Leveraging Unidentified Metabolic Features for Key Pathway Discovery: Chemical Classification-driven Network Analysis in Untargeted Metabolomics.

Untargeted metabolomics using liquid chromatography-electrospray ionization-high-resolution tandem mass spectrometry (UPLC-ESI-MS/MS) provides comprehensive insights into the dynamic changes of metabolites in biological systems. However, numerous unidentified metabolic features limit its utilization. In this study, a novel approach, the Chemical Classification-driven Molecular Network (CCMN), was proposed to unveil key metabolic pathways by leveraging hidden information within unidentified metabolic features. The method was demonstrated by using the herbivore-induced metabolic response in corn silk as a case study. Untargeted metabolomics analysis using UPLC-MS/MS was performed on wild corn silk and two genetically modified lines (pre- and postinsect treatment). Global annotation initially identified 256 (ESI-) and 327 (ESI+) metabolites. MS/MS-based classifications predicted 1939 (ESI-) and 1985 (ESI+) metabolic features into the chemical classes. CCMNs were then constructed using metabolic features shared classes, which facilitated the structure- or class annotation for completely unknown metabolic features. Next, 844/713 significantly decreased and 1593/1378 increased metabolites in ESI-/ESI+ modes were defined in response to insect herbivory, respectively. Method validation on a spiked maize sample demonstrated an overall class prediction accuracy rate of 95.7%. Potential key pathways were prescreened by a hypergeometric test using both structure- and class-annotated differential metabolites. Subsequently, CCMN was used to deeply amend and uncover the pathway metabolites deeply. Finally, 8 key pathways were defined, including phenylpropanoid (C6-C3), flavonoid, octadecanoid, diterpenoid, lignan, steroid, amino acid/small peptide, and monoterpenoid. This study highlights the effectiveness of leveraging unidentified metabolic features. CCMN-based key pathway analysis reduced the bias in conventional pathway enrichment analysis. It provides valuable insights into complex biological processes.

Enhancing Metabolome Annotation by Electron Impact Excitation of Ions from Organics-Molecular Networking.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) is widely used in untargeted metabolomics, but large-scale and high-accuracy metabolite annotation remains a challenge due to the complex nature of biological samples. Recently introduced electron impact excitation of ions from organics (EIEIO) fragmentation can generate information-rich fragment ions. However, effective utilization of EIEIO tandem mass spectrometry (MS/MS) is hindered by the lack of reference spectral databases. Molecular networking (MN) shows great promise in large-scale metabolome annotation, but enhancing the correlation between spectral and structural similarity is essential to fully exploring the benefits of MN annotation. In this study, a novel approach was proposed to enhance metabolite annotation in untargeted metabolomics using EIEIO and MN. MS/MS spectra were acquired in EIEIO and collision-induced dissociation (CID) modes for over 400 reference metabolites. The study revealed a stronger correlation between the EIEIO spectra and metabolite structure. Moreover, the EIEIO spectral network outperformed the CID spectral network in capturing structural analogues. The annotation performance of the structural similarity network for untargeted LC-MS/MS was evaluated. For the spiked NIST SRM 1950 human plasma, the annotation coverage and accuracy were 72.94 and 74.19%, respectively. A total of 2337 metabolite features were successfully annotated in NIST SRM 1950 human plasma, which was twice that of LC-CID MS/MS. Finally, the developed method was applied to investigate prostate cancer. A total of 87 significantly differential metabolites were annotated. This study combining EIEIO and MN makes a valuable contribution to improving metabolome annotation.

Deep Characterization of Serum Metabolome Based on the Segment-Optimized Spectral-Stitching Direct-Infusion Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Approach.

Direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (DI-FTICR MS) shows great promise for metabolomic analysis due to ultrahigh mass accuracy and resolution. However, most of the DI-FTICR MS approaches focused on high-throughput metabolomics analysis at the expense of sensitivity and resolution and the potential for metabolome characterization has not been fully explored. Here, we proposed a novel deep characterization approach of serum metabolome using a segment-optimized spectral-stitching DI-FTICR MS method integrated with high-confidence and database-independent formula assignments. With varied acquisition parameters for each segment, a highly efficient acquisition was achieved for the whole mass range with sub-ppm mass accuracy. In a pooled human serum sample, thousands of features were assigned with unambiguous formulas and possible candidates based on highly accurate mass measurements. Furthermore, a reaction network was used to select confidently unique formulas from possible candidates, which was constructed by unambiguous formulas and possible candidates connected by the formula differences resulting from biochemical and MS transformation. Compared with full-range and conventional segment acquisition, 8- and 1.2-fold increases in observed features were achieved, respectively. Assignment accuracy was 93-94% for both a standard mixture containing 190 metabolites and a spiked serum sample with the root mean square mass error of 0.15-0.16 ppm. In total, 3534 unequivocal neutral molecular formulas were assigned in the pooled serum sample, 35% of which are contained in the HMDB. This method offers great enhancement in the deep characterization of serum metabolome by DI-FTICR MS.

Effects of posterior lumbar plexus block on anesthesia and sedation in postmenopausal patients with osteoporotic subtrochanteric comminuted fractures.

To study the effect of posterior lumbar plexus nerve block on anaesthesia and sedation in postmenopausal patients with osteoporotic subtrochanteric femoral comminuted fractures. The research subjects selected 48 patients with postmenopausal osteoporotic subtrochanteric comminuted fractures who were hospitalized between January 2020 and January 2022, and were allocated to clusters according to the random number TBL approach. The controlling cluster (24 situations) underwent dura mater Under external anesthesia, the test cluster (24 situations) underwent posterior lumbar plexus block, and the block effect, anesthesia effect, sedation effect, hemodynamics, vital signs and reactions of adverse nature were contrasted involving the two clusters. In comparison to the control group, the test group had a longer duration of anesthesia and motor block, higher oxygenation indices but lower ITBVI, GEDVI, and ScrO2 values, lower MAP levels, and lower BIS contraction values at 5, 15, and 30 minutes following anesthesia (P < 0.05). The test group had shorter induction time and block onset time compared to the control group (P < 0.05), and a lower incidence of adverse reactions (16.67% vs. 29.17% in the control group), but the variation was not noTBL (P < 0.05). Posterior lumbar plexus nerve block in postmenopausal patients with osteoporotic subtrochanteric femoral comminuted fractures has a better sedative effect, shortens the induction time of anaesthesia and the onset of block, promotes sTBL haemodynamic indexes and has fewer adverse effects to ensure safety.

A Strategy for Uncovering the Serum Metabolome by Direct-Infusion High-Resolution Mass Spectrometry.

Direct infusion nanoelectrospray high-resolution mass spectrometry (DI-nESI-HRMS) is a promising tool for high-throughput metabolomics analysis. However, metabolite assignment is limited by the inadequate mass accuracy and chemical space of the metabolome database. Here, a serum metabolome characterization method was proposed to make full use of the potential of DI-nESI-HRMS. Different from the widely used database search approach, unambiguous formula assignments were achieved by a reaction network combined with mass accuracy and isotopic patterns filter. To provide enough initial known nodes, an initial network was directly constructed by known metabolite formulas. Then experimental formula candidates were screened by the predefined reaction with the network. The effects of sources and scales of networks on assignment performance were investigated. Further, a scoring rule for filtering unambiguous formula candidates was proposed. The developed approach was validated by a pooled serum sample spiked with reference standards. The coverage and accuracy rates for the spiked standards were 98.9% and 93.6%, respectively. A total of 1958 monoisotopic features were assigned with unique formula candidates for the pooled serum, which is twice more than the database search. Finally, a case study of serum metabolomics in diabetes was carried out using the developed method.

Effect of Dietary Supplemental Zinc on Laying Performance, Egg Quality, and Plasma Hormone Levels of Breeding Pigeons.

This study aimed to evaluate the dietary zinc requirement of parental pigeons for better laying and reproductive performance, egg quality, sex hormones, and mineral content in eggs. A total of 160 pairs of healthy American Silver King pigeons were randomly assigned to five treatments of eight replicate cages each with four pairs of birds per cage, and fed a basal diet without zinc supplementation or the basal diet supplemented with 30, 60, 90, and 120 mg of zinc/kg (ZnSO4·7H2O). The experiment lasted for 45 days, including two laying cycles. Results indicated the egg production rate (P = 0.081), egg shape index (P = 0.038), egg eggshell percentage (P = 0.070), and zinc and calcium contents (P < 0.01) tended to be affected or significantly affected by zinc addition. They increased quadratically with dietary zinc levels (P < 0.05). Besides, shell thickness (P = 0.069), plasma testosterone (P = 0.008), LH, and carbonic anhydrase contents (P < 0.05) tended to be affected or significantly affected by zinc addition. They increased linearly as dietary zinc level increased (P < 0.05). Compared with the control, 60 mg/kg zinc addition increased egg production rate, egg shape index, zinc and calcium contents in eggshell, and plasma testosterone concentration in pigeons (P < 0.05), and tended to increase the eggshell percentage (P = 0.07). Besides, supplemental 120 mg/kg zinc had higher shell thickness and LH content than control (P < 0.05), but had no difference with 60 mg/kg zinc addition. In conclusion, the supplementation of zinc at the level of 60 mg/kg to basal diet improved laying performance by increasing eggshell quality and sex hormone levels of breeding pigeons.

Novel Method for Comprehensive Annotation of Plant Glycosides Based on Untargeted LC-HRMS/MS Metabolomics.

Glycosides are a large family of secondary metabolites in plants, which play a critical role in plant growth and development. Due to the complexity and diversity in structures and the limited availability of authentic standards, comprehensive annotation of the glycosides remains a great challenge. In this study, using maize as an example, a deep annotation method of glycosides was proposed based on untargeted liquid chromatography-high-resolution tandem mass spectrometry metabolomics analysis. First, knowledge-based in silico aglycone and glycosyl/acyl-glycosyl libraries were built. A total of 1240 known and potential aglycones from databases and literature were recorded. Next, the MS parameters beneficial to aglycone ion-rich MS/MS were explored using 1782 high-resolution MS/MS spectra of glycosides from the MassBank of North America (MoNA) and confirmed by 52 authentic glycoside standards. Then, screening rules for aglycon ions in MS/MS were recommended. Glycoside candidates were further filtered by MS/MS-based chemical classification and MS/MS similarity of aglycon-glycoside pairs. Finally, the glycosylation sites of flavonoid mono-O-glycosides were recommended by characteristic fragmentation patterns. The developed method was validated using glycosides and nonglycosides from the MoNA library. The annotation accuracy rates were 96.8, 94.9, and 98.0% in negative ion mode (ESI-), positive ion mode (ESI+), and the combined ESI- & ESI+, respectively. The annotation specificity was 99.6% (ESI-), 99.6% (ESI+), and 99.2% (ESI- & ESI+). A total of 274 glycosides (including 34 acyl-glycosides) were tentatively annotated in maize by the developed method. The method enables effective and reliable annotation for plant glycosides.

In-depth characterization of nitrogen heterocycles of petroleum by liquid chromatography-energy-resolved high resolution tandem mass spectrometry.

With the increased attention to processing heavy crude oils, a detailed description of chemical composition is critical for the petroleum refining industry. The current analytical technique such as ultrahigh resolution mass spectrometry has been successfully applied for the molecular level characterization of complex petroleum fractions. But the structural characterization of heavy petroleum feedstock is still a great challenge. In this study, a novel in-depth characterization method of nitrogen heterocycles (N-heterocycles) in heavy petroleum mixtures was proposed by online liquid chromatography coupled with electrospray ionization high resolution energy-resolved mass spectrometry. A series of typical basic aromatic, neutral aromatic and naphtheno-aromatic nitrogen heterocyclic model compounds were synthesized to investigate energy-resolved fragmentation behaviors in high energy collision-induced dissociation at 10-100 eV. Energy-dependent fragmentation pathways were elucidated. Notably, characteristic double bond equivalent (DBE) versus carbon number distributions of N1 ions and all CH ions were discovered, which were closely related to their core structure. Then a workflow to assign core structures of alkyl-substituted N-heterocycles in petroleum was proposed and validated. The developed method was applied to investigate the structural isomers in feed and product vacuum gas oil (VGO) fractions. Core structural differences in feed VGO and subtle structural variations between feed and product VGOs were recognized. This work can distinguish structural isomers of N-heterocycles with the subtle difference in their core structure in heavy petroleum fractions based on global energy dimensional fragmentation characteristics.

Influence of dietary phosphorus concentrations on the performance of rearing pigeons (Columba livia), and bone properties of squabs.

The objective of this study was to investigate the effects of dietary P levels on the performance of rearing pigeons, and bone characteristics of squabs from 7 to 21 d of age. A total of 192 pairs of adult Silver King pigeons (40 wk of age) were used. The pigeons were randomly allocated to one of 4 treatment groups, each consisting of eight replicates of 6 pigeon pairs per replicate. Dietary treatments included the basal diet (containing 0.3% of P), the basal diet supplemented with 0.2, 0.4, or 0.8% inorganic P. And the dietary Ca content was kept at 1.40% across all treatments. The experimental diets were fed to parent pigeons as corn-soybean complete pellet feed, and squabs fed with crop milk secreted by parent pigeons. Pigeons in the group of 0.4% supplemental non-phytate phosphorus (NPP) had shorter (P = 0.045) oviposition interval than those in the control group and group of 0.8% NPP. When the diet was supplemented with 0.8% of NPP, the least average egg weight was observed (P = 0.006). Female breeding birds had much higher (P < 0.01) Ca, P, and ALP in serum than male ones. At 7-d of age, dietary P supplementation influenced P and Ca content in tibia ash of squabs (P < 0.05). The tibia ash Ca content in the group of 0.2% NPP was the highest among the treatments (P = 0.007). At d 21 of age, both the birds in the group of 0.4 and 0.8% NPP had higher tibia breaking strength (P < 0.01) and tibia ash contents (P < 0.001) compared to the ones in the control group. In conclusion, the P deficiency in the diet of parent pigeons could cause poor bone mineralization of squabs, especially impaired the bone-breaking strength and bone ash content. The 0.8% of NPP supplementation in the diet has a positive influence on mineralization of squabs although production depression was observed. Both P and Ca metabolism of female breeding birds were more active than male ones at earlier time points of rearing period. The desirable supplemental NPP level in diet for breeding pigeon was 0.4% according to the performance data in the present trial. The recommended Ca: P ratio for pigeons, which was different from the optimum value for broilers, needs to be studied in the future.

Protective Effects of Zinc on Salmonella Invasion, Intestinal Morphology and Immune Response of Young Pigeons Infected with Salmonella enterica Serovar Typhimurium.

The study aimed to determine the effects of orally supplemental zinc on body weight, Salmonella invasion, serum IgA, intestinal histomorphology, and immune response of Salmonella enterica serovar Typhimurium (S. typhimurium)-challenged young pigeons. A total of 72 healthy White King pigeons (25 days old) with similar weight were randomly assigned to 3 treatments with six replicate cages. The 3 treatments were unchallenged, S. typhimurium-challenged, and S. typhimurium-challenged orally supplemented with 1 mg zinc per bird. Salmonella infection decreased (P < 0.05) the body weight, the bursa index, the serum IgA content, and the villus height/crypt depth ratio in the ileum, but increased the neutrophil proportion (P < 0.001) and the mRNA expressions of IL-1ß and IL-8 in the jejunum (P < 0.05). Orally supplemental zinc reduced (P = 0.007) the bacterial load in the liver and improved (P < 0.05) the body weight, the bursa index, the serum IgA content, the villus height/crypt depth ratio, and the NOD-like receptor family pyrin domain containing 3 (NLRP3) protein expression, as well as tended to increase (P = 0.064) the protein abundance of caspase-1 of the jejunum, but did not alleviate the high level of neutrophil proportion and IL-1ß mRNA expression of the jejunum (P > 0.05). The results indicated that oral zinc supplementation improved the intestinal mucosal morphology and enhanced the immune response, as well as activated caspase-1-dependent cell pyroptosis pathways in the jejunal epithelium, thereby restricting Salmonella invasion of the challenged young pigeons.

Effects of Yucca schidigera extract on serum biochemical parameters, humoral immune response, and intestinal health in young pigeons.

Introduction: It is of great importance to find antibiotic alternatives that can improve poultry performance and enhance immunity. Plant-derived extracts and their concentrates are natural bioactive compounds that are widely and effectively applied as the antibiotic alternatives in animal industries. This study was conducted to investigate the effects of Yucca schidigera extract (YSE) on growth performance, serum biochemical parameters, immune function, intestinal morphology, and microbiota diversity of young pigeons. Methods: A total of 120 healthy White King pigeons (28 days old) with similar weight were randomly assigned to 4 treatments with six replicate cages. Each of the pigeons from 4 treatments was orally administrated with 0 (control), 5, 10, and 15 mg YSE per day, respectively. Results: The results showed that orally supplemental YSE had no significant effects (P > 0.05) on the growth performance and immune organ index of pigeons. The serum total protein and IgM contents in the 10 mg YSE group were significantly higher (P < 0.05) than those in the control group. Supplemental 10 and 15 mg YSE significantly lowered the level of serum total cholesterol (P < 0.05) and increased (P < 0.05) the villi height in the jejunum compared with the control group. Supplemental 5 and 10 mg YSE significantly decreased (P < 0.05) the level of serum alanine aminotransferase and the crypt depth in the ileum compared with the control group. The beta diversity showed a distinct difference in the ileum microbial composition between the control and the 10 mg YES group. YSE supplementation enriched the bacterial genera Sulfurospirillum, Solobacterium, Desulfovibrio, Desulfobulbus, Lactococcus, Parabacteroides, Acidaminococcus, Acetobacter, and Streptococcus. Additionally, Enterococcus genus showed a significantly negative correlation with serum alanine aminotransferase (R = -0.618, P = 0.043). Actinomyces genus showed a significantly negative correlation with cholesterol (R = -0.633, P = 0.036). Turicibacter genus showed a significantly positive correlation with villi height in the jejunum (R = 0.751, P = 0.008). Discussion: In conclusion, orally supplemental YSE could improve serum biochemistry, immunoglobulin contents, and intestinal morphology by regulating the composition of microbial community in the ileum of young pigeons.

Exploration of Proteomics Analysis of Crop Milk in Pigeons (Columba livia) during the Lactation Period.

Pigeon milk is a curdlike substance separated from the mature crop epithelium of breeders, associated with the rapid growth and development of squabs. The aim of this study was to investigate in detail the variations in the content of several important ingredients in crop milk. In this study, we utilized proteomic techniques to investigate the composition and changing pattern of crop milk protein of squabs on days 1 (D1), 3 (D3), and 7 (D7). Our results indicated that the crude protein contents in crop milk decreased with age, and they were up to 50% during the first 3 days. The proteomic data showed that a total of 2558 proteins were identified in all samples from three stages, and the top 15% crop milk proteins were ribosomal protein, keratin, peroxiredoxin, annexin, heat shock protein, and eukaryotic translation protein based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis and normalized spectral abundance factors (NSAFs) calculation. Furthermore, the compositions of crop milk protein between D1 and D3 were quite similar [51 differentially expressed proteins (DEPs)], while great proteomic differences were observed between D1/D3 and D7 (more than 240 DEPs). Additionally, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that up-regulated DEPs mainly participate in immune response, while down-regulated DEPs were involved in cell differentiation and development as well as tRNA aminoacylation biosynthesis. In conclusion, DEPs were mainly related to protein synthesis, immunity, and antioxidation, which provided effective information for the development of artificial squab milk products in the future.

Strategy for Nontargeted Metabolomic Annotation and Quantitation Using a High-Resolution Spectral-Stitching Nanoelectrospray Direct-Infusion Mass Spectrometry with Data-Independent Acquisition.

Direct-infusion nanoelectrospray ionization high-resolution mass spectrometry (DI-nESI-HRMS) is an alternative approach to chromatography-MS-based techniques for nontargeted metabolomics, offering a high sample throughout. However, its annotation accuracy of analytes is still full of challenges. In this study, we proposed a strategy for the annotation and quantitation of nontargeted metabolomic data using a spectral-stitching DI-nESI-HRMS with data-independent acquisition. The metabolite annotation strategy included the isotopic distribution, MS/MS spectrum similarity, and precursor and product ion correlation as well as matching of the extracted metabolite features along with the targeted metabolite precursors. Two groups of mixed standard solutions containing 40 and 79 metabolites were, respectively, used to establish the metabolite annotation strategy and validate its reliability. The results showed that the detected standards could be well annotated at top three explanations and total qualitative percentages were 100% (40 of 40) for the standard solution and 94.9% (74 of 78) for the standards spiked into the serum matrix. The intensity of the precursor ions was used for quantitation except for isomers, which were quantified by the intensities of the characteristic product ions if available. Finally, the strategy was applied to study serum metabolomics in diabetes, and the results demonstrated that it is promising for a large-scale cohort metabolomic study.

Protein profiling analysis based on matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry and its application in typing Streptomyces isolates.

Marine Streptomyces is a potential source of novel bioactive natural products in medicine and agriculture. The current discrimination and screening method of Streptomyces isolates is not accurate and time-consuming, and a novel method is necessary. In this study, a protein profiling method based on an ultrahigh resolution 15 T Matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR MS) was established and applied for differentiation and bioactivity screening of marine Streptomyces isolates. To obtain robust protein profiling, the effects of the protein extraction method, the matrix-solvent, the sample deposition mode, and the culture time of isolates on protein profiling were thoroughly studied, the optimal conditions were obtained. To evaluate the performance of the developed MALDI-FTICR MS method, MALDI-time of flight (TOF) MS and 16S rRNA were applied in parallel to analyze 25 marine Streptomyces isolates. We found that the clustering result of MALDI-FTICR MS was more similar to that of 16S rRNA than MALDI-TOF MS. And MALDI-FTICR MS could effectively indicate the antibacterial activity of Streptomyces isolates against three plant pathogenic bacteria including Xanthomonas campestris, Xanthomonas oryzae and Erwinia carotovora. Furthermore, a differential protein/peptide was defined and successfully applied to predict antibacterial activity of blind samples. This study demonstrated that MALDI-FTICR MS has great potential to discriminate and screen complex microorganisms, especially those closely related strains.

Carboxymethylated polyethylenimine modified magnetic nanoparticles specifically for purification of His-tagged protein.

Employing immobilized metal-ion affinity chromatography and magnetic separation could ideally provide a useful analytical strategy for purifying His-tagged protein. In the current study, a facile route was designed to prepare CMPEI-Ni2+ @SiO2 @Fe3 O4 (CMPEI=carboxymethylated polyethyleneimine) magnetic nanoparticles composed of a strong magnetic core of Fe3 O4 and a Ni2+ -immobilized carboxymethylated polyethyleneimine coated outside shell, which was formed by electrostatic interactions between polyanionic electrolyte of carboxymethylated polyethyleneimine and positively charged surface of 3-(trimethoxysilyl)propylamin modified SiO2 @Fe3 O4 . The resulting CMPEI-Ni2+ @SiO2 @Fe3 O4 composite nanoparticles displayed well-uniform structure and high magnetic responsiveness. Hexa His-tagged peptides and purified His-tagged recombinant retinoid X receptor alpha were chosen as the model samples to evaluate the adsorption, capacity, and reusability of the composite nanoparticles. The results demonstrated the CMPEI-Ni2+ @SiO2 @Fe3 O4 nanoparticles possessed rapid adsorption, large capacity, and good recyclability. The obtained nanoparticles were further used to purify His-tagged protein in practical environment. It was found that the nanoparticles could selectively capture His-tagged recombinant retinoid X receptor protein from complex cell lysate. Owing to its easy synthesis, large binding capacity, and good reusability, the prepared CMPEI-Ni2+ @SiO2 @Fe3 O4 magnetic nanoparticles have great potential for application in biotechnological fields.

Deep Annotation of Hydroxycinnamic Acid Amides in Plants Based on Ultra-High-Performance Liquid Chromatography-High-Resolution Mass Spectrometry and Its In Silico Database.

Hydroxycinnamic acid amides (HCAAs), diversely distributed secondary metabolites in plants, play essential roles in plant growth and developmental processes. Most current approaches can be used to analyze a few known HCAAs in a given plant. A novel method for comprehensive detection of plant HCAAs is urgently needed. In this study, a deep annotation method of HCAAs was proposed on the basis of ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) and its in silico database of HCAAs. To construct an in silico UHPLC-HRMS HCAAs database, a total of 846 HCAAs were generated from the most common phenolic acid and polyamine/aromatic monoamine substrates according to possible biosynthesis reactions, which represent the structures of plant-specialized HCAAs. The characteristic MS/MS fragmentation patterns of HCAAs were extracted from reference mixtures. Four quantitative structure-retention relationship (QSRR) models were developed to predict retention times of mono-trans-HCAAs (aromatic amines conjugates), mono-trans-HCAAs (aliphatic amines conjugates), bis-HCAAs, and tris-HCAAs. The developed method was applied for identifying HCAAs in seeds (maize, wheat, and rice), roots (rice), and leaves (rice and tobacco). A total of 79 HCAAs were detected: 42 of them were identified in these plants for the first time, and 20 of them have never been reported to exist in plants. The results showed that the developed method can be used to identify HCAAs in a plant without prior knowledge of HCAA distributions. To the best of our knowledge, it is the first UHPLC-HRMS database developed for effective deep annotation of HCAAs from nontargeted UHPLC-HRMS data. It is useful for the identification of novel HCAAs in plants.

Propofol Promotes Activity and Tumor-Killing Ability of Natural Killer Cells in Peripheral Blood of Patients with Colon Cancer.

BACKGROUND We investigated the effect of propofol on activities and tumor-killing ability of natural killer (NK) cells in patients with colon cancer. MATERIAL AND METHODS Twenty colon cancer patients and 20 healthy subjects were included. Peripheral blood (5 ml) was collected from all patients and healthy subjects. NK cells in peripheral blood were separated by negative screening using immunomagnetic beads. Flow cytometry was used to determine expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces. After in vitro treatment with propofol for 24 h, expression of activated receptors, inhibitory receptors, killing effector molecules, and proliferation-associated markers on NK cell surfaces was examined again. In addition, the tumor-killing effect of NK cells was studied by co-culture with K562 cells or colon cancer SW620 cells at a ratio of 1: 1. RESULTS The number of NK cells in peripheral blood from colon cancer patients was increased compared with healthy subjects, but activities and proliferation ability of the NK cells were decreased. The tumor-killing effect of NK cells isolated from colon cancer patients was decreased. Of note, propofol promoted activation of NK cells from colon cancer patients. In addition, propofol increased expression of tumor-killing effector molecules by NK cells and the proliferation ability of NK cells. Propofol also enhanced the killing effect of NK cells on colon cancer cells. CONCLUSIONS The present study demonstrates that propofol promotes the activity and tumor-killing ability of NK cells in peripheral blood of patients with colon cancer.