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J. Biol. Chem. 286, 19478–19488. Thrombomodulin is silenced in malignant mesothelioma by a poly(ADP-ribose) polymerase-1-mediated epigenetic mechanism. 2011

Nocchi, L., Tomasetti, M., Amati, M., Neuzil, J., Santarelli, L. and Saccucci, F.

Notes: Thrombomodulin (TM) expression was examined by isolating genomic DNA from biopsies of human malignant mesothelioma and normal mesothelial tissue, and cultured cell lines with or without PARP1 silencing treated with 5-aza-2´-deoxycytidine and trichostatin alone or in combination and then subjected to biosulfide modification. To analyze methylation of TM, a CpG island in the promoter, 5´ UTR and an exon region containing 44 CpG dinucleotides were PCR amplified, cloned into the pGEM®-T Easy Vector, transformed and positive clones selected using IPTG/X-Gal and analyzed by PCR. Colonies were cultured, the plasmids isolated using the Wizard® Plus SV Minipreps DNA Purification System then 10 clones
from each sample type were sequenced. (4132)

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J. Gen. Mol. Virol. 3, 18–26. Molecular epidemiology of human enterovirus71 (HEV71) strains isolated in Peninsular Malaysia and Sabah from year 2001 to 2009 2011

Yusof, M.A., Rais, F., Abdullah, M.A., Zamri, L.A., Ali, H.M., Kassim, F.M. and Saat, Z.

Notes: This study characterized the strains of hand, foot and mouth disease (HFMD) that circulated in Peninsular Malaysia and Sabah during the last ten years. Viral RNA was extracted from vesicle, throat and rectal swabs, and 5µl of purified RNA was amplified in a 50µl reaction using the AccessQuick™ RT-PCT System with primers for VP4 or VP1. The amplified products were then sequenced and compared to known HFMD sequences. (4341)

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J. Biol. Chem. 286, 37196–206. 5-Aza-2'-deoxycytidine activates iron uptake and heme biosynthesis by increasing c-myc nuclear localization and binding to the e-boxes of transferrin receptor 1 (TfR1) and ferrochelatase (Fech) genes. 2011

Ning, B., Liu., G., Liu, Y., Su, X., Anderson, G.J., Zheng, X., Chang, Y., Guo, M., Liu, Y., Zhao, Y. and Nie, G.

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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DNA Research 18, 93–105. The complete chloroplast genome of 17 individuals of pest species Jacobaea vulgaris: SNPs, microsatellites and barcoding markers for population and phylogenetic studies 2011

Doorduin, L., Gravendeel, B., Lammers, Y., Ariyurek, Y., Chin-A-Woeng, T. and Vrieling, K.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to extract and purify DNA fragments that had been amplified by long-range PCR and separated on an agarose gel prior to next-generation sequencing on an Illumina platform. (4535)

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Genome Res. 21, 1738-45. Application of microdroplet PCR for large-scale targeted bisulfite sequencing 2011

Komori, H.K., LaMere, S.A., Torkamani, A., Hart, G.T., Kotsopoulos, S., Warner, J., Samuels, M.L., Olson, J., Head, S.R., Ordoukhanian, P., Lee, P.L., Link, D.R. and Salomon, D.R.

Notes: The authors of this study sought to correlate promoter methylation with gene expression. Gene expression data was generated by RNA-seq of Jurkat cells. Amplified cDNA was prepared from total RNA, the cDNA was treated with S1 nuclease to remove single stranded nucleic acids and used as template for the sequencing libraries. (4536)

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S1 Nuclease

Blood Cells Mol. Dis. 46, 139-144. Application of MLPA assay to characterize unsolved α-globin gene rearrangements. 2011

Colosimo, A,. Gatta, V., Guida, V., Leodori, E., Foglietta, E., Rinaldi, S., Cappabianca, M.P., Amato, A., Stuppia, L., and Dallapiccola, B.

Notes: These authors used the Maxwell® 16 Blood DNA Purification Kit to isolate genomic DNA from leukocytes. (4210)

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mBio. 2(6), e00275-11. Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. 2011

Chen, J., Rood, J.I., and McClane, B.A.

Notes: These authors investigated whether ETX toxin production in C. perfringens type D is regulated by the Agr-like quorum sensing system, the VirS/VirR system, or both. They demonstrated that an agr mutant lacked ETX expression, and showed that lack of VirR had no effect on ETX production. The AccessQuick™ System was used in RT-PCR analysis of RNA isolated from mutant, wildtype and reconstituted (complemented) strains to confirm absence of agr transcripts in mutant strains. RT-PCR was also used to confirm the presence of etx transcripts in wild type strains, and their absence in the agr mutant. Previous studies had shown that toxin production is upregulated upon contact with enterocyte-like Caco-2 cells. This study showed that that the agr operon is required for such upregulation. (4289)

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PLos ONE 6(9), e24417. Metagenome plasticity of the bovine abomasal microbiota in immune animals in response to Ostertagia ostertagi infection. 2011

Li, R.W., Wu, S., Li, W., Huang, Y., and Gasbarre, L.C.

Notes: These authors performed metagenomic analysis to investigate any changes in composition of the microbial flora of the abomasa (ruminant 4th stomach compartment) in immune cattle after reinfection with the nematode Ostertagia ostertagi. DNA was extracted from abomasal contents using a QIAamp stool kit and the integrity verified using an Agilent Bioanalyzer 2000. PCR was then performed using primers targeting hypervariable regions of 16S rRNA genes. The amplified material was gel-purified and sequenced using the Roche 454 pyrosequencing system. DNA concentration was measured before and after PCR using the QuantiFluor™ Fluorometer.


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Proc. Natl. Acad. Sci. USA 108, 17159–64. Identification of the bacterial protein FtsX as a unique target of chemokine-mediated antimicrobial activity against Bacillus anthracis. 2011

Crawford, M.A., Lowe, D.E., Fisher, D.J., Stibitz, S., Plaut, R.D., Beaber, J.W., Zemansky, J., Mehrad, B., Glomski, I.J., Strieter, R.M. and Hughes, M.A.

Notes: The authors identified three genetic loci involved in chemokine-mediated antimicrobial effects against Bacillus anthracis using a transposon mutant library in which a transposon is randomly inserted into the B. anthracis genome, then treating the mutant cells with chemokine to select for resistant cells. To identify the transposon insertion site, and thus the resistance-conferring loci, the authors amplified regions flanking the transposon by PCR using the GoTaq® Green Master Mix. (4167)

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J. Exp. Bot. 62, 5217–31. The barley amo1 locus is tightly linked to the starch synthase IIIa gene and negatively regulates expression of granule-bound starch synthetic genes. 2011

Li, Z., Li, D., Du, X., Wang, H., Larroque, O., Jenkins, C.L., Jobling, S.A. and Morell, M.K.

Notes: The authors investigated starch synthesis in barley (Hordeum vulgare) by examining mutations in class I, class II and class III starch synthases (ssI, ssII and ssIII, respectively). Mutations of ssIIa and ssIIIa were detected by PCR using the GoTaq® Hot Start Polymerase. (4162)

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PLos ONE 6(7), E22438. Krüppel-Like Factor 6 Expression Changes during Trophoblast Syncytialization and Transactivates ßhCG and PSG Placental Genes. 2011

Racca A.C., Camolotto S.A., Ridano M.E., Bocco J.L., Genti-Raimondi S., and Panzetta-Dutari, G.M.

Notes: These authors studied KLF6 expression during human trophoblast cell differentiation, and its role in the regulation of genes associated with placental development and pregnancy maintenance. They used immunofluorescence microscopy, RT-qPCR and luciferase reporter assays to investigate cellular localization, mRNA expression, and transcriptional activation. Reporter assays were performed using various luciferase reporter constructs, the Dual-Luciferase® Assay, and the GloMax®-Multi Detection System. KLF6 was shown to play a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as βhCG and PSG. (4197)

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PLos ONE 6, e25263. Analysis of 16S rRNA amplicon sequencing options on the Roche/454 next-generation titanium sequencing platform 2011

Tamaki, H., Wright, C.L., Li, X., Lin, Q., Hwang, C., Wang, S., Timmapuram, J., Kamagata, Y. and Liu, W.T.

Notes: DNA was isolated from a variety of environmental samples including surface soil, drinking water biofilm, sludge from an anaerobic digester, bioreactor samples, ground water, peat soil and glacial deposit soil. The 16S rRNA gene was amplified from the DNA. PCR amplifications were run on agarose gels, and bands of the predicted sizes excised and purified using the Wizard® SV Gel and PCR Clean-Up System before pooling for pyrosequencing. (4554)

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Nucl. Acids Res. [Epub ahead of print]. RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage. 2011

Dai, W., Zhang, G. and Makeyev, E.V.

Notes: The full-length 3´ UTR of mouse RNA-binding protein HuR was amplified and cloned into psiCHECK™-1 Vector to create pEM429 plasmid. To generate radiolabeled RNA substrates for use in cleavage assays, RNA was synthesized from 1µg of linearized plasmid using 50µCi of [α-32P]UTP, 0.8mM Ribo m7G Cap Analog and T7 RNA Polymerase by incubating for 1.5 hours at 37°C. The RNAs were then treated with 1 unit of RQ1 RNase-Free DNase per 1µg of template DNA for 15 minutes at 37°C before extracting with phenol:chloroform, precipitating with ethanol and resuspending in DEPC-treated water. The cleavage assay used 60fmol of 32P-labeled substrate RNA with 10U Recombinant RNAsin Ribonuclease Inhibitor in a reaction incubated for 2.5 hours at 30°C. RNA probes were labeled with biotin using T7 or T3 RNA Polymerases with a biotin-UTP labeling NTP mixture and incubated for 2 hours at 37°C. The biotinylation reaction was then treated with RQ1 RNase-Free DNase following the same protocol used for radiolabeled RNA. To form HuR/RNA complexes, 2µg of biotinylated RNA was mixed with 100µg nuclear extract and 40 units Recombinant RNAsin® Ribonuclease Inhibitor in a total volume of 20µl, and incubated for 30 minutes at room temperature. For CstF-64/RNA complexes, 1µg of biotinylated RNA was used and the complexes were stabilized by UV crosslinking using 10U Recombinant RNAsin Ribonuclease Inhibitor during the UV treatment. NIH 3T3 cells were UV crosslinked and either total or nuclear RNA used for immunoprecipitation. After extraction the RNAs were treated with RQ1 RNase-Free DNase for 15 minutes at 37°C before RT-PCR using HuR-specific primers. Total RNA purified from cultured cells were incubated with 50U/ml RQ1 RNase-Free DNase at 37°C for 30 minutes before use in RT-qPCR. (4187)

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Proc. Natl. Acad. Sci. USA 108, 18488–93. Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy. 2011

Allen, J.A., Yost, J.M., Setola, V., Chen, X., Sassano, M.F., Chen, M., Peterson, S., Yadav, P.N., Huang, X.P., Feng, B., Jensen, N.H., Che, X., Bai, X., Frye, S.V., Wetsel, W.C., Caron, M.G., Javitch, J.A., Roth, B.L. and Jin, J.

Notes: This paper explored potential compounds as agonists of dopamine D2 receptor (D2R) with a bias toward β-arrestin signaling. Based on the aripiprazole scaffold, compounds were synthesized and tested in a D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R transfected with pGloSensor™-22F cAMP Plasmid. Assessing β-arrestin recruitment to agonist-stimulated receptors was determined using HTLA cells stably expressing β-arrestin-TEV protease and a tetracycline transactivator-driven luciferase exposed to agonist or D2 test ligand with or without reference agonist. After 18 hours, medium was removed from the cells, 1X Bright-Glo™ Reagent added and luminescence measured. (4518)

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J. Microbiol. Methods 81, 127-134. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis. 2011

Salonen, A., Nikkilä, J., Jalanka-Tuovinen, J., Immonen, O., Rajilić-Stojanović, M., Kekkonen, R.A., Palva, A., and de Vos, W.M.

Notes: These authors compared the performance of four DNA purification methods for recovery of bacterial and archaeal DNA from fecal material. One of the methods tested was the Wizard® Genomic DNA Purification Kit, which uses a solution-based, enzymatic method for extraction. The Wizard® Genomic method was rated highly on extraction speed, and gave the highest DNA yields. A second method involving mechanical disruption (repeated bead beating) was rated more highly on extraction efficiency from archaea and some bacterial species. The criteria for performance comparison are described fully in the paper. (4219)

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Appl. Environ. Microbiol. 77, 2113–21. General suppression of Escherichia coli O157:H7 in sand-based dairy livestock bedding. 2011

Westphal, A., Williams, M.L., Baysal-Gurel, F., LeJeune, J.T. and McSpadden Gardener, B.B.

Notes: The authors investigated the suppression of E. coli O157:H7 in sand-based livestock bedding and hypothesized that suppression of E. coli O157:H7 growth was mediated by an environmentally stable population of pathogen-suppressing bacteria. These bacteria were identified by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA gene sequences isolated from used bedding followed by cloning and sequencing of the most abundant terminal restriction fragments. Amplifications were performed using the GoTaq® Flexi DNA Polymerase, then PCR products were cloned into the pGEM®-T Easy Vector. The PureYield™ Plasmid Miniprep System was used to purify plasmids for sequencing. (4165)

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Nucl. Acids Res. Dec 8, Epub ahead of print. Protein-mediated protection as the predominant mechanism for defining processed mRNA termini in land plant chloroplasts. 2011

Zhelyazkhova, P., Hammani, K., Rojas, M., Voelker, R., Vargas-Suárez, M., Börner, T., and Barkan, A.

Notes: Pentatricopeptide repeat (PPR) proteins are helical repeat proteins that bind specific RNA segments and protect the adjacent RNA by serving as a barrier to exoribonucleases. This study showed that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. The authors used RNasin® Ribonuclease Inhibitor in binding reactions between labeled RNA and PPR proteins prior to Gel mobility shift assays. They also used the pGEM®-T Vector to clone various 3´ RNA terminal sequences amplified by RT-PCR. (4185)

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J. Biol. Chem. 286, 42690-42703. Alternative Splicing Produces Nanog Protein Variants with Different Capacities for Self-renewal and Pluripotency in Embryonic Stem Cells. 2011

Das, S., Jena, S., and Levasseur, D.N.

Notes: The transcription factor Nanog is required for the maintenance of embryonic stem (ES) cell pluripotency. These authors showed that the Nanog N-terminal domain is regulated by post-transcriptional modification, and that alternative splicing generates Nanog variants with different capacities for maintaining an undifferentiated cell state. As part of their study, the authors used GoScript® Reverse Transcriptase to to generate cDNA from RNA extracted from cell lines expressing different Nanog variants. The cDNA was used in RT-qPCR to quantify relative expression levels.

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J. Clin. Microbiol. 49, 281–291. Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers. 2011

Stressmann, F.A., Rogers, G.B., Klem, E.R., Lilley, A.K., Donaldson, S.H., Daniels, T.W., Carroll, M.P., Patel, N., Forbes, B., Boucher, R.C., Wolfgang, M.C. and Bruce, K.D.

Notes: Sputum samples were collected from cystic fibrosis patients and 16S rRNA sequences amplified by PCR. These products were cloned into a T-vector, transformed into competent cells and the resulting colonies grown in 2ml LB broth in 96-deep-well plate for 20 hours. Of this culture, 1.9ml was pelleted and the clones isolated using the Wizard® SV Plasmid Purification System. The purified plasmid DNA was subjected to agarose gel electrophoresis and sequenced. (4133)

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Biochem. J. 436, 387–397. The novel Nrf2-interacting factor KAP1 regulates susceptibility to oxidative stress by promoting the Nrf2-mediated cytoprotective response. 2011

Maruyama, A., Nishikawa, K., Kawatani, Y., Mimura, J., Hosoya, T., Harada, N., Yamamato, M. and Itoh, K.

Notes: These authors first used a FLAG-tagged protein (nfr2) with a HeLa Nuclear extract and captured interacting proteins via SDS-PAGE and in-gel digests of bands to identify (Krüppel-associated box)-associated protein 1 (KAP1) as a potential interacting partner. Human KAP1 was purchased as a HaloTag® CMV Flexi® Vector from Kazusa and used in a Mammalian PullDown scenario (with HaloLink™ Resin) to demonstrate interaction between the two proteins. A reporter assay was used to show that KAP1 facilitates Nrf2 transactivation in a dose-dependent manner. The authors defined the interaction sites using GST-tagged nrf2 and various forms of KAP1-HaloTag® Fusions expressed in TNT® SP6 High-Yield Wheat Germ Extract. GST-tagged proteins were expressed in E. coli and bound to glutathione-Sepharose beads. These bound proteins were mixed with the KAP1 from the cell-free expression system, incubated for 4 hours at 4°C, washed and stained with the HaloTag® TMR Ligand for 30 minutes. The proteins from the pull-down assay were subjected to SDS-PAGE and the HaloTag® proteins detected by phosphorimaging and the GST proteins by Coomassie Brilliant Blue Staining. A two-hybrid system consisting of the pRL-TK Vector with a firefly luciferase reporter with Gal4 UAS, mouse Nrf-2 N-terminal domain and KAP1 was also used. The vectors were transfected into Nrf2 knockout MEFs for 4 hours then incubated for 36 hours before luciferase expression was determined using the Dual-Luciferase® Reporter Assay System. (4123)

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Nucl. Acids Res. 38, 522–33. An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes 2010

Jex, A.R., Hall, R.S., Littlewood, D.T. and Gasser, R.B.

Notes: Wizard® SV Gel and PCR Clean-Up System was used to clean up genomic DNA isolated from parasitic nematodes isolated from a variety of animals. Species identification of each nematode specimen was determined via PCR amplification of specific nuclear DNA followed by purification of the amplified product using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. Wizard® SV Gel and PCR Clean-Up System was also used to prepare amplicons generated by long-PCR of mt genomes from the nematodes before NGS sequencing. Results from NGS were confirmed using PCR-based sequencing of short mt DNA tracts. Short mtDNA regions were amplified by conventional PCR. Amplicons were purified using Wizard® PCR Preps DNA Purification System before sequencing using BigDye chemistry. (4533)

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Vet. Pathol. 47, 163–6. Peliosis hepatis in cats is not associated with Bartonella henselae infections. 2010

Buchmann, A.U., Kempf, V.A., Kershaw, O. and Gruber, A.D.

Notes: The vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl2 and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

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J. Biomol. Scr. 15, 418–26. Epitope mapping of antibodies using a cell array-based polypeptide library. 2010

Maier, R.H., Maier, C.J., Rid, R., Hintner, H., Bauer, J.W. and Onder, K.

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

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J. Exp. Bot. 61, 395–404. CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis. 2010

Ruhlmann, J.M., Kram, B.W. and Carter C.J.

Notes: The authors used microarray analysis to identify genes that are differentially expressed in nectaries of Arabidopsis and may be involved in nectar synthesis and secretion. One of these genes was cell wall invertase 4 (cwinv4). The authors generated two Arabidopsis cwinv4 mutant lines to study gene function and used GoTaq® Green Master Mix to confirm the mutant genotype. Expression patterns of cwinv4 in wildtype Arabidopsis and an ortholog from Brassica rapa were examined in various tissues by RT-PCR. The reverse transcription step was performed using the Reverse Transcription System and 0.1µg of RNA. (4093)

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Appl. Environ. Microbiol. 76, 829–42. Lysogeny and sporulation in Bacillus isolates from the Gulf of Mexico. 2010

Mobberley, J., Authement, R.N., Segall, A.M., Edwards, R.A., Slepecky, R.A. and Paul, J.H.

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

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