Genetically Encoded RNA Sensors for Ratiometric and Multiplexed Imaging of Small Molecules in Living Cells.

Genetically encoded sensors afford powerful tools for studying small molecules and metabolites in live cells. However, genetically encoded sensors with a general design remain to be developed. Here we develop genetically encoded RNA sensors with a modular design for ratiometric and multiplexed imaging of small molecules in live cells. The sensor utilizes aptazyme as a recognition module and the light-up RNA aptamer as a signal reporter. The conformation of light-up aptamers is abrogated by a blocking sequence, and aptazyme-mediated cleavage restores the correct conformation, delivering activated fluorescence for small molecule imaging. We first developed a genetically encoded ratiometric sensor using Mango aptamer as a reference and SRB2 as a reporter. It is shown that the sensor allows quantitative imaging and detection of theophylline in live cells. The generality of the design is further demonstrated for imaging other small molecules by replacing the aptazymes. Its ability for multiplexed imaging of small molecules is further explored via the integration of different small-molecule responsive aptazymes and light-up RNA aptamers. This modular design could offer a versatile platform for imaging diverse molecules in living cells.

Genetically Encoded Light-Up RNA Amplifier Dissecting MicroRNA Activity in Live Cells.

Live cell dissection of microRNA activities is crucial for basic and translational medicine, but current hybridization-based strategies may fail to dissect surrounding-dependent activities. Here, we develop a genetically encoded miRNA-induced light-up RNA amplifier (iLAMP) that enables fast-activated, signal-amplified, fluorogenic imaging of miRNA activities in live cells. iLAMP responds to miRNA targets in the mode of "activation upon cleavage", in which the light-up RNA aptamer restores its fluorescence rapidly upon cleavage by the RNA-induced silencing complex. We demonstrate that iLAMP affords substantial signal amplification of ∼100-fold and high specificity in single nucleotide discrimination because of the miRNA-mediated cyclic cleavage. Combined with a Mango RNA aptamer reference module and a pseudoknot terminal stabilizer, iLAMP is shown for quantitative ratiometric imaging and dynamic monitoring of miRNA activities under exogenous stimulations. iLAMP is featured by a modular "plug and play" design and can be readily adapted to the detection of other miRNAs, highlighting its potential in tracking cell differentiation and screening miRNA therapeutics.

Genetically Encoded Dual-Color Light-Up RNA Sensor Enabled Ratiometric Imaging of MicroRNA.

MicroRNAs (miRNAs) play essential roles in regulating gene expression and cell fate. However, it remains a great challenge to image miRNAs with high accuracy in living cells. Here, we report a novel genetically encoded dual-color light-up RNA sensor for ratiometric imaging of miRNAs using Mango as an internal reference and SRB2 as the sensor module. This genetically encoded sensor is designed by expressing a splittable fusion of the internal reference and sensor module under a single promoter. This design strategy allows synchronous expression of the two modules with negligible interference. Live cell imaging studies reveal that the genetically encoded ratiometric RNA sensor responds specifically to mir-224. Moreover, the sensor-to-Mango fluorescence ratios are linearly correlated with the concentrations of mir-224, confirming their capability of determining mir-224 concentrations in living cells. Our genetically encoded light-up RNA sensor also enables ratiometric imaging of mir-224 in different cell lines. This strategy could provide a versatile approach for ratiometric imaging of intracellular RNAs, affording powerful tools for interrogating RNA functions and abundance in living cells.

[Investigation of metabolism of HN-1 isolated from Millettia pachyloba in vivo and in vitro].

Ultra-fast performance liquid chromatography-mass spectrometry( UFLC-MS/MS) was used to study the anti-inflammatory active ingredient of Millettia pachyloba,6-methoxy-8,8-dimethyl-3-( 2,4,5-trimethoxyphenyl)-4 H,8 H-pyrano[2,3-f]chromen-4-one( HN-1),in liver microsomes of rats,mice,rhesus monkeys,Beagle dogs and humans metabolic stability,and compare the metabolic differences between different species. The metabolic phenotype in human liver microsomes was determined by chemical inhibitor method. Using UPLC-Q-TOF-MS/MS detection method,the in vitro metabolites of various liver microsomes were preliminarily inferred by comparing the samples incubated for 0 min and 60 min in vitro. The metabolites of HN-1 in SD rats were presumed by comparing feces,urine,plasma blanks and samples after administration. The results showed that the metabolism of HN-1 in various liver microsomes was stable,and the metabolic properties of dog and human liver microsomes were the closest. It is mainly catabolized by CYP1 A1,CYP2 D6 and CYP3 A4 isoenzymes in human liver microsomes. The metabolites of HN-1 in vitro and in vivo,including 3 in vitro metabolites and5 in vivo metabolites,were preliminarily estimated. The results laid the foundation for further pharmacological studies of HN-1.

Cell Surface-Anchored DNA Nanomachine for Dynamically Tunable Sensing and Imaging of Extracellular pH.

DNA nanodevices that mimic natural biomolecular machines changing configurations in response to external inputs have enabled smart sensors to live cell imaging. We report for the first time the development of a dynamic DNA nanomachine that is anchored on a cell's surface and undergoes pH-responsive triplex-duplex conformation switching, allowing tunable sensing and imaging of extracellular pH. Results reveal that the DNA nanomachine can be stably anchored on the cell surface via multiple anchors, and the adjustment of C+G-C content in the switch element confers tunability of pH response windows. The anchored DNA nanomachine also demonstrates desirable sensitivity, excellent reversibility, and quantitative ability for extracellular pH detection and imaging. This cell surface-anchored pH-responsive DNA nanomachine can provide a useful platform for pH sensing in extracellular microenvironments and diagnostics of different pH-related diseases.

Honokiol Ameliorates Post-Myocardial Infarction Heart Failure Through Ucp3-Mediated Reactive Oxygen Species Inhibition.

Post-myocardial infarction heart failure (post-MI HF) is one of the leading global causes of death, and current prevention and treatment methods still cannot avoid the increasing incidence. Honokiol (HK) has previously been reported to improve myocardial ischemia/reperfusion injury and reverse myocardial hypertrophy by activating Sirt1 and Sirt3. We suspect that HK may also have a therapeutic effect on post-MI HF. In this study, we aimed to investigate the efficacy and mechanism of HK in the treatment of post-MI HF. We found that HK inhibited myocardial reactive oxygen species (ROS) production, reduced myocardial fibrosis, and improved cardiac function in mice after MI. HK also reduced the abnormality of mitochondrial membrane potential (MMP) and apoptosis of cardiomyocytes caused by peroxide in neonatal cardiomyocytes. RNAseq results revealed that HK restored the transcriptome changes to a certain extent and significantly enhanced the expression of mitochondrial inner membrane uncoupling protein isoform 3 (Ucp3), a protein that inhibits the production of mitochondrial ROS, protects cardiomyocytes, and relieves heart failure after myocardial infarction (MI). In cardiomyocytes with impaired Ucp3 expression, HK cannot protect against the damage caused by peroxide. More importantly, in Ucp3 knockout mice, HK did not change the increase in the ROS level and cardiac function damage after MI. Taken together, our results suggest that HK can increase the expression of the cardioprotective protein Ucp3 and maintain MMP, thereby inhibiting the production of ROS after MI and ameliorating heart failure.

Corrigendum: Honokiol ameliorates post-myocardial infarction heart failure through Ucp3-mediated reactive oxygen species inhibition.

[This corrects the article DOI: 10.3389/fphar.2022.811682.].

Clostridium butyricum Protects IPEC-J2 Cells from ETEC K88-Induced Oxidative Damage by Activating the Nrf2/ARE Signaling Pathway.

Clostridium butyricum (CB) is a naturally occurring probiotic compound that can alleviate the oxidative damage induced by enterotoxigenic Escherichia coli K88 (ETEC K88) in porcine intestinal epithelial (IPEC-J2) cells. In this study, we investigate the molecular mechanism underlying this effect. Based on cell viability, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPX) assessments, the optimal concentration of ETEC K88 was determined to be 1 × 103 cfu/mL. Viable bacteria counts in cells pretreated with CB and then infected with ETEC K88 show that CB can adhere to IPEC-J2 cells and that optimal adhesion is achieved at the multiple infection index (MOI) of 50 at 3 h of pretreatment. The results of qPCR indicate that although ETEC significantly decreases the expression levels of antioxidant enzymes regulated by NF-E2-related factor 2 (Nrf2) compared to the control group, CB reverses this effect. To confirm that Nrf2 is directly involved in the mechanism by which CB alleviates oxidative stress, siRNA was used to silence the expression of Nrf2 gene in IPEC-J2 cells. Compared to the NC+ETEC and siRNA+ETEC groups, the expressions of SOD1, SOD2, GPX1, and GPX2 in the NC+CB+ETEC and siRNA+CB+ETEC groups are significantly increased at 12 h and 24 h. This shows that CB can reduce ETEC K88-induced oxidative damage in IPEC-J2 cells by activating the expression of antioxidant enzymes implicated in the Kelch-like ECH-associated protein-1- (Keap1-) Nrf2/antioxidant response element (ARE) signaling pathway.

Design, synthesis and anti-inflammatory study of novel N-heterocyclic substituted Aloe-emodin derivatives.

A novel series of Aloe-emodin derivatives containing N-heterocyclic moieties was designed and synthesized. The structure-activity relationship studies (SARs) indicated that the replacement of hydroxyethyl and benzhydryl piperazine groups could improve efficacy. Compounds 12r and 14a-14c exhibited a higher inhibitory effect on LPS-induced nitric oxide (NO) production in RAW264.7 macrophages than Aloe-emodin did. Among them, 12r showed the most potent inhibition with an IC50 value of 5.66 ± 0.47 µM. Further toxicity and pharmacokinetic studies were carried out and 12r was found to be the most active structure with low toxicity risk and good metabolic properties. It could also decrease the levels of IL-1ß, TNF-α, PGE2 and inhibit the activation of nuclear factor-κB signalling pathway. Importantly, 12r showed oral bioavailability of up to 55.16% and attenuated the inflammatory symptoms in an ulcerative colitis mouse model in vivo. These results indicate that 12r is suitable for development as an anti-inflammatory agent.

Identification of Pyrrolo[2,3- d]pyrimidine-Based Derivatives as Potent and Orally Effective Fms-like Tyrosine Receptor Kinase 3 (FLT3) Inhibitors for Treating Acute Myelogenous Leukemia.

A series of pyrrolo[2,3- d]pyrimidine derivatives were prepared and optimized for cytotoxic activities against FLT3-ITD mutant cancer cells. Among them, compound 9u possessed nanomolar FLT3 inhibitory activities and subnanomolar inhibitory activities against MV4-11 and Molm-13 cells. It also showed excellent inhibitory activities in FLT3-ITD-D835V and FLT3-ITD-F691L cells which were resistant to quizartinib. Furthermore, 9u exhibited over 40-fold selectivity toward FLT3 relative to c-Kit kinase, which might reduce myelosuppression toxicity. Cellular assays demonstrated that 9u inhibited phosphorylated FLT3 and downstream signaling factors and also induced cell cycle arrest in the G0/G1 stage and apoptosis in MV4-11 and Molm-13 cells. Oral administration of 9u at 10 mg/kg could achieve rapid tumor extinction in the MV4-11 xenograft model and significantly inhibit the tumor growth in the MOLM-13 xenograft model with a tumor growth inhibitory rate of 96% without obvious toxicity. Additionally, 9u demonstrated high bioavailability ( F = 59.5%) and suitable eliminated half-life time ( T1/2 = 2.06 h), suggesting that 9u may be a potent candidate for treating acute myelogenous leukemia.