Challenges and opportunities for circRNA identification and delivery.

Circular RNAs (circRNAs) are evolutionarily conserved noncoding RNAs with tissue-specific expression patterns, and exert unique cellular functions that have the potential to become biomarkers in therapeutic applications. Therefore, accurate and sensitive detection of circRNA with facile platforms is essential for better understanding of circRNA biological processes and circRNA-related disease diagnosis and prognosis; and precise regulation of circRNA through efficient delivery of circRNA or siRNA is critical for therapeutic purposes. Here, we reviewed the current development of circRNA identification methodologies, including overviewing the purification steps, summarizing the sequencing methods of circRNA, as well as comparing the advantages and disadvantages of traditional and new detection methods. Then, we discussed the delivery and manipulation strategies for circRNAs in both research and clinic treatment. Finally, the challenges and opportunities of analyzing circRNAs were addressed.

A DNAzyme-enhanced nonlinear hybridization chain reaction for sensitive detection of microRNA.

As a typical biomarker, the expression of microRNA is closely related to the occurrence of cancer. However, in recent years, the detection methods have had some limitations in the research and application of microRNAs. In this paper, an autocatalytic platform was constructed through the combination of a nonlinear hybridization chain reaction and DNAzyme to achieve efficient detection of microRNA-21. Fluorescently labeled fuel probes can form branched nanostructures and new DNAzyme under the action of the target, and the newly formed DNAzyme can trigger a new round of reactions, resulting in enhanced fluorescence signals. This platform is a simple, efficient, fast, low-cost, and selective method for the detection of microRNA-21, which can detect microRNA-21 at concentrations as low as 0.004 nM and can distinguish sequence differences by single-base differences. In tissue samples from patients with liver cancer, the platform shows the same detection accuracy as real-time PCR but with better reproducibility. In addition, through the flexible design of the trigger chain, our method could be adapted to detect other nucleic acid biomarkers.

CPSF3 Promotes Pre-mRNA Splicing and Prevents CircRNA Cyclization in Hepatocellular Carcinoma.

CircRNAs are crucial in tumorigenesis and metastasis, and are comprehensively downregulated in hepatocellular carcinoma (HCC). Previous studies demonstrated that the back-splicing of circRNAs was closely related to 3'-end splicing. As a core executor of 3'-end cleavage, we hypothesized that CPSF3 modulated circRNA circularization. Clinical data were analyzed to establish the prognostic correlations. Cytological experiments were performed to determine the role of CPSF3 in HCC. A fluorescent reporter was employed to explore the back-splicing mechanism. The circRNAs regulated by CPSF3 were screened by RNA-seq and validated by PCR, and changes in downstream pathways were explored by molecular experiments. Finally, the safety and efficacy of the CPSF3 inhibitor JTE-607 were verified both in vitro and in vivo. The results showed that CPSF3 was highly expressed in HCC cells, promoting their proliferation and migration, and that a high CPSF3 level was predictive of a poor prognosis. A mechanistic study revealed that CPSF3 enhanced RNA cleavage, thereby reducing circRNAs, and increasing linear mRNAs. Furthermore, inhibition of CPSF3 by JET-607 suppressed the proliferation of HCC cells. Our findings indicate that the increase of CPSF3 in HCC promotes the shift of pre-mRNA from circRNA to linear mRNA, leading to uncontrolled cell proliferation. JTE-607 exerted a therapeutic effect on HCC by blocking CPSF3.

Clay nanoparticles as pharmaceutical carriers in drug delivery systems.

INTRODUCTION: Clay minerals are a class of silicates with chemical inertness, colloid, and thixotropy, which have excellent physicochemical properties, good biocompatibility, low toxicity, and have high application potential in biomedical fields. These inorganic materials have been widely used in pharmaceutical excipients and active substances. In recent years, nanoclay mineral materials have been used as drug vehicles for the delivery of a variety of drugs based on their broad specific surface area, rich porosity, diverse morphology, good adsorption performance, and high ion exchange capacity. AREAS COVERED: This review introduces the structures, properties, and applications of various common natural and synthetic nanoclay materials as drug carriers. Natural nanoclays have different morphologies including nanoplates, nanotubes, and nanofibers. Synthetic materials have controllable sizes and flexible structures, where mesoporous silica nanoparticles, laponite, and imogolite are typical ones. These inorganic nanoparticles are often linked to polymers to form multifunctional drug delivery systems for better pharmaceutical performance. EXPERT OPINION: The clay nanomaterials have typical properties, including enhanced solubility of insoluble drugs, targeting therapeutic sites, controlled release, and stimulation of responsive drug delivery systems.

CPSF4 regulates circRNA formation and microRNA mediated gene silencing in hepatocellular carcinoma.

CircRNAs play essential roles in various physiological processes and involves in many diseases, in particular cancer. Global downregulation of circRNA expression has been observed in hepatocellular carcinoma (HCC) in many studies. Previous studies revealed that the pre-mRNA 3' end processing complex participates in circRNA cyclization and plays an important role in HCC tumorigenesis. Therefore, we explored the role of CPSF4, for 3' end formation and cleavage, in circRNA formation. Clinical research has shown that CPSF4 expression is upregulated in HCC and that high expression of CPSF4 is associated with poor prognosis in HCC patients. Mechanistic studies have demonstrated that CPSF4 reduces the levels of circRNAs, which possess a polyadenylation signal sequence and this decrease in circRNAs reduces the accumulation of miRNA and disrupts the miRNA-mediated gene silencing in HCC. Experiments in cell culture and xenograft mouse models showed that CPSF4 promotes the proliferation of HCC cells and enhances tumorigenicity. Moreover, CPSF4 antagonizes the tumor suppressor effect of its downstream circRNA in HCC. In summary, CPSF4 acts as an oncogene in HCC through circRNA inhibition and disruption of miRNA-mediated gene silencing.

Specific and sensitive detection of CircRNA based on netlike hybridization chain reaction.

Circular RNA (circRNA), as a new class of biomarker, plays an important role in the occurrence and development of cancer. However, the limitations of detection methods in recent years have severely restricted the related research of circRNA. Here, we have developed an effective circRNA detection method based on the thermostatic netlike hybridization chain reaction (HCR). It combines reverse transcription-rolling circle amplification (RT-RCA) with well-designed netlike HCR to achieve dual selection and dual signal amplification, which can eliminate the interference of linear isomers. This two-dimensional netlike HCR is composed of an ingeniously designed trigger chain and two hairpin fuel probes, which can generate a stable network structure with RT-RCA products containing multiple sets of repeats at a constant temperature, thereby producing enhanced fluorescent signals. Systematic studies reveal that the optimized netlike HCR system has higher detection efficiency for DNA strands containing multiple sets of repetitive sequences, can detect circRNA as low as 0.1 pM, and has excellent selectivity. By using human tumor cell lines and tissues, it has been verified that the netlike HCR-based method can accurately detect specific circRNA in real biological samples without RNase R enrichment, which provides a simple and useful platform for detecting low-abundance circRNA. Furthermore, the proposed strategy is also a potential method for detecting some genes containing repetitive sequences, such as telomere DNA, centromere DNA and ribosomal DNA (rDNA).

Natural deep eutectic solvents as tailored and sustainable media for the extraction of five compounds from compound liquorice tablets and their comparison with conventional organic solvents.

An efficient and environmentally friendly ultrasound-assisted (UAE) natural deep eutectic solvent (NADES) extraction method was applied for the extraction of five bioactive compounds (liquiritin, isoliquiritin, liquiritigenin, glycyrrhizic acid and isoliquiritigenin) from compound liquorice tablets (CPLTs), and the antioxidant activities of these compounds were evaluated. In this study, eighteen different NADES systems based on either two or three components were tested and a 1,4-butanediol-levulinic acid system (1 : 3 molar ratio) was selected as a topgallant solvent for maximizing analyte extraction yields. Various extraction parameters, such as water content, liquid/solid ratio, extraction time and temperature, were systematically optimized by single-factor and response surface methodology (RSM) experiments. The results indicated that the optimum extraction conditions for the analytes featured a water content of 17%, a liquid/solid ratio of 42 mL g-1 and an extraction time of 30 min. The extracted amounts of liquiritin, isoliquiritin, liquiritigenin, glycyrrhizic acid and isoliquiritigenin reached 5.60, 3.17, 1.27, 74.62 and 1.34 mg g-1, respectively, under optimized conditions, which were much higher than those extracted using conventional organic solvents. In addition, antioxidant tests revealed that the NADES extracts showed higher DPPH and hydroxyl radical-scavenging capacity than the conventional solvent extracts used for comparison. This study provides a suitable approach for efficiently extracting the bioactive compounds of CPLTs. Meanwhile, NADESs can be extended to other natural products as green extraction media.

Investigation of pathogenesis and therapeutic targets of acute myeloid leukemia based on untargeted plasma metabolomics and network pharmacology approach.

Acute myeloid leukemia (AML) is a malignant disease originating from bone marrow hematopoietic stem cells, characterized by anemia, hemorrhage, fever, and infection, with low survival rate. However, the pathogenesis of AML is not fully understood at present. In this work, an integrated approach based untargeted metabolomics and network pharmacology was adopted to elucidate the pathogenesis of AML. Metabolic profiling of plasma samples from 14 patients and 16 healthy individuals were performed based on UHPLC-MS platform. As a result, 23 metabolites were identified by using the human metabolite database based on PLS-DA (partial least squares discriminant analysis) and independent sample test. And metabolic pathways related to AML mainly included fatty acid metabolism, amino acid metabolism, energy metabolism and lipid metabolism. Meanwhile, biomarkers-targets-pathways-disease network was constructed, 75 biomarker targets and 122 disease targets were identified. Furthermore, 30 pathways were predicted, some of which were consistent with these in metabolomics. This is the first time that metabolomics and network pharmacology approach have been combined to investigate the pathogenesis and therapeutic targets of AML. ALDH, CYP2E1 and CYP3A4 were potential therapeutic targets for AML, which provide available way to elucidate the pathogenesis and treatment of AML.

Magnetic solid-phase extraction based on magnetic amino modified multiwalled carbon nanotubes for the fast determination of seven pesticide residues in water samples.

A rapid and simple analytical method based on magnetic solid-phase extraction with magnetic amino modified multiwalled carbon nanotubes with ultra-high performance liquid chromatography-tandem mass spectrometry is reported for the determination of seven pesticides (futriafol, metalaxyl, myclobutanil, napropamide, epoxiconazole, fipronil and diniconazole) in water samples. In this study, magnetic amino modified multi-walled carbon nanotubes were synthesized and selected as a new kind of material to adsorb pesticides in the water samples. Various magnetic solid-phase extraction parameters, such as the amount and type of adsorbent, extraction methods, extraction time, the type and volume of desorption solvent, desorption time and solution ionic strength, were systematically optimized. Under optimum conditions, the method validation results showed good linearity and recoveries. The calibration curves were in the range of 1.0-100 ng mL-1 for napropamide, epoxiconazole, metalaxyl, and fipronil, while they were 5.0-500 ng mL-1 for futriafol, myclobutanil, and diniconazole, with determination coefficients (R2) higher than 0.9909. The limits of quantification were 1.0-5.0 ng mL-1 and the limits of detection were 0.3-1.5 ng mL-1. The recoveries of the seven pesticides ranged from 80.4% to 103.2%. This developed method, which is more convenient and effective in comparison with traditional methods, has been successfully applied for the analysis of pesticides in water samples qualitatively and quantitatively.

Effects of mongolian medicine Terminalia chebula Retz. on 6 CYP450 enzymes in rats.

Terminalia chebula Retz. (TCR) is a medicinal material commonly used in Mongolian medicine. After consulting the literature at home and abroad, current research on TCR focuses on chemical composition, pharmacodynamics, and fingerprints. The pharmacokinetics of TCR has not been reported. Cytochrome P450 (CYP450) is the main drug-metabolizing enzyme, and its activity may be induced or inhibited by certain drugs, resulting in drug interactions in clinical applications. The objective of this study was to establish a high performance liquid chromatography (HPLC) method that can simultaneously detect multiple probe drugs to determine the effect of TCR on the activities of CYP450 enzymes CYP2C19, CYP2E1, CYP2D6, CYP2C9, CYP3A4, and CYP1A2. Wistar rats (male) were divided into 5 groups according to the randomization principle, namely the control group, the positive group, and the high, medium and low dose group. After 15 days of continuous administration, the mixed probe drug was injected into the vein, and then a small amount of blood was collected from the orbital vein at different time points. After the samples were processed, the blood concentration of each probe drug was measured by the established HPLC method. The pharmacokinetic parameters of each probe drug were calculated using DAS software. Compared with the control group, the plasma clearance (CL) of chlorzoxazone and omeprazole decreased, and the maximum plasma concentration (Cmax) and area under the curve (AUC) increased in the TCR group. The pharmacokinetic parameters of theophylline, midazolam, metoprolol, and tolbutamide did not differ significantly. The results indicated that TCR mainly inhibited the activities of CYP2E1 and CYP2C19, but had no effect on the activities of CYP1A2, CYP2C9, CYP3A4 and CYP2D6. Extra care should be taken when drugs metabolized by CYP2C19 and CYP2E1 enzymes are used in combination with TCR, as drug-herb interactions may occur. These results can guide the clinical application of related drugs and provide valuable information for drug interactions. The main component that affects enzyme activity may be tannins in the water extract.