One microliter of the isolated DNA was used in PCR for the QRDR genes (gyrA, gyrB, parC and parE). (3939)

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J. Biol. Chem. May 6, Epub (ahead of print). Mechanism mediating the enhanced transcription of P2X3 receptor gene by calcitonin gene related peptide in trigeminal sensory neurons. 2008

Simonetti, M., Giniatullin, R., and Fabbretti, E.

Notes: These authors investigated the mechanism of action of the migraine mediator calcitonin gene-related peptide (CGRP), which is known to sensitize the P2X3 pain receptors to increase impulse flow to brain stem trigeminal nuclei. They showed that CAM KII inhibitors prevented CGRP-induced upregulation of P2X3 mRNA using real-time RT-PCR, and then confirmed that active CAM KII was involved in the signaling mechanism by staining with Anti-ACTIVE® CAM KII Antibodies. The immunoreactivity was upregulated by CGRP treatment of trigeminal neurons, and the distribution of the active CAM KII was localized to the membrane region. They then examined the mechanism of transcriptional activation of the P2X3 pain receptor genes and showed that the transcription factor CREB, which is known to be dependent on CAM KII for activation and nuclear localization, was involved. The authors also investigated the role of BDNF in the process, because CGRP is known to promote BDNF expression by trigeminal neurons, and because BDNF is thought to be involved in pain processing. They used the BDNF Emax Immunoassay System to measure BDNF levels in culture media after application of CGRP to neuronal cell cultures, and demonstrated that there was a fourfold increase in BDNF after CGRP exposure. They also showed that BDNF promoted CAMKII and CREB activation. (3873)

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Stem Cells 26, 1841-1849. Embryonic Stem Cells as a Platform for Analyzing Neural Gene Transcription 2008

Zhang, X., Horrell, S.A., Delaney, D., Gottlieb, D.I.

Notes: The authors note that while spatially and temporally specific gene transcription is a fundamental process in the normal development of mammalian stem cells, transcription in stem cells is currently studied by a set of methodologies with significant limitations. For instance, transient transfections analyze gene regulatory elements in nonchromosomal context. Using transgenic mice places transgenes in chromosomal context, however the chromosomal site where the transgene is inserted strongly influences the transgenes expression. As well, the need to make transgenic mice limits the number of experiments that can be done. ESCs can overcome these limitation. Undifferentiated stem cells are suitable for genetic engineering approaches such as gene targeting and recombinase-mediated cassette exchanges. By using such techniques, precisely planned alteration of native genes such as insertion of reporters, deletions of nearby or distant DNA sequences and mutational substitutions can be made. The authors wanted to analyze the Olig2 gene, a helix-loop-helix transcription factor expressed in the developing nervous system. Because Olig2 plays a central role in differentiation, understanding how it is regulated is important to understanding the larger transcriptional network controlling development. To this end, the authors used vectors for transient transfection experiments, constructed by amplifying regions of the Olig2 gene by PCR using primers tailed with appropriate restrictions sites and cloning the fragments into a pGL3 Luciferase Reporter Vector (Cat. # E1741, E1751, E1761, E1771). Promoter-reporter DNA was transfected into ESCs, cells were cultured 24 hours, then luciferase assays (Promega, type not specified) used to measure transgene expression. (3918)

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Phytopathology 97, 865-872. Multiplex real-time quantitative PCR to detect and quantify Verticillium dahliae colonization in potato lines that differ in response to Verticillium wilt. 2007

Atallah, Z.K., Bae, J., Jansky, S.H., Rouse, D.I., and Stevenson, W.R.

Notes: These authors developed a quantitative, real-time PCR method for the detection of Verticillium dahliae in potato cultivars. V. dahiae is the causative agent of Verticillium wilt, also known as potato early dying (PED). The standard detection method is a plating assay that takes 2 weeks to complete. The authors of this study performed qPCR assays using the V. dahliae beta tubulin-2 gene as a target. Initially, they amplified, subcloned and sequenced seven different genes in order to identify targets that were polymorphic among the genus Verticillium, but monoprphic in V. dahliae. After selection of beta-tubulin 2 as a suitable target, monoplex and duplex qPCR assays were performed using a Bio-Rad iCycler thermal cycler and 1ng DNA from various cultivars. For the duplex assays, the Plexor® qPCR System was used to amplify both beta-tubulin 2 and beta-actin genes simultaneously. One beta-tubulin primer was labeled with FAM, and one actin primer was labeled with Redmond Red phopsphoramidite. Amplifications were performed in 25µl reactions with 200nM each primer, 1ng DNA, and the Plexor® Master Mix. Cycling conditions were as follows: 2 minutes at 95°C; 40 cycles of 5s at 95°C, 35s at 61°C. Melt curve analysis was performed to confirm the specificity of the amplification products. The qPCR assays were shown to be faster and more sensitive than the standard plating technique, and were one order of magnitude more sensitive than other PCR-based assays. (3673)

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Nucl. Acids Res. 35, 2060–73. In vivo and in vitro investigation of bacterial type B RNase P interaction with tRNA 3'-CCA. 2007

Wegscheid, B. and Hartmann, R.K.

Notes: The authors used RT-PCR to quantitate the level of rnpB expression after complementing the Bacillus subtilis rnpB mutant strain SSB318 with wildtype or mutant rnpB genes from S. aureus or B. subtilis. Preliminary experiments with decreasing amounts of RNA template were performed to show that rnpB RT-PCR product yields were linearly dependent on RNA template amounts after 12 PCR cycles. RT-PCR was performed using the Access RT-PCR System. (3794)

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Emerging Infect. Dis. 13, 1756-1758. Human Bocavirus infection in children with gastroenteritis, Brazil. 2007

Albuquerque, M.C., Rocha, L.N., Benati, F.J., Soares, C.C., Maranhão, A.G., Ramírez, M.L, Erdman, D., and Santos, N.

Notes: In this study, the Wizard® Genomic DNA Purification Kit was used to extract DNA from diluted fecal samples. The extracted DNA was used in PCR with specific primers to detect viral sequences. PCR fragments were gel-purified using the SV Gel and PCR Clean-Up System prior to sequencing to confirm the Bocavirus DNA identity. (4221)

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