Please note that Figure 3 should read as follows (left-hand segment 24 and not 14; right-hand segment 37 and not 26): Figure 3. Venn diagram of proteins overlapping between the two trials and the number of proteins identified in each trial. Of the total proteins from the first and second trials, 76.9 and 68.4%, respectively, were common to both trials.

Noncoding RNAs have drawn significant attention in biology recently. Whereas the current research is highly inclined to microRNAs, research on other noncoding RNAs has lagged behind. Here, we investigated a novel noncoding RNA that has been known as precursor microRNA miR-886 (pre-miR-886). Pre-miR-886 has been proposed also as a vault RNA, a component of the vault complex implicated in cancer drug resistance. We identified pre-miR-886 as a 102-nucleotide-long, abundant cytoplasmic RNA that is neither a genuine pre-microRNA nor a vault RNA. Pre-miR-886 is physically associated with PKR (Protein Kinase RNA-activated), an interferon-inducible and double-stranded RNA dependent kinase. The suppression of pre-miR-886 activates PKR and its downstream pathways, eIF2a phosphorylation and the NF-kB pathway, leading to impaired cell proliferation. We also found that pre-miR-886 is suppressed in a wide-range of cancer cell lines and in clinical specimens. This study is the first intense characterization of pre-miR-886 as well as the initial report on its function as a PKR regulator, which suggests a critical role in tumorigenesis.

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC-MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer.

Age-related macular degeneration (AMD) describes the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris and is the leading cause of blindness in people over 50. The molecular mechanisms underlying this multifactorial disease remain largely unknown. To uncover novel secretory biomarkers related to the pathogenesis of AMD, we adopted an integrated approach to compare the proteins identified in the conditioned medium (CM) of cultured RPE cells and the exosomes derived from CM and from the aqueous humor (AH) of AMD patients by LC-ESI-MS/MS. Finally, LC-MRM was performed on the AH from patients and controls, which revealed that cathepsin D, cytokeratin 8, and four other proteins increased in the AH of AMD patients. The present study has identified potential biomarkers and therapeutic targets for AMD treatment, such as proteins related to the autophagy-lysosomal pathway and epithelial-mesenchymal transition, and demonstrated a novel and effective approach to identifying AMD-associated proteins that might be secreted by RPE in vivo in the form of exosomes. The proteomics-based characterization of this multifactorial disease could help to match a particular marker to particular target-based therapy in AMD patients with various phenotypes.

Somatic cell nuclear transfer (scNT) is a useful way to create cloned animals. However, scNT clones exhibit high levels of phenotypic instability. This instability may be due to epigenetic reprogramming and/or genomic damage in the donor cells. To test this, we produced transgenic pig fibroblasts harboring the truncated human thrombopoietin (hTPO) gene and used them as donor cells in scNT to produce firstgeneration (G1) cloned piglets. In this study, 2,818 scNT embryos were transferred to 11 recipients and five G1 piglets were obtained. Among them, a clone had a dimorphic facial appearance with severe hypertelorism and a broad prominent nasal bridge. The other clones looked normal. Second-generation (G2) scNT piglets were then produced using ear cells from a G1 piglet that had an abnormal nose phenotype. We reasoned that, if the phenotypic abnormality of the G1 clone was not present in the G2 and third-generation (G3) clones, or was absent in the G2 clones but reappeared in the G3 clones, the phenotypic instability of the G1 clone could be attributed to faulty epigenetic reprogramming rather than to inherent/accidental genomic damage to the donor cells. Blastocyst rates, cell numbers in blastocyst, pregnancy rates, term placenta weight and ponderal index, and birth weight between G1 and G2 clones did not differ, but were significantly (P < 0.05) lower than control age-and sex-matched piglets. Next, we analyzed global methylation changes during development of the preimplantation embryos reconstructed by donor cells used for the production of G1 and G2 clones and could not find any significant differences in the methylation patterns between G1 and G2 clones. Indeed, we failed to detect the phenotypic abnormality in the G2 and G3 clones. Thus, the phenotypic abnormality of the G1 clone is likely to be due to epigenetic dysregulation. Additional observations then suggested that expression of the hTPO gene in the transgenic clones did not appear to be the cause of the phenotypic abnormality in the G1 clones and that the abnormality was acquired by only a few of the G1 clone's cells during its gestational development. Developmental Dynamics 236:3369 -3382, 2007.

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Retinoic acid (RA; all-trans RA and 9-cis RA) enhances embryo developmental competence and quality through multiple mechanisms affecting the oocyte and preimplantation embryo. Folliculogenesis and oocyte maturation are influenced by tumor necrosis factor-α (TNF-α) via inhibition of aromatase activity and estradiol secretion in granulosa cells. Retinoic acid inhibits TNF-α production in various cell lines. The aim of the present study was to determine whether oocyte TNF-α concentrations regulate developmental competence and embryo quality and if the beneficial effects of 9-cis RA are mediated through attenuation of oocyte TNF-α production. Bovine cumulus oocyte complexes collected from abattoir ovaries were matured in maturation medium in the absence (control) or presence of 5 nM 9-cis RA (RA), 100 ng/mL of recombinant bovine TNF-α (TNF), or 5 nM 9-cis RA + 100 ng/mL of recombinant bovine TNF-α (RA+TNF). Oocytes were subsequently collected for gene expression analysis or subjected to in vitro fertilization and culture. Apoptosis and gene expression were analyzed in d-8 blastocysts. Results indicated that 9-cis RA downregulated (P < 0.01) both basal and TNF-α-induced TNF-α mRNA in oocytes (1.0-fold in control, 0.4-fold in RA, 2.1-fold in TNF, and 0.7-fold in RA+TNF). The 9-cis RA increased (P < 0.001) blastocyst development rates (37.1 ± 6.9 vs. 23.6 ± 8.0%) and total cell number (138.4 ± 19.2 vs. 120.2 ± 24.5) and reduced (P < 0.001) the percentage of apoptotic cells (3.3 ± 2.0 vs. 5.6 ± 2.3%) compared with controls. Expression of caspase 3 (0.4- vs. 1.0-fold) and TNF-α (0.4- vs. 1.0-fold) mRNA was downregulated (P < 0.05) in RA-treated blastocysts compared with controls. Moreover, 9-cis RA rescued (P < 0.001) development rates (24.5 ± 11.1 vs. 15.6 ± 9.0%), increased total cell number (124.6 ± 36.5 vs. 106.9 ± 31.1), and reduced apoptosis (5.8 ± 2.0 vs. 8.1 ± 3.1%) in blastocysts exposed to TNF-α (TNF group). Caspase 3 (0.8-fold in RA+TNF vs. 2.2-fold in TNF) and TNF-α (0.3-fold in RA+TNF vs. 2.8-fold in TNF) mRNA expression was attenuated (P < 0.05) in TNF-α-treated blastocysts. In conclusion, the present study suggests that 9-cis RA exerts its beneficial roles on oocyte developmental competence and embryo quality by attenuating oocyte TNF-α mRNA expression.

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