In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the invasiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in invasive cell lines when compared to normal and less invasive cell lines. Transfection of miR-200c, miR-205, and miR-375 mimics into MDA-MB-231 cells led to the inhibition of in vitro cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that miR-200c plays a pivotal role in regulating the invasiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer invasiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

The catalytic promiscuity of the novel benzophenone C-glycosyltransferase, MiCGT, which is involved in the biosynthesis of mangiferin from Mangifera indica, was explored. MiCGT exhibited a robust capability to regio- and stereospecific C-glycosylation of 35 structurally diverse druglike scaffolds and simple phenolics with UDP-glucose, and also formed O- and N-glycosides. Moreover, MiCGT was able to generate C-xylosides with UDP-xylose. The OGT-reversibility of MiCGT was also exploited to generate C-glucosides with simple sugar donor. Three aryl-C-glycosides exhibited potent SGLT2 inhibitory activities with IC50  values of 2.6×, 7.6×, and 7.6×10(-7)  M, respectively. These findings demonstrate for the first time the significant potential of an enzymatic approach to diversification through C-glycosidation of bioactive natural and unnatural products in drug discovery.

We conducted a genome-wide DNA methylation analysis in CD19 (+) B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.

Four new polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid metabolites, pericoannosin A (1), with an unusual hexahydro-1H-isochromen-5-isobutylpyrrolidin-2-one skeleton, and three cytochalasans, periconiasins D-F (2-4), were isolated from the endophytic fungus Periconia sp. F-31. Their structures and absolute configurations were elucidated through extensive spectroscopic analyses, calculated ECD, and single-crystal X-ray diffraction (Cu Kα). A possible biogenetic pathway is proposed. Compounds 1 and 4 showed anti-HIV activity with IC50s of 69.6 and 29.2 μM, respectively.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

C-Glycosyltransferases (CGTs) are powerful tools for the C-glycosylation of natural and unnatural products. However, CGTs able to catalyze bis-C-glycosylation are very rare and the key amino acids of which have not been uncovered. Here, we discovered a C-glycosyltransferase MiCGTb from Mangifera indica that has the capacity for bis-C-glycosylation. Further studies on active-site motifs revealed that I152 of MiCGTb was the critical amino acid residue for the second C-glycosylation and its S60/V100/T104 residues were pivotal for bis-C-glycosylation activity. Moreover, we developed a panel of variants with acceptor and donor promiscuity by site-directed mutagenesis. Among these variants, a mutant MiCGT-E152L displayed a broader acceptor scope for bis-C-glycosylation, and three mutants of MiCGTb exhibited sugar donor promiscuity toward structurally varied α-Dand β-Lglycosyl donors. Our work provides insights into the pivotal amino acid residues of CGTs for bis-C-glycosylation and biocatalytic tools to efficiently produce structurally diverse bis-C-glycosides with two identical or different sugar moieties in drug discovery.

Nine new polyoxygenated eremophilane sesquiterpenes, periconianones C-K (1-9), including one unusual isoeremophilane sesquiterpene, periconianone C (1), and four trinor-eremophilane sesquiterpenes, periconianones H-K (6-9), were isolated from the endophytic fungus Periconia sp. F-31. Compound 1 is the first furan-type isoeremophilane reported containing a linkage of C-8/C-11 and a 7,12-epoxy moiety. These compound structures, including absolute configurations, were elucidated through extensive spectroscopic data analysis, electronic circular dichroism, Mo2(AcO)4-induced circular dichroism, and single-crystal X-ray diffraction (Cu Kα). Compounds 2, 5, and 9 showed inhibition effects on lipopolysaccharide-induced NO production in BV2 cells by 10.2%, 18.3%, and 16.1% at a concentration of 1.0 μM, respectively, which is comparable to the positive control curcumin (12.9% at 1.0 μM).

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