Clinical value of coagulation parameters in predicting the severity of severe fever with thrombocytopenia syndrome.

Introduction: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by a novel bunyavirus infection with a high lethality rate. The purpose of this study was to investigate the changes in coagulation parameters in patients with SFTS, aiming to provide clinical evidence for early diagnosis, treatment, and disease analysis. Methods: A total of 40 patients with SFTS attended from April 1, 2020 to May 21, 2022 in Nanjing Drum Tower Hospital were selected and grouped according to the duration of the disease, mild and severe disease, cure and death, with 50 healthy physical examiners as controls, and the risk of severe and death disease was predicted using ROC curves. Results: Comparison between the healthy, mild and severe groups revealed that PT, INR, APTT, TT, D-D and vWF levels were higher than those in the healthy control group, and FII, FIX, FX, FXI, FXII, PC and PS levels were lower than those in the healthy control group, the differences were statistically significant (p < 0.05). Comparing the results of SFTS patients with different course times, the results of Fib, FV, FVII, FVIII, FIX, FX, FXI were statistically significant (p < 0.05). Among the survived and deceased patients, the PT, INR, DD and PS results of the deceased patients were higher than those of the survived patients, and the FVIII, FIX, FXI, FXII and PC were lower than those of the survived patients. The area under the ROC curve showed that D-D had higher predictive ability for the risk of severe disease (AUROC 0.93, sensitivity and specificity at a Cut-off value of 1.50 mg/L were 90.0 and 86.5%, respectively) and the risk of death occurring (AUROC 0.84, sensitivity and specificity at a Cut-off value of 3.39 mg/L were 87.5 and 80.0%, respectively). Discussion: The monitoring of the coagulation parameters in patients with SFTS is great significance for identifying the severity and death of the patient's condition, and it is of great clinical value to provide early attention, timely intervention and maximum reduction of the mortality rate for patients at risk of severe disease.

Association of low HDL-c levels with severe symptoms and poor clinical prognosis in patients with severe fever and thrombocytopenia syndrome.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by a novel bunyavirus, characterized by high fever, thrombocytopenia, and multiple organ damage. Disturbances in lipid metabolism often occur during viral infections, but the changes and clinical significance of lipid profiles in SFTS patients remain unclear. This study aimed to investigate the alterations in lipid profiles and their clinical significance in SFTS patients. Methods: A total of 157 SFTS patients and 157 healthy controls were enrolled in this study. Serum lipid levels were collected and analyzed among different groups and prognosis categories. Receiver operating characteristic (ROC) curve analysis was performed to assess the ability of lipid levels in distinguishing between severe and mild cases, as well as surviving and non-surviving patients. Pearson correlation analysis was used to examine the associations between lipid levels and clinical laboratory parameters. Results: SFTS patients exhibited significantly lower levels of HDL-c, LDL-c, cholesterol, APoAI, and ApoB compared to healthy controls, while triglyceride levels were significantly higher. Serum HDL-c and ApoAI demonstrated good performance as indicators for distinguishing between survivors and non-survivors (AUC of 0.87 and 0.85, respectively). Multivariate regression analysis indicated that HDL-c independently acts as a protective factor in patients with SFTS. HDL-c levels showed decline in non-survivors but recovered in survivors. Moreover, HDL-c exhibited significant correlations with various clinical laboratory parameters (IL-6, CRP, AST, TT, APTT, PLT, ALB, and CD4). Conclusion: This study identified abnormalities in serum lipid metabolism among SFTS patients. HDL-c and ApoAI levels hold potential as biomarkers for distinguishing survivors from non-survivors. Additionally, HDL-c and ApoAI may serve as therapeutic targets for the management of SFTS patients.

Evaluation of reverse transcription-polymerase chain reaction and simultaneous amplification and testing for quantitative detection of serum hepatitis B virus RNA.

Background: Chronic hepatitis B virus (HBV) infection is one of the common infectious diseases in the world. HBV covalently closed circular DNA (cccDNA) is the initial template of HBV replication, which can exist in human hepatocytes for a long time and is difficult to be completely removed. It has been shown that HBV RNA can directly respond to the levels and transcriptional activity of cccDNA in hepatocytes and can be used as a surrogate marker of cccDNA transcriptional activity. At present, the detection techniques used for quantitative HBV RNA mainly include reverse transcription quantitative polymerase chain reaction (RT-qPCR) and simultaneous amplification and testing (SAT). Methods: In this study, we verified the performance of the SAT method for detecting HBV RNA and the clinical effectiveness of SAT and RT-qPCR, and compared the correlation and consistency of the two detection methods for HBV RNA detection. Results: The results showed that the limit of detection for HBV RNA by SAT method was 50 copies/mL, with a linear range of 1 × 102-1 × 108 copies/mL. There was no difference in HBV RNA levels detected by the two methods. The correlation and consistency of the results were good, with the coefficient of determination of 0.7787 in HBeAg positive group and 0.8235 in HBeAg negative group. Conclusions: Therefore, this study confirmed that the SAT method and RT-qPCR for detecting HBV RNA have good agreement, which are both reliable methods to detect HBV RNA and can replace each other.

A Washing-Free and Easy-to-Operate Fluorescent Biosensor for Highly Efficient Detection of Breast Cancer-Derived Exosomes.

Traditional detection methods for protein tumor markers in the early screening of breast cancer are restricted by complicated operation procedures and unstable reproducibility. As one of alternative emerging tumor markers, exosomes play an important role in diagnosing and treating cancers at the early stage due to traceability of their origins and great involvement in occurrence and development of cancers. Herein, a washing-free and efficient fluorescent biosensor has been proposed to realize simple and straightforward analysis of breast cancer cell-derived exosomes based on high affinity aptamers and G quadruplex-hemin (G4-hemin). The whole reaction process can be completed by several simple steps, which realizes washing-free and labor-saving. With simplified operation procedures and high repeatability, the linear detection range for this developed fluorescent biosensing strategy to breast cancer cell-derived exosomes is from 2.5 × 105 to 1.00 × 107 particles/ml, and the limit of detection is down to 0.54 × 105 particles/ml.

Combined Detection of Exosome Concentration and Tumor Markers in Gastric Cancer.

It has been an established fact that exosomes act as a mediator in tumor microenvironment as well as participate actively in intercellular communication between cancer cells. Exosomes carry a variety of molecular cargoes that prevent cyclic degradation and represent the cells of their origin. In this study, the difference in expression levels of exosomes was measured for diagnosis of gastric cancer. We isolated exosomes from plasma by size-selective method. The morphology of the exosomes was characterized by transmission electron microscopy, and the particle size and concentration of the exosomes were detected by NanoSight's Nanoparticle Tracking Analysis. Results indicated that the expression level of exosomes in gastric cancer patients was higher than that in healthy individuals. The specificity and sensitivity were 65.2% and 73.1%, respectively. Currently, clinical tumor markers for gastric cancer detection mainly included Carbohydrate antigen 72-4 (CA72-4), Alpha-fetoprotein, Carbohydrate antigen 125, Carbohydrate antigen 19-9 (CA19-9), Carcinoembryonic Antigen, Carbohydrate antigen 242. When we combined positive rate for combined gastric cancer biomarkers, results showed that exosomes concentration +CA19-9 and exosomes concentration +CA72-4 in the two-combined test can provide enough positive rate. Therefore, it can be concluded that for gastric cancer, the concentration of exosomes may be regarded as a diagnostic indicator, eventually.

Progress in exosome associated tumor markers and their detection methods.

Exosomes are secreted by cells and are widely present in body fluids. Exosomes contain various molecular constituents of their cells of origin such as proteins, mRNA, miRNAs, DNA, lipid and glycans which are very similar as the content in tumor cells. These contents play an important role in various stages of tumor development, and make the tumor-derived exosome as a hot and emerging biomarker for various cancers diagnosis and management in non-invasive manner. The present problems of exosome isolation and detection hinder the application of exosomes. With the development of exosome isolation and detection technology, the contents of exosomes can be exploited for early cancer diagnosis. This review summarizes the recent progress on exosome-associated tumor biomarkers and some new technologies for exosome isolation and detection. Furthermore, we have also discussed the future development direction in exosome analysis methods.

Shiga-like toxin I exerts specific and potent anti-tumour efficacy against gastric cancer cell proliferation when driven by tumour-preferential Frizzled-7 promoter.

OBJECTIVES: Tumour-targeted gene therapy is a promising approach for effective control of gastric cancer cell proliferation. Our study aims to develop a cancer therapy which combines tumour-targeting promoters with cytotoxins. METHODS: The expression of globotriaosylceramide (Gb3), which is a Shiga-like toxin I (Stx1) receptor, was verified in gastric cancer compared with normal stomach tissues as assessed by flow cytometry and immunohistochemical analysis. We therefore constructed the recombinant pFZD7-Stx1 plasmid vectors with tumour-preferential Frizzled-7 promoter and Stx1. pFZD7-Stx1 was used to treat gastric cancer in vitro and in vivo. The gastric cancer cell proliferation and tumour growth were identified after the transfection with the pFZD7-Stx1. RESULTS: Globotriaosylceramide was obviously increased in gastric cancer compared with normal stomach. The gastric cancer cell proliferation and tumour growth decreased significantly after the transfection with the pFZD7-Stx1. CONCLUSION: Frizzled-7 promoter is preferentially active, and Gb3 is abundant in gastric cancer cells. Frizzled-7 promoter and Stx1 may be used to determine a novel and relatively specific and potent gastric cancer therapeutic strategy.

A Sensitive Aptasensor Based on a Hemin/G-Quadruplex-Assisted Signal Amplification Strategy for Electrochemical Detection of Gastric Cancer Exosomes.

Emerging evidence indicates that exosomes derived from gastric cancer cells enhance tumor migration and invasion through the modulation of the tumor microenvironment. However, it remains a major problem to detect cancer-specific exosomes due to technical and biological challenges. Most of the methods reported could not achieve efficient detection of tumor-derived exosomes in the background of normal exosomes. Herein, a label-free electrochemical aptasensor is presented for specific detection of gastric cancer exosomes. This platform contains an anti-CD63 antibody modified gold electrode and a gastric cancer exosome specific aptamer. The aptamer is linked to a primer sequence that is complementary to a G-quadruplex circular template. The presence of target exosomes could trigger rolling circle amplification and produce multiple G-quadruplex units. This horseradish peroxidase mimicking DNAzyme could catalyze the reduction of H2 O2 and generate electrochemical signals. This aptasensor exhibits high selectivity and sensitivity toward gastric cancer exosomes with a detection limit of 9.54 × 102 mL-1 and a linear response range from 4.8 × 103 to 4.8 × 106 exosomes per milliliter. Therefore, this electrochemical aptasensor is expected to become a useful tool for the early diagnosis of gastric cancer.

Establishment and Evaluation of a Simple Size-Selective Method for Exosome Enrichment and Purification.

Procedures for enrichment, isolation and purification of exosomes from complex biological samples are difficult, tedious, non-standardized, and require bulky instrumentation such as ultracentrifugation (UC). In this article, a simple method for isolating exosomes. Size-Selective Method (SSM) was established based on commercially available materials, and the UC and ExoQuick-TC kits (EQkit) methods were compared in terms of morphology, particle size, quantity, Western Blot (WB), and extraction time. Results showed that all three different exosome separation methods could obtain circular membranous vesicles, with a diameter of 30-110 nm. There were more non-exosome components in the samples extracted by SSM, such as large microvesicles, with a lower purity. UC obtained a large number of exosomes with a higher purity, but it required an ultracentrifuge, costed much time and had low yield. Both EQkit and SSM were easy to operate, but EQkit tended to aggregate exosomes and consume much time. WB results showed that exosomes extracted by all the three methods expressed CD63 protein. The SSM had the highest CD63 protein content and at the same protein concentration. The above evidences showed that SSM was fast and had high recovery, low cost and high protein concentration, but had more non-exosome protein components, which can be a choice for exosome separation.

Golgi protein 73 and its diagnostic value in liver diseases.

Golgi protein 73 (GP73, also referred to as Golph 2) with 400 amino acids is a 73 kDa transmembrane glycoprotein typically found in the cis-Golg complex. It is primarily expressed in epithelial cells, which has been found upregulated in hepatocytes in patients suffering from both viral and non-viral liver diseases. GP73 has drawn increasing attention for its potential application in the diagnosis of liver diseases such as hepatitis, liver cirrhosis and liver cancer. Herein, we reviewed the discovery history of GP73 and summarized studies by many groups around the world, aiming at understanding its structure, expression, function, detection methods and the relationship between GP73 and liver diseases in various settings.

Magnetic Nanoparticle-Based Automatic Chemiluminescent Enzyme Immunoassay for Golgi Protein 73 and the Clinical Assessment.

Golgi protein 73 (GP73) is an independent diagnostic indicator of cirrhosis. However, it lacks automatic detection techniques to meet the large-scale clinical requirement in physical examination. In this paper, an automatic approach was established based on the ACL2800 automatic chemiluminescent analyzer, using magnetic nanoparticles (MNPs) and chemiluminescence. This method depended on sandwich strategy among biotin-labeled capture monoclonal antibody, target GP73 and acridinium ester (AE)-labeled reporter monoclonal antibody, conjugation of streptavidin-labeled MNPs to biotin-labeled capture monoclonal antibody, and chemiluminescent detection of AE-linked targets. Optimal conditions were investigated and clinical assessment was processed. Detection of GP73 demonstrated a high sensitivity of 1.19 ng/mL with a wide range from 1.34 ng/mL to 684.38 ng/mL. The quantitative detection was achieved with the repeatability of 2.69%, the coefficient of variation of 3.55% and the percent recovery of 93.46%-107.82%. Therefore, an automatic quantitative detection method was successfully developed, which was a potential screening method in physical examination.

Concentration cell-based potentiometric analysis for point-of-care testing with minimum background.

One of the most critical problems of point-of-care testing is how to reduce the interference of background, especially under resource-limited conditions when sample pretreatment is not available. In this work we report a potentiometric method for point-of-care testing with minimum background. The method is based on the principles of a concentration cell which is a type of galvanic cells. It is an electrochemical cell having two carbon electrodes. The potential of each electrode is determined by ratio of a redox couple (i.e. Fe(CN)64-/3-) on the electrode surface. On one electrode, the adsorbed enzyme catalyzes the oxidation of analyte by Fe(CN)63- which produces Fe(CN)64-. The shift of the potential was because of the analyte as well as the background. In the other channel, no enzyme was present so that the shift of the potential, if any, is owing to the background. By measuring the potential difference between the two electrodes (i.e. voltage of the concentration cell), analyte can be quantitatively determined with most of the background eliminated. As the proof-of-concept analyte, blood glucose is quantitatively detected using a voltammeter with acceptable selectivity and accuracy. Noble metal electrodes that are indispensable for conventional electrochemical sensing are not required. All these features simplify the fabrication procedure and reduce the cost for the detection. Therefore, we believe it is promising for electrochemical point-of-care testing.

Effects of RNA Splicing Inhibitors on Amyloid Precursor Protein Expression.

U1 small ribonucleoproteins demonstrate proteopathy in Alzheimer's disease, and their inhibition modulates the expression of the amyloid precursor protein (APP). We sought to determine whether this effect on the APP expression is a universal result of different kinds of RNA splicing inhibitions. We treated cells with two chemical RNA splicing inhibitors: isoginkgetin (IGK) and spliceostatin A (SSA), in which SSA reduced the APP expression, whereas IGK substantially increased it. The following western blot and reverse transcription polymerase chain reaction analyses showed that the APP expression under the IGK treatment has distinct protein forms, but the total mRNA level was nearly unchanged despite a slight switch within its three major transcripts. Further analysis revealed that the APP-increasing effect of IGK depended on protein translation and might involve inhibition in the degradation system. By immunocytochemistry, the APP likely redistributed from Golgi to endoplasmic reticulum (ER) in cells treated with IGK. When compared to the well-characterized ER-to-Golgi transport inhibitor brefeldin A, IGK showed similar APP expression patterns on the western blot. In summary, we not only determined the diverse effects of RNA splicing inhibition on the APP expression but also found the additional function of IGK on protein subcellular traffic.

A sensitive three monoclonal antibodies based automatic latex particle-enhanced turbidimetric immunoassay for Golgi protein 73 detection.

Golgi protein 73 (GP73) is a novel and potential marker for diagnosing hepatocellular carcinoma (HCC) that has been found to be abnormally elevated in liver disease. A latex particle-enhanced turbidimetric immunoassay (LTIA) was recently introduced and licensed for application in a variety of automated clinical chemistry analyzers. However, no studies have reported sufficient data on analytical performance of this method when using 3 monoclonal antibodies for GP73 measurement. The experimental conditions were firstly optimized and range of linearity, diagnostic potential, clinical relevance were compared with the LTIA based on polyclonal antibodies and ELISA. Dilution tests for the LTIA using 3 monoclonal antibodies produced a calibration curve from 10 to 350 ng/mL while the polyclonal antibodies produced the curve from 20 to 320 ng/mL. The detection limit was achieved at 1.82 ng/mL concentration. Within-run CV was obtained in the range of 1.5-2.9% and ROC curves indicated sensitivity and specificity of the LTIA based on 3 monoclonal antibodies were 96.7% and 93.3%, respectively, higher than for the polyclonal antibodies (94.6% and 72.4%) and ELISA (70.0% and 83.3%). Therefore, the LTIA assay based on 3 monoclonal antibodies is thus applicable in quantification of GP73 concentration in automated biochemistry analyzers.

Detection of Hepatocellular Carcinoma Biomarker Golgi Protein 73 Based on Magnetic Enzyme-Linked Immunoassay.

The magnetic enzyme-linked immunoassay (MEIA) based on magnetic nanoparticles as the solid phase was reported in this work. Magnetic nanoparticles (MNPs) have represented perfectly suitable materials for a variety of biomedical and biotechnological applications. Therefore, we used MEIA based on magnetic nanoparticles to provide a screening method for fast analysis of serum Golgi protein 73 (GP73) in hepatocellular carcinoma (HCC) and healthy subjects and comparison was made with the enzyme-linked immunosorbent assay (ELISA) method. Several relevant conditions, including the concentration of anti-GP73 monoclonal antibody and HRP-anti-human GP73 monoclonal antibody, amount of immunomagnetic beads, and the incubation time, were determined and optimized. Finally, the MEIA was successfully established and validated by 79 HCC and 64 healthy subjects. The results showed this method achieved a detection limit of 0.78 ng/mL, which was more sensitive than ELISA. Furthermore, the sensitivity and specificity of the MEIA were 78.43% and 91.47%, respectively, which were higher than ELISA. The MEIA based on MNPs proved to be simple, sensitive, specific and time-saving, therefore holds great potential for development of a commercial kit in the future.

Fabrication techniques for microfluidic paper-based analytical devices and their applications for biological testing: A review.

Paper is increasingly recognized as a user-friendly and ubiquitous substrate for construction of microfluidic devices. Microfluidic paper-based analytical devices (µPADs) provide an alternative technology for development of affordable, portable, disposable and low-cost diagnostic tools for improving point of care testing (POCT) and disease screening in the developing world, especially in those countries with no- or low-infrastructure and limited trained medical and health professionals. We in this review present fabrication techniques for microfluidic devices and their respective applications for biological detection as reported to date. These include: (i) fabrication techniques: examples of devices fabricated by using two-dimensional (2D) and three-dimensional (3D) methods; (ii) detection application: biochemical, immunological and molecular detection by incorporating efficient detection methods such as, colorimetric detection, electrochemical detection, fluorescence detection, chemiluminescence (CL) detection, electrochemiluninescence (ECL) detection, photoelectrochemi (PEC) detection and so on. In addition, main advantages, disadvantages and future trends for the devices are also discussed in this review.

Frizzled-7 promoter is highly active in tumors and promoter-driven Shiga-like toxin I inhibits hepatocellular carcinoma growth.

Frizzled-7 protein plays a significant role in the formation of several malignant tumors. Up regulation of the Frizzled-7 in cancer cell lines is associated with nuclear accumulation of wild-type ß-catenin from the Wnt/ß-catenin pathway which is frequently activated in tumors. To analyze activity of the Frizzled-7 promoter in tumor cells, we constructed two recombinant plasmid vectors in which the Frizzled-7 promoter was used to drive the expression of green fluorescent protein (GFP) and Shiga-like toxin I (Stx1) (pFZD7-GFP/Stx1) genes. The Frizzled-7 protein was found to be expressed in the cancer cell lines but not in the normal cell lines. The GFP expression was restricted to the cancer cell lines and xenografts in the BALB/C mice but not to normal cell lines. Moreover, cell proliferation and tumor growth decreased significantly after transfection with the pFZD7-Stx1. Results from this study will help determine a highly effective strategy for gene therapy of tumors.