Regulation of post-translational modification of PD-L1 and associated opportunities for novel small-molecule therapeutics.

PD-L1 is overexpressed on the surface of tumor cells and binds to PD-1, resulting in tumor immune escape. Therapeutic strategies to target the PD-1/PD-L1 pathway involve blocking the binding. Immune checkpoint inhibitors have limited efficacy against tumors because PD-L1 is also present in the cytoplasm. PD-L1 of post-translational modifications (PTMs) have uncovered numerous mechanisms contributing to carcinogenesis and have identified potential therapeutic targets. Therefore, small molecule inhibitors can block crucial carcinogenic signaling pathways, making them a potential therapeutic option. To better develop small molecule inhibitors, we have summarized the PTMs of PD-L1. This review discusses the regulatory mechanisms of small molecule inhibitors in carcinogenesis and explore their potential applications, proposing a novel approach for tumor immunotherapy based on PD-L1 PTM.

[Box: see text].

A novel halloysite nanotubes-based hybrid monolith for in-tube solid-phase microextraction of polar cationic pesticides.

The accurate determination of polar cationic pesticides in food poses a challenge due to their high polarity and trace levels in complex matrices. This study hypothesized that the use of halloysite nanotubes (HNTs) can significantly enhance the extraction efficiency and sensitivity of these analytes because of their rich hydroxyl groups and cation exchange sites. Therefore, we chemically incorporated HNTs with organic polymer monoliths for in-tube solid-phase microextraction (SPME). This novel hybrid monolith extended service life, improved adsorption capacity, and exhibited excellent extraction performance for polar cationic pesticides. Based on these advancements, a robust and sensitive in-tube SPME-HILIC-MS/MS method was constructed to determine trace levels of polar cationic pesticides in complex food matrices. The method achieved limits of detection of 1.9, 2.1, and 0.1 µg/kg for maleic hydrazide, amitrole, and cyromazine, respectively. The spiked recoveries in five food samples ranged from 80.2 to 100.8%, with relative standard deviations below 10.7%.

[Transformation of scientific research results into teaching resources to improve the teaching effect: taking the teaching of the chromatographic method in the pharmaceutical analysis course as an example].

The pharmaceutical analysis course is a three-dimensional knowledge network that connects several courses to form a new comprehensive knowledge node involving a large knowledge system and flexible knowledge structure. In this course, the subject of chromatography covers a wide range of topics. However, because accurate content is challenging to present, the teaching effect of this subject is poor. In this work, we sought to achieve the educational purpose of establishing morality and cultivating talent, as well as the goal of training highly skilled professionals, by taking the teaching of chromatography in the pharmaceutical analysis course as an example of transforming scientific research results into teaching resources. The resources obtained are integrated into the teaching process to provide innovative and scientific research ideas to students with the aim of not only helping them understand and master technical knowledge but also exercise their ability to raise and solve problems. Furthermore, we expound on how to introduce scientific development frontiers and formulate scientific problems through curriculum design. We also describe how our strategy can promote the teaching effect and achieve teaching objectives. Based on the characteristics of rapid knowledge update and equal emphasis on theory and practice in pharmaceutical analysis, the course is designed by introducing new advances in scientific development, formulating scientific problems, and adopting question- and problem-based learning methods for teaching. The teaching effect is then evaluated through diversified assessment, student feedback, and self-evaluation. The results show that the transformation of scientific research results into teaching resources plays a significant role in stimulating students' interest in learning, improving students' ability to solve problems, and achieving curriculum objectives, all of which greatly improve the teaching effect.

High Throughput and Noninvasive Exosomal PD-L1 Detection for Accurate Immunotherapy Response Prediction via Tim4-Functionalized Magnetic Core-Shell Metal-Organic Frameworks.

Exosomal PD-L1 has been increasingly considered a noninvasive and accurate predictive marker for immunotherapy treatment response. However, the clinical monitoring of exosomal PD-L1 expression is still limited by its complex biological environment as well as the lack of a robust isolation strategy. Here, a Tim4-functionalized magnetic core-shell metal-organic framework (denoted as Fe3O4@SiO2-ILI-01@Tim4) was facilely constructed via layer-by-layer assembly. Owing to the strongly hydrophilic organic ligand of 1,3-bis(4-carboxybutyl)imidazolium bromide (ILI), magnetic Fe3O4@SiO2-ILI-01@Tim4 was endowed with the merits of low nonspecific adsorption and quick, easy, and convenient isolation of exosomes. The capture efficiency of Fe3O4@SiO2-ILI-01@Tim4 reached as high as 90.3 ± 0.5% and the recovery rate for exosomes was up to 93.0 ± 6.1%. The purity of the isolated exosomes was 7.5 times higher than that via the ultracentrifugation (UC) method. By further combination with immunofluorescence assay, high throughput and noninvasive exosomal PD-L1 detection for accurate immunotherapy response prediction was achieved. The prognosis accuracy of the developed Fe3O4@SiO2-ILI-01@Tim4-based strategy reached 85.7%, whereas the prognosis accuracy of the clinical gold standard, the PD-L1 combined positive score (CPS) test, was only 57.1%. Most interestingly, the developed method is especially suitable for those patients receiving false negative results in the CPS test. The proposed Fe3O4@SiO2-ILI-01@Tim4 is a highly efficient and robust technique showing great potential in high throughput and noninvasive exosomal PD-L1 detection for accurately predicting immunotherapy efficacy.

[Modified styrene-maleic anhydride copolymer-based chromatographic stationary phase for phospholipid separation and analysis].

Phospholipids are important signaling molecules, and their metabolism is closely related to various diseases, such as neurodegenerative diseases and cancers. Phospholipids are typically characterized with extreme complexity and structural diversity. For example, phospholipids present in many different forms, such as sn position isomers, double-bond position isomers, double-bond stereochemical isomers, and enantiomers. Therefore, further research on novel separation and analytical techniques for phospholipids is of great importance. As an amphiphilic alternating copolymer, styrene-maleic anhydride copolymer (SMA) can be inserted into the phospholipid bilayer of biofilms to form lipid nanodisks with membrane proteins as the centers, thereby solubilizing membrane proteins and phospholipids. Thus, the introduction of SMA into a chromatographic stationary phase can potentially improve the separation and analysis of phospholipids. In this paper, SMA was successfully grafted onto the surface of silica gel via the "click" reaction and free radical polymerization. After further ring-opening modification of SMA with methyl methionine hydrochloride (MME·HCl), a novel SMA-modified stationary phase material (Sil-SMA-MME) was fabricated. The Sil-SMA-MME stationary phase was characterized using thermogravimetric analysis and Fourier transform infrared spectroscopy (FT-IR), and the results indicated the successful fabrication of the target material. The retention mechanism of the packed Sil-SMA-MME chromatographic column was investigated using hydrophilic nucleosides and nucleic acid bases via high performance liquid chromatography (HPLC) and UV detection. According to the retention characteristics of the nucleosides and nucleic acid bases in different mobile phases, the Sil-SMA-MME chromatographic column exhibited a typical hydrophilic-interaction-based retention mechanism, similar to that of a commercially available amino (SiO2-NH2) column. The separation performance of the Sil-SMA-MME column was evaluated using three types of small-molecule substances, including amides, nucleoside/nucleic acid bases, and phenols. Cyanoacetamide, 2-iodoacetamide, benzamide, p-aminobenzamide, and nicotinamide were used to evaluate the chromatographic performance of the developed Sil-SMA-MME column. When acetonitrile-H2O (96∶4, v/v) was used as the mobile phase, the five compounds exhibited good peak shapes and could be baseline-separated within 8 min. The highest column efficiency achieved was 90900 N/m. By contrast, under the same chromatographic conditions, the test substances were not separated effectively on the SiO2-NH2 column. Regardless of the mobile phase ratio, the peaks of benzamide and 2-iodoacetamide overlapped. These results demonstrate that the developed Sil-SMA-MME column has good separation selectivity. The separation performance of the Sil-SMA-MME column for phospholipid samples was also investigated by HPLC and evaporative light scattering detection (ELSD) to explore its feasibility for phospholipid separation and analysis. Different phospholipid standards were used to evaluate the separation performance of the column. Under certain mobile phase conditions, baseline separation could be achieved for dipalmityl phosphatidyl serine sodium (DPPS), diolyl phosphatidyl choline (DOPC), and dipalmityl phosphatidyl ethanolamine (DPPE), as well as four phosphatidyl choline (PC) standards, namely, lysophosphatidylcholine (LysoPC), dimyristoyl phosphatidyl choline (DMPC), distearyl phosphatidyl choline (DSPC), and dipalmitoyl phosphatidyl choline (DPPC). The separation potential of the developed Sil-SMA-MME column was further evaluated by separating and analyzing phospholipid extracts from Antarctic krill oil and human serum. The results showed that the developed Sil-SMA-MME column has good potential for phospholipid separation and analysis.

Sodium hexametaphosphate-treated halloysite based solid-phase extraction of biguanides from dietary supplements.

Raw halloysite was purified by using sodium hexametaphosphate and utilized as the solid-phase extraction sorbent for the determination of biguanides from dietary supplements. The purified halloysite was characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The purified halloysite interacted with biguanides through hydrophilic interaction and ion exchange on account of its abundant hydroxyl groups and negative charge. Compared with traditional extraction methods based on hydrophobic interaction and/or ion exchange, the purified halloysite adsorbed more biguanides due to hydrophilicity and ion exchange, with a sample loading volume of up to 100 mL at least. Excellent reproducibility of halloysite purification was achieved, with within-batch (n = 3) and batch-to-batch (n = 3) relative standard deviations in the ranges of 1.5-4.2% and 5.6-8.8%, respectively. Coupled with reversed-phase liquid chromatography-tandem mass spectrometry, a low limit of detection of 0.3 µg kg-1 was obtained. The intra- and inter-day mean recoveries of the biguanides spiked at three levels in dietary supplements were within the ranges of 88.5-107.2% and 86.4-102.0%, respectively. The intra- and inter-day precisions were within the ranges of 1.5-6.4% and 5.4-9.9%, respectively. These results indicated that the developed method is efficient for the determination of trace biguanides in dietary supplements.

Design, synthesis and biological activity evaluation of novel covalent S-acylation inhibitors.

In order to obtain diverse S-acylation inhibitors and address the defects of existing S-acylation inhibitors, a series of novel covalent S-acylation inhibitors are designed through synthesis. According to the results of MTT assay, most compounds produce a better anti-proliferation effect on MCF-7, MGC-803 and U937 cell lines than 2-BP. Among them, 8d, 8i, 8j and 10e exert a significant inhibitory effect on MCF-7 cell, with the IC50 values falling below 20 µM. Besides, the toxic effects of some compounds on 3T3 cell line are less significant than 2-BP. According to the results of acyl-biotin exchange (ABE) experiment, most of them could inhibit S-acylation, and 8i performs best in this respect, with the inhibitory rate reaching 89.3% at the concentration of 20 µM. The results of molecular docking show the conjugation of 8i with surrounding amino acids. Additionally, 8i could not only suppress the migration of MCF-7 cell line, but also cause it to stagnate in G0/G1 phase, thus promoting cell apoptosis.

Design, synthesis and biological evaluation of dual Topo II/HDAC inhibitors bearing pyrimido[5,4-b]indole and pyrazolo[3,4-d]pyrimidine motifs.

Both topoisomerase II (Topo II) and histone deacetylase (HDAC) are important therapeutic targets for cancer. In this study, two series of novel compounds containing pyrimido[5,4-b]indole and pyrazolo[3,4-d]pyrimidine motifs were designed and synthesized as dual Topo II/HDAC inhibitors. MTT assay indicated that all the compounds displayed potential antiproliferative activity against three cancer cell lines (MGC-803, MCF-7 and U937) and low cytotoxicity on normal cell line (3T3). In the enzyme activity inhibition experiments, compounds 7d and 8d exhibited excellent dual inhibitory activities against Topo II and HDAC. Cleavage reaction assay showed that 7d was a Topo II poison, which was consistent with the docking results. Further experimental results revealed that compounds 7d and 8d could promote apoptosis and significantly inhibit the migration in MCF-7 cells. Molecular docking showed that compounds 7d and 8d bind Topo II and HDAC at the active sites. Molecular dynamics simulation showed that 7d can stably bind to Topo II and HDAC.

Preparation and chromatographic performance of chiral peptide-based stationary phases for enantiomeric separation.

This study presents the development of three new chiral stationary phases. They are based on silica modified with peptides containing phenylalanine and proline. Successful analyses and characterizations were conducted using Fourier transform infrared spectra, elemental analysis, and thermogravimetric analysis. After this, the enantioselective performance of the three chiral peptide-based columns was evaluated. The evaluation used 11 racemic compounds under normal-phase high performance liquid chromatography mode. Optimized enantiomeric separation conditions were established. Under these conditions, the enantiomers of flurbiprofen and naproxen were successfully separated on CSP-1 column: the separation factor of these was 1.27 and 1.21, respectively. In addition, the reproducibility of the CSP-1 column was also investigated. The results of the investigation illustrated that the stationary phases have good reproducibility (RSD = 0.73%, n = 5).

Determination of neotame in various foods by high-performance liquid chromatography coupled with ultraviolet and mass spectrometric detection.

A new analytical technique involving protein precipitation, heating, lipid degreasing, and SPE procedures combined with HPLC-UV and HPLC-MS/MS has been developed for the determination of neotame in a variety of food samples. This method is applicable for high-protein, high-lipid, or gum-based solid samples. The limit of detection of the HPLC-UV method was 0.5 µg/mL, while that of the HPLC-MS/MS method was 3.3 ng/mL. The spiked recoveries of neotame in 73 kinds of foods were in the range of 81.1-107.2 % with UV detection. The spiked recoveries obtained by HPLC-MS/MS in 14 kinds of foods ranged from 81.6 % to 105.8 %. This technique was successfully used to determine the contents of neotame in two positive samples, indicating its applicability in food analysis.

PEG-modified halloysite as a hydrophilic interaction and cation exchange mixed-mode sorbent for solid-phase extraction of biogenic amines in fish samples.

A novel type of PEG-modified halloysite was prepared and used as a hydrophilic interaction and cation exchange mixed-mode sorbent for solid-phase extraction of biogenic amines in fish samples. The eluates were analyzed by high-performance liquid chromatography-ultraviolet detection after the derivatization with benzoyl chloride. The developed sorbent was characterized by scanning electron microscopy, infrared spectroscopy, X-ray diffraction, zeta potential analyzer, and thermo-gravimetric analysis. After the optimization of various parameters influencing the extraction efficiency, the PEG-modified halloysite-based SPE method was evaluated. The adsorption capacities of putrescine, spermine, phenethylamine, and histamine were as high as 9.3, 8.5, 5.7, and 5.6 mg g-1, respectively. Satisfactory reproducibility of sorbent preparation was obtained with within-batch and batch-to-batch relative standard deviations (RSDs) lower than 3.9% and 8.6%, respectively. The biogenic amine spiking recoveries in fish samples ranged from 84.3 to 105.5% with good RSDs lower than 7.8%. Intra-day and inter-day precision, expressed as RSDs, were better than 8.8%. The limits of detection of histamine, putrescine, phenethylamine, and spermine were 9.4, 1.9, 0.5, and 0.9 µg L-1, respectively. This work provides a new hydrophilic interaction and cation exchange mixed-mode sorbent and is successfully applied to the extraction of trace biogenic amines from fish samples.

Amino acid ionic liquid-based magnetic dispersive solid-phase extraction for benzimidazole residue analysis in fruit juice and human serum based on theoretical screening.

The theoretical screening and design of green solvents to reduce organic solvents and to develop green and efficient sample pretreatment methods have attracted extensive attention. Here, benzimidazoles (BZDs) with potential reproductive toxicity were used as target compounds, and amino acid ionic liquids (AAILs) were designed and selected via a theoretical calculation. With the functionalized AAIL as monomers, and POSS as the crosslinking agent, novel specific magnetic nanoparticles (Fe3O4@SiO2@MAPs@AAIL-POSS) were prepared and used to develop the magnetic dispersive solid-phase extraction (MDSPE) of trace BZDs from fruit juice and human serum. In the evaluation of the method, Fe3O4@SiO2@MAPs@AAIL-POSS showed specific adsorption, recyclable ability, high adsorption efficiency, a good recovery rate, and a low coefficient of variation. In addition, the adsorption behavior of Fe3O4@SiO2@MAPs@AAIL-POSS on BZDs was verified by investigating the kinetic and thermodynamic of the adsorption process. The study design provides ideas for green rapid detection methods of other food pollutants.

Label-Free Small Extracellular Vesicles Capturing Strategy for Lung Cancer Diagnosis and Typing Based on a Natural Polyphenol-Metal Three-Dimensional Network.

Small extracellular vesicles (sEVs) have been increasingly recognized as circulating biomarkers and prognosticators for disease diagnosis. However, the clinical applications of sEVs are seriously limited by the lack of a robust and easy scale-up isolation technique. Herein, the feasibility of a polyphenol-metal three-dimensional (3D) network for label-free sEV isolation was explored. As a proof-of-concept, with tannic acid (TA) as the polyphenolic ligand and Fe(III) as the coordinated metal, the TA-Fe(III) 3D network coating mesoporous silica beads (SiO2@BSA@Fe-TA6) was designed and fabricated via a coordination-driven layer-by-layer self-assembly approach. The successful fabrication of SiO2@BSA@Fe-TA6 was validated by Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis. With the low-cost TA (as low as US$ 0.18/g) as the probe, SiO2@BSA@Fe-TA6 achieved universal capture toward sEVs in different cells and plasma samples. The capture efficiency reached 85.4 ± 1.5%, which is comparable to the antibody-based capture techniques and significantly higher than the ultracentrifugation (UC) method. The purity of sEVs isolated by SiO2@BSA@Fe-TA6 from the H1299 cell culture supernatant was measured as (1.07 ± 0.14) × 1011 particles/µg, which is 3.1 times higher than that via the UC method. Another important superiority of SiO2@BSA@Fe-TA6 is the facile self-assembly approach, which can harvest a yield of up to grams, allowing simultaneous processing of more than 500 plasma samples. The SiO2@BSA@Fe-TA6-based strategy was further successfully employed to distinguish nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC) with an accuracy of 87.1%. The developed SiO2@BSA@Fe-TA6 is a label-free, universal, low cost, and easy scale-up technique for sEV-based liquid biopsy in lung cancer diagnosis and typing.

Fabrication of edge-curled petals-like covalent organic frameworks and their properties for extracting indole alkaloids from complex biological samples.

In this study, a functionalized covalent-organic framework (COF) was first synthesized using porphyrin as the fabrication unit and showed an edge-curled, petal-like and well-ordered structure. The synthesized COF was then introduced to prepare porous organic polymer monolithic materials (POPMs). Two composite POPM/COF monolithic materials with rod shapes, referred to as sorbent A and sorbent B, were prepared in stainless steel tubes using different monomers. Sorbents A and B exhibited relatively uniform porous structures and enhanced specific surface areas of 153.14 m2/g and 80.01 m2/g, respectively. The prepared composite monoliths were used as in-tube solid-phase extraction (SPE) sorbents combined with HPLC for the on-line extraction and quantitative analytical systems. Indole alkaloids (from Catharanthus roseus G. Don and Uncaria rhynchophylla (Miq.) Miq. Ex Havil.) contained in mouse plasma were extracted and quantitatively analyzed using the online system. The two composite multifunctional monoliths showed excellent clean-up ability for complex biological matrices, as well as superior selectivity for target indole alkaloids. Method validation showed that the RSD values of the repeatability (n=6) were ≤ 3.46%, and the accuracy expressed by the spiked recoveries was in the ranges of 99.38%-100.91% and 96.39%-103.50% for vinca alkaloids and Uncaria alkaloids, respectively. Furthermore, sorbents A and B exhibited strong reusability, with RSD values ≤ 5.32%, which were based on the peak area of the corresponding alkaloids with more than 100 injections. These results indicate that the composite POPM/COF rod-shaped monoliths are promising media as SPE sorbents for extracting trace compounds in complex biological samples.

Ionic liquid-based magnetic nanoparticles for magnetic dispersive solid-phase extraction: A review.

Due to their highly tunable nature and outstanding physicochemical properties, ionic liquids (ILs) have been widely reported for use in the synthesis of multitudinous magnetic nanoparticles (MNPs). IL-based magnetic nanoparticles (IL-MNPs) have great potential in magnetic dispersive solid-phase extraction (MDSPE). At present, IL-MNPs have been successfully applied in the pretreatment of MDSPE samples from medicines, pesticides, veterinary drugs, heavy metals, dyes, additives, and proteins in agricultural products, foods and beverages, environmental water, and biological samples. In this review, the preparation of IL-MNPs and their application in MDSPE are comprehensively summarized. The structural characteristics of the introduced ILs used to prepare the IL-MNPs and the synthetic routes employed to obtain the IL-MNPs are described, including physical coating and chemical bonding methods. The IL-MNPs are then classified and described according to different modified materials, including silica-based materials, carbon-based materials, metal-organic frameworks, molecularly imprinted polymers and other interesting large/small molecules. Finally, the research prospects and development directions of IL-MNPs in the context of MDSPE are further identified.

[Determination of the enantiomers of salmeterol xinafoate in salmeterol fluticasone powder inhalant by chiral nonaqueous capillary electrophoresis].

Salmeterol xinafoate (SalX) is one of the ideal drugs used for the treatment of nocturnal asthma attacks and daily maintenance. The molecular structure of SalX contains a chiral carbon atom, and thus, SalX has two enantiomers, viz. (R)-SalX and (S)-SalX. It is clinically administered in the racemic form. Related studies have shown that the two enantiomers of SalX are quite different in pharmacology, toxicology, and other aspects. Therefore, it is of great significance to establish an analytical method for the chiral separation and determination of the SalX enantiomers to guarantee their quality and ensure their safety and effectiveness in clinical use. In this study, a chiral nonaqueous capillary electrophoresis (NACE) method, using a L(+)-tartaric acid-boric acid complex as the chiral selector, was established to determine the enantiomers of SalX in salmeterol fluticasone powder inhalant. The L(+)-tartaric acid-boric acid complex was synthesized in situ by the reaction of L(+)-tartaric acid and boric acid in methanol solution. The ion pair principle was considered the enantioseparation mechanism, and the non-aqueous system was found to be more favorable for ion pair formation, which is useful for chiral recognition. Chiral separation is based on the reversible formation of diastereomeric ion pairs between the negatively charged L(+)-tartaric acid-boric acid complex and the positively charged salmeterol enantiomers. Due to the difference in ion-pair binding ability between different enantiomers, the apparent electrophoretic mobilities of different enantiomers were also different, resulting in chiral separation in NACE. To achieve good resolution, the effects of L(+)-tartaric acid concentration, boric acid concentration, and apparent pH (pH* ) on the chiral separation were investigated. The optimized buffer solution (pH* 0.93) contained 120.0 mmol/L L(+)-tartaric acid and 120.0 mmol/L boric acid in methanol. Other experimental conditions were as follows: uncoated fused-silica capillary with an I. D. of 50.0 µm, a total length (Ltot) of 64.5 cm, and an effective length (Leff) of 55.5 cm, along with gravity injection of 17.5 cm×10.0 s, detection wavelength of 225 nm, room temperature, and operating voltage of 20.0 kV. Under these experimental conditions, the two enantiomers of SalX achieved a resolution of 2.18 within 18.0 min. Both enantiomers showed a good linear relationship of the peak area in the concentration range of 27.5-800.0 mg/L, the correlation coefficient (r) being greater than 0.9990. The detection limit (S/N=3) and quantitative limit (S/N=10) were 7.5 mg/L and 25.0 mg/L, respectively; the standard recovery was 98.1%-101.9%, with relative standard deviations (RSDs) of 1.2%-1.9%. The intra- and inter-day precisions were examined, and the RSDs of the peak area and migration time were found to be below 4.9% and 1.9%, respectively, indicating good repeatability (inter-day) and reproducibility (inter-day) of the method. The established chiral NACE method was used to determine the two SalX enantiomers in a random salmeterol fluticasone powder inhalant purchased from a local market. The results showed that the percentage of labeled quantities was 98.7% for both enantiomer 1 and enantiomer 2, with RSDs of 2.5% and 2.7%, respectively. Thus, this method is simple, feasible, accurate, and inexpensive, and can be applied for the determination of SalX enantiomers in commercially available salmeterol fluticasone powder inhalants.

Polymerized phosphonium ionic liquid functionalized silica microspheres as mixed-mode stationary phase for liquid chromatographic separation of phospholipids.

There is a large and growing demand for the vigorous development of new high performance liquid chromatography stationary phases in order to solve complex phospholipids separation. Herein, phosphonium-based ionic liquid trioctyl(allyl)phosphonium bromide ([P888Allyl]Br) was first synthesized with trioctylphosphine and allyl bromide. With [P888Allyl]Br as the polymerizable monomer, polymerized phosphonium ionic liquid functionalized silica microsphere (PIL@SiO2) was further synthetized via click chemistry reaction. Significantly, based on the inherent amphiphilic nature of the introduced [P888Allyl]Br, the packed PIL@SiO2 column displayed hydrophilic/hydrophobic mixed-mode retention mechanisms. The PIL@SiO2 column can achieve separation of nucleic acid bases and nucleosides, sulfonamides, amides and anilines with excellent selectivity in a shorter separation time. The column efficiency reached 109,700 N/m for 2-iodoacetamide. One of the important characteristics of the PIL@SiO2 column is that both phospholipid classes and species can be efficiently separated via the same column, outperforming that of the commercial amino column. Furthermore, the application potential of the PIL@SiO2 column was further verified via separation of phospholipids extracted from soy lecithin. The proposed PIL@SiO2 column provides a promising candidate for separation of complex phospholipid samples.

Removal of trace DNA toxic compounds using a Poly(deep eutectic solvent)@Biomass based on multi-physical interactions.

DNA toxic compounds (DNA-T-Cs), even in trace amounts, seriously threaten human health and must be completely eliminated. However, the currently used separation media face great challenges in removing trace DNA-T-Cs. Based on the functional advantages of deep eutectic solvents (DESs) and the natural features of biomass (BioM), a series of Poly(DES)@BioMs functioning as adsorbents were prepared for the removal of aromatic/hetero-atomic DNA-T-Cs at the ppm level. After optimisation of experimental conditions, the removal efficiency for DNA-T-Cs ranged from 92.4% to 96.0% with an initial concentration of 20.0 ppm, a temperature of 30 °C, duration of 30 min, and pH of 7.0. The removal processes between the DNA-T-Cs and Poly(DES)@BioMs are well described in the Temkin equilibrium and second-order kinetic adsorption models, and the desorption processes are well shown in the Korsmeryer-Peppas equilibrium and zero-order kinetic models. Molecular simulations revealed that the removal interactions include hydrogen bonding, π-π stacking, and hydrophobic/hydrophilic effects. The removal efficiency for the DNA-T-Cs at 8.0 ppm in industrial sewage ranged from 69.7% to 102%, while the removal efficiency for the DNA-T-Cs standing alone at 20.0 ppm in a methyl violet drug solution was 95.4%, confirming that the Poly(DES)@BioMs effectively removed trace DNA-T-Cs in field samples.

Highly Efficient Enrichment of O-GalNAc Glycopeptides by Using Immobilized Metal Ion Affinity Chromatography.

Proteomics analysis of O-GalNAc glycosylation is important for the screening of biomarkers and the assessment of therapeutic responses. However, its analysis still faces challenges due to the poor performance of currently available enrichment methods. In this study, an enrichment method was established on the basis of Ti-IMAC(IV) materials, which could enrich the intact O-GalNAc glycopeptides via both the hydrophilic interaction and affinity interaction. This method enabled nearly 200 intact O-GalNAc glycopeptides identified from only 0.1 µL of human serum. This was nearly 2-fold different from that of the HILIC method. An in-depth analysis of the O-GalNAc glycosylation was performed, and 2093 intact glycopeptides were identified from 7.2 µL of human serum samples. This is the largest O-GalNAc glycosylation database of human serum from a trace amount of sample. Furthermore, 52 significantly changed intact O-GalNAc glycopeptides were determined by the quantitative analysis of hepatocellular carcinoma (HCC) and control serum samples, indicating the potential applications of this enrichment method in biomarker discovery.

Anti-Tim4 Grafting Strongly Hydrophilic Metal-Organic Frameworks Immunoaffinity Flake for High-Efficiency Capture and Separation of Exosomes.

Exosomes have become the most ideal analysis target for liquid biopsy since they carry a large amount of genetic materials. The study on exosomes has great significance for cancer diagnosis and prognosis. However, the extremely low concentration renders the development of a robust exosomes enrichment technique, with the merits of low nonspecific cell adhesion, high-capture efficiency, and easy nondestructive release of captured exosomes, of vital significance. We successfully designed and developed a novel Tim4@ILI-01 immunoaffinity flake material. First, a strongly hydrophilic ILI-01 MOFs matrix material was fabricated with cationic ionic liquid 1,3-bis(4-carboxybutyl)imidazolium bromide as the organic ligand. The nonspecific adsorption of the ILI-01 MOFs material was only 0.7% after two washings with a neutral buffer. Moreover, based on the inherent abundant carboxyl groups on the ILI-01 MOFs flake, they can be facilely functionalized with an anti-Tim4 antibody with the bonding efficiency of 82.4%. The capture efficiency of the developed Tim4@ILI-01 immunoaffinity material for exosomes reached 85.2%, which is 5.2 times higher than that via the gold standard ultracentrifugation method. Furthermore, based on the Ca2+-dependent characteristic of the binding between the Tim4@ILI-01 immunoaffinity material and phosphatidylserine (PS) on the surfaces of exosomes, the captured exosomes can be easily released with the addition of a chelating agent under neutral eluent conditions. Thus, the captured exosomes maintained good biological activity. The developed Tim4@ILI-01 immunoaffinity flake was successfully applied for enrichment of exosomes from serums of healthy persons and lung adenocarcinoma patients. The levels of the expressed CD44 gene significantly changed under different stages of lung adenocarcinoma cancer. All these results demonstrate that the Tim4@ILI-01 immunoaffinity flake is a robust enrichment material and has a good potential in practical clinical applications.