The cytokinin receptor OHK4/OsHK4 regulates inflorescence architecture in rice via an IDEAL PLANT ARCHITECTURE1/WEALTHY FARMER'S PANICLE-mediated positive feedback circuit.

Inflorescence architecture is important for rice (Oryza sativa) grain yield. The phytohormone cytokinin (CK) has been shown to regulate rice inflorescence development; however, the underlying mechanism mediated by CK perception is still unclear. Employing a forward genetic approach, we isolated an inactive variant of the CK receptor OHK4/OsHK4 gene named panicle length1, which shows decreased panicle size due to reduced inflorescence meristem (IM) activity. A 2-amino acid deletion in the long α-helix stalk of the sensory module of OHK4 impairs the homodimerization and ligand-binding capacity of the receptor, even though the residues do not touch the ligand-binding domain or the dimerization interface. This deletion impairs CK signaling that occurs through the type-B response regulator OsRR21, which acts downstream of OHK4 in controlling inflorescence size. Meanwhile, we found that IDEAL PLANT ARCHITECTURE1(IPA1)/WEALTHY FARMER'S PANICLE (WFP), encoding a positive regulator of IM development, acts downstream of CK signaling and is directly activated by OsRR21. Additionally, we revealed that IPA1/WFP directly binds to the OHK4 promoter and upregulates its expression through interactions with 2 TCP transcription factors, forming a positive feedback circuit. Altogether, we identified the OHK4-OsRR21-IPA1 regulatory module, providing important insights into the role of CK signaling in regulating rice inflorescence architecture.

Deep learning-enabled discovery and characterization of HKT genes in Spartina alterniflora.

Spartina alterniflora is a halophyte that can survive in high-salinity environments, and it is phylogenetically close to important cereal crops, such as maize and rice. It is of scientific interest to understand why S. alterniflora can live under such extremely stressful conditions. The molecular mechanism underlying its high-saline tolerance is still largely unknown. Here we investigated the possibility that high-affinity K+ transporters (HKTs), which function in salt tolerance and maintenance of ion homeostasis in plants, are responsible for salt tolerance in S. alterniflora. To overcome the imprecision and unstable of the gene screening method caused by the conventional sequence alignment, we used a deep learning method, DeepGOPlus, to automatically extract sequence and protein characteristics from our newly assemble S. alterniflora genome to identify SaHKTs. Results showed that a total of 16 HKT genes were identified. The number of S. alterniflora HKTs (SaHKTs) is larger than that in all other investigated plant species except wheat. Phylogenetically related SaHKT members had similar gene structures, conserved protein domains and cis-elements. Expression profiling showed that most SaHKT genes are expressed in specific tissues and are differentially expressed under salt stress. Yeast complementation expression analysis showed that type I members SaHKT1;2, SaHKT1;3 and SaHKT1;8 and type II members SaHKT2;1, SaHKT2;3 and SaHKT2;4 had low-affinity K+ uptake ability and that type II members showed stronger K+ affinity than rice and Arabidopsis HKTs, as well as most SaHKTs showed preference for Na+ transport. We believe the deep learning-based methods are powerful approaches to uncovering new functional genes, and the SaHKT genes identified are important resources for breeding new varieties of salt-tolerant crops.

Enhanced Electron Transport and Mitigated Voltage Loss in Perovskite Photovoltaics Using Sb2O5@SnO2 Composite Electron Transport Layer.

The efficacy of electron transport layers (ETLs) is pivotal for optimizing the device performance of perovskite photovoltaic applications. However, colloidal dispersions of SnO2 are prone to aggregation and possess structural defects, such as terminal-hydroxyls (OHT) and oxygen vacancies (VOs), which can degrade the quality of ETLs, impede charge extraction and transport, and affect the nucleation and growth processes of the perovskite layer. In this study, the Sb(OH)4 - ions hydrolyzed from SbCl3 in colloidal dispersion can bind to defect sites and effectively stabilize the SnO2 nanocrystals are demonstrated. Upon oxidative annealing, a Sb2O5@SnO2 composite film is formed, in which the Sb2O5 not only mitigates the aforementioned defects but also broadens the energy range of unoccupied states through its dispersed conduction band. The increased electron affinity (EA) facilitates more efficient capture of photoexcited electrons from the perovskite layer, thus augmenting electron extraction and minimizing electron-hole recombination. As a result, a significant improvement in power conversion efficiency (PCE) from 22.60% to 24.54% is achieved, with an open circuit voltage (VOC) of up to 1.195 V, along with excellent stability of unsealed devices under various conditions. This study provides valuable insights for the understanding and design of ETLs in perovskite photovoltaic applications.

The Spartina alterniflora genome sequence provides insights into the salt-tolerance mechanisms of exo-recretohalophytes.

Spartina alterniflora is an exo-recretohalophyte Poaceae species that is able to grow well in seashore, but the genomic basis underlying its adaptation to salt tolerance remains unknown. Here, we report a high-quality, chromosome-level genome assembly of S. alterniflora constructed through PacBio HiFi sequencing, combined with high-throughput chromosome conformation capture (Hi-C) technology and Illumina-based transcriptomic analyses. The final 1.58 Gb genome assembly has a contig N50 size of 46.74 Mb. Phylogenetic analysis suggests that S. alterniflora diverged from Zoysia japonica approximately 21.72 million years ago (MYA). Moreover, whole-genome duplication (WGD) events in S. alterniflora appear to have expanded gene families and transcription factors relevant to salt tolerance and adaptation to saline environments. Comparative genomics analyses identified numerous species-specific genes, significantly expanded genes and positively selected genes that are enriched for 'ion transport' and 'response to salt stress'. RNA-seq analysis identified several ion transporter genes including the high-affinity K+ transporters (HKTs), SaHKT1;2, SaHKT1;3 and SaHKT1;8, and high copy number of Salt Overly Sensitive (SOS) up-regulated under high salt conditions, and the overexpression of SaHKT2;4 in Arabidopsis thaliana conferred salt tolerance to the plant, suggesting specialized roles for S. alterniflora to adapt to saline environments. Integrated metabolomics and transcriptomics analyses revealed that salt stress activate glutathione metabolism, with differential expressions of several genes such as γ-ECS, GSH-S, GPX, GST and PCS in the glutathione metabolism. This study suggests several adaptive mechanisms that could contribute our understanding of evolutional basis of the halophyte.

KLF4 regulates trophoblast function and associates with unexplained recurrent spontaneous abortion.

BACKGROUND: Recurrent spontaneous abortion (RSA) is defined as two or more consecutive spontaneous abortions before 20 weeks with the same spouse [1]. However, approximately 50% of RSA cases of unknown cause are classified as unexplained recurrent spontaneous abortion (URSA). Potential factors include decreased trophoblast cell migration and invasion, leading to impaired placental implantation and maintenance of the normal maternal-fetal interface. However, the mechanism of this pathogenesis remains unknown. In this study, we investigated the potential role and mechanism of KLF4 in regulating URSA by influencing the invasion and migration ability of trophoblast cells. METHODS: We firstly identified 817 differentially expressed genes by performing a difference analysis of the dataset GSE121950 [2] related to recurrent abortion, and intersected the top 10 genes obtained respectively by the three algorithms: DMNC, MNC, and EPC using Venn Diagram.To detect the expression levels of core genes, villi samples were obtained from normal pregnant women and patients with URSA. RT-qPCR analysis revealed a significant difference in KLF4 mRNA expression and KLF4 was then analyzed. Trophoblast cell lines HTR8 and JEG3 were used to investigate the effect of KLF4 on trophoblastic function. Wound healing and transwell assays was performed to detect the invasion and migration of trophoblast cells. The expression of epithelial-mesenchymal transition(EMT) molecules were detected by RT-qPCR and western blot. Promoter detection and epigenetic modification were detected by chromatin immunoprecipitation (ChIP) assay. Molecular nuclear localization was detected by immunofluorescence and subcellular fractionation. Miscarried mice model was used to study the effects of KLF4 on URSA induced by reduced trophoblast invasion and migration. RESULTS: KLF4 is highly expressed in the villi of patients with URSA. KLF4 inhibits the expression level of H3R2ME2a in trophoblast cells by regulating the transcriptional level and nuclear translocation of PRMT6, thereby inhibiting the possible regulatory mechanism of trophoblastic invasion and providing a potential treatment strategy for URSA in vivo. CONCLUSIONS: The KLF4/PRMT6/H3R2ME2a axis regulates mechanisms associated with unexplained recurrent spontaneous abortion by regulating trophoblast function.

DEAD-BOX RNA HELICASE 27 regulates microRNA biogenesis, zygote division, and stem cell homeostasis.

After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.

Screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Poria Cum Radix Pini and mechanism of action in the treatment of gout disease.

Poria Cum Radix Pini is a rare medicinal fungus that contains several potential therapeutic ingredients. On this basis, a particle swarm mathematical model was used to optimize the extraction process of total triterpenes from P. Cum Radix Pini, and xanthine oxidase inhibitors were screened using affinity ultrafiltration mass spectrometry. Meanwhile, the accuracy of the ultrafiltration assay was verified by molecular docking experiments and molecular dynamics analysis, and the mechanism of action of the active compounds for the treatment of gout was analyzed by enzymatic reaction kinetics and network pharmacology. A high-speed countercurrent chromatography method combined with the consecutive injection and the economical two-phase solvent system preparation using functional activity coefficient of universal quasichemical model (UNIFAC) mathematical model was developed for increasing the yield of target compound. In addition, dehydropachymic acid and pachymic acid were used as competitive inhibitors, and 3-O-acetyl-16alpha-hydroxydehydrotrametenolic acid and dehydrotrametenolic acid were used as mixed inhibitors. Then, activity-oriented separation and purification were performed by high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography and the purity of the four compounds isolated was higher than 90%. It will help to provide more opportunities to discover and develop new potential therapeutic remedies from health care food resources.

Rapid screening and isolation of 5-lipoxygenase inhibitors in Inonotus obliquus and mechanism of action in the treatment of asthma.

Accurate screening and targeted preparative isolation of active substances in natural medicines have long been two technical challenges in natural medicine research. This study outlines a new approach to improve the efficiency of natural product preparation, focusing on rapidly and accurately screening potential active ingredients in Inonotus obliquus as well as efficiently preparing 5-lipoxidase (5-LOX) inhibitors, to provide new ideas for the treatment of asthma with Inonotus obliquus. First, we used ultrafiltration (UF) mass spectrometry to screen for three potential inhibitors of 5-LOX in Inonotus obliquus. Subsequently, the inhibitory effect of the active ingredients screened in the UF assay on 5-LOX was verified using the molecular docking technique, and the potential role of the active compounds in Inonotus obliquus for the treatment of asthma was analyzed by network pharmacology. Finally, based on the above activity screening guidelines, we used semi-preparative liquid chromatography and consecutive high-speed countercurrent chromatography to isolate three high-purity 5-LOX inhibitors such as betulin, lanosterol, and quercetin. Obviously, through the above approach, we have seamlessly combined rapid discovery, screening, and centralized preparation of the active ingredient with molecular-level interactions between the active ingredient and the protease.

High-Speed Countercurrent Chromatography Isolation of Active Components from Evodia Rutaecarpa and Affinity Ultrafiltration Screening for Their Acetylcholinesterase Inhibitor Activity.

Acetylcholinesterase inhibitors from Evodia rutaecarpa were screened, prepared, and evaluated. To screen the lipophilic alkaloid active constituents in E. rutaecarpa, we improved and optimized an ultrafiltration system. Three acetylcholinesterase (AChE) inhibitors, dehydroevodiamine, evodiamine, and rutecarpine, were screened. Addressing the limitations of the traditional response surface methodology (RSM) for multiobjective screening, we integrated RSM with the Non-dominated Sorting Genetic Algorithm III to achieve the optimal extraction of these active ingredients. High-speed countercurrent chromatography was used to isolate the active components using a two-phase solvent system: n-hexane/ethyl acetate/methanol/water (3.0:2.5:3.5:2.0, v/v/v/v) and ethyl acetate/methanol/water (3.0:1.0:4.0, v/v/v). The nuclear magnetic resonance spectroscopy confirmed the structures of the compounds, and molecular docking and dynamics simulations assessed the inhibitory effects of the chemical components on AChE, which were consistent with the findings of the ultrafiltration experiments. We also confirmed the neuroprotective properties of these compounds against glutamate-induced apoptosis in PC12 cells. Overall, we achieved the systematic optimization of multitarget compound extraction and lipophilic alkaloid ultrafiltration screening, as well as preparation and activity validation, laying the groundwork for the development of AChE inhibitors from lipophilic alkaloids.

Genomic screening methodology not requiring barcoding: Single nucleotide polymorphism-based, mixed-cell screening (SMICS).

Although high-throughput, cancer cell-line screening is a time-honored, important tool for anti-cancer drug development, this process involves the testing of each, individual drug in each, individual cell-line. Despite the availability of robotic liquid handling systems, this process remains a time-consuming and costly investment. The Broad Institute developed a new method called Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) to screen a mixture of barcoded, tumor cell-lines. Although this methodology significantly improved the efficiency of screening large numbers of cell-lines, the barcoding process itself was tedious that requires gene transfection and subsequent selection of stable cell-lines. In this study, we developed a new, genomic approach for screening multiple cancer cell-lines using endogenous "tags" that did not require prior barcoding: single nucleotide polymorphism-based, mixed-cell screening (SMICS). The code for SMICS is available at https://github.com/MarkeyBBSRF/SMICS.

Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Efficient and fast screening and separation based on computer-aided screening and complex chromatography methods for lipoxygenase inhibitors from Ganoderma lucidum.

INTRODUCTION: Accurate screening and targeted preparative isolation of active substances from natural medicines have long been technical challenges in natural medicine research. OBJECTIVES: This study outlines a new approach for improving the efficiency of natural product preparation, focusing on the rapid and accurate screening of potential active ingredients in Ganoderma lucidum and efficient preparation of lipoxidase inhibitors, with the aim of providing new ideas for the treatment of Alzheimer's disease with G. lucidum. METHODS: The medicinal plant G. lucidum was selected through ultrafiltration coupled with liquid chromatography and mass spectrometry (UF-LC-MS) and computer-assisted screening for lipoxygenase (LOX) inhibitors. In addition, the inhibitory effect of the active compounds on LOX was studied using enzymatic reaction kinetics, and the underlying mechanism is discussed. Finally, based on the earlier activity screening guidelines, the identified ligands were isolated and purified through complex chromatography (high-speed countercurrent chromatography and semi-preparative high-performance liquid chromatography). RESULTS: Five active ingredients, ganoderic acids A, B, C2, D2, and F, were identified and isolated from G. lucidum. We improved the efficiency and purity of active compound preparation using virtual computer screening and enzyme inhibition assays combined with complex chromatography. CONCLUSION: The innovative methods of UF-LC-MS, computer-aided screening, and complex chromatography provide powerful tools for screening and separating LOX inhibitors from complex matrices and provide a favourable platform for the large-scale production of bioactive substances and nutrients.

Quaternity method for integrated screening, separation, extraction optimization, and bioactivity evaluation of acetylcholinesterase inhibitors from Sophora flavescens Aiton.

INTRODUCTION: Sophora flavescens Aiton (Fabaceae), a ubiquitous plant species in Asia, contains a wide range of pharmacologically active compounds, such as flavonoids, with potential anti-Alzheimer's disease (anti-AD) effects. OBJECTIVES: The objective of the study is to develop a quaternity method for the screening, isolation, extraction optimization, and activity evaluation of acetylcholinesterase (AChE)-inhibiting compounds from S. flavescens to realize high-throughput screening of active substances in traditional Chinese medicine and to provide experimental data for the development of anti-AD drugs. METHODS: With AChE as the target molecule, affinity ultrafiltration and liquid chromatography-mass spectrometry were applied to screen for potential inhibitors of the enzyme in S. flavescens. Orthogonal array experiments combined with the multi-objective Non-Dominated Sorting Genetic Algorithm III was used for the first time to optimize the process for extracting the active substances. Enzyme inhibition kinetics and molecular docking studies were performed to verify the potential anti-AD effects of the active compounds. RESULTS: Five AChE-inhibiting compounds were identified: kushenol I, kurarinone, sophoraflavanone G, isokurarinone, and kushenol E. These were successfully separated at purities of 72.88%, 98.55%, 96.86%, 96.74%, and 95.84%, respectively, using the n-hexane/ethyl acetate/methanol/water (4.0/5.0/4.0/5.0, v/v/v/v), n-hexane/ethyl acetate/methanol/water (5.0/5.0/6.0/4.0, v/v/v/v), and n-hexane/ethyl acetate/methanol/water (4.9/5.1/5.7/4.3, v/v/v/v) mobile phase systems. Enzyme inhibition kinetics revealed that kushenol E had the best inhibitory effect. CONCLUSION: This study elucidates the mechanism of action of five active AChE inhibitors in S. flavescens and provides a theoretical basis for the screening and development of anti-AD and other therapeutic drugs.

Fluorinated Polyimide Tunneling Layer for Efficient and Stable Perovskite Photovoltaics.

Despite the remarkable progress of perovskite solar cells (PSCs), challenges remain in terms of finding effective and viable strategies to enhance their long-term stability while maintaining high efficiency. In this study, a new insulating and hydrophobic fluorinated polyimide (FPI: 6FDA-6FAPB) was used as the interface layer between the perovskite layer and the hole transport layer (HTL) in PSCs. The functional groups of FPI play a pivotal role in passivating interface defects within the device. Due to its high work function, FPI demonstrates field-effect passivation (FEP) capabilities as an interface layer, effectively mitigating non-radiative recombination at the interface. Notably, the FPI insulating interface layer does not impede carrier transmission at the interface, which is attributed to the presence of hole tunneling effects. The optimized PSCs achieve an outstanding power conversion efficiency (PCE) of 24.61 % and demonstrate excellent stability, showcasing the efficacy of FPI in enhancing device performance and reliability.

ARGONAUTE2 Enhances Grain Length and Salt Tolerance by Activating BIG GRAIN3 to Modulate Cytokinin Distribution in Rice.

Maintaining stable, high yields under fluctuating environmental conditions is a long-standing goal of crop improvement but is challenging due to internal trade-off mechanisms, which are poorly understood. Here, we identify ARGONAUTE2 (AGO2) as a candidate target for achieving this goal in rice (Oryza sativa). Overexpressing AGO2 led to a simultaneous increase in salt tolerance and grain length. These benefits were achieved via the activation of BIG GRAIN3 (BG3), encoding a purine permease potentially involved in cytokinin transport. AGO2 can become enriched on the BG3 locus and alter its histone methylation level, thus promoting BG3 expression. Cytokinin levels decreased in shoots but increased in roots of AGO2-overexpressing plants. While bg3 knockout mutants were hypersensitive to salt stress, plants overexpressing BG3 showed strong salt tolerance and large grains. The knockout of BG3 significantly reduced grain length and salt tolerance in AGO2-overexpressing plants. Both genes were transcriptionally suppressed by salt treatment. Salt treatment markedly increased cytokinin levels in roots but decreased them in shoots, resulting in a hormone distribution pattern similar to that in AGO2-overexpressing plants. These findings highlight the critical roles of the spatial distribution of cytokinins in both stress responses and grain development. Therefore, optimizing cytokinin distribution represents a promising strategy for improving both grain yield and stress tolerance in rice.

Extraction and preparation of 5-lipoxygenase and acetylcholinesterase inhibitors from Astragalus membranaceus stems and leaves.

In this study, an efficient method that employs 5-lipoxygenase and acetylcholinesterase as biological target molecules in receptor-ligand affinity ultrafiltration-liquid chromatography was developed for the screening of enzyme inhibitors derived from the Astragalus membranaceus stems and leaves. The effects of the extraction time, number of extraction cycles, ethanol concentration, and liquid-solid ratio on the total yield of the target compounds were investigated using response surface methodology, and the bioactive components were isolated using a combination of semi-preparative high-performance liquid chromatography and high-speed countercurrent chromatography via a two-phase solvent system consisting of n-hexane-ethyl acetate-methanol-water (1:6:2:6, v/v/v/v). Subsequently, 10 naturally-occurring bioactive components in the Astragalus membranaceus stems and leaves, including wogonin, ononin, isoquercitrin, calycosin-7-glucoside, 3-hydroxy-9,10-dimethoxyptercarpan, hyperoside, 7,2'-dihydroxy-3',4'-dimethoxyisoflavan, baicalein, calycosin, and soyasaponin, were screened using affinity ultrafiltration to determine their potential effects against Alzheimer's disease. Consequently, all target compounds had purities higher than 95.0%, and the potential anti-Alzheimer's disease effect of the obtained bioactive compounds was verified using molecular docking analysis. Based on the results, the back-to-back screening of complex enzyme inhibitors and separation of the target bioactive compounds using complex chromatography could provide a new approach to the discovery and preparation of natural active ingredients.

In-depth analysis of the xanthine oxidase inhibitors of Cicer arietinum L.-based receptor-ligand affinity coupled with complex chromatography.

INTRODUCTION: Cicer arietinum L. is the choice of health food for people with diabetes, hypertension, and hyperlipidemia. As an essential source of high-nutrition legumes, it is also an important source of dietary isoflavones. OBJECTIVES: In order to improve the preparation efficiency of natural plants, a rapid biological activity screening and preparation of xanthine oxidase inhibitors from C. arietinum L. was established. METHODS: Xanthine oxidase (XOD) inhibitors were rapidly screened using ultrafiltration liquid chromatography-mass spectrometry (UF-LC-MS) based on receptor-ligand affinity. The change in XOD activity was evaluated by enzymatic reaction kinetics measurement. The potential bioactive compounds were verified through molecular docking. In addition, the biological activity of ligands screened was separated and purified by complex chromatography. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Three active ingredients, namely daidzin, daidzein, calycosin with XOD binding affinities were identified and isolated from the raw plant materials via semi-preparative high-performance liquid chromatography (HPLC), 0-60 min, 5-50% B and countercurrent chromatography (CCC) (ethyl acetate/acetic acid/water [5:0.8:10, v/v/v]). CONCLUSION: This study will help to elucidate the mechanisms of action of natural plants of interest at the molecular level and could also provide more opportunities for the discovery and development of new nutritional value from other natural resources.

Modeling and optimization of the protocol of complex chromatography separation of cyclooxygenase-2 inhibitors from Ganoderma lucidum spore.

INTRODUCTION: The spores of the medicinal fungus Ganoderma lucidum possess hepatoprotective properties. The main components, triterpenes, are particularly beneficial, making the screening and preparation of active triterpenes from Ganoderma lucidum significant. OBJECTIVES: We aimed to screen and verify cyclooxygenase-2 inhibitors from G. lucidum spores, establish a rapid online hyphenated technique for the preparation of active ingredients, and analyze the structures of the active ingredients. METHODS: Ultrafiltration LC combined with an enzyme inhibition assay and molecular docking was employed to screen and evaluate cyclooxygenase-2 ligands, which were prepared by pressurized liquid extraction coupled online with countercurrent chromatography and semi-preparative LC. The structures of the compounds were identified by nuclear magnetic resonance spectroscopy. RESULTS: Six cyclooxygenase-2 inhibitors, namely, ganoderic acids I, C2 , G, B, and A and ganoderenic acid A, were screened and evaluated. They were prepared using the online hyphenated technique, following which their structures were identified. CONCLUSION: This study provides opportunities for the discovery and development of new therapeutic drugs from other natural resources, as the present instrumental setup achieved efficient and systematic extraction and isolation of natural products compared with reference separation methods, thus exhibiting significant potential for industrial applications.

TMT and PRM Based Quantitative Proteomics to Explore the Protective Role and Mechanism of Iristectorin B in Stroke.

Stroke is a serious disease caused by the rupture or blockage of the cerebrovascular system. Its pathogenesis is complex and involves multiple mechanisms. Iristectorin B is a natural isoflavone that has certain anti stroke effects. In this study, an in vitro stroke injury model of glyoxylate deprivation was established using PC12 cells, which was used to evaluate the anti-stroke activity of Iristectorin B in ejecta stem. The results showed that Iristectorin B, a natural isoflavone derived from Dried Shoot, significantly reduced the damage to PC12 cells caused by oxygen glucose deprivation/reoxygenation, decreased apoptosis, enhanced cell survival and reduced Ca2+, LDH and ROS levels. The results showed that Iristectorin B had a significant protective effect on Na2S2O4-injured PC12 cells, and the mechanism may be related to the protective effect of neurons in the brain. After protein extraction and various analyses were performed, a series of cutting-edge technologies were organically combined to study the quantitative proteome of each group. Differential proteins were then analyzed. According to the protein screening principle, ferroptosis-related proteins were most closely associated with stroke. The differential proteins associated with ferroptosis screened were SLC3A2, TFR1 and HMOX1, with HMOX1 being the most significantly elevated and reduced via dosing. Iristectorin B may act as a protective agent against stroke by regulating ferroptosis, and SLC3A2, TFR1 and HMOX1 may serve as potential diagnostic biomarkers for stroke, providing additional evidence to support the importance of ferroptosis in stroke.

Efficient Combination of Complex Chromatography, Molecular Docking and Enzyme Kinetics for Exploration of Acetylcholinesterase Inhibitors from Poria cocos.

Poria cocos (P. cocos) is a traditional Chinese medicinal product with the same origin as medicine and food. It has diuretic, anti-inflammatory and liver protection properties, and has been widely used in a Chinese medicine in the treatment of Alzheimer's disease (AD). This study was conducted to explore the activity screening, isolation of acetylcholinesterase inhibitors (AChEIs), and in vitro inhibiting effect of P. cocos. The aim was to develop a new extraction process optimization method based on the Matlab genetic algorithm combined with a traditional orthogonal experiment. Moreover, bio-affinity ultrafiltration combined with molecular docking was used to screen and evaluate the activity of the AChEIs, which were subsequently isolated and purified using high-speed counter-current chromatography (HSCCC) and semi-preparative high-performance liquid chromatography (semi-preparative HPLC). The change in acetylcholinesterase (AChE) activity was tested using an enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on AChE and explore its mechanism of action. Five potential AChEIs were screened via bio-affinity ultrafiltration. Molecular docking results showed that they had good binding affinity for the active site of AChE. Meanwhile, the five active compounds had reversible inhibitory effects on AChE: Polyporenic acid C and Tumulosic acid were non-competitive inhibitors; 3-Epidehydrotumulosic acid was a mixed inhibitor; and Pachymic acid and Dehydrotrametenolic acid were competitive inhibitors. This study provided a basis for the comprehensive utilization of P. cocos and drug development for the treatment of AD.