Extraction-free, immuno-RPA-CRISPR/Cas13a-based one-pot detection of glypican-3 directly from extracellular vesicles.

BACKGROUND: Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI). It is not found in healthy adult liver but is overexpressed in human hepatocellular carcinoma (HCC). The protein marker GPC3 on extracellular vesicles (GPC3+ EVs) is also useful for HCC detection. Nevertheless, the absence of practical and dependable quantitative techniques to evaluate EVs proteins prevents their clinical implementation. RESULTS: Here, using an immuno-recombinase polymerase amplification (immuno-RPA) process and dual amplification of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, we firstly create an extraction-free one-pot immuno-RPA-CRISPR (opiCRISPR) for the direct and extremely sensitive detection of EVs proteins. The EVs protein-targeted detection probe is amplified by RPA to generate a long repetitive sequence containing multiple CRISPR RNA (crRNA) targeting barcodes, and the signal is further amplified by the CRISPR-Cas13a side-chain cleavage activity to generate a fluorescent signal. The results show that circulating extracellular vesicle GPC3 (eGPC3) levels are a reliable marker for GPC3 expression in tumor, opening up new avenues for tumor diagnosis. SIGNIFICANCE AND NOVELTY: We created an eGPC3 assay based on the CRISPR-Cas13a system, and successfully study the significance of extracellular vesicle GPC3 markers in hepatocellular carcinoma.

Current methods for the detection of glypican-3.

Glypican-3 (GPC3) is a heparan sulfate proteoglycan (HSPG) that binds to the cell membrane via glycosylphosphatidylinositol (GPI), widely expressed in human embryos, and is undetectable in healthy adult liver but overexpressed in human hepatocellular carcinoma (HCC). Therefore, accurate and sensitive detection of GPC3 is critical for disease diagnosis. In recent years, a series of methods have been developed for the highly sensitive detection of GPC3, but there is a lack of reviews on recent advances in GPC3-related assays. In this review, we provide the recent advances in GPC3 detection and GPC3 concentration detection, mainly in terms of various optical sensor-based assays and electrochemical assays, and also provide new insights into the challenges and future directions of the field.

Roles and regulatory mechanisms of KIN17 in cancers (Review).

KIN17, which is known as a DNA and RNA binding protein, is highly expressed in numerous types of human cancers and was discovered to participate in several vital cell behaviors, including DNA replication, damage repair, regulation of cell cycle and RNA processing. Furthermore, KIN17 is associated with cancer cell proliferation, migration, invasion and cell cycle regulation by regulating pathways including the p38 MAPK, NF-κB-Snail and TGF-ß/Smad2 signaling pathways. In addition, knockdown of KIN17 was found to enhance the sensitivity of tumor cells to chemotherapeutic agents. Immunohistochemical analysis revealed that there were significant differences in the expression of KIN17 between cancer tissues and adjacent tissues. Both the Kaplan-Meier survival analysis and multivariate Cox regression analysis indicated that KIN17 is aberrantly high expressed in various tumor tissues and is also associated with poor prognosis in patients with various tumor types. Taken together, KIN17 has key roles in tumorigenesis and cancer development. Investigating the relationship between KIN17 and neoplasms will provide a vital theoretical basis for KIN17 to serve as a diagnostic and prognostic biomarker for cancer patients and as a potential target for cancer therapy.

KIN17 promotes cell migration and invasion through stimulating the TGF-ß/Smad2 pathway in hepatocellular carcinoma.

KIN17 DNA and RNA binding protein (Kin17) is involved in the regulation of tumorigenesis of diverse human cancers. However, its role in the cancer progression and metastasis in hepatocellular carcinoma (HCC) remains largely unknown. Bioinformatics and immunohistochemistry staining were used to investigate the expression pattern of KIN17 and its prognostic value in HCC patients. The transwell, wound-healing assay was employed to determine the effects of KIN17 on migration and invasion of HCC cells in vitro. The tail veins model was employed to determine the effects of KIN17 on lung metastasis in vivo. The biological mechanisms involved in cell migration and invasion regulated by KIN17 were determined with Western blot analysis method. KIN17 expression was significantly increased in HCC tissues compared with adjacent normal tissues, with particularly higher in portal vein tumor thrombus and intrahepatic metastasis tissues. Patients with higher KIN17 expression experienced poor overall and disease free survival. KIN17 knockdown in HuH7 and HepG2 cells significantly reduced cell migration and invasion abilities, whereas its overexpression promoted migration and invasion in MHCC-97L and HepG2 cells in vitro and in vivo. In HuH7 and HepG2 cells, KIN17 knockdown inhibited the TGF-ß/Smad2 pathway. In contrast, KIN17 overexpression stimulated TGF-ß/Smad2 pathway in MHCC-97L and HepG2 cells, along with the genes involved in the epithelial-mesenchymal transition. These findings suggest that KIN17 promotes migration and invasion in HCC cells by stimulating the TGF-ß/Smad2 pathway. KIN17 could be a promising prognostic biomarker, as well as a potential therapeutic target in HCC.

Gut microbiota mediate melatonin signalling in association with type 2 diabetes.

AIMS/HYPOTHESIS: It has been shown that melatonin plays a general beneficial role in type 2 diabetes in rodents but its role in humans is controversial. In the present study, we investigated the association between serum melatonin and type 2 diabetes risk in a southern Chinese population in a case-control study. We also examined the role of gut microbiota in this relationship. METHODS: Individuals with type 2 diabetes (cases) and healthy individuals (controls) (n=2034) were recruited from a cross-sectional study and were matched for age and sex in a case-control study. The levels of serum melatonin were measured and the association between serum melatonin and type 2 diabetes risk was examined using a multivariable logistic regression model. We further conducted a rigorously matched case-control study (n=120) in which gut microbial 16S rRNA was sequenced and metabolites were profiled using an untargeted LC-MS/MS approach. RESULTS: Higher levels of serum melatonin were significantly associated with a lower risk of type 2 diabetes (OR 0.82 [95% CI 0.74, 0.92]) and with lower levels of fasting glucose after adjustment for covariates (ß -0.25 [95% CI -0.38, -0.12]). Gut microbiota exhibited alteration in the individuals with type 2 diabetes, in whom lower levels of serum melatonin, lower α- and ß-diversity of gut microbiota (p<0.05), greater abundance of Bifidobacterium and lower abundance of Coprococcus (linear discriminant analysis [LDA] >2.0) were found. Seven genera were correlated with melatonin and type 2 diabetes-related traits; among them Bifidobacterium was positively correlated with serum lipopolysaccharide (LPS) and IL-10, whereas Coprococcus was negatively correlated with serum IL-1ß, IL-6, IL-10, IL-17, TNF-α and LPS (Benjamini-Hochberg-adjusted p value [false discovery rate (FDR)] <0.05). Moreover, altered metabolites were detected in the participants with type 2 diabetes and there was a significant correlation between tryptophan (Trp) metabolites and the melatonin-correlated genera including Bifidobacterium and Coprococcus (FDR<0.05). Similarly, a significant correlation was found between Trp metabolites and inflammation factors, such as IL-1ß, IL-6, IL-10, IL-17, TNF-α and LPS (FDR<0.05). Further, we showed that Trp metabolites may serve as a biomarker to predict type 2 diabetes status (AUC=0.804). CONCLUSIONS/INTERPRETATION: A higher level of serum melatonin was associated with a lower risk of type 2 diabetes. Gut microbiota-mediated melatonin signalling was involved in this association; especially, Bifidobacterium- and Coprococcus-mediated Trp metabolites may be involved in the process. These findings uncover the importance of melatonin and melatonin-related bacteria and metabolites as potential therapeutic targets for type 2 diabetes.

Dietary variety relates to gut microbiota diversity and abundance in humans.

PURPOSE: We aim to investigate the relationship between gut microbiota and dietary variety in a Chinese population using Dietary Variety Score (DVS), an index of dietary variety, as little has studied the relationship of dietary variety and gut microbiota in a general population. METHODS: In this cross-sectional study, recruited participants were conducted with face-to-face interview to collect information on 24-h food intake and dietary consumption using a valid food frequency questionnaire. Subjects (n = 128) were divided as high and low DVS groups by the median of DVS after rigorously matching for confounding factors. The gut microbiota was assessed by 16S rRNA sequencing and the correlations between key phylotypes and DVS, Index of Nutritional Quality (INQ) and clinical indices were examined using generalized linear model in negative binomial regression. RESULTS: Higher score of DVS, INQVB6, INQVE and INQZn exhibited higher α-diversity. DVS was correlated with INQ and six genera. Among the DVS-correlated genera, Turicibacter, Alistipes and Barnesiella were positively correlated with INQVE, INQZn and INQCu, individually or in combination, while Cetobacterium was negatively correlated with INQCu, INQZn and INQVE. The abundance of Coprococcus and Barnesiella increased with the elevated cumulative scores of INQVE, INQVB6 and INQZn. The combination of Alistipes, Roseburia and Barnesiella could moderately predict dietary variety status. CONCLUSION: Higher DVS was correlated with higher microbial diversity and more abundance of some potentially beneficial bacteria but with less some potentially pathogenic bacteria. A high variety dietary, therefore, should be recommended in our daily life.