Application status and optimization suggestions of tumor organoids and CAR-T cell co-culture models.

Tumor organoids, especially patient-derived organoids (PDOs) exhibit marked similarities in histopathological morphology, genomic alterations, and specific marker expression profiles to those of primary tumour tissues. They are applied in various fields including drug screening, gene editing, and identification of oncogenes. However, CAR-T therapy in the treatment of solid tumours is still at an exploratory stage. Tumour organoids offer unique advantages over other preclinical models commonly used for CAR-T therapy research, which the preservation of the biological characteristics of primary tumour tissue is critical for the study of early-stage solid tumour CAR-T therapies. Although some investigators have used this co-culture model to validate newly targeted CAR-T cells, optimise existing CAR-T cells and explore combination therapy strategies, there is still untapped potential in the co-culture models used today. This review introduces the current status of the application of tumour organoid and CAR-T cell co-culture models in recent years and commented on the limitations of the current co-cultivation model. Meanwhile, we compared the tumour organoid model with two pre-clinical models commonly used in CAR-T therapy research. Eventually, combined with the new progress of organoid technologies, optimization suggestions were proposed for the co-culture model from five perspectives: preserving or reconstructing the tumor microenvironment, systematization, vascularization, standardized culture procedures, and expanding the tumor organoids resource library, aimed at assisting related researchers to better utilize co-culture models.

Inhibitory effect of quercetin-3-O-α-rhamnoside, p-coumaric acid, phloridzin and 4-O-ß-glucopyranosyl-cis-coumaric acid on rats liver microsomes cytochrome P450 enzyme activities.

P-coumaric acid, phloridzin, quercetin-3-O-α-rhamnoside and 4-O-ß-glucopyranosyl-cis-coumaric acid isolated in Malus micromalus Makino fruit were investigated the inhibitory activity of cytochrome CYP450 enzyme by the probe test method of rat liver microsomes in vitro, and determined the role in drug metabolism and/or toxicology. Enzymatic kinetics method was used to determine the inhibition type of these components and corresponding inhibition constants. The results demonstrated that all the 4 compounds had no significance to inhibit the activities of CYP2E1 and CYP2C11. P-coumaric acid, phloridzin and quercetin-3-O-α-rhamnoside had a weak inhibitory effect on CYP3A4, which belonged to the competitive inhibitory type with inhibitory constants of 10.56, 30.79 and 40.29 µmol L-1, respectively. 4-O-ß-glucopyranosyl-cis-coumaric acid had a moderate inhibitory effect on CYP3A4, which belonged to the anti-competitive inhibition type and the inhibition constant was 5.56 µmol L-1. The CYP1A2 could be weakly inhibited by p-coumaric acid in the competitive type, and the inhibition constant is 25.20 µmol L-1 4-O-ß-glucopyranosyl-cis-coumaric acid exhibited anti-competitive inhibition of CYP1A2 with an inhibition constant of 19.91 µmol L-1, and the inhibition effect was weak. The results will be useful to optimize the clinical dosage regimen and avoid drug-drug interactions when it is utilized comminating with other medicines in the clinic.

Exploring ancestral phenotypes and evolutionary development of the mammalian middle ear based on Early Cretaceous Jehol mammals.

We report a new Cretaceous multituberculate mammal with 3D auditory bones preserved. Along with other fossil and extant mammals, the unequivocal auditory bones display features potentially representing ancestral phenotypes of the mammalian middle ear. These phenotypes show that the ectotympanic and the malleus-incus complex changed notably during their retreating from the dentary at various evolutionary stages and suggest convergent evolution of some features to extant mammals. In contrast, the incudomalleolar joint was conservative in having a braced hinge configuration, which narrows the morphological gap between the quadroarticular jaw joint of non-mammalian cynodonts and the incudomalleolar articulations of extant mammals. The saddle-shaped and abutting malleus-incus complexes in therians and monotremes, respectively, could have evolved from the braced hinge joint independently. The evolutionary changes recorded in the Mesozoic mammals are largely consistent with the middle ear morphogenesis during the ontogeny of extant mammals, supporting the relation between evolution and development.

Fossoriality and evolutionary development in two Cretaceous mammaliamorphs.

Mammaliamorpha comprises the last common ancestor of Tritylodontidae and Mammalia plus all its descendants1. Tritylodontids are nonmammaliaform herbivorous cynodonts that originated in the Late Triassic epoch, diversified in the Jurassic period2-5 and survived into the Early Cretaceous epoch6,7. Eutriconodontans have generally been considered to be an extinct mammalian group, although different views exist8. Here we report a newly discovered tritylodontid and eutriconodontan from the Early Cretaceous Jehol Biota of China. Eutriconodontans are common in this biota9, but it was not previously known to contain tritylodontids. The two distantly related species show convergent features that are adapted for fossorial life, and are the first 'scratch-diggers' known from this biota. Both species also show an increased number of presacral vertebrae, relative to the ancestral state in synapsids or mammals10,11, that display meristic and homeotic changes. These fossils shed light on the evolutionary development of the axial skeleton in mammaliamorphs, which has been the focus of numerous studies in vertebrate evolution12-17 and developmental biology18-28. The phenotypes recorded by these fossils indicate that developmental plasticity in somitogenesis and HOX gene expression in the axial skeleton-similar to that observed in extant mammals-was already in place in stem mammaliamorphs. The interaction of these developmental mechanisms with natural selection may have underpinned the diverse phenotypes of body plan that evolved independently in various clades of mammaliamorph.

A rapid method and mechanism to identify the active compounds in Malus micromalus Makino fruit with spectrum-effect relationship, components knock-out and molecular docking technology.

Fingerprints of 20 batches of Malus micromalus Makino fruit were established by HPLC coupled with hierarchical cluster analysis (HCA) and principal component analysis (PCA) to estimate the common peaks on the basis of traditional similarity evaluation methods. Chromatographic peaks were identified as p-coumaric acid (P2), ferulic acid glycoside (P6), 4-O-ß-Glucopyranosyl-cis-coumaric acid (P8), phloretin-2'-xyloglucoside (P10), phloridzin (P11) and quercetin-3-O-α-rhamnoside (P12) by UPLC-MS/MS method. The results of tyrosinase kinetics experiments showed that: P2 and the concentration of P11 was greater than 0.50 mmol/L mainly had a competitive inhibitory effect on tyrosinase, and the concentration of phlorizin was less than at 0.25 mmol/L, it has a mixed inhibitory effect. P8 was mainly a non-competitive activation type in the concentration range, while P12 was a mixed activation type. The results of tyrosinase molecular docking showed that: P2, P8, P11, P12 was located in the active center of the hydrophobic pocket of the enzyme. They bound to tyrosinase residues by hydrogen bonds and interacted with many hydrophobic residues around them to maintain the structure of the complex. This research provides a rapid method to determine the active compounds in edible plants with the technology of spectrum-effect relationship, component knock-out and molecular docking.

Anti-Hepatoma Compound Determination by the Method of Spectrum Effect Relationship, Component Knock-Out, and UPLC-MS2 in Scheflera heptaphylla (L.)Frodin Harms and Its Mechanism.

Scheflera heptaphylla (L.)Frodin, a kind of Traditional Chinese Medicine, is commonly used in anti-inflammatory, analgesic, anti-viral, anti-tumor, and hemostasis. This study aimed to determine the anti-hepatoma components and its mechanism from the leaves of S. heptaphylla. The spectrum-effect relationships were analyzed by the method of Partial least squares, indicating that P1, P2, and P10 were positively correlated to inhibitory activity of Huh7 cells. Whereas others were negatively correlated. The technologies of component knock-out and UPLC-MS2 were used to determine compounds as 3,4-Dicaffeoylquinic acid (P6), 3,5-Dicaffeoylquinic acid (P7), 3α-Hydroxy-lup-20(29)-ene-23,28-dioic acid (P10, named Compound A). The results forecasted that Compound A had the best correlation with inhibitory activity. The effects of Compound A on the activities of human hepatoma cells (Huh7, SMMC-7721, HepG 2) and normal hepatocytes (L0-2, Chang liver) were evaluated. Cell apoptosis was observed with inverted microscope and flow cytometer. In addition, the proteins, related to apoptosis, were detected by Western blot. The results showed that Compound A (400 nM) could significantly inhibit the activity of three hepatoma cells (P < 0.001) with slight toxicity to normal hepatocytes, and the IC50 values were 285.3 and 315.1 nM, respectively, which were consistent with the prediction of spectrum-effect relationships. After treatment with Compound A, the number of hepatoma cells decreased significantly. And the apoptosis rate of Huh7 cells increased significantly (P < 0.001) in Compound A (200, 400 nM) groups, SMMC-7721 and HepG 2 were directly necrotic. Compound A groups could significantly improve the level of intracellular reactive oxygen species (ROS) (P < 0.05, P < 0.001) in Huh7 with no effect on normal hepatocytes. The content of apoptotic protein (Bax and Bim) in mitochondria was significantly increased in Compound A groups (P < 0.001). On the contrary, the content of anti-apoptotic protein (Bcl-xL and Mcl-1) decreased significantly (P < 0.001). These results demonstrated that Compound A was the main anti-hepatoma active component in the S. heptaphylla leaves. It achieved the effect of promoting apoptosis of Huh7 cells by regulating the levels of ROS and Bcl-2 family protein in mitochondrial apoptosis pathway.

Ionic Liquid-Based Ultrasonic-Assisted Extraction Coupled with HPLC and Artificial Neural Network Analysis for Ganoderma lucidum.

Ganoderma lucidum is widely used in traditional Chinese medicine (TCM). Ganoderic acid A and D are the main bioactive components with anticancer effects in G. lucidum. To obtain the maximum content of two compounds from G. lucidum, a novel extraction method, an ionic liquid-based ultrasonic-assisted method (ILUAE) was established. Ionic liquids (ILs) of different types and parameters, including the concentration of ILs, ultrasonic power, ultrasonic time, rotational speed, solid-liquid ratio, were optimized by the orthogonal experiment and variance analysis. Under these optimal conditions, the total extraction yield of the two compounds in G. lucidum was 3.31 mg/g, which is 36.21% higher than that of the traditional solvent extraction method. Subsequently, an artificial neural network (ANN) was developed to model the performance of the total extraction yield. The Levenberg-Marquardt back propagation algorithm with the sigmoid transfer function (logsig) at the hidden layer and a linear transfer function (purelin) at the output layer were used. Results showed that single hidden layer with 9 neurons presented the best values for the mean squared error (MSE) and the correlation coefficient (R), with respectively corresponding values of 0.09622 and 0.93332.

CYPs-mediated drug-drug interactions on psoralidin, isobavachalcone, neobavaisoflavone and daidzein in rats liver microsomes.

The incubation system of CYP2E1 and CYP3A4 enzymes in rat liver microsomes was established to investigate the effects of psoralidin, isobavachalcone, neobavaisoflavone and daidzein from Fructus Psoraleae in vitro. The relevant metabolites were measured by the method of high performance liquid chromatography (HPLC), after probe substrates of 4-nitrophenol, testosterone and the drugs at different concentrations were added to the incubation systems. In addition, real-time RT-PCR was performed to determine the effect of psoralidin, neobavaisoflavone and daidzein on the mRNA expression of CYP3A4 in rat liver. The results suggested that psoralidin, isobavachalcone and neobavaisoflavone were Medium-intensity inhibitors of CYP2E1 with Ki values of 2.58, 1.28 and 19.07 µM, respectively, which could inhibit the increase of CYP2E1 and reduce diseases caused by lipid peroxidation. Isobavachalcone (Ki = 37.52 µM) showed a weak competitive inhibition on CYP3A4. Psoralidin and neobavaisoflavone showed obvious induction effects on CYP3A4 in the expression level of mRNA, which could accelerate the effects of drug metabolism and lead to the risk of inducing DDIs and serious adverse reactions. The results could be used for guideline of Fructus Psoraleae in clinic, which aimed to calculate the drug toxicity by studying the drug-drug interactions caused by the induction and inhibition of CYP450.

Ionic Liquid-Based Ultrasonic-Assisted Extraction to Analyze Seven Compounds in Psoralea Fructus Coupled with HPLC.

Psoralea Fructus is widely used in traditional Chinese medicine (TCM), and the content of psoralen, isopsoralen, neobavaisoflavone, bavachin, psoralidin, isobavachalcone, and bavachinin A is the main quality control index of Psoralea Fructus because of its clinical effects. Thus, a fast and environmentally-benign extraction method of seven compounds in Psoralea Fructus is necessary. In this work, an ionic liquid-based ultrasonic-assisted method (ILUAE) for the extraction of seven compounds from Psoralea Fructus was proposed. Several ILs of different types and parameters, including the concentration of ILs, concentration of ethanol (EtOH), solid-liquid ratio, particle size, ultrasonic time, centrifugal speed, and ultrasonic power, were optimized by the Placket-Burman (PB) design and Box-Behnken response surface analysis. Under this optimal condition, the total extraction yield of the seven compounds in Psoralea Fructus was 18.90 mg/g, and significantly greater than the conventional 75% EtOH solvent extraction.