Chemoenzymatic Labeling of Extracellular Vesicles for Visualizing Their Cellular Internalization in Real Time.

Extracellular vesicles (EVs) are intercellular communicators that are heavily implicated in diverse pathological processes. However, it is poorly understood how EVs interact with recipient cells due to the lack of appropriate tracking techniques. Here, we report a robust chemoenzymatic labeling technique for visualizing the internalization process of EVs into target cells in real time. This method uses phospholipase D (PLD) to catalyze the in situ exchange of choline by alkyne in the native EV phosphatidylcholine. Subsequent alkyne-azide click chemistry allows conjugation of Cy5 dyes for visualizing EVs internalization by confocal fluorescence microscopy. The fluorescent labeling of EVs was accomplished in an efficient and biocompatible way, without affecting both the morphology and biological activity of EVs. We applied this chemoenzymatic labeling strategy to monitor the cellular uptake of cancer cell-derived EVs in real time and to further reveal multiple internalization mechanisms. This robust, biocompatible labeling strategy provides an essential tool for EV-related studies ranging from chemical biology to drug delivery.

Click RNA for Rapid Capture and Identification of Intracellular MicroRNA Targets.

Rapid capture and identification of the intracellular target genes of microRNAs (miRNAs) are the key to understanding miRNA functions and development of RNA-based therapeutics. However, developing biochemical tools that can fish out the target genes of miRNAs in live cells is a significant technical challenge. Here, we report a remarkably simple yet powerful technology capable of loading virtually any miRNA into Ago2 of the RNA-induced silencing complexes (RISCs). This surprising discovery enables rapid capture and identification of target mRNAs and long noncoding RNAs. It is achieved by linking dibenzocyclooctyne (DBCO), a classical chemical moiety in copper-free click chemistry, to the 3' end of miRNAs. DBCO serves as a high-affinity tag to the Ago2 protein, thus boosting the formation of RISCs with miRNA target genes in living cells. Upon cell lysing, DBCO's routine function in click chemistry allows rapid enrichment of target genes for analysis without the need of additional molecular handles. A series of miR-21 and miR-27a target genes that were previously unknown were pulled down from various cell lines and identified with qRT-PCR, demonstrating the utility of this innovative technology in both transcriptomic research and RNA-based studies.

General Approach to Engineering Extracellular Vesicles for Biomedical Analysis.

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.

Two-step signal amplification for high-sensitivity detection of biomarkers using gold nanoparticle-based conjugates.

The measurement of biomarkers in bodily fluids is extremely important for diagnosing disease, monitoring disease progression, and evaluating treatment efficacy. In this paper, we present a highly sensitive and compatible gold nanoparticle (AuNP)-based, two-step signal amplification system for biomarker detection. First, AuNPs were coated onto the surfaces of 96-well plates to generate rough surfaces, which enable immobilization of many more capture antibodies than a smooth substrate. As a result, detection sensitivity was enhanced significantly. Second, the horseradish peroxidase (HRP)-conjugated detection antibodies were labeled on large-size AuNPs, which increase the localized amounts of HRP and thus further lower the detection limit. Based on the consecutive signal amplification system, a high-sensitivity assay was achieved, with a LOD of 0.07 ng/mL for prostate-specific antigen (PSA). This assay was allowed to detect the PSA levels in clinical samples without changing the current standard immunoassay setups, showing great potential in many settings where immunoassays are needed.

Cross-Linked Poly(ethylene glycol) Shells for Nanoparticles: Enhanced Stealth Effect and Colloidal Stability.

Preventing protein corona formation and macrophage uptake is the key to improving the delivery efficiency of nanocarriers. Herein, we present a kind of cross-linking poly(ethylene glycol) (CL-PEG) shell-wrapped gold nanoparticles (namely, Au@CL-PEG NPs), which show much enhanced stealth effect and colloidal stability in physiological environments. Compared to the AuNPs coated with conventional linear PEGs (namely, Au@PEG NPs), Au@CL-PEG NPs have a greater ability to resist protein adsorption and thus show reduced cellular uptake by macrophages. In addition, the Au@CL-PEG NPs show higher chemical and colloidal stability under different extreme conditions than the conventional Au@PEG NPs. The CL-PEGylation strategy provides a new window for the surface functionalization of nanomaterials, indicating great promise for the development of high-performance nanomedicines.

In Vivo Tracking of Multiple Tumor Exosomes Labeled by Phospholipid-Based Bioorthogonal Conjugation.

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.

MiR-200 modulates coelomocytes antibacterial activities and LPS priming via targeting Tollip in Apostichopus japonicus.

In order to explore the potential roles of microRNAs (miRNAs) in regulating Toll-like receptor (TLR) pathways, we identified Toll interacting protein as a putative target of miR-200 in Apostichopus japonicus coelomocytes by RNA-seq screening (denoted as AjTollip). The positive expression profiles of miR-200 and AjTollip were detected in both LPS exposure primary coelomocytes and Vibrio splendidus challenge sea cucumber. Co-infection miR-200 mimics significantly elevated the expression of AjTollip and its down-stream molecules. In contrast, miR-200 inhibitor significantly repressed the expression of these TLR-pathway members. More importantly, miR-200 displayed not only to enhance coelomocytes antibacterial activities, but to suppress LPS priming in vitro. Overall, all these results will enhance our understanding on miR-200 regulatory roles in anti-bacterial process in sea cucumber.

MiR-31 modulates coelomocytes ROS production via targeting p105 in Vibrio splendidus challenged sea cucumber Apostichopus japonicus in vitro and in vivo.

MiR-31 is a critical regulator of gene expression in many pathogenic processes in vertebrates. In this study, we identified p105 as a novel target of miR-31 in Apostichopus japonicus and investigated their regulatory roles in vitro and in vivo. The negative expression profiles between miR-31 and Ajp105 were detected in both LPS-exposed primary coelomocytes and Vibrio splendidus-challenged sea cucumber. Co-infection miR-31 mimics significantly depressed the expression of Ajp105 and increased ROS production in vitro. In contrast, miR-31 inhibitor significantly elevated the expression of Ajp105 and decreased ROS level. Consistently, miR-31 over-expression or Ajp105 silencing in vivo both greatly promoted ROS accumulation. Taken together, our findings confirmed that miR-31 could modulate respiratory burst via targeting Ajp105 during sea cucumber pathological development.

Identification and characterization of miR-92a and its targets modulating Vibrio splendidus challenged Apostichopus japonicus.

miR-92a is a kind of disease related fine-tuning regulator which is not only related with tumorigenesis and tumor development but also participates in host-pathogen interaction in vertebrates. In present study, the potential targets of miR-92a in Apostichopus japonicus coelomocytes were screened by high-throughout sequencing and PCR approaches. Total of 10 annotated candidates were identified by hybrid PCR, and 23 were verified from RNA-seq, in which SMURF, PCBP and MEGF were found in both methods. The expression patterns of miR-92a and some putative targets were further characterized by qPCR at cell and individual levels. Vibrio splendidus and LPS exposure could significantly increase the expression level of sea cucumber miR-92a at all examined time points. Accordingly, strictly negative correlation expression profiles were detected in two candidates genes of MEGF and SMURF, suggesting that these two genes showed higher possibilities to be the targets of miR-92a in sea cucumber. Overall, the present work will enhance our understanding in the context of miR-92a modulating the interaction of sea cucumber upon pathogen challenged.

The value of DWI in predicting the response to synchronous radiochemotherapy for advanced cervical carcinoma: comparison among three mathematical models.

BACKGROUND: Diffusion weighted imaging(DWI) mode mainly includes intravoxel incoherent motion (IVIM), stretched exponential model (SEM) and Gaussian diffusion model, but it is still unclear which mode is the most valuable in predicting the response to radiochemotherapy for cervical cancer. This study aims to compare the values of three mathematical models in predicting the response to synchronous radiochemotherapy for cervical cancer. METHODS: Eighty-four patients with cervical cancer were enrolled into this study. They underwent DWI examination by using 12 b-values prior to treatment. The imaging parameters were calculated on the basis of IVIM, SEM and Gaussian diffusion models respectively. The imaging parameters derived from three mathematical modes were compared between responders and non-responders groups. The repeatability of each imaging parameter was assessed. RESULTS: The ADC, D or DDC value was lower in responders than in non-responders groups (P = 0.03, 0.02, 0.01). The α value was higher in responders group than in non-responders group (P = 0.03). DDC had the largest area under curves (AUC) (=0.948) in predicting the response to treatment. The imaging parameters derived from SEM had better repeatability (CCC for DDC and α were 0.969 and 0.924 respectively) than that derived from other exponential models. CONCLUSION: Three exponential modes of DWI are useful for predicting the response to radiochemotherapy for cervical cancer, and SEM may be used as a potential optimal model for predicting treatment effect.

Assembly of DNA Probes into Superstructures for Dramatically Enhancing Enzymatic Stability and Signal-to-Background Ratio.

DNA fluorescent probes are versatile tools that are widely used for biological detection in tubes. Using DNA probes in living systems, however, represents a significant challenge because of the endogenous nuclease-induced DNA degradation and strong background fluorescence in complex biological environments. Here, we show that assembling DNA probes into core-satellite gold nanoparticle (AuNP) superstructures could unprecedentedly enhance enzymatic stability and reduce background interference. The embedded DNA probes are protected from interaction with nuclease, eliminating the enzymatic degradation. In the meantime, the AuNP superstructures show extremely high quenching efficiency (>98%) toward the embedded DNA probes, whose fluorescence can be instantly turned on by the target binding, resulting in high signal-to-background ratio. To demonstrate these distinct properties, we made use of the assembled nanoprobes to monitor the ATP levels under different stimuli in living cells. The assembly strategy leads to a new opportunity for accurately sensing targets in living systems.

miR-31 Links Lipid Metabolism and Cell Apoptosis in Bacteria-Challenged Apostichopus japonicus via Targeting CTRP9.

The biological functions of microRNAs (miRNAs) have been studied in a number of eukaryotic species. Recent studies on vertebrate animals have demonstrated critical roles of miRNA in immune and metabolic activities. However, studies on the functions of miRNA in invertebrates are very limited. Here, we demonstrated that miR-31 from Apostichopus japonicus disrupts the balance of lipid metabolism, thus resulting in cell apoptosis by targeting complement C1q tumor necrosis factor-related protein 9 (AjCTRP9), a novel adipokine with pleiotropic functions in immunity and metabolism. Lipidomic analysis suggested that the intercellular lipid metabolites were markedly altered, and three ceramide (Cer) species synchronously increased in the AjCTRP9-silenced coelomocytes. Moreover, exogenous Cer exposure significantly induced apoptosis in the coelomocytes in vivo, in agreement with findings from miR-31 mimic- or AjCTRP9 small-interfering RNA-transfected coelomocytes. Furthermore, we found that the imbalance in sphingolipid metabolism triggered by the overproduction of Cers ultimately resulted in the activation of the apoptosis initiator caspase-8 and executioner caspase-3. Our findings provide the first direct evidence that miR-31 negatively modulates the expression of AjCTRP9 and disturbance of Cer channels, thus leading to caspase-3- and caspase-8-dependent apoptosis, during the interactions between pathogens and host.

The Roles of Two miRNAs in Regulating the Immune Response of Sea Cucumber.

MicroRNAs (miRNAs) have emerged as key regulators in many pathological processes by suppressing the transcriptional and post-transcriptional expression of target genes. MiR-2008 was previously found to be significantly up-regulated in diseased sea cucumber Apostichopus japonicus by high-through sequencing, whereas the reads of miR-137, a well-documented tumor repressor, displayed no significant change. In the present study, we found that miR-137 expression was slightly attenuated and miR-2008 was significantly enhanced after Vibrio splendidus infection or Lipopolysaccharides application. Further target screening and dual-luciferase reporter assay revealed that the two important miRNAs shared a common target gene of betaine-homocysteine S-methyltransferase (AjBHMT), which exhibited noncorrelated messenger RNA and protein expression patterns after bacterial challenge. In order to fully understand their regulatory mechanisms, we conducted the functional experiments in vitro and in vivo. The overexpression of miR-137 in sea cucumber or primary coelomocytes significantly decreased, whereas the inhibition of miR-137 increased the mRNA and protein expression levels of AjBHMT. In contrast, miR-2008 overexpression and inhibition showed no effect on AjBHMT mRNA levels, but the concentration of AjBHMT protein displayed significant changes both in vitro and in vivo. Consistently, the homocysteine (Hcy) contents were also accordingly altered in the aberrant expression analysis of both miRNAs, consistent with the results of the AjBHMT silencing assay in vitro and in vivo. More importantly, small interfering RNA mediated AjBHMT knockdown and Hcy exposure analyses both significantly increased reactive oxygen species (ROS) production and decreased the number of surviving invasive pathogen in sea cucumber coelomocytes. Taken together, these findings confirmed the differential roles of sea cucumber miR-137 and miR-2008 in regulating the common target AjBHMT to promote ROS production and the clearance of pathogenic microorganisms through Hcy accumulation.

Characterization of two regulators of the TNF-α signaling pathway in Apostichopus japonicus: LPS-induced TNF-α factor and baculoviral inhibitor of apoptosis repeat-containing 2.

The TNF-α signaling cascade is involved in the regulation of a variety of biological processes, including cell proliferation, differentiation, apoptosis and the immune response in vertebrates. Here, two regulatory genes, lipopolysaccharide-induced tumor necrosis factor α factor (LITAF) and baculoviral inhibitor of apoptosis repeat-containing 2 (BIRC2), were identified in coelomocytes from the sea cucumber Apostichopus japonicus by RNA-seq and RACE (denoted as AjLITAF and AjBIRC2, respectively). The full-length cDNA of AjLITAF was 1417 bp, with a 5' untranslated region (UTR) of 189 bp, a 3' UTR of 637 bp with one cytokine RNA instability motif (ATTTA) and an open reading frame (ORF) of 591 bp encoding a polypeptide of 196 amino acid residues and a predicted molecular weight of 22.1 kDa. The partial AjBIRC2 cDNA was 2324 bp with a 5' UTR of 145 bp, a 3' UTR of 469 bp and a complete ORF of 1710 bp encoding a polypeptide of 569 amino acid residues. Analysis of the deduced amino acid sequences revealed that both genes shared a remarkably high degree of structural conservation with their mammalian orthologs, including a highly conserved LITAF domain in AjLITAF and three types of BIR domains in AjBIRC2. Spatial expression analysis revealed that AjLITAF and AjBIRC2 were expressed at a slightly lower level in the intestine and tentacle tissues compared with the other four tissues examined. After challenging the sea cucumbers with Vibrio splendidus, the expression levels of AjLITAF and AjBIRC2 in coelomocytes were increased by 2.65-fold at 6 h and 1.76-fold at 24 h compared with the control group. In primary cultured coelomocytes, a significant increase in the expression of AjLITAF and AjBIRC2 was detected after 6 h of exposure to 1 µg mL(-1) LPS. Together, these results suggest that AjLITAF and AjBIRC2 might be involved in the sea cucumber immune response during the course of a pathogenic infection or exposure to pathogen-associated molecular pattern (PAMP) molecules.

Cloning and characterization of two lipopolysaccharide-binding protein/bactericidal permeability-increasing protein (LBP/BPI) genes from the sea cucumber Apostichopus japonicus with diversified function in modulating ROS production.

Lipopolysaccharide-binding protein and bactericidal permeability-increasing protein (LBP/BPI) play crucial role in modulating cellular signals in response to Gram-negative bacteria infection. In the present study, two isoforms of LBP/BPI genes (designated as AjLBP/BPI1 and AjLBP/BPI2, respectively) were cloned from the sea cucumber Apostichopus japonicus by RACE approach. The full-length cDNAs of AjLBP/BPI1 and AjLBP/BPI2 were of 1479 and 1455 bp and encoded two secreted proteins of 492 and 484 amino acid residues, respectively. Signal peptide, two BPI/LBP/CETP and one central domain were totally conserved in the deduced amino acid of AjLBP/BPI1 and AjLBP/BPI2. Phylogentic analysis further supported that AjLBP/BPI1 and AjLBP/BPI2 belonged to new members of invertebrates LBP/BPI family. Spatial expression analysis revealed that both AjLBP/BPI1 and AjLBP/BPI2 were ubiquitously expressed in all examined tissues with the larger magnitude in AjLBP/BPI1. The Vibrio splenfidus challenge and LPS stimulation could significantly up-regulate the mRNA expression of both AjLBP/BPI1 and AjLBP/BPI2, with the increase of AjLBP/BPI2 expression occurred earlier than that of AjLBP/BPI1. More importantly, we found that LPS induced ROS production was markedly depressed after AjLBP/BPI1 knock-down, but there was no significant change by AjLBP/BPI2 silencing. Consistently, the expression level of unclassified AjToll, not AjTLR3, was tightly correlated with that of AjLBP/BPI1. Silencing the AjToll also depressed the ROS production in the cultured coelomocytes. All these results indicated that AjLBP/BPI1 and AjLBP/BPI2 probably played distinct roles in bacterial mediating immune response in sea cucumber, and AjLBP/BPI1 depressed coelomocytes ROS production via modulating AjToll cascade.

miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.

In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK-1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3'UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates.

iTRAQ-based proteomics reveals novel members involved in pathogen challenge in sea cucumber Apostichopus japonicus.

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.

Proteomic identification of differentially expressed proteins in sea cucumber Apostichopus japonicus coelomocytes after Vibrio splendidus infection.

Skin ulceration syndrome (SUS) was the main limitation in the development of Apostichopus japonicus culture industries. To better understand how Vibrio splendidus modulates SUS outbreak, the immune response of A. japonicus coelomocytes after the pathogen challenge were investigated through comparative proteomics approach, and differentially expressed proteins were screened and characterized in the present study. A total of 40 protein spots representing 30 entries were identified at 24, 72 and 96 h post-infection. Of these proteins, 32 were up-regulated and 8 were down-regulated in the V. splendidus challenged samples compared to those of control. These differentially expressed proteins were mainly classified into four categories by GO analysis, in which approximate 33% of proteins showed to be related to immunity response. The mRNA expression levels of 6 differentially expressed proteins were further validated by qRT-PCR. Similar protein-mRNA-level expression patterns were detected in genes of phospholipase (spot 4), G protein (spot 20), annexin (spot 30) and filamin (spot 31). Whilst the levels of ficolin (spot 12) and calumenin (spot 14) transcripts were not corresponded with those of their translation products. These data provide a new insight to understand the molecular immune mechanism of sea cucumber responsive towards pathogen infection.

De novo assembly of the sea cucumber Apostichopus japonicus hemocytes transcriptome to identify miRNA targets associated with skin ulceration syndrome.

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 Apostichopusjaponicus. Target identification for these differentially expressed miRNAs is a major challenge for this non-model organism. METHODOLOGY/PRINCIPAL FINDINGS: To 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. CONCLUSION: This 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.