Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE.

Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE's ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.

MicroRNA miR-199a-3p alleviates liver fibrosis by targeting CDK17 in activated hepatic stellate cells.

Liver fibrosis, a common feature of most chronic liver diseases, poses significant health risks and results from various etiologies. While microRNAs (miRNAs) have demonstrated promising anti-fibrotic potential through the direct regulation of target genes, their therapeutic mechanisms remain incompletely understood. In this study, we identified miR-199a, initially discovered in anti-liver fibrotic exosomes, as a key modulator that alleviates thioacetamide-induced liver fibrosis in a mouse model. Consistent with its in vivo effects, treatment with an miR-199a mimic effectively inhibited the activation and function of human hepatic stellate cells (HSCs)-central drivers of liver fibrosis-as well as HSC proliferation and viability in vitro. Notably, miR-199a-3p exerted these anti-fibrotic effects by directly downregulating its biologically relevant target, cyclin-dependent kinase 17 (CDK17). Depletion of CDK17 alone in activated HSCs was sufficient to suppress their activation, function, proliferation, and viability, mirroring the effects of miR-199a mimic treatment. Conversely, overexpression of CDK17 reversed all cellular effects induced by miR-199a mimic treatment. Our findings highlight the miR-199a-3p-CDK17 regulatory axis and suggest that targeting CDK17 in activated HSCs could be a promising therapeutic strategy for liver fibrosis.

CRISPR/Cas13a-assisted amplification-free miRNA biosensor via dark-field imaging and magnetic gold nanoparticles.

MicroRNAs (miRNAs) are short (about 18-24 nucleotides) non-coding RNAs and have emerged as potential biomarkers for various diseases, including cancers. Due to their short lengths, the specificity often becomes an issue in conventional amplification-based methods. Next-generation sequencing techniques could be an alternative, but the long analysis time and expensive costs make them less suitable for routine clinical diagnosis. Therefore, it is essential to develop a rapid, selective, and accurate miRNA detection assay using a simple, affordable system. In this work, we report a CRISPR/Cas13a-based miRNA biosensing using point-of-care dark-field (DF) imaging. We utilized magnetic-gold nanoparticle (MGNPs) complexes as signal probes, which consist of 200 nm-sized magnetic beads and 60 nm-sized gold nanoparticles (AuNPs) linked by DNA hybridization. Once the CRISPR/Cas13a system recognized the target miRNAs (miR-21-5p), the activated Cas13a cleaved the bridge linker containing RNA sequences, releasing 60 nm-AuNPs detected and quantified by a portable DF imaging system. The combination of CRISPR/Cas13a, MGNPs, and DF imaging demonstrated amplification-free detection of miR-21-5p within 30 min at a detection limit of 500 attomoles (25 pM) and with single-base specificity. The CRISPR/Cas13a-assisted MGNP-DF assay achieved rapid, selective, and accurate detection of miRNAs with simple equipment, thus providing a potential application for cancer diagnosis.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detection of multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. First, we characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles are required.

A multi-layered network model identifies Akt1 as a common modulator of neurodegeneration.

The accumulation of misfolded and aggregated proteins is a hallmark of neurodegenerative proteinopathies. Although multiple genetic loci have been associated with specific neurodegenerative diseases (NDs), molecular mechanisms that may have a broader relevance for most or all proteinopathies remain poorly resolved. In this study, we developed a multi-layered network expansion (MLnet) model to predict protein modifiers that are common to a group of diseases and, therefore, may have broader pathophysiological relevance for that group. When applied to the four NDs Alzheimer's disease (AD), Huntington's disease, and spinocerebellar ataxia types 1 and 3, we predicted multiple members of the insulin pathway, including PDK1, Akt1, InR, and sgg (GSK-3ß), as common modifiers. We validated these modifiers with the help of four Drosophila ND models. Further evaluation of Akt1 in human cell-based ND models revealed that activation of Akt1 signaling by the small molecule SC79 increased cell viability in all models. Moreover, treatment of AD model mice with SC79 enhanced their long-term memory and ameliorated dysregulated anxiety levels, which are commonly affected in AD patients. These findings validate MLnet as a valuable tool to uncover molecular pathways and proteins involved in the pathophysiology of entire disease groups and identify potential therapeutic targets that have relevance across disease boundaries. MLnet can be used for any group of diseases and is available as a web tool at http://ssbio.cau.ac.kr/software/mlnet.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.

Molecular fingerprinting of biological nanoparticles with a label-free optofluidic platform.

Label-free detecting multiple analytes in a high-throughput fashion has been one of the long-sought goals in biosensing applications. Yet, for all-optical approaches, interfacing state-of-the-art label-free techniques with microfluidics tools that can process small volumes of sample with high throughput, and with surface chemistry that grants analyte specificity, poses a critical challenge to date. Here, we introduce an optofluidic platform that brings together state-of-the-art digital holography with PDMS microfluidics by using supported lipid bilayers as a surface chemistry building block to integrate both technologies. Specifically, this platform fingerprints heterogeneous biological nanoparticle populations via a multiplexed label-free immunoaffinity assay with single particle sensitivity. Herein, we first thoroughly characterise the robustness and performance of the platform, and then apply it to profile four distinct ovarian cell-derived extracellular vesicle populations over a panel of surface protein biomarkers, thus developing a unique biomarker fingerprint for each cell line. We foresee that our approach will find many applications where routine and multiplexed characterisation of biological nanoparticles is required.

Electrokinetically enhanced label-free plasmonic sensing for rapid detection of tumor-derived extracellular vesicles.

ANALYSIS: of rare circulating extracellular vesicles (EV) from early cancers or different types of host cells requires extremely sensitive EV sensing technologies. Nanoplasmonic EV sensing technologies have demonstrated good analytical performances, but their sensitivity is often limited by EVs' diffusion to the active sensor surface for specific target EV capture. Here, we developed an advanced plasmonic EV platform with electrokinetically enhanced yields (KeyPLEX). The KeyPLEX system effectively overcomes diffusion-limited reactions with applied electroosmosis and dielectrophoresis forces. These forces bring EVs toward the sensor surface and concentrate them in specific areas. Using the keyPLEX, we showed significant improvements in detection sensitivity by ∼100-fold, leading to the sensitive detection of rare cancer EVs from human plasma samples in 10 min. The keyPLEX system could become a valuable tool for point-of-care rapid EV analysis.

CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles.

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures and remain as bulk analysis. Here, the authors introduce an amplification-free and extraction-free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome-EV fusion. This allows for accurately quantify specific miRNA-positive EV counts using 1 × 108  EVs. The authors show that miR-21-5p-positive EV counts are in the range of 2%-10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold-standard method, RT-qPCR. The authors also demonstrate multiplexed protein-miRNA analysis in tumor-derived EVs by capturing EpCAM-positive EVs and quantifying miR-21-5p-positive ones in the subpopulation, which show significantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.

Gold-Nanoparticle-Coated Magnetic Beads for ALP-Enzyme-Based Electrochemical Immunosensing in Human Plasma.

A simple and sensitive AuNP-coated magnetic beads (AMB)-based electrochemical biosensor platform was fabricated for bioassay. In this study, AuNP-conjugated magnetic particles were successfully prepared using biotin-streptavidin conjugation. The morphology and structure of the nanocomplex were characterized by scanning electron microscopy (SEM) with energy-dispersive X-ray analysis (EDX) and UV-visible spectroscopy. Moreover, cyclic voltammetry (CV) was used to investigate the effect of AuNP-MB on alkaline phosphatase (ALP) for electrochemical signal enhancement. An ALP-based electrochemical (EC) immunoassay was performed on the developed AuNP-MB complex with indium tin oxide (ITO) electrodes. Subsequently, the concentration of capture antibodies was well-optimized on the AMB complex via biotin-avidin conjugation. Lastly, the developed AuNP-MB immunoassay platform was verified with extracellular vesicle (EV) detection via immune response by showing the existence of EGFR proteins on glioblastoma multiforme (GBM)-derived EVs (108 particle/mL) spiked in human plasma. Therefore, the signal-enhanced ALP-based EC biosensor on AuNP-MB was favorably utilized as an immunoassay platform, revealing the potential application of biosensors in immunoassays in biological environments.

Physisorption of Affinity Ligands Facilitates Extracellular Vesicle Detection with Low Non-Specific Binding to Plasmonic Gold Substrates.

Plasmonic biosensors are increasingly being used for the analysis of extracellular vesicles (EVs) originating from disease areas. However, the high non-specific binding of EVs to a gold-sensing surface has been a critical problem and hindered the true translational potential. Here, we report that direct antibody immobilization on the plasmonic gold surface via physisorption shows excellent capture of cancer-derived EVs with ultralow non-specific binding even at very high concentrations. Contrary to commonly used methods that involve thiol-based linker attachment and an EDC/sulfo-NHS reaction, we show a higher specific capture rate and >50-fold lower non-specific on citrate-capped plain and nanopatterned gold surfaces. The method provides a simple, fast, and reproducible means to functionalize plasmonic gold surfaces with antibodies for robust EV biosensing.

Stasis and diversity in living fossils: Species delimitation and evolution of lingulid brachiopods.

The Lingulidae are often considered living fossils, because they have shown little morphological change since the Paleozoic. Limited morphological variation has also made the taxonomic study of living lingulids challenging. We investigated species diversity and phylogenetic relationships of extant lingulids and show that they are substantially more diverse than realized, demonstrating that morphological stasis was commonly accompanied by speciation. Species delimitation based on cytochrome c oxidase subunit I (COI) gene sequences from 194 specimens sampled from East Asia, Australia, Oceania, and the Americas suggested 14-22 species in the lingulids (9-17 species in Lingula and 4-5 species in Glottidia), in contrast to the 11-12 species currently recognized globally in the family. Four-gene phylogenetic analyses supported the sister relationship between Lingula and Glottidia. Within Lingula, L. adamsi, which possesses large, brownish shells, was recovered as sister to all remaining Lingula species, which have more or less greenish shells. Within the greenish Lingula clade, the 'L. anatina' complex was sister to the clade that includes the 'L. reevei' complex. The 'L. anatina' complex was further separated into two major clades with partly separate ranges centered on (i) temperate East Asia, and (ii) the tropical west-central Pacific. Within Glottidia, Pacific species were nested within Atlantic species. Time-calibrated phylogenetic analyses suggested that Lingula likely originated in the early Cretaceous contrary to a previously proposed hypothesis advocating a Cenozoic origin. The separation of Lingula and Glottidia appears to date from the Mesozoic, not from the Carboniferous, contrary to a previous hypothesis. Overall, our results uncovered substantial cryptic diversity in lingulids, which will form the basis for conservation and further taxonomic revision.

Plasmonic Sensors for Extracellular Vesicle Analysis: From Scientific Development to Translational Research.

Extracellular vesicles (EVs), actively shed from a variety of neoplastic and host cells, are abundant in blood and carry molecular markers from parental cells. For these reasons, EVs have gained much interest as biomarkers of disease. Among a number of different analytical methods that have been developed, surface plasmon resonance (SPR) stands out as one of the ideal techniques given its sensitivity, robustness, and ability to miniaturize. In this Review, we compare different SPR platforms for EV analysis, including conventional SPR, nanoplasmonic sensors, surface-enhanced Raman spectroscopy, and plasmonic-enhanced fluorescence. We discuss different surface chemistries used to capture targeted EVs and molecularly profile their proteins and RNAs. We also highlight these plasmonic platforms' clinical applications, including cancers, neurodegenerative diseases, and cardiovascular diseases. Finally, we discuss the future perspective of plasmonic sensing for EVs and their potentials for commercialization and clinical translation.

Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles.

Extracellular vesicles (EVs)-nanoscale phospholipid vesicles secreted by cells-present new opportunities for molecular diagnosis from non-invasive liquid biopsies. Single EV protein analysis can be extremely valuable in studying EVs as circulating cancer biomarkers, but it is technically challenging due to weak detection signals associated with limited amounts of epitopes and small surface areas for antibody labeling. Here, a new, simple method that enables multiplexed analyses of EV markers with improved sensitivities is reported. Specifically, plasmon-enhanced fluorescence detection is implemented that amplifies fluorescence signals using surface plasmon resonances excited by periodic gold nanohole structures. It is shown that fluorescence signals in multiple channels are amplified by one order of magnitude, and both transmembrane and intravesicular markers can be detected at the single EV level. This approach can offer additional insight into understanding subtypes, heterogeneity, and production dynamics of EVs during disease development and progression.

Roles for Drosophila cap1 2'-O-ribose methyltransferase in the small RNA silencing pathway associated with Argonaute 2.

Cap1 2'-O-ribose methyltransferase (CMTR1) modifies RNA transcripts containing the 7-methylguanosine cap via 2'-O-ribose methylation of the first transcribed nucleotide, yielding cap1 structures. However, the role of CMTR1 in small RNA-mediated gene silencing remains unknown. Here, we identified and characterized a Drosophila CMTR1 gene (dCMTR1) mutation. We found that the catalytic activity of dCMTR1 was involved in the biogenesis of small interfering RNAs (siRNAs) but not microRNAs. Additionally, dCMTR1 interacted with R2D2, a key component for the assembly of the RNA-induced silencing complex (RISC) containing Argonaute 2 (Ago2). Consistent with this finding, loss of dCMTR1 function impaired RISC assembly by inhibiting the unwinding of Ago2-bound siRNA duplexes, thus preventing the retention of the guide strand. Moreover, dCMTR1 is unlikely to modify siRNAs during RISC assembly. Collectively, our data indicate that dCMTR1 is a positive regulator of the small RNA pathway associated with Ago2 with roles in both siRNA biogenesis and RISC assembly.

Ecdysone-responsive microRNA-252-5p controls the cell cycle by targeting Abi in Drosophila.

The steroid hormone ecdysone has a central role in the developmental transitions of insects through its control of responsive protein-coding and microRNA (miRNA) gene expression. However, the complete regulatory network controlling the expression of these genes remains to be elucidated. In this study, we performed cross-linking immunoprecipitation coupled with deep sequencing of endogenous Argonaute 1 (Ago1) protein, the core effector of the miRNA pathway, in Drosophila S2 cells. We found that regulatory interactions between miRNAs and their cognate targets were substantially altered by Ago1 in response to ecdysone signaling. Additionally, during the larva-to-adult metamorphosis, miR-252-5p was up-regulated via the canonical ecdysone-signaling pathway. Moreover, we provide evidence that miR-252-5p targets Abelson interacting protein ( Abi) to decrease the protein levels of cyclins A and B, controlling the cell cycle. Overall, our data suggest a potential role for the ecdysone/miR-252-5p/Abi regulatory axis partly in cell-cycle control during metamorphosis in Drosophila.-Lim, D.-H., Lee, S., Han, J. Y., Choi, M.-S., Hong, J.-S., Seong, Y., Kwon, Y.-S., Lee, Y. S. Ecdysone-responsive microR-252-5p controls the cell cycle by targeting Abi in Drosophila.

MicroRNA signatures associated with thioacetamide-induced liver fibrosis in mice.

Multiple etiologies of liver injury are associated with fibrosis in which the key event is the activation of hepatic stellate cells (HSCs). Although microRNAs (miRNAs) are reportedly involved in fibrogenesis, the complete array of miRNA signatures associated with the disease has yet to be elucidated. Here, deep sequencing analysis revealed that compared to controls, 80 miRNAs were upregulated and 21 miRNAs were downregulated significantly in the thioacetamide (TAA)-induced mouse fibrotic liver. Interestingly, 58 of the upregulated miRNAs were localized to an oncogenic miRNA megacluster upregulated in liver cancer. Differential expression of some of the TAA-responsive miRNAs was confirmed, and their human orthologs were similarly deregulated in TGF-ß1-activated HSCs. Moreover, a functional analysis of the experimentally validated high-confidence miRNA targets revealed significant enrichment for the GO terms and KEGG pathways involved in HSC activation and liver fibrogenesis. This is the first comprehensive report of miRNAs profiles during TAA-induced mouse liver fibrosis.

Purification of serine protease from polychaeta, Lumbrineris nipponica, and assessment of its fibrinolytic activity.

Ischemic stroke and cardiovascular disease can occur from blockage of blood vessels by fibrin clots formed naturally in the body. Therapeutic drugs of anticoagulant or thrombolytic agents have been studied; however, various problems have been reported such as side effects and low efficacy. Thus, development of new candidates that are more effective and safe is necessary. The objective of this study is to evaluate fibrinolytic activity, anti-coagulation, and characterization of serine protease purified from Lumbrineris nipponica, polychaeta, for new thrombolytic agents. In the present study, we isolated and identified a new fibrinolytic serine protease from L. nipponica. The N-terminal sequence of the identified serine protease was EAMMDLADQLEQSLN, which is not homologous with any known serine protease. The size of the purified serine protease was 28 kDa, and the protein purification yield was 12.7%. The optimal enzyme activity was observed at 50°C and pH 2.0. A fibrin plate assay confirmed that indirect fibrinolytic activity of the purified serine protease was higher than that of urokinase-PA, whereas direct fibrinolytic activity, which causes bleeding side effects, was relatively low. The serine protease did not induce any cytotoxicity toward the endothelial cell line. In addition, anticoagulant activity was verified by an in vivo DVT animal model system. These results suggest that serine protease purified from L. nipponica has the potential to be an alternative fibrinolytic agent for the treatment of thrombosis and use in various biomedical applications.

Assessment of Neuronal Cell-Based Cytotoxicity of Neurotoxins from an Estuarine Nemertean in the Han River Estuary.

A heteronemertean, Yininemertes pratensis, was collected in Han River Estuary, South Korea. This estuarine nemertean has been known by the local fishermen for harmful effects to the glass eels, juveniles of Japanese eel Anguilla japonica, migrating to fresh water. The present study confirmed the neurotoxic effects of this heteronemertean ribbon worm at the cellular level. Derivative types of neurotoxic tetrodotoxin (TTX), 5,11-dideoxy TTX (m/z 288) and 11-norTTX-6(S)-01 (m/z 305.97), were identified through HPLC and MALDI-TOF MS. However, significant neurotoxicity was confirmed in the fraction containing an undefined molecule corresponding to the 291.1 (m/z) peak, when tested in rat primary astrocytes and dorsal ganglion cells. This study is the first to report neurotoxins of the estuarine nemertean, fairly abundant in the Han River estuary, and suggests the long-term monitoring of population dynamics and surveillance of the toxicity in this river estuary.

Effect of the Hebei Spirit oil spill on intertidal meiofaunal communities in Taean, Korea.

In December 2007, approximately 10,900tons of oil from a crude carrier spread rapidly onto the coast of South Korea. We studied the effects of oil on meiofauna by comparing two contaminated intertidal sites with an uncontaminated site. During 2008-2009, the density of meiofauna fluctuated among the contaminated sites but did not vary by season. Seasonal changes in density were observed at contaminated sites 3years after the oil accident. Meiofauna appeared to be more sensitive to oil pollution stress at the more heavily contaminated site than at the less contaminated site. CLUSTER analysis showed that meiofauna communities in the 3years immediately following the accident significantly differed from those sampled later. A non-metric multidimensional scaling analysis showed that nematode species composition in the first month after the accident significantly differed from those sampled later. Long-term monitoring is needed to assess the effects of oil on the meiofaunal community.