Synergistic Incorporation of Two ssDNA Activators Enhances the Trans-Cleavage of CRISPR/Cas12a.

CRISPR/Cas12a has been believed to be powerful in molecular detection and diagnostics due to its amplified trans-cleavage feature. However, the activating specificity and multiple activation mechanisms of the Cas12a system are yet to be elucidated fully. Herein, a "synergistic activator effect" is discovered, which supports an activation mechanism that a synergistic incorporation of two short ssDNA activators can promote the trans-cleavage of CRISPR/Cas12a, while either of them is too short to work independently. As a proof-of-concept example, the synergistic activator-triggered CRISPR/Cas12a system has been successfully harnessed in the AND logic operation and the discrimination of single-nucleotide variants, requiring no signal conversion elements or other amplified enzymes. Moreover, a single-nucleotide specificity has been achieved for the detection of single-nucleotide variants by pre-introducing a synthetic mismatch between crRNA and the "helper" activator. The finding of "synergistic activator effect" not only provides deeper insight into CRISPR/Cas12a but also may facilitate its expanded application and power the exploration of the undiscovered properties of other CRISPR/Cas systems.

Compute-and-Release Logic-Gated DNA Cascade Circuit for Accurate Cancer Cell Imaging.

Accurate identification of cancer cells is an essential prerequisite for cancer diagnosis and subsequent effective curative interventions. The logic-gate-assisted cancer imaging system that allows a comparison of expression levels between biomarkers, rather than just reading biomarkers as inputs, returns a more comprehensive logical output, improving its accuracy for cell identification. To fulfill this key criterion, we develop a compute-and-release logic-gated double-amplified DNA cascade circuit. This novel system, CAR-CHA-HCR, consists of a compute-and-release (CAR) logic gate, a double-amplified DNA cascade circuit (termed CHA-HCR), and a MnO2 nanocarrier. CAR-CHA-HCR, a novel adaptive logic system, is designed to logically output the fluorescence signals after computing the expression levels of intracellular miR-21 and miR-892b. Only when miR-21 is present and its expression level is above the threshold CmiR-21 > CmiR-892b, the CAR-CHA-HCR circuit performs a compute-and-release operation on free miR-21, thereby outputting enhanced fluorescence signals to accurately image positive cells. It is capable of comparing the relative concentrations of two biomarkers while sensing them, thus allowing accurate identification of positive cancer cells, even in mixed cell populations. Such an intelligent system provides an avenue for highly accurate cancer imaging and is potentially envisioned to perform more complex tasks in biomedical studies.

A Potential Fungicide against Pseudoperonospora Cubensis: Design, Synthesis and 3D-QSAR of New Pyrazole-5-Carboxamide Derivatives.

BACKGROUND: Downy mildew is one of the major fungi causing significant economic losses to crops. The resistance of this fungus to current fungicides is increasing and new fungicides with a unique mode of action are needed. OBJECTIVE: To find a novel pyrazole amide derivative as a potential fungicide. METHODS: A series of pyrazole-5-carboxamide derivatives containing a diaryl ether were designed and synthesized by the Intermediate derivatization method (IDM). Their fungicidal activities against Pseudoperonospora cubensis (P. cubensis, cucumber downy mildew) were evaluated in the greenhouse. RESULTS: Bioassays indicated that several compounds exhibited excellent fungicidal activity against P. cubensis in vivo. In particular, T24 (EC50 = 0.88 mg L-1) had the highest activity compared with Dimethomorph and Fluazinam and other analogues. The relationship between the activity and the structure of these derivatives was analyzed, and an accurate and reliable three-dimensional quantitative structure-activity relationship (3D-QSAR) model was established to determine that electrostatic and steric fields had important effects on the improvement of fungicidal activity. CONCLUSION: The novel pyrazole-5-carboxamide derivative T24 can be considered a potential fungicide for P. cubensis control.

A protein enzyme-free strategy for fluorescence detection of single nucleotide polymorphisms using asymmetric MNAzymes.

To establish protein enzyme-free and simple approach for sensitive detection of single nucleotide polymorphisms (SNPs), the nucleic acid amplification reactions were developed to reduce the dependence on protein enzymes (polymerase, endonuclease, ligase). These methods, while enabling highly amplified analysis for the short sequences, cannot be generalized to long genomic sequences. Herein, we develop a protein enzyme-free and general SNPs assay based on asymmetric MNAzyme probes. The multi-arm probe (MNAzyme-9M-13) with two asymmetric recognition arms, containing a short (9 nt) and a long (13 nt) arm, is designed to detect EGFR T790 M mutation (MT). Owing to the excellent selectivity of short recognition arm, MNAzyme-9M-13 probe can efficiently avoid interferences from wild-type target (WT) and various single-base mutations. Through a one-pot mixing, MNAzyme-9M-13 probe enables the sensitive detection of MT, without protein enzyme or multi-step operation. The calculated detection limit for MT is 0.59 nM and 0.83%. Moreover, this asymmetric MNAzyme strategy can be applied for SNPs detection in long genomic sequences as well as short microRNAs (miRNAs) only by changing the low-cost unlabeled recognition arms. Therefore, along with simple operation, low-cost, protein enzyme-free and strong versatility, our asymmetric MNAzyme strategy provides a novel solution for SNPs detection and genes analysis.

Design, Synthesis, and Structure-Activity Relationship of New Arylpyrazole Pyrimidine Ether Derivatives as Fungicides.

To explore a novel fungicide effectively against cucumber downy mildew (CDM), a series of new arylpyrazole containing pyrimidine ether derivatives were designed and synthesized by employing the intermediate derivatization method (IDM). The structures of synthesized compounds were identified by 1H NMR, 13C NMR, elemental analyses, MS, and X-ray diffraction. Bioassays demonstrated that some of the title compounds exhibited excellent fungicidal activities against CDM. Especially, compound 7 (EC50 = 1.22 mg/L) displayed significantly higher bioactivity than that of commercial fungicides diflumetorim and flumorph and nearly equal effect to that of cyazofamid. The relationship between the structure and fungicidal activity of the synthesized compounds was discussed as well. The study showed that compound 7 was a promising fungicide candidate for further development.

Discovery of pyridine-based agrochemicals by using Intermediate Derivatization Methods.

Pyridine-based compounds have been playing a crucial role as agrochemicals or pesticides including fungicides, insecticides/acaricides and herbicides, etc. Since most of the agrochemicals listed in the Pesticide Manual were discovered through screening programs that relied on trial-and-error testing and new agrochemical discovery is not benefiting as much from the in silico new chemical compound identification/discovery techniques used in pharmaceutical research, it has become more important to find new methods to enhance the efficiency of discovering novel lead compounds in the agrochemical field to shorten the time of research phases in order to meet changing market requirements. In this review, we selected 18 representative known agrochemicals containing a pyridine moiety and extrapolate their discovery from the perspective of Intermediate Derivatization Methods in the hope that this approach will have greater appeal to researchers engaged in the discovery of agrochemicals and/or pharmaceuticals.

Effects of quercetin derivatives from mulberry leaves: Improved gene expression related hepatic lipid and glucose metabolism in short-term high-fat fed mice.

BACKGROUND/OBJECTIVES: Mulberry leaves contain quercetin derivatives, which have the effects of reducing obesity and improving lipid and glucose metabolism in mice with obesity. It is not clear whether or not mulberry leaves can directly affect metabolic disorders, in the presence of obesity, because of the interaction between obesity and metabolic disorders. The aim of the current study was to assess the direct action of quercetin derivatives on metabolic disorders in non-obese conditions in short-term high-fat diet fed mice. MATERIALS/METHODS: C57BL/6N mice were fed a high-fat diet, supplemented with either 0% (control), 1%, or 3% mulberry leaf powder (Mul) or 1% catechin powder for five days. Anthropometric parameters and blood biochemistry were determined, and hepatic gene expression associated with lipid and glucose metabolism was analyzed. RESULTS: Body and white fat weights did not differ among the four groups. Plasma triglycerides, total cholesterol, and free fatty acids in the 1%, 3% Mul and catechin groups did not differ significantly from those of the controls, however, plasma glucose and 8-isoprostane levels were significantly reduced. Liver gene expression of gp91phox, a main component of NADPH oxidase, was significantly down-regulated, and PPAR-α, related to ß-oxidation, was significantly up-regulated. FAS and GPAT, involved in lipid metabolism, were significantly down-regulated, and Ehhadh was significantly up-regulated. Glucose-metabolism related genes, L-PK and G6Pase, were significantly down-regulated, while GK was significantly up-regulated in the two Mul groups compared to the control group. CONCLUSIONS: Our results suggest that the Mul quercetin derivatives can directly improve lipid and glucose metabolism by reducing oxidative stress and enhancing ß-oxidation. The 1% Mul and 1% catechin groups had similar levels of polyphenol compound intake (0.4 × 10(-5) vs 0.4 × 10(-5) mole/5 days) and exhibited similar effects, but neither showed dose-dependent effects on lipid and glucose metabolism or oxidative stress.

Anti-obesity effects of hot water extract from Wasabi (Wasabia japonica Matsum.) leaves in mice fed high-fat diets.

The anti-obesity effects of a hot water extract from wasabi (Wasabia japonica Matsum.) leaves (WLE), without its specific pungent constituents, such as allyl-isothiocyanate, were investigated in high fat-diet induced mice. C57J/BL mice were fed a high-fat diet (control group) or a high-fat diet supplemented with 5% WLE (WLE group). Physical parameters and blood profiles were determined. Gene expression associated with lipid metabolism in liver and white adipose tissue were analyzed. After 120 days of feeding, significantly lower body weight gain, liver weight and epididymal white adipose tissue weight was observed in the WLE group compared to the control group. In liver gene expression within the WLE group, PPARα was significantly enhanced and SREBP-1c was significantly suppressed. Subsequent downstream genes controlled by these regulators were significantly suppressed. In epididymal white adipose tissue of the WLE group, expression of leptin, PPARγ, and C/EBPα were significantly suppressed and adiponectin was significantly enhanced. Acox, related to fatty acid oxidization in adipocytes, was also enhanced. Our results demonstrate that the WLE dietary supplement induces mild suppression of obesity in a high-fat diet induced mice, possibly due to suppression of lipid accumulation in liver and white adipose tissue.

Antiobesity effect of polyphenolic compounds from molokheiya (Corchorus olitorius L.) leaves in LDL receptor-deficient mice.

BACKGROUND: Dietary supplementation with polyphenolic compounds is associated with reduced diet-induced obesity and metabolic disorders in humans. The antioxidative properties of polyphenolic compounds contribute to their antiobesity effect in animal experiments and human studies. AIM: The aim of the study was to investigate the antiobesity effect of polyphenolic compounds from molokheiya leaves in LDLR-/- mice fed high-fat diet and to elucidate the mechanism of this effect. METHODS: Three groups of LDLR-/- mice were fed with a high-fat diet, supplemented with 0% (control), 1 or 3% molokheiya leaf powder (MLP). Gene expression in the liver associated with lipid and glucose metabolism was analyzed, and physical parameters and blood biochemistry were determined. RESULTS: Compared to controls, mice body weight gain (P = 0.003), liver weight (P = 0.001) and liver triglyceride levels (P = 0.005) were significantly lower in the two MLP groups. Epididymal adipose tissue weight (P = 0.003) was reduced in the 3% MLP group. Liver tissue gene expression of gp91phox (NOX2), involved in oxidative stress, was significantly down-regulated (P = 0.005), and PPARα and CPT1A, related to the activation of ß-oxidation, were significantly up-regulated (P = 0.025 and 0.006, respectively) in the 3% MLP group compared to the control group. CONCLUSIONS: Our results demonstrate an antiobesity effect of polyphenolic compounds from molokheiya leaves and that this effect is associated with reduction in oxidative stress and enhancement of ß-oxidation in the liver. Consumption of molokheiya leaves may be beneficial for preventing diet-induced obesity.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 6: Opioid receptor binding properties of cyclic variants of 8-carboxamidocyclazocine.

A series of 7,8- and 8,9-fused pyrimidinone, aminopyrimidine and pyridone derivatives of 8-carboxamidocyclazocine (8-CAC) have been prepared and evaluated in opioid receptor binding assays. Targets were designed to corroborate a pharmacophore hypothesis regarding the bioactive conformation of the carboxamide of 8-CAC. In addition to the results from this study strongly supporting this pharmacophore hypothesis, a number of novel compounds with high affinity to opioid receptors have been identified.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 3: 8-Thiocarboxamido and 8-thioformamido derivatives of cyclazocine.

8-Position variants of cyclazocine have been made where the phenolic 8-OH was replaced by thioamide, amidine, guanidine, urea and thiourea groups. High affinity for opioid receptors was observed for the 8-CSNH2 and 8-NHCHS analogues indicating that these groups are isosteric with not only the 8-OH but with the previously synthesized 8-CONH2 and 8-NHCHO cyclazocine derivatives.

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. Part 2: 8-formamidocyclazocine analogues.

High affinity binding for mu and kappa opioid receptors has been observed in analogues of cyclazocine, ethylketocyclazocine and naltrexone where the prototypic (of opiates) phenolic OH group was replaced with a formamide (-NHCHO) group. For the 8-formamide analogue of cyclazocine, binding is highly enantiospecific (eudismic ratios approximately 2000 for mu and kappa) with K(i) values

Syntheses and opioid receptor binding affinities of 8-amino-2,6-methano-3-benzazocines.

8-Amino-2,6-methano-3-benzazocine derivatives have been made using Pd-catalyzed amination procedures, and their affinities for opioid receptors were assessed. The 8-amino group was hypothesized to be a replacement for the prototypic 8-OH substituent for 2,6-methano-3-benzazocines and related opiates. This OH group is generally required for binding yet is implicated in unfavorable pharmacokinetic characteristics such as low oral bioavailability and rapid clearance via O-glucuronidation. The core structures in which the 8-OH group was replaced were cyclazocine and its enantiomers, ethylketocyclazocine and its enantiomers, ketocyclazocine, and Mr2034. Many new analogues had high affinity for opioid receptors with several in the subnanomolar range. Highest affinity was seen in analogues with secondary 8-(hetero)arylamino appendages. Binding to opioid receptors was enantioselective with the (2R,6R,11R)-configuration preferred and high selectivity for mu and kappa over delta opioid receptors was observed within the series. Several derivatives were shown to have intrinsic opioid-receptor-mediated activity in [(35)S]GTPgammaS assays.