Integrated OMICs approach reveals energy metabolism pathway is vital for Salmonella Pullorum survival within the egg white.

Eggs, an important part of a healthy daily diet, can protect chicken embryo development due to the shell barrier and various antibacterial components within the egg white. Our previous study demonstrated that Salmonella Pullorum, highly adapted to chickens, can survive in the egg white and, therefore, be passed to newly hatched chicks. However, the survival strategy of Salmonella Pullorum in antibacterial conditions remains unknown. The overall transcripts in the egg white showed a large-scale shift compared to LB broth. The expression of common response genes and pathways, such as those involved in iron uptake, biotin biosynthesis, and virulence, was significantly changed, consistent with the other transovarial transmission serovar Enteritidis. Notably, membrane stress response, amino acid metabolism, and carbohydrate metabolism were specifically affected. Additional upregulated functionally relevant genes (JI728_13095, JI728_13100, JI728_17960, JI728_10085, JI728_15605, and nhaA) as mutants confirmed the susceptible phenotype. Furthermore, fim deletion resulted in an increased survival capacity in the egg white, consistent with the downregulated expression. The second-round RNA-Seq analysis of the Δfim mutant in the egg white revealed significantly upregulated genes compared with the wild type in the egg white responsible for energy metabolism located on the hyc and hyp operons regulated by FhlA, indicating the Δfim mutant cannot receive enough oxygen and switched to fermentative growth due to its inability to attach to the albumen surface. Together, this study provides a first estimate of the global transcriptional response of Salmonella Pullorum under antibacterial egg white and highlights the new potential role of fim deletion in optimizing energy metabolism pathways that may assist vertical transmission. IMPORTANCE: Pullorum disease, causing serious embryo death and chick mortality, results in substantial economic losses worldwide due to transovarial transmission. Egg-borne outbreaks are frequently reported in many countries. The present study has filled the knowledge gap regarding how the specific chicken-adapted pathogen Salmonella Pullorum behaves within the challenging environment of egg white. The deletion of the fim fimbrial system can increase survival in the albumen, possibly by reprogramming metabolism-related gene products, which reveals a new adaptive strategy of pathogens. Moreover, the comparison, including previous research on Salmonella Enteritidis, capable of vertical transmission, aims to provide diversified data sets in the field and further help to implement reasonable and effective measures to improve both food safety and animal health.

Adaptive sampling physics-informed neural network method for high-order rogue waves and parameters discovery of the (2 + 1)-dimensional CHKP equation.

Rogue waves are important physical phenomena, which have wide applications in nonlinear optics, hydrodynamics, Bose-Einstein condensates, and oceanic and atmospheric dynamics. We find that when using the original PINNs to study rogue waves of high dimensional PDEs, the prediction performance will become very poor, especially for high-order rogue waves due to that the randomness of selection of sample points makes insufficient use of the physical information describing the local sharp regions of rogue waves. In this paper, we propose an adaptive sampling physics-informed neural network method (ASPINN), which renders the points in local sharp regions to be selected sufficiently by a new adaptive search algorithm to lead to a prefect prediction performance. To valid the performance of our method, the (2+1)-dimensional CHKP equation is taken as an illustrative example. Experimental results reveal that the original PINNs can hardly be able to predict dynamical behaviors of the high-order rogue waves for the CHKP equation, but the ASPINN method can not only predict dynamical behaviors of these high-order rogue waves, but also greatly improve the prediction efficiency and accuracy to four orders of magnitude. Then, the data-driven inverse problem for the CHKP equation with different levels of corrupted noise is studied to show that the ASPINN method has good robustness. Moreover, some main factors affecting the neural network performance are discussed in detail, including the size of training data, the number of layers of the neural network, and the number of neurons per layer.

Mobilome-driven partitions of the resistome in Salmonella.

IMPORTANCE: Antimicrobial resistance (AMR) has become a significant global challenge, with an estimated 10 million deaths annually by 2050. The emergence of AMR is mainly attributed to mobile genetic elements (MGEs or mobilomes), which accelerate wide dissemination among pathogens. The interaction between mobilomes and AMR genes (or resistomes) in Salmonella, a primary cause of diarrheal diseases that results in over 90 million cases annually, remains poorly understood. The available fragmented or incomplete genomes remain a significant limitation in investigating the relationship between AMR and MGEs. Here, we collected the most extensive closed Salmonella genomes (n = 1,817) from various sources across 58 countries. Notably, our results demonstrate that resistome transmission between Salmonella lineages follows a specific pattern of MGEs and is influenced by external drivers, including certain socioeconomic factors. Therefore, targeted interventions are urgently needed to mitigate the catastrophic consequences of Salmonella AMR.

Myricetin served as antagonist for negatively regulate MRGPRX2 mediated pseudo-allergic reactions through CD300f/SHP1/SHP2 phosphorylation.

BACKGROUND: Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a vital role in mast cells (MCs) degranulation and pseudo-allergic reactions. Leukocyte mono-immunoglobulin-like receptor 3 (CD300f) can negatively regulate MCs degranulation. Identification of drug candidates which target CD300f represents a promising prospect in drug development. Myricetin is widely distributed in plants and has been reported to inhibit allergic reactions in OVA-induced murine models. OBJECTIVE: This study aims to determine whether myricetin can activate CD300f to arrest MCs degranulation mediated by MRGPRX2. RESULTS: Myricetin inhibited the allergic mediator and cytokine release triggered by MRGPRX2 in vivo and in vitro. Under C48/80 stimulation, the release of ß-hexosaminidase, TNF-α, IL-8 and MCP-1 in CD300f knockdown in LAD2 cells was significantly increased compared with NC-LAD2 cells. Myricetin displayed good structural affinity (KD = 7.21 × 10-5) with CD300f by SPR. Molecular docking results showed that hydrogen bonds were formed between myricetin and CD300f, indicating high binding ability (5.6653). Myricetin can upregulate the phosphorylation of SHP-1 and SHP-2 and dephosphorylation in the MRGPRX2 signaling pathway, involving PLCγ1, AKT, P38, and ERK1/2. CONCLUSION: In the present study, myricetin is identified as an exogenous ligand for CD300f, which negatively regulates MRGPRX2-mediated MCs activation via CD300f to inhibit MCs degranulation and pseudo-allergic reactions.

Myricetin alleviated immunologic contact urticaria and mast cell degranulation via the PI3K/Akt/NF-κB pathway.

Immunologic contact urticaria (ICU) is characterized by the wheal and flare reaction from direct contact with a chemical or protein agent, which involves a type I hypersensitivity mediated by allergen-specific immunoglobulin E (sIgE). Myricetin (Myr), a bioactive flavonoid, exhibits antiinflammatory activities. Our results showed that treatment with Myr could alleviate ICU symptoms, including a decrease in the number of wheals and scratching, and inhibit ear swelling in the IgE/DNFB-induced mice. The serum level of IgE, histamine, interleukin (IL)-4, TNF-α, and MCP-1 were reduced in Myr-treated mice. Myr also attenuated mast cells (MCs) degranulation and H-PGDS, TSLP, IL-33, PI3K, Akt, and NF-κB mRNA levels in ICU model. The IgE-mediated anaphylaxis mouse models demonstrated anti-allergic effects of Myr. In vitro analysis showed that Myr reduced IgE-induced calcium (Ca2+ ) influx, suppressed degranulation, and chemokine release in LAD2 cells (human primary mast cells). Myr can significantly inhibited PLCγ1, Akt, NF-κB, and p38 phosphorylation. In conclusion, the study demonstrated that Myr alleviate ICU symptoms and inhibit mast cell activation via PI3K/Akt/NF-κB signal pathway.

Artemisinic acid attenuated symptoms of substance P-induced chronic urticaria in a mice model and mast cell degranulation via Lyn/PLC-p38 signal pathway.

BACKGROUND: Chronic urticaria (CU) is a common skin disease that affects about 1% of the world's population of all ages and seriously affects patients' quality of life. Therefore, further safe and effective treatments are urgently needed. Therefore, artemisinic acid was investigated in the present study due to its pharmacologic effect on inhibiting mast cell degranulation and chronic urticaria in a mouse model. RESULTS: 4Artemisinic acid decreased the symptoms of substance P-induced chronic urticaria in the mouse model and alleviated secretagogue-induced local cutaneous and systemic anaphylaxis through the Lyn-PLC-p38-NF-κB signaling pathway. Artemisinic acid inhibited mast cell degranulation and pro-inflammatory cytokine production in vitro. Mechanism analysis demonstrated that it could arrest mast cell activation through the Lyn-PLC-p38/ERK1/2/AKT-NF-κB signaling pathway. Based on the results of in vitro kinase assay of Lyn and PLC, artemisinic acid was a potential small molecule inhibitor of Lyn. Artemisinic acid displayed good structural affinity (KD = 2.64 × 10-6) with Lyn SPR results. CONCLUSION: Artemisinic acid can attenuate substance P/MRGPRX2-mediated chronic urticaria and mast cell activation. Artemisinic acid is an antagonist of Lyn kinase and can be developed as a drug candidate to treat allergic diseases.

Surface-Modified Approach to Fabricate Nafion Membranes Covalently Bonded with Polyhedral Oligosilsesquioxane for Vanadium Redox Flow Batteries.

An aminopropyl isobutyl polyhedral oligosilsesquioxane (NH2-POSS) surface-modified Nafion membrane has been designed by chemical grafting for vanadium redox flow batteries (VRFBs). NH2-POSS is a cage-like macromer consisting of an inorganic Si8O12 core surrounded by seven inert isobutyl groups and one active aminopropyl group. The sulfonic acid groups on the surface of Nafion can be activated by 1,1-carbonyldiimidazole for further modification with NH2-POSS. Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) prove that NH2-POSS has been successfully grafted on the surface of a Nafion 115 membrane. Although the proton conductivity decreases slightly, the organic-inorganic hybrid membranes display enhanced ion selectivity and excellent dimensional stability with lower water uptake and swelling ratio than Nafion 115. Moreover, two-dimensional-grazing incidence X-ray diffraction (2D-GIXRD) reveals that the introduction of NH2-POSS forms a POSS layer on the surface of the membrane and narrows the space of Nafion clusters, which helps to block VO2+ permeation. A VRFB with the surface-modified Nafion membrane displays an outstanding performance with an average Coulombic efficiency (CE) of 98.7% and energy efficiency (EE) of 84.5% at a current density of 80 mA cm-2, superior to those of the Nafion 115 membrane (CE = 95.7%, EE = 81.7%). Furthermore, the cell holds a high capacity retention of 49.2% after 1000 charge-discharge cycles, in contrast to that of 41.9% for the cell with Nafion 115 after only 200 cycles. The results suggest that the surface-modified hybrid membrane is a promising strategy to overcome the vanadium ion crossover in VRFBs.

Alpha-linolenic acid inhibits IgE-mediated anaphylaxis by inhibiting Lyn kinase and suppressing mast cell activation.

Excessive reactions to allergens can induce systemic, life-threatening physiological dysfunction (anaphylaxis) in humans. The surface of mast cells expresses high-affinity IgE receptors that play a vital role during anaphylaxis. Alpha-linolenic acid (ALA) is an essential non-toxic fatty acid in humans. Since it has been reported having potential to regulate pro-inflammatory reactions, we postulated that ALA could inhibit anaphylaxis by down-regulating Lyn kinase phosphorylation. We found that local and systematic inflammation induced by albumin from chicken egg white (OVA) were attenuated by ALA in vivo. Furthermore, ALA inhibited IgE-mediated Ca2+ mobilization, degranulation, and cytokine release in Laboratory of Allergic Disease 2 (LAD2) cells. The western blot results showed that ALA down-regulate the FcεRI/Lyn/Syk signaling pathway by suppressing Lyn kinase activity. Therefore, ALA could serve as a therapeutic drug candidate for preventing IgE-mediated anaphylaxis.

Roles of Endogenous Melatonin in Resistance to Botrytis cinerea Infection in an Arabidopsis Model.

Melatonin is an important bioactive molecule in plants. Two synthetases, N-acetylserotonin methyltransferase (ASMT) and serotonin N-acetyltransferase (SNAT) are involved in the final two steps of melatonin synthesis. Melatonin participates in responses to a variety of biotic and abiotic stresses in plants, but few studies have addressed the roles of endogenous melatonin in pathogen resistance. We investigated the role of endogenous melatonin in resistance to Botrytis cinerea infection in an Arabidopsis thaliana model system. Plant lines that overexpressed ASMT or SNAT through genetic manipulation showed upregulated expression of resistance genes PR1 and PR5, transcription factor gene WRKY33, and jasmonic acid (JA) defense pathway marker gene PDF1.2, and downregulated transcription factor gene MYC2 in JA signaling pathway. Higher melatonin content also enhanced the activity of antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD), increased JA content, reduced plant disease symptoms, and reduced lesion size in leaves. These findings indicate that endogenous melatonin enhances plant resistance to B. cinerea infection. In contrast, ASMT and SNAT gene silencing lines showed opposite results and were more susceptible to B. cinerea. Thus, it can be demonstrated that melatonin functions as an effective regulator of plant stress resistance at the genetic level. A schematic model is presented for its role in resistance to B. cinerea infection. Our findings also helped to elucidate the associated signal transduction pathways and interactions between melatonin and other plant hormones.

α-Linolenic acid attenuates pseudo-allergic reactions by inhibiting Lyn kinase activity.

BACKGROUND: Pseudo-allergic reactions are potentially fatal hypersensitivity responses caused by mast cell activation. α-linolenic acid (ALA) is known for its anti-allergic properties. However, its potential anti-pseudo-allergic effects were not much investigated. PURPOSE: To investigate the inhibitory effects of ALA on IgE-independent allergy in vitro, and in vivo, as well as the mechanism underlying its effects. METHODS/STUDY DESIGNS: The anti-anaphylactoid activity of ALA was evaluated in passive cutaneous anaphylaxis reaction (PCA) and systemic anaphylaxis models. Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of Lyn-PLCγ-IP3R-Ca2+ and Lyn-p38/NF-κB signaling pathway. RESULTS: ALA (0, 1.0, 2.0, and 4.0 mg/kg) dose-dependently reduced serum histamine, chemokine release, vasodilation, eosinophil infiltration, and the percentage of degranulated mast cells in C57BL/6 mice. In addition, ALA (0, 50, 100, and 200 µM) reduced Compound 48/80 (C48/80) (30 µg/ml)-or Substance P (SP) (4 µg/ml)-induced calcium influx, mast cell degranulation and cytokines and chemokine release in Laboratory of Allergic Disease 2 (LAD2) cells via Lyn-PLCγ-IP3R-Ca2+ and Lyn-p38/NF-κB signaling pathway. Moreover, ALA (0, 50, 100, and 200 µM) inhibited C48/80 (30 µg/ml)- and SP (4 µg/ml)-induced calcium influx in Mas-related G-protein coupled receptor member X2 (MrgX2)-HEK293 cells and in vitro kinase assays confirmed that ALA inhibited the activity of Lyn kinase. In response to 200 µM of ALA, the activity of Lyn kinase by (7.296 ± 0.03751) × 10-5 units/µl and decreased compared with C48/80 (30 µg/ml) by (8.572 ± 0.1365) ×10-5 units/µl. CONCLUSION: Our results demonstrate that ALA might be a potential Lyn kinase inhibitor, which could be used to treat pseudo-allergic reaction-related diseases such as urticaria.

Kaempferol ameliorates secretagogue-induced pseudo-allergic reactions via inhibiting intracellular calcium fluctuation.

OBJECTIVES: To investigate the inhibitory effects of Kaempferol, a natural flavonol active compound, on pseudo-allergic reactions (in vivo and in vitro), particularly on the mechanism underlying its effect in human mast cells. METHODS: Compound 48/80 (C48/80)-induced immunoglobulin E (IgE)-independent passive cutaneous anaphylaxis (PCA) model and systemic anaphylaxis were applied to investigate the anti-allergic activity of Kaempferol. The degranulation assay, calcium imaging and the secretion of cytokines and chemokines were used to evaluate the inhibitory effect on mast cell activation. Western blot analysis was performed to investigate intracellular calcium fluctuation-related signalling pathways. KEY FINDINGS: Kaempferol dose-dependently attenuated C48/80-induced mice hind paw swelling, dye extravasation and skin mast cell degranulation, and rehabilitated the hypothermia, as well as reduced the serum concentrations of histamine, tryptase, tumour necrosis factor-alpha (TNF-α), interleukin-8 (IL-8) and monocyte chemo-attractant protein-1 (MCP-1). Furthermore, Kaempferol suppressed C48/80-triggered human MC degranulation and calcium fluctuations by inhibiting phospholipase Cγ (PLCγ) phosphorylation and subsequent cytokines synthesis pathways. CONCLUSIONS: The inhibition of the process of PLCγ phosphorylation to Ca2+ mobilization represents a major strategy in Kaempferol-suppressed pseudo-allergic reactions. Thus, Kaempferol could be considered as a therapeutic drug candidate for non-IgE-mediated allergic reactions or inflammations.

Inhibitory function of Shikonin on MRGPRX2-mediated pseudo-allergic reactions induced by the secretagogue.

BACKGROUND: Mast cells (MCs) are crucial effectors in allergic disorders by secreting inflammatory mediators. The Mas-related G-protein-coupled receptor X2 (Mrgprx2) was shown to have a key role in IgE-independent allergic reactions. Therefore, potential drug candidates that directly target Mrgprx2 could be used to treat pseudo-allergic diseases. Shikonin, an active ingredient derived from Lithospermum erythrorhizon Sieb. et Zucc has been used for its anti-inflammatory properties since ancient China. PURPOSE: To investigate the inhibitory effects of Shikonin on IgE-independent allergy both in vitro and in vivo, as well as the mechanism underlying its effects. METHODS/STUDY DESIGNS: The anti-anaphylactoid activity of Shikonin was evaluated in PCA and systemic anaphylaxis models, Calcium imaging was used to assess intracellular Ca2+ mobilization. The release of cytokines and chemokines was measured using enzyme immunoassay kits. Western blot analysis was conducted to investigate the molecules of PLCγ-PKC-IP3 signaling pathway. The analytical method of surface plasmon resonance was employed to study the interaction between Shikonin and potential target protein Mrgprx2. RESULTS: Shikonin can suppress compound 48/80 (C48/80)-induced PCA, active systemic anaphylaxis, and MCs degranulation in mice in a dose-dependent manner. In addition, Shikonin reduced C48/80-induced calcium flux and suppressed LAD2 cell degranulation via PLCγ-PKC-IP3 signaling pathway. Moreover, Shikonin was found to inhibit C48/80-induced Mrgprx2 expression in HEK cells, displaying specific interactions with the Mrgprx2 protein. CONCLUSION: Shikonin could be a potential antagonist of Mrgprx2, thereby inhibiting pseudo-allergic reactions through Ca2+ mobilization.

Magnolin inhibits IgE/Ag-induced allergy in vivo and in vitro.

Mast cells (MCs) play critical roles in allergic reactions and modulating the activation of MCs could be an effective strategy to treat allergic diseases, which cause a rapidly increasing threat to the public health. Herein, we described that Magnolin, a major component from Flos magnoliae could inhibit IgE-dependent MCs activation. We found Magnolin inhibited IgE/Ag-induced calcium mobilization, degranulation, and cytokines release in LAD2 cells. Magnolin was also found to attenuate IgE/Ag-induced mice paw swelling in a dose-dependent manner. Further mechanistic studies suggested a possible anti-allergic and anti-inflammatory effects of Magnolin in IgE/Ag-induced anaphylactic reactions. Thereby, Magnolin could be a potential therapeutic agent for preventing mast cell-related immediate and delayed allergic diseases.

A mast-cell-specific receptor mediates Iopamidol induced immediate IgE-independent anaphylactoid reactions.

Iopamidol is a radiographic contrast media which caused a very high incidence of anaphylactic reactions. Mast cells are sentinel cells in host defense reactions during immediate hypersensitivity responses and anaphylactic responses. Mas-related G protein-coupled receptor X2 (MRGPRX2) is a kind of mast cell specific receptor, which triggers mast cell degranulation in anaphylactic reactions. Mice MrgprB2 is a homologous gene of MRGPRX2. We sought to better understand the anaphylactic reactions induced by Iopamidol and the mechanisms involving MRGPRX2. The MRGPRX2-related anaphylactic reactions induced by Iopamidol were investigated using the hindpaw swelling and extravasation assay in vivo and a calcium imaging assay was used for mast cell intracellular calcium responses detection and mast cell release of anaphylactic mediators, such as ß-hexosaminidase, histamine and TNF-α, was also detected in vitro. The mast cell deficient KitW-sh/W-sh mice and MrgprB2 knockout mice exhibited a reduced Iopamidol-induced inflammation effect compared with wild type mice. Furthermore, human mast cells that express MRGPRX2 were activated by Iopamidol in a dose-dependent manner, meanwhile MRGPRX2 knockdown mast cells showed reduced intracellular calcium responses and anaphylactic mediators release effect. It could be concluded that Iopamidol-induced anaphylactoid reactions were MRGPRX2 mediated to provoke mast cells Ca2+ mobilization and degranulation.

Neohesperidin suppresses IgE-mediated anaphylactic reactions and mast cell activation via Lyn-PLC-Ca2+ pathway.

Mast cells play an essential role in IgE-FcεR1-mediated allergic diseases. Citrus aurantium is a prolific source of flavonoids with various biological activities, including anti-inflammatory, antioxidant, and anti-tumor efficacies. Neohesperidin is a novel flavonoid isolated from the leaves of C. aurantium. In this study, the anti-allergic and anti-inflammatory potentials of neohesperidin were investigated along with its molecular mechanism. The anti-anaphylactic activity of neohesperidin was evaluated through hind paw extravasation study in mice. Calcium imaging was used to assess intracellular Ca2+ mobilization. The levels of cytokines and chemokines were measured using enzyme immunoassay kits. Western blotting was used to explore the related molecular signaling pathways. Neohesperidin suppressed IgE-induced mast cell activations, including degranulation and secretion of cytokines and eicosanoids through inhibiting phosphorylation of Lyn kinase. Neohesperidin inhibited the release of histamine and other proinflammatory cytokines through a mast cell-dependent passive cutaneous anaphylaxis animal model. Histological studies demonstrated that neohesperidin substantially inhibited IgE-induced cellular infiltration and attenuated mast cell activation in skin tissue. In conclusion, our study revealed that neohesperidin could inhibit allergic responses in vivo and in vitro, and the molecule may be regarded as a novel agent for preventing mast cell-immediate and delayed allergic diseases.

Dehydroandrographolide inhibits IgE-mediated anaphylactic reactions via calcium signaling pathway.

The classical mast cells degranulation pathway is mediated by FcεRI aggregation and varies in strength among subjects. Dehydroandrographolide (DA) is one of principal components of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) and considered the main contributors of its therapeutic properties, such as anti-tumor. In this study, inhibition of IgE-mediated anaphylactic reactions and anti-inflammatory potential of DA were investigated. The anti-anaphylactic activity of DA was investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. The release of cytokines was measured using ELISA kits. Human Phospho-Kinase Array kit and western blotting were used to explore the related molecular signaling pathways. DA inhibited IgE-mediated mast cell activation, including degranulation and release of cytokines in vitro. Moreover, DA reduced the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner by inhibiting mast cell degranulation. DA obviously reduced the concentrations of histamine, TNF-α, MCP-1, IL-8, IL-13, and IL-4 in mice serum and inhibited IgE-mediated anaphylactic reactions as a potential P-PLCγ inhibitor. Our study reveals that DA can inhibit allergic responses in vivo and in vitro, and it may be regarded as a novel P-PLCγ inhibitor for preventing mast cell-immediate and delayed allergic diseases.

Levistolide A synergistically enhances doxorubicin­induced apoptosis of k562/dox cells by decreasing MDR1 expression through the ubiquitin pathway.

Multidrug resistance (MDR) is one of the main reasons underlying failure of cancer chemotherapy. Certain natural compounds may help prevent MDR, and may be used in combination with chemotherapeutic agents to enhance their efficacy. Levistolide A is a natural product that is extracted from the rhizome of Angelicae sinensis (Oliv.), which has been used as an essential component of antitumor formulas since ancient times in China. The present study conducted the following experiments: MTT assay, apoptosis analysis, cellular doxorubicin accumulation assay, immunoblotting and reverse transcription­quantitative polymerase chain reaction, to investigate whether levistolide A enhance doxorubicin­induced apoptosis of k562/dox cells and to determine the molecular mechanisms involved. When combined with doxorubicin, levistolide A exhibited a synergistic effect and induced cytotoxicity in k562/dox cells. Drug accumulation studies revealed that levistolide A increased the intracellular concentration of doxorubicin in a dose­dependent manner. Cell apoptosis experiments indicated that levistolide A increased the sensitivity of k562/dox cells to doxorubicin. Furthermore, detection of reactive oxygen species (ROS) revealed that levistolide A enhanced doxorubicin­induced cell death by increasing the levels of ROS. Mitochondrial potential detection with JC­1 staining also indicated that levistolide A synergistically enhanced doxorubicin­induced cell death. Immunoblotting demonstrated that levistolide A enhanced doxorubicin­induced cell death by decreasing the expression levels of B­cell lymphoma 2 and increasing caspase 3 expression. Furthermore, multidrug resistance protein 1 (MDR1) expression in k562/dox cells was downregulated by levistolide A in a dose­dependent manner, thus suggesting that levistolide A may modulate MDR1 during cancer therapy. Therefore, the combination of levistolide A with doxorubicin could result in more effective and less toxic anticancer regimens.

Quercetin inhibits Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R related Ca2+ fluctuations.

An allergic reaction is a potentially fatal hypersensitivity response caused by mast cell activation, particularly histamine and lipid mediators. Histamine release caused by reaction to drugs is considered a pseudo-allergic reaction. Quercetin is known for its anti-allergic immune effect. However, at present, its anti-pseudo-allergic effect and its mechanism are less investigated. Therefore, the purpose of this study was to evaluate the anti-pseudo-allergic effect of Quercetin in vivo and to explore the mechanism in vitro. The anti-pseudo-allergic activity of Quercetin was evaluated in vivo using a mouse model, while Quercetin mechanism of action was examined in vitro using HEK293 cells expressing Mrgprx2, a mast cell specific receptor, and LAD2 mast cell line. Our in vivo results showed that Quercetin could attenuate Evans blue leakage in the paws and hind paw thickness in C57BL/6 mice in a dose-dependent manner, and could significantly inhibit serum histamine and chemokines release. In addition, it suppressed calcium mobilization and attenuated the release of histamine and MCP-1 in peritoneal mast cells in a dose-dependent manner. Furthermore, it inhibited the vasodilation due to histamine, the release of eosinophils, and the percentage of degranulated mast cells, indicating that Quercetin antagonized mast cell mediators in vivo, histamine-induced vasodilation and eosinophil release. In vitro results showed that Quercetin reduced pseudo-allergic induced calcium influx, suppressed degranulation and chemokines release in a similar way as dexamethasone (100 µM) (mast cell stabilizer) in LAD2 mast cell line. In addition, Quercetin inhibited Mrgprx2-induced both calcium influx and pseudo-allergic reaction in HEK293 cells expressing Mrgprx2. C48/80, a histamine promoter, and Substance P (a neuropeptide) EC50 was higher when combined with Quercetin compared to the EC50 of these compounds alone, suggesting that Quercetin could inhibit Mrgprx2-induced pseudo-allergic reaction. Furthermore, Quercetin decreased PLCγ-IP3R signaling pathway activation induced by C48/80 in LAD2 mast cell line. In Mrgprx2 knockdown LAD2 cells, the effect of Quercetin (200 µM) reduced C48/80 induced calcium flux and the release of ß­hexosaminidase, histamine, MCP-1 and IL-8 compared with non-atopic control (NC) transfected LAD2 human mast cells, suggesting that Quercetin anti-pseudo-allergic effect was related to Mrgprx2. The docking results showed that Quercetin had a good binding affinity with Mrgprx2 similar to the one of Substance P and C48/80. Therefore, Quercetin inhibited Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations. Our results validated Quercetin as an effective small molecule inhibiting Mrgprx2-induced pseudo-allergic reaction via PLCγ-IP3R associated Ca2+ fluctuations, thus highlighting a potential candidate to suppress Mrgprx2 induced pseudo-allergic related diseases.

Protein kinase C mediates juvenile hormone-dependent phosphorylation of Na+/K+-ATPase to induce ovarian follicular patency for yolk protein uptake.

In oviparous animals, vitellogenesis is prerequisite to egg production and embryonic growth after oviposition. For successful insect vitellogenesis and oogenesis, vitellogenin (Vg) synthesized in the fat body (homologue to vertebrate liver and adipose tissue) must pass through the intercellular channels, a condition known as patency in the follicular epithelium, to reach the surface of oocytes. This process is controlled by juvenile hormone (JH) in many insect species, but the underlying mechanisms remain elusive. Previous work has suggested the possible involvement of Na+/K+-ATPase in patency initiation, but again, the regulatory cascade of Na+/K+-ATPase for patency initiation has been lacking. Using the migratory locust Locusta migratoria as a model system, we report here that RNAi-mediated knockdown of gene coding for Na+/K+-ATPase, inhibition of its phosphorylation, or suppression of its activity causes loss of patency, resulting in blocked Vg uptake, arrested oocyte maturation, and impaired ovarian growth. JH triggers G protein-coupled receptor (GPCR), receptor tyrosine kinase (RTK), phospholipase C (PLC), inositol trisphosphate receptor (IP3R), and protein kinase C (PKC) to phosphorylate Na+/K+-ATPase α-subunit at amino acid residue Ser8, consequently activating Na+/K+-ATPase for the induction of patency in vitellogenic follicular epithelium. Our results thus point to a previously unidentified mechanism by which JH induces the phosphorylation and activation of Na+/K+-ATPase via a signaling cascade of GPCR, RTK, PLC, IP3R, and PKC. The findings advance our understanding of JH regulation in insect vitellogenesis and oogenesis.