Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Zebrafish phosvitin-derived peptide Pt5 inhibits melanogenesis via cAMP pathway.

Zebrafish phosvitin-derived peptide Pt5, consisting of the C-terminal 55 residues of phosvitin, has been shown to have an antimicrobial-immunomodulatory activity comparable to phosvitin. Here, we showed clearly that Pt5 had the capacity to inhibit tyrosinase (TYR) activity and melanin biosynthesis, and this inhibition was independent of cell proliferation and cytotoxic effects. Incubation of fluorescein isothiocyanate (FITC)-labeled Pt5 with B16F10 melanoma cells revealed that Pt5 was localized in the cytoplasm of the cells. In addition, Pt5 inhibited the expression of TYR, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), and microphthalmia-associated transcription factor (MITF) in B16F10 melanoma cells and reduced the intracellular cyclic adenosine monophosphate (cAMP) concentration in the cells, but it did not affect the cellular contents of pERK1/2 and ß-catenin, suggesting that Pt5 regulates melanin biosynthesis via cAMP signaling pathway rather than Wnt and MAPK pathways. Collectively, these data indicate that Pt5 has the potential to be used as a melanogenesis inhibitor in medical and cosmetic industry, a novel role ever reported.

[Antitumor effect of anuoning].

BACKGROUND & OBJECTIVE: The authors' previous studies have demonstrated that anuoning bullatacin and squamocin have anticancer activity in vitro. Squamocin could induce apoptosis of HL-60 cells. The purpose of this paper was to investigate cytotoxicity and antitumor effect of anuoning. METHODS: MTT assay was used to examine the growth inhibition of anuoning on human colon carcinoma cell line (HT-29), human nasopharyngeal carcinoma cell line (SUNE1, CNE2), human liver carcinoma (bel-7402), human breast adenocarcinoma cell line (MCF-7) and human lung adenocarcinoma cell line (GLC-82). The models of mice S-180 sarcoma and HepS were used for in vivo antitumor test. RESULTS: The IC50 of anuoning on CNE2, bel-7402, HT-29, SUNE1 cell were 0.044, 0.068, 0.446, and 1.617 micrograms/ml, respectively. The IC50 of anuoning on MCF-7 cell and GLC-82 cell were 1.857 and 3.481 micrograms/ml, respectively. Under the doses of 15, 30, and 60 micrograms/kg, i.p., qd x 10 d, the average tumor inhibitory rates of anuoning to mice tumor HepS were 36.9%, 51.8%, and 57.9%, respectively (P < 0.05). Under the same concentration, the average tumor inhibitory rates of anuoning on mice S-180 sarcoma were 43.0%, 52.1%, and 61.0%, respectively (P < 0.05). CONCLUSIONS: The results indicate that the anuoning possess cell growth inhibition activity to various human tumor cells in vitro and antitumor effect in vivo.