OBJECTIVE: To observe the effect of electroacupuncture (EA) on behavior and hippocampal protein phosphorylation in rats with chronic fatigue syndrome (CFS), so as to explore its mechanisms underlying improvement of CFS.
METHODS: Male SD rats were randomly divided into control, model and EA groups (n=12 rats in each group). The CFS model was established by chronic multifactor combined with stress stimulation (treadmill training + restraint stress + sleep disturbance + crowded environment). For rats of the EA group, EA (1 mA, frequency of 10 Hz) was applied to \"Shenting\" (GV24) (with an acupuncture needle penetrated from GV24 to \"Baihui\" ［GV20］) and \"Dazhui\" (GV14) for 15 min, once daily for 28 days. After treatment, the body weight, food intake and water intake of rats in each group were observed. The fatigue degree of rats was evaluated by Semi-quantitative score observation table of the general condition of experimental rats.The open field test (OFT) was used to assess the rats\'anxiety severity by detecting the total number of grid-crossing and the times of the central area entered in 5 min, and Morris water maze test was employed to assess the rats\' learning-memory ability by detecting the escape latency in 1 min, and the times of the original platform quadrant crossing in 1 min. The hippocampaus was taken for phosphorylated Label-free quantitative proteomics analysis by using Maxquant technology based on full scan mode to calculate the integral of each peptide signal of liquid chromatography-mass spectrometry(LC-MS). The differentially-expressed proteins (>1.5 folds for up-regulation or <0.67 folds for down-regulation) were evaluated by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis.
RESULTS: Compared with the control group, the body weight, food intake, and the times of original-platform quadrant crossing of spatial exploring of Morris water maze test were significantly decreased (P<0.01, P<0.05) , and the score of general conditions, times of grid-crossing and center area-entering of OFT, and the escape latency of navigation task were apparently increased (P<0.01) in rats of the model group. After EA intervention, the decreased original-platform quadrant crossing, and the increased score of general conditions, times of grid-crossing and the escape latency of navigation task were all reversed (P<0.01, P<0.05). Outcomes of proteomics analysis indicated that compared with the model group, there were 297 differentially expressed peptide (48 up-regulated and 249 down-regulated) segments in the control group, and there were 245 differentially expressed peptide (185 up-regulated and 60 down-regulated) segments in the EA group, in which, 25 overlapping peptide segments were reversed after EA treatment, corresponding to 24 proteins, mainly involving cytoskeletal structure. GO function annotation analysis showed that the top three differentially expressed phosphorylated proteins involved in the effect of EA intervention were the actin filament polymerization, protein depolymerization and cytoskeletal tissue in the biological process, the actin binding, structural molecular activity and cytoskeletal protein binding in the molecular function, and the cytoskeleton, dendrites and dendritic trees in the cellular component, respectively. The KEGG pathway annotation analysis for differentially expressed phosphorylated proteins showed that theinsulin secretion, axon guidance, phosphatidylinositol signaling system and lysine biosynthesis, etc. were involved in the effect of EA intervention.
CONCLUSIONS: EA of GV24-GV20 and GV14 can improve the general state, anxiety and learning-memory ability of CFS model rats, which may be related to its functions in regulating the hippocampal protein phosphorylation level, and repairing the structure and function of synapses in hippocampus.
目的: 观察电针对慢性疲劳综合征（CFS）大鼠行为学和海马组织蛋白磷酸化的影响，探讨电针治疗CFS的作用机制。方法: 将雄性SD大鼠随机分为空白组、模型组和电针组，每组12只。采用多因素慢性复合应激法制备CFS模型。电针组大鼠给予电针“神庭”（透刺“百会”）和“大椎”治疗，每日1次，每次15 min，连续28 d。治疗结束后观察各组大鼠体质量、摄食量和饮水量，采用实验大鼠一般情况半定量评分观察表评价大鼠的疲劳程度，采用旷场实验及Morris水迷宫实验评价大鼠焦虑程度和学习记忆能力。取各组大鼠海马组织进行磷酸化Label-free定量蛋白质组学检测。结果: 治疗结束后，与空白组比较，模型组大鼠体质量降低（P<0.01），摄食量减少（P<0.05），一般情况半定量评分升高（P<0.01），旷场实验总穿格次数和进入中央区次数均增多（P<0.01），Morris水迷宫实验逃避潜伏期延长（P<0.01）、穿越原平台次数减少（P<0.01）。与模型组比较，电针组大鼠一般情况半定量评分降低（P<0.01），旷场实验总穿格次数减少（P<0.05），Morris水迷宫实验逃避潜伏期缩短（P<0.01）、穿越原平台次数增加（P<0.01）。与空白组比较，模型组有297个差异表达肽段，对应255个蛋白，与模型组比较，电针组有245个差异表达肽段，对应198个蛋白，其中共有24个重合蛋白在电针治疗后表达回调。GO分析表明，电针干预的作用在生物学过程方面主要为对蛋白质聚合和细胞骨架组织的调节，在分子功能聚类上表现为对蛋白质聚合的调节，在细胞组分聚类方面表现为对突触功能的影响。KEGG分析结果显示，电针干预对胰岛素分泌、轴突导引、磷脂酰肌醇信号系统及赖氨酸生物合成等对中枢神经系统组织功能有重要作用的信号通路产生了影响。结论: 电针可以改善CFS模型大鼠的疲劳状态，缓解焦虑情绪，提高学习记忆能力，其机制可能与调节海马组织蛋白磷酸化水平有关。.

Background: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune central nervous system (CNS) inflammatory and demyelinating disorder that can lead to serious disability and mortality. Humoral fluid biomarkers with specific, convenient, and efficient profiles that could characterize and monitor disease activity or severity are very useful. We aimed to develop a sensitive and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS)/MS-based analytical method for novel biomarkers finding in NMOSD patients and verified its function tentatively. Methods: Serum samples were collected from 47 NMOSD patients, 18 patients with other neurological disorders (ONDs), and 35 healthy controls (HC). Cerebrospinal fluid (CSF) samples were collected from 18 NMOSD and 17 OND patients. Three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) and nine important metabolites that included phenylacetylglutamine (PAGln), indoleacrylic acid (IA), 3-indole acetic acid (IAA), 5-hydroxyindoleacetic acid (HIAA), hippuric acid (HA), I-3-carboxylic acid (I-3-CA), kynurenine (KYN), kynurenic acid (KYNA), and quinine (QUIN) were analyzed by using the liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method. The profile of IA was further analyzed, and its function was verified in an astrocyte injury model stimulated by NMO-IgG, which represents important events in NMOSD pathogenesis. Results: In the serum, tyrosine and some of the tryptophan metabolites IA and I-3-CA decreased, and HIAA increased significantly in NMOSD patients. The CSF levels of phenylalanine and tyrosine showed a significant increase exactly during the relapse stage, and IA in the CSF was also increased markedly during the relapse and remission phases. All conversion ratios had similar profiles with their level fluctuations. In addition, the serum IA levels negatively correlated with glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) levels in the serum of NMOSD patients were measured by using ultra-sensitive single-molecule arrays (Simoa). IA showed an anti-inflammatory effect in an in vitro astrocyte injury model. Conclusion: Our data suggest that essential aromatic amino acid tryptophan metabolites IA in the serum or CSF may serve as a novel promising biomarker to monitor and predict the activity and severity of NMOSD disease. Supplying or enhancing IA function can promote anti-inflammatory responses and may have therapeutic benefits.

An immobilized metal affinity (IMAC) adsorbent was prepared for selective enrichment of adenine type CKs, via grafting polymer chain pendant with iminodiacetic acid (IDA) from polydopamine (PDA)/polyethyleneimine (PEI)-coated magnetic graphene oxide (magGO) via surface-initiated-atom transfer radical polymerization (SI-ATRP). The prepared IMAC sorbent exhibited remarkable adsorption performances and good selectivity for adenine-type CKs and was utilized as a sorbent of magnetic solid-phase extraction (MSPE) for effective enrichment of four adenine-type CKs in bean sprouts. Under the optimized extraction conditions, an analytical method for four adenine type CKs in bean sprouts was established by combining the MSPE combined with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The recoveries of the analytes were between 80.4 ± 1.9% and 114.6 ± 1.5% (n = 3). The limits of detection (LODs) range from 0.63 to 2.30 pg⋅mL-1. The relative standard deviations of intra-day and inter-day were less than 12.6%. The established method was successfully applied to the selective extraction and sensitive detection of trace adenine-type CKs in plant samples.

BACKGROUND: Takeda G protein receptor 5 (TGR5) is widely recognized as a potential drug target for the treatment of metabolic diseases. TGR5 is not only a metabolic regulator, but also has a potential role that participating in developing and progressing of gastrointestinal cancer. We aimed to investigate the potential role of TGR5 in pancreatic cancer by utilizing molecular experiments and the liquid chromatography mass spectrometry (LC-MS) based metabolomics.
METHODS: Herein, we assessed pancreatic cancer proliferation, migration and invasion in response to TGR5 antagonist SBI-115 in vitro experiments. Cell death was examined by using TUNEL assay on agarose-embedded sections. Then we investigated the effects of TGR5 on PANC-1 and BXPC3 cells via transmission electron microscopy (TEM). Moreover, LC-MS-based metabolomics was performed to explore the potential underlying mechanisms of TGR5 in pancreatic cancer. The correlations between TGR5 and the metabolism-related genes were further analysed by GEPIA 2 database.
RESULTS: We found the proliferation capacities were decreased significantly in PANC-1 and BXPC3 cells after the treatment of SBI-115 for 48 h. The results of TUNEL assay showed that antagonism of TGR5 by SBI-115 had a remarkable effect on inducing cell death. Analysis of TEM demonstrated that SBI-115 treatment could impair the morphology of mitochondria in most PANC-1 and BXPC3 cells. The LC-MS-based analyses revealed that antagonism of TGR5 could alter the metabolic profiles of PANC-1 cells in vitro. Moreover, TGR5 was associated with some metabolism-related genes in pancreatic cancer.
CONCLUSIONS: Our data suggests that antagonism of TGR5 may suppress cell proliferation and induce apoptosis in pancreatic cancer cells. TGR5 may affect the metabolism of pancreatic cancer, and TGR5 would be an attractive target for pancreatic cancer treatment.

Liquid chromatography-mass spectrometry was used to study the changes of lipids in salmon muscle stored at 4 °C for different storage times to explore the relationship between lipid composition and salmon freshness. Ninety-two kinds of lipid changes were observed at three different storage times (5, 10, and 15 days) compared with the fresh control group (0 day). Bioinformatics analysis revealed that the contents of four lipids were significantly increased from the tenth day, namely, lysophosphatidylcholine (LPC) (17:0), LPC (18:0), LPC (22:2), and phosphatidylcholine (PC) (18:4/16:1). LPC (17:0) and LPC (18:0) are produced by PC (18:4/16:1) hydrolysis. The traditional freshness index also showed that the salmon slices were in the initial state of spoilage on the tenth day. Therefore, they may be indicators of raw salmon freshness.

The correlation among stationary phases, ion-pairing reagents (IPR) and sequences for ion-pair reversed-phase liquid chromatography mass spectrometry (IP-RP LC-MS) analysis of oligonucleotide (ODN) remains unclear. The present study aimed to evaluate such correlation using particle-packed C18 columns in order to search for the optimal combination among them. Five C18 columns packed with core-shell silica, polymer, porous silica and hybrid particles, respectively, were evaluated for the analysis of synthetic and chemically modified ODNs with six different IPRs. Our results showed that silica-based porous particles, compared to other particles, retained ODN the strongest no matter which IPR was used. Meanwhile, among the six IPRs hexylamine (HA) produced the longest retention for all ODNs, regardless of the types of C18 particles. For the separation of ODNs, C18 columns performed similarly under identical LC conditions. However, the separation ability of C18 columns is highly dependent on the type of IPR and ODN sequences. Moreover, the type of particles has little impact on the signals of ODNs for the majority of synthetic sequences, but such impact could be dramatic for chemically modified sequences. On the other hand, both the type of IPR and ODN sequence have a significant effect on MS signals for synthetic and chemically modified ODNs.

Sodium taurocholate cotransporting polypeptide (NTCP) is an important hepatocyte transporter, while its physiological functions require further investigation. In our study, an integrated plasma and liver GC-MS- and LC-MS-based metabolomics strategy with an optimized two-step liquid-liquid extraction was utilized to explore the physiological functions of NTCP via a knockout (KO) mouse model. The present study found that NTCP deficiency resulted in obvious metabolic change in the plasma and liver of mice. Totally, 102 and 87 differential metabolites were discovered in the liver and plasma, respectively. Pathway analysis revealed that the metabolism of tyrosine, glycine, taurine, fatty acid and glycerophospholipid as well as the biosynthesis of tryptophan, pantothenate and CoA were significantly dysregulated in the Ntcp KO mice, indicating that NTCP is closely involved in these metabolic pathways. Moreover, L-tryptophan, cadaverine and D-pantothenic acid could serve as the diagnostic biomarker for NTCP deficiency. Our study provided deep insights into the physiological functions of NTCP, and the findings would hold the great potential to be used for the discovery of new therapeutic and diagnostic strategies for NTCP deficiency clinically.

The incidence of thyroid cancer is increasing worldwide. So far, still no non-invasive clinical test biomarkers were developed for the diagnosis of thyroid cancer. The diiodothyronines (T2s) are precursors and metabolites of thyroid hormone (T4). Some reports predict that T2s may be associated with several thyroid diseases, especially the thyroid cancer. Detecting free T2s in human serum may help the diagnosis of thyroid cancer. However, few works have reported the detection of T2s due to their trace amounts. Here we developed a novel hyper organic cross-linked poly ionic liquid (PIL) material for the enrichment of three main compounds in T2s family, including 3,5- diiodothyronine (3,5-T2), 3\',5\'-diiodothyronine (3\',5\'-T2), and 3,5-diiodothyronamine (3,5-T2AM). This PIL material provided specific enrichment superiority for three T2s. After enrichment, the signal intensity of 3,5-T2, 3\',5\'-T2, and 3,5-T2AM increased 14, 132 and 1.6 folds, respectively, with LOQ of 76, 87, and 107 fM, respectively. Finally, we successfully applied PIL material coupled with HPLC-ESI-MS/MS in enrichment and quantitative determination of free 3,5-T2, 3\',5\'-T2, and 3,5-T2AM in human serum of 45 thyroid cancer patients and 15 healthy people. We also used free thyroid hormone (FT4) as the calibration reference to eliminate individual differences. We found that the levels of 3,5-T2 (P < 0.001), and 3\',5\'-T2 (P = 0.001) in patients with thyroid cancer were significantly higher than those in healthy people. Additionally, we further investigated the power of different T2 thyroid hormones divided FT4 to classify thyroid cancer patients and healthy people. And 3,5-T2/FT4 had the highest classification performance for discriminating thyroid cancer patients from healthy people at certain threshold, indicating that 3,5-T2/FT4 in human serum can act as potential biomarkers for \"non-invasive\" clinical diagnosis of thyroid cancer.

BACKGROUND: This article aims to compare and analyze the contents of ginsenosides in ginseng of different plant ages from different localities in China.
METHODS: In this study, 77 fresh ginseng samples aged 2-4 years were collected from 13 different cultivation regions in China. The content of eight ginsenosides (Rg3, Rc, Rg1, Rf, Rb2, Rb1, Re, and Rd) was determined using rapid resolution liquid chromatography coupled with quadrupole-time-of-flight tandem mass spectrometry (RRLC-Q-TOF MS/MS) to comparatively evaluate the influences of cultivation region and age.
RESULTS: Ginsenoside contents differed significantly depending on age and cultivation region. The contents of ginsenosides Re, Rc, Rg1, Rg3, and Rf increased with cultivation age, whereas that of ginsenoside Rb1 peaked in the third year of cultivation. Moreover, the highest ginsenoside content was obtained from Changbai (19.36 mg/g) whereas the lowest content was obtained from Jidong (12.05 mg/g). Ginseng from Jilin Province contained greater total ginsenosides and was richer in ginsenoside Re than ginseng of the same age group in Heilongjiang and Liaoning provinces, where Rb1 and Rg1 contents were relatively high.
CONCLUSIONS: In this study, RRLC-Q-TOF MS/MS was used to analyze ginsenoside contents in 77 ginseng samples aged 2-4 years from different cultivation regions. These patterns of variation in ginsenoside content, which depend on harvesting location and age, could be useful for interested parties to choose ginseng products according to their needs.

Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine, and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long-chain bases, steroids, and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun-based and liquid chromatography mass spectrometry-based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past 5 years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents, especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods.