A one-pot isothermal Fluorogenic Mango II arrays-based assay for label-free detection of miRNA.

The capability to detect a small number of miRNAs in clinical samples with simplicity, selectivity, and sensitivity is immensely valuable, yet it remains a daunting task. Here, we described a novel Mango II aptamers-based sensor for the one-pot, sensitive and specific detection of miRNAs. Target miRNA-initiated mediated catalyzed hairpin assembly (CHA) would allow for the production of plenty of DNA duplexes and the formation of the complete T7 promoter, motivating the rolling circle transcription (RCT). Then, the subsequent RCT process efficiently generates a huge number of repeating RNA Mango II aptamers, brightened by the incorporation of fluorescent dye TO1-B for miRNA quantification, realizing label-free and high signal-to-background ratio. Moreover, this assay possesses a remarkable ability to confer high selectivity, enabling the distinction of miRNAs among family members with mere 1- or 2- nucleotide (nt) differences. By employing the proposed assay, we have successfully achieved a sensitive evaluation of miR-21 content in diverse cell lines and clinical serum samples. This offers a versatile approach for the sensitive assay of miRNA biomarkers in molecular diagnosis.

Sensitive Detection of Escherichia coli O157:H7 Using Allosteric Probe and Hairpin Switches-Based Isothermal Transcription Amplification.

Pathogens pose a serious threat to public and population health, leading to serious outbreak and spread of diseases irrespective of the region. The capability to directly, sensitively, and specifically detect viable pathogens in low numbers in food and clinical samples is very desirable but remains a challenge. In this work, we present a novel assay of a combination of an aptamer-based allosteric probe and hairpin switch-controlled T7 RNA polymerase-based isothermal transcription amplification, which enables rapid, ultrasensitive, label-free detection of direct pathogens. It can detect Escherichia coli as low as 73.2 CFU/mL. Moreover, with the usage of the proposed assay, sensitive quantification of E. coliO157:H7 in milk samples has been achieved, showing significant potential as a simple and sensitive tool to quantify pathogens in milk and other foods.

Mobile phone-assisted imprinted nanozyme for bicolor colorimetric visual detection of erythromycin in river water and milk samples.

The residues of erythromycin (ERY) may have negative impacts on the ecological environment, health, and food safety. How to detect ERY effectively and visually is a challenging issue. Herein, we synthesized a molecularly imprinted polymer based nanozymes for selective detection of erythromycin (ERY-MIPNs) at neutral pH, and developed a mobile phone-assisted bicolor colorimetric detection system. This system produced a wide range of color changes from blue to pinkish purple as the ERY concentration increased, making it easy to capture the visualization result. Also, the system showed good sensitivity to ERY ranging from 15 to 135 µM, with a detection limit of 1.78 µM. In addition, the system worked well in the detection of ERY in river water and milk, with the recoveries of 95.57% âˆ¼ 103.20%. These data suggests that this strategy is of considerable potential for practical applications and it provides a new idea for visual detection with portable measurement.

Structural characterization and anti-inflammatory activities of a purified polysaccharide from fruits remnants of Alpinia zerumbet (Pers.) Burtt. et Smith.

We reported here an interesting source of Alpinia zerumbet Polysaccharides (named AZPs) from the residues after extracting essential oil by steam distillation from Alpinia zerumbet fructus. After a series of purifications, a homogeneous polysaccharide (AZP-2) of molecular weight 1.25 × 105 Da was obtained. Structure, anti-inflammatory activity, and anti-inflammatory mechanism were investigated. AZP-2 was mainly composed of galactose, arabinose, xylopyranose, glucose, and galacturonic acid. The main linkage structure of AZP-2 was determined after integrating the nuclear magnetic resonance (NMR) and methylation analysis, and the structure was comparatively complex. The results indicated that AZP-2 significantly decreased the production of NO and ROS in the inflammatory model established by lipopolysaccharide (LPS) stimulated RAW264.7, particularly at the concentration of 200 µg/mL. Furthermore, AZP-2 significantly modulated the secretion of both pro-inflammatory and anti-inflammatory cytokines. Notably, the mechanism of AZP-2 exhibiting inhibitory effects was related to regulating the NF-κB signaling pathway. Overall, AZP-2 could be used as a potential anti-inflammatory agent for further in-depth studies.

Fluorophore Label-Free Light-up Near Infrared Deoxyribonucleic Acid Nanosensor for Monitoring Extracellular Potassium Levels.

Fluorescent DNA nanosensors have been widely used due to their unique advantages, among which the near-infrared (NIR) imaging mode can provide deeper penetration depth and lower biological background for the nanosensors. However, efficient NIR quenchers require ingenious design, complex synthesis, and modification, which severely limit the development of NIR DNA nanosensors. Label-free strategies based on G-quadruplex (G4) and NIR G4 dyes were first introduced into in situ extracellular imaging, and a novel NIR sensing strategy for the specific detection of extracellular targets is proposed. The strategy avoids complex synthesis and site-specific modification by controlling the change of the NIR signal through the formation of a G4 nanostructure. A light-up NIR DNA nanosensor based on potassium ion (K+)-sensitive G4 chain PS2.M was constructed to verify the strategy. PS2.M forms a stable G4 nanostructure in the presence of K+ and activates the NIR G4 dye CSTS, thus outputting NIR signals. The nanosensor can rapidly respond to K+ with a linear range of 5-50 mM and has good resistance to interference. The nanosensor with cholesterol can provide feedback on the changes in extracellular K+ concentration in many kinds of cells, serving as a potential tool for the study of diseases such as epilepsy and cancer, as well as the development of related drugs. The strategy can be potentially applied to the NIR detection of a variety of extracellular targets with the help of functional DNAs such as aptamer and DNAzyme.

Chemical Composition and Potential Antimicrobial and Anti-Inflammatory Activities of Essential Oil from Fruits of Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm.

Alpinia zerumbet (Pers.) B.L.Burtt & R.M.Sm. was extensively used in traditional medicine for its several properties, but continuous investigation is needed to discover the properties of its essential oils (EOs). This work evaluated the properties of an EO obtained by steam distillation (named ESD) as well as extracts obtained by petroleum ether (named EP) both from Alpinia zerumbet fruits. Gas chromatography-mass spectrometry (GC-MS) was chosen to identify the composition, and eleven compounds were identified as the main components of the EO and EP of Alpinia zerumbet fruits. The antimicrobial properties were investigated by minimum inhibitory concentration (MIC) and the inhibition area. The results identified the differences in antimicrobial activities attributed to different extraction methods. Enzyme-linked immunosorbent assay (ELISA) and Western Blot (WB) assay were conducted to assess the anti-inflammatory effects of ESD. In conclusion, our study suggested that EO from Alpinia zerumbet fruits might be a prospective candidate for antimicrobial and anti-inflammatory therapy.

Hairpin Switches-Based Isothermal Transcription Amplification for Simple, Sensitivity Detection of MicroRNA.

The ability to simply, selectively, and sensitively detect low numbers of miRNAs in clinical samples is highly valuable but remains a challenge. In this work, we present a novel miRNA detection system by using the elaborately designed hairpin switch, where the T7 primer, template, target recognize sequence, and light-up RNA aptamer template are edited and embedded in one single-stranded DNA hairpin structure. In the beginning, the hairpin switch maintained the hairpin structure 1, in which the ds promoter of T7 polymerase was disrupted, thus the transcription reaction of T7 polymerase was inhibited. After binding to the target, the hairpin switch 1 was unfolded and turned to the hairpin structure 2. This switch initiates the in vitro T7 transcription reaction, producing plenty of RNA transcripts containing RNA aptamers. Consequently, transcribed tremendous RNA aptamers lighted up the fluorophore for quantitative analysis. Compared with the existing T7 polymerase-based amplification system, this strategy exhibits several advantages, including simplicity, convenience, and high selectivity and sensitivity. The experimental results demonstrated that we could achieve the quantification of miRNA in buffer and complex biological samples.

CRISPR-Cas12a coupled with cyclic reverse transcription for amplified detection of miRNA.

In this work, we present a highly sensitive, specific, and versatile method to quantify miRNA expression by coupling CRISPR-Cas12a with cyclic reverse transcription (CRT), termed as CRISPR-CRT. Each miRNA target was first converted and amplified into multiple hairpin RT products via CRT. Afterward, the hairpin RT products could serve as activators to initiate the collateral cleavage activity of CRISPR-Cas12a. Due to the above two-stage amplification, this assay could detect miRNA at sub-femtomolar level (LOD, 0.201 fM). Since the sequence of target miRNA is double checked: first in the CRT and then in the CRISPR system, the proposed assay also shows an excellent specificity in detecting miR-21. Finally, with the usage of this assay, the sensitive assessment of miR-21 levels in human serum samples has been achieved and the disease human serum has been detected. Conclusively, CRISPR-CRT holds a great application prospective in the field of clinical molecular diagnosis.

Self-assembly of DNA nanospheres with controllable size and self-degradable property for enhanced antitumor chemotherapy.

Controllable size, self-degradability and targeting property are important for a precise improvement of anticancer effects and reduction of side effects of drug vehicles. Here, a series of DNA nanospheres with controllable size and self-degradation ability were constructed through the hybridization of two i-motif strands and two linker strands for targeted cancer therapy. DNA nanospheres with different sizes were fabricated by regulating the linker sequence, and their pH-responsive self-degradation property was realized by the introduction of the i-motif strand. Moreover, the ZY11 aptamer was introduced to endow the DNA nanospheres with targeting property toward SMMC-7721 cancer cells. The results revealed that the appropriate size of DNA nanospheres (80 nm) highly promoted the internalization by mammalian cells. The results of DLS, AFM and CD spectra showed that the DNA nanospheres were stable in a physiological environment but they self-degraded in a slightly acidic environment due to the existence of the i-motif strand. Moreover, the fluorescence of DOX@AP-NSs2 was triple at pH = 5.0 than at pH = 7.4, which further confirmed the pH-responsive drug release performance. The above results proved that the use of DOX@AP-NSs2 is a promising approach to accelerate the rapid release of drugs into the tumors and avoid drug leakage into the normal tissue. The results at a cellular level and in vivo confirmed the pH-responsive targeted antitumor effect. Hence, the novel DNA nanospheres with controllable size and self-degradable property represent a potential tool for targeted drug delivery and cancer therapy.

Nanoflare Couple: Multiplexed mRNA Imaging and Logic-Controlled Combinational Therapy.

Theranostics, which combines both diagnostic and therapeutic capabilities in one dose, has always been an intractable challenge in personalized cancer treatment. Herein, a versatile nanotheranostic platform "nanoflare couple (NC)" has been developed for in situ multiplex cancer-related mRNA imaging and subsequent logic-controlled aggregation of gold nanoparticles, leading to gene therapy and photothermal therapy upon irradiation with infrared light. As a proof of concept, TK1 and survivin mRNAs that are highly expressed in most tumor tissues are selected as endogenous cancer indicators and therapy triggers to design the NC. Mice bearing breast cancer cells MCF-7 are prepared as a model to test its efficacy. The in vitro and in vivo assays validate that the NC show the capability for multiplexed mRNA imaging and high efficiency for logic-controlled combinational therapy of breast cancer.

Fabrication, GSH-responsive drug release, and anticancer properties of thioctic acid-based intelligent hydrogels.

Injectable hydrogels are potential local drug delivery systems since they contain plenty of water and soft like biological tissues. Such hydrogels could be injected directly into the tumor site where the drug is released under the tumor microenvironment. However, drug loaded hydrogels for cancer treatment based on lipoic acid (natural small molecule) have not been exploited. Here, a novel poly(lipoic acid)-poly(ethylene glycol) (PEG-PTA) hydrogels were prepared through a two-step reaction. The hydrogels contained disulfide bonds, so they could be degraded via the thiol exchange reaction with the abundant GSH in the tumor microenvironment, and subsequently release the drug. The results in vitro and at cellular level showed that the hydrogels were degraded and released the drugs only in the presence of GSH. Therefore, the injectable GSH-responsive hydrogels are promising to be served as an intelligent drug delivery system for cancer treatment.

Waste coal cinder catalyst enhanced electrocatalytic oxidation and persulfate advanced oxidation for the degradation of sulfadiazine.

Waste coal cinder, a kind of waste cinder discharged from coal combustion of thermal power plants, industrial and civil boilers, and other equipment, was rich in metal oxides with catalytic activity. In this work, waste coal cinder was used to enhance electrochemical coupling peroxymonosulfate (PMS) advanced oxidation degradation of sulfadiazine (SD). The surface morphology, elemental composition, and electrocatalytic activity of waste coal cinder were characterized by various characterization instruments. The results show that compared with simple electrocatalytic oxidation, electrocatalytic oxidation + waste coal cinder and electrocatalytic coupled persulfate oxidation, electrocatalytic oxidation + PMS advanced oxidation + waste coal cinder has the largest removal efficiency (99.95%) and mineralization rates (90.16%) of SD in 90 min, indicating that the introduction of waste coal cinder greatly increases the degradation efficiency. •OH and SO4-• were detected during the process of degradation. The optimal degradation process parameters were explored through different voltage, pH, plate spacing, aeration flow rate, potassium peroxymonosulfate sulfate complex salt dose, and Na2SO4 dosage. Cycling experiments show waste coal cinder has good structural stability. Through the analysis of triple quadrupole liquid chromatography-mass spectrometry (LC-MS), we put forward three possible ways of SD degradation. This research will provide a novel vision for water treatment.

Designing a CRISPR/Cas12a- and Au-Nanobeacon-Based Diagnostic Biosensor Enabling Direct, Rapid, and Sensitive miRNA Detection.

Direct, rapid, sensitive, and selective detection of nucleic acids in complex biological fluids is crucial for medical early diagnosis. We herein combine the trans-cleavage ability of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with Au-nanobeacon to establish a CRISPR-based biosensor, providing rapid miRNA detection with high speed and attomolar sensitivity. In this strategy, we first report that the trans-cleavage activity of CRISPR/cas12a, which was previously reported to be triggered only by target ssDNA or dsDNA, can be activated by the target miRNA directly. Therefore, this method is direct, i.e., does not need the conversion of miRNA into its complementary DNA (cDNA). Meanwhile, as compared to the traditional ssDNA reporters and molecular beacon (MB) reporters, the Au-nanobeacon reporters exhibit improved reaction kinetics and sensitivity. In this assay, the miRNA-21 could be detected with very high sensitivity in only 5 min. Finally, the proposed strategy enables rapid, sensitive, and selective miRNA determination in complex biological samples, providing a potential tool for medical early diagnosis.

The Main Structural Unit Elucidation and Immunomodulatory Activity In Vitro of a Selenium-Enriched Polysaccharide Produced by Pleurotus ostreatus.

In recent years, the structure of selenium-enriched polysaccharides and their application in immunomodulation have attracted much attention. In previous studies, we extracted and purified a novel selenium-enriched Pleurotus ostreatus polysaccharide called Se-POP-21, but its structure and immunomodulatory activity were still unclear. In this study, the main structural unit formula of Se-POP-21 was characterized by methylation analysis and an NMR experiment. The results showed that the backbone of Se-POP-21 was →[2,6)-α-D-Galp-(1→6)-α-D-Galp-(1]4→2,4)-ß-L-Arap-(1→[2,6)-α-D-Galp-(1→6)-α-D-Galp-(1]4→, branched chain of ß-D-Manp-(1→ and ß-D-Manp-(1→4)-ß-L-Arap-(1→ connected with →2,6)-α-D-Galp-(1→ and →2,4)-ß-L-Arap-(1→,respectively, through the O-2 bond. In vitro cell experiments indicated that Se-POP-21 could significantly enhance the proliferation and phagocytosis of RAW264.7 cells, upregulate the expression of costimulatory molecules CD80/CD86, and promote RAW264.7 cells to secrete NO, ROS, TNF-α, IL-1ß, and IL-6 by activating the NF-κB protein. The results of this study indicate that Se-POP-21 can effectively activate RAW264.7 cells. Thus, it has the potential to be used in immunomodulatory drugs or functional foods.

Bimetallic modified halloysite particle electrode enhanced electrocatalytic oxidation for the degradation of sulfanilamide.

The treatment of antibiotics wastewater by electrocatalytic oxidation has attracted much attention. In the paper, a novel halloysite bimetallic (HLS-Cu-Mn) particle electrode material was prepared and a bench-scale electrocatalytic reaction tank was designed. A three-dimensional electrocatalytic oxidation reactor composed of HLS-Cu-Mn and a bench-scale electrocatalytic reaction tank was used to degrade Sulfanilamide (SA) wastewater. Characterization of the synthesized material was conducted with Scanning electron microscopy (SEM), X-ray polycrystalline powder diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), and Brunauer-Emmett-Teller (BET). The electron spin resonance spectroscopy test results confirmed that HLS-Cu-Mn produced a large number of •OH. The electrochemical workstation confirmed that HLS-Cu-Mn had strong electrocatalytic activity and repolarization ability. Under the optimum preparation conditions and degradation process parameters, the removal efficiency of SA and TOC was 99.84% and 88.95% respectively. The method also has good degradation efficiency for aniline, phenol, herbicides, antibiotics, and dyeing wastewater. It was found that 4 main intermediates appeared in the degradation process by Ultra-high performance liquid chromatography/triple tandem quadrupole mass spectrometry (LC-MS). In sum, it was believed that this work provides a new vision and idea for water treatment.

Structural characterization and anti-tumor activity in vitro of a water-soluble polysaccharide from dark brick tea.

Recently, more and more attention has been paid to the structure and application of tea polysaccharides. Herein, a water-soluble homogeneous polysaccharide (DTP-1) from dark brick tea was purified, characterized, and investigated its anti-tumor activity in vitro. The DTP-1 with a molecular weight of 11,805 Da is mainly composed of glucose, galactose and arabinose. It has a backbone, which is composed of →4)-α-D-Glcp-(1→, →5)-α-L-Araf-(1→, →6)-ß-D-Galp-(1→, →2)-α-L-Araf-(1→, →3)-ß-D-Galp-(1→, with →4,6)-ß-D-Galp-(1 â†’ as branching point and →1)-ß-D-Glcp as terminal. In addition, DTP-1 could significantly affect the viability of A549 and SMMC7721 cells with an inhibition rate of 31.71% and 33.38% (600 µg/mL, 24 h), respectively, by inducing apoptosis and inhibiting cell migration. Moreover, DTP-1 had no effect on corresponding normal cells. Therefore, DTP-1 showed great potential to become a functional food and an anti-tumor drug.

A natural selenium polysaccharide from Pleurotus ostreatus: Structural elucidation, anti-gastric cancer and anti-colon cancer activity in vitro.

The development and application of new natural selenium polysaccharides with relatively clear structure and excellent activity have become hot and difficult issues. This study used GC-MS and 2D NMR to characterize the detailed chain structure information of selenium polysaccharide (Se-POP-3) from Selenium-enriched Pleurotus ostreatus, and then explored its anti-gastric cancer and anti-colon cancer effects in vitro. Results showed that the main chain of Se-POP-3 was →[3)-ß-D-Glcp-(1]2 â†’ 6)-ß-D-Glcp-(1 â†’ 3,6)-ß-D-Glcp-(1 â†’ 3)-ß-D-Glcp-(1→, and the branch was α-D-Glcp-(1 â†’ [4)-α-D-Glcp-(1]4→, which was connected to the main chain through the O-3 bond of →3,6)-ß-D-Glcp-(1 â†’ glycosidic bond. In addition, Se-POP-3 could reduce viability, induce apoptosis, inhibit migration and invasion, destroy the Bax/Bcl-2 ratio, and inhibit the epithelial-to-mesenchymal transition of MGC-803 and HCT-116 cells in vitro. Moreover, this study also showed that within the concentration range set in this study, Se-POP-3 had no significant effect on the growth of normal cells (NCM460 cells). This study can provide a theoretical basis for the potential application of Se-POP-3 as an anti-gastrointestinal cancer drug or functional food.

Discovery of the Rnase activity of CRISPR-Cas12a and its distinguishing cleavage efficiency on various substrates.

We, herein, indicated for the first time the Rnase activities of LbCas12a on linear ssRNA above 11 bases, and hairpin RNA substrates. Meanwhile, the LbCas12a bound to ssDNA or ssRNA exhibited different cleavage efficiencies on various substrates, including short ssDNA, hairpin DNA, linear ssRNA and hairpin RNA. With hairpin DNA as a reporter, we attained a detection limit of 5 pM and 50 pM for the ssDNA and ssRNA targets, respectively. We believe that these findings will pave a new avenue for expanding the reporter toolbox for Cas12a-based diagnostics in biosensing and biochemistry.

Cell-Surface-Anchored DNA Sensors for Simultaneously Monitoring Extracellular Sodium and Potassium Levels.

The K+ and Na+ levels in cells have a synergistic effect on many biological processes (BPs); therefore, the simultaneous detection of them is important. Here, we propose a novel Y-shaped DNA sensor for simultaneous monitoring of Na+ and K+ in extracellular microenvironments. The designed sensor contributed to the selective response to the above two ions. In addition, it performed the imaging of the above two ions on the cell surface in a real-time, on-site manner, which would shed more light on the association of the Na+/K+ content with regulatory BPs. We believe that this new strategy will be a promising tool to investigate the synergy of Na+/K+ in regulating different BPs.