CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

Electrostatic assemblies of molecularly imprinted polymers on the surface of electrospun nanofiber membranes for the point-of-care detection of thiodiglycol, a sulfur mustard poisoning metabolic marker.

In this study, molecularly imprinted polymers (MIPs) were assembled on the surface of ethylene imine polymer (PEI)/poly(vinyl alcohol) (PVA) electrospun nanofiber membranes for the point-of-care testing (POCT) of thiodiglycol (TDG), a sulfur mustard poisoning metabolic marker, using concentrated gold nanoparticles (AuNPs) as the signal reporting units. The MIPs/PEI/PVA nanofiber membranes could capture TDG specifically through the recognition interaction between MIPs and TDG. Then, AuNPs were adsorbed onto the MIPs/PEI/PVA nanofiber membranes through the Au-S interaction between TDG and AuNPs to produce a visible red color. In order to improve the sensitivity, the silver-enhanced solutions were used to deepen the color of the nanofiber membranes and the software Image J was used to read the gray value as the signal response for subsequent analysis. There was a good linear relationship between the color change of the MIPs/PEI/PVA nanofiber membranes and the TDG concentration from 0.1 ng mL-1 to 1.0 µg mL-1, and the limit of detection was 38 pg mL-1. This method was applied for the selective detection of TDG in urine, showing great potential for the clinical diagnosis of mustard gas poisoning.

Trait value and phenotypic integration contribute to the response of exotic Rhus typhina to heterogeneous nitrogen deposition: A comparison with native Rhus chinensis.

The temporal heterogeneity of nitrogen availability in soils is increasing due to agricultural deposition. We here compared the effects of gradually increasing nitrogen deposition rate and its increasing temporal heterogeneity patterns on the functional traits of seedlings of exotic species Rhus typhina and the native species Rhus chinensis. Nitrogen deposition rates of 0, 8, 20 g N m-2 year-1 and constant, single-peak, and double-peak nitrogen were added to simulate deposition rate and temporal heterogeneity. After 60 days of treatment, R. typhina seedlings had several advantageous growth trait values, such as higher total biomass production, but lower phenotypic plasticity than R. chinensis seedlings. R. typhina seedlings also had higher phenotypic integration, measured as the correlation among functional traits. The increased nitrogen deposition rate affected several traits of the two species differently. Thus, while R. chinensis seedlings allocated more biomass to leaves and less to roots with increasing N deposition, R. typhina seedlings had stable biomass allocation among all N treatments. Chlorophyll content, leaf phosphorus concentration, and water use efficiency increased, but the maximum net photosynthetic rate decreased, with N availability in R. chinensis, but not in R. typhina. Temporal heterogeneity had no significant effect on the total biomass of R. typhina and R. chinensis seedlings. Overall, the performance of R. typhina is better than that of R. chinensis seedlings under different nitrogen deposition treatments, which is due to the significantly advantageous trait values and greater phenotypic integration of R. typhina seedlings, whereas R. chinensis seedlings have higher phenotypic plasticity.

Assessment on the binding characteristics of dasatinib, a tyrosine kinase inhibitor to calf thymus DNA: insights from multi-spectroscopic methodologies and molecular docking as well as DFT calculation.

The binding characteristics of calf thymus DNA (ct-DNA) with dasatinib (DSTN), a tyrosine kinase inhibitor was assessed through multi-spectroscopic methodologies and viscosity measurement combined with molecular docking as well as DFT calculation to understand the binding mechanism, affinity of DSTN onto ct-DNA, effect of DSTN on ct-DNA conformation, and among others. The results confirmed DSTN bound onto ct-DNA, leading to forming the DSTN-ct-DNA complex with the binding constant of 4.82 × 103 M-1 at 310 K. DSTN preferentially inserted to the minor groove of ct-DNA with rich A-T region, that was the binding mode of DSTN onto ct-DNA was groove binding. The enthalpic change (ΔH0) and entropic change (ΔS0) during the binding process of DSTN with ct-DNA were 128.9 kJ mol-1 and 489.2 J mol-1 K-1, respectively, confirming clearly that the association of DSTN with ct-DNA was an endothermic process and the dominative driven-force was hydrophobic interaction. Meanwhile, the results also indicated that there was a certain extent of electrostatic force and hydrogen bonding, but they maybe play an auxiliary role. The CD measurement results confirmed the alteration in the helical configuration of ct-DNA but almost no change in the base stacking after binding DSTN. The results revealed that there was the obvious change in the conformation, the dipole moment, and the atomic charge distribution of DSTN in the B-DNA complexes, compared with free DSTN, to satisfy the conformational adaptation. From the obtained fronitier molecular orbitals of DSTN, it can be inferred that the nature of DSTN alters with the change of the environment around DSTN. Communicated by Ramaswamy H. Sarma.

Induction of Antiphytopathogenic Metabolite and Squalene Production and Phytotoxin Elimination by Adjustment of the Mode of Fermentation in Cocultures of Phytopathogenic Nigrospora oryzae and Irpex lacteus.

The investigation of the metabolites from different cocultures of Nigrospora oryzae and Irpex lacteus in solid medium revealed two new squalenes (1 and 2); one new azaphilone (3); two new tremulane sesquiterpenes (4 and 5); and three known compounds, conocenol B (6), conocenol C (7), and 4-(4-dihydroxymethylphenoxy)benzaldehyde (8). The antagonistic relationship was examined by studying metabolite production. The production of compounds 6 and 8 by I. lacteus after the induction of coculture indicated significant selectivity for antifungal activity against phytopathogenic N. oryzae, with MICs of 16 µg/mL; compounds 6 and 8 also exhibited antifungal activities in vivo against Cerasus cerasoides infected by N. oryzae at concentrations of 100 µg/mL. New compounds 2 and 4 showed antifungal activities against Colletotrichum gloeosporioides, with MICs of 8 µg/mL, and compound 4 showed antifungal activity against Didymella glomerata with an MIC of 1 µg/mL. These results indicate that the mutually antagonistic relationship in the coculture of the phytopathogen and the endophyte can result in antibiotics that inhibit the phytopathogen and downregulate the production of phytotoxins by phytopathogenic N. oryzae. New compound 5 from I. lacteus showed weak activity against acetylcholinesterase (AChE), with an inhibition ratio of 16% at a concentration of 50 µM.

[Overexpression of miR-30a Promotes Apoptosis of Leukemia K562 Cells].

OBJECTIVE: To investigate the pro-apoptotic effect and mechanism of miR-30a overexpression on chronic myeloid leukemia K562 cells. METHODS: The k562 cells were transfected with the recombinant plasmid pEGFP-pre-miR-30a, the real-time quantitative PCR was used to detect the level of miR-30a and BCR/ABL, and then the cell apoptosis was assessed by flow cytometry with AnnexinV-FITC/PI double staining. Western blot was used to detect the expression of BCR/ABL protein,apoptosis-related protein BCL-2 and BAX, PTEN, AKT and p-AKT. RESULTS: Sequencing and digestion map indicated that the recombinant plasmid was constructed successfully. Compared with 2 control groups, the miR-30a expression in k562 cells transfected with recombinant plasmid pEGFP-pre-miR-30a was obviously up-regulated. The expression of BCR/ABL mRNA and BCR/ABL protein was both significantly down-regulated. Apoptotic rate was significantly enhanced (both P<0.05),and the expression of anti-apoptotic protein BCL-2 was down-regulated while the expression of pro-apoptotic protein BAX was up-regulated. The level of PTEN was significantly up-regulated in omparison with control groups,no variation was found in total AKT, but the expression of p-AKT was down-regulated. CONCLUSION: The overexpression of miR-30a is abled to down-regulate the level of BCR/ABL mRNA and BCR/ABL protein, and increase apoptotic rate, its mechanism may be related with inhibition of the activity of BCR/ABL-PTEN/AKT signaling pathway.

The role of medical gas in stroke: an updated review.

Medical gas is a large class of bioactive gases used in clinical medicine and basic scientific research. At present, the role of medical gas in neuroprotection has received growing attention. Stroke is a leading cause of death and disability in adults worldwide, but current treatment is still very limited. The common pathological changes of these two types of stroke may include excitotoxicity, free radical release, inflammation, cell death, mitochondrial disorder, and blood-brain barrier disruption. In this review, we will discuss the pathological mechanisms of stroke and the role of two medical gases (hydrogen and hydrogen sulfide) in stroke, which may potentially provide a new insight into the treatment of stroke.