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.

Characterization and comparison of proteomes of albino sea cucumber Apostichopus japonicus (Selenka) by iTRAQ analysis.

Sea cucumber is a commercially important marine organism in China. Of the different colored varieties sold in China, albino sea cucumber has the greatest appeal among consumers. Identification of factors contributing to albinism in sea cucumber is therefore likely to provide a scientific basis for improving the cultivability of these strains. In this study, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification labeling was used for the first time to quantitatively define the proteome of sea cucumbers and reveal proteomic characteristics unique to albino sea cucumbers. A total of 549 proteins were identified and quantified in albino sea cucumber and the functional annotations of 485 proteins have been exhibited based on COG database. Compared with green sea cucumber, 12 proteins were identified as differentially expressed in the intestine and 16 proteins in the body wall of albino sea cucumber. Among them, 5 proteins were up-regulated in the intestine and 8 proteins were down-regulated in body wall. Gene ontology annotations of these differentially expressed proteins consisted mostly of 'biological process'. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying albinism in sea cucumber.

[Protective effects and anticoagulation effect of polysaccharides from the sea cucumber on acute incomplete cerebral ischemia in rats].

OBJECTIVE: To investigate the protective effect and anticoagulation effect of polysaccharides from the sea cucumber (PSU) on acute incomplete cerebral ischemia (AICI). METHODS: Adult SD rats were randomly divided into 5 group (n = 12): sham operation group, model group, low-dose (30 mg/(kg x d)), middle-dose (60 mg/(kg x d)) and high-dose (120 mg/(kg x d)) groups. The cerebral ischemia rat model was established by permanently ligating the common carotid arteries on both sides of rats. We observed the change of behavior disturbance, brain water content, the levels of serum C-reactive protein (CRP) as well as the effects on prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen(FIB). RESULTS: Due to the use of polysaccharides, the behavioral disturbance was improved, brain water content and the levels of serum CRP were significantly decreased. Also PSU significantly prolonged APTT, TT and reduced the content of FIB (P < 0.05). CONCLUSION: PSU has protective effect on acute incomplete cerebral ischemia injured rats and significant anticoagulation effect.

[Effects of NO3- stress on photosynthetic rate, photochemical efficiency of PS II and light energy allocation in cucumber seedling leaves].

This paper studied the effects of different NO3- concentration on the photosynthetic rate, photochemical efficiency, and absorbed light energy allocation in cucumber seedling leaves. The results indicated that when the available NO3- concentration in the medium was low (14-98 mmol NO3- x L(-1)), an appropriate supplement of NO3- could enhance the capability of cucumber leaves in capturing light energy, and promote the photosynthesis. However, with further increase of NO3-, the photochemical efficiency of PS II decreased, electron transfer restrained, and net photosynthetic rate as well as the absorbed light energy used in photochemical reaction of PS II decreased. At the same time, the light energy used in antenna heat dissipation increased, while the photochemical efficiency decreased. After treated with 140 and 182 mmol NO3- x L(-1) for 6 days, the photosynthetic rate (P(n)) was decreased by 35% and 78%, respectively, maximal PS II efficiency at open centers in the absence of NPQ (F(v)/F(m)), antenna efficiency at open centers in the presence of NPQ (F(v)'/F(m)'), actual PS II efficiency (phi (PSII ) and photochemical quenching (q(P)) were lower, non-photochemical quenching (NPQ) was higher, and the deviation from full balance between PS I and PS II (beta/alpha - 1) was improved significantly, compared with the control. The fluctuant ranges of these chlorophyll fluorescence parameters were increased at higher NO3- concentration, compared with those at lower NO3- concentration. The absorbed light energy allocated to the photochemical reaction of PS II (P) was reduced by high light intensity and high NO3- concentration. Meanwhile, the proportion allocated in antenna heat dissipation (D) increased significantly. Antenna heat dissipation was the main way for excessive energy dissipation.

[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.