Dense Polarized Positrons from Beam-Solid Interaction.

Relativistic positron sources with high spin polarization have important applications in nuclear and particle physics and many frontier fields. However, it is challenging to produce dense polarized positrons. Here we present a simple and effective method to achieve such a positron source by directly impinging a relativistic high-density electron beam on the surface of a solid target. During the interaction, a strong return current of plasma electrons is induced and subsequently asymmetric quasistatic magnetic fields as high as megatesla are generated along the target surface. This gives rise to strong radiative spin flips and multiphoton processes, thus leading to efficient generation of copious polarized positrons. With three-dimensional particle-in-cell simulations, we demonstrate the production of a dense highly polarized multi-GeV positron beam with an average spin polarization above 40% and nC-scale charge per shot. This offers a novel route for the studies of laserless strong-field quantum electrodynamics physics and for the development of high-energy polarized positron sources.

Visual screening of CRISPR/Cas9 editing efficiency based on micropattern arrays for editing porcine cells.

CRISPR/Cas9 technology, combined with somatic cell nuclear transplantation (SCNT), represents the primary approach to generating gene-edited pigs. The inefficiency in acquiring gene-edited nuclear donors is attributed to low editing and delivery efficiency, both closely linked to the selection of CRISPR/Cas9 forms. However, there is currently no direct method to evaluate the efficiency of CRISPR/Cas9 editing in porcine genomes. A platform based on fluorescence reporting signals and micropattern arrays was developed in this study, to visually assess the efficiency of gene editing. The optimal specifications for culturing porcine cells, determined by the quantity and state of cells grown on micropattern arrays, were a diameter of 200 µm and a spacing of 150 µm. By visualizing the area of fluorescence loss and measuring the gray value of the micropattern arrays, it was quickly determined that the mRNA form targeting porcine cells exhibited the highest editing efficiency compared to DNA and Ribonucleoprotein (RNP) forms of CRISPR/Cas9. Subsequently, four homozygotes of the ß4GalNT2 gene knockout were successfully obtained through the mRNA form, laying the groundwork for the subsequent generation of gene-edited pigs. This platform facilitates a quick, simple, and effective evaluation of gene knockout efficiency. Additionally, it holds significant potential for swiftly testing novel gene editing tools, assessing delivery methods, and tailoring evaluation platforms for various cell types.

One-step in vivo gene knock-out in porcine embryos using recombinant adeno-associated viruses.

Introduction: Gene-edited pigs have become prominent models for studying human disease mechanisms, gene therapy, and xenotransplantation. CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (CRISPR/Cas9) technology is a widely employed tool for generating gene-edited pigs. Nevertheless, delivering CRISPR/Cas9 to pre-implantation embryos has traditionally posed challenges due to its reliance on intricate micromanipulation equipment and specialized techniques, resulting in high costs and time-consuming procedures. This study aims to introduce a novel one-step approach for generating genetically modified pigs by transducing CRISPR/Cas9 components into pre-implantation porcine embryos through oviductal injection of recombinant adeno-associated viruses (rAAV). Methods: We first used rAAV-1, rAAV-6, rAAV-8, rAAV-9 expressing EGFP to screen for rAAV serotypes that efficiently target porcine embryos, and then, to achieve efficient expression of CRISPR/Cas9 in vivo for a short period, we packaged sgRNAs targeting the GHR genes to self-complementary adeno-associated virus (scAAV), and Cas9 proteins to single-stranded adeno-associated virus (ssAAV). The efficiency of porcine embryos -based editing was then validated in vitro. The feasibility of this one-step method to produce gene-edited pigs using rAAV-CRISPR/Cas9 oviductal injection into sows within 24 h of conception was then validated. Results: Our research firstly establishes the efficient delivery of CRISPR/Cas9 to pig zygotes, both in vivo and in vitro, using rAAV6. Successful gene editing in pigs was achieved through oviductal injection of rAAV-CRISPR/Cas9. Conclusion: This method circumvents the intricate procedures involved in in vitro embryo manipulation and embryo transfers, providing a straightforward and cost-effective approach for the production of gene-edited pigs.

Construction and applications of the EOMA spheroid model of Kaposiform hemangioendothelioma.

BACKGROUND: Kaposiform hemangioendothelioma (KHE) is a rare intermediate vascular tumor with unclear pathogenesis. Recently, three dimensional (3D) cell spheroids and organoids have played an indispensable role in the study of many diseases, such as infantile hemangioma and non-involuting congenital hemangiomas. However, few research on KHE are based on the 3D model. This study aims to evaluate the 3D superiority, the similarity with KHE and the ability of drug evaluation of EOMA spheroids as an in vitro 3D KHE model. RESULTS: After two days, relatively uniform morphology and high viability of EOMA spheroids were generated by the rotating cell culture system (RCCS). Through transcriptome analysis, compared with 2D EOMA cells, focal adhesion-related genes such as Itgb4, Flt1, VEGFC, TNXB, LAMA3, VWF, and VEGFD were upregulated in EOMA spheroids. Meanwhile, the EOMA spheroids injected into the subcutaneous showed more obvious KMP than 2D EOMA cells. Furthermore, EOMA spheroids possessed the similar characteristics to the KHE tissues and subcutaneous tumors, such as diagnostic markers (CD31 and LYVE-1), cell proliferation (Ki67), hypoxia (HIF-1α) and cell adhesion (E-cadherin and N-cadherin). Based on the EOMA spheroid model, we discovered that sirolimus, the first-line drug for treating KHE, could inhibit EOMA cell proliferation and downregulate the VEGFC expression. Through the extra addition of VEGFC, the effect of sirolimus on EOMA spheroid could be weakened. CONCLUSION: With a high degree of similarity of the KHE, 3D EOMA spheroids generated by the RCCS can be used as a in vitro model for basic researches of KHE, generating subcutaneous tumors and drug screening.

Geographical distribution-based differentiation of cultivated Angelica dahurica, exploring the relationship between the secretory tract and the quality.

Based on geographical distribution, cultivated Chinese Angelica dahurica has been divided into Angelica dahurica cv. 'Hangbaizhi' (HBZ) and Angelica dahurica cv. 'Qibaizhi' (QBZ). Long-term geographical isolation has led to significant quality differences between them. The secretory structure in medicinal plants, as a place for accumulating effective constituents and information transmission to the environment, links the environment with the quality of medicinal materials. However, the secretory tract differences between HBZ and QBZ has not been revealed. This study aimed to explore the relationship between the secretory tract and the quality of two kinds of A. dahurica. Root samples were collected at seven development phases. High-Performance Liquid Chromatography (HPLC) and Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI) were used for the content determination and spatial location of coumarins. Paraffin section was used to observe and localize the root secretory tract. Origin, CaseViewer, and HDI software were used for data analysis and image processing. The results showed that compared to QBZ, HBZ, with better quality, has a larger area of root secretory tracts. Hence, the root secretory tract can be included in the quality evaluation indicators of A. dahurica. Additionally, DESI-MSI technology was used for the first time to elucidate the temporal and spatial distribution of coumarin components in A. dahurica root tissues. This study provides a theoretical basis for the quality evaluation and breeding of improved varieties of A. dahurica and references the DESI-MSI technology used to analyze the metabolic differences of various compounds, including coumarin and volatile oil, in different tissue parts of A. dahurica.

Tissue-specific micropattern array chips fabricated via decellularized ECM for 3D cell culture.

Multicellular three-dimensional (3D) in vitro models, such as cell spheroids and organoids, can significantly improve the viability, histomorphology, genotype stability, function and drug metabolism of cells [1], [2], [3]. In general, several culture methods of 3D models, including the hanging drop, microwell-mesh and hydrogel encapsulating methods, have difficulty building a standard mode and controlling the size and arrangement of cell spheroids or organoids, which could severely affect the authenticity and repeatability of experimental results [4]. Another key factor in 3D in vitro models is the extracellular matrix (ECM), which can determine cell viability, proliferation, differentiation, function, migration and organization [5]. In this study, micropattern array chips combined with decellularized ECM (dECM) not only provide tissue-specific ECM but also control the size and arrangement of 3D models. •Methods have been established to demonstrate the use of dECM as a bioink to generate dECM-coated micropattern array chips by microcontact printing.•The micropattern can limit cell growth and migration, and cells spontaneously assemble into cell spheroids with uniform size and orderly arrangement.

Strengthening Mechanism of Rotary-Forged Deformable Biodegradable Zn-0.45Li Alloys.

The use of zinc (Zn) alloys as a biodegradable metal for medical purposes has been a popular research topic. This study investigated the strengthening mechanism of Zn alloys to enhance their mechanical properties. Three Zn-0.45Li (wt.%) alloys with different deformation amounts were prepared by rotary forging deformation. Their mechanical properties and microstructures were tested. A simultaneous increase in strength and ductility was observed in the Zn-0.45Li alloys. Grain refinement occurred when the rotary forging deformation reached 75.7%. The surface average grain size reached 1.19 ± 0.31 µm, and the grain size was uniformly distributed. Meanwhile, the maximum elongation of the deformed Zn-0.45Li was 139.2 ± 18.6%, and the ultimate tensile strength reached 426.1 ± 4.7 MPa. In situ tensile tests showed that the reinforced alloys still broke from the grain boundary. Continuous and discontinuous dynamic recrystallization during severe plastic deformation produced many recrystallized grains. During deformation, the dislocation density of the alloy first increased and then decreased, and the texture strength of the (0001) direction increased with deformation. Analysis of the mechanism of alloy strengthening showed that the strength and plasticity enhancement of Zn-Li alloys after macro deformation was a combination of dislocation strengthening, weave strengthening, and grain refinement rather than only fine-grain strengthening as observed in conventional macro-deformed Zn alloys.

Metabolomics analysis reveals metabolite changes during freeze-drying and oven-drying of Angelica dahurica.

Angelica dahurica (Angelica dahurica Fisch. ex Hoffm.) is widely used as a traditional Chinese medicine and the secondary metabolites have significant pharmacological activities. Drying has been shown to be a key factor affecting the coumarin content of Angelica dahurica. However, the underlying mechanism of metabolism is unclear. This study sought to determine the key differential metabolites and metabolic pathways related to this phenomenon. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) based targeted metabolomics analysis was performed on Angelica dahurica that were freeze-drying (- 80 °C/9 h) and oven-drying (60 °C/10 h). Furthermore, the common metabolic pathways of paired comparison groups were performed based on KEEG enrichment analysis. The results showed that 193 metabolites were identified as key differential metabolites, most of which were upregulated under oven drying. It also displayed that many significant contents of PAL pathways were changed. This study revealed the large-scale recombination events of metabolites in Angelica dahurica. First, we identified additional active secondary metabolites apart from coumarins, and volatile oil were significantly accumulated in Angelica dahurica. We further explored the specific metabolite changes and mechanism of the phenomenon of coumarin upregulation caused by temperature rise. These results provide a theoretical reference for future research on the composition and processing method of Angelica dahurica.

Effect of Mg Contents on the Microstructure, Mechanical Properties and Cytocompatibility of Degradable Zn-0.5Mn-xMg Alloy.

The effect of magnesium (Mg) content on the microstructure, mechanical properties, and cytocompatibility of degradable Zn-0.5Mn-xMg (x = 0.05 wt%, 0.2 wt%, 0.5 wt%) alloys was investigated. The microstructure, corrosion products, mechanical properties, and corrosion properties of the three alloys were then thoroughly characterized by scanning electron microscopy (SEM), electron back-scattered diffraction (EBSD), and other methods. According to the findings, the grain size of matrix was refined by the addition of Mg, while the size and quantity of Mg2Zn11 phase was increased. The Mg content could significantly improve the ultimate tensile strength (UTS) of the alloy. Compared with the Zn-0.5Mn alloy, the UTS of Zn-0.5Mn-xMg alloy was increased significantly. Zn-0.5Mn-0.5Mg exhibited the highest UTS (369.6 MPa). The strength of the alloy was influenced by the average grain size, the solid solubility of Mg, and the quantity of Mg2Zn11 phase. The increase in the quantity and size of Mg2Zn11 phase was the main reason for the transition from ductile fracture to cleavage fracture. Moreover, Zn-0.5Mn-0.2Mg alloy showed the best cytocompatibility to L-929 cells.

The CRISPR/Cas9 System Delivered by Extracellular Vesicles.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems can precisely manipulate DNA sequences to change the characteristics of cells and organs, which has potential in the mechanistic research on genes and the treatment of diseases. However, clinical applications are restricted by the lack of safe, targeted and effective delivery vectors. Extracellular vesicles (EVs) are an attractive delivery platform for CRISPR/Cas9. Compared with viral and other vectors, EVs present several advantages, including safety, protection, capacity, penetrating ability, targeting ability and potential for modification. Consequently, EVs are profitably used to deliver the CRISPR/Cas9 in vivo. In this review, the advantages and disadvantages of the delivery form and vectors of the CRISPR/Cas9 are concluded. The favorable traits of EVs as vectors, such as the innate characteristics, physiological and pathological functions, safety and targeting ability of EVs, are summarized. Furthermore, in terms of the delivery of the CRISPR/Cas9 by EVs, EV sources and isolation strategies, the delivery form and loading methods of the CRISPR/Cas9 and applications have been concluded and discussed. Finally, this review provides future directions of EVs as vectors of the CRISPR/Cas9 system in clinical applications, such as the safety, capacity, consistent quality, yield and targeting ability of EVs.

Influence of Enzymes on the In Vitro Degradation Behavior of Pure Zn in Simulated Gastric and Intestinal Fluids.

Zinc (Zn) alloys are being developed as the degradable biomaterial. However, the corrosion mechanism of Zn in the gastrointestinal environment is seldom investigated and needs to be addressed. In this study, the impacts of enzymes on the degradation of pure Zn via electrochemical measurements and immersion were investigated. Pepsin and pancreatin affected the degradation of pure Zn. In contrast with the solutions without enzymes, the degradation rates declined with the addition of enzymes in solutions. However, localized corrosion was observed because the adsorption of pepsin was not a perfect barrier to prevent corrosion. The adsorbed pancreatin protected the samples from corrosion mainly at the initial stage of immersion. With immersion in the simulated intestinal fluid, adsorption and desorption of pancreatin occurred simultaneously on the sample surface. These findings allow the development of Zn alloy-implanted devices for the digestive tract as well as the understanding of the Zn corrosion mechanism in the gastrointestinal environment.

Three-Dimensional Microtumor Formation of Infantile Hemangioma-Derived Endothelial Cells for Mechanistic Exploration and Drug Screening.

Infantile hemangioma (IH) is the most prevalent type of vascular tumor in infants. The pathophysiology of IH is unknown. The tissue structure and physiology of two-dimensional cell cultures differ greatly from those in vivo, and spontaneous regression often occurs during tumor formation in nude mice and has severely limited research into the pathogenesis and development of IH. By decellularizing porcine aorta, we attempted to obtain vascular-specific extracellular matrix as the bioink for fabricating micropattern arrays of varying diameters via microcontact printing. We then constructed IH-derived CD31+ hemangioma endothelial cell three-dimensional microtumor models. The vascular-specific and decellularized extracellular matrix was suitable for the growth of infantile hemangioma-derived endothelial cells. The KEGG signaling pathway analysis revealed enrichment primarily in stem cell pluripotency, RAS, and PI3KAkt compared to the two-dimensional cell model according to RNA sequencing. Propranolol, the first-line medication for IH, was also used to test the model's applicability. We also found that metformin had some impact on the condition. The three-dimensional microtumor models of CD31+ hemangioma endothelial cells were more robust and efficient experimental models for IH mechanistic exploration and drug screening.

Study on the potential mechanism, therapeutic drugs and prescriptions of insomnia based on bioinformatics and molecular docking.

Insomnia is a very common disease worldwide. It seriously affects the quality of human life and even endangers health. Traditional Chinese medicine (TCM) has unique advantages in the intervention and treatment of insomnia. However, its underlying mechanism has yet to be elucidated. This study was performed to explore the potential biomarkers and mechanisms of insomnia, and treatment TCM and classical prescriptions. The gene microarray data of insomnia is downloaded and preprocessed. Differentially expressed genes (DEGs) and GO and KEGG enrichment analyses were performed. The protein-protein interaction network (PPI) was constructed. Small molecule drugs for curing insomnia were identified using cMap and CTD databases. We searched the TCM corresponding to small molecule drugs and the classic prescriptions corresponding to TCM by the TCMSP database. We constructed a network of "ingredient-TCM-classic prescriptions". The molecular docking was performed to validate the screening results. We obtained a total of 124 DEGs, including 78 up-regulated genes, 46 down-regulated genes, 10 Hub genes and 3 key modules. A total of 125 significant GO entries and 15 significant KEGG were enriched (P < 0.05). The main biological processes involve neuronal apoptosis, autophagy, cell growth and apoptosis, etc. These signaling pathways may be involved in molecular regulatory mechanisms of insomnia, such as autophagy regulation, Alzheimer's disease, pathways to neurodegenerative diseases and neurotrophic factor signaling pathways. We identified 10 traditional Chinese medicines and 2 classical prescriptions of potential value. In addition, the molecular docking results indicated that small molecule ligands were nicely bound to the Hub gene, and the binding affinity ranged from -7.6 to -9.7 kcal/mol. This study provides a foundation for the clinical treatment of insomnia, explains the molecular mechanisms, and efficiently develops TCM and classical prescriptions.

Autoclaving pHEMA-Based Hydrogels Immersed in Deionized Water has No Effect on Physicochemical Properties and Cell Behaviors.

Hydrogels based on poly-(2-hydroxyethyl methacrylate) (pHEMA) have been widely used as biomaterials in tissue engineering due to their biocompatibility, hydrophilicity, and low friction coefficient. The terminal sterilization of hydrogels is a critical step in clinical applications. However, regulations and standardization for the sterilization of hydrogels based on pHEMA are still lacking. In this study, we explored six sterilization methods on pHEMA-based materials (A1: pHEMA, A2: pHEMA copolymerizes with acrylic acid, and A3: pHEMA copolymerizes with acrylic acid and further coordinated with iron ions), such as gamma irradiation, 75% ethanol, ultraviolet (UV), ethylene oxide (EtO), and autoclaving with or without deionized water (autoclaving-H2O or autoclaving-dry). Combining results from the multifaceted approaches with assessment, pHEMA-based hydrogels can be completely sterilized via the autoclaving-H2O method analyzed by sterilized testing. The physicochemical properties and cell behavior of sterilized hydrogels were not influenced by this sterilization approach, validated by Fourier transform infrared (FT-IR) spectroscopy and tensile tests. The pHEMA-based hydrogel sterilized by the autoclaving-H2O method also had no effect on the cell behavior evaluated by in vitro cytotoxicity experiments and caused no evident inflammatory reaction in tissue in vivo implantation experiments. However, it was also found that there were still some defects in the A2 and A3 groups as biomaterials possibly because of an inappropriate proportion of formulations or raw material used in exploring sterilization methods. These findings have implications for the improvement and clinical application of pHEMA-based hydrogels.

Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres.

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

Ordered micropattern arrays fabricated by lung-derived dECM hydrogels for chemotherapeutic drug screening.

Aims: This study aims to evaluate ECM-coated micropattern arrays derived from decellularization of native porcine lungs as a novel three-dimensional cell culture platform. Methods: ECM derived from decellularization of native porcine lungs was exploited to prepare hydrogels. Then, dECM-coated micropattern arrays were fabricated at four different diameters (50, 100, 150 and 200 â€‹µm) using polydimethylsiloxane (PDMS). Two lung cancer cell lines, A549 and H1299, were tested on a dECM-coated micropattern array as a novel culture platform for cell adhesion, distribution, proliferation, viability, phenotype expression, and drug screening to evaluate the cytotoxicity of paclitaxel, doxorubicin and cisplatin. Results: The ECM derived from decellularization of native porcine lungs supported cell adhesion, distribution, viability and proliferation better than collagen I and Matrigel as the coated matrix on the surface. Moreover, the optimal diameter of the micropattern arrays was 100-150 â€‹µm, as determined by measuring the morphology, viability, proliferation and phenotype of the cancer cell spheroids. Cell spheroids of A549 and H1299 on dECM-coated micropattern arrays showed chemoresistance to anticancer drugs compared to that of the monolayer. The different distributions of HIF-1α, MCL-1 (in the center) and Ki-67 and MRP2 (in the periphery) of the spheroids demonstrated the good establishment of basal-lateral polarity and explained the chemoresistance phenomenon of spheroids. Conclusions: This novel three-dimensional cell culture platform is stable and reliable for anticancer drug testing. Drug screening in dECM-coated micropattern arrays provides a powerful alternative to existing methods for drug testing and metabolic profiling in the drug discovery process.

Effective removal of hydrogen sulfide from landfill gases using a modified iron pentacarbonyl desulfurization agent and the desulfurization mechanism.

High-efficiency desulfurization is key to the recovery and use of landfill gases. In this study, a nano­iron oxide desulfurization agent modified from iron pentacarbonyl was prepared in n-decane (DE) and hexadecane (HE) by ultrasonic disruption without any supporting materials and its hydrogen sulfide removal ability and desulfurization mechanism were studied. The yield of the desulfurization agent was higher when HE was used as the solvent; however, the products generated by both solvents had the same crystal type and similar properties. The efficiency of the desulfurization agent was significantly improved at 150-200 °C, exceeding 90% at 150 °C with single sulfur production. The maximum sulfur adsorption capacity of the desulfurization agent produced after 3 h of DE ultrasonic treatment at 200 °C (DE3) was 492 mg/g (desulfurization efficiency = 97.33%), while that of the agent produced after 3 h of HE ultrasonic treatment at 250 °C (HE3) was 522 mg/g (desulfurization efficiency = 99.30%). The desulfurization reaction involved both chemical adsorption and catalytic decomposition and the catalytic decomposition reaction rate was lower than that of chemical adsorption. Therefore, the more FexSy produced in the chemical adsorption process, the better catalytic performance was.

The complete mitochondrial genome of snailfish Liparis tanakae Gilbert & Burke, 1912 (Perciformes: Cottioidei: Liparidae).

Mitochondrial genome is maternal inheritance that provides higher resolution in taxonomic and phylogenetic research. The absence of complete mitogenome becomes an obstacle to further research. Here, we reported the complete mitogenome of Liparis tanakae Gilbert & Burke, 1912 (Perciformes: Cottioidei: Liparidae), which has a length of 17,860 bp. It comprised 39 genes, including 13 protein-coding genes (PCGs), 23 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and 1 control region (D-loop). The 23 tRNAs in this mitogenome included two tRNA-Ala genes on the light strand. Maximum-likelihood (ML) phylogenetic analysis based on 14 available mitogenomes of 10 species in suborder Cottioidei confirmed L. tanakae as monophyletic with other snailfishes. This study would provide important genetic resources and could be useful for evolutionary analysis.

[Composition changes reveal relationship between color and enzymatic reaction of Magnoliae Officinalis Cortex during "sweating" process].

In this study, we employed Q Exactive to determine the main differential metabolites of Magnoliae Officinalis Cortex du-ring the "sweating" process. Further, we quantified the color parameters and determined the activities of polyphenol oxidase(PPO), peroxidase(POD), and tyrosinase of Magnoliae Officinalis Cortex during the "sweating" process. Gray correlation analysis was performed for the color, chemical composition, and enzyme activity to reveal the effect of enzymatic reaction on the color of Magnoliae Officinalis Cortex during the "sweating" process. Magnoliae Officinalis Cortex sweating in different manners showed similar metabolite changes. The primary metabolites that changed significantly included amino acids, nucleotides, and sugars, and the secondary metabolites with significant changes were phenols and phenylpropanoids. Despite the different sweating methods, eleven compounds were commonly up-regulated, including L-glutamic acid, acetylarginine, hypoxanthine, and xanthine; six compounds were commonly down-re-gulated, including L-arginine, L-aspartic acid, and phenylalanine. The brightness value(L~*), red-green value(a~*), and yellow-blue value(b~*) of Magnoliae Officinalis Cortex kept decreasing during the "sweating" process. The changes in the activities of PPO and POD during sweating were consistent with those in the color parameter values. The gray correlation analysis demonstrated that the main differential metabolites such as amino acids and phenols were closely related to the color parameters L~*, a~* and b~*; POD was correlated with amino acids and phenols; PPO had strong correlation with phenols. The results indicated that the color change of Magnoliae Officinalis Cortex during "sweating" was closely related to the reactions of enzymes dominated by PPO and POD. The study analyzed the correlations among the main differential metabolites, color parameters, and enzyme activities of Magnoliae Officinalis Cortex in the "sweating" process. It reveals the common law of material changes and ascertains the relationship between color changes and enzymatic reactions of Magnoliae Officinalis Cortex during "sweating". Therefore, this study provides a reference for studying the "sweating" mechanism of Magnoliae Officinalis Cortex and is of great significance to guarantee the quality of Magnoliae Officinalis Cortex.

Fabrication of Size-Controllable and Arrangement-Orderly HepG2 Spheroids for Drug Screening via Decellularized Liver Matrix-Derived Micropattern Array Chips.

Three-dimensional (3D) culture via micropattern arrays to generate cellular spheroids seems a promising in vitro biomimetic system for liver tissue engineering applications, such as drug screening. Recently, organ-derived decellularized extracellular matrix emerges as arguably the most biomimetic bioink. Herein, decellularized liver matrix (DLM)-derived micropattern array chips were developed to fabricate size-controllable and arrangement-orderly HepG2 spheroids for drug screening. The porcine DLM was obtained by the removal of cellular components and then ground into powder, followed by enzymolysis. DLM as a coating substrate was compared with collagen type I (Col I) and Matrigel in terms of biological performance for enhancing cell adhesion, proliferation, and functions. Subsequently, we used poly(dimethylsiloxane) (PDMS) to adsorb DLM as the bioink to fabricate micropattern array chips. The optimal shape and size of micropattern were determined by evaluating the morphology, viability, and functions of HepG2 3D cellular aggregates. In addition, drug-susceptibility testing (paclitaxel, doxorubicin HCl, and disulfiram) was performed on this novel platform. The DLM provided the tissue-specific microenvironment that provided suitable supports for HepG2 cells, compared to Col I and Matrigel. A circular micropattern with a diameter of 100 µm was the optimal processing parameter to rapidly fabricate large-scale, size-controllable, and arrangement-orderly HepG2 cellular aggregates with 3D spheroid's shape and high cell viability. Drug screening testing showed that the effect of a drug could be directly demonstrated on-chip by confocal microscopy measuring the viability of spheroids. We provide a novel platform for the large-scale generation of HepG2 spheroids with uniform size and arrangement, thus bringing convenience, reducing error, and increasing reproducibility for a rapid drug discovery by fluorescence quantitative analysis. This methodology may be possible to apply in advancing personalized medicine and drug discovery.