Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Iristectorigenin C suppresses LPS-induced macrophages activation by regulating mPGES-1 expression and p38/JNK pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used clinically to treat inflammatory diseases clinically. However, the adverse effects of NSAIDs cannot be ignored. Therefore, it is critical for us to find alternative anti-inflammatory drugs that can reduce adverse reactions to herbal medicine, such as Iris tectorum Maxim., which has therapeutic effects and can treat inflammatory diseases and liver-related diseases. AIM OF THE STUDY: This study aimed to isolate active compounds from I. tectorum and investigate their anti-inflammatory effects and action mechanisms. MATERIALS AND METHODS: Fourteen compounds were isolated from I. tectorum using silica gel column chromatography, Sephadex LH-20, ODS and high performance liquid chromatography, and their structures were identified by examining physicochemical properties, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Classical inflammatory cell models were established using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and rat primary peritoneal macrophages to examine the effect of these compounds. To examine the action mechanisms, the nitric oxide (NO) levels were measured by Griess reagent and the levels of inflammatory cytokines in the supernatant were measured by ELISA; The expressions of major proteins in prostaglandin E2 (PGE2) synthesis and the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were examined by Western blotting, and the mRNA expression levels were measured by quantitative real-time polymerase chain reaction; and the nuclear translocation of p65 was examined by high content imaging. Molecular docking was used to predict the binding of active compound to target protein. RESULTS: Our findings revealed that Iristectorigenin C (IT24) significantly inhibited the levels of NO and PGE2 without affecting cyclooxygenase (COX)-1/COX-2 expression in LPS-induced RAW264.7 cells and rat peritoneal macrophages. Furthermore, IT24 was shown to decrease the expression of microsomal prostaglandin synthetase-1 (mPGES-1) in LPS-induced rat peritoneal macrophages. IT24 did not suppress the phosphorylation and nuclear translocation of proteins in the NF-κB pathway, but it inhibited the phosphorylation of p38/JNK in LPS-stimulated RAW264.7 cells. Additionally, molecular docking analysis indicated that IT24 may directly bind to the mPGES-1 protein. CONCLUSION: IT24 might inhibit mPGES-1 and the p38/JNK pathway to exert its anti-inflammatory effects and could be also developed as an inhibitor of mPGES-1 to prevent and treat mPGES-1-related diseases, such as inflammatory diseases, and holds promise for further research and drug development.

Visual Assay for Methicillin-Resistant Staphylococcus aureus Based on Rolling Circular Amplification Triggering G-Quadruplex/Hemin DNAzyme Proximity Assembly.

Nowadays, infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have constituted a new challenge for anti-infective treatment. Precise identification and rapid clinical diagnostics of MRSA from other methicillin-sensitive strains entail assays with robust diagnostic efficiency and simple operation steps. Sensitive detection of MecA gene is promising to indicate MRSA infection, but it is challenged by the lack of isothermal and simple strategies. A visual assay based on isothermal rolling circular amplification and G-quadruplex/hemin (G4/hemin) DNAzyme proximity assembly was proposed for the immediate, efficient, and cost-effective detection of MecA in simple operation steps and in a single tube. The presence of MecA specifically drove the formation of circular templates, which further triggered isothermal amplification. The amplified product offered abundant binding sites for DNA-grafted hemin probes to form a novel proximity-assembled G4/hemin DNAzyme structure for colorimetric changing diagnosis. This tandem-repeated novel DNAzyme possessed higher catalytic activity and a lower background signal than traditional G4/hemin DNAzyme, ensuring sensitive discrimination of MRSA (limit of detection: 9.6 pM). Assay stability and antimatrix interference capability enable clinical application, which shows compared diagnostic ability with classic methods (100% sensitivity and 100% specificity) but possesses more simplified procedures and shorter turnaround time (<6 h). This colorimetric strategy in a nonsite-specific and hypersensitive manner holds foreseeable prospects in clinical diagnostic and research applications.

3, 4-seco-Labdane diterpenoids from the leaves of Callicarpa nudiflora with anti-inflammatory effects.

Four new 3, 4-seco-labdane diterpenoids, nudiflopenes J-M, were isolated from the leaves of Callicarpa nudiflora along with six known compounds. The structures of these diterpenoids were determined by comprehensive spectroscopic analysis. All the isolated compounds were evaluated for their inhibitory effects on NO production in LPS-stimulated RPMs and RAW264.7 cells. The results suggest that nudiflopenes J-M and other four known compounds showed significant inhibitory effects against NO production comparable to the positive control dexamethasone.

Common functional polymorphism within miR-146a and miR-196a-2 as susceptibility loci for hepatocellular carcinoma: An updated meta-analysis.

BACKGROUND: Mutations or single nucleotide polymorphisms (SNPs) within the gene region of microRNAs play an important role for the development of hepatocellular carcinoma (HCC). Extensive studies have tried to investigate the susceptibility role of miR-146a rs2819164 and miR-196a-2 rs11614913. However, these results are still inconsistent and inconclusive. We undertook a meta-analysis containing primarily Asian studies to assess the associations of the two SNPs with HCC risk. METHODS: 19 studies including miR-146a (7170 cases and 9443 controls) and 15 studies including miR-196a-2 (6417 cases and 7627 controls) were used for meta-analysis. Odds ratios and 95% CI were calculated to assess the association in five different genetic models. RESULTS: For the rs2910164 polymorphism of miR-146a, significantly increased risks for HCC were observed when all studies were pooled under two models (CG vs CC: OR = 1.11, 95% CI = 1.02-1.21, P = 0.021; GG + CG vs CC: OR = 1.11, 95% CI = 1.01-1.22, P = 0.035). For the rs11614913 polymorphism of miR-196a-2, significant increased risks for HCC development were observed when all studies were pooled under four models (C vs T: OR = 1.14, 95% CI = 1.06-1.23, P = 0.001; CC vs TT: OR = 1.31, 95% CI = 1.12-1.53, P = 0.001; CC + TC vs TT: OR = 1.16, 95% CI = 1.03-1.31, P = 0.018; CC vs TC + TT: OR = 1.14, 95% CI = 1.00-1.30, P = 0.043). CONCLUSION: Our results show that the two common SNPs within the miRNAs were associated with modest increased risk of HCC (OR < 1.6), especially in the Asian population. Larger population-based studies validating these results are needed.

An electrochemical aptasensor for detection of IFN-γ using graphene and a dual signal amplification strategy based on the exonuclease-mediated surface-initiated enzymatic polymerization.

Tuberculosis is one of the major health problems in the world. The cytokine interferon γ (IFN-γ) is associated with the disease-specific immune responses and is used as a tuberculosis diagnosis marker. In this study, a novel electrochemical aptasensor was developed for IFN-γ detection based on the exonuclease-catalyzed target recycling and the TdT-mediated cascade signal amplification. To construct the aptasensor, a previously hybridized double-stranded DNA (capture probe hybridization with a complementary IFN-γ binding aptamer) was immobilized on a gold nanoparticle-graphene (Au-Gra) nanohybrid film-modified electrode. In the presence of IFN-γ, the formation of an aptamer-IFN-γ complex leads to the liberation of the aptamer from the double-stranded DNA (dsDNA). Using exonuclease, the aptamer was selectively digested, and IFN-γ was released for the target recycling. A large amount of single-stranded capture probes formed and led to the hybridization with signal probe-labelled Au@Fe3O4. Then, the labelled signal probe sequences were catalyzed at the 3'-OH group by terminal deoxynucleotidyl transferase (TdT) to form a long single-stranded DNA structure. As a result, the electron mediator hexaammineruthenium(III) chloride ([Ru(NH3)6](3+)) electrostatically adsorbed onto DNA producing a strong electrochemical signal which can be used to quantitatively measure the IFN-γ levels. With the conducting nanomaterial Au-Gra as a substrate and the target recycling-based surface-initiated enzymatic polymerization-mediated signal amplification strategy, the proposed aptasensor displayed a broad linearity with a low detection limit of 0.003 ng mL(-1). Moreover, the resulting aptasensor exhibited good specificity, acceptable reproducibility and stability, which makes this method versatile and suitable for detecting IFN-γ and other biomolecules.

Ultrasensitive strategy based on PtPd nanodendrite/nano-flower-like@GO signal amplification for the detection of long non-coding RNA.

Highly up-regulated in liver cancer (HULC) is a novel promising noninvasive biomarker for hepatocellular carcinoma (HCC), which is a kind of long non-coding RNAs (lncRNAs). But traditional methods limited HULC clinical detection for ownself drawbacks. Development a new HULC detection approach is urgent and necessary. Electrochemical nucleic acid sensor based on different signal amplification strategies with high sensitivity, fast, simple, and convenient, may solve this problem. Herein, we propose a novel strategy based on Pt-Pd bimetallic nanodendrites/nanoflower-like clusters on graphene oxide/Au/horseradish peroxidase (PtPd BND/BNF@GO/Au/HRP) to enhance the catalytic efficiency and sensitivity. And Au particles were simultaneously and separately capped with thionine or detection probe, which increase the binding amount of detection probe and decrease the electronic background. The results indicated that the catalytic effect was noticeably elevated and that the biosensor provides ultrasensitive detection for the lncRNA HULC. The linear calibration of the biosensor ranged from 1.00×10(-3) to 1.00×10(3) pM/mL, and the limit of detection was 0.247 fM/mL. The lncRNA biosensor based on the PtPd BND/BNF@GO/Au/HRP/Au/thionine exhibited acceptable reproducibility and clear selectivity. This strategy may provide a new alternative for clinical HCC diagnosis through the detection of HULC.

A novel ultrasensitive ECL sensor for DNA detection based on nicking endonuclease-assisted target recycling amplification, rolling circle amplification and hemin/G-quadruplex.

In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL) detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease)-assisted target recycling amplification (TRA), rolling circle amplification (RCA) and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form "Y" junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O8(2-) system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM) detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

Procalcitonin sensitive detection based on graphene-gold nanocomposite film sensor platform and single-walled carbon nanohorns/hollow Pt chains complex as signal tags.

Septicemia is a serious disease that requires early diagnosis, and procalcitonin (PCT) serves as a diagnostic biomarker for this disease. Traditional clinical detection (via immune-gold chips) remains difficult and expensive. An electrochemical immunosensor based on new nanomaterials may provide a solving approach. Herein, an ultrasensitive sandwich electrochemical strategy for PCT detection was developed. Firstly, reduced graphene oxide (rGO)-gold (Au) nano-composite film was used as the immunosensor platform to increase the amount of PCT antibody 1(Ab1) immobilized. Next, single-walled carbon nanohorns (SWCNHs)/hollow Pt chains (HPtCs) complex was firstly utilized to label PCT Ab2 as signal tags. For SWCNHs with few side effects, high surface area and HPtCs with higher specific surface, better catalytic activity, complex synthesized from both may provide more advantages. Moreover, to amplify signal, HPtC catalytic activity with H2O2 was enhanced by horseradish peroxidase (HRP) for dual synergy amplification. The whole results demonstrated that the proposed immunosensor exhibited fast operation, high sensitivity, good reproducibility, acceptable stability and ideal selectivity compared with traditional method. The linear calibration of the immunosensor ranged from 1.00 pg/mL to 2.00 × 10(1)ng/mL with a detection limit of 0.43 pg/mL. Analytical application results revealed that the immunosensor matched with the real concentrations of serum samples. Overall this immunosensor may provide a new alternative strategy for PCT detection.

LBP and CD14 polymorphisms correlate with increased colorectal carcinoma risk in Han Chinese.

AIM: To explore the associations of polymorphisms of lipopolysaccharide binding protein (LBP), cluster of differentiation 14 (CD14), toll-like receptor 4 (TLR-4), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) with the colorectal carcinoma (CRC) risk in Han Chinese. METHODS: Polymorphisms of LBP (rs1739654, rs2232596, rs2232618), CD14 (rs77083413, rs4914), TLR-4 (rs5030719), IL-6 (rs13306435) and TNF-α (rs35131721) were genotyped in 479 cases of sporadic colorectal carcinoma and 486 healthy controls of Han Chinese in a case-control study. Single-nucleotide polymorphisms (SNPs) between cases and controls were analyzed by unconditional logistic regression. RESULTS: GA and GG genotypes of LBP rs2232596 were associated with a significantly increased risk of CRC [odds ratio (OR) = 1.51, 95% confidence interval (CI) 1.15-1.99, P = 0.003; OR = 2.49, 95% CI 1.16-5.38, P = 0.016, respectively]. A similar association was also observed for the CG genotype of CD14 rs4914 (OR= 1.69, 95% CI 1.20-2.36, P = 0.002). In addition, a combination of polymorphisms in LBP rs2232596 and CD14 rs4914 led to a 3.4-fold increased risk of CRC (OR = 3.44, 95% CI 1.94-6.10, P = 0.000). CONCLUSION: This study highlights the LBP rs2232596 and CD14 rs4914 polymorphisms as biomarkers for elevated CRC susceptibility in the Chinese Han population.

Preliminary validation of ERBB2 expression regulated by miR-548d-3p and miR-559.

ERBB2 overexpression occurs in numerous types of primary human tumors and alterations in microRNA (miRNA) expression have been associated with tumor suppression or tumorigenesis in human cancer, nevertheless, little is known about natural miRNAs acting on ERBB2. In this study, bioinformatical analysis of the 3'-UTRs of ERBB2 revealed the target elements for miR-548d-3p and miR-559. Moreover, a predicted miRNA/mRNA interaction experimental validation showed that both miR-548d-3p and miR-559 can interact specifically with the 3'-UTR of the ERBB2 mRNA. And miR-548d-3p plus miR-559 transfection showed a cooperative regulation of translationally repressing ERBB2 mRNA rather than by either miR-548d-3p or miR-559 alone. These results not only support the idea that different miRNAs can simultaneously and cooperatively repress a given target mRNA but also preliminarily validate the role of miR-548d-3p and miR-559 in regulating the ERBB2 expression. These data provide molecular basis for the application of miRNAs in ERBB2-targeted therapy.