Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

The detection of microRNA expression profiles plays an important role in early diagnosis of different cancers. On the basis of the employment of redox labels with distinct potential positions and duplex specific nuclease (DSN)-assisted target recycling signal amplifications, we have developed a multiplexed and convenient electronic sensor for highly sensitive detection of microRNA (miRNA)-141 and miRNA-21. The sensor is constructed by self-assembly of thiol-modified, redox species-labeled hairpin probes on the gold sensing electrode. The hybridizations between the target miRNAs and the surface-immobilized probes lead to the formation of RNA/DNA duplexes, and DSN subsequently cleaves the redox-labeled hairpin probes of the RNA/DNA duplexes to recycle the target miRNAs and to generate significantly amplified current suppression at different potentials for multiplexed detection of miRNA-141 and miRNA-21 down to 4.2 and 3.0 fM, respectively. The sensor is also highly selective toward the target miRNAs and can be employed to monitor miRNAs from human prostate carcinoma (22Rv1) and breast cancer (MCF-7) cell lysates simultaneously. The sensor reported here thus holds great potential for the development of multiplexed, sensitive, selective, and simple sensing platforms for simultaneous detection of a variety of miRNA biomarkers for clinic applications with careful selection of the labels.

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, on the basis of a new miRNA-triggered molecular machine for enzyme-free target recycling signal amplification, the development of a simple electronic sensor for highly sensitive detection of miRNA-21 from human breast cancer cells is described. The three-stand DNA duplex probes are self-assembled on the gold electrode surface to fabricate the sensor. The miRNA-21 target binds to the terminal toehold region of the probes, displaces one of the short strands through toehold-mediated strand displacement reactions, and exposes the secondary toehold region for subsequent hybridization with the methylene blue (MB)-modified DNA fuel strand, which further displaces both the miRNA-21 target and the other short strand to activate the operation of the molecular machine. As a result, the miRNA-21 target is cyclically reused, and many MB-DNA fuel strands are attached to the sensor surface, leading to a significantly amplified current response for sensitive detection of miRNA-21 down to 1.4 fM. The developed sensor also shows high sequence discrimination capability and can be used to monitor miRNA-21 expression levels in cancer cells. Moreover, this sensor avoids the involvement of any enzymes for target recycling amplification and features with highly minimized background noise for miRNA detection, which makes this method hold great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.

On the basis of the use of silver nanoclusters (AgNCs) in situ synthesized by cytosine (C)-rich loop DNA templates as signal amplification labels, the development of a label-free and highly sensitive method for electrochemical detection of microRNA (miRNA-199a) is described. The target miRNA-199a hybridizes with the partial dsDNA probes to initiate the target-assisted polymerization nicking reaction (TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction (HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets.

Phthalic acid esters (PAEs) are a class of lipophilic chemicals widely used as plasticizers and additives to improve various products’ mechanical extensibility and flexibility. At present, synthesized PAEs, which are considered to cause potential hazards to ecosystem functioning and public health, have been easily detected in the atmosphere, water, soil, and sediments; PAEs are also frequently discovered in plant and microorganism sources, suggesting the possibility that they might be biosynthesized in nature. In this review, we summarize that PAEs have not only been identified in the organic solvent extracts, root exudates, and essential oils of a large number of different plant species, but also isolated and purified from various algae, bacteria, and fungi. Dominant PAEs identified from natural sources generally include di-n-butyl phthalate, diethyl phthalate, dimethyl phthalate, di(2-ethylhexyl) phthalate, diisobutyl phthalate, diisooctyl phthalate, etc. Further studies reveal that PAEs can be biosynthesized by at least several algae. PAEs are reported to possess allelopathic, antimicrobial, insecticidal, and other biological activities, which might enhance the competitiveness of plants, algae, and microorganisms to better accommodate biotic and abiotic stress. These findings suggest that PAEs should not be treated solely as a “human-made pollutant” simply because they have been extensively synthesized and utilized; on the other hand, synthesized PAEs entering the ecosystem might disrupt the metabolic process of certain plant, algal, and microbial communities. Therefore, further studies are required to elucidate the relevant mechanisms and ecological consequences.

No abstract

Charcot–Marie–Tooth disease (CMT), also known as hereditary motor and sensory neuropathy, is the most common inherited peripheral nerve disorder. Missense mutations, such as K141N, in the small heat shock protein HSPB8 are known to cause distal hereditary motor neuropathy 2A (dHMN2A) or Charcot-Marie-Tooth neuropathy type 2L (CMT2L). However, of critical clinical significance, very few specific therapies for this disease exist. In the present study, we investigated the impact of mutant K141N HSPB8 on mitochondrial distribution and function in a cellular model of CMT2L. Our results indicate that K141N HSPB8 induced mitochondrial aggregation and caused increased oxidative stress injury. As an extraction from Chinese celery Apium graveolens Linn seeds, L-3-n-Butylphthalide (NBP), has been reported to exert many neuroprotective effects, we interrogated whether NBP could elicit a protective effect on the cell injury typically caused by HSPB8 K141N mutations. We found NBP could reverse the pathological processes induced by HSPB8 K141N mutation via an antioxidant effect, modulation of the Bax/Bcl-2 mitochondrial apoptotic and Nrf2 pathways. We propose a novel function of HSPB8, highlighting the consequence of the K141N pathogenic mutation. Furthermore, we suggest NBP may have promising therapeutic potential in the treatment of CMT2L.

No abstract