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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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Mol. Med. Reports Atenas 1, 123–129. HPV infection in Brazilian oral squamous cell carcinoma patients and its correlation with clinicopathological outcomes 2008

Oliveira, L.R., Ribeiro-Silva, A., Ramalho, L.N.Z., Simões, A.L. and Zucoloto, S.

Notes: In this study of the frequency of human papilloma virus (HPV) in patients with oral squamous cell carcinoma (OSCC), the MagneSil® Genomic, Fixed Tissue System was used to isolate DNA from formalin-fixed paraffin-embedded tissue samples from primary tumors and matched samples. Five microliters of genomic DNA was amplified using PCR Master Mix and primers for both a 110 bp fragment of human ß-globin gene and HPV genotype. After PCR, the product was analyzed on an 8% nondenaturing polyacrylamide gel and stained with AgNO3. (3938)

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Cancer Res. 2007, 6146–54. Incorporation of an internal ribosome entry site-dependent mechanism in arsenic-induced GADD45 alpha expression. 2008

Chang, Q,. Bhatia, D., Zhang, Y., Meighan, T., Castranova, V., Shi, X. and Chen, F.

Notes: Trivalent arsenic (As3+) induces expression of growth arrest- and DNA damage-induced gene 45α (GADD45α), which interacts with intracellular signaling molecules involved in cell cycle regulation, apoptosis and immune response. To characterize GADD45α mRNA expression patterns, total RNA was isolated from untreated (growing) and As3+-treated (arrested) BEAS-2B cells, and GADD45α mRNA was amplified using the AccessQuick™ RT-PCR System. GADD45α mRNA levels were undetectable in growing cells but increased in a time-dependent manner in arrested cells. Reverse transcription was carried out at 45°C for 50 minutes, followed by 35 cycles of PCR. (3766)

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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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J. Cell Sci. 121, 504–13. Integrin alpha9 beta1 is a receptor for nerve growth factor and other neurotrophins. 2008

Staniszewska, I., Sariyer, I.K., Lecht, S., Brown, M.C., Walsh, E.M., Tuszynski, G.P., Safak, M., Lazarovici, P. and Marcinkiewicz, C.

Notes: The authors investigated the ability of α9β1 integrin to act as a neurotrophin receptor and affect cell signaling pathways. As part of the study, RT-PCR was used to detect the presence of other neurotrophin receptors in their model cell line, SW480. Reverse transcription was performed using the Reverse Transcription System and 1µg of total RNA isolated using the SV Total RNA Isolation System. The resulting cDNA (5µg) was amplified for 35 cycles (β-actin as a control) or 40 cycles (TrkA and p75NTR) using GoTaq® Green Master Mix. RT-PCR results were confirmed by Western blot analysis. (3884)

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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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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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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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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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Clin. Can. Res. 14, 5033–42. Midkine enhances soft-tissue sarcoma growth: a possible novel therapeutic target. 2008

Jin, Z., Lahat, G., Korchin, B., Nguyen, T., Zhu, Q.S., Wang, X., Lazar, A.J., Trent, J., Pollock, R.E. and Lev D.

Notes: Increased expression of midkine (MK), a growth factor normally involved in neural development, is associated with several human malignancies. The authors used quantitative RT-PCR to examine mRNA levels for MK and MK receptors in various soft-tissue sarcoma (STS) cell lines. Reverse transcription was performed using 1µg of total RNA, and 2µl of cDNA was used in qPCR using the PCR Master Mix, primers specific to MK and either glyceraldehyde-3-phosphate or β-actin primers for normalization, and EvaGreen® dye. To examine the protumorigenic effects of MK, the authors incubated HT1080 and SW684 STS cell lines, which express low levels of MK, with human recombinant MK and measured cell proliferation using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay. In addition, the authors examined cell proliferation in MK-stably transfected HT1080 cells. The plasmid used for stable transfections was created by reverse transcribing the MK-coding region using the ImProm-II™ Reverse Transcription System, then cloning the resulting cDNA into an expression vector. (3895)

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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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Haematologica 93, 1505–1513. Molecular characterization of three novel splicing mutations causing factor V deficiency and analysis of the F5 gene splicing pattern. 2008

Dall'Osso, C., Guella, I., Duga, S., Locatelli, N., Paraboschi, E.M., Spreafico, M., Afrasiabi, A., Pechlaner, C., Peyvandi, F., Tenchini, M.L. and Asselta, R.

Notes: To examine the causes of Factor V (FV) deficiency, the authors examined transcript splicing and its mutated variations. Three regions of human FV (F5) were amplified from a healthy individual and the PCR products cloned into the pTargeT™ Mammalian Expression Vector. Three identified mutations from people with FV deficiency were introduced by site-directed mutatgenesis. All constructs were sequenced before transfection into HeLa cells. After 48 hours, the total RNA was purified and the splicing pattern of the wild type and mutant constructs were analyzed by RT-PCR. The mutant constructs were also transfected into HepG2 cells and tested for nonsense-mediated mRNA decay (NMD) with or without NMD inhibitors (puromycin, cycloheximide, and wortmannin) using RT-PCR. (3992)

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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, 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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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, 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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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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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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J. Clin. Microbiol. 46, 3752–3758. Plastic polymers for efficient DNA microarray hybridization: application to microbiological diagnostics. 2008

Zhao, Z., Peytavi, R., Diaz-Quijada, G.A., Picard, F.J., Huletsky, A., Leblanc, E., Frenette, J., Boivin, G., Veres, T., Dumoulin, M.M. and Bergeron, M.G.

Notes: A plastic support suitable for use in microfluidic systems for highly sensitive DNA microarray hybridizations was developed and tested. Human DNA from Hsap-11 cells was isolated using the MagneSil® KF, Genomic System on a KingFisher ML instrument. Ten nanograms of the isolated DNA was used in RT-PCR. (4021)

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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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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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Biochem. Biophys. Res. Commun. 373, 48–52. Selection of mRNA 5´-untranslated region sequence with high translation efficiency through ribosome display. 2008

Mie, M., Shimizu, S., Takahashi, F. and Kobatake, E.

Notes: The authors developed an in vitro selection system that is based on ribosome display and favors identification of 5´-untranslated regions (UTRs) with high translation efficiencies. A 5´-UTR random library was created in which the 5´-UTRs were upstream of a polyhistidine-tag/Renilla luciferase-coding region. In vitro transcripts from this library were translated in vitro using the Flexi® Rabbit Reticulocyte Lysate System. The authors preferentially selected mRNAs with high translational efficiencies by shortening the translation time and capturing ternary complexes of mRNA, ribosome and nascent proteins. These complexes were captured using MagneHis™ Ni Particles. RNA was extracted from these complexes and used as a template in RT-PCR for the next round of selection. Before and after each round of selection, 9µl of RNA was translated in vitro, and 20µl of translated product was removed every 5 minutes to measure Renilla luciferase activity and monitor translation efficiency. Renilla luciferase was measured using the Renilla Luciferase Assay System. After two rounds of selection, RT-PCR products were cloned into a pUC18 vector, the sequences of the resulting plasmids were confirmed, and 0.5µg of plasmid was translated in vitro using the TNT® T7 Coupled Rabbit Reticulocyte Lysate System to further evaluate translation efficiency. (3963)

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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. Biol. Chem. 283, 23514–23. Snail regulates cell-matrix adhesion by regulation of the expression of integrins and basement membrane proteins. 2008

Haraguchi, M., Okubo, T., Miyashita, Y., Miyamoto, Y., Hayashi, M., Crotti, T.N., McHugh, K.P. and Ozawa, M.

Notes: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

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J. Bacteriol. 190, 1649–1657. Structural and biological characterization of a capsular polysaccharide produced by Staphylococcus haemolyticus. 2008

Flahaut, S., Vinogradov, E., Kelley, K.A., Brennan, S., Hiramatsu, K. and Lee, J.C.

Notes: The authors wanted to purify and characterize the capsular polysaccharide (CP) produced by Staphylococcus haemolyticus strain JCSC1435. S. haemolyticus strains grown in TSB cultures were harvested, lysed with lysozyme and lysostaphin and genomic DNA isolated using the Wizard® Genomic DNA Purification Kit. The DNA was then used in CP gene PCR. Total RNA was isolated from exponential, postexponential, and stationary phases of S. haemolyticus growth and used in RT-PCR using the Access RT-PCR System. (3977)

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