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The antigenicity of β-lactoglobulin (β-LG) can be influenced by pH values and reduced by epigallocatechin-3-gallate (EGCG). However, a detailed mechanism concerning EGCG decreasing the antigenicity of β-LG at different pH levels lacks clarity. Here, we explore the inhibition mechanism of EGCG on the antigenicity of β-LG at pH 6.2, 7.4 and 8.2 using enzyme-linked immunosorbent assay, multi-spectroscopy, mass spectrometry and molecular simulations. The results of Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) elucidate that the noncovalent binding of EGCG with β-LG induces variations in the secondary structure and conformations of β-LG. Moreover, EGCG inhibits the antigenicity of β-LG the most at pH 7.4 (98.30 %), followed by pH 6.2 (73.18 %) and pH 8.2 (36.24 %). The inhibitory difference is attributed to the disparity in the number of epitopes involved in the interacting regions of EGCG and β-LG. Our findings suggest that manipulating pH conditions may enhance the effectiveness of antigenic inhibitors, with the potential for further application in the food industry. [Display omitted] • EGCG inhibited the antigenicity of β-LG in the order of pH 7.4 > 6.2 > 8.2. • EGCG reduced the antigenicity of β-LG by noncovalent binding. • EGCG binding to β-LG changed the secondary structure and conformation of β-LG. • EGCG binds to the outer surface of β-LG under three pH conditions. • The inhibitory effect of EGCG on β-LG antigenicity hinged on different binding sites. [ABSTRACT FROM AUTHOR]

Quercetin is widely distributed in nature and abundant in the human diet, which exhibits diverse biological activities and potential medical benefits. However, there remains a lack of comprehensive understanding about its cellular targets, impeding its in-depth mechanistic studies and clinical applications. This study aimed to profile protein targets of quercetin at the proteome level. A label-free CETSA-MS proteomics technique was employed for target enrichment and identification. The R package Inflect was used for melting curve fitting and target selection. D3Pocket and LiBiSco tools were used for binding pocket prediction and binding pocket analysis. Western blotting, molecular docking, site-directed mutagenesis and pull-down assays were used for target verification and validation. We curated a library of direct binding targets of quercetin in cells. This library comprises 37 proteins that show increased thermal stability upon quercetin binding and 33 proteins that display decreased thermal stability. Through Western blotting, molecular docking, site-directed mutagenesis and pull-down assays, we validated CBR1 and GSK3A from the stabilized protein group and MAPK1 from the destabilized group as direct binding targets of quercetin. Moreover, we characterized the shared chemical properties of the binding pockets of quercetin with targets. Our findings deepen our understanding of the proteins pivotal to the bioactivity of quercetin and lay the groundwork for further exploration into its mechanisms of action and potential clinical applications. [Display omitted] • Identification of quercetin's cellular protein targets using CETSA-MS. • Creation of a curated library with 37 stabilized and 33 destabilized proteins. • Identification of CBR1, GSK3A, and MAPK1 as direct quercetin binding targets. • Revelation of shared chemical properties in quercetin-target binding pockets. [ABSTRACT FROM AUTHOR]

A porous aromatic framework (PAF) is shown to be a viable sorbent for the adsorption of phenols. To overcome the difficulty of quick adsorption and enrichment by phenols from the matrix, a sorbent material consisting of porous aromatic framework magnetic nanoparticles (PAF MNPs) with a core–shell structure was fabricated by an in situ growth method. The PAF MNP sorbent was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and other techniques. The factors affecting enrichment performance including the amount of PAF-6 MNPs, sample pH, extraction time and elution conditions were optimized. Under the optimal conditions, a method utilizing high-performance liquid chromatography with an ultraviolet detector (HPLC-UV) was developed to quantify phenols in urine. The method showed good linearity (r ≥ 0.998), good precision (RSD ≤ 9.9%, n = 6) and a low limit of detection (1.0–2.0 ng/mL, S/N = 3), with recoveries performed in urine matrix ranging from 76.7% to 113.2%. The method is simple, time-saving and sensitive. Moreover, compared with traditional mass spectrometry detection methods, this method has advantages in terms of low cost and repeatability. [ABSTRACT FROM AUTHOR]

[Display omitted] • Conjugates of rutin and β-LG were prepared by alkaline and free radical methods. • The β-LG-rutin prepared by the alkaline method showed lower IgE binding capacity. • The conjugates exhibited higher intestinal digestibility relative to unmodified β-LG. • The covalent binding of rutin amplified the antioxidant efficacy of β-LG. β-lactoglobulin (β-LG) is an essential nutrient in milk, but it is the primary allergen causing dairy allergy in humans. Currently, researchers are focusing on using flavonoids to covalently modify β-LG for improving its functionality. However, the impact and underlying mechanisms of rutin covalent modification on the functional properties and allergenicity of β-LG remain unclear. Here, we aim to investigate the changes in allergenicity, digestive characteristics, and antioxidant properties of β-LG after covalent modification using a combination of spectroscopy, enzyme-linked immunosorbent assay (ELISA), simulated digestion, and antioxidant assays. The results indicate that rutin forms covalent bonds with the free amino group, sulfhydryl group, and tryptophan of β-LG, leading to alterations in the secondary structure of β-LG. Furthermore, the modified β-LG exhibits improved antioxidant capacity and decreased allergenicity, along with reduced resistance to pancreatin digestion in vitro. This study provides novel insights and strategies to expand the functional application of β-LG. [ABSTRACT FROM AUTHOR]

[Display omitted] • Conjugates of rutin and β-LG were prepared by alkaline and free radical methods. • The β-LG-rutin prepared by the alkaline method showed lower IgE binding capacity. • The conjugates exhibited higher intestinal digestibility relative to unmodified β-LG. • The covalent binding of rutin amplified the antioxidant efficacy of β-LG. β-lactoglobulin (β-LG) is an essential nutrient in milk, but it is the primary allergen causing dairy allergy in humans. Currently, researchers are focusing on using flavonoids to covalently modify β-LG for improving its functionality. However, the impact and underlying mechanisms of rutin covalent modification on the functional properties and allergenicity of β-LG remain unclear. Here, we aim to investigate the changes in allergenicity, digestive characteristics, and antioxidant properties of β-LG after covalent modification using a combination of spectroscopy, enzyme-linked immunosorbent assay (ELISA), simulated digestion, and antioxidant assays. The results indicate that rutin forms covalent bonds with the free amino group, sulfhydryl group, and tryptophan of β-LG, leading to alterations in the secondary structure of β-LG. Furthermore, the modified β-LG exhibits improved antioxidant capacity and decreased allergenicity, along with reduced resistance to pancreatin digestion in vitro. This study provides novel insights and strategies to expand the functional application of β-LG. [ABSTRACT FROM AUTHOR]

To obtain a better understanding as to whether concentration alterations of metals and metalloids in urine were related to Henoch-Schonlein purpura nephritis (HSPN), the profiles of as many as 29 elements in urine were compared among three groups, the Henoch-Schonlein purpura (HSP), HSPN and a healthy control group. To this end, a reliable method has been developed for the simultaneous quantification of multiple elements including Li, Be, B, Al, Sc, Ti, V, Cr, Mn, Fe, Ni, Co, Cu, Zn, Ga, Ge, As, Se, Rb, Sr, Mo, Cd, Sn, Sb, Cs, Ba, Tl, Pb and Bi in urine using inductively coupled plasma orthogonal acceleration time-of-flight mass spectrometry (ICP-oa-TOF-MS). The process of sample pre-treatment used a direct 20-fold dilution method with centrifuged urine. The internal standard element used for quantification was 103 Rh, and 1,4-butanediol was chosen as a matrix matching reagent. The method detection limits of these 29 elements were in the range of 0.04–12 ng mL −1 . Results of statistical analysis revealed that the concentrations of 15 elements and the element homeostasis were significantly different among these three groups. Our study provides a potential method for HSPN metal and metalloid biomarker discovery. [ABSTRACT FROM AUTHOR]

A two-dimensional ion chromatography system was developed for the determination of γ-hydroxybutyrate (GHB) in human urine samples. Ion exclusion chromatography was used in the first dimensional separation for elimination of urine matrices and detection of GHB above 10 mg L −1 , ion exchange chromatography was used in the second dimensional separation via column-switching technique for detection of GHB above 0.08 mg L −1 . Under the optimized chromatographic conditions, the ion exclusion and ion exchange chromatography separation system exhibited satisfactory repeatability (RSD < 3.1%, n = 6) and good linearity in the range of 50–1000 mg L −1 and 0.5–100 mg L −1 , respectively. By this method, concentrations of GHB in the selected human urine samples were detected in the range of 0–1.57 mg L −1 . The urine sample containing 0.89 mg L −1 GHB was selected to evaluate the accuracy; the spiked recoveries of GHB were 95.9-102.8%. The results showed that the two-dimensional ion chromatography system was convenient and practical for the determination of GHB in human urine samples. [ABSTRACT FROM AUTHOR]

This work demonstrates the successful application of an on-cartridge derivatisation procedure for facile, fast and sensitive determination of formaldehyde in beer by HPLC-UV. The derivatisation and solid-phase extraction (SPE) were integrated into a novel calixarene SPE sorbent: tetraazacalix[2]arene[2]triazine bonded silica gel. Specifically, 2,4-dinitrophenylhydrazine was adsorbed onto the sorbent in advance, based on the charge-transfer interaction between the macrocyclic molecule and nitrobenzenes. The method was optimised and validated: under the optimal conditions of derivatisation, SPE and HPLC separation, good linearity was obtained in the range of 0.080–3.2 μg mL −1 with a correlation coefficient of 0.9939, the limit of detection was 3.0 ng mL −1 (S/N = 3), the limit of quantification was 10 ng mL −1 (S/N = 10), and the recovery level using this method was desirable at 75–84%. The developed method was successfully applied to determine formaldehyde content in real beer samples; the results were in the range of 0.11–1.1 μg mL −1 . [ABSTRACT FROM AUTHOR]

A new stationary phase for high-performance liquid chromatography was prepared by covalently bonding a heteroatom-bridged cyclophane onto silica gel using 3-aminopropyltriethoxysilane as the coupling reagent. The structure of the new material was characterized by infrared spectroscopy, elemental analysis, and thermogravimetric analysis. The linear solvation energy relationship method was successfully employed to evaluate the new phase with a set of 25 solutes, and compared with octadecylsilyl and p- tert-butyl-calix[4]arene bonded stationary phases. The retention characteristics of the new phase are similar to the octadecylsilyl and conventional calixarene phases, and it also has distinctive features. In addition, the chromatographic behavior of the phase was illustrated by eluting alkylbenzenes and inorganic anions in the reversed-phase mode and anion-exchange mode, respectively. Thus, multi-interaction mechanisms and mixed-mode separation of the new phase can very likely guarantee its promising application in the analysis of complex samples. The column has been successfully employed for the analysis of triazines in milk, and it is demonstrated to be a competitive alternative analytical method for the determination of triazine herbicide residues. [ABSTRACT FROM AUTHOR]

Two new kinds of alanine-substituted calix[4]arene stationary phases of 5,11,17,23- p-tert-butyl-25,27-bis( l-alanine-methylester- N-carbonyl-methoxy)-26,28-dihyroxycalix[4]arene-bonded silica gel stationary phase (BABS4) and 5, 11, 17, 23- p-tert-butyl-25,26,27,28-tetra( l-alanine-methylester- N-carbonyl-methoxy)-calix[4]arene-bonded silica gel stationary phase (TABS4) were prepared and characterized in the present study. They were compared with each other and investigated in terms of their chromatographic performance by using polycyclic aromatic hydrocarbons, disubstituted benzene isomers, and mono-substituted benzenes as solute probes. The results indicated that both BABS4 and TABS4 exhibited multiple interactions with analytes. In addition, the commonly used Tanaka characterization protocol for the evaluation of commercially available stationary phases was applied to evaluate the properties of these two new functionalized calixarene stationary phases. The Tanaka test results were compared with Zorbax Eclipse XDB C18 and Kromasil phenyl columns, respectively. BABS4 has stronger hydrogen-bonding capacity and ion-exchange capacity than TABS4, and features weaker hydrophobicity and hydrophobic selectivity. Both of them behave similarly in stereoselectivity. Both BABS4 and TABS4 are weaker than C18 and phenyl stationary phases in hydrophobicity and hydrophobic selectivity. [ABSTRACT FROM AUTHOR]

Objectives Oral squamous cell carcinoma (OSCC) is a common malignancy in the oral and maxillofacial regions with an increasing incidence rate. Circular RNA (circRNA) is a recently discovered long-chain non-coding RNA family member. The objective of this study was to analyze the role of circ_0068162 in OSCC development. Methods We downloaded sample data GSE145608 from the Gene Expression Omnibus database. Online databases Starbase, TargetScan and miRDB were used to predict the target microRNAs (miRNAs) and genes. Cell viability and proliferation were assessed using the CCK-8 and EdU assays, respectively. Cell migration and invasion abilities were detected using transwell assay. The double luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the interaction relationship between the identified target molecules. RNase R and actinomycin D treatment were performed to analyze the stability of circ_0068162. Results We found that circ_0068162 was overexpressed in the cytoplasm of OSCC cells and clinical OSCC tissues. Knockdown of circ_0068162 inhibited the growth, migration and invasion of OSCC cells. We also identified miR-186 as the target miRNA of circ_0068162, and JAG1 and JAG2 as the target genes of miR-186. The miR-186 inhibitor rescued the effects of sh-circ_0068162 and JAG1/JAG2 overexpression rescued the effects of miR-186 mimic in OSCC cells. Furthermore, ESRP1 promoted the biosynthesis of circ_0068162. Conclusions The circ_0068162/miR-186/JAGs/ESRP1 feedback loop is closely related to OSCC development. [ABSTRACT FROM AUTHOR]

The five anthraquinones compounds (including aloe-emodin, emodin, physcion, chrysophanol, and rhein) are regarded as the main effective ingredients in rhubarb (Dahuang in Chinese, one of the commonly used Chinese herbal medicines). In this work, a simple and effective solid phase extraction (SPE) method based on bis(tetraoxacalix[2]arene[2]triazine) modified silica gel as adsorbent was developed. Coupled with UHPLC-FLD, the developed method was successfully applied for the measuring of main anthraquinones in human urine after oral administration of the extracts of rhubarb. To obtain the highest recoveries of the five anthraquinones in the SPE process, the main parameters which may affect extraction efficiency were optimized. The optimized sorbent amount, sample loading pH, sample loading rate, washing solution, and eluent condition were obtained. The developed method showed good linearity in 0.012–1.800 μg mL−1 for the five anthraquinones with correlation coefficients more than 0.9993. The investigated LOD values ranged from 3.9 to 5.7 ng mL−1, while the LOQs were between 12.0 and 18.2 ng mL−1. The recoveries of the method were also investigated, which were in the range of 94.8–106.6%. The application of the mixed-mode SPE materials in the proposed method was feasible and simple, and suitable for the enrichment of anthraquinones in urine samples. [ABSTRACT FROM AUTHOR]

Background: The purpose of this study was to evaluate the correlation and consistency between urine protein/creatinine ratio (UPCR) and 24-h urine protein (24HUPr) in children, and to determine cutoff values of UPCR relative to 24HUPr at 100 mg/m2/d (≥ 100 mg/m2/d as pathological proteinuria) and 1000 mg/m2/d (≥ 1000 mg/m2/d as nephrotic-range proteinuria). Methods: Three hundred sixty-six children were enrolled, including 81 controls, 109 with Henoch-Schönlein purpura nephritis, 167 with nephrotic syndrome, 5 with IgA nephropathy, and 4 with lupus nephritis. Patients were divided into three groups: normal group; non-nephrotic-range proteinuria group; nephrotic-range proteinuria group. The cutoff values of UPCR in predicting the different levels of proteinuria were determined using ROC curve analysis. Results: UPCR was positively correlated with 24HUPr (r = 0.915, p < 0.01). Bland-Altman diagrams showed that UPCR and 24HUPr had good consistency, and > 95% spots of UPCR and 24HUPr were within 95% confidence intervals. Relative to 24HUPr at 100 mg/m2/d, the cutoff value of UPCR (0.18 g/g Cr) had the highest sensitivity (94%) and specificity (98.8%) which is close to 0.2 g/g Cr as proposed by the American College of Rheumatology. Relative to 24HUPr at 1000 mg/m2/d, the cutoff value of UPCR (2.09 g/g Cr) had the highest sensitivity (92.1%) and specificity (92.1%) which is close to the 2.0 g/g Cr proposed in KDIGO guidelines. Conclusions: UPCR showed strong correlation and consistency with 24HUPr for evaluating levels of proteinuria in children. UPCR < 0.2 g/g Cr can be considered a criterion for normal-range proteinuria. Instead of 24HUPr ≥ 1000 mg/m2/d, UPCR ≥ 2.0 g/g Cr can be considered a criterion for nephrotic-range proteinuria or nephrotic syndrome in children. [ABSTRACT FROM AUTHOR]

Abstract Calixarenes and their derivatives have been widely used in chromatographic studies due to unique host-guest recognition properties. However, stationary phases in hydrophilic interaction chromatography associated with them have yet to be studied because of their general hydrophobicity. In this paper, a tetra-proline modified calix[4]arene bonded stationary phase (DTPCSP) was prepared and characterized by FT-IR spectra, elemental analysis, solid state 13C NMR, SEM, EDS and thermogravimetric analysis. The chromatographic performance and retention mechanism of the developed stationary phase were validated in hydrophilic interaction mode and compared with a commercial column using a variety of hydrophilic or hydrophobic compounds including phenols, nucleosides and sulfonamides. The developed stationary phase exhibited better selectivity than conventional calixarene phases, and the retention behaviors of phenols on DTPCSP column were elucidated by the calculation of quantum chemistry using ODS as contrast. All the results indicate that the developed DTPCSP stationary phase can be beneficial for simultaneously separating complex hydrophilic samples with high selectivity. Graphical abstract fx1 Highlights • A novel tetra-proline modified calix[4]arene bonded stationary phase (DTPCSP) was prepared and characterized. • DTPCSP exhibited better selectivity and separation capacity than commercial HILIC column. • DTPCSP can be beneficial for simultaneously separating complex hydrophilic samples based on a multiple interaction. [ABSTRACT FROM AUTHOR]

This study reports a novel strategy for the preparation of porous aromatic framework (PAF-6) coated magnetic nanoparticles (PAF-6 MNPs) using cyanuric chloride as a planar trigonal basis upon which to build a linear piperazine linker unit. The PAF-6 MNPs were examined as an efficient solid-phase extraction (SPE) sorbent for enrichment of trace organic pollutants including phenol, 2,4,6-trinitrophenol, naphthalene, naphthol, bisphenol A, 2,4-dichlorophenol and 3-nitrochlorobenzene in water. The high-performance liquid chromatography detection limits of such analytes were in the range of 0.08–5.02 ng/mL and recoveries were found to be 84.0–94.0% in well water, tap water, river water and wastewater. The main toxic components of cigarette smoke, including phenolic compounds and benzo[a]pyrene, are efficiently adsorbed by PAF-6 MNPs, and over 50% of such toxins are removed. Theoretical computations were performed to understand the molecular interaction mechanism between PAF-6 and such analytes. The results demonstrate that the PAF-6 MNPs sorbents show excellent adsorption of phenols, polycyclic aromatic hydrocarbons and nitroaromatics based on multiple π-π stacking and hydrogen-bond interactions. These results suggest that the PAF-6 MNPs can be applied to extraction, removal and determination of diverse trace multi-target analytes from complex media. [ABSTRACT FROM AUTHOR]

• Proposing a DFI and NL dependent annotation algorithm. • Authenticating S. tetrandra by LMJ-HRMS. Stephania tetrandra S. Moore, a widely used traditional antirheumatic herbal medicine (HM), is a rich source of isoquinoline alkaloids. With the exception of the two recognized isoquinolines, viz. tetrandrine and fangchinoline, the other isoquinoline alkaloids present in S. tetrandra have not been clearly clarified. In addition, due to their similar names and morphological similarities, S. tetrandra is often mistakenly substituted and adulterated with the nephrotoxic Aristolochia fangchi. In this study, ultra-high-performance liquid chromatography-triple time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) was initially employed to comprehensively profile the isoquinolines from S. tetrandra. To overcome the complexities arising due to the similar mass behaviors of the isoquinolines, a stepwise diagnostic fragment ion (DFI) and neutral loss (NL)-dependent structure annotation algorithm was proposed, and this accelerated the identification of 393 isoquinolines distributed over twenty classes. Consequently, liquid microjunction surface sampling-high-resolution mass spectrometry (LMJ-HRMS) was deployed in an attempt to directly authenticate S. tetrandra by the chemical profiling of its crude slice. By matching the 393 isoquinolines, the 87 peaks detected by LMJ-HRMS were assigned to 270 isoquinolines, including the recognized tetrandrine and fangchinoline. The absence of aristolochic acid-related mass signals confirmed the authentication of S. tetrandra. In summary, LMJ-HRMS can be considered a direct, nondestructive, high-throughput, and environment-friendly analytical method for the authentication of HMs. Moreover, the stepwise DFI- and NL-dependent structure annotation algorithm-based UHPLC-Q-TOF-MS method allowed high-coverage detection and high-quality data processing of the inherent structural similarity and complexity of isoquinolines or other phytochemical compounds. [ABSTRACT FROM AUTHOR]