Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.     

A novel long non-coding RNA AC073352.1 promotes metastasis and angiogenesis via interacting with YBX1 in breast cancer

Breast cancer is the major cause of cancer death worldwide in women. Patients with metastasis have poor prognosis and the mechanisms of breast cancer metastasis are not completely understood. Long non-coding RNAs (lncRNAs) have been shown to have crucial roles in breast cancer development and progression. However, the underlying mechanisms by which lncRNA-driven breast cancer metastasis are unknown. The main objective of this paper is to explore a functional lncRNA and its mechanisms in breast cancer. Here we identified a novel lncRNA AC073352.1 that was significantly upregulated in breast cancer tissues and was associated with advanced TNM stages and poor prognosis in breast cancer patients. In addition, AC073352.1 was found to promote the migration and invasion of breast cancer cells in vitro and enhance breast cancer metastasis in vivo. Mechanistically, we elucidated that AC073352.1 interacted with YBX1 and stabilized its protein expression. Knock down of YBX1 reduced breast cancer cell migration and invasion and could partially reverse the stimulative effects of AC073352.1 overexpressed on breast cancer metastasis. Moreover, AC073352.1 might be packaged into exosomes by binding to YBX1 in breast cancer cells resulting in angiogenesis. Collectively, our results demonstrated that AC073352.1 promoted breast cancer metastasis and angiogenesis via binding YBX1, and it could serve as a promising, novel biomarker for prognosis and a therapeutic target in breast cancer.Subject terms: Breast cancer, Cell invasion, Long non-coding RNAs     

Role of the progressive ankylosis gene (ank) in cartilage mineralization

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.     

Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions

Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.     

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79 U ml⁻¹. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.     

Inhibitory effect of 14-3-3 proteins on serum-induced proliferation of cardiac fibroblasts

Proliferation in cardiac fibroblasts (CFs) can be induced by a wide variety of growth factors that recruit multiple signal transduction pathways, including mitogen-activated protein kinase, phosphatidylinositol 3-kinase and protein kinase C. As a family of dimeric phophoserine-binding proteins, 14-3-3s are associated with a multitude of proteins that regulate signal transduction, apoptosis and checkpoint control pathways. However, it remains unknown whether the 14-3-3 proteins play an active role in cardiac proliferation and alter their expression patterns in response to growth factors in CFs. R18 peptide, an isoform-independent 14-3-3 inhibitor, was used to disrupt 14-3-3 function by adenovirus-mediated transfer of R18-EYFP (AdR18). Our results demonstrate that the 14-3-3 isoforms gamma, zeta and epsilon were highly expressed in CFs and the expression of 14-3-3 epsilon was elevated following serum stimulation. Inhibition of 14-3-3 proteins by AdR18 potentiated mitogen-induced DNA synthesis in CFs. This potentiation was presumably due to the increased inactivated glycogen synthase kinase-3 beta by Ser9 phosphorylation and nuclear factor of activated T-cell nuclear accumulation. However, AdR18 had no effect on extracellular signal-regulated kinase phosphorylation and reduced p70 S6 kinase (p70S6K) phosphorylation upon mitogenic stimulation. Furthermore, though R18 can block 14-3-3 binding abilities, it did not affect the serum-induced upregulation of 14-3-3 epsilon protein. Collectively, these findings reveal that the expression of 14-3-3 epsilon can be upregulated by serum in CFs and 14-3-3s may exert an inhibitory effect on serum-induced proliferation.     

Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks

Hypoxia has generally been reported to impair learning and memory. Here we established a hypoxia-enhanced model. Intermittent hypoxia (IH) was simulated at 2 km (16.0% O2) or 5 km (10.8% O2) in a hypobaric chamber for 4 h/day from birth to 1, 2, 3, or 4 week(s), respectively. Spatial learning and memory ability was tested in the Morris water maze (MWM) task at ages of postnatal day 36 (P36)-P40 and P85-89, respectively, and in the 8-arm maze task at P60-68. The long-term potentiation (LTP), synaptic density, and phosphorylated cAMP-responsive element-binding protein (p-CREB) level in the hippocampus were measured in mice at P36 under the IH for 4 weeks (IH-4w). The results showed that IH for 3 weeks (IH-3w) and IH-4w at 2 km significantly reduced the escape latencies of mice at P36-40 in the MWM task with significantly enhanced retention, and this spatial enhancement was further confirmed by the 8-arm maze test in mice at P60-68. The improvement in MWM induced by IH-4w at 2 km was still maintained in mice at P85-89. IH-4w at 2 or 5 km significantly increased amplitude of LTP, the number of synapse, and the p-CREB level in the hippocampus of P36 mice. These results indicated that IH (4 h/day) exposure to neonatal mice at 2 km for 3 or 4 weeks enhanced mice spatial learning and memory, which was related to the increased p-CREB, LTP, and synapses of hippocampus in this model.     

Quenching of the intrinsic fluorescence of bovine serum albumin by phenylfluorone-Mo(VI) complex as a probe

In this paper, the binding characteristics of bovine serum albumin (BSA) and phenylfluorone (PF)-molybdenum (Mo(VI)) complex have been studied by fluorophotometry. The binding constants are calculated at different temperatures. The binding distance and the energy transfer efficiency between PF-Mo(VI) complex and protein are obtained on the basis of the theory of Forster energy transfer. DeltaH and DeltaS are calculated to be -7.11 kJ mol-1 and 70.30 J mol-1 K-1, which indicate that electrostatic force plays major role in the interaction of PF-Mo(VI) complex and BSA. The experimental results show that BSA and PF-Mo(VI) complex have strong interactions and the mechanism of quenching belongs to static quenching.