Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

Competition-Based Two-Dimensional Photonic Crystal Dually Cross-Linked Supramolecular Hydrogel for Colorimetric and Fluorescent Dual-Mode Sensing of Bisphenol A.

Bisphenol A (BPA), one of the most abundantly produced endocrine disrupting chemicals, is widely used in everyday plastic products and thus must be monitored. Multimode sensing platforms are able to combine the advantages of different strategies while solving the issues of inaccurate test results of single signal sensing. However, the exploration in this field is limited due to the compromise of sensing conditions and inevitable mutual interferences of different systems. Herein, we constructed a two-dimensional photonic crystal dually cross-linked supramolecular hydrogel (2DPCDCSH) by utilizing a host-guest pair of ß-cyclodextrin (ß-CD) and tert-butyl (t-Bu) as the second cross-linking for colorimetric and fluorescent dual-mode sensing of BPA. Based on the fact that BPA can act as a competitive guest to break the host-guest interaction between ß-CD and t-Bu, the cross-linking density decreased and an expansion-induced structural color change occurred. Sensitive and selective BPA detection can be easily achieved by measuring the Debye diffraction ring diameter or observing the color change of 2DPC with a detection limit of 1 µg mL-1. Moreover, the formation of the ß-CD/BPA complex gave a significant enhancement of the intrinsic fluorescence of BPA, obtaining a detection limit of 0.001 µg mL-1. The two sensing systems can share the same reaction condition and yield a wider dynamic response range than the single signal strategy. Overall, the proposed method presented an efficient, rapid, cost-effective, and regenerative dual-mode method for BPA analysis and shed new insights for the design of diversified sensing platforms.

Label-free fluorescence detection of microRNA based on target induced adenosine2-coralyne-adenosine2 formation.

This study develops a simple and label-free biosensor for sensitive and selective detection of microRNA (miRNA) based on the formation of the adenosine2-coralyne-adenosine2 complex mediated by miRNA-specific polyadenosine extension.

A highly sensitive strategy for base excision repair enzyme activity detection based on graphene oxide mediated fluorescence quenching and hybridization chain reaction.

A novel fluorescent nanosensor has been developed by combining super fluorescence quenching ability of graphene oxide and hybridization chain reaction amplification, which enables highly sensitive detection of base excision repair enzyme activity with a wide dynamic range from 0.0001 to 100 U mL(-1) and a detection limit of 0.00006 U mL(-1).

Isothermal nucleic acid amplification strategy by cyclic enzymatic repairing for highly sensitive microRNA detection.

Technologies enabling highly sensitive and selective detection of microRNAs (miRNAs) are critical for miRNA discovery and clinical theranostics. Here we develop a novel isothermal nucleic acid amplification technology based on cyclic enzymatic repairing and strand-displacement polymerase extension for highly sensitive miRNA detection. The enzymatic repairing amplification (ERA) reaction is performed via replicating DNA template using lesion bases by DNA polymerase and cleaving the DNA replicate at the lesions by repairing enzymes, uracil-DNA glycosylase, and endonuclease IV, to prime a next-round replication. By utilizing the miRNA target as the primer, the ERA reaction is capable of producing a large number of reporter sequences from the DNA template, which can then be coupled to a cyclic signal output reaction mediated by endonuclease IV. The ERA reaction can be configured as a single-step, close-tube, and real-time format, which enables highly sensitive and selective detection of miRNA with excellent resistance to contaminants. The developed technology is demonstrated to give a detection limit of 0.1 fM and show superb specificity in discriminating single-base mismatch. The results reveal that the ERA reaction may provide a new paradigm for efficient nucleic acid amplification and may hold the potential for miRNA expression profiling and related theranostic applications.

ß-cyclodextrin/spiropyran-functionalized optical-driven hydrogel film for bisphenol A detection in food packaging.

Bisphenol A (BPA), an endocrine disruptor, is widely used in food packaging materials, including drink containers. Sensitive detection of BPA is crucial to food safety. Herein, we have developed a novel optical-driven hydrogel film sensor for sensitive BPA detection based on the displacement of spiropyran (SP) from ß-cyclodextrin (ß-CD) cavity by BPA followed by the photochromism of the released SP. The released SP converts to the ring-opened merocyanine form which shows an enhanced red fluorescence in the dark. The sensor demonstrates a linear detection range from 0.1 to 20 µg mL-1 with a limit of detection at 0.027 µg mL-1 and a limit of quantification at 0.089 µg mL-1. Notably, the proposed ß-CD/SP hydrogel can be reused due to the reversible isomerization of SP and the reversible host-guest interaction. This sensor also shows good performance for BPA determination in real samples, indicating its great potential for food safety monitoring.

Induction Chemotherapy Followed by Primary Tumor Resection Did Not Bring Survival Benefits in Colon Cancer Patients With Asymptomatic Primary Lesion and Synchronous Unresectable Metastases.

BACKGROUND: It is still controversial whether primary tumor resection (PTR) improves survival in colorectal cancer (CRC) patients with unresectable metastases. METHODS: Colon cancer patients were enrolled and randomly allocated to with or without PTR after induction chemotherapy with XELOX or mFOLFOX6, and those with chemotherapy failure were excluded. The primary endpoint was TTF (time to strategy failure) on an intent-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02291744. RESULTS: Between April 2015 and July 2020, 140 patients were enrolled, and 54 patients were excluded due to colon obstruction (16), perforation (1), disease progression (22), death (1), radical resection (3), or other reasons (11). After induction chemotherapy, 86 patients were randomized into group A (the resection group, n = 42) or group B (chemotherapy-alone group, n = 44). The median TTF was 143 days (95% CI: 104.9-181.1) in group A and 196 days (95% CI: 96.5-295.5) in group B (HR: 0.930 95% CI: 0.589-1.468, p = 0.755), and there was no significant difference in PFS, OS, and incidence of chemotherapy-related adverse events between two groups. The primary lesion-related events after PTR in group A were significantly fewer than those in group B. Patients with a tumor regression grade (TRG) score of 2 had longer TTF and PFS than those with score of 3. CONCLUSION: PTR after induction chemotherapy could not bring survival benefits for colon cancer patients with unresectable metastases, and it is not recommended routinely. However, it also requires individualized treatment as colon obstruction or perforation occurred in some patients and PTR could reduce primary tumor-related events, and the TRG score might help for selection of beneficial patients.

EZH2: a novel target for cancer treatment.

Enhancer of zeste homolog 2 (EZH2) is enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2) that can alter downstream target genes expression by trimethylation of Lys-27 in histone 3 (H3K27me3). EZH2 could also regulate gene expression in ways besides H3K27me3. Functions of EZH2 in cells proliferation, apoptosis, and senescence have been identified. Its important roles in the pathophysiology of cancer are now widely concerned. Therefore, targeting EZH2 for cancer therapy is a hot research topic now and different types of EZH2 inhibitors have been developed. In this review, we summarize the structure and action modes of EZH2, focusing on up-to-date findings regarding the role of EZH2 in cancer initiation, progression, metastasis, metabolism, drug resistance, and immunity regulation. Furtherly, we highlight the advance of targeting EZH2 therapies in experiments and clinical studies.

Dual-Signal Amplification Strategy for Sensitive MicroRNA Detection Based on Rolling Circle Amplification and Enzymatic Repairing Amplification.

MicroRNAs (miRNAs) play crucial regulatory roles as post-transcriptional regulators for gene expression and serve as promising biomarkers for diagnosis and prognosis of diseases. Herein, a dual-signal amplification method has been developed for sensitive and selective detection of miRNA based on rolling circle amplification (RCA) and enzymatic repairing amplification (ERA) with low nonspecific background. This strategy designs a padlock probe that can be cyclized in the presence of target miRNA to initiate the RCA reaction, after which the TaqMan probes that are complementary to the RCA products can be cyclically cleaved to produce obvious fluorescence signals with the help of endonuclease IV (Endo IV). Attributed to the dual-signal amplification procedure and the high fidelity of Endo IV, the RCA-ERA method allows quantitative detection of miR-21 in a dynamic range from 2 pM to 5 nM with a low background signal. Moreover, it has the ability to discriminate single-base difference between miRNAs and shows good performance for miRNA detection in complex biological samples. The results demonstrate that the RCA-ERA assay holds a great promise for miRNA-based diagnostics.

Single-step, high-specificity detection of single nucleotide mutation by primer-activatable loop-mediated isothermal amplification (PA-LAMP).

Loop-mediated isothermal amplification (LAMP) is a useful platform for nucleic acids detection in point-of-care (POC) situations, and development of single-step, close-tube LAMP reactions for specific detection of single nucleotide mutations (SNMs) remains a challenge. We develop a novel primer-activatable LAMP (PA-LAMP) strategy that enables highly specific and sensitive SNM detection using single-step, close-tube reactions. This strategy designs a terminal-blocked inner primer with a ribonucleotide insertion, which is cleaved and activated specifically to perfectly matched targets by ribonuclease (RNase) H2, to realize efficient amplification of mutant genes. It has shown dynamic responses of mutant target in a linear range from 220 aM to 22 pM with a lowest detectable concentration of 22 aM. It also demonstrates very high specificity in identifying the mutant in a large excess of the wild-type with a discrimination ratio as high as ∼10,000. It has been successfully applied to mutation detection of genomic DNA in tumor cells. The PA-LAMP strategy provides a useful, portable and affordable POC platform for highly sensitive and specific detection of genetic mutations in clinical applications.

Target induced reconstruction of DNAzymatic amplifier nanomachines in living cells for concurrent imaging and gene silencing.

A novel DNAzymatic amplifier nanomachine that is reconstructed in response to an mRNA input has been developed to enable the functions of concurrent sensitive mRNA imaging and activatable gene therapy in living cells.

A label-free and highly sensitive strategy for uracil-DNA glycosylase activity detection based on stem-loop primer-mediated exponential amplification (SPEA).

Uracil-DNA glycosylase (UDG) plays essential roles in base excision repair (BER) pathway by eliminating uracil from DNA to sustain the genome integrity. Sensitive detection of UDG activity is of great significance in the study of many fundamental biochemical processes and clinical applications. We develop a label-free method for UDG activity detection using stem-loop primer-mediated exponential amplification (SPEA). In the presence of active UDG, the uracil base in helper hairpin probe (HP) can be excised to generate an abasic site (AP site), which can be cleaved by endonuclease IV (Endo IV) with a blocked primer released. This primer then triggers the strand displacement reaction to produce a dumb-bell structure DNA, which can initiate a loop-mediated isothermal amplification (LAMP) reaction. This reaction generates a large number of long double-strand DNA replicates, which can be stained by SYBR Green (SG) I to deliver enhanced fluorescence for quantitative detection of UDG activity. A linear range from 0.001 U/mL to 1 U/mL and a detection limit down to 0.00068 U/mL are achieved. This strategy has also been demonstrated for UDG assay in complex cell lysates, implying its great potential for UDG based clinical diagnostics and therapeutics.

A ligation-based loop-mediated isothermal amplification (ligation-LAMP) strategy for highly selective microRNA detection.

A novel ligation-based loop-mediated isothermal amplification (ligation-LAMP) method has been developed for miRNA detection, which enables highly selective and sensitive quantitative detection of miR-21 in a dynamic range from 1 fM to 1 nM with the ability to discriminate single-base mismatches.