SPARC promoter hypermethylation in colorectal cancers can be reversed by 5-Aza-2′deoxycytidine to increase SPARC expression and improve therapy response

Poor clinical outcomes in cancer can often be attributed to inadequate response to chemotherapy. Strategies to overcome either primary or acquired chemoresistance may ultimately impact on patients' survival favourably. We previously showed that lower levels of SPARC were associated with therapy-refractory colorectal cancers (CRC), and that upregulating its expression enhances chemo-sensitivity resulting in greater tumour regression in vivo. Here, we examined aberrant hypermethylation of the SPARC promoter as a potential mechanism for repressing SPARC in CRCs and whether restoration of its expression with a demethylating agent 5-Aza-2′deoxycytidine (5-Aza) could enhance chemosensitivity. Initially, the methylation status of the SPARC promoter from primary human CRCs were assessed following isolation of genomic DNA from laser capture microdissected specimens by direct DNA sequencing. MIP101, RKO, HCT 116, and HT-29 CRC cell lines were also used to evaluate the effect of 5-Aza on: SPARC promoter methylation, SPARC expression, the interaction between DNMT1 and the SPARC promoter (ChIP assay), cell viability, apoptosis, and cell proliferation. Our results revealed global hypermethylation of the SPARC promoter in CRCs, and identified specific CpG sites that were consistently methylated in CRCs but not in normal colon. We also demonstrate that SPARC repression in CRC cell lines could be reversed following exposure to 5-Aza, which resulted in increased SPARC expression, leading to a significant reduction in cell viability (by an additional 39% in RKO cells) and greater apoptosis (an additional 18% in RKO cells), when combined with 5-FU in vitro (in comparison to 5-FU alone). Our exciting findings suggest potential diagnostic markers of CRCs based on specific methylated CpG sites. Moreover, the results reveal the therapeutic utility of employing demethylating agents to improve response through augmentation of SPARC expression

Transcriptomics of diapause in an isogenic self-fertilizing vertebrate.

BackgroundMany vertebrate species have the ability to undergo weeks or even months of diapause (a temporary arrest of development during early ontogeny). Identification of diapause genes has been challenging due in part to the genetic heterogeneity of most vertebrate animals.ResultsHere we take the advantage of the mangrove rivulus fish (Kryptolebias marmoratus or Kmar)-the only vertebrate that is extremely inbred due to consistent self-fertilization-to generate isogenic lineages for transcriptomic dissection. Because the Kmar genome is not publicly available, we built de novo genomic (642 Mb) and transcriptomic assemblies to serve as references for global genetic profiling of diapause in Kmar, via RNA-Seq. Transcripts unique to diapause in Kmar proved to constitute only a miniscule fraction (0.1 %) of the total pool of transcribed products. Most genes displayed lower expression in diapause than in post-diapause. However, some genes (notably dusp27, klhl38 and sqstm1) were significantly up-regulated during diapause, whereas others (col9a1, dspp and fmnl1) were substantially down-regulated, compared to both pre-diapause and post-diapause.ConclusionKmar offers a strong model for understanding patterns of gene expression during diapause. Our study highlights the importance of using a combination of genome and transcriptome assemblies as references for NGS-based RNA-Seq analyses. As for all identified diapause genes, in future studies it will be critical to link various levels of RNA expression with the functional roles of the coded products

Complete mitochondrial genome of a self-fertilizing fish Kryptolebias marmoratus (Cyprinodontiformes, Rivulidae) from Florida.

The complete mitochondrial genome was sequenced in a mangrove rivulus Kryptolebias marmoratus from western Florida using next-generation sequencing. The 17 329 bp-long genome was identical in length and 99.8% similar to a previously published genome of this species from a specimen of unknown geographic origin. Gene arrangement in K. marmoratus is similar to other cyprinodontiform fishes, except for the presence of a second copy of the control region inserted upstream of the nad1 gene

A Competitive Pricing Model

This paper is principally concerned with the development of a competitive pricing model aimed at predicting the impact of a vector of price increases on sales of a given brand of a low priced, frequently purchased product. The model has its base in a set of assumptions describing the purchasing behavior of the individual consumer. A method for testing the model against empirical data is described and illustrated. The competitive pricing model is found to perform somewhat better than a simple regression model in the application reported.marketing: pricing, marketing: buyer behavior

Pengaruh Gaya Mengajar Komando terhadap Gerak Dasar Menendang Bola

Dari hasil pengamatan, banyak mahasiswa Pendidikan Jasmani Kesehatan & Rekreasi (PJKR) Fakultas Ilmu Keoalhragaan (FIK) Unima yang belum bisa melakuakn gerak dasar menendang bola dengan baik dan benar. Penelitian ini bertujuan untuk mengetahui seberapa besar pengaruh gaya mengajar komando tehadap gerak dasar menendang bola menggunakan kaki bagian dalam pada pemainan sepak bola. Metode yang digunakan yaitu metode eksperimen. Sampel berasal dari Mahasiswa PJKR FIK Unima yang berjumlah 20 orang yang diambil secara acak (simple random sampling). Instrumen yang digunakan yaitu tes gerka dasar menendang bola menggunakan kaki bagian dalam pada permaianan sepak bola dari awal sampai akhir. Teknik analisa data yang digunakan yaitu teknis analisis statistik Uji-t. Adapun hasil penelitian yaitu thitung = 3,830 ttabel = 2,101. Kesimpulan penelitian ini adalah terdapat pengaruh penerapan gaya mengajar komando terhadap gerak dasar dasar menendang bola menggunakan kaki bagian dalam pada permainan sepak bola Mahasiswa PJKR FIK Unima

Hundreds of SNPs vs. dozens of SSRs: which dataset better characterizes natural clonal lineages in a self-fertilizing fish?

For more than two decades, mitochondrial DNA sequences and simple sequence repeats (SSRs, or microsatellite loci) have served as gold standards in population genetics. More recently, next generation sequencing (NGS) has enabled researchers to address biological questions that can benefit from hundreds or even thousands of nuclear single-nucleotide polymorphisms (SNPs) generated by restriction-site associated DNA sequencing (RAD-seq). Here we compare the performance of SSR and RAD-seq SNP methods to characterize clonal patterns in a self-fertilizing and highly inbred killifish, Kryptolebias marmoratus (mangrove rivulus) in Florida. RAD-seq analyses conducted on 18 inbred lineages of mangrove rivulus obtained from western Florida and a distant location in eastern Florida unveiled 481 polymorphic RAD loci of which 129 were homozygous within individuals and 352 loci were heterozygous in at least one individual. An initial UPGMA phenogram was constructed, based on 32 microsatellite loci, and used as a benchmark for comparisons with SNP-based phenograms, using a number of different criteria for SNP selection. A phenogram produced by the homozygous SNPs was in excellent agreement with the one generated from 32 microsatellite loci. However, heterozygous SNP data and RAD loci with more than one polymorphic site contributed more noise than usable signal and were unable to resolve clades consistently. This is likely due to errors in identifying homologous loci in the absence of a reference genome. In summary, although the RAD data were powerful in distinguishing the clonal lineages identified by SSR analyses, they also carried considerable phylogenetic noise. Our results suggest that RAD-seq methods should be used with caution for inferring fine population structure, and that stringent quality controls are necessary to reduce false phylogenetic signals