A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

The hidden diet: Synthetic antioxidants in packaged food and their impact on human exposure and health.

Synthetic antioxidants (AOs) are commonly used in everyday items and industrial products to inhibit oxidative deterioration. However, the presence of AOs in food packaging and packaged foods has not been thoroughly documented. Moreover, studies on human exposure to AOs through skin contact with packaging or ingesting packaged foods are limited. In this study, we analyzed twenty-three AOs-including synthetic phenolic antioxidants (SPAs) and organophosphite antioxidants (OPAs)-along with six transformation products in various food samples and their packaging materials. We found AOs in food products at concentrations ranging from 1.30 × 103 to 1.77 × 105 ng/g, which exceeded the levels in both outer packaging (6.05 × 102-3.07 × 104 ng/g) and inner packaging (2.27 × 102-1.09 × 105 ng/g). The most common AOs detected in foodstuffs were tris(2,4-di-tert-butylphenyl) phosphate (AO168O), butylated hydroxytoluene (BHT), and octadecyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (AO1076), together constituting 95.7 % of the total AOs found. Our preliminary exposure assessment revealed that dietary exposure-estimated at a median of 2.55 × 104 ng/kg body weight/day for children and 1.24 × 104 ng/kg body weight/day for adults-is a more significant exposure route than dermal contact with packaging. Notably, four AOs were identified in food for the first time, with BHT making up 76.8 % and 67.6 % of the total BHT intake for children and adults, respectively. These findings suggest that food consumption is a significant source of BHT exposure. The estimated daily intakes of AOs via consumption of foodstuffs were compared with the recommended acceptable daily intake to assess the risks. This systematic investigation into AOs contributes to understanding potential exposure and health risks associated with AOs in packaged foods. It emphasizes the need for further evaluation of human exposure to these substances.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

Actinidia eriantha polysaccharide exerts adjuvant activity by targeting linc-AAM.

Actinidia eriantha polysaccharide (AEPS) is a potent adjuvant with dual Th1 and Th2 potentiating activity. linc-AAM has been previously proved to facilitate the expression of immune response genes (IRGs) in AEPS-activated RAW264.7 macrophages. However, its role in mediating adjuvant activity of AEPS remains to be elucidated. In this study, bone marrow-derived macrophages (BMDMs) from wide-type (WT) and linc-AAM knockout C57BL/6J mice treated with AEPS were subjected to transcriptome sequencing and bioinformatic analysis. linc-AAM deficiency inhibited M1 and M2 immune responses in BMDMs induced by AEPS. In mechanisms, AEPS facilitated the expression of IRGs and activated BMDMs through NF-κB-linc-AAM-JAK/STAT axis. Furthermore, linc-AAM knockout inhibited cytokine and chemokine production, immune cell recruitment as well as immune cell migration to draining lymph nodes at peritoneal cavity in mice induced by AEPS. More importantly, linc-AAM deletion reduced the adjuvant activity of APES on antigen-specific cellular and humoral immune responses to ovalbumin in mice. This study has for the first time demonstrated the role of lncRNAs in regulating the adjuvant activity of polysaccharides and its mechanisms. These findings expanded current knowledge on the mechanism of action of adjuvant and provide a new target for the design and development of vaccine adjuvants.

Boron nitride quantum dots-enhanced laser desorption/ionization mass spectrometry analysis and imaging of bisphenol A.

Bisphenol A (BPA) displays harmful effects on the human health, including potent endocrine activity and potential impact on the development of cancer. Analysis BPA residues in water and plastic products attracted considerable attention in the past decades. However, dominantly used conventional analysis techniques are unable to directly and non-destructively identify the correct species of BPA in plastic products. Hence, this study demonstrates the effective utilisation of boron nitride quantum dots (BNQDs) as an inorganic matrix in matrix-assisted laser desorption/ionization mass spectrometry analysis and imaging (MALDI-MS & MSI) for BPA. The presence of abundant hydroxyl and amino groups on the BNQDs' surface is favourable for the formation of hydrogen bonds with BPA, and increases their ionization and chemoselectivity. Intriguingly, the BNQDs matrix offers a distinct signal for phenolic hazardous molecules featuring different hydroxyl groups. The method was applied to detect BPA at nanomolar level in environmental water, and also allowed non-destructive and in situ mapping of BPA in plastics and pacifiers. This research provides a novel strategy for adapting nanomaterials as inorganic matrices for analysis of small molecular pollutants in environmentally relevant samples using MALDI-MS & MSI.

Association of embryo aneuploidy and sperm DNA damage in unexplained recurrent implantation failure patients under NGS-based PGT-A cycles.

PURPOSE: Recurrent implantation failure (RIF) is one of the most common conditions affecting In Vitro Fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) outcomes. Aneuploidy embryos, one of the main types of embryos-related factors, was reported to be a major contributor to RIF. The present study aimed to examine the association between sperm DNA fragmentation index (DFI) and outcomes of next-generation sequencing (NGS)-based preimplantation genetic testing for aneuploidy (PGT-A) in unexplained RIF patients. METHODS: This study analyzed 119 couples with unexplained RIF who underwent 119 PGT-A cycles between January, 2017 and March, 2022. The 119 males were divided into 3 groups according to their sperm DFI levels: Group1 (low, DFI ≤ 15%, n = 50), Group2 (medium, 15% < DFI < 30%, n = 41) and Group3 (high, DFI ≥ 30%, n = 28). Sperm DFI was measured by sperm chromatin structure analysis (SCSA) technique. Trophectoderm biopsy on day 5 or 6 were performed with NGS technique. The following outcomes of PGT-A were analyzed and compared: fertilization, good-quality embryos, aneuploidy rate, miscarriage, live birth and newborn defects. RESULTS: The component of aneuploidy embryos was significantly higher in high DFI group (42.71%) than that of medium group (28.39%) and low group (27.80%). The miscarriage rate of high DFI group (27.27%) and medium group (14.29%) is significantly higher than that of low group (0.00%). No significant differences were found regarding fertility, good-quality embryo rate, pregnancy rate, live birth rate or newborn defects among three groups. CONCLUSION: The sperm DNA damage is associated with blastocyst aneuploidy and miscarriage rate in unexplained RIF cases. Embryo selection by PGT-A and efforts to decrease sperm DFI before IVF/ICSI treatments should be considered for those male patients with high DFI.

Peptide Oxidation Induced by Liquid-Solid Contact Electrification as Revealed in Liquid Microjunction-Surface Sampling Probe Mass Spectrometry.

Reproducible peptide oxidation was observed using a homebuilt liquid microjunction-surface sampling probe (LMJ-SSP) platform for analyzing peptide standards. Although electrochemical oxidation and corona discharges have previously been associated with analyte oxidation in electrospray ionization (ESI) and ESI-related ambient ionization mass spectrometry (MS) methods, they were unlikely the causes for the peptide oxidation observed in the LMJ-SSP studies. A systematic investigation demonstrated that analyte oxidation was induced during the droplet drying on a solid surface through liquid-solid electrification processes. To minimize unwanted analyte oxidation, the water content in the sample solution should be decreased and the use of hydroxyl-functionalized substrates, such as glass slides, should be avoided. In addition, if water is an essential solvent component, adding an antioxidant, such as ascorbic acid, to the sample solution before droplet evaporation on the solid surface could lower the percentage of analyte oxidation. The present findings apply to all the MS methods that involve drying microliters of sample solution onto a suitable substrate in their sample preparation protocols.

Adjuvant activity of tubeimosides by mediating the local immune microenvironment.

Rhizoma Bolbostemmatis, the dry tuber of Bolbostemma paniculatum, has being used for the treatment of acute mastitis and tumors in traditional Chinese medicine. In this study, tubeimoside (TBM) I, II, and III from this drug were investigated for the adjuvant activities, structure-activity relationships (SAR), and mechanisms of action. Three TBMs significantly boosted the antigen-specific humoral and cellular immune responses and elicited both Th1/Th2 and Tc1/Tc2 responses towards ovalbumin (OVA) in mice. TBM I also remarkably facilitated mRNA and protein expression of various chemokines and cytokines in the local muscle tissues. Flow cytometry revealed that TBM I promoted the recruitment and antigen uptake of immune cells in the injected muscles, and augmented the migration and antigen transport of immune cells to the draining lymph nodes. Gene expression microarray analysis manifested that TBM I modulated immune, chemotaxis, and inflammation-related genes. The integrated analysis of network pharmacology, transcriptomics, and molecular docking predicted that TBM I exerted adjuvant activity by interaction with SYK and LYN. Further investigation verified that SYK-STAT3 signaling axis was involved in the TBM I-induced inflammatory response in the C2C12 cells. Our results for the first time demonstrated that TBMs might be promising vaccine adjuvant candidates and exert the adjuvant activity through mediating the local immune microenvironment. SAR information contributes to developing the semisynthetic saponin derivatives with adjuvant activities.

[Imine-linked porous covalent organic framework used for the solid-phase extraction of estrogens from honey prior to liquid chromatography-tandem mass spectrometry].

This study aimed to establish a method for the rapid determination of trace estrogens in honey samples by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) using imine-linked porous covalent organic framework material (IL-COF-1) as the adsorbent for solid-phase extraction (SPE). Estradiol (E1), diethylstilbestrol (DES), estriol (E3), ß-estradiol (E2), and ethinylestradiol (EE2) were used as the target analytes. A single factor optimization method was performed to optimize the extraction effect by adding estrogens to honey samples. The optimal conditions were as follows. A total of 30 mg IL-COF-1 was filled in the SPE column. The sample pH was adjusted to 7. The sample was loaded at a flow rate of 3 mL/min and eluted with 5 mL of a 1% (v/v) NH3·H2O-methanol solution. The flow rate of the eluent was 0.4 mL/min. NaCl was not added in the extraction process. HPLC coupled to electrospray ionization and triple quadrupole mass spectrometry was introduced to quantify the estrogens in the extracts. The estrogens were separated on a Thermo Fisher Scientific C18 analytical column (100 mm×2.1 mm, 5 µm). Acetonitrile and 5 mmol/L ammonium acetate solution were used as the mobile phases for gradient elution. The column temperature was set at 40 ℃, and the autosampler temperature was maintained at 10 ℃. The rapid qualitative and quantitative analysis of the five estrogens in the honey samples was operated under multiple reaction monitoring mode in a negative electrospray ion source mode. IL-COF-1 prepared in six batches was used as a filler for the SPE column. The relative standard deviations (RSDs) of the recoveries of the estrogens among different batches were 5.2%-9.1%. The reusability of IL-COF-1 material was assessed. After six SPE cycles on the same solid-phase extraction column, the RSDs of the estrogen recoveries were 2.5%-6.1%, indicating that IL-COF-1 has good reusability. The recoveries of estrogens obtained on an IL-COF-1 solid-phase extraction column within 6 days (tested once a day) were 95.1%-107.4%, and the RSDs were 6.2%-8.9%. These results confirmed that the SPE filler had good stability. The method validation results showed that the linear detection ranges were 1-500 ng/g for E3, E2, and EE2, and 0.1-100 ng/g for E1 and DES withe the correlation coefficients of 0.9934-0.9972. The limits of detection (LODs, S/N=3) were 0.01-0.30 ng/g, and the limits of quantification (LOQs, S/N=10) were 0.05-0.95 ng/g. Five estrogens were added (50 ng/g) for the repeated experiments. The RSDs of the intra-day precision were 3.2%-6.6%. The RSDs of the inter-day precision were 4.2%-7.9%. This method was applied to determine the estrogen levels in four honey samples, and no estrogen was found. The recoveries of the five estrogens in sample spiked at three levels including low, middle, and high levels were investigated, and satisfactory recoveries (80.1%-115.2%) were obtained. The SPE-HPLC-MS/MS method based on IL-COF-1 is rapid, accurate, and sensitive, making it suitable for analyzing and detecting estrogen in honey. Further exploration of the use of IL-COF-1 for the extraction processes is in progress.

Global Pattern and Trends in Penile Cancer Incidence: Population-Based Study.

BACKGROUND: Penile cancer is a relatively rare genital malignancy whose incidence and mortality are rising in many countries. OBJECTIVE: This study aims to assess the recent incidence and mortality patterns and incidence trends of penile cancer. METHODS: The age-standardized incidence and mortality rates (ASIR and ASMR, respectively) of penile cancer in 2020 were estimated from the Global Cancer Registries (GLOBOCAN) database. Incidence trends of penile cancer from 1973 to 2012 were assessed in 44 populations from 43 countries using the Cancer Incidence in Five Continents plus (CI5plus) and the Nordic Cancer Registries (NORDCAN) databases. Average annual percentage change was calculated to quantify trends in ASIR using joinpoint regression. RESULTS: Globally, the estimated ASIR and ASMR of penile cancer were 0.80 (per 100,000) and 0.29 (per 100,000) in 2020, equating to 36,068 new cases and 13,211 deaths in 2020, respectively. There was no significant correlation between the ASIR (P=.05) or ASMR (P=.90) and Human Development Index. In addition, 15 countries saw increasing ASIR for penile cancer, 13 of which were from Europe (United Kingdom, Lithuania, Norway, Estonia, Finland, Sweden, Cyprus, Netherlands, Italy, Croatia, Slovakia, Russia, and the Czech), and 2 from Asia (China and Israel). CONCLUSIONS: Although the developing countries still bear the higher incidence and mortality of penile cancer, the incidence is on the rise in most European countries. To mitigate the disease burden resulting from penile cancer, measures to lower the risk for penile cancers, including improving penile hygiene and male human papillomavirus vaccination, may be warranted.

Tandem Mass Spectrometry for Structural Characterization of Doubly-Charged N-Linked Glycopeptides.

Three dissociation methods, including collision-induced dissociation (CID), electron capture dissociation (ECD), and electronic excitation dissociation (EED), were systematically compared for structural characterization of doubly charged glycopeptide. CID produced distinctively different tandem mass spectra for glycopeptide adducted with different charge carriers. Protonated species produced mainly glycosidic cleavages in high abundance. CID of magnesiated glycopeptide formed more cross-ring cleavages, whereas doubly sodiated species produced cleavages at both glycan and peptide moieties. The effect of charge carriers on the fragmentation in ECD and EED was lower than that in CID. ECD produced mainly peptide backbone cleavages but limited cleavages at the glycan moiety, whereas EED of glycopeptide resulted in extensive fragmentation throughout the molecular ion regardless of the charge carriers. Magnesiated species gave, however, more cross-ring cleavages than other charge carriers did. These results demonstrated that EED of magnesiated species could be used as a one-step dissociation method for comprehensive structural analysis of glycopeptides.

Paper-Based Flow Sensor for the Detection of Hyaluronidase via an Enzyme Hydrolysis-Induced Viscosity Change in a Polymer Solution.

Hyaluronidase (HAase) is implicated in inflammation, cancer development, and allergic reaction. The detection of HAase is significantly important in clinical diagnosis and medical treatment. Herein, we propose a new principle for the development of equipment-free and label-free paper-based flow sensors based on the enzymatic hydrolysis-induced viscosity change in a stimuli-responsive polymer solution, which increases the water flow distance on the pH indicator paper. The detection of HAase is demonstrated as an example. This facile and versatile method can overcome the potential drawbacks of traditional hydrogel-based sensors, including complex preparation steps, slow response time, or low sensitivity. Moreover, it can also avoid the use of specialized instruments, labeled molecules, or functionalized nanoparticles in the sensors developed using the polymer solutions. Using this strategy, the detection of HAase is achieved with a limit of detection as low as 0.2 U/mL. Also, it works well in human urine. Additionally, the detection of tannic acid, which is an inhibitor of HAase, is also fulfilled. Overall, a simple, efficient, high-throughput, and low-cost detection method is developed for the rapid and quantitative detection of HAase and its inhibitor without the use of labeled molecules, synthetic particles, and specialized instruments. As only minimal reagents of HAase, HA, and paper are used, it is very promising in the development of commercial kits for point-of-care testing.

Fate and ecological risks of current-use pesticides in seawater and sediment of the Yellow Sea and East China Sea.

With the frequent use of chemical pesticides, the current-use pesticides (CUPs) emerge and concentrate in the sea. The partition between the sediment and seawater is essential for understanding the environmental fate of CUPs. However, there is little research on this topic. In the present study, seventeen CUPs were screened in seawater and sediment samples collected from the Yellow Sea and East China Sea. Total concentration of 17 CUPs in surface seawater samples ranged from 9.5 to 267.3 ng/L, with 6 CUPs presenting 100% detection frequency. Carbendazim, tricyclazole, tebuconazole, atrazine and imidacloprid accounted for >80% of all CUPs, which was due to their large application in the local agriculture and fishing activities. Higher concentration sites were located near the shore and Yangtze river estuary, indicating intense human activities and riverine input that elevated the level of CUPs in marginal sea. The pesticides in seawater were mainly found in the surface followed by the bottom layer, which indicated that atmospheric deposition and re-suspension played key roles for their vertical distribution characteristics. The high fugacity fraction ratios (ff > 0.5) indicated the non-equilibrium state of pesticides that might have been transferred from sediment to seawater at most sites. These 17 detectable pesticides in seawater were at low levels, presenting ignorable or low toxic effects to aquatic organisms.

Rationally Designed, Self-Assembling, Multifunctional Hydrogel Depot Repairs Severe Spinal Cord Injury.

Following severe spinal cord injury (SCI), dysregulated neuroinflammation causes neuronal and glial apoptosis, resulting in scar and cystic cavity formation during wound healing and ultimately the formation of an atrophic microenvironment that inhibits nerve regrowth. Because of this complex and dynamic pathophysiology, a systemic solution for scar- and cavity-free wound healing with microenvironment remodeling to promote nerve regrowth has rarely been explored. A one-step solution is proposed through a self-assembling, multifunctional hydrogel depot that punctually releases the anti-inflammatory drug methylprednisolone sodium succinate (MPSS) and growth factors (GFs) locally according to pathophysiology to repair severe SCI. Synergistically releasing the anti-inflammatory drug MPSS and GFs in the hydrogel depot throughout SCI pathophysiology protects spared tissues/axons from secondary injury, promotes scar boundary- and cavity-free wound healing, and results in permissive bridges for remarkable axonal regrowth. Behavioral and electrophysiological studies indicate that remnants of spared axons, not regenerating axons, mediate functional recovery, strongly suggesting that additional interventions are still required to render the rebuilt neuronal circuits functional. These findings pave the way for the development of a systemic solution to treat acute SCI.

linc-AAM Facilitates Gene Expression Contributing to Macrophage Activation and Adaptive Immune Responses.

Although various long noncoding RNAs (lncRNAs) are specifically expressed in activated macrophages, their in vivo functions and mechanisms of action are largely unexplored. Here, we identify a long intergenic noncoding RNA associated with activated macrophage (linc-AAM) and elucidate its function and mechanisms. linc-AAM is highly expressed in activated macrophages. In vitro function analysis reveals that linc-AAM facilitates macrophage activation and promotes the expression of immune response genes (IRGs). In mechanisms, linc-AAM interacts with heterogeneous nuclear ribonucleoprotein L (hnRNPL) via two CACACA motifs, resulting in its dissociation from histone H3 to activate chromatin and facilitate transcription of IRGs. Of note, linc-AAM knockout (KO) mice manifest impaired antigen-specific cellular and humoral immune responses to ovalbumin (OVA) in vivo. Altogether, the results uncover a mechanism of lncRNA in modulating hnRNPL function and confirm that linc-AAM acts as a transcription enhancer to activate macrophages and promote adaptive immunity.

Integration of proteomics and metabolomics reveals promotion of proliferation by exposure of bisphenol S in human breast epithelial MCF-10A cells.

Bisphenol S (BPS) has been reported to have similar estrogenic effects as bisphenol A (BPA). Considering the endocrine disrupting effects of BPS, in this study, we investigated the effects of BPS exposure on normal human breast epithelial cell line MCF-10A by using mass spectrometry (MS)-based metabolomics and quantitative proteomics. We found that exposure to BPS for 24 h altered the proliferation of MCF-10A cells in a hormetic manner with the highest proliferation rate at the dosage of 1 µM. A total of 200 proteins were identified to be significantly changed by 1 µM of BPS exposure. The upregulation of epidermal growth factor receptor (EGFR) and Ras/mTOR-related proteins implied that EGFR-mediated pathways were involved in BPS-induced proliferation of MCF-10A cells. In addition, several proliferation-related protein markers were found to be elevated, such as MKI67 and CDH1, further indicating the promotion of proliferation by low dose of BPS exposure. Besides, 35 endogenous metabolites were found to be significantly changed. The joint pathway analysis of the altered metabolites and proteins suggested changes in pathways of tricarboxylic acid (TCA) cycle, purine metabolism, pyruvate metabolism and lipid metabolism, which were involved in sustaining cell proliferation and cellular signal transduction. Taken together, this study provides insights into the effects and the potential mechanisms of BPS on estrogen receptor α-negative normal breast cell line MCF-10A, broadening our knowledge about the risk of using BPS as the alternative of BPA.

Activation of RAW264.7 macrophages by active fraction of Albizia julibrissin saponin via Ca2+-ERK1/2-CREB-lncRNA pathways.

The saponin active fraction from the stem bark of Albizia julibrissin (AJSAF) is an ideal vaccine adjuvant, but its mechanism of action remains unclear. The recent evidences indicate that long noncoding RNAs (lncRNAs) play essential roles in regulating the activation and function of macrophages. The current experiments were designed to investigate the effects of AJSAF on the activation of RAW264.7 macrophages and to explore its intracellular molecular mechanisms using a global gene expression microarray. AJSAF could significantly enhance phagocytic activity, induce reactive oxygen species (ROS), promote surface molecule expression, and up-regulate the mRNA and protein expression of cytokines and chemokines in RAW264.7 cells. AJSAF induced the differential expression of 223 mRNAs and 103 lncRNAs in RAW264.7 cells. Bioinformatics were used to predict the potential target mRNAs and function of up-regulated lncRNA A_30_P01018532 in RAW264.7 cells induced by AJSAF. The total 99 co-expressed mRNAs were classified as putative target genes of A_30_P01018532. A_30_P01018532 was associated with the inflammatory and immune response. AJSAF significantly increased the intracellular free Ca2+ levels and induced the phosphorylation of ERK1/2 and CREB in RAW264.7 cells. Moreover, Ca2+ chelator BAPTA-AM, ERK1/2 inhibitor PD98059 and CREB inhibitor KG-501 significantly inhibited the up-regulation of TNF-α, CCL2, CXCL2, CCL22, and A_30_P01018532 in RAW264.7 cells induced by AJSAF. These results suggested that AJSAF could activate RAW264.7 cells via Ca2+-ERK1/2-CREB pathways and that A_30_P01018532 might be an important regulator of mRNA expression in AJSAF-activated macrophage. This study may provide insights into the molecular mechanisms of action of AJSAF.

Rapid Differentiation of Asian and American Ginseng by Differential Ion Mobility Spectrometry-Tandem Mass Spectrometry Using Stepwise Modulation of Gas Modifier Concentration.

This study reports a rapid and robust method for the differentiation of Asian and American ginseng samples based on differential ion mobility spectrometry-tandem mass spectrometry (DMS-MS/MS). Groups of bioactive ginsenoside/pseudo-ginsenoside isomers, including Rf/Rg1/F11, Rb2/Rb3/Rc, and Rd/Re, in the ginseng extracts were sequentially separated using DMS with stepwise changes in the gas modifier concentration prior to MS analysis. The identities of the spatially separated ginsenoside/pseudo-ginsenoside isomers were confirmed by their characteristic compensation voltages at specific modifier loading and MS/MS product ions. As expected, Asian ginseng samples contained some Rf and an insignificant amount of F11, whereas American ginseng samples had a high level of F11 but no Rf. The origin of the whole and sliced ginseng could further be confirmed using the quantitative ratios of three sets of ginsenoside markers, namely, Rg1/Re, Rb1/Rg1, and Rb2/Rc. Based on our results, new benchmark ratios of Rg1/Re < 0.15, Rb1/Rg1 > 2.15, and Rb2/Rc < 0.26 were proposed for American ginseng (as opposed to Asian ginseng).

Comprehensive analysis of lncRNA and mRNA expression profiles in macrophages activated by Actinidia eriantha polysaccharide.

The polysaccharide from the roots of Actinidia eriantha (AEPS) is a potent antitumor agent and immunological adjuvant. The recent evidences indicate that long noncoding RNAs (lncRNAs) play essential roles in regulating the activation and function of immune cells. The objective of this study was to investigate the expression profiles of lncRNAs and mRNAs and explore the role of lncRNAs in AEPS-activated RAW264.7 cells using whole gene expression microarray. AEPS induced the differential expression of 1,807 mRNAs and 506 lncRNAs in RAW264.7 cells. The mRNA expression levels of both M1 and M2 specific cytokines and chemokines were significantly upregulated in RAW264.7 cells by AEPS. NF-κB inhibitors and shRNA-NF-κB p65 significantly decreased the up-regulation of IL-1ß expression in RAW264.7 cells induced by AEPS. Five AEPS-induced lncRNAs (D730047E02Rik, Gm14047, A_30_P01020139, A_30_P01026293 and A_30_P01032196) were identified and predicted to locally regulate mRNA expressions of immune response genes in RAW264.7 cells through the specific interaction with NF-κB p65. The inhibition of NF-κB could significantly suppress the expression of these lncRNAs induced by AEPS. These results indicated that AEPS induced the activation of macrophages via lncRNAs/NF-κB networks. This study further expanded current knowledge on the mechanisms of plant polysaccharides.