Optimization of Physical Refining Process of Camellia Oil for Reduction of 3-Monochloropropane-1,2-Diol (3-MCPD) Ester Formation Using Response Surface Methodology on a Laboratory Scale.

Refined and deodorized camellia oil has been reported to contain a high amount of 3-monochloropropane-1,2-diol esters (3-MCPDE) due to the high-temperature deodorization step. To reduce 3-MCPDE in camellia oil, the physical refining process of camellia oil was simulated on a laboratory scale. Response surface methodology (RSM) was designed to modify and optimize the refining process with five processing parameters (water degumming dosage, degumming temperature, activated clay dosage, deodorization temperature and deodorization time). The optimized new refining approach achieved a 76.9% reduction in 3-MCPDE contents, in which the degumming moisture was 2.97%, the degumming temperature was 50.5 °C, the activated clay dosage was 2.69%, the deodorizing temperature was 230 °C, and the deodorizing time was 90 min. A significance test and analysis of variance results demonstrated that the deodorization temperature and deodorization time contributed significantly to the reduction of 3-MCPD ester. The joint interaction effects of activated clay dosage and deodorization temperature were significant for 3-MCPD ester formation.

Determination of ß2-Agonist Residues in Fermented Ham Using UHPLC-MS/MS after Enzymatic Digestion and Sulfonic Resin Solid Phase Purification.

ß2-agonists are a class of synthetic sympathomimetic drugs with acute poisoning effects if consumed as residues in foods. To improve the efficiency of sample preparation and to overcome matrix-dependent signal suppression in the quantitative analysis of four ß2-agonists (clenbuterol, ractopamine, salbutamol, and terbutaline) residues in fermented ham, an enzyme digestion coupled cation exchange purification method for sample preparation was established using ultra-high performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS). Enzymatic digests were subject to cleanup treatment on three different solid phase extraction (SPE) columns and a polymer-based strong cation resin (SCR) cartridge containing sulfonic resin was found to be optimal compared with silica-based sulfonic acid and polymer sulfonic acid resins based SPEs. The analytes were investigated over the linear range of 0.5 to 10.0 µg/kg with recovery rates of 76.0-102.0%, and a relative standard deviation of 1.8-13.3% (n = 6). The limit of detection (LOD) and the limit of quantification (LOQ) were 0.1 µg/kg and 0.3 µg/kg, respectively. This newly developed method was applied to the detection of ß2-agonist residues in 50 commercial ham products and only one sample was found to contain ß2-agonist residues (clenbuterol at 15.2 µg/kg).

[Optimization and application of determination method of cholesterol in various dishes].

OBJECTIVE: To establish a rapid and accurate method for the determination of cholesterol in a variety of finished dishes. METHODS: The samples were saponified with ethanol and potassium hydroxide solution at 80 ℃ for 0.5 h, then vortexed and mixed fully with ultrapure water, and extracted twice with 25 mL n-hexane. The extracting solution were evaporated to dryness under vacuum and redissolved by ethanol, finally determined by gas chromatograph. The sample loading solution was separated by HP-5 capillary column(30 m×0.25 mm, 0.25 µm) and quantified with external standard method. RESULTS: The method had good linearity in the concentration range of 2.5-250 µg/mL for cholesterol and the correlation coefficient was 0.9999. The detection limit was 0.25 mg/100 g edible part, and the limit of quantification was 0.75 mg/100 g edible part. Three representative samples were picked for spiked recovery experiments with three concentration levels according to their content of cholesterol. The average recovery rates ranged from 91.1% to 101%, and the relative standard deviations were not more than 5%. The content range of cholesterol in 15 vegan dishes was from negative to 10.3 mg/100 g edible part, in 14 dishes contained meat and vegetable was from 2.34 to 80.2 mg/100 g edible part and in 9 pure meat dishes was from 25.4 to 288 mg/100 g edible part. CONCLUSION: Compared with the national standard method, the method is simple to operate and more friendly to the experimenter while ensuring the accuracy and sensitivity requirements. It can meet the needs of efficient batch determination of cholesterol in a variety of finished dishes.

Determination of Polycyclic Aromatic Hydrocarbons in Traditional Chinese Medicine Raw Material, Extracts, and Health Food Products.

The four polycyclic aromatic hydrocarbon markers (PAH4) of benzo[a]anthracene (BaA), chrysene (Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP) are indicators showing polycyclic aromatic hydrocarbon (PAH) contamination levels in Chinese medicine raw materials (CMRMs), extracts and health food products; Samples of herbal medicine, herbal extracts, and food supplements were extracted with n-hexane, then cleaned up sequentially on Florisil and EUPAH solid-phase extraction (SPE) columns. A gas chromatography-mass spectrometry method for the determination of four polycyclic aromatic hydrocarbon markers in Chinese medicine raw material, extracts, and health food products was established; In spiked-recovery experiments, the average recovery was about 78.6-107.6% with a precision of 2.3-10.5%. The limit of quantification (LOQ) and limit of detection (LOD) of the PAH4 markers in this method were 2.0 µg/kg and 0.7 µg/kg, respectively. When the developed method was utilized to determine PAH4 contents in 12 locally available health food products, 3 samples contained over 10.0 µg/kg BaP, and 5 samples contained over 50.0 µg/kg PAH4. The European Union (EU) limits for BaP and PAH4 are 10 and 50.0 µg/kg, respectively; therefore, more attention must be drawn to the exposure risk of BaP and PAH4 in CMRMs, their extracts, and health food products. According to the risk assessment based on the Margin of Exposure (MOE) method, it is recognized that the products mentioned in this study pose a low risk.

Interleukin-1ß as clinically predictive risk marker for recurrent pregnancy loss in women positive for antinuclear antibody.

AIMS: Antinuclear antibody (ANA) was found to be associated with recurrent pregnancy loss (RPL). This study was designed to explore the immunological predictive indicators of RPL in women with positive ANA. METHODS: A retrospective case-control study in a university hospital from March 2020 to January 2021 was performed, including 56 cases of women with RPL and 56 controls matched for age, all of which were positive for ANA. Levels of cytokines, lymphocyte subsets, immune globulin and complement in peripheral blood among two groups were compared. Statistical analyses in this study were performed using SPSS 25.0. RESULTS: The level of IL-1ß was higher in RPL women compared with controls according to univariable analysis and multivariable regression logistic analysis (7.67 [5.86-11.35] vs 5.00 [5.00-5.00], P = .000). After the cut-off value of IL-1ß was set as 5.66 pg/mL, the prevalence of RPL was significantly elevated in the high IL-1ß group as compared with the low IL-1ß group (P < .05). The sensitivity and specificity of IL-1ß predicting RPL in ANA-positive women were 76.8% and 91.1%, respectively. CONCLUSION: Increased IL-1ß may play roles in the occurrence of RPL among women with positive ANA. The level of the IL-1ß has the potential to be a predictive indicator of RPL in women with positive ANA.

[Characterization and investigation of 16 European priority polycyclic aromatic hydrocarbons in commercial infant formula milk powder in Hangzhou].

OBJECTIVE: A gas chromatography-mass spectrometry(GC-MS) method for the determination of 16 European priority polycyclic aromatic hydrocarbons(16 EUPAHs) in infant formula milk powder was established, and the characterization and investigation of 16 EUPAHs in 70 milk formula powders were carried out in 2020. METHODS: After hydrolysis, extraction, saponification and solid phase extraction, infant formula milk powder was detected by GC-MS using DB-EUPAH capillary column(20 m×0.18 mm, 0.14 µm)and quantified by internal standard method. RESULTS: The average recoveries ranged from 67.8% to 116.2% and the relative standard deviations ranged from 2.0% to 15.1%(n=6). The limits of quantification and detection of the method were 0.5 and 0.2 µg/kg, respectively. The content of 16 EUPAHs was <0.2-0.48 µg/kg, including PAH4 content in the range of <0.2-0.91 µg/kg, the characterization and investigation of infant formula powder in 16 EUPAHs mainly chrysene, cyclopenta[c, d]pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[g, h, i]perylene. CONCLUSION: The method is simple, accurate and suitable for the determination of 16 EUPAHs in infant formula milk powder. The result showed that the content of 16 EUPAHs in commercially available infant formula milk powder in Hangzhou was low and all of them met the limit requirement of European Union.

[Detection and content investigation of benzo[a]pyrene in infant milk powder].

OBJECTIVE: To develop a method for determination of benzo[a]pyrene and to analysis of benzo[a]pyrene content in commercially available infant milk powder. METHODS: Firstly, infant milk powder was extracted with ether-petroleum ether(1∶1, V/V) under alkaline conditions, then saponified with 1. 5 mol/L potassium hydroxide ethanol solution. After purification by solid phase extraction column, it was separated on DB-EUPAH chromatographic column(20 m×0. 18 mm×0. 14 µm) and detected by gas chromatography-mass spectrometry, and quantified using D_(12)-benzo[a]pyrene internal standard. RESULTS: When the benzo[a]pyrene in infant formula was spiked 0. 3, 1. 0, 5. 0 µg/kg, the average recoveries were 116. 7%、86. 0% and 96. 4%, respectively, and the relative standard deviations were 10. 5%、4. 2% and 4. 4%, respectively(n= 6). The limit of quantification was 0. 3 µg/kg, and the detection limit was 0. 1 µg/kg. A total of 40 domestic and imported infant milk powders sold in Hangzhou supermarkets were analyzed. The range of benzo[a]pyrene was <0. 1-0. 25 µg/kg, and the detection rate was 32. 5%. CONCLUSION: The method has the advantages of rapid operation, good purifying effect, sensitiveness and accuracy, and meets the requirements for detection of benzo[a]pyrene in infant milk powder. The investigation reveals that the benzo[a]pyrene content in the currently marketed infant milk powder is at a low level.

[Determination of 22 antibiotics in disinfection products by ultra-performance liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of 5 nitroimidazole antibiotics and 17 sulfonamides antibiotics in disinfection products by ultra-performance liquid chromatography tandem mass spectrometry(UPLC-MS/MS). METHODS: Samples were spiked with isotope internal standard, and then extracted by 2% formic acid solution and acetonitrile with ultrasonic, followed by MCX column to remove matrix interference and for enrichment. The supernatants were diluted with 2% formic acid solution before loading on the columns, then washed with 2% formic acid solution and methanol respectively, eluted with 5% ammonium methanol. The separation was performed on a CORTECS~(TM) UPLC C_(18)(100 mm×2. 1 mm, 1. 6 µm) column by using 0. 1% formic acid solution and 0. 1% formic acid acetonitrile as mobile phase with gradient elution. The antibiotics were analyzed by UPLC-MS/MS. RESULTS: The linear ranges of 22 antibiotics were 0. 05-5. 0 ng/mL and the correlation coefficients were greater than 0. 999. The limits of detection(LODs) were 0. 03-0. 15 µg/kg and the recoveries were 84. 3%-121. 2% with the relative standard deviations were 1. 13%-8. 43%(n=6). The method was successfully used to detect the content of antibiotics in 20 disinfection products, 45% of samples had been detected positively. CONCLUSION: This method is simple, sensitive, selective and accurate, and could be applied for simultaneous detection of antibiotics in disinfection products.

[Determination of zearalenone and α-zearalenol in vegetable oil and grain products by C_(18)-Al_2O_3 solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry].

OBJECTIVE: To develop a method for simultaneous determination of zearalenone( ZEN) and α-zearalenol( α-ZEL) in vegetable oil and grain products by solid phase extraction column purification coupled with ultra-performance liquid chromatography tandem mass spectrometry. METHODS: Firstly, ZEN and α-ZEL in grain products were extracted by hexane/ethyl acetate( 50 : 50, V/V), and then extracted as vegetable oil by acetonitrile-water solution( 90: 10, V/V), and purified by C_(18)-Al_2O_3 solid phase extraction column. ZEN and α-ZEL was separated by UPLC with acetonitrile-water gradient elution on C_(18) column( 2. 1 mm × 100 mm, 1. 6 µm), and qualified/quantified by mass spectrometry with ESI negative MRM mode with ~(13)C_(18)-zearalenone as internal standard. RESULTS: The linearity of ZEN and α-ZEL ranged from 1. 0-500 ng/mL. The limit of detection for ZEN and α-ZEL in vegetable oil and grain products was 0. 3 and 0. 2 µg/kg, respectively. The limit of quantification for ZEN and α-ZEL in vegetable oil and grain products was 1. 0 and 0. 5 µg/kg. The average recoveries of ZEN and α-ZEL for spiked samples of 1. 0-100 µg/kg were 93. 5%-108. 0% and 92. 0%-105. 0%. The relative standard deviations of ZEN and α-ZEL were 3. 2%-8. 5% and 4. 6%-7. 8%( n = 6). 55 samples sold in Hangzhou supermarkets were analyzed. ZEN was detected in all corn germ oil with median and maximum contents of 126. 2 and 453. 1 µg/kg. α-ZEL was detected in 50% corn germ oil with median and maximum contents of 2. 0 and 5. 0µg/kg. CONCLUSION: The method possesses several advantages including sensitivity, precision, good efficiency of purification, simplicity and economy, and it is applicable to the batch analysis of zearalenone and α-zearalenol in vegetable oil and grain products.

[Determination of phthalate ester metabolites in urine by solid-phase extraction and gas chromatography-mass spectrometry].

OBJECTIVE: A method for the determination of 5 kinds of phthalate monoesters in urine by solid-phase extraction and gas chromatography-mass spectrometry was developed. METHODS: After urine enzymatic hydrolysis by ß-glucuronidase, the sample was pretreated by SPE. Phthalate monoesters were eluted by boron trifluoridemethanol complex, and determined by gas chromatograph-mass spectrometer with DB-5 ms Phenomenex( 30 m × 0. 25 mm, 0. 25 µm). RESULTS: Within the concentration range of25. 0-1000 µg/L, the relationship between peak area and concentration of 5 kinds of phthalate monoesters presented linear relation. The correlation coefficient was 0. 9989-0. 9996, and the determination limit was 10. 0-15. 0 µg/L. The recoveries of phthalate monoesters were in the range of 80. 2%-97. 8% when set at 15. 0, 200 and 500 µg/L concentration levels, and the relative standard deviation was in the range of 4. 2%-10. 2%( n = 6). A total of 81 pregnant women were detected by this method and thedetection rate was 98. 7%. CONCLUSION: This method could be used in the simultaneous determination of 5 kinds of phthalate monoesters.

Isotope dilution gas chromatography with mass spectrometry for the analysis of 4-octyl phenol, 4-nonylphenol, and bisphenol A in vegetable oils.

By the combination of solid-phase extraction as well as isotope dilution gas chromatography with mass spectrometry, a sensitive and reliable method for the determination of endocrine-disrupting chemicals including bisphenol A, 4-octylphenol, and 4-nonylphenol in vegetable oils was established. The application of a silica/N-(n-propyl)ethylenediamine mixed solid-phase extraction cartridge achieved relatively low matrix effects for bisphenol A, 4-octylphenol, and 4-nonylphenol in vegetable oils. Experiments were designed to evaluate the effects of derivatization, and the extraction parameters were optimized. The estimated limits of detection and quantification for bisphenol A, 4-octylphenol, and 4-nonylphenol were 0.83 and 2.5 µg/kg, respectively. In a spiked experiment in vegetable oils, the recovery of the added bisphenol A was 97.5-110.3%, recovery of the added 4-octylphenol was 64.4-87.4%, and that of 4-nonylphenol was 68.2-89.3%. This sensitive method was then applied to real vegetable oil samples from Zhejiang Province of China, and none of the target compounds were detected.

[Determination of eleven fluorescent whitening agents in paper food packaging materials by UPLC-FLD/PDA with series double-detector].

OBJECTIVE: To establish a new qualitative and quantitative ultraperformance liquid chromatography-fluorescence detector / photodiode array detector with series double-detector method for the determination of eleven fluorescent whitening agents in paper food packaging materials. METHODS: The sample was extracted with 40%acetonitrile water solution, separated by Waters ACQUITY UPLC BEH C_(18)column( 1. 7µm, 2. 1 mm × 100 mm) and eluted gradient. The excitation wavelength and emission wavelength of fluorescence detector( FLD) were 350 nm and 430 nm, and the wavelength of photodiode array detector( PDA) was 350 nm. The detectors were used in series to achieve qualitative and quantitative detection. RESULTS: In the substrates of paper cups, paper bowls, paper trays and paper boxes, those eleven fluorescent whitening agents were separated properly. For both detectors, in the linear range of 25- 1000 ng / m L, the correlation coefficient was greater than 0. 99, and the recoveries of spiked recoveries were between 82. 2%- 104. 1% with the RSD less than 10%( n = 6). The detection limits ofthose eleven fluorescent whitening agents were 0. 20- 0. 28 mg / kg for FLD and 1. 4- 2. 5mg / kg for PDA. CONCLUSION: The eleven fluorescent whitening agents could be separated properly with complete separation, good shapes and high recovery rate. This method is easy to operate also. Thus it's an effective method to detect the fluorescent whitening agents in paper food packaging materials.

Determination of three kinds of banned drugs in milk powder by hybrid solid-phase extraction purification combined with gas chromatography and mass spectrometry.

A gradient clean-up method for the quantification of five kinds of banned drugs (two hormones, two sedatives, and one chloramphenicol) in milk powder was developed. We used the combination of solid-phase extraction purification with gas chromatography and mass spectrometry. Milk powder was initially hydrolyzed by ß-glucuronidase/arylsulfatase, and then the hydrolyzed solution was concentrated and purified using a C8 and cation resin solid-phase extraction column. To isolate hormones and chloramphenicol drugs, products from the previous step were diluted with methanol and further purified using a silica and diatomite solid-phase extraction column. After derivatization, the drugs were analyzed by gas chromatography with mass spectrometry, and the hydrolyzed solution was diluted with 5% ammoniated methanol to purify sedatives before gas chromatography with mass spectrometry analysis. Results showed that after adding the banned drugs at concentrations of 0.3-10.0 µg/kg, the average recovery range was 78.2-97.3% with relative standard deviations of 5.3-12.5%. The limit of quantification of the banned drugs (S/N ≥ 10) was 0.3-5.0 µg/kg, whereas the limit of detection (S/N ≥ 3) was 0.1-2.0 µg/kg. The solid-phase extraction gradient purification system was simple, rapid, and accurate, and could satisfy the detection requirements of hormone, sedatives, and chloramphenicol drugs when used together with gas chromatography and mass spectrometry.

Identification of chemoresistance-related cell-surface glycoproteins in leukemia cells and functional validation of candidate glycoproteins.

Chemoresistance remains the most significant obstacle to successful chemotherapy for leukemia, and its exact mechanism is still unknown. In this work, we used the cell-surface capturing method together with quantitative proteomics to investigate differences in the glycoproteomes of adriamycin-sensitive and adriamycin-resistant leukemia cells. Two quantitative methods, isotopic dimethyl labeling and SWATH, were used to quantify glycoproteins, and 35 glycoproteins were quantified by both methods. High correlation was observed between the glycoproteins quantified by the above two methods, and 15 glycoproteins displayed a consistent significant change trend in both sets of quantitative results. These 15 proteins included classical multidrug resistance-related glycoproteins such as ABCB1 as well as a set of novel glycoproteins that have not previously been reported to be associated with chemoresistance in leukemia cells. Further validation with quantitative real-time PCR and Western blotting confirmed the proteomic screening results. Subsequent functional experiments based on RNA interference technology showed that CTSD, FKBP10, and SLC2A1 are novel genes that participate in the acquisition and maintenance of the adriamycin-resistant phenotype in leukemia cells.

Effect of oral glucose tolerance test-based insulin resistance on embryo quality in women with/without polycystic ovary syndrome.

Objective: To explore the effects of insulin resistance (IR) on embryo quality and pregnancy outcomes in women with or without polycystic ovary syndrome (PCOS) undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI). Methods: A retrospective cohort study concerning patients with/without PCOS who received gonadotropin-releasing hormone (GnRH)-antagonist protocol for IVF/ICSI from January 2019 to July 2022 was conducted. All the patients included underwent oral glucose tolerance test plus the assessment of insulin release within 6 months before the controlled ovarian stimulation. The Matsuda Index was calculated to diagnose IR. Two populations (PCOS and non-PCOS) were included and each was divided into IR and non-IR groups and analyzed respectively. The primary outcome was the high-quality day 3 embryo rate. Results: A total of 895 patients were included (751 with PCOS and 144 without PCOS). For patients with PCOS, the IR group had a lower high-quality day 3 embryo rate (36.8% vs. 39.7%, p=0.005) and available day 3 embryo rate (67.2% vs. 70.6%, p<0.001). For patients without PCOS, there was no significant difference between the IR and non-IR groups in high-quality day 3 embryo rate (p=0.414) and available day 3 embryo rate (p=0.560). There was no significant difference in blastocyst outcomes and pregnancy outcomes for both populations. Conclusion: Based on the diagnosis by the Matsuda Index, IR may adversely affect the day 3 embryo quality in patients with PCOS but not pregnancy outcomes. In women without PCOS, IR alone seems to have less significant adverse effects on embryo quality than in patients with PCOS. Better-designed studies are still needed to compare the differences statistically between PCOS and non-PCOS populations.

Investigations of the highly efficient processing technique, chemical constituents, and anti-inflammatory effect of N-ethyl-2-pyrrolidinone-substituted flavan-3-ol (EPSF)-enriched white tea.

N-Ethyl-2-pyrrolidinone-substituted flavan-3-ols (EPSFs) are a newly discovered compound class in tea with various bioactivities. This study aimed to develop a novel processing technique to enhance EPSF contents in white tea efficiently. Using optimal processing parameters of 125 °C and 30 min in a high-temperature sterilizing oven, total EPSF content significantly increased by 1.42-18.80-fold to 1.57-6.22 mg/g without impacting sensory characteristics. Metabolomics analysis revealed elevated levels of nucleosides, nucleotides, bases, theaflavins, flavonol aglycones, EPSFs, and most flavone-C-glycosides, as well as decreased levels of amino acids, procyanidins, theasinensins, several flavanols, and flavonol-O-glycosides after EPSF-enrichment treatment. Furthermore, the EPSF-enriched white tea exhibited notable anti-inflammatory effects, mitigating xylene-induced ear edema in mice and carrageenan-induced paw edema and cotton ball-induced granulomas in rats. This study developed a new processing technique for highly efficient enhancement of EPSFs in white tea and demonstrated that EPSF-enriched white tea has a potential to serve as effective anti-inflammatory dietary supplement.

Effects of different gonadotropin preparations in GnRH antagonist protocol for patients with polycystic ovary syndrome during IVF/ICSI: a retrospective cohort study.

Purpose: To compare the effects of recombinant FSH alfa (rFSH-alfa), rFSH-beta, highly purified human menopausal gonadotropin (HP-hMG) and urinary FSH (uFSH) in women with polycystic ovarian syndrome who have undertaken the GnRH antagonist protocol during IVF/ICSI treatment. Method: A single-center retrospective cohort study including women with PCOS who received the GnRH antagonist protocol from January 2019 to July 2022 was conducted. Patients were divided into rFSH-alfa group, HP-hMG group, uFSH group, and rFSH-beta group, and the number of oocytes retrieved, clinical pregnancy rate of the fresh cycle (primary outcomes), embryo quality, and severe OHSS rate (secondary outcomes) were compared. Results: No statistical differences were found among the four groups in fresh cycle clinical pregnancy rate (p=0.426), nor in the subgroup analyses. The HP-hMG group had a smaller number of oocytes retrieved and a higher high-quality D3 embryo rate than the three FSH groups (p<0.05). No statistical differences were found among the four groups in the severe OHSS rate (p=0.083). Conclusion: For women with PCOS undergoing the GnRH antagonist protocol, the clinical pregnancy rates of fresh IVF/ICSI-ET cycle are similar for all four types of Gn. With a lower risk of OHSS and a similar number of high-quality and available embryos, HP-hMG may have an advantage in the PCOS population.

Sesamin ameliorates nonalcoholic hepatic steatosis by inhibiting CD36-mediated hepatocyte lipid accumulation in vitro and in vivo.

Hepatic steatosis is a critical factor in the development of nonalcoholic steatohepatitis (NASH). Sesamin (Ses), a functional lignan isolated from Sesamum indicum, possesses hypolipidemic, liver-protective, anti-hypertensive, and anti-tumor properties. Ses has been found to improve hepatic steatosis, but the exact mechanisms through which Ses achieves this are not well understood. In this study, we observed the anti-hepatic steatosis effects of Ses in palmitate/oleate (PA/OA)-incubated primary mouse hepatocytes, AML12 hepatocytes, and HepG2 cells, as well as in high-fat, high-cholesterol diet-induced NASH mice. RNA sequencing analysis revealed that cluster of differentiation 36 (CD36), a free fatty acid (FA) transport protein, was involved in the Ses-mediated inhibition of hepatic fat accumulation. Moreover, the overexpression of CD36 significantly increased hepatic steatosis in both Ses-treated PA/OA-incubated HepG2 cells and NASH mice. Furthermore, Ses treatment suppressed insulin-induced de novo lipogenesis in HepG2 cells, which was reversed by CD36 overexpression. Mechanistically, we found that Ses ameliorated NASH by inhibiting CD36-mediated FA uptake and upregulation of lipogenic genes, including FA synthase, stearoyl-CoA desaturase 1, and sterol regulatory element-binding protein 1. The findings of our study provide novel insights into the potential therapeutic applications of Ses in the treatment of NASH.

Cell nucleus targeting for living cell extraction of nucleic acid associated proteins with intracellular nanoprobes of magnetic carbon nanotubes.

Since nanoparticles could be ingested by cells naturally and target at a specific cellular location as designed, the extraction of intracellular proteins from living cells for large-scale analysis by nanoprobes seems to be ideally possible. Nucleic acid associated proteins (NAaP) take the crucial position during biological processes in maintaining and regulating gene structure and gene related behaviors, yet there are still challenges during the global investigation of intracellular NAaP, especially from living cells. In this work, a strategy to extract intracellular proteins from living cells with the magnetic carbon nanotube (oMWCNT@Fe3O4) as an intracellular probe is developed, to achieve the high throughput analysis of NAaP from living human hepatoma BEL-7402 cells with a mass spectrometry-based proteomic approach. Due to the specific intracellular localization of the magnetic carbon nanotubes around nuclei and its strong interaction with nucleic acids, the highly efficient extraction was realized for cellular NAaP from living cells, with the capability of identifying 2383 intracellular NAaP from only ca. 10,000 living cells. This method exhibited potential applications in dynamic and in situ analysis of intracellular proteins.

Polyacrylamide gel with switchable trypsin activity for analysis of proteins.

Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG-trypsin-aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline pH, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low pH. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis.