(http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Amphioxus microRNAs

An analysis of amphioxus miRNAs suggests an expansion of miRNAs played a key role in the evolution of chordates to vertebrates

UCP2 and UCP3, two novel uncoupling proteins, are important regulators of energy expenditure and thermogenesis in various organisms. The striking disparity between UCP2 mRNA and protein levels in muscle tissues prompted initial speculation that microRNAs are implicated in the regulatory pathway of UCP2. We found, for the first time, that the repression of UCP2 expression in cardiac and skeletal muscle resulted from its targeting by a muscle-specific microRNA, miR-133a. Moreover, our findings illustrate a novel function of UCP2 as a brake for muscle development. We also show that MyoD can remove the braking role of UCP2 via direct up-regulation of miR-133a during myogenic differentiation. Taken together, our current work delineates a novel regulatory network employing MyoD, microRNA, and uncoupling proteins to fine-tune the balance between muscle differentiation and proliferation during myogenesis.

Ophiocordyceps sinensis has been used as a traditional medicine or healthy food in China for thousands of years. Hirsutella sinensis was reported as the only correct anamorph of O. sinensis. It is reported that the laboratory-grown H. sinensis mycelium has similar clinical efficacy and less associated toxicity compared to the wild O. sinensis. The research of the H. sinensis is becoming more and more important and urgent. To gain deeper insight into the biological and pharmacological mechanisms, we sequenced the genome of H. sinensis. The genome of H. sinensis (102.72 Mb) was obtained for the first time, with > 99% coverage. 10,200 protein-encoding genes were predicted based on the genome sequence. A detailed secondary metabolism analysis and structure verification of the main ingredients were performed, and the biosynthesis pathways of seven ingredients (mannitol, cordycepin, purine nucleotides, pyrimidine nucleotides, unsaturated fatty acid, cordyceps polysaccharide and sphingolipid) were predicted and drawn. Furthermore, infection process and mechanism of H. sinensis were studied and elaborated in this article. The enzymes involved in the infection mechanism were also predicted, cloned and expressed to verify the mechanism. The genes and proteins were predicted and annotated based on the genome sequence. The pathways of several active components in H. sinensis were predicted and key enzymes were confirmed. The work presented here would improve the understanding of the genetic basis of this organism, and contribute to further research, production and application of H. sinensis.

Carbonyl reductase (CR)-catalyzed asymmetric reduction offers an approach for producing t-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate ((3R,5R)-1b), which serves as a key building block in atorvastatin (Liptor). However, controlling the stereopreference of CR with the desired selectivity remains challenging because natural CRs usually exhibit Prelog preference. Moreover, transferring knowledge from engineered anti-Prelog CRs to other CRs is difficult. Herein, the key residues that regulate the stereopreference of a CR from Kluyveromyces marxianus (KmCR) were identified by a semirational engineering toward t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((5R)-1a). A structural switch that consists of the key residues was discovered, and related structural features were summarized to predict the stereopreference by analyzing the structural information and multiple-sequence alignment of structure-available CRs carefully. According to the obtained knowledge, the simultaneous mutation of four key residues enabled the conversion of Prelog-selectivity of KmCR into a complete anti-Prelog selectivity (de p > 99% (R) for (3R,5R)-1b). Moreover, the stereopreferences of 11 CRs that share 20–40% sequence identities with KmCR were predicted successfully and engineered experimentally. The knowledge gained from this protein engineering study on KmCR has universal significance for CRs toward (5R)-1a.

Lumen formation of breast epithelium is rapidly lost during tumorigenesis along with expression of cell adhesion molecule CEACAM1. CEACAM1 induces lumena in a three-dimensional culture of MCF7/CEACAM1 cells that otherwise fail to form lumena. We hypothesized miRNAs may be involved because >400 genes were up-or down-regulated in MCF7/CEACAM1 cells and miRNAs may modify global expression patterns. Comparative analysis of miRNA expression in MCF7 versus MCF7/ CEACAM1 cells revealed two miRNAs significantly down-regulated (hsa-miR-30a-3p by 6.73-fold and hsa-miR-342-5p by 5.68-fold). Location of miR-342 within an intron of the EVL gene, hypermethylated and involved in tumorigenesis, suggested that miR-342 overexpression may block lumen formation. In fact, overexpression of miR-342 in MCF7/CEACAM1 cells significantly blocked lumen formation (p < 0.001). ID4, a dominant-negative inhibitor of basic helix-loop-helix transcription factors, up-regulated in MCF7/CEACAM1 cells, down-regulated in breast cancer, and containing a miR-342 binding site, was tested as a potential target of miR-342. The ratio of ID4 to miR-342 increased from 1:2 in MCF7 cells to 30:1 in MCF7/ CEACAM1 cells and a miR-342 inhibitor was able to induce 3-UTR ID4 reporter activity in MCF7 cells. Because 5-methylcytosine methyltransferase DNMT1 is also a potential target of miR-342, we inhibited miR-342 in MCF7 cells and found DNMT1 was up-regulated with no change in EVL expression, suggesting that miR-342 regulates DNMT1 expression but DNMT1 does not affect the EVL expression in these cells. We conclude that the regulation of lumen formation by miR-342 involves at least two of its known targets, namely ID4 and DNMT1.Lumen formation of mammary epithelial cells in a threedimensional culture is an in vitro assay that phenocopies mammary epithelial gland formation (1-3). The loss of lumen formation as exhibited by most, if not all, breast cancer cells in the three-dimensional culture assay phenocopies the premalignant stage of breast cancer know as ductal carcinoma in situ. One approach to identify genes that may govern the change from normal to (pre)malignancy is to transfect breast cancer cells with genes down-regulated in breast cancer and assay their regain of function in the lumen formation assay. A second approach is to block suspect gene expression in breast cancer cells for regain of function, or block gene expression in normal breast epithelial cells for loss of function. One gene that stands out in these assays is the cell-cell adhesion molecule CEACAM1 that is highly expressed in normal breast with a luminal expression pattern (4, 5), and is consistently down-regulated in premalignant and invasive breast cancer (4, 5).Previously, we have shown that transfection of CEACAM1 into MCF7 breast cancer cells resulted in lumen formation when the cells were cultured three-dimensionally (6), whereas anti-CEACAM1 antibodies blocked lumen formation of the normal breast epithelial cell line MCF10F that express CEACAM1 (5). Although CEACAM1 is expressed as mu...