Exosome detection via the ultrafast-isolation system: EXODUS.

Exosomes have shown great potential in disease diagnostics and therapeutics. However, current isolation approaches are burdensome and suffer from low speed, yield and purity, limiting basic research and clinical applications. Here, we describe an efficient exosome detection method via the ultrafast-isolation system (EXODUS) that allows automated label-free purification of exosomes from varied biofluids. We obtained the ultra-efficient purification of exosomes by negative pressure oscillation and double coupled harmonic oscillator-enabled membrane vibration. Our two coupled oscillators generate dual-frequency transverse waves on the membranes, enabling EXODUS to outperform other isolation techniques in speed, purity and yield. We demonstrated EXODUS by purifying exosomes from urine samples of 113 patients and validated the practical relevance in exosomal RNA profiling with the high-resolution capability and high-throughput analysis.

Efficient preparation of high-purity and intact mesenchymal stem cell-derived extracellular vesicles.

Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great promise for regeneration and immunomodulation. However, efficient and scalable methods for their preparation are still lacking. In this study, we present the adoption of a label-free technique known as "EXODUS" to isolate and purify MSC-EVs from the conditioned medium. Our findings indicate that EXODUS can rapidly isolate EVs from 10 mL of conditioned medium with a 5-fold higher yield compared to conventional approaches, including ultracentrifugation (UC) and polyethylene glycol precipitation (PEG) methods. Additionally, pre-storing the conditioned medium at 4°C for 1 week resulted in a ~2-fold higher yield of MSC-EVs compared to the freshly prepared medium. However, storing the purified EV particles at 4°C for 1 month led to a 2-fold reduction in particle concentration. Furthermore, we found that MSC-EVs isolated using EXODUS exhibit higher expression levels of EV markers such as Alix, Flotillin1, CD81, and TSG101 in comparison to PEG and UC methods. We also discovered that MSC-EVs isolated using EXODUS are enriched in response to cytokine, collagen-containing extracellular matrix, and calcium ion binding compared to PEG method and enriched in extracellular structure organization, extracellular matrix, and extracellular matrix structure constituents compared to UC. Finally, we demonstrated that MSC-EVs isolated using EXODUS exhibit greater potential in animal organ development, tissue development, and anatomical structure morphogenesis compared to the UC. These findings suggest that EXODUS is a suitable method for the large-scale preparation of high-quality MSC-EVs for various clinical applications.

The genetic source tracking of human urinary exosomes.

The genetic origins of nanoscale extracellular vesicles in our body fluids remains unclear. Here, we perform a tracking analysis of urinary exosomes via RNA sequencing, revealing that urine exosomes mostly express tissue-specific genes for the bladder and have close cell-genetic relationships to the endothelial cell, basal cell, monocyte, and dendritic cell. Tracking the differentially expressed genes of cancers and corresponding enrichment analysis show urine exosomes are intensively involved in immune activities, indicating that they may be harnessed as reliable biomarkers of noninvasive liquid biopsy in cancer genomic diagnostics and precision medicine.

A pH-Mediated Highly Selective System Enabling Simultaneous Analysis of Circulating RNAs Carried by Extracellular Vesicles and Lipoproteins.

Extracellular vesicles (EVs) and lipoproteins (LPPs) serve as important carriers of circulating miRNAs in peripheral blood, offering immense potential for disease diagnosis and therapeutic interventions. Due to their shared physicochemical attributes, EVs and LPPs are frequently coisolated, potentially leading to misunderstandings regarding their distinct functional roles in physiological and pathological processes. Here, we report a highly selective magnetic system based on the pH-mediated affinity displayed by cibacron blue (CB) toward EVs and LPPs, enabling successful separation and collection of these two nanoparticles without cross-contamination for subsequent circulating RNA analysis. First, we found that CB-modified magnetic beads (CBMBs) exhibit a strong affinity toward LPP particles while displaying little interaction with EVs in standard samples under physiological pH conditions. We further demonstrate that the affinity between CB molecules and bionanoparticles in plasma samples is highly pH-dependent. Specifically, CBMBs show affinities for both LPP and EV particles under neutral and acidic conditions. However, at basic pH levels, CB molecules selectively bind only to LPP particles. Consequently, the remaining EV particles present in plasma are subsequently isolated by using titanium dioxide-modified beads (TiMBs) through phospholipid affinity. The simultaneous analysis of the transcriptomic contents of EV and LPP reveals clear differences in their small RNA profiles, with the differentially expressed RNAs reflecting distinct biological processes. Significantly, in a proof-of-concept study, we successfully demonstrated a strong correlation between miRNAs carried by both EV and LPP particles with the occurrence of ocular neovascularization during the progression of diabetic retinopathy. The involved miRNAs may serve as potential biomarkers for DR diagnostics and severity classification. To sum up, this pH-mediated separation system is not only user-friendly but also highly compatible, rendering it a potent tool for probing the molecular compositions, biomarkers, and underlying biological mechanisms of EVs and LPPs.

Metabolomic investigation of urinary extracellular vesicles for early detection and screening of lung cancer.

Lung cancer is a prevalent cancer type worldwide that often remains asymptomatic in its early stages and is frequently diagnosed at an advanced stage with a poor prognosis due to the lack of effective diagnostic techniques and molecular biomarkers. However, emerging evidence suggests that extracellular vesicles (EVs) may promote lung cancer cell proliferation and metastasis, and modulate the anti-tumor immune response in lung cancer carcinogenesis, making them potential biomarkers for early cancer detection. To investigate the potential of urinary EVs for non-invasive detection and screening of patients at early stages, we studied metabolomic signatures of lung cancer. Specifically, we conducted metabolomic analysis of 102 EV samples and identified metabolome profiles of urinary EVs, including organic acids and derivatives, lipids and lipid-like molecules, organheterocyclic compounds, and benzenoids. Using machine learning with a random forest model, we screened for potential markers of lung cancer and identified a marker panel consisting of Kanzonol Z, Xanthosine, Nervonyl carnitine, and 3,4-Dihydroxybenzaldehyde, which exhibited a diagnostic potency of 96% for the testing cohort (AUC value). Importantly, this marker panel also demonstrated effective prediction for the validation set, with an AUC value of 84%, indicating the reliability of the marker screening process. Our findings suggest that the metabolomic analysis of urinary EVs provides a promising source of non-invasive markers for lung cancer diagnostics. We believe that the EV metabolic signatures could be used to develop clinical applications for the early detection and screening of lung cancer, potentially improving patient outcomes.

Highly Selective Purification of Plasma Extracellular Vesicles Using Titanium Dioxide Microparticles for Depicting the Metabolic Signatures of Diabetic Retinopathy.

Extracellular vesicle (EV) cargos with regular fluctuations hold the potential for providing chemical predictors toward clinical diagnosis and prognosis. A plasma sample is one of the most important sources of circulating EVs, yet the technical barrier and cost consumption in plasma-EV isolation still limit its application in disease diagnosis and biomarker discovery. Here, we introduced an easy-to-use strategy that allows selectively purifying small EVs (sEVs) from human plasma and detecting their metabolic alternations. Fe3O4@TiO2 microbeads with a rough island-shaped surface have proven the capability of performing efficient and reversible sEV capture owing to the phospholipid affinity, enhanced binding sites, and size-exclusion-like effect of the rough TiO2 shell. The proposed system can also shorten the separation procedure from hours to 20 min when compared with the ultracentrifugation method and yield approximately 108 sEV particles from 100 µL of plasma. Metabolome variations of sEVs among progressive diabetic retinopathy subjects were finally studied, observing a cluster of metabolites with elevated levels and suggesting potential roles of these sEV chemicals in diabetic retinopathy onset and progression. Such a scalable and flexible EV capture system can be seen as an effective analytical tool for facilitating plasma-based liquid biopsies.

Quantitative metabolic analysis of plasma extracellular vesicles for the diagnosis of severe acute pancreatitis.

BACKGROUND: Severe acute pancreatitis (SAP) is the most common gastrointestinal disease and is associated with unpredictable seizures and high mortality rates. Despite improvements in the treatment of acute pancreatitis, the timely and accurate diagnosis of SAP remains highly challenging. Previous research has shown that extracellular vesicles (EVs) in the plasma have significant potential for the diagnosis of SAP since the pancreas can release EVs that carry pathological information into the peripheral blood in the very early stages of the disease. However, we know very little about the metabolites of EVs that might play a role in the diagnosis of SAP. METHODS: Here, we performed quantitative metabolomic analyses to investigate the metabolite profiles of EVs isolated from SAP plasma. We also determined the metabolic differences of EVs when compared between healthy controls, patients with SAP, and those with mild acute pancreatitis (MAP). RESULTS: A total of 313 metabolites were detected, mainly including organic acids, amino acids, fatty acids, and bile acids. The results showed that the metabolic composition of EVs derived from SAP and MAP was significantly different from those derived from healthy controls and identified specific differences between EVs derived from patients with SAP and MAP. On this basis, we identified four biomarkers from plasma EVs for SAP detection, including eicosatrienoic acid (C20:3), thiamine triphosphate, 2-Acetylfuran, and cis-Citral. The area under the curve (AUC) was greater than 0.95 for both discovery (n = 30) and validation (n = 70) sets. CONCLUSIONS: Our data indicate that metabolic profiling analysis of plasma EVs and the screening of potential biomarkers are of significant potential for improving the early diagnosis and severity differentiation of acute pancreatitis.

Lipidomic identification of urinary extracellular vesicles for non-alcoholic steatohepatitis diagnosis.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a usual chronic liver disease and lacks non-invasive biomarkers for the clinical diagnosis and prognosis. Extracellular vesicles (EVs), a group of heterogeneous small membrane-bound vesicles, carry proteins and nucleic acids as promising biomarkers for clinical applications, but it has not been well explored on their lipid compositions related to NAFLD studies. Here, we investigate the lipid molecular function of urinary EVs and their potential as biomarkers for non-alcoholic steatohepatitis (NASH) detection. METHODS: This work includes 43 patients with non-alcoholic fatty liver (NAFL) and 40 patients with NASH. The EVs of urine were isolated and purified using the EXODUS method. The EV lipidomics was performed by LC-MS/MS. We then systematically compare the EV lipidomic profiles of NAFL and NASH patients and reveal the lipid signatures of NASH with the assistance of machine learning. RESULTS: By lipidomic profiling of urinary EVs, we identify 422 lipids mainly including sterol lipids, fatty acyl lipids, glycerides, glycerophospholipids, and sphingolipids. Via the machine learning and random forest modeling, we obtain a biomarker panel composed of 4 lipid molecules including FFA (18:0), LPC (22:6/0:0), FFA (18:1), and PI (16:0/18:1), that can distinguish NASH with an AUC of 92.3%. These lipid molecules are closely associated with the occurrence and development of NASH. CONCLUSION: The lack of non-invasive means for diagnosing NASH causes increasing morbidity. We investigate the NAFLD biomarkers from the insights of urinary EVs, and systematically compare the EV lipidomic profiles of NAFL and NASH, which holds the promise to expand the current knowledge of disease pathogenesis and evaluate their role as non-invasive biomarkers for NASH diagnosis and progression.

Deep dive on the proteome of salivary extracellular vesicles: comparison between ultracentrifugation and polymer-based precipitation isolation.

Salivary extracellular vesicles (EVs), as novel functional carriers and potential biomarkers, are usually obtained by ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation methods. However, salivary EVs obtained by these two methods have not been systematically compared. Here, we perform an in-depth analysis on EVs isolated by these two methods using proteomics. Both methods obtain EVs ranging from 40 to 210 nm, with the PEG method resulting in a wider size distribution. PEG-separated products were irregularly shaped and aggregated, while UC-separated ones were monodispersed and teacup-shaped. Additionally, the expression of EV-specific markers was higher in UC-separated EVs. Using tandem mass spectrometry proteomics, we identified and quantified 1217 kinds of saliva exosomal proteins and 361 kinds of differential proteins, showing that UC can isolate more EV-related proteins. These results offer some guidance for EV separating and provide potential direction for the use of EVs in non-invasive diagnosis.

High-Efficiency Separation of Extracellular Vesicles from Lipoproteins in Plasma by Agarose Gel Electrophoresis.

Isolation and purification of extracellular vesicles (EVs) from plasma is essential to understand the EV circulation mechanism and discover biomarkers for the early detection of diseases. However, the size range of lipoprotein particles such as high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) overlap that of EVs, making it difficult to remove lipoproteins from EVs. Here, we propose a method for the high efficiency separation of EVs in plasma using agarose gel electrophoresis based on their differences in size and zeta potential properties. Electrophoresis track assays revealed that EVs propagate more slowly than HDL but more quickly than LDL and VLDL in 1% agarose gel with pH 7.4 Tris-Acetate-EDTA (TAE) buffer. The size and morphology of the electrophoresis-recovered products were characterized to be consistent with typical EVs. In addition, the biological function of recovered EVs was investigated with cell uptake tests. The feasibility of this method was further verified with human plasma samples. In summary, this technique has the potential to become a convenient and efficient approach for high-purity EV separation.

Rapid determination of free prolyl dipeptides and 4-hydroxyproline in urine using flow-gated capillary electrophoresis.

Unhydrolyzed prolyl hydroxyproline (Pro-Hyp) and total 4-hydroxyproline (Hyp) in urine have been suggested as disease biomarkers for bone turnover and osteoporosis. Here, a rapid method was developed to accurately and selectively determine free prolyl compounds in unhydrolyzed urine samples. Urine samples were treated with o-phthalaldehyde to block primary amines followed by selective fluorogenic derivatization of secondary amines using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) at room temperature. The derivatized mixture was then directly analyzed and quantitated on a flow-gated capillary electrophoresis system. Six prolyl compounds: Pro-Hyp, Pro-Pro, Pro-Gly, Pro-Leu, Hyp, and Pro in unhydrolyzed urine samples were separated in 30 s, which was > 60-fold faster than the reported HPLC method, using the separation buffer (pH 9.2) composed of tetraborate, cholate, and deoxycholate at 40 mM each. The limits of detection were ~ 20 nM for the dipeptides and ~ 60 nM for Hyp and Pro. The levels of these prolyl compounds in fresh urine samples were determined by using the one-point standard addition method with nipecotic acid as the internal standard. The present protocol was significantly simplified compared with reported techniques, which could improve accuracy and analytical speed. This method is potentially useful in the determination of prolyl dipeptides and Hyp in biological fluids. Graphical abstract Rapid quantitative analysis of prolyl dipeptides in urine using flow-gated capillary electrophoresis coupled with laser-induced fluorescence detection.

Stereospecific capillary electrophoresis assays using pentapeptide substrates for the study of Aspergillus nidulans methionine sulfoxide reductase A and mutant enzymes.

Stereospecific capillary electrophoresis-based methods for the analysis of methionine sulfoxide [Met(O)]-containing pentapeptides were developed in order to investigate the reduction of Met(O)-containing peptide substrates by recombinant Aspergillus nidulans methionine sulfoxide reductase A (MsrA) as well as enzymes carrying mutations in position Glu99 and Asp134. The separation of the diastereomers of the N-acetylated, C-terminally 2,4-dinitrophenyl (Dnp)-labeled pentapeptides ac-Lys-Phe-Met(O)-Lys-Lys-Dnp, ac-Lys-Asp-Met(O)-Asn-Lys-Dnp and ac-Lys-Asn-Met(O)-Asp-Lys-Dnp was achieved in 50 mM Tris-HCl buffers containing sulfated ß-CD in fused-silica capillaries, while the diastereomer separation of ac-Lys-Asp-Met(O)-Asp-Lys-Dnp was achieved by sulfated ß-CD-mediated MEKC. The methods were validated with regard to range, linearity, accuracy, limits of detection and quantitation as well as precision. Subsequently, the substrates were incubated with wild-type MsrA and three mutants in the presence of dithiothreitol as reductant. Wild-type MsrA displayed the highest activity towards all substrates compared to the mutants. Substitution of Glu99 by Gln resulted in the mutant with the lowest activity towards all substrates except for ac-Lys-Asn-Met(O)-Asp-Lys-Dnp, while replacement Asn for Asp134 lead to a higher activity towards ac-Lys-Asp-Met(O)-Asn-Lys-Dnp compared with the Glu99 mutant. The mutant with Glu instead of Asp134 was the most active among the mutant enzymes. Molecular modeling indicated that the conserved Glu99 residue is buried in the Met-S-(O) groove, which might contribute to the correct placing of substrates and, consequently, to the catalytic activity of MsrA, while Asp134 did not form hydrogen bonds with the substrates but only within the enzyme.

Stereospecific electrophoretically mediated microanalysis assay for methionine sulfoxide reductase enzymes.

An electrophoretically mediated microanalysis assay (EMMA) for the determination of the stereoselective reduction of L-methionine sulfoxide diastereomers by methionine sulfoxide reductase enzymes was developed using fluorenylmethyloxycarbonyl (Fmoc)-L-methionine sulfoxide as substrate. The separation of the diastereomers of Fmoc-L-methionine sulfoxide and the product Fmoc-L-methionine was achieved in a successive multiple ionic-polymer layer-coated capillary using a 50 mM Tris buffer, pH 8.0, containing 30 mM sodium dodecyl sulfate as background electrolyte and an applied voltage of 25 kV. 4-Aminobenzoic acid was employed as internal standard. An injection sequence of incubation buffer, enzyme, substrate, enzyme, and incubation buffer was selected. The assay was optimized with regard to mixing time and mixing voltage and subsequently applied for the analysis of stereoselective reduction of Fmoc-L-methionine-(S)-sulfoxide by human methionine sulfoxide reductase A and of the Fmoc-L-methionine-(R)-sulfoxide by human methionine sulfoxide reductase B. The Michaelis-Menten constant, K m, and the maximum velocity, v max, were determined. Essentially identical data were determined by the electrophoretically mediated microanalysis assay and the analysis of the samples by CE upon offline incubation. Furthermore, it was shown for the first time that Fmoc-methionine-(R)-sulfoxide is a substrate of human methionine sulfoxide reductase B.

Capillary electrophoresis separation of peptide diastereomers that contain methionine sulfoxide by dual cyclodextrin-crown ether systems.

A dual-selector system employing achiral crown ethers in combination with cyclodextrins has been developed for the separation of peptide diastereomers that contain methionine sulfoxide. The combinations of the crown ethers 15-crown-5, 18-crown-6, Kryptofix® 21 and Kryptofix® 22 and ß-cyclodextrin, carboxymethyl-ß-cyclodextrin, and sulfated ß-cyclodextrin were screened at pH 2.5 and pH 8.0 using a 40/50.2 cm, 50 µm id fused-silica capillary and a separation voltage of 25 kV. No diastereomer separation was observed in the sole presence of crown ethers, while only sulfated ß-cyclodextrin was able to resolve some peptide diastereomers at pH 8.0. Depending on the amino acid sequence of the peptide and the applied cyclodextrin, the addition of crown ethers, especially the Krpytofix® diaza-crown ethers, resulted in significantly enhanced chiral recognition. Keeping one selector of the dual system constant, increasing concentrations of the second selector resulted in increased peak resolution and analyte migration time for peptide-crown ether-cyclodextrin combinations. The simultaneous diastereomer separation of three structurally related peptides was achieved using the dual selector system.

Separation of high-purity plasma extracellular vesicles for investigating proteomic signatures in diabetic retinopathy.

Extracellular vesicles (EVs) play a multifaceted role in intercellular communication and hold significant promise as bio-functional indicators for clinical diagnosis. Although plasma samples represent one of the most critical sources of circulating EVs, the existing technical challenges associated with plasma-EV isolation have restricted their application in disease diagnosis and biomarker discovery. In this study, we introduce a two-step purification method utilizing ultracentrifugation (UC) to isolate crude extracellular vesicle (EV) samples, followed by a phospholipid affinity-based technique for the selective isolation of small EVs, ensuring a high level of purity for downstream proteomic analysis. Our research demonstrates that the UC & TiO2-coated magnetic bead (TiMB) purification system significantly improves the purity of EVs when compared to conventional UC or TiMB along. We further revealed that proteomic alterations in plasma EVs effectively reflect key gene ontology components associated with diabetic retinopathy (DR) pathogenesis, including the VEGF-activated neuropilin pathway, positive regulation of angiogenesis, angiogenesis, cellular response to vascular endothelial growth factor stimulus, and immune response. By employing a comprehensive analytical approach, which incorporates both time-series analysis (cluster analysis) and differential analysis, we have identified three potential protein signatures including LGALS3, MYH10, and CPB2 that closely associated with the retinopathy process. These proteins exhibit promising diagnostic and severity-classification capabilities for DR disease. This adaptable EV isolation system can be regarded as an effective analytical tool for enhancing plasma-based liquid biopsies toward clinical applications.

Metabolic signatures of tear extracellular vesicles caused by herpes simplex keratitis.

PURPOSE: Herpes simplex keratitis (HSK), caused by type 1 herpes simplex virus (HSV) reactivation, is a severe infectious disease that leads to vision loss. HSV can trigger metabolic reprogramming in the host cell and change the extracellular vesicles (EV) cargos; however, little is known about the EV metabolic signatures during ocular HSV infection. Here, we aimed to depict the EV-associated metabolic landscape in HSK patients' tears. METHODS: We collected 82 samples from 41 participants with unilateral HSK (contralateral unaffected tears were set as negative control), including subtype cohorts of 13 epithelial, 20 stromal, and 8 endothelial HSK. We isolated tear EVs via our previously established platform and conducted metabolic analysis using LC-MS/MS. The metabolic signatures for recognizing HSK and subtypes were assessed through differential analysis and machine learning algorithms. RESULTS: Hypopsia and increased extracellular CD63 levels were observed in affected eyes. We identified 339 metabolites based on sEVs isolated from tears. Differential analysis revealed alterations in energy and amino acid metabolism, as well as the infectious microenvironment. Furthermore, we observed dysregulated metabolite such as methyldopa, which is associated with inappropriate neovascularization and corneal sensation loss, contributing to the HSK severity particularly in the stromal subtype. Moreover, machine learning classification also suggested a set of EV metabolic signatures that have potential for pan-keratitis detection. CONCLUSIONS: Our findings demonstrate that tear EV metabolites can serve as valuable indicators for comprehending the underlying pathological mechanisms. This knowledge is expected to facilitate the development of liquid biopsy means and therapeutic target discovery.

Stereospecific micellar electrokinetic chromatography assay of methionine sulfoxide reductase activity employing a multiple layer coated capillary.

A micellar electrokinetic chromatography method for the analysis of the l-methionine sulfoxide diastereomers employing a successive multiple ionic-polymer layer coated fused-silica capillary was developed and validated in order to investigate the stereospecificity of methionine sulfoxide reductases. The capillary coating consisted of a first layer of hexadimethrine and a second layer of dextran sulfate providing a stable strong cathodic EOF and consequently highly repeatable analyte migration times. The methionine sulfoxide diastereomers, methionine as product as well as ß-alanine as internal standard were derivatized by dabsyl chloride and separated using a 35 mM sodium phosphate buffer, pH 8.0, containing 25 mM SDS as BGE and a separation voltage of 25 kV. The method was validated in the range of 0.15-2.0 mM with respect to linearity and precision. The LODs of the analytes ranged between 0.04 and 0.10 mM. The assay was subsequently applied to determine the stereospecificity of methionine sulfoxide reductases as well as the enzyme kinetics of human methionine sulfoxide reductase A. Monitoring the decrease of the l-methionine-(S)-sulfoxide Km = 411.8 ± 33.8 µM and Vmax = 307.5 ± 10.8 µM/min were determined.

Identification and detection of plasma extracellular vesicles-derived biomarkers for esophageal squamous cell carcinoma diagnosis.

Esophageal cancer is a malignant tumor with two-thirds of patients having a local recurrence or distant metastasis. To date, diagnostic biomarkers with high sensitivity and specificity are lacking. Extracellular vesicles (EVs) have shown their potential values as disease biomarkers as they carry specific proteins and RNAs derived from cancer cells. In this study, we investigate ESCC precision diagnostics from the insights of circulating EVs, and integrate the ultrafast EV isolation approach (EXODUS) and ELISA for fast detection and screening of ESCC patients. First, we isolate and characterize the high-purity plasma EVs with EXODUS and identify 401 proteins and 372 proteins from ESCC patient and healthy individuals, respectively. Further looking into the differentially expressed proteins (DEPs) of ESCC patients and enriched KEGG pathways, we discover EV-CD14 as a potential diagnostic biomarker for ESCC, which has been further validated as a significantly differentially expressed protein by Western Blot and immunogold labelling TEM. For fast screening and detection of ESCC towards clinical applications, we apply ELISA method to diagnose ESCC from 60 clinical samples based on circulating EV-CD14, which shows a high AUC value up to 96.0% for detection of ESCC in a test set (30 samples), and displays a high accuracy rate up to 90% for prediction of ESCC in a screening test (30 samples). Our results suggest that the circulating EV-CD14 may highly be related to the initiation and progression of ESCC, providing a novel method for the diagnosis and prognosis of ESCC towards clinical translations.

Robust Acute Pancreatitis Identification and Diagnosis: RAPIDx.

The occurrence of acute pancreatitis (AP) is increasing significantly worldwide. However, current diagnostic methods of AP do not provide a clear clinical stratification of severity, and the prediction of complications in AP is still limited. Here, we present a robust AP identification and diagnosis (RAPIDx) method by the proteomic fingerprinting of intact nanoscale extracellular vesicles (EVs) from clinical samples. By tracking analysis of circulating biological nanoparticles released by cells (i.e., EVs) via bottom-up proteomics, we obtain close phenotype connections between EVs, cell types, and multiple tissues based on their specific proteomes and identify the serum amyloid A (SAA) proteins on EVs as potential biomarkers that are differentially expressed from AP patients significantly. We accomplish the quantitative analysis of EVs fingerprints using MALDI-TOF MS and find the SAA proteins (SAA1-1, desR-SAA1-2, SAA2, SAA1-2) with areas under the curve (AUCs) from 0.92 to 0.97, which allows us to detect AP within 30 min. We further realize that SAA1-1 and SAA2, combined with two protein peaks (5290.19, 14032.33 m/z), can achieve an AUC of 0.83 for classifying the severity of AP. The RAPIDx platform will facilitate timely diagnosis and treatment of AP before severity development and persistent organ failure and promote precision diagnostics and the early diagnosis of pancreatic cancer.

Study on Key Technologies of Virtual Interactive Surgical Simulation for 3D Reconstruction of Medical Images.

Medical computed tomography (CT), nuclear magnetic resonance imaging (MRI), and other imaging facility produce a large amount of medical image data which has great diagnosis value. Traditional three-dimensional reconstruction of medical images has high requirements for graphics acceleration hardware and the processing speed. In this study, VxWorks embedded real-time process feature is used for CT or MRI DICOM data to real restoration and establishment of virtual three-dimensional model for realizing volume reconstruction, maximum density projection, multiplane reconstruction, dynamic interactive cutting of any surface, dynamic display of three-dimensional model and two-dimensional sectional image, surgical path planning and interactive surgical simulation, and determine the best surgical scheme. The practical application shows that the virtual simulation environment supports the seamless transplantation of code, function debugging, and interaction and solves the issue of high requirements for hardware. It can meet the needs of scientific research and teaching for clinicians and medical imaging workers and can be widely used in the training of virtual surgical anatomy for medical students.