The ForenSeq™ FGx System (Illumina, San Diego, CA) was initially evaluated in concordance with SWGDAM guidelines for internal validation to determine the quality of the system's components: the ForenSeq™ DNA Signature Prep Kit reagents, the MiSeq FGx™ instrument, and the ForenSeq™ Universal Analysis Software, for the analysis of targeted, forensically informative single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). This multiplex consisted of STRs (autosomal, X, and Y) and SNPs (identity, ancestry, and phenotypic) that were run using one preparation process. Overall, the ForenSeq™ FGx System performed as well as the traditional capillary electrophoresis-based method in producing usable profile information, along with additional information that could aid in investigative leads. The MiSeq FGx™ System was validated using DNA samples in studies testing reproducibility, repeatability, concordance, sensitivity, and mock case single donor samples. Overall, genotyping results for STRs and SNPs were concordant with the profiles generated from conventional STR analysis using Identifiler and SNPs typed by 23andMe analysis. Genotypes of the ForenSeq™ aSNPs were used to evaluate biogeographical ancestry estimations using ForenSeq™ Universal Analysis Software, FROG-kb database (KIDD aiSNP 55 panel), and 23andMe. The system was shown to provide reproducible genotypes and reliable results were obtained at levels as low as 50 pg. All mock case samples were concordant with the donor profile. The results support consideration of the ForenSeq™ FGx System as an acceptable alternative to current STR and SNP analysis, pending formal developmental and internal validation studies.
pathways. Our CRPC miRNA expression signature is a landmark of the miRNA research for CRPC and novel CRPC pathways will be found based on this signature.
Epidemiological data indicate that dietary consumption of cruciferous vegetables may reduce the risk of many cancers including breast cancer. Cruciferous vegetables contain high levels of glucosinolates; stable phytochemicals that when metabolized produce cancer-preventing active isothiocyanates. Isothiocyanates, such as sulforaphane (SFN) found in broccoli sprouts, are thought to prevent tumorigenesis through epigenetic mechanisms controlling the expression of tumor-related genes. Ingestion of broccoli sprouts results in rapid distribution of SFN to breast epithelium, however, the effect of chronic exposure to low levels of SFN on the growth of breast cells has not been fully explored. In the present study we assessed the effect of SFN on the growth of an immortalized breast cell line (MCF10A), and two breast cancer cell lines; the estrogen receptor positive MCF-7 cell line, and the estrogen receptor negative TMX2-28 cell line. We first determined the seeding densities for each cell line that resulted in 80% confluence after three days of growth in T-25 flasks: MCF10A (1 x 106), MCF-7 (8 x 105), and TMX2-28 (5 x 105) per 5 mL. Next, cells from each line were plated at the predetermined seeding densities in T-25 flasks and treated with either SFN (5 μM) or vehicle (0.02% DMSO) for up to 40 days. Every third day, cell cultures were photographed, after which the cells were resuspended in culture medium, counted using a hemocytometer, and the predetermined numbers of cells (seeding numbers) were returned to T-25 flasks and treated with SFN or vehicle. Cells not needed for seeding the flasks were archived for future analysis of gene expression and DNA methylation. Treatment with SFN decreased cell growth. After 22 days of treatment with SFN, mean proliferation of both the MCF10A and MCF-7 cell cultures was inhibited by 62% (t = 2.8; p = 0.02) and 72% (t = 2.6; p = 0.02), respectively. In contrast, the decrease in proliferation of TMX2-28 cells treated with SFN was not statistically significant (t = 1.2; p = 0.255) from the vehicle control at 22 days. However, by day 40, TMX2-28 cell cultures treated with SFN showed a significant decrease in mean proliferation 36% (t = 2.2; p = 0.04). Ongoing studies are examining the epigenetic mechanisms by which SFN decreased cell proliferation. Citation Format: Ashley L. Silvia, Eva P. Browne, Kathleen F. Arcaro. Chronic, low-dose treatment with sulforaphane inhibits growth of breast cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5366. doi:10.1158/1538-7445.AM2013-5366
Background Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG expansion mutation in huntingtin (HTT). Mutant HTT induces cell death modulating several molecular pathways, including cell signalling, transcriptional machineries, and neurotrophins production and release. Recently, we have reported an early defect in transforming growth factor (TGF)β1 production in HD subjects and in two HD mouse models (R6/2 and YAC128). TGFβ1 is a pleiotropic cytokine with an established neuroprotective function as well as a powerful anti-inflammatory role. Aim Since TGFβ1 is predominantly expressed in the immune system and since myeloid derived immune cells (ie, glial cells) are implicated in neurodegeneration, we investigated the levels of this cytokine in immortalised human astroglial cell lines expressing wild-type (SVGp12-25Q) and mutant (SVGp12-72Q) HTT-exon1, and in macrophages from HD subjects and R6/2 mice. Methods We analysed the levels of TGFβ1 within cell lines by flow cytometry and by quantitative real time PCR. All HD subjects (n=28) were clinically and genetically evaluated and age matched with health controls (n=14). Results Our analysis showed significant reduced levels of TGFβ1 in human and mouse HD macrophages, and in human immortalised HD glial, compared with wild-type cells. Interestingly, TGFβ1 levels in human macrophages depended on the disease stage. Such scenario was confirmed by experiments in HD mice who showed significant low levels of the cytokine at the presymptomatic stage. Conclusion While confirming a TGFβ1 related dysfunction in peripheral and brain tissues in HD, we remark the potentiality for searching novel possible progression related markers in HD.
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