"On/off"-switchable crosslinked PTX-nanoformulation with improved precise delivery for NSCLC brain metastases and restrained adverse reaction over nab-PTX.

Non-small cell lung cancer (NSCLC) brain metastases present a significant treatment challenge due to limited drug delivery efficiency and severe adverse reactions. In this study, we address these challenges by designing a "on/off" switchable crosslinked paclitaxel (PTX) nanocarrier, BPM-PD, with novel ultra-pH-sensitive linkages (pH 6.8 to 6.5). BPM-PD demonstrates a distinct "on/off" switchable release of the anti-cancer drug paclitaxel (PTX) in response to the acidic extratumoral microenvironment. The "off" state of BPM-PD@PTX effectively prevents premature drug release in the blood circulation, blood-brain barrier (BBB)/blood-tumor barrier (BTB), and normal brain tissue, surpassing the clinical PTX-nanoformulation (nab-PTX). Meanwhile, the "on" state facilitates precise delivery to NSCLC brain metastases cells. Compared to nab-PTX, BPM-PD@PTX demonstrates improved therapeutic efficacy with a reduced tumor area (only 14.6%) and extended survival duration, while mitigating adverse reactions (over 83.7%) in aspartate aminotransferase (AST) and alanine aminotransferase (ALT), offering a promising approach for the treatment of NSCLC brain metastases. The precise molecular switch also helped to increase the PTX maximum tolerated dose from 25 mg/kg to 45 mg/kg This research contributes to the field of cancer therapeutics and has significant implications for improving the clinical outcomes of NSCLC patients.

Establishment of cluster of differentiation 20 immobilized cell membrane chromatography for the screening of active antitumor components in traditional Chinese medicine.

Non-Hodgkin lymphoma (NHL) is a heterogeneous group of malignant tumors occurring in B or T lymphocytes, and no small molecule-positive drugs to treat NHL have been marketed. Cluster of differentiation 20 (CD20) is an important molecule regulating signaling for the life and differentiation of B lymphocytes and possesses the characteristics of a drug target for treating NHL. 2-Methoxyestradiol induces apoptosis in lymphoma Raji cells and CD20 protein is highly expressed by Raji lymphoma cells. Therefore, in this study, a CD20-SNAP-tag/CMC model was developed to validate the interaction of 2-methoxyestradiol with CD20. 2-Methoxyestradiol was used as a small molecule control compound, and the system was validated for good applicability. The cell membrane chromatography model was combined with high-performance liquid chromatography ion trap time-of-flight mass spectroscopy (HPLC-IT-TOF-MS) in a two-dimensional system to successfully identify, analyze, and characterize the potential active compounds of Schisandra chinensis (Turcz.) Baill. extract and Lysionotus pauciflorus Maxim. extract, including Schisandrin A, Schizandrol A, Schizandrol B, Schisantherin B, and Nevadensin, which can act on CD20 receptors. The five potential active compounds were analyzed by non-linear chromatography. The thermodynamic and kinetic parameters of their interaction with CD20 were also analyzed, and the mode of interaction was simulated by molecular docking. Their inhibitory effects on lymphoma cell growth were assessed using a Cell Counting Kit-8 (CCK-8). Nevadensin and Schizandrin A were able to induce apoptosis in Raji cells within a certain concentration range. In conclusion, the present experiments provide some bases for improving NHL treatment and developing small molecule lead compounds targeting CD20 with low toxicity and high specificity.

Revealing receptor-ligand interactions of atypical antipsychotic drugs and screening anti-schizophrenia ingredients in Magnolia officinalis based on 5-HTR2A-SNAP-Tag/CMC and DRD2-SNAP-Tag/CMC models.

Schizophrenia is a serious mental illness with unknown etiology, and shows increasing incidence and high lifetime prevalence rate. The main receptors related to the disease are DRD2 and 5-HTR2A. Thus, a comprehensive understanding of the interaction mode between antipsychotic drugs with relevant receptors is very important for developing more effective drugs. 5-HTR2A-SNAP-Tag/CMC and DRD2-SNAP-Tag/CMC models constructed in this work provided a new method for studying the interaction between atypical antipsychotics and the two receptors. The results of comparative experiments showed that the new models not only met the high selectivity and specificity of the screening requirements but were also more stable and long-lasting than the traditional CMC model. Binding assays showed that the effects of three atypical antipsychotics (including clozapine, olanzapine, and quetiapine) on 5-HTR2A were stronger than their effects on DRD2. Additionally, two potentially active components, magnolol and honokiol, were screened in Magnolia officinalis methanol extract using the 5-HTR2A-SNAP-Tag/CMCHPLC-MS system. Nonlinear chromatographic analysis and molecular docking were conducted to study the interactions between screened compounds and the two receptors. The binding constants (KA) of magnolol and honokiol with 5-HTR2A were 17,854 ± 1,117 M-1 and 38,858 ± 4,964 M-1, respectively, and KA values with DRD2 were 4,872 ± 1,618 M-1 and 20,692 ± 10,267 M-1, respectively. We concluded that the established models are reliable for studying receptor-ligand interactions and screening antagonists of schizophrenia.

KIT-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-GIST compound screening from Evodia rutaecarpa.

Gastrointestinal mesenchymal tumors, as the most common mesenchymal tumors in the gastrointestinal tract, are adjuvantly treated with multi-targeted tyrosine kinase inhibitors, such as imatinib and sunitinib, but there are problems of drug resistance and complex methods of monitoring therapeutic agents. The pathogenesis of this disease is related to mutations in tyrosine kinase (KIT) or platelet-derived growth factor receptor α, an important target for drug therapy. In recent years, the screening of relevant tyrosine kinase inhibitors from traditional Chinese medicine has become a hotspot in antitumor drug research. In the current study, the KIT-SNAP-tag cell membrane chromatography (KIT-SNAP-tag/CMC) column was prepared with satisfying specificity, selectivity, and reproducibility by chemically bonding high KIT expression cell membranes to the silica gel surface using the SNAP-tag technology. The KIT-SNAP-tag/CMC-HPLC-MS two-dimensional coupling system was investigated using the positive drug imatinib, and the results showed that the system was a reliable model for screening potential antitumor compounds from complex systems. This system screened and identified three potential active compounds of evodiamine (EVO), rutaecarpin (RUT), and dehydroevodiamine (DEVO), which possibly target the KIT receptor, from the alcoholic extract of the traditional Chinese medicine Evodia rutaecarpa. Then, the KD values of the interaction of EVO, RUT, and DEVO with KIT receptors measured using nonlinear chromatography were 7.75 (±4.93) × 10-6, 1.42 (±0.71) × 10-6, and 2.34 (±1.86) × 10-6 mol/L, respectively. In addition, the methyl thiazolyl tetrazolium assay validated the active effects of EVO and RUT in inhibiting the proliferation of high KIT-expressing cells in the ranges of 0.1-10 µmol/L and 0.1-50 µmol/L, respectively. In conclusion, the KIT-SNAP-tag/CMC could be a reliable model for screening antitumor components from complex systems.

Interferon-regulatory factor-1 boosts bevacizumab cardiotoxicity by the vascular endothelial growth factor A/14-3-3γ axis.

AIM: Myocardial injury is a significant cause of death. This study investigated the role and underlying mechanism of interferon-regulatory factor-1 (IRF1) in bevacizumab (BVZ)-induced cardiomyocyte injury. METHODS AND RESULTS: HL-1 cells and C57BL/6 mice receiving BVZ treatment were used to establish in vitro and in vivo models of myocardial injury. The relationship between VEGFA and 14-3-3γ was verified through co-immunoprecipitation and Glutathione S Transferase (GST) pull-down assay. Cell viability and apoptosis were analysed by MTT, propidium iodide (PI) staining and flow cytometry. The release of lactate dehydrogenase (LDH), cardiac troponins T (cTnT), and creatine kinase MB (CK-MB) was measured using the enzyme linked immunosorbent assay. The effects of knocking down IRF1 on BVZ-induced mice were analysed in vivo. IRF1 levels were increased in BVZ-treated HL-1 cells. BVZ treatment induced apoptosis, inhibited cell viability, and promoted the release of LDH, cTnT, and CK-MB. IRF1 silencing suppressed BVZ-induced myocardial injury, whereas IRF1 overexpression had the opposite effect. IRF1 regulated VEGFA expression by binding to its promoter, with the depletion of VEGFA or 14-3-3γ reversing the effects of IRF1 knockdown on the cell viability and apoptosis of BVZ-treated HL-1 cells. 14-3-3γ overexpression promoted cell proliferation, inhibited apoptosis, and reduced the release of LDH, cTnT, and CK-MB, thereby alleviating BVZ-induced HL-1 cell damage. In vivo, IRF1 silencing alleviated BVZ-induced cardiomyocyte injury by regulating the VEGFA/14-3-3γ axis. CONCLUSION: The IRF1-mediated VEGFA/14-3-3γ signalling pathway promotes BVZ-induced myocardial injury. Our study provides evidence for potentially new target genes for the treatment of myocardial injury.

Application of SNAP-tag-EGFR cell membrane chromatography model in screening antitumor active components of Silybum marianum (L.) Gaertn.

The SNAP-tag-epidermal growth factor receptor (SNAP-tag-EGFR) cell membrane chromatography (CMC) model is a powerful tool for investigating ligand-receptor interactions and screening active ingredients in traditional Chinese medicine. Most tyrosine kinase inhibitors (TKIs) target epidermal growth factor receptors. However, TKIs associated with significant side effects and drug resistance must be addressed immediately. Therefore, there is an urgent need to develop new TKIs with high efficiency and low toxicity. Because of its low toxicity and side effects, traditional Chinese medicine has been widely employed to treat various diseases, including cancer. Hence, this study aimed to use the SNAP-tag-EGFR/CMC-high-performance liquid chromatography-mass spectrometry (HPLC-MS) two-dimensional system model as the research tool to screen and identify potential EGFR antagonists from the Chinese medicine Silybum marianum (L.) Gaertn. The applicability of the system was verified using the positive control drug osimertinib. Four potential EGFR antagonists were screened from the Chinese medicine Silybum marianum (L.) Gaertn.. They were identified as silydianin, silychristin, silybin, and isosilybin. Additionally, their pharmacological activity was preliminarily verified using a CCK-8 assay. The kinetic parameters of the four active ingredients interacting with EGFR and their binding modes with EGFR were analyzed using nonlinear chromatography (NLC) and molecular docking. This study identified silydianin, silychristin, silybin, and isosilybin from Silybum marianum (L.) Gaertn. and verified their potential antitumor effects on EGFR.

Single-cell RNA sequencing unveils Lrg1's role in cerebral ischemia‒reperfusion injury by modulating various cells.

BACKGROUND AND PURPOSE: Cerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown. METHODS: Wild-type (WT) and Lrg1 knockout (Lrg1-/-) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout. RESULTS: Lrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1-/- mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin ß5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death. CONCLUSIONS: Our results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

Background and roles: myosin in autoimmune diseases.

The myosin superfamily is a group of molecular motors. Autoimmune diseases are characterized by dysregulation or deficiency of the immune tolerance mechanism, resulting in an immune response to the human body itself. The link between myosin and autoimmune diseases is much more complex than scientists had hoped. Myosin itself immunization can induce experimental autoimmune diseases of animals, and myosins were abnormally expressed in a number of autoimmune diseases. Additionally, myosin takes part in the pathological process of multiple sclerosis, Alzheimer's disease, Parkinson's disease, autoimmune myocarditis, myositis, hemopathy, inclusion body diseases, etc. However, research on myosin and its involvement in the occurrence and development of diseases is still in its infancy, and the underlying pathological mechanisms are not well understood. We can reasonably predict that myosin might play a role in new treatments of autoimmune diseases.

Developing neural network diagnostic models and potential drugs based on novel identified immune-related biomarkers for celiac disease.

BACKGROUND: As one of the most common intestinal inflammatory diseases, celiac disease (CD) is typically characterized by an autoimmune disorder resulting from ingesting gluten proteins. Although the incidence and prevalence of CD have increased over time, the diagnostic methods and treatment options are still limited. Therefore, it is urgent to investigate the potential biomarkers and targeted drugs for CD. METHODS: Gene expression data was downloaded from GEO datasets. Differential gene expression analysis was performed to identify the dysregulated immune-related genes. Multiple machine algorithms, including randomForest, SVM-RFE, and LASSO, were used to select the hub immune-related genes (HIGs). The immune-related genes score (IG score) and artificial neural network (ANN) were constructed based on HIGs. Potential drugs targeting HIGs were identified by using the Enrichr platform and molecular docking method. RESULTS: We identified the dysregulated immune-related genes at a genome-wide level and demonstrated their roles in CD-related immune pathways. The hub genes (MR1, CCL25, and TNFSF13B) were further screened by integrating several machine algorithms. Meanwhile, the CD patients were divided into distinct subtypes with either high- or low-immunoactivity using single-sample gene set enrichment analysis (ssGSEA) and consensus clustering. By constructing IG score based on HIGs, we found that patients with high IG score were mainly attributed to high-immunoactivity subgroups, which suggested a strong link between HIGs and immunoactivity of CD patients. In addition, the novel constructed ANN model showed the sound diagnostic ability of HIGs. Mechanistically, we validated that the HIGs play pivotal roles in regulating CD's immune and inflammatory state. Through targeting the HIGs, we also found potential drugs for anti-CD treatment by using the Enrichr platform and molecular docking method. CONCLUSIONS: This study unveils the HIGs and elucidates the networks regulated by these genes in the context of CD. It underscores the pivotal significance of HIGs in accurately predicting the presence or absence of CD in patients. Consequently, this research offers promising prospects for the development of diagnostic biomarkers and therapeutic targets for CD.

Harringtonine: A more effective antagonist for Omicron variant.

Fusion with host cell membrane is the main mechanism of infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we propose that a new strategy to screen small-molecule antagonists blocking SARS-CoV-2 membrane fusion. Using cell membrane chromatography (CMC), we found that harringtonine (HT) simultaneously targeted SARS-CoV-2 S protein and host cell surface TMPRSS2 expressed by the host cell, and subsequently confirmed that HT can inhibit membrane fusion. HT effectively blocked SARS-CoV-2 original strain entry with the IC50 of 0.217 µM, while the IC50 in delta variant decreased to 0.101 µM, the IC50 in Omicron BA.1 variant was 0.042 µM. Due to high transmissibility and immune escape, Omicron subvariant BA.5 has become the dominant strain of the SARS-CoV-2 virus and led to escalating COVID-19 cases, however, against BA.5, HT showed a surprising effectiveness. The IC50 in Omicron BA.5 was even lower than 0.0019 µM. The above results revealed the effect of HT on Omicron is very significant. In summary, we characterize HT as a small-molecule antagonist by direct targeting on the Spike protein and TMPRSS2.

Integrated effect of bulk cations on nano-confined reactivity of clay-intercalated subnanoscale zero-valent iron in water-tetrahydrofuran mixtures.

Smectite clay-intercalated subnanoscale zero-valent iron (CSZVI) exhibits superior reactivity toward contaminants due to the small iron clusters (∼0.5 nm) under nano-confinement, which however is significantly influenced by the solution chemistry e.g., various cations, of polluted soil and water. This work was undertaken to elucidate the mechanisms of solution chemistry effects on dehalogenation ability of CSZVI in water-tetrahydrofuran solution using decabromodiphenyl ether as a model contaminant. By combined spectroscopic characterization and molecular dynamics simulation, it was revealed that bulk cations, i.e., Na+, K+, Mg2+ and Ca2+ collectively affected the interlayer distance, water content and Brønsted acidity of CSZVI and thus its degradation efficiency. Although causing inter-particle aggregation, Mg2+ induced optimal nano-confined interlayers at concentration of 20 mM, exhibiting a superior debromination efficiency with rate constant 9.84 times larger than that by the common nano-sized ZVI. Conversely, K+ rendered the interlayers less reactive, but protected CSZVI from corrosion loss with higher electron utilization efficiency, which was 1.7 times higher than CSZVI in presence of Mg2+. The findings provide new strategies to manipulate the reactivity of nano-confined CSZVI for effective wastewater and contaminated soil remediation.

Reaction of decabromodiphenyl ether with organo-modified clay-templated zero-valent iron in water-tetrahydrofuran solution: Nano- to micrometric scale effects.

Smectite clay-templated nanoscale zero-valent iron (CZVI) was modified with tetramethylammonium (TMA), trimethylphenylammonium (TMPA) and hexadecyltrimethylammonium (HDTMA) to achieve organoclay-templated ZVI (OCZVI). The reactivity of various OCZVIs was evaluated on the basis of degradation of decabromodiphenyl ether (DBDE) in tetrahydrofuran (THF)-water binary solution. Characterization of OCZVI interlayer at nanometric scale indicated that the clay particles had the domains with three basal spacings in the THF/water solution. In the 50 % THF solution TMPA modification promoted the formation of the domains with a basal spacing at 1.56 nm, which could promote the degradation of DBDE. At the micrometric scale, in the 90 % THF solution TMA and TMPA modification tended to enhance the aggregation of OCZVI particles, while the HDTMA modification reduced the aggregation, and high percentage of modification yielded viscous gel structures. The relatively rapid sedimentation processes in 90 % THF solution (compared to that in 50 % THF solution) and formation gel structures could reduce the access of DBDE to the interlayer reactive nZVIs, and lead to the significant reduction in reaction rate. These results provide important insights to the organo-modification on clays which could alter their orientations and dispersion in organic-water binary solution to achieve the desired reactivity on confined clay surfaces.

Establishment of angiotensin-converting enzyme 2 and cluster of differentiation 147 dual target cell membrane chromatography based on SNAP-tag technology for screening anti severe acute respiratory syndrome coronavirus 2 active components.

Patients have different responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and these may be life-threatening for critically ill patients. Screening components that act on host cell receptors, especially multi-receptor components, is challenging. The in-line combination of dual-targeted cell membrane chromatography and a liquid chromatography-mass spectroscopy (LC-MS) system for analyzing angiotensin-converting enzyme 2 (ACE2) and cluster of differentiation 147 (CD147) receptors based on SNAP-tag technology provides a comprehensive solution for screening multiple components in complex samples acting on the two receptors. The selectivity and applicability of the system were validated with encouraging results. Under the optimized conditions, this method was used to screen for antiviral components in Citrus aurantium extracts. The results showed that 25 µmol /L of the active ingredient could inhibit virus entry into cells. Hesperidin, neohesperidin, nobiletin, and tangeretin were identified as antiviral components. In vitro pseudovirus assays and macromolecular cell membrane chromatography further verified the interaction of these four components with host-virus receptors, showing good effects on some or all of the pseudoviruses and host receptors. In conclusion, the in-line dual-targeted cell membrane chromatography LC-MS system developed in this study can be used for the comprehensive screening of antiviral components in complex samples. It also provides new insight into small-molecule drug-receptor and macromolecular-protein-receptor interactions.

Construction and application of covalently bonded CD147 cell membrane chromatography model based on polystyrene microspheres.

In this study, a novel cell membrane chromatography (CMC) model was developed to investigate cluster of differentiation 147 (CD147) targeted anti-tumor drug leads for specific screening and ligand-receptor interaction analysis by SNAP-tagged CD147 fusion protein conjugation and polystyrene microspheres (PS) modification. Traditional Chinese medicines (TCMs) are widely used in the treatment of cancer. CD147 plays important roles in tumor progression and acts as an attractive target for therapeutic intervention; therapeutic drugs for CD147-related cancers are limited to date. Thus, a screening method for active components in TCMs is crucial for the further research and development of CD147 antagonists. However, improvement is still needed to perform specific and accurate drug lead screening using the CMC-based method. Recently, our group developed a covalently immobilized receptor-SNAP-tag/CMC model using silica gel as carrier. Besides the carboxyl group on multi-step modified silica particles, the amino group of benzyl-guanine (BG, substrate of SNAP-tag) also possesses reactivity towards the carboxyl group on available carboxyl-modified PS. Herein, we used PS as carrier and an extended SNAP-tag with CD147 receptor to construct the PS-BG-CD147/CMC model for active compound investigation coupled with HPLC/MS and applied this coupled PS-BG-CD147/CMC-HPLC/MS two-dimensional system to drug lead screening from Nelumbinis Plumula extract (NPE) sample. In addition, to comprehensively verify the pharmacological effects of screened ingredients, a cell proliferation inhibition assay was performed, and the interaction between the ingredients and CD147 was studied by the frontal analysis method. This study developed a high-throughput PS-based CMC screening platform, which could be widely applied and utilized in chromatographic separation and drug lead discovery.

Tamoxifen Regulates Epithelial-Mesenchymal Transition in Endometrial Cancer via the CANP10/NRP1 Signaling Pathway.

Tamoxifen, which is used to treat advanced gynecological tumors, has been associated with tumor cell metastasis. Herein, we investigated the effect of tamoxifen on epithelial-mesenchymal transition in endometrial cancer and the associated signaling mechanism. Wound healing and invasion chamber assays, respectively, were performed to determine the migrative capacity and invasiveness of tamoxifen-stimulated endometrial carcinoma (RL95-2) cells. Western blotting and immunofluorescence were used to evaluate the expression of vimentin, E-cadherin, calpain 10 (CANP10), and neuropilin-1 (NRP1). Transfection of a CAPN10-harboring plasmid was used to overexpress CANP10 in RL95-2 cells, and small interfering RNAs were used to silence CANP10 and NRP1 expression. Tamoxifen induced migration, invasion, and morphological changes in RL95-2 cells. It also downregulated E-cadherin expression and upregulated vimentin, CANP10, and NRP1 expression. CANP10 silencing inhibited tamoxifen-induced NRP1 upregulation, and CANP10 or NRP1 silencing inhibited the migration and invasion of RL95-2 cells. CANP10 overexpression upregulated vimentin expression and downregulated that of E-cadherin and also increased cell migration and invasion. Silencing NRP1 protein expression inhibited the induction effect of CANP10 overexpression. In conclusion, tamoxifen promotes the epithelial-mesenchymal transition of RL95-2 cells via the CANP10/NRP1 signaling pathway. Thus, targeting CANP10 or NRP1 may be a novel strategy for preventing tamoxifen-induced endometrial cancer metastasis.

Aneuploidy promotes intraspecific diversification of the endemic East Asian herb Lycoris aurea complex.

Polyploidy has received considerable interest in the past, but aneuploidy and partial rearrangements may also influence genomic divergence. In this study, we reported a comprehensive cytogeographic, morphological and genetic analysis of Lycoris aurea complex throughout its range and attempted to explore the association between aneuploidy and species diversification. The karyotypes of this complex presented aneuploidy variations mainly divided into four cytotypes: I (2n = 10m + 2T), II (2n = 8m + 6T), III (2n = 7m + 8T), and IV (2n = 6m + 10T). Cytotype distributions were highly structured geographically. Two main cytotypes, II and IV, are geographically allopatric. The populations with cytotype II are mainly distributed in central China and the southern islands of Japan. Cytotypes IV is disjunctly distributed in southwestern and southeastern China. The cytotypes with fewer chromosome numbers tend to occur at high latitudes. For analyzing the phylogeographic pattern and genetic structure of this complex, we sequenced four chloroplast DNA fragments (4,748 bp in total) of 241 individuals from 42 populations. Extremely high diversity of cpDNA haplotypes was found, with genetic diversity index (H d) being 0.932 and 98.61% of the genetic variation occurring among populations, indicating that this complex has undergone strong intraspecific differentiation. The cytotype II had the highest haplotype diversity (H d = 0.885), while cytotype IV harbored the highest nucleotide diversity (π = 4.09 × 10-3). We detected significant leaf morphological differences not only between cytotype II and IV but also between west lineage and east lineage within cytotype IV. These results illustrated that aneuploidy contributed to extensive morphological and genetic differentiation in L. aurea complex. It was suggested that L. aurea complex should comprise multiple independent evolutionary lineages, and accurate species delimitation needs to be established further in an integrative taxonomic approach.

Quantitative Analysis and Structural Elucidation of Fatty Acids by Isobaric Multiplex Labeling Reagents for Carbonyl-Containing Compound (SUGAR) Tags and m-CPBA Epoxidation.

In this study, a novel analytical method was developed to investigate fatty acids (FAs) for relative quantification, carbon-carbon double-bond localization, and cis-/trans-geometry differentiation by isobaric multiplex labeling reagents for carbonyl-containing compound (SUGAR) tag conjugation and meta-chloroperoxybenzoic acid (m-CPBA) epoxidation. FAs are essential components of cells and have diverse functions in energy storage and as complex lipid constituents. It has been reported that FAs play different roles in various biological processes such as the functional development of the brain. The comprehensive characterization and quantification of FAs are crucial to further elucidate their biological roles. However, it is challenging to perform relative quantification and structural elucidation of FAs using integrated mass spectrometry (MS)-based methods. Recently, our group developed isobaric multiplex SUGAR tags for quantitative glycomics. Besides aldehyde/ketone groups on glycans, hydrazide groups also possess reactivity toward carboxylic acids on FAs. In this study, we extended SUGAR tag labeling with FAs for the quantitative analysis by liquid chromatography (LC)-MS/MS in the positive ion mode and applied this strategy for the comparative analysis of FAs hydrolyzed from oil samples. In addition, to comprehensively elucidate the structures of unsaturated FAs, epoxidation by m-CPBA was performed before SUGAR tag labeling to enable carbon-carbon double-bond localization. Moreover, the cis- and trans-geometries of carbon-carbon double bonds in multiple pairs of monounsaturated FAs could also be differentiated in higher-energy collisional dissociation (HCD)-MS/MS. This study developed a high-throughput comprehensive FA analysis platform, which could be widely applied and utilized in biological and clinical studies.

MrgX2-SNAP-tag/cell membrane chromatography model coupled with liquid chromatography-mass spectrometry for anti-pseudo-allergic compound screening in Arnebiae Radix.

Pseudo-allergic reactions (PARs) are IgE-independent hypersensitivity reactions. Mas-related G protein-coupled receptor-X2 (MrgX2) was proved the key receptor of PAR. The anti-pseudo-allergic compound discovery based on MrgX2 was of great value. Cell membrane chromatography (CMC) based on MrgX2 provides a convenient and effective tool in anti-pseudo-allergic compound screening and discovery, and further improvements of this method are still needed. In this work, SNAP-tag was introduced at C-terminal of Mas-related G protein-coupled receptor (MrgX2-SNAP-tag), and an MrgX2-SNAP-tag/CMC model was then conducted using CMC technique. Comparative experiments showed that the new model not only satisfied the good selectivity and specificity of screening but also exhibited more stable and longer life span than traditional MrgX2/CMC model. By coupling with HPLC-MS, two compounds were screened out from Arnebiae Radix and identified as shikonin and acetylshikonin. Nonlinear chromatography was performed to study the interactions between two screened compounds and MrgX2, and binding constant (KA) of shikonin and acetylshikonin with MrgX2 were 2075.67 ± 0.34 M-1 and 32201.36 ± 0.35 M-1, respectively. Furthermore, ß-hexosaminidase and histamine release assay in vitro demonstrated that shikonin (1-5 µM) and acetylshikonin (2.5-10 µM) could both antagonize C48/80-induced allergic reaction. In conclusion, the MrgX2-SNAP-tag/CMC could be a reliable model for screening pseudo-allergy-related components from complex systems.

Purification and determination of antibody drugs in bio-samples by EGFR/cell membrane chromatography method.

With the rapid development of therapeutic monoclonal antibody drugs, it is increasingly difficult to meet clinical needs using traditional antibody purification techniques. In this study, epidermal growth factor receptor (EGFR)-SNAP-tag was expressed in HEK293 cells. Then we captured the EGFR-SNAP-tag from the cell membrane suspension onto a O6-benzylguanine-modified silica gel to prepare a new EGFR stationary phase as a bioactive material, which could specifically recognize its antibody in bio-samples. The EGFR stationary phase was systematically characterized via scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, and fourier transform infrared spectroscopy. Then we used EGFR stationary phase to establish a new EGFR cell membrane chromatography (CMC) model. The EGFR/CMC-online-ion exchange chromatography (IEC)/high performance liquid chromatography (HPLC) was established for the efficient purification and specific identification of cetuximab, nituzumab, and panizumab from cell culture medium and human serum. The results show that the EGFR stationary phase prepared by one-step immobilized technique can maintain biological activity and stability like EGFR in cell membrane. The EGFR/CMC-online-IEC/HPLC method has a high specificity, accuracy and sensitivity. Therefore, it will present a valuable method for the purification, identification, and analysis of monoclonal antibody drugs.

Enhanced stability designs of cell membrane chromatography for screening drug leads.

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.