Identification and expression analysis of miRNAs in germination and seedling growth of Tibetan hulless barley.

Germination and seedling growth are crucial for plant development and agricultural production. While, the regulatory mechanisms during these processes in Tibetan hulless barley (Hordeum vulgare L. var. nudum) are not well understood. Given the regulatory roles of microRNAs (miRNAs) in crop plants and the irreplaceability of barley in the highland area of China, we herein presented a genome-wide survey of miRNAs to reveal a potential regulatory network in the early developmental stages of two Tibetan hulless barleys, from which a total of 156 miRNAs was identified including 35 known and 121 novel ones. Six of the identified novel miRNAs were further experimentally validated. According to the evolutionary analysis, miR156, miR166, miR168, and miR171 were conserved across Tibetan hulless barleys and eight other seed plants. Expression profiles of ten known miRNAs showed that they were involved in phytohormone signaling, carbohydrate and lipid metabolism, as well as juvenile-adult transition during barley development. Moreover, a total of 1280 genes targeted by 101 miRNAs were predicted from both barley libraries. Three genes (PLN03212, MATE eukaryotic, and GRAS) were validated via the RNA ligase-mediated 5'-rapid amplification of cDNA ends (RLM-5' RACE) to be the targets of hvu-miR159a, hvu-miR166a, and hvu-miR171-3p, respectively. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of putative targets, the most abundant pathways were related to "metabolism". These results revealed that miRNA-target pairs participating in the regulation of multigene expression and the embryonic development of Tibetan hulless barleys were controlled by complex mechanisms involving the concordant expression of different miRNAs and feedback loops among miRNAs as well as their targets. The study provides insight into the regulatory network of barley miRNAs for better understanding of miRNA functions during germination and seedling growth.

Apigenin, flavonoid component isolated from Gentiana veitchiorum flower suppresses the oxidative stress through LDLR-LCAT signaling pathway.

Flower of Gentiana veitchiorum has traditionally been used as an herbal medicine in Tibet for treatment of variola, respiratory infection, and pneumonia. However, the effective components contained in flower are not identified yet, and the underlying mechanisms for anti-inflammatory, antibacterial, and antioxidative activities remain to be elucidated. Here, we first extracted the flavonoid mixture from G. veitchiorum flower. The mixture was then further isolated and the within compounds was identified through the high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The results showed that apigenin (4',5,7-trihydroxyflavone) was the most abundant flavonoid in G. veitchiorum flower. We next examined the antioxidative activity of the extracted apigenin using the ferric reducing/antioxidant power (FRAP), the 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) assays and found that a positive correlation between apigenin concentration and reactive oxygen species (ROS) scavenging rate. The biochemical assays further revealed that the levels of total cholesterol (TC), triglyceride (TG), and malondialdehyde (MDA) were reduced, while the activity of superoxide dismutase (SOD) was increased after apigenin treatment in hyperlipidemic rats. Moreover, we performed histopathological investigations and found that the lipidic deposition patterns were recovered and the amount of lipid vacuoles was significantly reduced in apigenin-treated hyperlipidemic rat liver. Western blotting assay showed that the expression of low-density lipoprotein receptor (LDLR) and lecithin-cholesterol acyltransferase (LCAT) were up-regulated in the apigenin-treated samples. Overall, our results demonstrated that the apigenin isolated from G. veitchiorum flower exhibited radical scavenging activities, and reversed the high fat diet-induced oxidative damage in rats. Its antioxidative activities are probably achieved via LDLR-LCAT signaling pathway.

Origins and Bioactivities of Natural Compounds Derived from Marine Ascidians and Their Symbionts.

Marine ascidians are becoming important drug sources that provide abundant secondary metabolites with novel structures and high bioactivities. As one of the most chemically prolific marine animals, more than 1200 inspirational natural products, such as alkaloids, peptides, and polyketides, with intricate and novel chemical structures have been identified from ascidians. Some of them have been successfully developed as lead compounds or highly efficient drugs. Although numerous compounds that exist in ascidians have been structurally and functionally identified, their origins are not clear. Interestingly, growing evidence has shown that these natural products not only come from ascidians, but they also originate from symbiotic microbes. This review classifies the identified natural products from ascidians and the associated symbionts. Then, we discuss the diversity of ascidian symbiotic microbe communities, which synthesize diverse natural products that are beneficial for the hosts. Identification of the complex interactions between the symbiont and the host is a useful approach to discovering ways that direct the biosynthesis of novel bioactive compounds with pharmaceutical potentials.

Identification and functional characterization of lactadherin, an agglutinating glycoprotein from the chordate Styela clava.

Lactadherin is an extracellular matrix glycoprotein with stimulating agglutination ability that plays crucial roles in animal immunology. In the present study, a novel lactadherin, Sc-lactadherin, was identified from the marine invertebrate chordate, Styela clava. Its full-length cDNA consisted of 579 bps, encoding 193 amino acids with a coagulation FA58C domain. Recombinant Sc-lactadherin via a prokaryotic expression system showed strong hemocyte fusion activity. Therefore, we further examined its effects on cell behaviors using human umbilical vein endothelial cells (HUVECs) and human cervical cancer (HeLa) cells. Recombinant Sc-lactadherin significantly increased the proliferation rate of HUVECs and HeLa cells and improved the cell migration rate of HUVECs. These results demonstrated that the lactadherin identified from the marine ascidian displayed the agglutinating activity. Functional characterization of the recombinant protein showed that it promoted cell proliferation and migration, indicating the potential roles of Sc-lactadherin in immunology and organogenesis in marine ascidians.

Dual Roles of Ascidian Chondromodulin-1: Promoting Cell Proliferation Whilst Suppressing the Growth of Tumor Cells.

Chondromodulin-1 (ChM-1) is an extracellular matrix protein that plays crucial roles in tumor cell growth and angiogenesis in vertebrates and humans. ChM-1 is highly expressed in the invertebrate Ciona savignyi, a marine ascidian chosen as a model. The effect of the recombinant Ciona mature ChM-1 peptide (Cs-mChM-1) on cell proliferation, migration and angiogenesis was evaluated on cultured cells. The results revealed that low concentrations of Cs-mChM-1 (12.5 nM) promoted osteoblastic cell (MC3T3-E1) growth and protected cells from H2O2-induced damage. However, a higher concentration of Cs-mChM-1 (i.e., 500 nM) not only suppressed both growth and migration of tumor cells, including human cervical cancer (HeLa) cells and human neuroblastoma (SH-SY5Y) cells, but also significantly inhibited proliferation and angiogenesis of human umbilical vein endothelial cells (HUVECs). The expression levels of cyclinD1 and mitogen-activated protein kinase 1 (MAPK1) were slightly increased in Cs-mChM-1 treated MC3T3-E1 cells, whereas these genes decreased in treated HeLa cells, SH-SY5Y cells and HUVECs. This result indicates that Cs-mChM-1 modifies cell behavior by regulating cell cycle and cell adhesion. Thus, the present results reveal that recombinant peptides of ChM-1 from invertebrates can play a dual role in cell proliferation and migration of different cell types. The inhibition effects on tumor cell growth and angiogenesis indicate potential pharmaceutical applications for recombinant Cs-mChM-1.

The anti-tumor activity of Mikania micrantha aqueous extract in vitro and in vivo.

Aqueous extract obtained from Mikania micrantha (MMAE) is commonly used as traditional medicine in some countries. We hypothesized that MMAE may inhibit tumor cell growth, both in an in vitro and in vivo setting. In in vitro experiments, two kinds of human cancer cell lines, K562 and Hela were used to test the anti-tumor activity. Inhibitory concentrations (IC50) were obtained from the inhibition curves fitted by regression analysis, inhibitory rates (%) were calculated by MTT assay, morphological changes were observed by transmission electron microscope (TEM), cell cycles were analyzed by flow cytometry (FCM), and DNA ladders were determined by agarose gel electrophoresis. The in vivo anti-tumor activity was evaluated by calculating the tumor inhibitory rates, thymus index and spleen index of S180-bearing mice. Paraffin-embedded sections were used to test the pathologic changes. The result displayed that the growth of K562 and Hela were enhanced when treated with MMAE at 20 µg/mL after 48 h. Other concentrations of MMAE (50, 100, 200, 400 µg/mL) inhibited the proliferation of both kinds of cells. The IC50 values of K562 and Hela at 48 h were 167.16 and 196.27 µg/mL and at 72 h 98.07 and 131.56 µg/mL, respectively. The effects showed time-dose dependence. MMAE led to damages of organelles and induced apoptosis. These results were confirmed by ladder DNA fragmentation profile. MMAE also increased the percentage of cells in G2/M phase and decreased the percentage of cells undergoing G0/G1 and S phase in in vivo tests using S180 cells. MMAE showed antitummor activity in vivo, with its tumor inhibitory rate ranging from 12.1 to 46.9 %. MMAE also induced necrosis, as shown by pathological examination of Hematoxilin-Eosin stained tumor sections. Meanwhile, compared with the control group, the changes of thymus index and spleen index in MMAE treated group were not obvious. This study suggests that MMAE may be an effective agent for cancer therapy with low toxicity.