查看更多>>摘要：In our previous study, activated carbon (AC) was employed in the purification process of therapeutic monoclonal antibody (mAb) as a replacement for Protein A affinity chromatography. In addition, we established an inno-vative column-free flow-through purification process using AC filter. In these investigations, the effective clearance of impurities (high-molecular-weight species, low-molecular-weight species, host cell proteins, and DNA) was observed compared to the conventional Protein A platform purification process. In this study, virus removal capability of our established AC process (AC filter) was investigated using two model viruses, Murine Leukemia Virus (MuLV) and Minute Virus of Mice (MVM) with the combination of two filtration methods (single -pass filtration and re-circulation filtration) using three kinds of mAbs. We found effective clearance of both MuLV and MVM (> 3 log reduction factor, LRF) in all mAbs. Not only filtration method but also re-circulation duration didn't affect LRF, and > 3 LRF of virus removal could be achieved by only single-pass filtration. From these results, it is expected that AC will be a promising candidate for the virus removal unit operation for mAb pu-rification processes.
查看更多>>摘要：Some natural products are important sources of treatments for hypertension based on their potential inhibitory effects on angiotensin-converting enzyme (ACE); however, it is difficult to identify natural ACE inhibitors (ACEIs) due to the complex secondary metabolite environment of natural products. Enzyme immobilization is an important method for screening active constituents in natural products, but this method can sometimes return false-positive and false-negative results. To improve the accuracy and reliability of ligand-fishing methods, we established a novel strategy based on enzyme-immobilized ligand fishing combined with active-site blocking and directional enrichment technologies. We first synthesized ACE-immobilized mesoporous magnetic beads and then verified the screened compounds by molecular docking and in vitro activity detection. We then used active-site blocking to exclude non-specific binding constituents and applied directional enrichment to enrich the low-content constituents for ligand fishing. The screening identified six potential ACEIs from Scutellariae Radix and eight potential ACEIs from Lonicerae japonicae flos, and their inhibitory activity was confirmed by molecular docking simulations and in vitro activity detection. This process screened six additional compounds and excluded two false-positive results as compared with results exclusively using enzyme immobilization. This strategy provides a feasible method for screening active compounds in natural products.
Hosny, Noha M. M.Abdelkarim, MahmoudGadallah, Mohamed I. I.Mousa, Heba Salah...
8页
查看更多>>摘要：A specific and sensitive thin layer chromatographic method coupled with fluorescence detection for determination of flibanserin (FLN) that treats woman hypoactive sexual desire disorder was developed. The proposed method depends on the enhancement of FLN native fluorescence intensity via the exposure of the developed TLC plate to concentrated hydrochloric acid vapors. Herein, an evaporation setup needed for HCl vapors exposure step was designed for the first time to ensure a uniform distribution of the vapors throughout the developed bands on the plate. Chloroform: methanol (9.5: 0.5, v/v) was the optimum mobile phase that gave a compact band (R-f= 0.44 +/- 0.02) using TLC aluminium plates precoated with silica gel G 60F254 as a stationary phase. After exposure of the developed TLC plate to HCl vapors, the FLN bands emission intensities were measured after excitation at 275 nm. Conferring ICH guidelines, the linearity range was 20.0 - 1500.0 ng/band with a good linear relationship (r= 0.9998). Detection and quantitation limits were 5.12 and 15.50 ng/band, respectively. Also, the method was validated for accuracy, precision, robustness, specificity and selectivity. Statistical analysis verified the suitability of the proposed method for estimation of FLN in tablets and in human plasma with acceptable recoveries (98.07-101.45%).
查看更多>>摘要：A new method involving gut microbiota biotransformation, spectrum-effect relationship analysis and metab-olomics analysis was developed to study the antitussive and expectorant microbial metabolites of platycosides fraction (MPFs) of Platycodonis Radix. Furthermore, their possible metabolic mechanisms were studied for the first time. The findings showed that the antitussive and expectorant effects of the platycosides fraction (PF) were significantly enhanced by the gut microbiota biotransformation. 11 active antitussive microbial metabolites and 12 active expectorant microbial metabolites, which shared 8 components, were successfully screened out via spectrum-effect relationship analysis. The prototypes of the active microbial metabolites could be reversely traced according to the gut microbiota biotransformation pathways. It was found out that one platycoside could produce several active microbial metabolites and several different platycosides could produce the same active microbial metabolite. In addition, the metabolomics analysis showed that both the PF and its active microbial metabolites could regulate the same metabolomic pathways of Linoleic acid metabolism, Arachidonic acid metabolism and Glycerophospholipid metabolism to exert antitussive activity, and regulate the same metab-olomic pathway of Arachidonic acid metabolism to exert expectorant activity. These findings suggested the microbial metabolites may be the active forms of the platycosides. Overall, the proposed approach was useful in screening the active microbial metabolites; this work explained the in vivo antitussive and expectorant metabolic mechanisms of multi-constituents, multi-targets and synergistic effects of PF of Platycodonis Radix.
查看更多>>摘要：Insulin is a peptide hormone that is secreted by the beta cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). Marmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin. Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled (C-13, N-15) synthetic marmoset insulin tryptic peptide was used as internal standard. The assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 15.49 pmol/L and the limit of quantitation was 140.78 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L. In summary, this paper presents a simple, reproducible method to measure marmoset insulin in serum using LC-MS/MS.
查看更多>>摘要：Mescaline, a natural alkaloid found in the peyote cactus (Lophophora williamsii) in the Americas, has gradually become a drug of abuse in China because of its psychedelic properties. Its intake may lead to hallucinations and confusion or even be life-threatening. Mescaline is classified as a class I psychotropic drug in China, which means its use in medicine or scientific research is under strict control of the government. However, studies on sur-veillance of mescaline abuse in the Chinese population are lacking. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the determination and quantification of mescaline in hair. The method had good linearity in the range from 10 to 1000 pg/mg, with the limit of detection (LOD) of 3 pg/mg and the limit of quantitation (LOQ) of 10 pg/mg. The total runtime was 5 min. Acceptable intraday and interday precision (RSD < 15%) and ac-curacy (bias,-11.2% ~ 6.8%) were achieved. The recovery was 85.0-101.0%, and the matrix effect was 92.0-105.0%. The validated method was successfully applied to 19 real forensic cases. The concentrations of mescaline in hair ranged from 10 to 784 pg/mg. The method has the benefits of simple sample preparation, high sensitivity, and short running time, making it suitable for large-scale quantitative surveillance analysis of mescaline in forensic toxicology.
查看更多>>摘要：Various snake species and snake predators have natural neutralization against snake toxins, which their antidotal abilities are commonly attributed to the intrinsic inhibitors produced by the liver, e.g., phospholipase A(2)inhibitor (PLI) and metalloproteinase inhibitor (SVMPI). Sinonatrix annularis was found to possess broad-spectrum neutralization to different snake venoms in our lab. Although the anti-venom compound PLI gamma has been previously characterized in our laboratory, the mechanism of resistance of S. annularis to snake venoms remains obscure. In this research, a venom affinity chromatography was constructed by immobilizing D. acutus venom to NHSagarose beads and applied for antitoxins mining from S. annularis. The binding capacity of the venom column was validated using a self-prepared rabbit antivenom against D. acutus. Serum and liver homogenate of S. annularis were then applied to the column, the bound components were profiled using SDS-PAGE and mass spectrometry. PLIs, snake venom metalloproteins inhibitor (SVMPI), small serum protein (SSP), heat shock proteins, etc were identified. To identify their toxin targets in D. acutus venom, a reverse separation was conducted by coupling the fractionated S. annularis serum proteins to NHS-agarose beads. Fifteen toxins of five families were captured and identified as follows: PLA(2s), metalloproteinases, cysteine-rich secretory proteins, snake venom serine proteinases, and C-type lectins. These discoveries increased our understanding of the capacity and mechanism of the natural neutralization of S. annularis to snake venom. These natural inhibitors are medically significant due to their powerful and broad antidotal activities, which may provide alternative and promising drug candidates for snakebite treatment.
查看更多>>摘要：Untargeted lipidomics using liquid chromatography high-resolution mass spectrometry (LC-HRMS) was performed using polarity switching, and in positive and negative polarity separately on the same set of serum samples, and the performances of the methods were evaluated. Polarity switching causes an increase in the cycle time of the HRMS measurements (1.18 s/cycle vs 0.27 s/ cycle), resulting in fewer data points across chromatographic peaks. The coefficient of variation (CV) was on average lower for the added isotopically labelled standards in pooled samples (QC) and patient samples using separate polarities (QC = 5.6%, samples = 12.5%) compared to polarity switching (QC = 8.5%, samples = 13.4%), but the difference was not statistically significant. For the endogenous features measured in the QCs polarity switching resulted in on average significantly higher CVs (3.80 (p = 4.25e-30) and 3.3 percentage points (p = 6.84e-40), for positive and negative modes, respectively) however still acceptable for an untargeted method (mean CVs of 17.9% and 12.2% in positive and negative modes respectively). A slightly larger number of endogenous features were detected using the separate polarities, but the large majority of features (> 95%) were detected with both methodologies. The overlap of features detected in both positive and negative polarities was low (4.1%) demonstrating the importance of using both polarities for untargeted lipidomics. When investigating the effects of a treatment on multiple sclerosis patients it was found that both methodologies gave highly similar biological results, further confirming the applicability of polarity switching.
NSTL
Elsevier