MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases. This study describes a comparison between the miRNA profile of the systemic lupus erythematosus (SLE) patients and the controls to develop further understanding of the pathogenesis of SLE. Peripheral blood mononuclear cells were isolated from blood samples of 23 SLE patients, 10 idiopathic thrombocytopenic purpura patients and 10 healthy controls. The miRNA microarray chip analysis identified 16 miRNAs differentially expressed in SLE. The chip results were confirmed by northern blot analysis. This work indicates that miRNAs are potential diagnosis biomarkers and probable factors involved in the pathogenesis of SLE.

In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nt that regulate the expression of target genes in a post-transcriptional manner. Evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases. This study describes a comparison between the miRNA profile of kidney biopsies from lupus nephritis (LN) patients and the controls, to develop further understanding of the pathogenesis of LN. Kidney biopsies were taken from five LN patients detected LN Class II and three normal controls. The miRNA microarray chip analysis identified 66 miRNAs differentially expressed in LN. The chip results were confirmed by QRT-PCR. This work indicates that miRNAs are potential diagnosis biomarkers and probable factors involved in the pathogenesis of LN.

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Our studies in HUVECs show that ox-LDL induced autophagy and damaged mtDNA leading to TLR9 expression. LOX-1 antibody or the ROS inhibitor apocynin attenuated ox-LDL-mediated autophagy, mtDNA damage and TLR9 expression, suggesting that these events are LOX-1 and ROS-dependent phenomena. Experiments using siRNA to DNase II indicated that DNase II digests mtDNA to protect the tissue from inflammation. Next, we studied and found intense autophagy, TLR9 expression and inflammatory signals (CD45 and CD68) in the aortas of LDLR knockout mice fed high cholesterol diet. Deletion of LOX-1 (LDLR/LOX-1 double knockout mice) attenuated autophagy, TLR9 expression as well as CD45 and CD68. Damaged mtDNA signal was also very high in LDLR knockout mice aortas, and this signal was attenuated by LOX-1 deletion. Thus, it appears that oxidative stress-mediated damaged mtDNA that escapes autophagy induces a potent inflammatory response in atherosclerosis.

Systemic lupus erythematosus (SLE) is a chronic inflammatory disease characterized by multi-system involvement, diverse clinical presentation, and alterations in circulating metabolites. In this study, a (1)H NMR spectroscopy-based metabolomics approach was applied to establish a human SLE serum metabolic profile. Serum samples were obtained from patients with SLE (n = 64), patients with rheumatoid arthritis (RA) (n = 30) and healthy controls (n = 35). The NOESYPR1D spectrum combined with multi-variate pattern recognition analysis was used to cluster the groups and establish a disease-specific metabolites phenotype. Principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA) models were capable of distinguishing SLE or RA patients from healthy subjects. The OPLS-DA model was able to predict diagnosis of SLE with a sensitivity rate of 60.9% and a specificity rate of 97.1%. For diagnosing RA, the model has much higher sensitivity (96.7%) and specificity (91.4%). The SLE serum samples were characterized by reduced concentrations of valine, tyrosine, phenylalanine, lysine, isoleucine, histidine, glutamine, alanine, citrate, creatinine, creatine, pyruvate, high-density lipoprotein, cholesterol, glycerol, formate and increased concentrations of N-acetyl glycoprotein, very low-density lipoprotein and low-density lipoprotein in comparison with the control population. The results not only indicated that serum NMR-based metabolomic methods had sufficient sensitivity and specificity to distinguish SLE and RA from healthy controls, but also have the potential to be developed into a clinically useful diagnostic tool, and could also contribute to a further understanding of disease mechanisms.

Embryonic stem cell (ES cell) lines were first generated by culturing mouse inner cell mass (ICM) on feeder layers in 1981 [1]. However, in large domestic animals, attempts to establish ES cell lines from ICM of blastocysts or the later epiblast have not been successful. This has hindered the efficient production of genetically modified livestocks by using ES-based approaches. Recently, it was found that ectopic expression of various combinations of transcription factors is able to reprogram somatic cells to a pluripotent state [2][3][4][5]. These induced pluripotent stem (iPS) cells show similarities to embryo-derived ES cells and can be used to produce viable mice through tetraploid complementation [6,7]. So far, iPS cells of several mammalian species have been successfully generated [2,3,[8][9][10][11][12]. In this letter, we report the first establishment of bovine iPS cells using defined transcription factors and a modified culture medium.cDNAs coding for the bovine OCT4 (also named POU5F1), SOX2, KLF4, MYC, LIN28, and NANOG genes were cloned into pMXs retroviral vector. The pMXs plasmids containing human OCT4, SOX2, KLF4, and c-MYC genes were all purchased from Addgene. GP2-293 cells were used as the packaging cell line for retroviral production. Bovine fibroblasts used in this study were derived from an E55 Western Shandong Yellow cattle fetus. Three sets of factors, termed b4TF, b6TF, and h4TF, were used to transduce cells by overnight retroviral infection, respectively. Whereas the former two included only bovine factors (b4TF: bOCT4, bSOX2, bMYC, bKLF4; b6TF: bOCT4, bSOX2, bKLF4, bMYC, bLIN28, bNANOG), the latter employed only human factors (hOCT4, hSOX2, hc-MYC, hKLF4). On day 2, the infected cells were harvested by trypsinization and plated onto mitomycin C-treated MEF feeders at a density of 1 × 10 4 cells per 100-mm dish. The next day after being seeded onto feeders, growing cells were cultured in iPS media ( Figure 1A and Supplementary information, Table S1). Bovine iPS (hereinafter named biPS) cells with a mouse ES-like morphology were detected after ~3 Figure 1B). On day 21-35, colonies were isolated mechanically using a 200 µl pipette and transferred to feeder-coated tissue culture dishes. The biPS cells were split with trypsin at a ratio of 1:10 every 4-5 days afterwards (Supplementary information, Data S1). A total of 26 b6TF-derived colonies have been expanded into biPS cell lines. These lines maintained good ES-like morphology for more than 16 passages. However, none of the colonies generated by b4TF or h4TF could be passaged more than six times. Importantly, we showed that the combination of six transcription factors (b6TF set) significantly increased the efficiency of iPS cell generation (by three-fold) compared to the other two combinations ( Figure 1C).We tested eight different types of biPS culture media (Supplementary information, Table S1) by assessing the numbers of ES-like colonies obtained from b6TF-transduced BEFs on day 28. Three out of the eight media could efficiently generat...

Large-scale production of biopharmaceuticals by current bioreactor techniques is limited by low transgenic efficiency and low expression of foreign proteins. In general, a bacterial artificial chromosome (BAC) harboring most regulatory elements is capable of overcoming the limitations, but transferring BAC into donor cells is difficult. We describe here the use of cattle mammary bioreactor to produce functional recombinant human lactoferrin (rhLF) by a novel procedure of transgenic cloning, which employs microinjection to generate transgenic somatic cells as donor cells. Bovine fibroblast cells were co-microinjected for the first time with a 150-kb BAC carrying the human lactoferrin gene and a marker gene. The resulting transfection efficiency of up to 15.79×10−2 percent was notably higher than that of electroporation and lipofection. Following somatic cell nuclear transfer, we obtained two transgenic cows that secreted rhLF at high levels, 2.5 g/l and 3.4 g/l, respectively. The rhLF had a similar pattern of glycosylation and proteolytic susceptibility as the natural human counterpart. Biochemical analysis revealed that the iron-binding and releasing properties of rhLF were identical to that of native hLF. Importantly, an antibacterial experiment further demonstrated that rhLF was functional. Our results indicate that co-microinjection with a BAC and a marker gene into donor cells for somatic cell cloning indeed improves transgenic efficiency. Moreover, the cattle mammary bioreactors generated with this novel procedure produce functional rhLF on an industrial scale.