Explore the vast range of titles available.

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. An amplification‐free and extraction‐free extracellular vesicle (EV) miRNA detection method using CRISPR/Cas13a technology is reported. The CRISPR/Cas13a sensing components are encapsulated in liposomes and deliver them into EVs through liposome‐EV fusion. The new approach enables multiplexed protein and RNA analysis in intact EVs, quantifying specific miRNA‐positive tumor‐derived EVs in human plasma samples for more accurate cancer diagnosis.

So far, although various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Here, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridization assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturized NMR device. Ultimately, the magneto-DNA platform will allow both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay is robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 h. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications. A magnetic detection assay based on nucleic acid probes and nanoparticles can rapidly identify a variety of bacterial species in clinical specimens with sensitivity down to single bacteria, offering a useful technology platform for point-of-care diagnostics.

A nanosensor allows sensitive, high-throughput detection of the protein content of exosomes from cancer cells. Exosomes show potential for cancer diagnostics because they transport molecular contents of the cells from which they originate. Detection and molecular profiling of exosomes is technically challenging and often requires extensive sample purification and labeling. Here we describe a label-free, high-throughput approach for quantitative analysis of exosomes. Our nano-plasmonic exosome (nPLEX) assay is based on transmission surface plasmon resonance through periodic nanohole arrays. Each array is functionalized with antibodies to enable profiling of exosome surface proteins and proteins present in exosome lysates. We show that this approach offers improved sensitivity over previous methods, enables portable operation when integrated with miniaturized optics and allows retrieval of exosomes for further study. Using nPLEX to analyze ascites samples from ovarian cancer patients, we find that exosomes derived from ovarian cancer cells can be identified by their expression of CD24 and EpCAM, suggesting the potential of exosomes for diagnostics.

Addressing the global disparity in cancer care necessitates the development of rapid and affordable nucleic acid (NA) testing technologies. This need is particularly critical for cervical cancer, where molecular detection of human papillomavirus (HPV) has emerged as an accurate screening method. However, implementing this transition in low- and middle-income countries has been challenging due to the high costs and centralized facilities required for current NA tests. Here, we present CreDiT (CRISPR Enhanced Digital Testing) for on-site NA detection. The CreDiT platform integrates i) a one-pot CRISPR strategy that simultaneously amplifies both target NAs and analytical signals and ii) a robust fluorescent detection based on digital communication (encoding/decoding) technology. These features enable a rapid assay (<35 minutes) in a single streamlined workflow. We demonstrate the sensitive detection of cell-derived HPV DNA targets down to single copies and accurate identification of HPV types in clinical cervical brushing specimens ( n  = 121). Developing rapid and affordable HPV tests is imperative for effective cervical cancer screening in resource-limited regions. Here, the authors demonstrate an advanced HPV diagnostic system that integrates CRISPR technology with digital signal processing.

Purpose We compared the effects of suspension training (ST) with traditional resistance training (TRT) on muscle mass, strength and functional performance in older adults. Methods Forty-two untrained older adults were randomized in TRT, ST (both performed 3 sets of whole body exercises to muscle failure) or control group (CON). Muscle thickness (MT) of biceps brachii (MT BB ) and vastus lateralis (MT VL ), maximal dynamic strength test (1RM) for biceps curl (1RM BC ) and leg extension exercises (1RM LE ), and functional performance tests (chair stand [CS], timed up and go [TUG] and maximal gait speed [MGS]) were performed before and after 12 weeks of training. Results MT BB increased significantly and similarly for all training groups (TRT 23.35%; ST 21.56%). MT VL increased significantly and similarly for all training groups (TRT 13.03%; ST 14.07%). 1RM BC increased significantly and similarly for all training groups (TRT 16.06%; ST 14.33%). 1RM LE increased significantly and similarly for all training groups (TRT 14.89%; ST 18.06%). MGS increased significantly and similarly for all groups (TRT 6.26%; ST 5.99%; CON 2.87%). CS decreased significantly and similarly for all training groups (TRT − 20.80%; ST − 15.73%). TUG decreased significantly and similarly for all training groups (TRT − 8.66%; ST − 9.16%). Conclusion Suspension training (ST) promotes similar muscle mass, strength and functional performance improvements compared to TRT in older adults.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.

Cholangiocarcinoma (CCA) is a fatal disease often detected late in unresectable stages. Currently, there are no effective diagnostic methods or biomarkers to detect CCA early with high confidence. Analysis of tumor‐derived extracellular vesicles (tEVs) harvested from liquid biopsies can provide a new opportunity to achieve this goal. Here, an advanced nanoplasmonic sensing technology is reported, termed FLEX (fluorescence‐amplified extracellular vesicle sensing technology), for sensitive and robust single EV analysis. In the FLEX assay, EVs are captured on a plasmonic gold nanowell surface and immunolabeled for cancer‐associated biomarkers to identify tEVs. The underlying plasmonic gold nanowell structures then amplify EVs’ fluorescence signals, an effective amplification process at the single EV level. The FLEX EV analysis revealed a wide heterogeneity of tEVs and their marker levels. FLEX also detected small tEVs not detected by conventional EV fluorescence imaging due to weak signals. Tumor markers (MUC1, EGFR, and EPCAM) are identified in CCA, and this marker combination is applied to detect tEVs in clinical bile samples. The FLEX assay detected CCA with an area under the curve of 0.93, significantly better than current clinical markers. The sensitive and accurate nanoplasmonic EV sensing technology can aid in early CCA diagnosis. An advanced nanoplasmonic sensing technology is developed for the molecular analysis of single extracellular vesicles (EVs). Periodic gold nanowell arrays, fabricated by wafer‐scale interference lithography, significantly enhance fluorescence signals of tumor‐derived EVs, enabling sensitive detection of tumor‐derived EVs in human bile samples for cholangiocarcinoma diagnosis.

CRISPR/Cas systems offer a powerful sensing mechanism to transduce sequence‐specific information into amplified analytical signals. However, performing multiplexed CRISPR/Cas assays remains challenging and often requires complex approaches for multiplexed assays. Here, a hydrogel‐based CRISPR/Cas12 system termed CLAMP (Cas‐Loaded Annotated Micro‐Particles) is described. The approach compartmentalizes the CRISPR/Cas reaction in spatially‐encoded hydrogel microparticles (HMPs). Each HMP is identifiable by its face code and becomes fluorescent when target DNA is present. The assay is further streamlined by capturing HMPs inside a microfluidic device; the captured particles are then automatically recognized by a machine‐learning algorithm. The CLAMP assay is fast, highly sensitive (attomolar detection limits with preamplification), and capable of multiplexing in a single‐pot assay. As a proof‐of‐concept clinical application, CLAMP is applied to detect nucleic acid targets of human papillomavirus in cervical brushing samples. A new strategy is demonstrated for multiplexed CRISPR/Cas assays. In this approach, each Cas reaction takes place inside a spatially‐encoded hydrogel microparticle (HMP). A machine‐learning (ML) algorithm then identifies coded HMPs and quantifies their intended molecular targets. The method is scalable to detect many targets in a single‐pot format, facilitating high‐throughput diagnoses.

Extracellular vesicles (EVs) carry information inherited from parental cells, having significant potential for disease diagnosis. In blood, however, EVs are outnumbered >10 -fold by low density lipoproteins (LDLs), yet similar in size and density. These fundamental disadvantages often cause LDL spillover into EV isolates, thus confounding assay results. We hypothesized that EVs can be further separated from LDLs based on electric charge: EVs and LDLs have different lipid composition, which can lead to differential surface charge densities. To test this hypothesis, we modeled and quantified the surface charge of EVs and LDLs, and used the information to optimally separate EVs from LDLs ion-exchange chromatography. We built an enhanced dual-mode chromatography (eDMC) device which performed i) size-exclusion to remove particles smaller than EVs and LDLs and ii) cation-exchange in an acidic elution to retain LDLs longer than EVs. The performance of the eDMC, in comparison to size-exclusion only, was evaluated by analyzing the yield and purity of the isolated EVs. By measuring and modeling zeta potentials at different buffer pH, we estimated surface charge densities of EVs (-6.2 mC/m ) and LDLs (-3.6 mC/m ), revealing that EVs are more negatively charged than LDLs. Furthermore, the charge difference between EVs and LDLs was maximal at a weak acidic condition (pH = 6.4). By applying these findings, we optimized eDMC operation to enrich EVs directly from plasma, depleting >99.8% of LPPs within 30 min. Minimizing LDL contamination improved analytical signals in EV molecular assays, including single vesicle imaging, bulk protein measurements, and mRNA detection. These developments will promote the translational value of the dual-mode separation - a fast, equipment-free, and non-biased way for EV isolation from plasma samples.

Assays for cancer diagnosis via the analysis of biomarkers on circulating extracellular vesicles (EVs) typically have lengthy sample workups, limited throughput or insufficient sensitivity, or do not use clinically validated biomarkers. Here we report the development and performance of a 96-well assay that integrates the enrichment of EVs by antibody-coated magnetic beads and the electrochemical detection, in less than one hour of total assay time, of EV-bound proteins after enzymatic amplification. By using the assay with a combination of antibodies for clinically relevant tumour biomarkers (EGFR, EpCAM, CD24 and GPA33) of colorectal cancer (CRC), we classified plasma samples from 102 patients with CRC and 40 non-CRC controls with accuracies of more than 96%, prospectively assessed a cohort of 90 patients, for whom the burden of tumour EVs was predictive of five-year disease-free survival, and longitudinally analysed plasma from 11 patients, for whom the EV burden declined after surgery and increased on relapse. Rapid assays for the detection of combinations of tumour biomarkers in plasma EVs may aid cancer detection and patient monitoring. A device that integrates the enrichment and electrochemical detection, in less than one hour, of tumour extracellular vesicles bearing clinically relevant tumour biomarkers accurately classifies patients with colorectal cancer.