Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially samples without reference genomes. Differentially expressed miRNAs have been previously identified in hemocytes collected from healthy skin and from skin affected by skin ulceration syndrome (SUS) in Apostichopus japonicus . Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism.Methodology/Principal FindingsTo thoroughly understand the function of miRNAs, a normalized cDNA library was sequenced with the Illumina Hiseq2000 technology. A total of 91,098,474 clean reads corresponding to 251,148 unigenes, each with an average length of 494bp, were obtained. Blastx analysis against a nonredundant (nr) NCBI protein database revealed that in this set, 52,680 unigenes coded for 3,893 annotated proteins. Two digital gene expression (DGE) libraries from healthy and SUS samples showed that 4,858 of the unigenes were expressed at significantly different levels; 2,163 were significantly up-regulated, while 2,695 were significantly down-regulated. The computational prediction of miRNA targets from these differentially expressed genes identified 732 unigenes as the targets of 57 conserved and 8 putative novel miRNA families, including spu-miRNA-31 and spu-miRNA-2008.ConclusionThis study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The DGE assembly data represent a substantial increase in the genomic resources available for this species and will provide insights into the gene expression profile analysis and the miRNAs function annotations of further studies.

Exosomes are cell-secreted nanoscale membrane vesicles that play critical roles in many pathophysiological processes. The clinical value of exosomes is under intense investigation, yet current knowledge regarding their in vivo properties is very limited because of the lack of efficient labeling techniques. Here, we report a phospholipid-based bioorthogonal labeling strategy to endow exosomes with optical probes without influencing their native biological functions. We investigated the dynamic in vivo biodistribution and organotropic uptake of multiple tumor exosomes in a single mouse. The results indicate that the exosomes derived from different cell lines show specific organotropic uptake. This phospholipid-based labeling strategy opens a new window to directly visualize and monitor exosome trafficking in living systems and holds great promise for exploring exosome-involved biological events such as cancer metastasis.

Skin ulceration syndrome (SUS) is considered to be a major constraint for the stable development of Apostichopus japonicus culture industries. In this study, we investigated protein changes in the coelomocytes of A. japonicus challenged by Vibrio splendidus using isobaric tags for relative and absolute quantification (iTRAQ) over a 96 h time course. Consequently, 228 differentially expressed proteins were identified in two iTRAQs. A comparison of the protein expression profiles among different time points detected 125 proteins primarily involved in response to endogenous stimuli at 24 h. At 48 h, the number of differentially expressed proteins decreased to 67, with their primary function being oxidation reduction. At the end of pathogen infection, proteins responsive to amino acid stimuli and some metabolic processes were classified as the predominant group. Fifteen proteins were differentially expressed at all time points, among which eight proteins related to pathologies in higher animals were shown to be down-regulated after V. splendidus infection: paxillin, fascin-2, aggrecan, ololfactomedin-1, nesprin-3, a disintegrin-like and metallopeptidase with thrombospondin type 1 motif (Adamts7), C-type lectin domain family 4 (Clec4g) and n-myc downstream regulated gene 1 (Ndrg1). To gain more insight into two SUS-related miRNA (miR-31 and miR-2008) targets at the protein level, all 129 down-regulated proteins were further analyzed in combination with RNA-seq. Twelve and eight proteins were identified as putative targets for miR-31 and miR-2008, respectively, in which six proteins (5 for miR-31 and 1 for miR-2008) displayed higher possibilities to be regulated at the level of translation. Overall, the present work enhances our understanding of the process of V. splendidus-challenged sea cucumber and provides a new method for screening miRNAs targets at the translation level.

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that play critical roles in numerous pathophysiological processes. Enrichment and detection of EVs are technically challenging due to the lack of appropriate modification strategies. Herein, we propose a general, facile, and robust approach to engineering EVs by installation of maleimide (Mal) moieties onto EV surfaces based on a hydrophobic insertion strategy. Mal serves as a high-efficiency clickable handle for functionalizing EVs without influencing their structural integrity and biological activity. The Mal-installed EVs were applied into three biomedical applications: (i) labeling with a fluorescent dye for monitoring the EV-mediated cellular communication, (ii) rapid enrichment by magnetic particles (MPs) for high-efficiency EVs isolation, and (iii) conjugation with gold nanoparticles (AuNPs) for Raman detection of the surface components of EVs in situ. This technique would greatly facilitate the applications of EVs in both basic studies and clinical uses.

No abstract